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Sample records for acid function biosynthesis

  1. [Biosynthesis of adipic acid].

    Science.gov (United States)

    Han, Li; Chen, Wujiu; Yuan, Fei; Zhang, Yuanyuan; Wang, Qinhong; Ma, Yanhe

    2013-10-01

    Adipic acid is a six-carbon dicarboxylic acid, mainly for the production of polymers such as nylon, chemical fiber and engineering plastics. Its annual demand is close to 3 million tons worldwide. Currently, the industrial production of adipic acid is based on the oxidation of aromatics from non-renewable petroleum resources by chemo-catalytic processes. It is heavily polluted and unsustainable, and the possible alternative method for adipic acid production should be developed. In the past years, with the development of synthetic biology and metabolic engineering, green and clean biotechnological methods for adipic acid production attracted more attention. In this study, the research advances of adipic acid and its precursor production are reviewed, followed by addressing the perspective of the possible new pathways for adipic acid production.

  2. Fatty Acid Biosynthesis IX

    DEFF Research Database (Denmark)

    Carey, E. M.; Hansen, Heinz Johs. Max; Dils, R.

    1972-01-01

    # 1. I. [I-14C]Acetate was covalently bound to rabbit mammary gland fatty acid synthetase by enzymic transacylation from [I-14C]acetyl-CoA. Per mole of enzyme 2 moles of acetate were bound to thiol groups and up to I mole of acetate was bound to non-thiol groups. # 2. 2. The acetyl-fatty acid...... synthetase complex was isolated free from acetyl-CoA. It was rapidly hydrolysed at 30°C, but hydrolysis was greatly diminished at o°C and triacetic lactone synthesis occurred. In the presence of malonyl-CoA and NADPH, all the acetate bound to fatty acid synthetase was incorporated into long-chain fatty acids....... Hydrolysis of bound acetate and incorporation of bound acetate into fatty acids were inhibited to the same extent by guanidine hydrochloride. # 3. 3. Acetate was also covalently bound to fatty acid synthetase by chemical acetylation with [I-14C]acetic anhydride in the absence of CoASH. A total of 60 moles...

  3. Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

    Science.gov (United States)

    Li, Rongfeng; Khaleeli, Nusrat; Townsend, Craig A.

    2000-01-01

    Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed. PMID:10869089

  4. Cloning and functional analysis of the second geranylgeranyl diphosphate synthase gene influencing helvolic acid biosynthesis in Metarhizium anisopliae.

    Science.gov (United States)

    Singkaravanit, Suthitar; Kinoshita, Hiroshi; Ihara, Fumio; Nihira, Takuya

    2010-07-01

    A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) gene was cloned from Metarhizium anisopliae NAFF635007. The ggs2 gene (1,239-bp open reading frame with no intron) encoded a protein of 412 amino acids, and the transcription occurred only after late log-phase during the growth. Gene disruption of ggs2, performed to clarify the function in M. anisopliae, resulted in decreased GGPP synthase activity together with a slight delay of sporulation. An high performance liquid chromatography (HPLC) comparison of compound profiles between the wild-type strain and the disruptant revealed that a compound was abolished by the ggs2 disruption. Purification and structural elucidation by 1H-NMR and mass spectrometry analyses revealed that the lost compound is helvolic acid. Furthermore, the pathogenicity assay against two species of insect larvae revealed that the ggs2-disruptant possessed much weaker toxicity than the wild-type strain. Based on these results, it was concluded that ggs2 encodes the GGPP synthase influencing the biosynthesis of secondary metabolites in various species, including helvolic acid in M. anisopliae. To the best of our knowledge, this is the first report to identify a GGPP synthase gene related to secondary metabolism in entomopathogenic fungi.

  5. Hyaluronic Acid--an "Old" Molecule with "New" Functions: Biosynthesis and Depolymerization of Hyaluronic Acid in Bacteria and Vertebrate Tissues Including during Carcinogenesis.

    Science.gov (United States)

    Tsepilov, R N; Beloded, A V

    2015-09-01

    Hyaluronic acid is an evolutionarily ancient molecule commonly found in vertebrate tissues and capsules of some bacteria. Here we review modern data regarding structure, properties, and biological functions of hyaluronic acid in mammals and Streptococcus spp. bacteria. Various aspects of biogenesis and degradation of hyaluronic acid are discussed, biosynthesis and degradation metabolic pathways for glycosaminoglycan together with involved enzymes are described, and vertebrate and bacterial hyaluronan synthase genes are characterized. Special attention is given to the mechanisms underlying the biological action of hyaluronic acid as well as the interaction between polysaccharide and various proteins. In addition, all known signaling pathways involving hyaluronic acid are outlined. Impaired hyaluronic acid metabolism, changes in biopolymer molecular weight, hyaluronidase activity, and enzyme isoforms often accompany carcinogenesis. The interaction between cells and hyaluronic acid from extracellular matrix that may be important during malignant change is discussed. An expected role for high molecular weight hyaluronic acid in resistance of naked mole rat to oncologic diseases and the protective role of hyaluronic acid in bacteria are discussed.

  6. 2-Oxoglutarate: linking TCA cycle function with amino acid, glucosinolate, flavonoid, alkaloid and gibberellin biosynthesis

    Directory of Open Access Journals (Sweden)

    Wagner L. Araújo

    2014-10-01

    Full Text Available The tricarboxylic acid (TCA cycle intermediate 2-oxoglutarate (2-OG is used as an obligatory substrate in a range of oxidative reactions catalyzed by 2-OG-dependent dioxygenases. These enzymes are widespread in nature being involved in several important biochemical processes. We have recently demonstrated that tomato plants in which the TCA cycle enzyme 2-OG dehydrogenase (2-ODD was antisense inhibited were characterized by early senescence and modified fruit ripening associated with differences in the levels of bioactive gibberellin (GA. Accordingly, there is now compelling evidence that the TCA cycle plays an important role in modulating the rate of flux from 2-OG to amino acid metabolism. Here we discuss recent advances in the biochemistry and molecular biology of 2-OG metabolism occurring in different biological systems indicating the importance of 2-OG and 2-OG dependent dioxygenases not only in glucosinolate, flavonoid and alkaloid metabolism but also in GA and amino acid metabolism. We additionally summarize recent findings regarding the impact of modification of 2-OG metabolism on biosynthetic pathways involving 2-ODDs.

  7. The biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis. Identification and functional analysis of CMAS-2.

    Science.gov (United States)

    George, K M; Yuan, Y; Sherman, D R; Barry, C E

    1995-11-10

    The major mycolic acid produced by Mycobacterium tuberculosis contains two cis-cyclopropanes in the meromycolate chain. The gene whose product cyclopropanates the proximal double bond was cloned by homology to a putative cyclopropane synthase identified from the Mycobacterium leprae genome sequencing project. This gene, named cma2, was sequenced and found to be 52% identical to cma1 (which cyclopropanates the distal double bond) and 73% identical to the gene from M. leprae. Both cma genes were found to be restricted in distribution to pathogenic species of mycobacteria. Expression of cma2 in Mycobacterium smegmatis resulted in the cyclopropanation of the proximal double bond in the alpha 1 series of mycolic acids. Coexpression of both cyclopropane synthases resulted in cyclopropanation of both centers, producing a molecule structurally similar to the M. tuberculosis alpha-dicyclopropyl mycolates. Differential scanning calorimetry of purified cell walls and mycolic acids demonstrated that cyclopropanation of the proximal position raised the observed transition temperature by 3 degrees C. These results suggest that cyclopropanation contributes to the structural integrity of the cell wall complex.

  8. Biosynthesis of Polyunsaturated Fatty Acids in Sea Urchins: Molecular and Functional Characterisation of Three Fatty Acyl Desaturases from Paracentrotus lividus (Lamark 1816)

    Science.gov (United States)

    Carboni, Stefano; Davie, Andrew; Oboh, Angela

    2017-01-01

    Sea urchins are broadly recognised as a delicacy and their quality as food for humans is highly influenced by their diet. Lipids in general and the long-chain polyunsaturated fatty acids (LC-PUFA) in particular, are essential nutrients that determine not only the nutritional value of sea urchins but also guarantee normal growth and reproduction in captivity. The contribution of endogenous production (biosynthesis) of LC-PUFA in sea urchins remained unknown. Using Paracentrotus lividus as our model species, we aimed to characterise both molecularly and functionally the repertoire of fatty acyl desaturases (Fads), key enzymes in the biosynthesis of LC-PUFA, in sea urchins. Three Fads, namely FadsA, FadsC1 and FadsC2, were characterised. The phylogenetic analyses suggested that the repertoire of Fads within the Echinodermata phylum varies among classes. On one hand, orthologues of the P. lividus FadsA were found in other echinoderm classes including starfishes, brittle stars and sea cucumbers, thus suggesting that this desaturase is virtually present in all echinoderms. Contrarily, the FadsC appears to be sea urchin-specific desaturase. Finally, a further desaturase termed as FadsB exists in starfishes, brittle stars and sea cucumbers, but appears to be missing in sea urchins. The functional characterisation of the P. lividus Fads confirmed that the FadsA was a Δ5 desaturase with activity towards saturated and polyunsaturated fatty acids (FA). Moreover, our experiments confirmed that FadsA plays a role in the biosynthesis of non-methylene interrupted FA, a group of compounds typically found in marine invertebrates. On the other hand, both FadsC desaturases from P. lividus showed Δ8 activity. The present results demonstrate that P. lividus possesses desaturases that account for all the desaturation reactions required to biosynthesis the physiological essential eicosapentaenoic and arachidonic acids through the so-called “Δ8 pathway”. PMID:28052125

  9. Biosynthesis and function of plant lipids

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, W.W.; Mudd, J.B.; Gibbs, M. (eds.)

    1983-01-01

    The Sixth Annual Symposium in Botany and Plant Physiology was held January 13-15, 1983, at the University of California, Riverside. This volume comprises the papers that were presented. Subjects discussed at the symposium covered a wide range in the field of plant lipids. Biosynthesis of lipids occupied an important fraction of the presentations at the symposium. Subjects included detailed studies of the enzymes of fatty acid synthesis, several discussions of the incorporation of fatty acids into glycerolipids and the further modification of the fatty acids, and the synthesis of glycerolipids and desaturation of fatty acids in both maturing oilseeds and chloroplasts. The physicochemical studies of glycerolipids and sterols in artificial membranes have led to distinct conclusions about their behaviour which must be relevant in the biological membrane. Results on the functional consequences of modifying the galactolipid composition in the chloroplast were an encouraging sign of progress in the attempts to relate membrane lipid composition to physiological function.

  10. Functional characterization of aromatic amino acid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts.

    Science.gov (United States)

    Hirata, Hiroshi; Ohnishi, Toshiyuki; Ishida, Haruka; Tomida, Kensuke; Sakai, Miwa; Hara, Masakazu; Watanabe, Naoharu

    2012-03-15

    In rose flowers, 2-phenylethanol (2PE) is biosynthesized from l-phenylalanine (l-Phe) via phenylacetaldehyde (PAld) by the actions of two enzymes, pyridoxal-5'-phosphate (PLP)-dependent aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). We here report that Rosa 'Yves Piaget' aromatic amino acid aminotransferase produced phenylpyruvic acid (PPA) from l-Phe in isolated petal protoplasts. We have cloned three full length cDNAs (RyAAAT1-3) of aromatic amino acid aminotransferase families based on rose EST database and homology regions. The RyAAATs enzymes were heterogeneously expressed in Escherichia coli and characterized biochemically. The recombinant RyAAAT3 showed the highest activity toward l-Phe in comparison with l-tryptophan, l-tyrosine, d-Phe, glycine, and l-alanine, and showed 9.7-fold higher activity with l-Phe rather than PPA as a substrate. RyAAAT3 had an optimal activity at pH 9 and at 45-55°C with α-ketoglutaric acid, and was found to be a PLP dependent enzyme based on the inhibition test using Carbidopa, an inhibitor of PLP-dependent enzymes. The transcript of RyAAAT3 was expressed in flowers as well as other organs of R. 'Yves Piaget'. RNAi suppression of RyAAAT3 decreased 2PE production, revealing the involvement of RyAAAT3 in 2PE biosynthesis in rose protoplasts and indicating that rose protoplasts have potentially two different 2PE biosynthetic pathways, the AADC route and the new route via PPA from l-Phe.

  11. Bile acid biosynthesis and its regulation

    Directory of Open Access Journals (Sweden)

    Areta Hebanowska

    2010-10-01

    Full Text Available Bile acid biosynthesis is the main pathway of cholesterol catabolism. Bile acids are more soluble than cholesterol so are easier to excrete. As amphipathic molecules they participate in lipid digestion and absorption in the intestine and they help to excrete free cholesterol with bile. They are also ligands for nuclear receptors regulating the expression of genes involved in cholesterol metabolism. Interconversion of cholesterol into bile acids is an important point of its homeostasis. Seventeen enzymes are engaged in this process and many of them are cytochromes P450. Bile acid synthesis initiation may proceed with the “classical” pathway (starting with cholesterol hydroxylation at the C7α position or the “alternative” pathway (starting with cholesterol hydroxylation at the C27 position. Two additional pathways are possible, though their quantitative significance is small (initiated with cholesterol hydroxylations of C24 and C25 positions. Oxysterols produced are not only intermediates of bile acid biosynthesis but also important regulators of metabolism. Bile acid biosynthesis takes place in the liver, but some enzymes are also present in other organs, where they participate in regulation of cholesterol metabolism. Those enzymes are potential targets for new drugs against cholesterol metabolism disturbances. This article is a brief description of the bile acid biosynthesis pathway and participating enzymes.

  12. Long-chain polyunsaturated fatty acid biosynthesis in the euryhaline herbivorous teleost Scatophagus argus: Functional characterization, tissue expression and nutritional regulation of two fatty acyl elongases.

    Science.gov (United States)

    Xie, Dizhi; Chen, Fang; Lin, Siyuan; You, Cuihong; Wang, Shuqi; Zhang, Qinghao; Monroig, Óscar; Tocher, Douglas R; Li, Yuanyou

    2016-08-01

    Both the spotted scat Scatophagus argus and rabbitfish Siganus canaliculatus belong to the few cultured herbivorous marine teleost, however, their fatty acyl desaturase (Fad) system involved in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is different. The S. argus has a △6 Fad, while the rabbitfish has △4 and △6/△5 Fads, which were the first report in vertebrate and marine teleost, respectively. In order to compare the characteristics of elongases of very long-chain fatty acids (Elovl) between them, two Elovl cDNAs were cloned from S. argus in the present study. One has 885bp of open read fragment (ORF) encoding a protein with 294 amino acid (aa) showing Elovl5 activity functionally characterized by heterologous expression in yeast, which was primarily active for the elongation of C18 and C20 PUFAs. The other has 915bp of ORF coding for a 305 aa protein showing Elovl4 activity, which was more efficient in the elongation of C20 and C22 PUFAs. Tissue distribution analyses by RT-PCR showed that elovl5 was highly expressed in the liver compared to other tissues determined, whereas elovl4 transcripts were only detected in the eye. The expression of elovl5 and elovl4 were significantly affected by dietary fatty acid composition, with highest expression of mRNA in the liver and eye of fish fed a diet with an 18:3n-3/18:2n-6 ratio of 1.7:1. These results indicated that the S. argus has a similar Elovl system in the LC-PUFA biosynthetic pathway to that of rabbitfish although their Fad system was different, suggesting that the diversification of fish LC-PUFA biosynthesis specificities is more associated with its Fad system. These new insights expand our knowledge and understanding of the molecular basis and regulation of LC-PUFA biosynthesis in fish.

  13. Pantothenic acid biosynthesis in zymomonas

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V.

    2014-07-01

    Zymomonas is unable to synthesize pantothenic acid and requires this essential vitamin in growth medium. Zymomonas strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pantothenic acid supplementation in seed culture and fermentation media.

  14. Plant Sterols: Diversity, Biosynthesis, and Physiological Functions.

    Science.gov (United States)

    Valitova, J N; Sulkarnayeva, A G; Minibayeva, F V

    2016-08-01

    Sterols, which are isoprenoid derivatives, are structural components of biological membranes. Special attention is now being given not only to their structure and function, but also to their regulatory roles in plants. Plant sterols have diverse composition; they exist as free sterols, sterol esters with higher fatty acids, sterol glycosides, and acylsterol glycosides, which are absent in animal cells. This diversity of types of phytosterols determines a wide spectrum of functions they play in plant life. Sterols are precursors of a group of plant hormones, the brassinosteroids, which regulate plant growth and development. Furthermore, sterols participate in transmembrane signal transduction by forming lipid microdomains. The predominant sterols in plants are β-sitosterol, campesterol, and stigmasterol. These sterols differ in the presence of a methyl or an ethyl group in the side chain at the 24th carbon atom and are named methylsterols or ethylsterols, respectively. The balance between 24-methylsterols and 24-ethylsterols is specific for individual plant species. The present review focuses on the key stages of plant sterol biosynthesis that determine the ratios between the different types of sterols, and the crosstalk between the sterol and sphingolipid pathways. The main enzymes involved in plant sterol biosynthesis are 3-hydroxy-3-methylglutaryl-CoA reductase, C24-sterol methyltransferase, and C22-sterol desaturase. These enzymes are responsible for maintaining the optimal balance between sterols. Regulation of the ratios between the different types of sterols and sterols/sphingolipids can be of crucial importance in the responses of plants to stresses.

  15. Flavonoids: Biosynthesis, Biological functions and Biotechnological applications

    Directory of Open Access Journals (Sweden)

    Maria Lorena eFalcone Ferreyra

    2012-09-01

    Full Text Available Flavonoids are widely distributed secondary metabolites with different metabolic functions in plants. The elucidation of the biosynthetic pathways, as well as their regulation by MYB, bHLH and WD40-type transcription factors, has allowed metabolic engineering of plants through the manipulation of the different final products with valuable applications. The present review describes the regulation of flavonoid biosynthesis, as well as the biological functions of flavonoids in plants, such as in defense against UV-B radiation and pathogen infection, nodulation, pollen fertility. In addition, we discuss different strategies and achievements through the genetic engineering of flavonoid biosynthesis with implication in the industry and the combinatorial biosynthesis in microorganisms by the reconstruction of the pathway to obtain high amounts of specific compounds.

  16. Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

    Science.gov (United States)

    Long, Michael C; Nagegowda, Dinesh A; Kaminaga, Yasuhisa; Ho, Kwok Ki; Kish, Christine M; Schnepp, Jennifer; Sherman, Debra; Weiner, Henry; Rhodes, David; Dudareva, Natalia

    2009-07-01

    Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

  17. Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis.

    Science.gov (United States)

    Božić, Dragana; Papaefthimiou, Dimitra; Brückner, Kathleen; de Vos, Ric C H; Tsoleridis, Constantinos A; Katsarou, Dimitra; Papanikolaou, Antigoni; Pateraki, Irini; Chatzopoulou, Fani M; Dimitriadou, Eleni; Kostas, Stefanos; Manzano, David; Scheler, Ulschan; Ferrer, Albert; Tissier, Alain; Makris, Antonios M; Kampranis, Sotirios C; Kanellis, Angelos K

    2015-01-01

    Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.

  18. Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis.

    Directory of Open Access Journals (Sweden)

    Dragana Božić

    Full Text Available Carnosic acid (CA is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage and Rosmarinus officinalis (Rosemary. To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC. Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.

  19. Plant Terpenoids: Biosynthesis and Ecological Functions

    Institute of Scientific and Technical Information of China (English)

    Ai-Xia Cheng; Yong-Gen Lou; Ying-Bo Mao; Shan Lu; Ling-Jian Wang; Xiao-Ya Chen

    2007-01-01

    Among plant secondary metabolites terpenoids are a structurally most diverse group; they function as phytoalexins in plant direct defense, or as signals in indirect defense responses which involves herbivores and their natural enemies. In recent years, more and more attention has been paid to the investigation of the ecological role of plant terpenoids. The biosynthesis pathways of monoterpenes, sesquiterpenes, and diterpenes include the synthesis of C5 precursor isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), the synthesis of the immediate diphosphate precursors, and the formation of the diverse terpenoids. Terpene synthases (TPSs) play a key role in volatile terpene synthesis. By expression of the TPS genes, significant achievements have been made on metabolic engineering to increase terpenoid production. This review mainly summarizes the recent research progress in elucidating the ecological role of terpenoids and characterization of the enzymes involved in the terpenoid biosynthesis. Spatial and temporal regulations of terpenoids metabolism are also discussed.

  20. Clavulanic acid biosynthesis and genetic manipulation for its overproduction.

    Science.gov (United States)

    Song, Ju Yeon; Jensen, Susan E; Lee, Kye Joon

    2010-10-01

    Clavulanic acid, a β-lactamase inhibitor, is used together with β-lactam antibiotics to create drug mixtures possessing potent antimicrobial activity. In view of the clinical and industrial importance of clavulanic acid, identification of the clavulanic acid biosynthetic pathway and the associated gene cluster(s) in the main producer species, Streptomyces clavuligerus, has been an intriguing research question. Clavulanic acid biosynthesis was revealed to involve an interesting mechanism common to all of the clavam metabolites produced by the organism, but different from that of other β-lactam compounds. Gene clusters involved in clavulanic acid biosynthesis in S. clavuligerus occupy large regions of nucleotide sequence in three loci of its genome. In this review, clavulanic acid biosynthesis and the associated gene clusters are discussed, and clavulanic acid improvement through genetic manipulation is explained.

  1. [Snake venom metalloproteinases: structure, biosynthesis and function(s)].

    Science.gov (United States)

    Limam, I; El Ayeb, M; Marrakchi, N

    2010-01-01

    The biochemical and the pharmacological characterization of snake venoms revealed an important structural and functional polymorphism of proteins which they contain. Among them, snake venom metalloproteases (SVMPs) constitute approximatively 20 to 60% of the whole venom proteins. During the last decades, a significant progress was performed against structure studies and the biosynthesis of the SVMPs. Indeed, several metalloproteases were isolated and characterized against their structural and pharmacological properties. In this review, we report the most important properties concerning the classification, the structure of the various domains of the SVMPs as well as their biosynthesis and their activities as potential therapeutic agents.

  2. Recent advances in the elucidation of enzymatic function in natural product biosynthesis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Tan Gao-Yi

    2015-12-01

    Full Text Available With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed.

  3. Recent advances in the elucidation of enzymatic function in natural product biosynthesis [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Gao-Yi Tan

    2016-02-01

    Full Text Available With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed.

  4. Inhibitors of amino acids biosynthesis as antifungal agents.

    Science.gov (United States)

    Jastrzębowska, Kamila; Gabriel, Iwona

    2015-02-01

    Fungal microorganisms, including the human pathogenic yeast and filamentous fungi, are able to synthesize all proteinogenic amino acids, including nine that are essential for humans. A number of enzymes catalyzing particular steps of human-essential amino acid biosynthesis are fungi specific. Numerous studies have shown that auxotrophic mutants of human pathogenic fungi impaired in biosynthesis of particular amino acids exhibit growth defect or at least reduced virulence under in vivo conditions. Several chemical compounds inhibiting activity of one of these enzymes exhibit good antifungal in vitro activity in minimal growth media, which is not always confirmed under in vivo conditions. This article provides a comprehensive overview of the present knowledge on pathways of amino acids biosynthesis in fungi, with a special emphasis put on enzymes catalyzing particular steps of these pathways as potential targets for antifungal chemotherapy.

  5. Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium.

    Science.gov (United States)

    Creelman, R A; Gage, D A; Stults, J T; Zeevaart, J A

    1987-11-01

    RESEARCH ON THE BIOSYNTHESIS OF ABSCISIC ACID (ABA) HAS FOCUSED PRIMARILY ON TWO PATHWAYS: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in (18)O(2). It was found that in stressed leaves three atoms of (18)O from (18)O(2) are incorporated into the ABA molecule, and that the amount of (18)O incorporated increases with time. One (18)O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in (18)O(2) shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more (18)O into the tertiary hydroxyl group at C-1' after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, (18)O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied (14)C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional (18)O incorporated during 8'-hydroxylation of ABA to phaseic acid.

  6. Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Thoden, James B.; Holden, Hazel M. (UW)

    2010-09-08

    2,3-Diacetamido-2,3-dideoxy-D-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-D-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3{prime} hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD{sup +} to NADH. This enzyme has been shown to use {alpha}-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and {alpha}-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both {alpha}-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3{prime}, respectively) abutting the re face of the cofactor. They are positioned {approx}3 {angstrom} from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.

  7. GENETIC ANALYSIS OF ABSCISIC ACID BIOSYNTHESIS

    Energy Technology Data Exchange (ETDEWEB)

    MCCARTY D R

    2012-01-10

    The carotenoid cleavage dioxygenases (CCD) catalyze synthesis of a variety of apo-carotenoid secondary metabolites in plants, animals and bacteria. In plants, the reaction catalyzed by the 11, 12, 9-cis-epoxy carotenoid dioxygenase (NCED) is the first committed and key regulated step in synthesis of the plant hormone, abscisic acid (ABA). ABA is a key regulator of plant stress responses and has critical functions in normal root and seed development. The molecular mechanisms responsible for developmental control of ABA synthesis in plant tissues are poorly understood. Five of the nine CCD genes present in the Arabidopsis genome encode NCED's involved in control of ABA synthesis in the plant. This project is focused on functional analysis of these five AtNCED genes as a key to understanding developmental regulation of ABA synthesis and dissecting the role of ABA in plant development. For this purpose, the project developed a comprehensive set of gene knockouts in the AtNCED genes that facilitate genetic dissection of ABA synthesis. These mutants were used in combination with key molecular tools to address the following specific objectives: (1) the role of ABA synthesis in root development; (2) developmental control of ABA synthesis in seeds; (3) analysis of ATNCED over-expressers; (4) preliminary crystallography of the maize VP14 protein.

  8. The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

    KAUST Repository

    Wang, Zhen-Yu

    2014-11-21

    Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.

  9. Molecular Basis for Mycophenolic Acid Biosynthesis in Penicillium brevicompactum

    DEFF Research Database (Denmark)

    Regueira, Torsten Ulrik Bak; Kildegaard, Kanchana Rueksomtawin; Hansen, Bjarne Gram;

    2011-01-01

    Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide...

  10. Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants.

    Science.gov (United States)

    de la Torre, Fernando; Cañas, Rafael A; Pascual, M Belén; Avila, Concepción; Cánovas, Francisco M

    2014-10-01

    In the chloroplasts and in non-green plastids of plants, aspartate is the precursor for the biosynthesis of different amino acids and derived metabolites that play distinct and important roles in plant growth, reproduction, development or defence. Aspartate biosynthesis is mediated by the enzyme aspartate aminotransferase (EC 2.6.1.1), which catalyses the reversible transamination between glutamate and oxaloacetate to generate aspartate and 2-oxoglutarate. Plastids contain two aspartate aminotransferases: a eukaryotic-type and a prokaryotic-type bifunctional enzyme displaying aspartate and prephenate aminotransferase activities. A general overview of the biochemistry, regulation, functional significance, and phylogenetic origin of both enzymes is presented. The roles of these plastidic aminotransferases in the biosynthesis of essential amino acids are discussed.

  11. Functional specialization in proline biosynthesis of melanoma.

    Directory of Open Access Journals (Sweden)

    Jessica De Ingeniis

    Full Text Available Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C, and then to proline via pyrroline-5-carboxylate reductases (PYCRs. Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of (13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.

  12. Functional Specialization in Proline Biosynthesis of Melanoma

    Science.gov (United States)

    Richardson, Adam D.; Scott, David A.; Aza-Blanc, Pedro; De, Surya K.; Kazanov, Marat; Pellecchia, Maurizio; Ronai, Ze'ev; Osterman, Andrei L.; Smith, Jeffrey W.

    2012-01-01

    Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis. PMID:23024808

  13. Molecular mechanisms of the coordination between astaxanthin and fatty acid biosynthesis in Haematococcus pluvialis (Chlorophyceae).

    Science.gov (United States)

    Chen, Guanqun; Wang, Baobei; Han, Danxiang; Sommerfeld, Milton; Lu, Yinghua; Chen, Feng; Hu, Qiang

    2015-01-01

    Astaxanthin, a red ketocarotenoid with strong antioxidant activity and high commercial value, possesses important physiological functions in astaxanthin-producing microalgae. The green microalga Haematococcus pluvialis accumulates up to 4% fatty acid-esterified astaxanthin (by dry weight), and is used as a model species for exploring astaxanthin biosynthesis in unicellular photosynthetic organisms. Although coordination of astaxanthin and fatty acid biosynthesis in a stoichiometric fashion was observed in H. pluvialis, the interaction mechanism is unclear. Here we dissected the molecular mechanism underlying coordination between the two pathways in H. pluvialis. Our results eliminated possible coordination of this inter-dependence at the transcriptional level, and showed that this interaction was feedback-coordinated at the metabolite level. In vivo and in vitro experiments indicated that astaxanthin esterification drove the formation and accumulation of astaxanthin. We further showed that both free astaxanthin biosynthesis and esterification occurred in the endoplasmic reticulum, and that certain diacylglycerol acyltransferases may be the candidate enzymes catalyzing astaxanthin esterification. A model of astaxanthin biosynthesis in H. pluvialis was subsequently proposed. These findings provide further insights into astaxanthin biosynthesis in H. pluvialis.

  14. Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana

    DEFF Research Database (Denmark)

    Qian, W; Yu, C; Qin, H

    2007-01-01

    -vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM......Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana......, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces...

  15. Branched-chain fatty acid biosynthesis in a branched-chain amino acid aminotransferase mutant of Staphylococcus carnosus

    DEFF Research Database (Denmark)

    Beck, Hans Christian

    2005-01-01

    Fatty acid biosynthesis by a mutant strain of Staphylococcus carnosus deficient in branched-chain amino acid aminotransferase (IlvE) activity was analysed. This mutant was unable to produce the appropriate branched-chain alpha-ketoacid precursors for branched-chain fatty acid biosynthesis from...... for 2-methylpropanoic acid production, revealing that the IlvE protein plays an important, but not essential role in the biosynthesis of branched-chain fatty acids and secondary metabolites in S. carnosus....

  16. Ceramide biosynthesis in keratinocyte and its role in skin function.

    Science.gov (United States)

    Mizutani, Yukiko; Mitsutake, Susumu; Tsuji, Kiyomi; Kihara, Akio; Igarashi, Yasuyuki

    2009-06-01

    The enucleate layer of the epidermis, i.e. the stratum corneum, is responsible for certain critical protective functions, such as epidermal permeability barrier function. Within the epidermal membrane lamella component, ceramides are the dominant lipid class by weight (over 50%) and exhibit the greatest molecular heterogeneity in terms of sphingoid base and fatty acid composition. It is now evermore important to understand how ceramide production and functions are controlled in the epidermis, since decreased epidermal ceramide content has been linked to water loss and barrier dysfunction. During the past several years, critical enzymes in ceramide biosynthesis have been identified, including ceramide synthases (CerS) and ceramide hydroxylase/desaturase. In this review, we describe the molecular heterogeneity of ceramides synthesized in the epidermis and their possible roles in epidermal permeability barrier functions. We also describe recent studies that identified the family of CerS (CerS1-CerS6) in mammals. We further focus on the roles of specific isoforms of these enzymes in synthesizing the epidermal ceramides, especially in relation to chain-length specificity. In addition, we provide experimental information, including our recent findings, as to how applying ceramide or ceramide-containing substances to skin, orally or directly, can benefit skin health.

  17. Structural and Functional Characterization of PseC, an Aminotransferase Involved in the Biosynthesis of Pseudaminic Acid, an Essential Flagellar Modification in Helicobacter Pylori

    Energy Technology Data Exchange (ETDEWEB)

    Schoenhofen,I.; Lunin, V.; Julien, J.; Li, Y.; Ajamian, E.; Matte, A.; Cygler, M.; Brisson, J.; Aubry, A.; et al.

    2006-01-01

    Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. As motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-a-D-GlcNAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-{beta}-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc product, the latter being converted to the external aldimine form in the enzyme's active site. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the {beta}-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and in conjunction with the recent functional characterization of this enzyme, will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.

  18. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Lei [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Xiao, Yongsheng [Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States); Wang, Yinsheng, E-mail: yinsheng.wang@ucr.edu [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States)

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  19. Biosynthesis and functions of sulfur modifications in tRNA

    Directory of Open Access Journals (Sweden)

    Naoki eShigi

    2014-04-01

    Full Text Available Sulfur is an essential element for a variety of cellular constituents in all living organisms. In tRNA molecules, there are many sulfur-containing nucleosides, such as the derivatives of 2‑thiouridine (s2U, 4-thiouridine (s4U, 2-thiocytidine (s2C, and 2-methylthioadenosine (ms2A. Earlier studies established the functions of these modifications for accurate and efficient translation, including proper recognition of the codons in mRNA or stabilization of tRNA structure. In many cases, the biosynthesis of these sulfur modifications starts with cysteine desulfurases, which catalyze the generation of persulfide (an activated form of sulfur from cysteine. Many sulfur-carrier proteins are responsible for delivering this activated sulfur to each biosynthesis pathway. Finally, specific modification enzymes activate target tRNAs and then incorporate sulfur atoms. Intriguingly, the biosynthesis of 2-thiouridine in all domains of life is functionally and evolutionarily related to the ubiquitin-like post-translational modification system of cellular proteins in eukaryotes. This review summarizes the recent characterization of the biosynthesis of sulfur modifications in tRNA and the novel roles of this modification in cellular functions in various model organisms, with a special emphasis on 2-thiouridine derivatives. Each biosynthesis pathway of sulfur-containing molecules is mutually modulated via sulfur trafficking, and 2-thiouridine and codon usage bias have been proposed to control the translation of specific genes.

  20. Effect of Methyl Jasmonic Acid on Baccatin Ⅲ Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jianfeng; GUO Zhigang

    2006-01-01

    As baccatin Ⅲ is an immediate diterpenoid precursor of taxol, the increase of baccatin Ⅲ is beneficial to the biosynthesis of taxol. Addition of methyl jasmonic acid (M J) enhances the activity of 10- deaceyle baccatin (DAB) Ⅲ acetyl transferase which catalyzes the bioconversion from 10-DAB Ⅲ to beccatin Ⅲ. In this paper, the baccatin Ⅲ content was increased by 174% by the addition of 100 μmol/L MJ in suspension cultures of Taxus cuspidate. Induction by MJ also increased the expression of a 49.0-kDa protein. This paper describes the cell free acetylation of 10-DAB Ⅲ in crude extracts of enzyme from the suspension cultures of Taxus cuspidate. The reaction product was confirmed by high performance liquid chromatograph (HPLC). About 25.0% of the 10-DAB Ⅲ was acetylized into baccatin Ⅲ on the 4th day with 100 μmol/L MJ.The 10-DAB Ⅲ acetyl transferase activity reached a peak on the 2nd day with 100 μmol/L M J, with 54.7% of the 10-DAB Ⅲ transformed into baccatin Ⅲ. The baccatin Ⅲ content increased with the increase of 10-DAB Ⅲ acetyl transferase activity, although the biosynthesis was delayed by more than 24 h. The remarkable induction of MJ on baccatin Ⅲ biosynthesis shows a promising way to increase the production of taxol.

  1. Kinetic Modeling of Sunflower Grain Filling and Fatty Acid Biosynthesis

    Science.gov (United States)

    Durruty, Ignacio; Aguirrezábal, Luis A. N.; Echarte, María M.

    2016-01-01

    Grain growth and oil biosynthesis are complex processes that involve various enzymes placed in different sub-cellular compartments of the grain. In order to understand the mechanisms controlling grain weight and composition, we need mathematical models capable of simulating the dynamic behavior of the main components of the grain during the grain filling stage. In this paper, we present a non-structured mechanistic kinetic model developed for sunflower grains. The model was first calibrated for sunflower hybrid ACA855. The calibrated model was able to predict the theoretical amount of carbohydrate equivalents allocated to the grain, grain growth and the dynamics of the oil and non-oil fraction, while considering maintenance requirements and leaf senescence. Incorporating into the model the serial-parallel nature of fatty acid biosynthesis permitted a good representation of the kinetics of palmitic, stearic, oleic, and linoleic acids production. A sensitivity analysis showed that the relative influence of input parameters changed along grain development. Grain growth was mostly affected by the specific growth parameter (μ′) while fatty acid composition strongly depended on their own maximum specific rate parameters. The model was successfully applied to two additional hybrids (MG2 and DK3820). The proposed model can be the first building block toward the development of a more sophisticated model, capable of predicting the effects of environmental conditions on grain weight and composition, in a comprehensive and quantitative way. PMID:27242809

  2. Isolation and characterization of Pseudomonas aeruginosa mutants requiring salicylic acid for pyochelin biosynthesis.

    OpenAIRE

    Ankenbauer, R G; Cox, C D

    1988-01-01

    Pseudomonas aeruginosa mutants requiring salicylic acid for pyochelin biosynthesis were isolated after chemical mutagenesis by plating on a siderophore detection medium. Like the wild type, these mutants incorporated 7-[14C]salicylic acid into pyochelin, demonstrating that salicylic acid is an intermediate in the biosynthesis pathway of pyochelin.

  3. An Integrative Analysis of the Effects of Auxin on Jasmonic Acid Biosynthesis in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Jun Liu; Xiu-Jie Wang

    2006-01-01

    Auxin and jasmonic acid (JA) are two plant phytohormones that both participate in the regulation of many developmental processes. Jasmonic acid also plays important roles in plant stress response reactions.Although extensive investigations have been undertaken to study the biological functions of auxin and JA,little attention has been paid to the cross-talk between their regulated pathways. In the few available reports examining the effects of auxin on the expression of JA or JA-responsive genes, both synergetic and antagonistic results have been found. To further investigate the relationship between auxin and JA, we adopted an integrative method that combines microarray expression data with pathway information to study the behavior of the JA biosynthesis pathway under auxin treatment. Our results showed an overall downregulation of genes involved in JA biosynthesis, providing the first report of a relationship between auxin and the JA synthesis pathway in Arabidopsis seedlings.

  4. Salicylic acid induces mitochondrial injury by inhibiting ferrochelatase heme biosynthesis activity.

    Science.gov (United States)

    Gupta, Vipul; Liu, Shujie; Ando, Hideki; Ishii, Ryohei; Tateno, Shumpei; Kaneko, Yuki; Yugami, Masato; Sakamoto, Satoshi; Yamaguchi, Yuki; Nureki, Osamu; Handa, Hiroshi

    2013-12-01

    Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECH-salicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.

  5. FUNCTIONAL SPECIALIZATION OF DUPLICATED FLAVONOID BIOSYNTHESIS GENES IN WHEAT

    Directory of Open Access Journals (Sweden)

    Khlestkina E.

    2012-08-01

    Full Text Available Gene duplication followed by subfunctionalization and neofunctionalization is of a great evolutionary importance. In plant genomes, duplicated genes may result from either polyploidization (homoeologous genes or segmental chromosome duplications (paralogous genes. In allohexaploid wheat Triticum aestivum L. (2n=6x=42, genome BBAADD, both homoeologous and paralogous copies were found for the regulatory gene Myc encoding MYC-like transcriptional factor in the biosynthesis of flavonoid pigments, anthocyanins, and for the structural gene F3h encoding one of the key enzymes of flavonoid biosynthesis, flavanone 3-hydroxylase. From the 5 copies (3 homoeologous and 2 paralogous of the Myc gene found in T. aestivum, only one plays a regulatory role in anthocyanin biosynthesis, interacting complementary with another transcriptional factor (MYB-like to confer purple pigmentation of grain pericarp in wheat. The role and functionality of the other 4 copies of the Myc gene remain unknown. From the 4 functional copies of the F3h gene in T. aestivum, three homoeologues have similar function. They are expressed in wheat organs colored with anthocyanins or in the endosperm, participating there in biosynthesis of uncolored flavonoid substances. The fourth copy (the B-genomic paralogue is transcribed neither in wheat organs colored with anthocyanins nor in seeds, however, it’s expression has been noticed in roots of aluminium-stressed plants, where the three homoeologous copies are not active. Functional diversification of the duplicated flavonoid biosynthesis genes in wheat may be a reason for maintenance of the duplicated copies and preventing them from pseudogenization.The study was supported by RFBR (11-04-92707. We also thank Ms. Galina Generalova for technical assistance.

  6. Effect of low temperature on highly unsaturated fatty acid biosynthesis in activated sludge.

    Science.gov (United States)

    He, Su; Ding, Li-Li; Xu, Ke; Geng, Jin-Ju; Ren, Hong-Qiang

    2016-07-01

    Low temperature is a limiting factor for the microbial activity of activated sludge for sewage treatment plant in winter. Highly unsaturated fatty acid (UFA) biosynthesis, phospholipid fatty acid (PLFA) constituents and microbial structure in activated sludge at low temperature were investigated. Over 12 gigabases of metagenomic sequence data were generated with the Illumina HiSeq 2000 platform. The result showed 43.11% of phospholipid fatty acid (PLFA) in the activated sludge participated in UFA biosynthesis, and γ-Linolenic could be converted to Arachidonic acid at low temperature. The highly UFA biosynthesis in activated sludge was n-6 highly UFA biosynthesis, rather than n-3 highly UFA biosynthesis. The microbial community structures of activated sludge were analyzed by PLFA and high-throughput sequencing (HiSeq) simultaneously. Acidovorax, Pseudomonas, Flavobacterium and Polaromonas occupied higher percentage at 5°C, and genetic changes of highly UFA biosynthesis derived from microbial community structures change.

  7. The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis

    KAUST Repository

    Wang, Zhenyu

    2011-05-01

    Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 genee xpression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxy genase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol)treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly thatCED1 encodes a putative a/b hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cut in biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling. © 2011 American Society of Plant Biologists. All rights reserved.

  8. Genetic Dissection of Tropodithietic Acid Biosynthesis by Marine Roseobacters

    DEFF Research Database (Denmark)

    Geng, Haifeng; Bruhn, Jesper Bartholin; Nielsen, Kristian Fog;

    2008-01-01

    formation is coincident with the production of an antibiotic and a yellow-brown pigment. In this report, we demonstrate that the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis...... by the bacteria, and mutation in any one of these results in a loss of antibiotic activity (Tda(-)) and pigment production. Unexpectedly, six of the genes, referred to as tdaA-F, could not be found on the annotated TM1040 genome and were instead located on a previously unidentified plasmid (ca. 130 kb; pSTM3......) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp. strain TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter sp. strain 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were...

  9. Function of laccases in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Larsen, Anders; Holm, Preben Bach; Andersen, Jeppe Reitan

    2011-01-01

    substrate specificities and expression patterns. As part of the strategic research centre Bio4Bio, the present project deals with laccase functions in relation to cell wall formation in grasses based on a study of the model species Brachypodium distachyon. Thirty-one isozymes have been retrieved from......Laccases are multicopper oxidases capable of polymerizing monolignols. Histochemical assays have shown temporal and spatial correlation with secondary cell wall formation in both herbs and woody perennials. However, in plants laccases constitutes a relatively large group of isoenzymes with unique...... hybridization. Specific isozymes that show high correlation with the process of secondary cell wall formation will be further studied in a reverse genetic study in which candidates will be knocked out using RNA interference. Phenotypes of knock-out mutants are to be described in relation to cell wall...

  10. Dietary Polyunsaturated Fatty Acids and Inflammation: The Role of Phospholipid Biosynthesis

    OpenAIRE

    Sordillo, Lorraine M.; William Raphael

    2013-01-01

    The composition of fatty acids in the diets of both human and domestic animal species can regulate inflammation through the biosynthesis of potent lipid mediators. The substrates for lipid mediator biosynthesis are derived primarily from membrane phospholipids and reflect dietary fatty acid intake. Inflammation can be exacerbated with intake of certain dietary fatty acids, such as some ω-6 polyunsaturated fatty acids (PUFA), and subsequent incorporation into membrane phospholipids. Inflammati...

  11. Co-expression Analysis Identifies CRC and AP1 the Regulator of Arabidopsis Fatty Acid Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Xinxin Han; Linlin Yin; Hongwei Xue

    2012-01-01

    Fatty acids (FAs) play crucial rules in signal transduction and plant development,however,the regulation of FA metabolism is still poorly understood.To study the relevant regulatory network,fifty-eight FA biosynthesis genes including de novo synthases,desaturases and elongases were selected as "guide genes" to construct the co-expression network.Calculation of the correlation between all Arabidopsis thaliana (L.) genes with each guide gene by Arabidopsis co-expression dating mining tools (ACT)identifies 797 candidate FA-correlated genes.Gene ontology (GO) analysis of these co-expressed genes showed they are tightly correlated to photosynthesis and carbohydrate metabolism,and function in many processes.Interestingly,63 transcription factors (TFs) were identified as candidate FA biosynthesis regulators and 8 TF families are enriched.Two TF genes,CRC and AP1,both correlating with 8 FA guide genes,were further characterized.Analyses of the ap1 and crc mutant showed the altered total FA composition of mature seeds.The contents of palmitoleic acid,stearic acid,arachidic acid and eicosadienoic acid are decreased,whereas that of oleic acid is increased in ap1 and crc seeds,which is consistent with the qRT-PCR analysis revealing the suppressed expression of the corresponding guide genes.In addition,yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) revealed that CRC can bind to the promoter regions of KCS7 and KCS15,indicating that CRC may directly regulate FA biosynthesis.

  12. Extra-gonadal sites of estrogen biosynthesis and function.

    Science.gov (United States)

    Barakat, Radwa; Oakley, Oliver; Kim, Heehyen; Jin, Jooyoung; Ko, CheMyong Jay

    2016-09-01

    Estrogens are the key hormones regulating the development and function of reproductive organs in all vertebrates. Recent evidence indicates that estrogens play important roles in the immune system, cancer development, and other critical biological processes related to human well-being. Obviously, the gonads (ovary and testis) are the primary sites of estrogen synthesis, but estrogens synthesized in extra- gonadal sites play an equally important role in controlling biological activities. Understanding non-gonadal sites of estrogen synthesis and function is crucial and will lead to therapeutic interventions targeting estrogen signaling in disease prevention and treatment. Developing a rationale targeting strategy remains challenging because knowledge of extra-gonadal biosynthesis of estrogens, and the mechanism by which estrogen activity is exerted, is very limited. In this review, we will summarize recent discoveries of extra-gonadal sites of estrogen biosynthesis and their local functions and discuss the significance of the most recent novel discovery of intestinal estrogen biosynthesis. [BMB Reports 2016; 49(9): 488-496].

  13. Characterization of a Pipecolic Acid Biosynthesis Pathway Required for Systemic Acquired Resistance.

    Science.gov (United States)

    Ding, Pingtao; Rekhter, Dmitrij; Ding, Yuli; Feussner, Kirstin; Busta, Lucas; Haroth, Sven; Xu, Shaohua; Li, Xin; Jetter, Reinhard; Feussner, Ivo; Zhang, Yuelin

    2016-10-01

    Systemic acquired resistance (SAR) is an immune response induced in the distal parts of plants following defense activation in local tissue. Pipecolic acid (Pip) accumulation orchestrates SAR and local resistance responses. Here, we report the identification and characterization of SAR-DEFICIENT4 (SARD4), which encodes a critical enzyme for Pip biosynthesis in Arabidopsis thaliana Loss of function of SARD4 leads to reduced Pip levels and accumulation of a Pip precursor, Δ(1)-piperideine-2-carboxylic acid (P2C). In Escherichia coli, expression of the aminotransferase ALD1 leads to production of P2C and addition of SARD4 results in Pip production, suggesting that a Pip biosynthesis pathway can be reconstituted in bacteria by coexpression of ALD1 and SARD4. In vitro experiments showed that ALD1 can use l-lysine as a substrate to produce P2C and P2C is converted to Pip by SARD4. Analysis of sard4 mutant plants showed that SARD4 is required for SAR as well as enhanced pathogen resistance conditioned by overexpression of the SAR regulator FLAVIN-DEPENDENT MONOOXYGENASE1. Compared with the wild type, pathogen-induced Pip accumulation is only modestly reduced in the local tissue of sard4 mutant plants, but it is below detection in distal leaves, suggesting that Pip is synthesized in systemic tissue by SARD4-mediated reduction of P2C and biosynthesis of Pip in systemic tissue contributes to SAR establishment.

  14. Biosynthesis of natural products containing β-amino acids.

    Science.gov (United States)

    Kudo, Fumitaka; Miyanaga, Akimasa; Eguchi, Tadashi

    2014-08-01

    Covering: up to January, 2014. We focus here on β-amino acids as components of complex natural products because the presence of β-amino acids produces structural diversity in natural products and provides characteristic architectures beyond those of ordinary α-L-amino acids, thus generating significant and unique biological functions in nature. In this review, we first survey the known bioactive β-amino acid-containing natural products including nonribosomal peptides, macrolactam polyketides, and nucleoside-β-amino acid hybrids. Next, the biosynthetic enzymes that form β-amino acids from α-amino acids and the de novo synthesis of β-amino acids are summarized. Then, the mechanisms of β-amino acid incorporation into natural products are reviewed. Because it is anticipated that the rational swapping of the β-amino acid moieties with various side chains and stereochemistries by biosynthetic engineering should lead to the creation of novel architectures and bioactive compounds, the accumulation of knowledge regarding β-amino acid-containing natural product biosynthetic machinery could have a significant impact in this field. In addition, genome mining of characteristic β-amino acid biosynthetic genes and unique β-amino acid incorporation machinery could lead to the discovery of new β-amino acid-containing natural products.

  15. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    Directory of Open Access Journals (Sweden)

    Daniel Braga

    2016-12-01

    Full Text Available Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

  16. Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA from renewable carbon

    Directory of Open Access Journals (Sweden)

    Müller Roland H

    2010-02-01

    Full Text Available Abstract Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert-butyl ether (MTBE in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB. This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source.

  17. PGC-1alpha activates CYP7A1 and bile acid biosynthesis.

    Science.gov (United States)

    Shin, Dong-Ju; Campos, Jose A; Gil, Gregorio; Osborne, Timothy F

    2003-12-12

    Cholesterol 7-alpha-hydroxylase (CYP7A1) is the key enzyme that commits cholesterol to the neutral bile acid biosynthesis pathway and is highly regulated. In the current studies, we have uncovered a role for the transcriptional co-activator PGC-1alpha in CYP7A1 gene transcription. PGC-1alpha plays a vital role in adaptive thermogenesis in brown adipose tissue and stimulates genes important to mitochondrial function and oxidative metabolism. It is also involved in the activation of hepatic gluconeogenesic gene expression during fasting. Because the mRNA for CYP7A1 was also induced in mouse liver by fasting, we reasoned that PGC-1alpha might be an important co-activator for CYP7A1. Here we show that PGC-1alpha and CYP7A1 are also co-induced in livers of mice in response to streptozotocin induced diabetes. Additionally, infection of cultured HepG2 cells with a recombinant adenovirus expressing PGC-1alpha directly activates CYP7A1 gene expression and increases bile acid biosynthesis as well. Furthermore, we show that PGC-1alpha activates the CYP7A1 promoter directly in transient transfection assays in cultured cells. Thus, PGC-1alpha is a key activator of CYP7A1 and bile acid biosynthesis and is likely responsible for the fasting and diabetes dependent induction of CYP7A1. PGC-1alpha has already been shown to be a critical activator of several other oxidative processes including adaptive thermogenesis and fatty acid oxidation. Our studies provide further evidence of the fundamental role played by PGC-1alpha in oxidative metabolism and define PGC-1alpha as a link between diabetes and bile acid metabolism.

  18. A 7-deoxyloganetic acid glucosyltransferase contributes a key step in secologanin biosynthesis in Madagascar periwinkle.

    Science.gov (United States)

    Asada, Keisuke; Salim, Vonny; Masada-Atsumi, Sayaka; Edmunds, Elizabeth; Nagatoshi, Mai; Terasaka, Kazuyoshi; Mizukami, Hajime; De Luca, Vincenzo

    2013-10-01

    Iridoids form a broad and versatile class of biologically active molecules found in thousands of plant species. In addition to the many hundreds of iridoids occurring in plants, some iridoids, such as secologanin, serve as key building blocks in the biosynthesis of thousands of monoterpene indole alkaloids (MIAs) and many quinoline alkaloids. This study describes the molecular cloning and functional characterization of three iridoid glucosyltransfeases (UDP-sugar glycosyltransferase6 [UGT6], UGT7, and UGT8) from Madagascar periwinkle (Catharanthus roseus) with remarkably different catalytic efficiencies. Biochemical analyses reveal that UGT8 possessed a high catalytic efficiency toward its exclusive iridoid substrate, 7-deoxyloganetic acid, making it better suited for the biosynthesis of iridoids in periwinkle than the other two iridoid glucosyltransfeases. The role of UGT8 in the fourth to last step in secologanin biosynthesis was confirmed by virus-induced gene silencing in periwinkle plants, which reduced expression of this gene and resulted in a large decline in secologanin and MIA accumulation within silenced plants. Localization studies of UGT8 using a carborundum abrasion method for RNA extraction show that its expression occurs preferentially within periwinkle leaves rather than in epidermal cells, and in situ hybridization studies confirm that UGT8 is preferentially expressed in internal phloem associated parenchyma cells of periwinkle species.

  19. Expanding the modular ester fermentative pathways for combinatorial biosynthesis of esters from volatile organic acids.

    Science.gov (United States)

    Layton, Donovan S; Trinh, Cong T

    2016-08-01

    Volatile organic acids are byproducts of fermentative metabolism, for example, anaerobic digestion of lignocellulosic biomass or organic wastes, and are often times undesired inhibiting cell growth and reducing directed formation of the desired products. Here, we devised a general framework for upgrading these volatile organic acids to high-value esters that can be used as flavors, fragrances, solvents, and biofuels. This framework employs the acid-to-ester modules, consisting of an AAT (alcohol acyltransferase) plus ACT (acyl CoA transferase) submodule and an alcohol submodule, for co-fermentation of sugars and organic acids to acyl CoAs and alcohols to form a combinatorial library of esters. By assembling these modules with the engineered Escherichia coli modular chassis cell, we developed microbial manufacturing platforms to perform the following functions: (i) rapid in vivo screening of novel AATs for their catalytic activities; (ii) expanding combinatorial biosynthesis of unique fermentative esters; and (iii) upgrading volatile organic acids to esters using single or mixed cell cultures. To demonstrate this framework, we screened for a set of five unique and divergent AATs from multiple species, and were able to determine their novel activities as well as produce a library of 12 out of the 13 expected esters from co-fermentation of sugars and (C2-C6) volatile organic acids. We envision the developed framework to be valuable for in vivo characterization of a repertoire of not-well-characterized natural AATs, expanding the combinatorial biosynthesis of fermentative esters, and upgrading volatile organic acids to high-value esters. Biotechnol. Bioeng. 2016;113: 1764-1776. © 2016 Wiley Periodicals, Inc.

  20. Metabolic engineering of chloroplasts for artemisinic acid biosynthesis and impact on plant growth

    Indian Academy of Sciences (India)

    Bhawna Saxena; Mayavan Subramaniyan; Karan Malhotra; Neel Sarovar Bhavesh; Shobha Devi Potlakayala; Shashi Kumar

    2014-03-01

    Chloroplasts offer high-level transgene expression and transgene containment due to maternal inheritance, and are ideal hosts for biopharmaceutical biosynthesis via multigene engineering. To exploit these advantages, we have expressed 12 enzymes in chloroplasts for the biosynthesis of artemisinic acid (precursor of artemisinin, antimalarial drug) in an alternative plant system. Integration of transgenes into the tobacco chloroplast genome via homologous recombination was confirmed by molecular analysis, and biosynthesis of artemisinic acid in plant leaf tissues was detected with the help of 13C NMR and ESI-mass spectrometry. The excess metabolic flux of isopentenyl pyrophosphate generated by an engineered mevalonate pathway was diverted for the biosynthesis of artemisinic acid. However, expression of megatransgenes impacted the growth of the transplastomic plantlets. By combining two exogenous pathways, artemisinic acid was produced in transplastomic plants, which can be improved further using better metabolic engineering strategies for commercially viable yield of desirable isoprenoid products.

  1. Functional Diversity of Genes for the Biosynthesis of Paeoniflorin and Its Derivatives in Paeonia

    Directory of Open Access Journals (Sweden)

    Luqi Huang

    2013-09-01

    Full Text Available The Paeonia root, with or without bark, are considered vital traditional Chinese medicine materials; the examples are those of Bai Shao, Chi Shao, and Dan Pi. In this study, we examine 24 genes and their expressions involved in the biosynthesis of paeoniflorin and its derivatives, which are active compounds of the Paeonia root, in Paeonia lactiflora and P. suffruticosa, as well as other related plants, Punica granatum, Rhus radicans, and Coriaria nepalensis. Our phylogenetic analyses suggest that these genes have functional diversity, and analysis of the transcriptional level shows paeoniflorin and gallic acid biosynthesis-related genes exhibit different transcription profiles in flowers, carpels, bark-free roots, and bark of P. lactiflora. The correlation analysis of gene expression and active compound contents support the idea that hydroxymethylglutaryl-CoA synthase and phosphomevalonate kinase in the mevalonate pathway and 3-dehydroquinate dehydratase/shikimate dehydrogenase in shikimate biosynthesis are potentially closely related to the accumulation of paeoniflorin and benzoylpaeoniflorin. Coupling gene diversity with chemical analysis, we show that paeoniflorin and its derived aromatic amino acids are predominant in bark.

  2. The Catalytic Machinery of a Key Enzyme in Amino Acid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Ronald E. Viola

    2011-01-01

    Full Text Available The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate β-semialdehyde dehydrogenase (ASADH catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

  3. The Catalytic Machinery of a Key Enzyme in Amino Acid Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Viola, Ronald E.; Faehnle, Christopher R.; Blanco, Julio; Moore, Roger A.; Liu, Xuying; Arachea, Buenafe T.; Pavlovsky, Alexander G. (Toledo); (Yale); (Cold Spring); (NIH)

    2013-02-28

    The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate {beta}-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

  4. Function and distribution of bilin biosynthesis enzymes in photosynthetic organisms.

    Science.gov (United States)

    Dammeyer, Thorben; Frankenberg-Dinkel, Nicole

    2008-10-01

    Bilins are open-chain tetrapyrrole molecules essential for light-harvesting and/or sensing in many photosynthetic organisms. While they serve as chromophores in phytochrome-mediated light-sensing in plants, they additionally function in light-harvesting in cyanobacteria, red algae and cryptomonads. Associated to phycobiliproteins a variety of bile pigments is responsible for the specific light-absorbance properties of the organisms enabling efficient photosynthesis under different light conditions. The initial step of bilin biosynthesis is the cleavage of heme by heme oxygenases (HO) to afford the first linear molecule biliverdin. This reaction is ubiquitously found also in non-photosynthetic organisms. Biliverdin is then further reduced by site specific reductases most of them belonging to the interesting family of ferredoxin-dependent bilin reductases (FDBRs)-a new family of radical oxidoreductases. In recent years much progress has been made in the field of heme oxygenases but even more in the widespread family of FDBRs, revealing novel biochemical FDBR activities, new crystal structures and new ecological aspects, including the discovery of bilin biosynthesis genes in wild marine phage populations. The aim of this review is to summarize and discuss the recent progress in this field and to highlight the new and remaining questions.

  5. Metazoan Remaining Genes for Essential Amino Acid Biosynthesis: Sequence Conservation and Evolutionary Analyses

    Directory of Open Access Journals (Sweden)

    Igor R. Costa

    2014-12-01

    Full Text Available Essential amino acids (EAA consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS and betaine-homocysteine S-methyltransferase (BHMT diverged from the expected Tree of Life (ToL relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  6. Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex

    Directory of Open Access Journals (Sweden)

    Lin Yuheng

    2012-04-01

    Full Text Available Abstract Background Caffeic acid (3,4-dihydroxycinnamic acid is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. Results We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H, is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. Conclusion We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis

  7. Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1)

    Science.gov (United States)

    Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; Allen, Eric E.; Kalyuzhnaya, Marina G.

    2017-01-01

    Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph. PMID:28119683

  8. Biosynthesis of pyruvic acid from glucose by Blastobotrys adeninivorans.

    Science.gov (United States)

    Kamzolova, Svetlana V; Morgunov, Igor G

    2016-09-01

    The ability of taxonomically different yeasts to synthesize pyruvic acid (PA) from glucose was studied. The study showed that many yeasts are able to produce PA from glucose under the condition of growth limitation by thiamine. This ability was found in the yeast Blastobotrys adeninivorans for the first time. The production (oversynthesis) of PA in this yeast can be explained by disturbance in the function of thiamine-dependent pyruvate dehydrogenase. Namely, the partial inhibition of this enzyme brings about the excretion of PA from the yeast cells. Due to incomplete inhibition of pyruvate dehydrogenase, the formation of acetyl-CoA continues, although at a lower level, maintaining the synthesis of α-ketoglutaric acid (KGA) in the tricarboxylic acid (TCA) cycle. KGA is no longer oxidized in the TCA cycle, because thiamine limitation inhibits α-ketoglutarate dehydrogenase. As a result, KGA is excreted from the yeast cells as a byproduct of PA oversynthesis. Furthermore, the increased level of KGA in the yeast cells inhibits NAD-dependent isocitrate dehydrogenase in the TCA cycle and enhances the production and excretion of citric acid, another byproduct of PA oversynthesis. During cultivation in a fermentor, the strain Blastobotrys adeninivorans VKM Y-2677 produced 43.2 g l(-1) PA from glucose with a product yield (YPA) of 0.77 g PA/g glucose. The proportion of PA to byproducts was 18:1 for KGA and 8:1 for citric acid.

  9. Phosphate limitation promotes unsaturated fatty acids and arachidonic acid biosynthesis by microalgae Porphyridium purpureum.

    Science.gov (United States)

    Su, Gaomin; Jiao, Kailin; Li, Zheng; Guo, Xiaoyi; Chang, Jingyu; Ndikubwimana, Theoneste; Sun, Yong; Zeng, Xianhai; Lu, Yinghua; Lin, Lu

    2016-07-01

    Polyunsaturated fatty acids (PUFAs) are highly appreciated on their nutritive value for human health and aquaculture. P. purpureum, one of the red microalgae acknowledged as a promising accumulator of ARA, was chosen as the target algae in the present research. Effects of sodium bicarbonate (0.04-1.2 g/L), temperature (25, 30 and 33 °C) and phosphate (0.00-0.14 g/L) on biomass yield, total fatty acids (TFA) and arachidonic acid (ARA) accumulation were investigated systemically. NaHCO3 dose of 0.8 g/L and moderate temperature of 30 °C were preferred. In addition, TFA and ARA production were significantly enhanced by an appropriate concentration of phosphate, and the highest TFA yield of 666.38 mg/L and ARA yield of 159.74 mg/L were obtained at a phosphate concentration of 0.035 g/L. Interestingly, with phosphate concentration continuing to fall, UFA/TFA and ARA/EPA ratios were increased accordingly, suggesting that phosphate limitation promoted unsaturated fatty acids and arachidonic acid biosynthesis. Low concentration of phosphate may be favored to increase the enzymatic activities of ∆6-desaturase, which played a key role in catalyzing the conversion of C16:0 to C18:2, and thus the selectivity of UFA increased. Meanwhile, the increase of ARA selectivity could be attributed to ω6 pathway promotion and ∆17-desaturase activity inhibition with phosphate limitation. Phosphate limitation strategy enhanced unsaturated fatty acids and ARA biosynthesis in P. purpureum, and can be applied in commercial scale manufacturing and commercialization of ARA.

  10. Evolution of the biosynthesis of the branched-chain amino acids

    Science.gov (United States)

    Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

    1995-06-01

    The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from α-ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

  11. Evolution of the biosynthesis of the branched-chain amino acids

    Science.gov (United States)

    Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

    1995-01-01

    The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

  12. Oxalic acid biosynthesis is encoded by an operon in Burkholderia glumae

    Science.gov (United States)

    Although the biosynthesis of oxalic acid is known to occur in a number of bacteria, the mechanism(s) regulating its production remains largely unknown. To date, there is no report on the identification of an oxalic acid biosynthetic pathway gene from bacteria. In an attempt to identify such a gene...

  13. Biosynthesis and biological functions of terpenoids in plants.

    Science.gov (United States)

    Tholl, Dorothea

    2015-01-01

    Terpenoids (isoprenoids) represent the largest and most diverse class of chemicals among the myriad compounds produced by plants. Plants employ terpenoid metabolites for a variety of basic functions in growth and development but use the majority of terpenoids for more specialized chemical interactions and protection in the abiotic and biotic environment. Traditionally, plant-based terpenoids have been used by humans in the food, pharmaceutical, and chemical industries, and more recently have been exploited in the development of biofuel products. Genomic resources and emerging tools in synthetic biology facilitate the metabolic engineering of high-value terpenoid products in plants and microbes. Moreover, the ecological importance of terpenoids has gained increased attention to develop strategies for sustainable pest control and abiotic stress protection. Together, these efforts require a continuous growth in knowledge of the complex metabolic and molecular regulatory networks in terpenoid biosynthesis. This chapter gives an overview and highlights recent advances in our understanding of the organization, regulation, and diversification of core and specialized terpenoid metabolic pathways, and addresses the most important functions of volatile and nonvolatile terpenoid specialized metabolites in plants.

  14. Different impacts of short-chain fatty acids on saturated and polyunsaturated fatty acid biosynthesis in Aurantiochytrium sp. SD116.

    Science.gov (United States)

    Song, Xiaojin; Tan, Yanzhen; Liu, Yajun; Zhang, Jingtao; Liu, Guanglei; Feng, Yingang; Cui, Qiu

    2013-10-16

    Aurantiochytrium is an important docosahexaenoic acid (DHA) producer containing two kinds of fatty acid synthesis pathways, that is, the fatty acid synthase pathway (FAS) for saturated fatty acid synthesis and the polyketide synthase pathway (PKS) for polyunsaturated fatty acid synthesis. To understand the regulation mechanism between the two pathways, the impacts of six short-chain fatty acids on the fatty acid synthesis of Aurantiochytrium sp. SD116 were studied. All short-chain fatty acids showed little effect on the cell growth, but some of them significantly affected lipid accumulation and fatty acid composition. Pentanoic acid and isovaleric acid greatly inhibited the synthesis of saturated fatty acids, whereas the polyunsaturated fatty acid synthesis was not affected. Analysis of malic enzyme activity, which supplied NADPH for saturated fatty acids biosynthesis, indicated that the two fatty acid synthesis pathways can utilize different substrates and possess independent sources of NADPH.

  15. Expression of D-myo-inositol-3-phosphate synthase in soybean. Implications for phytic acid biosynthesis.

    Science.gov (United States)

    Hegeman, C E; Good, L L; Grabau, E A

    2001-04-01

    Phytic acid, a phosphorylated derivative of myo-inositol, functions as the major storage form of phosphorus in plant seeds. Myo-inositol phosphates, including phytic acid, play diverse roles in plants as signal transduction molecules, osmoprotectants, and cell wall constituents. D-myo-inositol-3-phosphate synthase (MIPS EC 5.5.1.4) catalyzes the first step in de novo synthesis of myo-inositol. A soybean (Glycine max) MIPS cDNA (GmMIPS1) was isolated by reverse transcriptase-PCR using consensus primers designed from highly conserved regions in other plant MIPS sequences. Southern-blot analysis and database searches indicated the presence of at least four MIPS genes in the soybean genome. Northern-blot and immunoblot analyses indicated higher MIPS expression and accumulation in immature seeds than in other soybean tissues. MIPS was expressed early in the cotyledonary stage of seed development. The GmMIPS1 expression pattern suggested that it encodes a MIPS isoform that functions in seeds to generate D-myo-inositol-3-phosphate as a substrate for phytic acid biosynthesis.

  16. Vascular dysfunction induced in offspring by maternal dietary fat involves altered arterial polyunsaturated fatty acid biosynthesis.

    Directory of Open Access Journals (Sweden)

    Christopher J Kelsall

    Full Text Available Nutrition during development affects risk of future cardiovascular disease. Relatively little is known about whether the amount and type of fat in the maternal diet affect vascular function in the offspring. To investigate this, pregnant and lactating rats were fed either 7%(w/w or 21%(w/w fat enriched in either 18:2n-6, trans fatty acids, saturated fatty acids, or fish oil. Their offspring were fed 4%(w/w soybean oil from weaning until day 77. Type and amount of maternal dietary fat altered acetylcholine (ACh-mediated vaso-relaxation in offspring aortae and mesenteric arteries, contingent on sex. Amount, but not type, of maternal dietary fat altered phenylephrine (Pe-induced vasoconstriction in these arteries. Maternal 21% fat diet decreased 20:4n-6 concentration in offspring aortae. We investigated the role of Δ6 and Δ5 desaturases, showing that their inhibition in aortae and mesenteric arteries reduced vasoconstriction, but not vaso-relaxation, and the synthesis of specific pro-constriction eicosanoids. Removal of the aortic endothelium did not alter the effect of inhibition of Δ6 and Δ5 desaturases on Pe-mediated vasoconstriction. Thus arterial smooth muscle 20:4n-6 biosynthesis de novo appears to be important for Pe-mediated vasoconstriction. Next we studied genes encoding these desaturases, finding that maternal 21% fat reduced Fads2 mRNA expression and increased Fads1 in offspring aortae, indicating dysregulation of 20:4n-6 biosynthesis. Methylation at CpG -394 bp 5' to the Fads2 transcription start site predicted its expression. This locus was hypermethylated in offspring of dams fed 21% fat. Pe treatment of aortae for 10 minutes increased Fads2, but not Fads1, mRNA expression (76%; P<0.05. This suggests that Fads2 may be an immediate early gene in the response of aortae to Pe. Thus both amount and type of maternal dietary fat induce altered regulation of vascular tone in offspring though differential effects on vaso-relaxation, and

  17. Autoxidated linolenic acid inhibits aflatoxin biosynthesis in Aspergillus flavus via oxylipin species.

    Science.gov (United States)

    Yan, Shijuan; Liang, Yating; Zhang, Jindan; Chen, Zhuang; Liu, Chun-Ming

    2015-08-01

    Aflatoxins produced by Aspergillus species are among the most toxic and carcinogenic compounds in nature. Although it has been known for a long time that seeds with high oil content are more susceptible to aflatoxin contamination, the role of fatty acids in aflatoxin biosynthesis remains controversial. Here we demonstrate in A. flavus that both the saturated stearic acid (C18:0) and the polyunsaturated linolenic acid (C18:3) promoted aflatoxin production, while C18:3, but not C18:0, inhibited aflatoxin biosynthesis after exposure to air for several hours. Further experiments showed that autoxidated C18:3 promoted mycelial growth, sporulation, and kojic acid production, but inhibited the expression of genes in the AF biosynthetic gene cluster. Mass spectrometry analyses of autoxidated C18:3 fractions that were able to inhibit aflatoxin biosynthesis led to the identification of multiple oxylipin species. These results may help to clarify the role of fatty acids in aflatoxin biosynthesis, and may explain why controversial results have been obtained for fatty acids in the past.

  18. Effect of alpha-linolenic, capric and lauric acid on the fatty acid biosynthesis in Staphylococcus aureus.

    Science.gov (United States)

    Sado-Kamdem, Sylvain L; Vannini, Lucia; Guerzoni, M Elisabetta

    2009-02-28

    The antimicrobial activity of alpha-linolenic, capric and lauric acids on Staphylococcus aureus was studied in relation to their effect on the de novo fatty acid biosynthesis. Labelled acetate was used as integrated carbon source and traced in the de novo fatty acid by using a GC-Mass spectrometer and the single ion monitoring (SIM) technique. The detection of the incorporation of the labelled carbon into the individual cell fatty acids (FAs) provided an insight into the different effects of alpha-linolenic, capric and lauric acids on the FA biosynthesis. The results suggested that FAs pathway is the major target of alpha-linolenic acid and that other enzymes in addition to FabI are involved in S. aureus response mechanism when medium chain fatty acids are present.

  19. THE BIOSYNTHESIS OF HYDROXYBENZOIC ACIDS IN HIGHER PLANTS

    Science.gov (United States)

    methylating protocatechuic to vanillic acid or hydroxylating it to yield gallic acid . Demethoxylation of sinapic and dehydroxylation of caffeic acid occurred in...Radioactive para-hydroxybenzoic, vanillic and syringic acids were shown to be synthesized in a variety of plants from the corresponding...hydroxycinnamic acids labelled in the beta-position. Decarboxylation of the hydroxybenzoic acids indicated that nearly all the activity was contained in the

  20. P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination

    Science.gov (United States)

    Jiang, Jishan; Chen, Zhihong; Ban, Liping; Wu, Yudi; Huang, Jianping; Chu, Jinfang; Fang, Shuang; Wang, Zan; Gao, Hongwen; Wang, Xuemin

    2017-01-01

    P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling. PMID:28084442

  1. Dietary Polyunsaturated Fatty Acids and Inflammation: The Role of Phospholipid Biosynthesis

    Science.gov (United States)

    Raphael, William; Sordillo, Lorraine M.

    2013-01-01

    The composition of fatty acids in the diets of both human and domestic animal species can regulate inflammation through the biosynthesis of potent lipid mediators. The substrates for lipid mediator biosynthesis are derived primarily from membrane phospholipids and reflect dietary fatty acid intake. Inflammation can be exacerbated with intake of certain dietary fatty acids, such as some ω-6 polyunsaturated fatty acids (PUFA), and subsequent incorporation into membrane phospholipids. Inflammation, however, can be resolved with ingestion of other fatty acids, such as ω-3 PUFA. The influence of dietary PUFA on phospholipid composition is influenced by factors that control phospholipid biosynthesis within cellular membranes, such as preferential incorporation of some fatty acids, competition between newly ingested PUFA and fatty acids released from stores such as adipose, and the impacts of carbohydrate metabolism and physiological state. The objective of this review is to explain these factors as potential obstacles to manipulating PUFA composition of tissue phospholipids by specific dietary fatty acids. A better understanding of the factors that influence how dietary fatty acids can be incorporated into phospholipids may lead to nutritional intervention strategies that optimize health. PMID:24152446

  2. Analysis of putative nonulosonic acid biosynthesis pathways in Archaea reveals a complex evolutionary history.

    Science.gov (United States)

    Kandiba, Lina; Eichler, Jerry

    2013-08-01

    Sialic acids and the other nonulosonic acid sugars, legionaminic acid and pseudaminic acid, are nine carbon-containing sugars that can be detected as components of the glycans decorating proteins and other molecules in Eukarya and Bacteria. Yet, despite the prevalence of N-glycosylation in Archaea and the variety of sugars recruited for the archaeal version of this post-translational modification, only a single report of a nonulosonic acid sugar in an archaeal N-linked glycan has appeared. Hence, to obtain a clearer picture of nonulosonic acid sugar biosynthesis capability in Archaea, 122 sequenced genomes were scanned for the presence of genes involved in the biogenesis of these sugars. The results reveal that while Archaea and Bacteria share a common route of sialic acid biosynthesis, numerous archaeal nonulosonic acid sugar biosynthesis pathway components were acquired from elsewhere via various routes. Still, the limited number of Archaea encoding components involved in the synthesis of nonulosonic acid sugars implies that such saccharides are not major components of glycans in this domain.

  3. Dietary Polyunsaturated Fatty Acids and Inflammation: The Role of Phospholipid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Lorraine M. Sordillo

    2013-10-01

    Full Text Available The composition of fatty acids in the diets of both human and domestic animal species can regulate inflammation through the biosynthesis of potent lipid mediators. The substrates for lipid mediator biosynthesis are derived primarily from membrane phospholipids and reflect dietary fatty acid intake. Inflammation can be exacerbated with intake of certain dietary fatty acids, such as some ω-6 polyunsaturated fatty acids (PUFA, and subsequent incorporation into membrane phospholipids. Inflammation, however, can be resolved with ingestion of other fatty acids, such as ω-3 PUFA. The influence of dietary PUFA on phospholipid composition is influenced by factors that control phospholipid biosynthesis within cellular membranes, such as preferential incorporation of some fatty acids, competition between newly ingested PUFA and fatty acids released from stores such as adipose, and the impacts of carbohydrate metabolism and physiological state. The objective of this review is to explain these factors as potential obstacles to manipulating PUFA composition of tissue phospholipids by specific dietary fatty acids. A better understanding of the factors that influence how dietary fatty acids can be incorporated into phospholipids may lead to nutritional intervention strategies that optimize health.

  4. Precursor Amino Acids Inhibit Polymyxin E Biosynthesis in Paenibacillus polymyxa, Probably by Affecting the Expression of Polymyxin E Biosynthesis-Associated Genes

    Directory of Open Access Journals (Sweden)

    Zhiliang Yu

    2015-01-01

    Full Text Available Polymyxin E belongs to cationic polypeptide antibiotic bearing four types of direct precursor amino acids including L-2,4-diaminobutyric acid (L-Dab, L-Leu, D-Leu, and L-Thr. The objective of this study is to evaluate the effect of addition of precursor amino acids during fermentation on polymyxin E biosynthesis in Paenibacillus polymyxa. The results showed that, after 35 h fermentation, addition of direct precursor amino acids to certain concentration significantly inhibited polymyxin E production and affected the expression of genes involved in its biosynthesis. L-Dab repressed the expression of polymyxin synthetase genes pmxA and pmxE, as well as 2,4-diaminobutyrate aminotransferase gene ectB; both L-Leu and D-Leu repressed the pmxA expression. In addition, L-Thr affected the expression of not only pmxA, but also regulatory genes spo0A and abrB. As L-Dab precursor, L-Asp repressed the expression of ectB, pmxA, and pmxE. Moreover, it affected the expression of spo0A and abrB. In contrast, L-Phe, a nonprecursor amino acid, had no obvious effect on polymyxin E biosynthesis and those biosynthesis-related genes expression. Taken together, our data demonstrated that addition of precursor amino acids during fermentation will inhibit polymyxin E production probably by affecting the expression of its biosynthesis-related genes.

  5. Biosynthesis of 'essential' amino acids by scleractinian corals.

    Science.gov (United States)

    Fitzgerald, L M; Szmant, A M

    1997-02-15

    Animals rely on their diet for amino acids that they are incapable either of synthesizing or of synthesizing in sufficient quantities to meet metabolic needs. These are the so-called 'essential amino acids'. This set of amino acids is similar among the vertebrates and many of the invertebrates. Previously, no information was available for amino acid synthesis by the most primitive invertebrates, the Cnidaria. The purpose of this study was to examine amino acid synthesis by representative cnidarians within the Order Scleractinia. Three species of zooxanthellate reef coral, Montastraea faveolata, Acropora cervicornis and Porites divaricata, and two species of non-zooxanthellate coral, Tubastrea coccinea and Astrangia poculata, were incubated with 14C-labelled glucose or with the 14C-labelled amino acids glutamic acid, lysine or valine. Radiolabel tracer was followed into protein amino acids. A total of 17 amino acids, including hydroxyproline, were distinguishable by the techniques used. Of these, only threonine was not found radiolabelled in any of the samples. We could not detect tryptophan or cysteine, nor distinguish between the amino acid pairs glutamic acid and glutamine, or aspartic acid and asparagine. Eight amino acids normally considered essential for animals were made by the five corals tested, although some of them were made only in small quantities. These eight amino acids are valine, isoleucine, leucine, tyrosine, phenylalanine histidine, methionine and lysine. The ability of cnidarians to synthesize these amino acids could be yet another indicator of a separate evolutionary history of the cnidarians from the rest of the Metazoa.

  6. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    Science.gov (United States)

    Andre, Christelle M; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; Cooney, Janine M; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; Laing, William A

    2016-09-01

    Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis.

  7. Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239

    NARCIS (Netherlands)

    Boer, L. de; Vrijbloed, J.W.; Grobben, G.; Dijkhuizen, L.

    1989-01-01

    The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenyl

  8. A novel C(28)-hydroxylated lupeolic acid suppresses the biosynthesis of eicosanoids through inhibition of cytosolic phospholipase A(2).

    Science.gov (United States)

    Verhoff, Moritz; Seitz, Stefanie; Northoff, Hinnak; Jauch, Johann; Schaible, Anja M; Werz, Oliver

    2012-09-01

    Eicosanoids are potent lipid mediators derived from phospholipase (PL)-released arachidonic acid (AA) coupled to subsequent metabolism by cyclooxygenase (COX)-1/2 or lipoxygenases (LO) which are involved in a variety of homeostatic biological functions and inflammation. We have investigated three lupeolic acids (LA) from the gum resin of Boswellia carterii for their ability to interfere with eicosanoid biosynthesis in human blood cells. A novel, yet unknown C(28)-hydroxylated LA, that is, 3α-acetoxy-28-hydroxylup-20(29)-en-4β-oic acid (Ac-OH-LA) was found to inhibit the biosynthesis of COX-, 5-LO- and 12-LO-derived eicosanoids from endogenous AA in activated platelets, neutrophils, and monocytes from human blood with consistent IC(50) values of 2.3-6.9 μM. In contrast, two other LAs lacking the C(28)-OH moiety were essentially inactive in this respect. Inhibition of eicosanoids by Ac-OH-LA correlated with reduced release of AA in intact cells. When AA was exogenously provided as substrate for cellular eicosanoid biosynthesis the inhibitory effects of Ac-OH-LA were essentially reversed, even though some inhibition of 5-LO and COX-1 product formation still remained. Finally, by means of a cell-free phospholipid hydrolysis assay using human recombinant cytosolic PLA(2)α, we show that Ac-OH-LA may directly interfere with cPLA(2)α activity (IC(50) = 3.6 μM). Together, we identified a novel, naturally occuring C(28)-hydroxylated LA which acts as efficient inhibitor of cPLA(2)α and consequently suppresses eicosanoid biosynthesis in intact cells.

  9. Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants.

    Science.gov (United States)

    Rogalski, Marcelo; Carrer, Helaine

    2011-06-01

    The ability to manipulate plant fatty acid biosynthesis by using new biotechnological approaches has allowed the production of transgenic plants with unusual fatty acid profile and increased oil content. This review focuses on the production of very long chain polyunsaturated fatty acids (VLCPUFAs) and the increase in oil content in plants using molecular biology tools. Evidences suggest that regular consumption of food rich in VLCPUFAs has multiple positive health benefits. Alternative sources of these nutritional fatty acids are found in cold-water fishes. However, fish stocks are in severe decline because of decades of overfishing, and also fish oils can be contaminated by the accumulation of toxic compounds. Recently, there is also an increase in oilseed use for the production of biofuels. This tendency is partly associated with the rapidly rising costs of petroleum, increased concern about the environmental impact of fossil oil and the attractive need to develop renewable sources of fuel. In contrast to this scenario, oil derived from crop plants is normally contaminant free and less environmentally aggressive. Genetic engineering of the plastid genome (plastome) offers a number of attractive advantages, including high-level foreign protein expression, marker-gene excision and transgene containment because of maternal inheritance of plastid genome in most crops. Here, we describe the possibility to improve fatty acid biosynthesis in plastids, production of new fatty acids and increase their content in plants by genetic engineering of plastid fatty acid biosynthesis via plastid transformation.

  10. Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

    Science.gov (United States)

    Goodrich-Tanrikulu, M; Stafford, A E; Lin, J T; Makapugay, M I; Fuller, G; McKeon, T A

    1994-10-01

    New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

  11. Biosynthesis of vanillin via ferulic acid in Vanilla planifolia.

    Science.gov (United States)

    Negishi, Osamu; Sugiura, Kenji; Negishi, Yukiko

    2009-11-11

    (14)C-Labeled phenylalanine, 4-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, ferulic acid, and methionine were applied to disks of green vanilla pods 3 and 6 months after pollination (immature and mature pods), and the conversion of these compounds to vanillin or glucovanillin was investigated. In mature green vanilla pods, radioactivities of 11, 15, 29, and 24% from (14)C-labeled phenylalanine, 4-coumaric acid, ferulic acid, and methionine, respectively, were incorporated into glucovanillin within 24 h. In the incorporation processes of methionine and phenylalanine into glucovanillin, some of the (14)C labels were also trapped by the unlabeled ferulic acid. However, (14)C-labeled 4-hydroxybenzaldehyde and 4-hydroxybenzyl alcohol were not converted to glucovanillin. On the other hand, in immature green vanilla pods radioactivities of the above six compounds were not incorporated into glucovanillin. Although 4-coumaric acid, ferulic acid, 4-hydroxybenzaldehyde, and 4-hydroxybenzyl alcohol were converted to the respective glucose esters or glucosides and vanillin was converted to glucovanillin, their conversions were believed to be from the detoxication of the aglycones. These results suggest that the biosynthetic pathway for vanillin is 4-coumaric acid --> --> ferulic acid --> --> vanillin --> glucovanillin in mature vanilla pods.

  12. [Overexpression of four fatty acid synthase genes elevated the efficiency of long-chain polyunsaturated fatty acids biosynthesis in mammalian cells].

    Science.gov (United States)

    Zhu, Guiming; Saleh, Abdulmomen Ali Mohammed; Bahwal, Said Ahmed; Wang, Kunfu; Wang, Mingfu; Wang, Didi; Ge, Tangdong; Sun, Jie

    2014-09-01

    Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.

  13. Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus

    Directory of Open Access Journals (Sweden)

    Clark Shawn M

    2013-01-01

    Full Text Available Abstract Background Bitter acids (e.g. humulone are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP pathway. We used RNA sequencing (RNA-seq to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic and reverse (catabolic reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial and

  14. Biosynthesis of branched-chain amino acids in Schizosaccharomyces pombe: properties of acetohydroxy acid synthetase.

    Science.gov (United States)

    McDonald, R A; Satyanarayana, T; Kaplan, J G

    1973-04-01

    The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.

  15. Coordination of glycerol utilization and clavulanic acid biosynthesis to improve clavulanic acid production in Streptomyces clavuligerus.

    Science.gov (United States)

    Guo, Dekun; Zhao, Youbao; Yang, Keqian

    2013-07-01

    The glycerol utilization (gyl) operon is involved in clavulanic acid (CA) production by Streptomyces clavuligerus, and possibly supplies the glyceraldehyde-3-phosphate (G3P) precursor for CA biosynthesis. The gyl operon is regulated by GylR and is induced by glycerol. To enhance CA production in S. clavuligerus, an extra copy of ccaR expressed from Pgyl (the gyl promoter) was integrated into the chromosome of S. clavuligerus NRRL 3585. This construct coordinated the transcription of CA biosynthetic pathway genes with expression of the gyl operon. In the transformants carrying the Pgyl-controlled regulatory gene ccaR, CA production was enhanced 3.19-fold in glycerol-enriched batch cultures, relative to the control strain carrying an extra copy of ccaR controlled by its own promoter (PccaR). Consistent with enhanced CA production, the transcription levels of ccaR, ceas2 and claR were significantly up-regulated in the transformants containing Pgyl-controlled ccaR.

  16. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds.

    Science.gov (United States)

    Tagashira, Yusuke; Shimizu, Tomoe; Miyamoto, Masanobu; Nishida, Sho; Yoshida, Kaoru T

    2015-04-24

    The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP₆) biosynthesis-related genes, as InsP₆ is a major storage form of P in seeds. The rice (Oryza sativa L.) low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP₆ biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP₆ and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP₆ biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP₆ biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  17. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds

    Directory of Open Access Journals (Sweden)

    Yusuke Tagashira

    2015-04-01

    Full Text Available The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP6 biosynthesis-related genes, as InsP6 is a major storage form of P in seeds. The rice (Oryza sativa L. low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP6 biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP6 and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP6 biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP6 biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  18. Water stress responses of tomato mutants impaired in hormone biosynthesis reveal abscisic acid, jasmonic acid and salicylic acid interactions

    Directory of Open Access Journals (Sweden)

    Valeria eMuñoz

    2015-11-01

    Full Text Available To investigate the putative crosstalk between JA and ABA in Solanum lycopersicum plants in response to drought, suppressor of prosystemin-mediated responses2 (spr2, JA-deficient and flacca (flc, ABA-deficient mutants together with the naphthalene/salicylate hydroxylase (NahG transgenic (SA-deficient line were used. Hormone profiling and gene expression of key enzymes in ABA, JA and SA biosynthesis were analyzed during early stages of drought. ABA accumulation was comparable in spr2 and wild type (WT plants whereas expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1 and NCED2 was different, implying a compensation mechanism between NCED genes and an organ-specific regulation of NCED1 expression. JA levels and 12-oxo-phytodienoic acid reductase 3 (OPR3 expression in flc plants suggest that ABA regulates the induction of the OPR3 gene in roots. By contrast, ABA treatment to flc plants leads to a reduction of JA and SA contents. Furthermore, different pattern of SA accumulation (and expression of isochorismate synthase and phenylalanine ammonia lyase 1 was observed between WT seedlings and mutants, suggesting that SA plays an important role on the early response of tomato plants to drought and also that JA and ABA modulate its biosynthesis. Finally, hormone profiling in spr2 and NahG plants indicate a crosstalk between JA and SA that could enhance tolerance of tomato to water stress.

  19. Water Stress Responses of Tomato Mutants Impaired in Hormone Biosynthesis Reveal Abscisic Acid, Jasmonic Acid and Salicylic Acid Interactions

    Science.gov (United States)

    Muñoz-Espinoza, Valeria A.; López-Climent, María F.; Casaretto, José A.; Gómez-Cadenas, Aurelio

    2015-01-01

    To investigate the putative crosstalk between JA and ABA in Solanum lycopersicum plants in response to drought, suppressor of prosystemin-mediated responses2 (spr2, JA-deficient) and flacca (flc, ABA-deficient) mutants together with the naphthalene/salicylate hydroxylase (NahG) transgenic (SA-deficient) line were used. Hormone profiling and gene expression of key enzymes in ABA, JA and SA biosynthesis were analyzed during early stages of drought. ABA accumulation was comparable in spr2 and wild type (WT) plants whereas expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED2 was different, implying a compensation mechanism between NCED genes and an organ-specific regulation of NCED1 expression. JA levels and 12-oxo-phytodienoic acid reductase 3 (OPR3) expression in flc plants suggest that ABA regulates the induction of the OPR3 gene in roots. By contrast, ABA treatment to flc plants leads to a reduction of JA and SA contents. Furthermore, different pattern of SA accumulation (and expression of isochorismate synthase and phenylalanine ammonia lyase 1) was observed between WT seedlings and mutants, suggesting that SA plays an important role on the early response of tomato plants to drought and also that JA and ABA modulate its biosynthesis. Finally, hormone profiling in spr2 and NahG plants indicate a crosstalk between JA and SA that could enhance tolerance of tomato to water stress. PMID:26635826

  20. Effect of oxidoreduction potential on aroma biosynthesis by lactic acid bacteria in nonfat yogurt.

    Science.gov (United States)

    Martin, F; Cachon, R; Pernin, K; De Coninck, J; Gervais, P; Guichard, E; Cayot, N

    2011-02-01

    The aim of this study was to investigate the effect of oxidoreduction potential (Eh) on the biosynthesis of aroma compounds by lactic acid bacteria in non-fat yogurt. The study was done with yogurts fermented by Lactobacillus bulgaricus and Streptococcus thermophilus. The Eh was modified by the application of different gaseous conditions (air, nitrogen, and nitrogen/hydrogen). Acetaldehyde, dimethyl sulfide, diacetyl, and pentane-2,3-dione, as the major endogenous odorant compounds of yogurt, were chosen as tracers for the biosynthesis of aroma compounds by lactic acid bacteria. Oxidative conditions favored the production of acetaldehyde, dimethyl sulfide, and diketones (diacetyl and pentane-2,3-dione). The Eh of the medium influences aroma production in yogurt by modifying the metabolic pathways of Lb. bulgaricus and Strep. thermophilus. The use of Eh as a control parameter during yogurt production could permit the control of aroma formation.

  1. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68

    Directory of Open Access Journals (Sweden)

    Hamid-Reza Samadlouie

    2014-06-01

    Full Text Available The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

  2. The role of cis-carotenoids in abscisic acid biosynthesis.

    Science.gov (United States)

    Parry, A D; Babiano, M J; Horgan, R

    1990-08-01

    Evidence has been obtained which is consistent with 9'-cis-neoxanthin being a major precursor of abscisic acid (ABA) in higher plants. A mild, rapid procedure was developed for the extraction and analysis of carotenoids from a range of tissues. Once purified the carotenoids were identified from their light-absorbance properties, reactions with dilute acid, high-performance liquid chromatography Rts, mass spectra and the quasiequilibria resulting from iodine-catalysed or chlorophyllsensitised photoisomerisation. Two possible ABA precursors, 9'-cis-neoxanthin and 9-cis-violaxanthin, were identified in extracts of light-grown and etiolated leaves (of Lycopersicon esculentum, Phaseolus vulgaris, Vicia faba, Pisum sativum, Cicer arietinum, Zea mays, Nicotiana plumbaginifolia, Plantago lanceolata and Digitalis purpurea), and roots of light-grown and etiolated plants (Lycopersicon, Phaseolus and Zea). The 9,9'-di-cisisomer of violaxanthin was synthesised but its presence was not detected in any extracts. Levels of 9'-cis-neoxanthin and all-trans-violaxanthin were between 20- to 100-fold greater than those of ABA in light-grown leaves. The levels of 9-cis-violaxanthin were similar to those of ABA but unaffected by water stress. Etiolated Phaseolus leaves contained reduced amounts of carotenoids (15-20% compared with light-grown leaves) but retained the ability to synthesise large amounts of ABA. The amounts of ABA synthesised, measured as increases in ABA and its metabolites phaseic acid and dihydrophaseic acid, were closely matched by decreases in the levels of 9'-cis-neoxanthin and all-trans-violaxanthin. In etiolated seedlings grown on 50% D2O, deuterium incorporation into ABA was similar to that into the xanthophylls. Relative levels of carotenoids in roots and light-grown and etiolated leaves of the ABA-deficient mutants, notabilis, flacca and sitiens were the same as those found in wild-type tomato tissues.

  3. Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis

    DEFF Research Database (Denmark)

    Rodriguez Prado, Edith Angelica; Kildegaard, Kanchana Rueksomtawin; Li, Mingji;

    2015-01-01

    Aromatic amino acids are precursors of numerous plant secondary metabolites with diverse biological functions. Many of these secondary metabolites are already being used as active pharmaceutical or nutraceutical ingredients, and there are numerous exploratory studies of other compounds...... with promising applications. p-Coumaric acid is derived from aromatic amino acids and, besides being a valuable chemical building block, it serves as precursor for biosynthesis of many secondary metabolites, such as polyphenols, flavonoids, and some polyketides. Here we developed a p-coumaric acid...... was obtained, when overexpressing tyrosine ammonia-lyase TAL from Flavobacterium johnsoniaeu, DAHP synthase ARO4K229L, chorismate mutase ARO7G141S and E. coli shikimate kinase II (aroL) in Δpdc5Δaro10 strain background. To our knowledge this is the highest reported titer of an aromatic compound produced...

  4. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis.

    Science.gov (United States)

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  5. Cloning, sequencing and function of sanB, a gene related to nikkomycin biosynthesis of Streptomyces ansochromogenes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A 6.0 kb DNA fragment related to nikkomycin biosynthesis was cloned from nikkomycin- producing Streptomyces ansochromogenes 7100. Sequence analysis showed that the 1.9 kb Tth111Ⅰ fragment, a part of the 6.0 kb DNA fragment, contains one complete ORF designated sanB (GenBank accession No. AF224501), which is composed of 1740 bp encoding a protein consisting of 580 amino acid residues. Its start codon is GTG at 100 bp position and stop codon is TGA at 1840-bp position. Database searching indicated that the deduced protein of sanB is homologous to the histidinol-phosphate aminotransferase in Streptomyces coelicolor with 31% identities and 47% positives. Gene disruption was performed to study the function of sanB. It was found that disruptants of sanB lost the ability to synthesize nikkomycin, which reveals that sanB is a novel gene essential for nikkomycin biosynthesis.

  6. Acquisition and biosynthesis of saturated and unsaturated fatty acids by trypanosomatids.

    Science.gov (United States)

    Uttaro, Antonio D

    2014-08-01

    As components of phospholipids and glycosylphosphatidylinositol anchors, fatty acids are responsible for forming the core of biological membranes and the correct localization of proteins within membranes. They also contribute to anchoring proteins by direct acylation of specific amino acids. Fatty acids can be used as energy sources and serve as signaling molecules or precursors for their synthesis. All these processes highlight the important role of fatty acids in cell physiology, justifying the diverse strategies for their acquisition evolved by different organisms. This review describes several recent findings in the salvage and biosynthesis of fatty acids by parasitic protists belonging to the class Kinetoplastea. They include two biosynthetic routes, the mitochondrial one and a peculiar membrane-associated pathway, the synthesis of polyunsaturated fatty acids, and the scavenging of lysophospholipids and lipoproteins from host plasma. These different processes are also explored as putative targets for chemotherapy.

  7. BnWRI1 coordinates fatty acid biosynthesis and photosynthesis pathways during oil accumulation in rapeseed.

    Science.gov (United States)

    Wu, Xue-Long; Liu, Zhi-Hong; Hu, Zhang-Hua; Huang, Rui-Zhi

    2014-06-01

    Photosynthesis in "green" seeds, such as rapeseed, soybean, and Arabidopsis, plays a substantial role in the improved efficiency of oil accumulation. However, the molecular mechanism underpinning the coordinated expression of fatty acid (FA) biosynthesis- and photosynthesis-related genes in such developing seeds remains to be elucidated. Here, we found that seed-specific overexpression of BnWRI1, a WRI1 homolog from rapeseed (Brassica napus cv. ZGY2), results in enhanced chlorophyll content in developing seeds and increased oil content and seed mass in matured seeds. BnWRI1 was co-expressed with BnBCCP and BnCAB, two marker genes of FA biosynthesis and photosynthesis during seed development, respectively. Overexpression of BnWRI1 increased expression of both marker genes. Further, the nuclear-localized BnWRI1 protein was found to act as a transcription activator. It could bind to the GT1-element and/or GCC-box, which are widespread in the upstream regions of genes involved in FA biosynthesis and photosynthesis pathways. Accordingly, BnWRI1 could interact with promoters of BCCP2 and LHB1B2 in vivo. These results suggested that BnWRI1 may coordinate FA biosynthesis and photosynthesis pathways in developing seeds via directly stimulating expression of GT1-element and/or GCC-box containing genes.

  8. BnWRI1 coordinates fatty acid biosynthesis and photosynthesis pathways during oil accumulation in rapeseed

    Institute of Scientific and Technical Information of China (English)

    Xue-Long Wu; Zhi-Hong Liu; Zhang-Hua Hu; Rui-Zhi Huang

    2014-01-01

    Photosynthesis in“green”seeds, such as rapeseed, soybean, and Arabidopsis, plays a substantial role in the improved efficiency of oil accumulation. However, the molecular mecha-nism underpinning the coordinated expression of fatty acid (FA) biosynthesis-and photosynthesis-related genes in such develop-ing seeds remains to be elucidated. Here, we found that seed-specific overexpression of BnWRI1, a WRI1 homolog from rapeseed (Brassica napus cv. ZGY2), results in enhanced chlorophyl content in developing seeds and increased oil content and seed mass in matured seeds. BnWRI1 was co-expressed with BnBCCP and BnCAB, two marker genes of FA biosynthesis and photosynthesis during seed development, respectively. Over-expression of BnWRI1 increased expression of both marker genes. Further, the nuclear-localized BnWRI1 protein was found to act as a transcription activator. It could bind to the GT1-element and/or GCC-box, which are widespread in the upstream regions of genes involved in FA biosynthesis and photosynthesis pathways. Accordingly, BnWRI1 could interact with promoters of BCCP2 and LHB1B2 in vivo. These results suggested that BnWRI1 may coordinate FA biosynthesis and photosynthesis pathways in developing seeds via directly stimulating expression of GT1-element and/or GCC-box containing genes.

  9. The zinc finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato.

    Science.gov (United States)

    Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong; Xiao, Han

    2015-03-01

    Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening.

  10. Biosynthesis of car1ssol and carissic Acid.

    Science.gov (United States)

    Nizami, S S; Khan, M A; Naim, Z; Islam, M N; Azeem, S W

    1993-01-01

    Carissa carandas belongs to family apocynaceae which consists of 300 genera and 1000 species. It is a large shrub with simple thorn and commonly cultivated throughout Pakistan for hedges and is called "Kakronda". The different parts of this plant have been used for various systems of medicine (Kirtikar et al., 1993). Cardiotonic activity was found in root of this plant (Rastogi et al., 1966; Rastogi et al., 1967; Vohra et al., 1963). This plant has been mentioned in the old chemical literature as purgative, stomachic, antihelmintics and antidote for snake-bite (Kirtikar., 1933). The physical characteristics of oil from the fruits of Carissa carandas were determined by using standard methods. In addition to this a study of sugars and amino acids from the fruits of this plant was also undertaken by the present authors (Naim et al., 1986). Our studies in the chemical investigation on this plant had led to the isolation of two new triterpene carissol Ia (Naim et al., 1985) and carissic acid lb (Naim et al., 1988).

  11. Oxalic acid biosynthesis and oxalacetate acetylhydrolase activity in Streptomyces cattleya.

    Science.gov (United States)

    Houck, D R; Inamine, E

    1987-11-15

    In addition to producing the antibiotic thienamycin, Streptomyces cattleya accumulates large amounts of oxalic acid during the course of a fermentation. Washed cell suspensions were utilized to determine the specific incorporation of carbon-14 into oxalate from a number of labeled organic and amino acids. L-[U-14C]aspartate proved to be the best precursor, whereas only a small percentage of label from [1,5-14C]citrate was found in oxalate. Cell-free extracts catalyzed the formation of [14C]oxalate and [14C]acetate from L-[U-14C]aspartate. When L-[4-14C]aspartate was the substrate only [14C]acetate was formed. The cell-free extracts were found to contain oxalacetate acetylhydrolase (EC 3.7.1.1), the enzyme that catalyzes the hydrolysis of oxalacetate to oxalate and acetate. The enzyme is constitutive and is analogous to enzymes in fungi that produce oxalate from oxalacetate. Properties of the crude enzyme were examined.

  12. Oxalic acid biosynthesis and oxalacetate acetylhydrolase activity in Streptomyces cattleya

    Energy Technology Data Exchange (ETDEWEB)

    Houck, D.R.; Inamine, E.

    1987-11-15

    In addition to producing the antibiotic thienamycin, Streptomyces cattleya accumulates large amounts of oxalic acid during the course of a fermentation. Washed cell suspensions were utilized to determine the specific incorporation of carbon-14 into oxalate from a number of labeled organic and amino acids. L-(U-/sup 14/C)aspartate proved to be the best precursor, whereas only a small percentage of label from (1,5-/sup 14/C)citrate was found in oxalate. Cell-free extracts catalyzed the formation of (/sup 14/C)oxalate and (/sup 14/C)acetate from L-(U-/sup 14/C)aspartate. When L-(4-/sup 14/C)aspartate was the substrate only (/sup 14/C)acetate was formed. The cell-free extracts were found to contain oxalacetate acetylhydrolase, the enzyme that catalyzes the hydrolysis of oxalacetate to oxalate and acetate. The enzyme is constitutive and is analogous to enzymes in fungi that produce oxalate from oxalacetate. Properties of the crude enzyme were examined.

  13. A Functional Bikaverin Biosynthesis Gene Cluster in Rare Strains of Botrytis cinerea Is Positively Controlled by VELVET

    Science.gov (United States)

    Schumacher, Julia; Gautier, Angélique; Morgant, Guillaume; Studt, Lena; Ducrot, Paul-Henri; Le Pêcheur, Pascal; Azeddine, Saad; Fillinger, Sabine; Leroux, Pierre; Tudzynski, Bettina; Viaud, Muriel

    2013-01-01

    The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT). In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3) or missing (bcbik1) but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa) that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin) and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus. PMID:23308280

  14. A functional bikaverin biosynthesis gene cluster in rare strains of Botrytis cinerea is positively controlled by VELVET.

    Directory of Open Access Journals (Sweden)

    Julia Schumacher

    Full Text Available The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT. In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3 or missing (bcbik1 but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus.

  15. Identification of microRNAs actively involved in fatty acid biosynthesis in developing Brassica napus seeds using high-throughput sequencing

    Directory of Open Access Journals (Sweden)

    Jia Wang

    2016-10-01

    Full Text Available Seed development has a critical role during the spermatophyte life cycle. In Brassica napus, a major oil crop, fatty acids are synthesized and stored in specific tissues during embryogenesis, and understanding the molecular mechanism underlying fatty acid biosynthesis during seed development is an important research goal. In this study, we constructed three small RNA libraries from early seeds at 14, 21 and 28 days after flowering (DAF and used high-throughput sequencing to examine microRNA (miRNA expression. A total of 85 known miRNAs from 30 families and 1,160 novel miRNAs were identified, of which 24, including 5 known and 19 novel miRNAs, were found to be involved in fatty acid biosynthesis. bna-miR156b, bna-miR156c, bna-miR156g, novel_mir_1706, novel_mir_1407, novel_mir_173, and novel_mir_104 were significantly down-regulated at 21 DAF and 28 DAF, whereas bna-miR159, novel_mir_1081, novel_mir_19 and novel_mir_555 were significantly up-regulated. In addition, we found that some miRNAs regulate functional genes that are directly involved in fatty acid biosynthesis and that other miRNAs regulate the process of fatty acid biosynthesis by acting on a large number of transcription factors. The miRNAs and their corresponding predicted targets were partially validated by quantitative RT-PCR. Our data suggest that diverse and complex miRNAs are involved in the seed development process and that miRNAs play important roles in fatty acid biosynthesis during seed development.

  16. Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis

    Science.gov (United States)

    Rodrigues, Elisete P.; Soares, Cleiton de Paula; Galvão, Patrícia G.; Imada, Eddie L.; Simões-Araújo, Jean L.; Rouws, Luc F. M.; de Oliveira, André L. M.; Vidal, Márcia S.; Baldani, José I.

    2016-01-01

    Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and

  17. Identification of genes involved in indole-3-acetic acid biosynthesis by Gluconacetobacter diazotrophicus PAL5 strain using transposon mutagenesis

    Directory of Open Access Journals (Sweden)

    ELISETE PAINS RODRIGUES

    2016-10-01

    Full Text Available Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA; however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01, which exhibited 95% less indolic compounds that the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA and indole-3-lactate (ILA. In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO. GDI2456 (lao gene forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao, cccA and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with

  18. Arabidopsis Acetyl-Amido Synthetase GH3.5 Involvement in Camalexin Biosynthesis through Conjugation of Indole-3-Carboxylic Acid and Cysteine and Upregulation of Camalexin Biosynthesis Genes

    Institute of Scientific and Technical Information of China (English)

    Mu-Yang Wang; Xue-Ting Liu; Ying Chen; Xiao-Jing Xu; Biao Yu; Shu-Qun Zhang; Qun Li; Zu-Hua He

    2012-01-01

    Camalexin (3-thiazol-2'-yl-indole) is the major phytoalexin found in Arabidopsis thaliana.Several key intermediates and corresponding enzymes have been identified in camalexin biosynthesis through mutant screening and biochemical experiments.Camalexin is formed when indole-3-acetonitrile (IAN)is catalyzed by the cytochrome P450 monooxygenase CYP71A13.Here,we demonstrate that the Arabidopsis GH3.5 protein,a multifunctional acetyl-amido synthetase,is involved in camalexin biosynthesis via conjugating indole-3-carboxylic acid (ICA) and cysteine (Cys) and regulating camalexin biosynthesis genes.Camalexin levels were increased in the activation-tagged mutant gh3.5-1D in both Col-0 and cyp71A13-2 mutant backgrounds after pathogen infection.The recombinant GH3.5 protein catalyzed the conjugation of ICA and Cys to form a possible intermediate indole-3-acyl-cysteinate (ICA(Cys)) in vitro.In support of the in vitro reaction,feeding with ICA and Cys increased camalexin levels in Col-0 and gh3.5-1D.Dihydrocamalexic acid (DHCA),the precursor of camalexin and the substrate for PAD3,was accumulated in gh3.5-1Dlpad3-1,suggesting that ICA(Cys) could be an additional precursor of DHCA for camalexin biosynthesis.Furthermore,expression of the major camalexin biosynthesis genes CYP79B2,CYP71A12,CYP71A13 and PAD3 was strongly induced in gh3.5-1D.Our study suggests that GH3.5 is involved in camalexin biosynthesis through direct catalyzation of the formation of ICA(Cys),and upregulation of the major biosynthetic pathway genes.

  19. Biosynthesis of very long chain fatty acids in Trypanosoma cruzi.

    Science.gov (United States)

    Livore, Verónica I; Uttaro, Antonio D

    2015-01-01

    Trypanosoma brucei and Trypanosoma cruzi showed similar fatty acid (FA) compositions, having a high proportion of unsaturated FAs, mainly 18:2Δ9,12 (23-39%) and 18:1Δ9 (11-17%). C22 polyunsaturated FAs are in significant amounts only in T. brucei (12-20%) but represent a mere 2% of total FAs in T. cruzi. Both species have also similar profiles of medium- and long-chain saturated FAs, from 14:0 to 20:0. Interestingly, procyclic and bloodstream forms of T. brucei lack very long chain FAs (VLCFAs), whereas epimastigotes and trypomastigotes of T. cruzi contain 22:0 (0.1-0.2%), 24:0 (1.5-2%), and 26:0 (0.1-0.2%). This is in agreement with the presence of an additional FA elongase gene (TcELO4) in T. cruzi. TcELO4 was expressed in a Saccharomyces cerevisiae mutant lacking the endogenous ScELO3, rescuing the synthesis of saturated and hydroxylated C26 FAs in the yeast. Expression of TcELO4 also rescued the synthetic lethality of a ScELO2, ScELO3 double mutation, and the VLCFA profile of the transformed yeast was similar to that found in T. cruzi. By identifying TcELO4 as the enzyme responsible for the elongation of FA from 16:0 and 18:0 up to 26:0, with 24:0 being the preferred product, this work completed the characterization of FA elongases in Trypanosoma spp.

  20. Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R H; Woo, Y; Cantor, J; Shekhtman, A

    2007-04-05

    The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cyclotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

  1. Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain

    Directory of Open Access Journals (Sweden)

    Kang Sun-Young

    2012-12-01

    Full Text Available Abstract Background The phenylpropanoid metabolites are an extremely diverse group of natural products biosynthesized by plants, fungi, and bacteria. Although these compounds are widely used in human health care and nutrition services, their availability is limited by regional variations, and isolation of single compounds from plants is often difficult. Recent advances in synthetic biology and metabolic engineering have enabled artificial production of plant secondary metabolites in microorganisms. Results We develop an Escherichia coli system containing an artificial biosynthetic pathway that yields phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, from simple carbon sources. These artificial biosynthetic pathways contained a codon-optimized tal gene that improved the productivity of 4-coumaric acid and ferulic acid, but not caffeic acid in a minimal salt medium. These heterologous pathways extended in E. coli that had biosynthesis machinery overproducing tyrosine. Finally, the titers of 4-coumaric acid, caffeic acid, and ferulic acid reached 974 mg/L, 150 mg/L, and 196 mg/L, respectively, in shake flasks after 36-hour cultivation. Conclusions We achieved one gram per liter scale production of 4-coumaric acid. In addition, maximum titers of 150 mg/L of caffeic acid and 196 mg/L of ferulic acid were achieved. Phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, have a great potential for pharmaceutical applications and food ingredients. This work forms a basis for further improvement in production and opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from phenylpropanoic acids.

  2. Deciphering Carbamoylpolyoxamic Acid Biosynthesis Reveals Unusual Acetylation Cycle Associated with Tandem Reduction and Sequential Hydroxylation.

    Science.gov (United States)

    Qi, Jianzhao; Wan, Dan; Ma, Hongmin; Liu, Yuanzhen; Gong, Rong; Qu, Xudong; Sun, Yuhui; Deng, Zixin; Chen, Wenqing

    2016-08-18

    Polyoxin, produced by Streptomcyes cacaoi var. asoensis and Streptomyces aureochromogenes, contains two non-proteinogenic amino acids, carbamoylpolyoxamic acid (CPOAA) and polyoximic acid. Although the CPOAA moiety is highly unusual, its biosynthetic logic has remained enigmatic for decades. Here, we address CPOAA biosynthesis by reconstitution of its pathway. We demonstrated that its biosynthesis is initiated by a versatile N-acetyltransferase, PolN, catalyzing L-glutamate (1) to N-acetyl glutamate (2). Remarkably, we verified that PolM, a previously annotated dehydrogenase, catalyzes an unprecedented tandem reduction of acyl-phosphate to aldehyde, and subsequently to alcohol. We also unveiled a distinctive acetylation cycle catalyzed by PolN to synthesize α-amino-δ-hydroxyvaleric acid (6). Finally, we report that PolL is capable of converting a rare sequential hydroxylation of α-amino-δ-carbamoylhydroxyvaleric acid (7) to CPOAA. PolL represents an intriguing family of Fe(II)-dependent α-ketoglutarate dioxygenase with a cupin fold. These data illustrate several novel enzymatic reactions, and also set a foundation for rational pathway engineering for polyoxin production.

  3. Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway.

    Science.gov (United States)

    Valegård, Karin; Iqbal, Aman; Kershaw, Nadia J; Ivison, David; Généreux, Catherine; Dubus, Alain; Blikstad, Cecilia; Demetriades, Marina; Hopkinson, Richard J; Lloyd, Adrian J; Roper, David I; Schofield, Christopher J; Andersson, Inger; McDonough, Michael A

    2013-08-01

    Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

  4. Sphingolipid biosynthesis upregulation by TOR complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress.

    Science.gov (United States)

    Guerreiro, Joana F; Muir, Alexander; Ramachandran, Subramaniam; Thorner, Jeremy; Sá-Correia, Isabel

    2016-12-01

    Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks.

  5. Synthesis and study on biological activity of nitrogen-containing heterocyclic compounds – regulators of enzymes of nucleic acid biosynthesis

    Directory of Open Access Journals (Sweden)

    Alexeeva I. V.

    2013-07-01

    Full Text Available Results of investigations on the development of new regulators of functional activity of nucleic acid biosynthesis enzymes based on polycyclic nitrogen-containing heterosystems are summarized. Computer design and molecular docking in the catalytic site of target enzyme (T7pol allowed to perform the directed optimization of basic structures. Several series of compounds were obtained and efficient inhibitors of herpes family (simple herpes virus type 2, Epstein-Barr virus, influenza A and hepatitis C viruses were identified, as well as compounds with potent antitumor, antibacterial and antifungal activity. It was established that the use of model test systems based on enzymes participating in nucleic acids synthesis is a promising approach to the primary screening of potential inhibitors in vitro.

  6. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro.

    Science.gov (United States)

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-04-01

    Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell-cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase.

  7. [Effect of organic acids on the biosynthesis of macrotetralide antibiotics by an Actinomyces chrysomallus var. carotenoides strain].

    Science.gov (United States)

    Nefelova, M V; Sverdlova, A N; Silaev, A B

    1978-07-01

    The biosynthesis of macrotetrolides by Actinomyces chrysomalus var. carotenoides was stimulated by acetic, succinic, propionic, oxalic, malic, tartaric, citric, pyruvic, alpha-ketoglutaric and fumaric acids. Incorporation of 14C-acetate into the molecule of the antibiotic and the data on dependence of the stimulating effect upon the quantitative ratio and time of the organic acid addition were indicative of the role of acetic, succinic and propionic acids as precursors of macrotetrolides. The other organic acids increased the biosynthesis of macrotetolides when added to the culture within wide time ranges of the culture development and prolonged the period of the mycelium productive state.

  8. Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis.

    Science.gov (United States)

    Creelman, R A; Bell, E; Mullet, J E

    1992-07-01

    Several lines of evidence indicate that abscisic acid (ABA) is derived from 9'-cis-neoxanthin or 9'-cis-violaxanthin with xanthoxin as an intermediate. (18)O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11', 12') double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.

  9. Modulation of endogenous indole-3-acetic acid biosynthesis in bacteroids within Medicago sativa nodules.

    Science.gov (United States)

    Bianco, C; Senatore, B; Arbucci, S; Pieraccini, G; Defez, R

    2014-07-01

    To evaluate the dose-response effects of endogenous indole-3-acetic acid (IAA) on Medicago plant growth and dry weight production, we increased the synthesis of IAA in both free-living and symbiosis-stage rhizobial bacteroids during Rhizobium-legume symbiosis. For this purpose, site-directed mutagenesis was applied to modify an 85-bp promoter sequence, driving the expression of iaaM and tms2 genes for IAA biosynthesis. A positive correlation was found between the higher expression of IAA biosynthetic genes in free-living bacteria and the increased production of IAA under both free-living and symbiotic conditions. Plants nodulated by RD65 and RD66 strains, synthetizing the highest IAA concentration, showed a significant (up to 73%) increase in the shoot fresh weight and upregulation of nitrogenase gene, nifH, compared to plants nodulated by the wild-type strain. When these plants were analyzed by confocal microscopy, using an anti-IAA antibody, the strongest signal was observed in bacteroids of Medicago sativa RD66 (Ms-RD66) plants, even when they were located in the senescent nodule zone. We show here a simple system to modulate endogenous IAA biosynthesis in bacteria nodulating legumes suitable to investigate which is the maximum level of IAA biosynthesis, resulting in the maximal increase of plant growth.

  10. Nitric oxide metabolism and indole acetic acid biosynthesis cross-talk in Azospirillum brasilense SM.

    Science.gov (United States)

    Koul, Vatsala; Tripathi, Chandrakant; Adholeya, Alok; Kochar, Mandira

    2015-04-01

    Production of nitric oxide (NO) and the presence of NO metabolism genes, nitrous oxide reductase (nosZ), nitrous oxide reductase regulator (nosR) and nitric oxide reductase (norB) were identified in the plant-associated bacterium (PAB) Azospirillum brasilense SM. NO presence was confirmed in all overexpressing strains, while improvement in the plant growth response of these strains was mediated by increased NO and indole-3-acetic acid (IAA) levels in the strains. Electron microscopy showed random distribution to biofilm, with surface colonization of pleiomorphic Azospirilla. Quantitative IAA estimation highlighted a crucial role of nosR and norBC in regulating IAA biosynthesis. The NO quencher and donor reduced/blocked IAA biosynthesis by all strains, indicating their common regulatory role in IAA biosynthesis. Tryptophan (Trp) and l-Arginine (Arg) showed higher expression of NO genes tested, while in the case of ipdC, only Trp and IAA increased expression, while Arg had no significant effect. The highest nosR expression in SMnosR in the presence of IAA and Trp, along with its 2-fold IAA level, confirmed the relationship of nosR overexpression with Trp in increasing IAA. These results indicate a strong correlation between IAA and NO in A. brasilense SM and suggest the existence of cross-talk or shared signaling mechanisms in these two growth regulators.

  11. Biosynthesis of Polyunsaturated Fatty Acids in Marine Invertebrates: Recent Advances in Molecular Mechanisms

    Science.gov (United States)

    Monroig, Óscar; Tocher, Douglas R.; Navarro, Juan C.

    2013-01-01

    Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading, de novo synthesis of PUFA is possible in some lower animals. The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs. PMID:24152561

  12. Phylogenetic analysis of genes involved in mycosporine-like amino acid biosynthesis in symbiotic dinoflagellates.

    Science.gov (United States)

    Rosic, Nedeljka N

    2012-04-01

    Mycosporine-like amino acids (MAAs) are multifunctional secondary metabolites involved in photoprotection in many marine organisms. As well as having broad ultraviolet (UV) absorption spectra (310-362 nm), these biological sunscreens are also involved in the prevention of oxidative stress. More than 20 different MAAs have been discovered so far, characterized by distinctive chemical structures and a broad ecological distribution. Additionally, UV-screening MAA metabolites have been investigated and used in biotechnology and cosmetics. The biosynthesis of MAAs has been suggested to occur via either the shikimate or pentose phosphate pathways. Despite their wide distribution in marine and freshwater species and also the commercial application in cosmetic products, there are still a number of uncertainties regarding the genetic, biochemical, and evolutionary origin of MAAs. Here, using a transcriptome-mining approach, we identify the gene counterparts from the shikimate or pentose phosphate pathway involved in MAA biosynthesis within the sequences of the reef-building coral symbiotic dinoflagellates (genus Symbiodinium). We also report the highly similar sequences of genes from the proposed MAA biosynthetic pathway involved in the metabolism of 4-deoxygadusol (direct MAA precursor) in various Symbiodinium strains confirming their algal origin and conserved nature. Finally, we reveal the separate identity of two O-methyltransferase genes, possibly involved in MAA biosynthesis, as well as nonribosomal peptide synthetase and adenosine triphosphate grasp homologs in symbiotic dinoflagellates. This study provides a biochemical and phylogenetic overview of the genes from the proposed MAA biosynthetic pathway with a focus on coral endosymbionts.

  13. Oleanolic acid and ursolic acid inhibit peptidoglycan biosynthesis in Streptococcus mutans UA159

    Directory of Open Access Journals (Sweden)

    Soon-Nang Park

    2015-06-01

    Full Text Available In this study, we revealed that OA and UA significantly inhibited the expression of most genes related to peptidoglycan biosynthesis in S. mutans UA159. To the best of our knowledge, this is the first report to introduce the antimicrobial mechanism of OA and UA against S. mutans.

  14. Extrinsic functions of lectin domains in O-N-acetylgalactosamine glycan biosynthesis

    DEFF Research Database (Denmark)

    Lorenz, Virginia; Ditamo, Yanina; Cejas, Romina B;

    2016-01-01

    Glycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. We evaluated the extrinsic effect of lectin domains (β-trefoil fold) of polypeptide GalNAc-transferases (ppGalNAc-Ts) on catalytic activity of glycosyltransferases...

  15. Importance of the Long-Chain Fatty Acid Beta-Hydroxylating Cytochrome P450 Enzyme YbdT for Lipopeptide Biosynthesis in Bacillus subtilis Strain OKB105

    Science.gov (United States)

    Youssef, Noha H.; Wofford, Neil; McInerney, Michael J.

    2011-01-01

    Bacillus species produce extracellular, surface-active lipopeptides such as surfactin that have wide applications in industry and medicine. The steps involved in the synthesis of 3-hydroxyacyl-coenzyme A (CoA) substrates needed for surfactin biosynthesis are not understood. Cell-free extracts of Bacillus subtilis strain OKB105 synthesized lipopeptide biosurfactants in presence of l-amino acids, myristic acid, coenzyme A, ATP, and H2O2, which suggested that 3-hydroxylation occurs prior to CoA ligation of the long chain fatty acids (LCFAs). We hypothesized that YbdT, a cytochrome P450 enzyme known to beta-hydroxylate LCFAs, functions to form 3-hydroxy fatty acids for lipopeptide biosynthesis. An in-frame mutation of ybdT was constructed and the resulting mutant strain (NHY1) produced predominantly non-hydroxylated lipopeptide with diminished biosurfactant and beta-hemolytic activities. Mass spectrometry showed that 95.6% of the fatty acids in the NHY1 biosurfactant were non-hydroxylated compared to only ∼61% in the OKB105 biosurfactant. Cell-free extracts of the NHY1 synthesized surfactin containing 3-hydroxymyristic acid from 3-hydroxymyristoyl-CoA at a specific activity similar to that of the wild type (17 ± 2 versus 17.4 ± 6 ng biosurfactant min−1·ng·protein−1, respectively). These results showed that the mutation did not affect any function needed to synthesize surfactin once the 3-hydroxyacyl-CoA substrate was formed and that YbdT functions to supply 3-hydroxy fatty acid for surfactin biosynthesis. The fact that YbdT is a peroxidase could explain why biosurfactant production is rarely observed in anaerobically grown Bacillus species. Manipulation of LCFA specificity of YbdT could provide a new route to produce biosurfactants with activities tailored to specific functions. PMID:21673922

  16. Importance of the Long-Chain Fatty Acid Beta-Hydroxylating Cytochrome P450 Enzyme YbdT for Lipopeptide Biosynthesis in Bacillus subtilis Strain OKB105

    Directory of Open Access Journals (Sweden)

    Michael J. McInerney

    2011-03-01

    Full Text Available Bacillus species produce extracellular, surface-active lipopeptides such as surfactin that have wide applications in industry and medicine. The steps involved in the synthesis of 3-hydroxyacyl-coenzyme A (CoA substrates needed for surfactin biosynthesis are not understood. Cell-free extracts of Bacillus subtilis strain OKB105 synthesized lipopeptide biosurfactants in presence of L-amino acids, myristic acid, coenzyme A, ATP, and H2O2, which suggested that 3-hydroxylation occurs prior to CoA ligation of the long chain fatty acids (LCFAs. We hypothesized that YbdT, a cytochrome P450 enzyme known to beta-hydroxylate LCFAs, functions to form 3-hydroxy fatty acids for lipopeptide biosynthesis. An in-frame mutation of ybdT was constructed and the resulting mutant strain (NHY1 produced predominantly non-hydroxylated lipopeptide with diminished biosurfactant and beta-hemolytic activities. Mass spectrometry showed that 95.6% of the fatty acids in the NHY1 biosurfactant were non-hydroxylated compared to only ~61% in the OKB105 biosurfactant. Cell-free extracts of the NHY1 synthesized surfactin containing 3-hydroxymyristic acid from 3-hydroxymyristoyl-CoA at a specific activity similar to that of the wild type (17 ± 2 versus 17.4 ± 6 ng biosurfactant min−1·ng·protein−1, respectively. These results showed that the mutation did not affect any function needed to synthesize surfactin once the 3-hydroxyacyl-CoA substrate was formed and that YbdT functions to supply 3-hydroxy fatty acid for surfactin biosynthesis. The fact that YbdT is a peroxidase could explain why biosurfactant production is rarely observed in anaerobically grown Bacillus species. Manipulation of LCFA specificity of YbdT could provide a new route to produce biosurfactants with activities tailored to specific functions.

  17. Systematic unravelling of the biosynthesis of poly (L-diaminopropionic acid) in Streptomyces albulus PD-1

    Science.gov (United States)

    Xu, Zhaoxian; Sun, Zhuzhen; Li, Sha; Xu, Zheng; Cao, Changhong; Xu, Zongqi; Feng, Xiaohai; Xu, Hong

    2015-01-01

    Poly(L-diaminopropionic acid) (PDAP) is one of the four homopoly(amino acid)s that have been discovered in nature. However, the molecular mechanism of PDAP biosynthesis has yet to be described. In this work, the general layout of the PDAP biosynthetic pathway is characterised in Streptomyces albulus PD-1 by genome mining, gene disruption, heterologous expression and in vitro feeding experiments. As a result, L-diaminopropionic acid (L-DAP), which is the monomer of PDAP, is shown to be jointly synthesised by two protein homologues of cysteine synthetase and ornithine cyclodeaminase. Then, L-DAP is assembled into PDAP by a novel nonribosomal peptide synthetase (NRPS) with classical adenylation and peptidyl carrier protein domains. However, instead of the traditional condensation or thioesterase domain of NRPSs, this NRPS has seven transmembrane domains surrounding three tandem soluble domains at the C-terminus. As far as we know, this novel single-module NRPS structure has only been reported in poly(ε-L-lysine) synthetase. The similar NRPS structure of PDAP synthetase and poly(ε-L-lysine) synthetase may be a common characteristic of homopoly(amino acid)s synthetases. In this case, we may discover and/or design more homopoly(amino acid)s by mining this kind of novel NRPS structure in the future. PMID:26632244

  18. Acumulación de aminoácidos tipo micosporina (MAAs: biosíntesis, fotocontrol y funciones ecofisiológicas Accumulation of mycosporine-like amino acids (MAAs: biosynthesis, photocontrol and ecophysiological functions

    Directory of Open Access Journals (Sweden)

    NATHALIE KORBEE

    2006-03-01

    Full Text Available Los efectos nocivos que inducen la radiación UV en organismos acuáticos pueden ser aminorados a través de varios mecanismos de fotoprotección entre los que se encuentran la acumulación de sustancias que absorben radiación UV (fotoprotectores y activación de sistemas antioxidantes. Entre los fotoprotectores descritos se encuentran los aminoácidos tipo micosporina (MAAs. En esta revisión se enfatiza sobre la capacidad fotoprotectora y antioxidante de estas sustancias. La función fotoprotectora de los MAAs puede deducirse de las características fotofísicas de estos compuestos. Es sabido que actúan como pantalla pasiva disipando térmicamente la energía UV absorbida. La acumulación de MAAs se induce tanto por radiación UV (UV-A y UV-B como por luz azul dentro de la banda de la radiación activa fotosintética (PAR. Por otro lado, hay algunas referencias que indican otras funciones fisiológicas de los MAAs, incluyendo la actividad antioxidante, la regulación osmótica y la reproductora. Siendo los MAAs sustancias nitrogenadas, recientemente se ha comenzado a estudiar la influencia de la disponibilidad de nitrógeno en la acumulación de estos, en combinación con UV y/o alta irradiancia de PAR. El amonio se moviliza hacia los MAAs relativamente rápido, por lo que se ha propuesto también a los MAAs como reservorio de nitrógenoThe negative effects of UV radiation on aquatic organisms can be diminished by several photo-protective mechanisms such as the accumulation of UV absorbing substances (photoprotectors and the activation of antioxidant systems. The mycosporine-like amino acids (MAAs are one of the most known photoprotectors. In this review their photoprotective and antioxidant capabilities are described. The UV screening function of MAAs can be inferred from their photophysic characteristics. It is known that MAAs act as passive screen dissipating the UV energy absorbed as thermal form. The accumulation of MAAs is induced by

  19. Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-Acid-Derived Biofuels and Chemicals with Relieved Side-Pathway Competition

    DEFF Research Database (Denmark)

    Zhou, Yongjin J.; Buijs, Nicolaas A; Zhu, Zhiwei

    2016-01-01

    to peroxisomes can increase the production of fatty-acid-derived fatty alcohols, alkanes and olefins up to 700%. In addition, we demonstrate that biosynthesis of these chemicals in the peroxisomes results in significantly decreased accumulation of byproducts formed by competing enzymes. We further demonstrate...... environment for biosynthesis. Peroxisomes are cellular organelles where fatty acids are degraded, a process that is inhibited under typical fermentation conditions making them an interesting workhouse for production of fatty-acid-derived molecules. Here, we show that targeting synthetic pathways...

  20. Characterization of three novel desaturases involved in the delta-6 desaturation pathways for polyunsaturated fatty acid biosynthesis from Phytophthora infestans.

    Science.gov (United States)

    Sun, Quanxi; Liu, Jiang; Zhang, Qin; Qing, Xiaohe; Dobson, Gary; Li, Xinzheng; Qi, Baoxiu

    2013-09-01

    Phytophthora infestans is the causative agent of potato blight that resulted in the great famine in Ireland in the nineteenth century. This microbe can release large amounts of the C20 very long-chain polyunsaturated fatty acids arachidonic acid (ARA; 20:4Δ(5, 8, 11, 14)) and eicosapentaenoic acid (EPA; 20:5Δ(5, 8, 11, 14, 17)) upon invasion that is known to elicit a hypersensitive response to their host plant. In order to identify enzymes responsible for the biosynthesis of these fatty acids, we blasted the recently fully sequenced P. infestans genome and identified three novel putatively encoding desaturase sequences. These were subsequently functionally characterized by expression in Saccharomyces cerevisiae and confirmed that they encode desaturases with Δ12, Δ6 and Δ5 activity, designated here as PinDes12, PinDes6 and PinDes5, respectively. This, together with the combined fatty acid profiles and a previously identified Δ6 elongase activity, implies that the ARA and EPA are biosynthesized predominantly via the Δ6 desaturation pathways in P. infestans. Elucidation of ARA and EPA biosynthetic mechanism may provide new routes to combating this potato blight microbe directly or by means of conferring resistance to important crops.

  1. Deficient liver biosynthesis of docosahexaenoic acid correlates with cognitive impairment in Alzheimer's disease.

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    Giuseppe Astarita

    Full Text Available Reduced brain levels of docosahexaenoic acid (C22:6n-3, a neurotrophic and neuroprotective fatty acid, may contribute to cognitive decline in Alzheimer's disease. Here, we investigated whether the liver enzyme system that provides docosahexaenoic acid to the brain is dysfunctional in this disease. Docosahexaenoic acid levels were reduced in temporal cortex, mid-frontal cortex and cerebellum of subjects with Alzheimer's disease, compared to control subjects (P  =  0.007. Mini Mental State Examination (MMSE scores positively correlated with docosahexaenoic/α-linolenic ratios in temporal cortex (P =  0.005 and mid-frontal cortex (P  =  0.018, but not cerebellum. Similarly, liver docosahexaenoic acid content was lower in Alzheimer's disease patients than control subjects (P  =  0.011. Liver docosahexaenoic/α-linolenic ratios correlated positively with MMSE scores (r  =  0.78; P<0.0001, and negatively with global deterioration scale grades (P  =  0.013. Docosahexaenoic acid precursors, including tetracosahexaenoic acid (C24:6n-3, were elevated in liver of Alzheimer's disease patients (P  =  0.041, whereas expression of peroxisomal d-bifunctional protein, which catalyzes the conversion of tetracosahexaenoic acid into docosahexaenoic acid, was reduced (P  = 0.048. Other genes involved in docosahexaenoic acid metabolism were not affected. The results indicate that a deficit in d-bifunctional protein activity impairs docosahexaenoic acid biosynthesis in liver of Alzheimer's disease patients, lessening the flux of this neuroprotective fatty acid to the brain.

  2. Screening for the genes involved in bombykol biosynthesis: Identification and functional characterization of Bombyx mori acyl carrier protein (BmACP

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    Atsushi eOhnishi

    2011-12-01

    Full Text Available Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG via fatty acid synthesis (FAS. Biosynthesis of moth sex pheromones is usually regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN, a 33-aa peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets (LDs, which store the sex pheromone (bombykol precursor fatty acid, accumulate in PG cells prior to eclosion. PBAN activation of the PBAN receptor stimulates lipolysis of the stored LD triacylglycerols (TAGs resulting in release of the bombykol precursor for final modification. While we have previously characterized a number of molecules involved in bombykol biosynthesis, little is known about the mechanisms of PBAN signaling that regulate the TAG lipolysis in PG cells. In the current study, we sought to further identify genes involved in bombykol biosynthesis as well as PBAN signaling, by using a subset of 312 expressed sequence tag (EST clones that are in either our B. mori PG cDNA library or the public B. mori EST databases, SilkBase and CYBERGATE, and which are preferentially expressed in the PG. Using RT-PCR expression analysis and an RNAi screening approach, we have identified another 8 EST clones involved in bombykol biosynthesis. Furthermore, we have determined the functional role of a clone designated BmACP that encodes B. mori acyl carrier protein (ACP. Our results indicate that BmACP plays an essential role in the biosynthesis of the bombykol precursor fatty acid via the canonical FAS pathway during pheromonogenesis.

  3. Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate.

    Science.gov (United States)

    Song, Heng; Hu, Wen; Naowarojna, Nathchar; Her, Ampon Sae; Wang, Shu; Desai, Rushil; Qin, Li; Chen, Xiaoping; Liu, Pinghua

    2015-01-01

    Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.

  4. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    Science.gov (United States)

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans.

  5. Ergothioneine Biosynthesis and Functionality in the Opportunistic Fungal Pathogen, Aspergillus fumigatus

    Science.gov (United States)

    Sheridan, Kevin J.; Lechner, Beatrix Elisabeth; Keeffe, Grainne O’; Keller, Markus A.; Werner, Ernst R.; Lindner, Herbert; Jones, Gary W.; Haas, Hubertus; Doyle, Sean

    2016-01-01

    Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H2O2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and β-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H2O2 in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H2O2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis. PMID:27748436

  6. Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast

    Science.gov (United States)

    Scheler, Ulschan; Brandt, Wolfgang; Porzel, Andrea; Rothe, Kathleen; Manzano, David; Božić, Dragana; Papaefthimiou, Dimitra; Balcke, Gerd Ulrich; Henning, Anja; Lohse, Swanhild; Marillonnet, Sylvestre; Kanellis, Angelos K.; Ferrer, Albert; Tissier, Alain

    2016-10-01

    Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.

  7. Biosynthesis and possible functions of inositol pyrophosphates in plants

    Directory of Open Access Journals (Sweden)

    Sarah Phoebe Williams

    2015-02-01

    Full Text Available Inositol phosphates (InsPs are intricately tied to lipid signaling, as at least one portion of the inositol phosphate signaling pool is derived from hydrolysis of the lipid precursor, phosphatidyl inositol (4,5 bisphosphate. The focus of this review is on the inositol pyrophosphates, which are a novel group of InsP signaling molecules containing diphosphate or triphosphate chains (i.e. PPx attached to the inositol ring. These PPx-InsPs are emerging as critical players in the integration of cellular metabolism and stress signaling in non-plant eukaryotes. Most eukaryotes synthesize the precursor molecule, myo-inositol (1,2,3,4,5,6-hexakisphosphate (InsP6, which can serve as a signaling molecule or as storage compound of inositol, phosphorus, and minerals (referred to as phytic acid. Even though plants produce huge amounts of precursor InsP6 in seeds, almost no attention has been paid to whether PPx-InsPs exist in plants, and if so, what roles these molecules play. Recent work has delineated that Arabidopsis has two genes capable of PP-InsP5 synthesis, and PPx-InsPs have been detected across the plant kingdom. This review will detail the known roles of PPx-InsPs in yeast and animal systems, and provide a description of recent data on the synthesis and accumulation of these novel molecules in plants, and potential roles in signaling.

  8. Phenolic Phytoalexins in Rice: Biological Functions and Biosynthesis

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    Man-Ho Cho

    2015-12-01

    Full Text Available Phytoalexins are inducible secondary metabolites possessing antimicrobial activity against phytopathogens. Rice produces a wide array of phytoalexins in response to pathogen attacks and environmental stresses. With few exceptions, most phytoalexins identified in rice are diterpenoid compounds. Until very recently, flavonoid sakuranetin was the only known phenolic phytoalexin in rice. However, recent studies have shown that phenylamides are involved in defense against pathogen attacks in rice. Phenylamides are amine-conjugated phenolic acids that are induced by pathogen infections and abiotic stresses including ultra violet (UV radiation in rice. Stress-induced phenylamides, such as N-trans-cinnamoyltryptamine, N-p-coumaroylserotonin and N-cinnamoyltyramine, have been reported to possess antimicrobial activities against rice bacterial and fungal pathogens, an indication of their direct inhibitory roles against invading pathogens. This finding suggests that phenylamides act as phytoalexins in rice and belong to phenolic phytoalexins along with sakuranetin. Phenylamides also have been implicated in cell wall reinforcement for disease resistance and allelopathy of rice. Synthesis of phenolic phytoalexins is stimulated by phytopathogen attacks and abiotic challenges including UV radiation. Accumulating evidence has demonstrated that biosynthetic pathways including the shikimate, phenylpropanoid and arylmonoamine pathways are coordinately activated for phenolic phytoalexin synthesis, and related genes are induced by biotic and abiotic stresses in rice.

  9. Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

    Science.gov (United States)

    Cao, Peng-Fei; Wu, Chen-Gao; Dang, Zhi-Hao; Shi, Liang; Jiang, Ai-Liang; Ren, Ang; Zhao, Ming-Wen

    2017-01-01

    We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 μmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 μg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 μmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 μmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

  10. Evolution of Diterpene Metabolism: Sitka Spruce CYP720B4 Catalyzes Multiple Oxidations in Resin Acid Biosynthesis of Conifer Defense against Insects1[C][W][OA

    Science.gov (United States)

    Hamberger, Björn; Ohnishi, Toshiyuki; Hamberger, Britta; Séguin, Armand; Bohlmann, Jörg

    2011-01-01

    Diterpene resin acids (DRAs) are specialized (secondary) metabolites of the oleoresin defense of conifers produced by diterpene synthases and cytochrome P450s of the CYP720B family. The evolution of DRA metabolism shares common origins with the biosynthesis of ent-kaurenoic acid, which is highly conserved in general (primary) metabolism of gibberellin biosynthesis. Transcriptome mining in species of spruce (Picea) and pine (Pinus) revealed CYP720Bs of four distinct clades. We cloned a comprehensive set of 12 different Sitka spruce (Picea sitchensis) CYP720Bs as full-length cDNAs. Spatial expression profiles, methyl jasmonate induction, and transcript enrichment in terpenoid-producing resin ducts suggested a role of CYP720B4 in DRA biosynthesis. CYP720B4 was characterized as a multisubstrate, multifunctional enzyme by the formation of oxygenated diterpenoids in metabolically engineered yeast, yeast in vivo transformation of diterpene substrates, in vitro assays with CYP720B4 protein produced in Escherichia coli, and alteration of DRA profiles in RNA interference-suppressed spruce seedlings. CYP720B4 was active with 24 different diterpenoid substrates, catalyzing consecutive C-18 oxidations in the biosynthesis of an array of diterpene alcohols, aldehydes, and acids. CYP720B4 was most active in the formation of dehydroabietic acid, a compound associated with insect resistance of Sitka spruce. We identified patterns of convergent evolution of CYP720B4 in DRA metabolism and ent-kaurene oxidase CYP701 in gibberellin metabolism and revealed differences in the evolution of specialized and general diterpene metabolism in a gymnosperm. The genomic and functional characterization of the gymnosperm CYP720B family highlights that the evolution of specialized metabolism involves substantial diversification relative to conserved, general metabolism. PMID:21994349

  11. Stimulatory Effects of Acibenzolar-S-methyl on Chlorogenic Acids Biosynthesis in Centella asiatica Cells

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    Efficient N Ncube

    2016-09-01

    Full Text Available Centella asiatica is a perennial herb that grows in tropical regions with numerous medicinal properties, mostly attributed to the presence of pentacyclic triterpenoids. Interestingly, this plant also possess a significant amount of phenylpropanoid-derived chlorogenic acids (CGAs that have recently been reported to confer neuroprotective properties. In a biotechnological attempt to increase the biosynthesis of CGA-derivatives in cultured Centella cells, acibenzolar-S-methyl was applied as a xenobiotic inducer in combination with quinic acid and shikimic acid as precursor molecules. Applying a semi-targeted metabolomics-based approach, time and concentration studies were undertaken to evaluate the effect of the manipulation on cellular metabolism leading to CGA production. Phytochemical extracts were prepared using methanol and analysed using a UHPLC-qTOF-MS platform. Data was processed and analysed using multivariate data models. A total of four CGA-derivatives, annotated as trans-5-feruloylquinic acid, 3,5 di-caffeoylquinic acid, 3,5-O-dicaffeoyl-4-O-malonylquinic acid (irbic acid and 3-caffeoyl, 5-feruloylquinic acid, were found to be upregulated by the acibenzolar-S-methyl treatment. To the best of our knowledge, this is the first report on the induction of CGA derivatives in this species. Contrary to expectations, the precursor molecules had very little effects on the levels of the CGAs. However, a total of 16 metabolites, including CGA derivatives, were up-regulated by precursor treatment. Therefore, this study shows potential to biotechnologically manipulate C. asiatica cells to increase the production of these health beneficial CGAs.

  12. Improvement of Neutral Lipid and Polyunsaturated Fatty Acid Biosynthesis by Overexpressing a Type 2 Diacylglycerol Acyltransferase in Marine Diatom Phaeodactylum tricornutum

    Directory of Open Access Journals (Sweden)

    Ying-Fang Niu

    2013-11-01

    Full Text Available Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT is a key enzyme that catalyzes the last step of triacylglyceride (TAG biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.

  13. Bird Integumentary Melanins: Biosynthesis, Forms, Function and Evolution.

    Science.gov (United States)

    Galván, Ismael; Solano, Francisco

    2016-04-08

    Melanins are the ubiquitous pigments distributed in nature. They are one of the main pigments responsible for colors in living cells. Birds are among the most diverse animals regarding melanin-based coloration, especially in the plumage, although they also pigment bare parts of the integument. This review is devoted to the main characteristics of bird melanins, including updated views of the formation and nature of melanin granules, whose interest has been raised in the last years for inferring the color of extinct birds and non-avian theropod dinosaurs using resistant fossil feathers. The molecular structure of the two main types of melanin, eumelanin and pheomelanin, and the environmental and genetic factors that regulate avian melanogenesis are also presented, establishing the main relationship between them. Finally, the special functions of melanin in bird feathers are also discussed, emphasizing the aspects more closely related to these animals, such as honest signaling, and the factors that may drive the evolution of pheomelanin and pheomelanin-based color traits, an issue for which birds have been pioneer study models.

  14. Bird Integumentary Melanins: Biosynthesis, Forms, Function and Evolution

    Directory of Open Access Journals (Sweden)

    Ismael Galván

    2016-04-01

    Full Text Available Melanins are the ubiquitous pigments distributed in nature. They are one of the main pigments responsible for colors in living cells. Birds are among the most diverse animals regarding melanin-based coloration, especially in the plumage, although they also pigment bare parts of the integument. This review is devoted to the main characteristics of bird melanins, including updated views of the formation and nature of melanin granules, whose interest has been raised in the last years for inferring the color of extinct birds and non-avian theropod dinosaurs using resistant fossil feathers. The molecular structure of the two main types of melanin, eumelanin and pheomelanin, and the environmental and genetic factors that regulate avian melanogenesis are also presented, establishing the main relationship between them. Finally, the special functions of melanin in bird feathers are also discussed, emphasizing the aspects more closely related to these animals, such as honest signaling, and the factors that may drive the evolution of pheomelanin and pheomelanin-based color traits, an issue for which birds have been pioneer study models.

  15. Genetic analysis of pathway regulation for enhancing branched-chain amino acid biosynthesis in plants

    KAUST Repository

    Chen, Hao

    2010-08-01

    The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over-expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE-TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map-based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant-containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed. © 2010 Blackwell Publishing Ltd.

  16. Improvement of clavulanic acid production in Streptomyces clavuligerus by genetic manipulation of structural biosynthesis genes.

    Science.gov (United States)

    Jnawali, Hum Nath; Yoo, Jin Cheol; Sohng, Jae Kyung

    2011-06-01

    To enhance clavulanic acid production, four structural clavulanic acid biosynthesis genes, carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2) and proclavaminate amidinohydrolase (pah2), were amplified from Streptomyces clavuligerus genomic DNA. They were cloned in the pSET152 integration and pIBR25 expression vectors containing the strong ermE* promoter to generate pHN18 and pHN19, respectively, and both plasmids were introduced into S. clavuligerus by protoplast transformation. Clavulanic acid production was increased by 8.7-fold (to ~310 mg/l) in integrative pHN18 transformants and by 5.1-fold in pHN19 transformants compared to controls. Transcriptional analyses showed that the expression levels of ceas2, bls2, cas2 and pah2 were markedly increased in both transformants as compared with wild-type. The elevation of the ceas2, bls2, cas2 and pah2 transcripts was consistent with the enhanced production of clavulanic acid.

  17. The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival.

    Directory of Open Access Journals (Sweden)

    João Daniel Santos Fernandes

    Full Text Available Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR. We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8. The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i quality of nitrogen (Nitrogen Catabolism Repression, NCR and carbon sources (Carbon Catabolism Repression, CCR, (ii amino acid availability in the extracellular environment (SPS-sensing and (iii nutritional deprivation (Global Amino Acid Control, GAAC. This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro.

  18. Resistance to herbicides inhibiting the biosynthesis of very-long-chain fatty acids.

    Science.gov (United States)

    Busi, Roberto

    2014-09-01

    Herbicides that act by inhibiting the biosynthesis of very-long-chain fatty acids (VLCFAs) have been used to control grass weeds in major crops throughout the world for the past 60 years. VLCFA-inhibiting herbicides are generally highly selective in crops, induce similar symptoms in susceptible grasses and can be found within the herbicide groups classified by the HRAC as K3 and N. Even after many years of continuous use, only 12 grass weed species have evolved resistance to VLCFA-inhibiting herbicides. Here, the cases of resistance that have evolved in major grass weed species belonging to the Avena, Echinochloa and Lolium genera in three different agricultural systems are reviewed. In particular we explore the possible reasons why VLCFA herbicides have been slow to select resistant weeds, outline the herbicide mode of action and discuss the resistance mechanisms that are most likely to have been selected.

  19. Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sun, Ruhao; Gao, Lingchao; Yu, Xiaoping; Zheng, Yusheng; Li, Dongdong; Wang, Xinguang

    2016-10-10

    Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes.

  20. A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum

    Science.gov (United States)

    Taura, Futoshi; Iijima, Miu; Yamanaka, Eriko; Takahashi, Hironobu; Kenmoku, Hiromichi; Saeki, Haruna; Morimoto, Satoshi; Asakawa, Yoshinori; Kurosaki, Fumiya; Morita, Hiroyuki

    2016-01-01

    Rhododendron dauricum L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of R. dauricum. The primary structure of ORS shared relatively low identities to those of PKSs from other plants, and the active site of ORS had a unique amino acid composition. The bacterially expressed, recombinant ORS accepted acetyl-CoA as the preferable starter substrate, and produced orcinol as the major reaction product, along with four minor products including OSA. The ORS identified in this study is the first plant PKS that generates acetate-derived aromatic tetraketides, such as orcinol and OSA. Interestingly, OSA production was clearly enhanced in the presence of Cannabis sativa olivetolic acid cyclase, suggesting that the ORS is involved in OSA biosynthesis together with an unidentified cyclase in R. dauricum. PMID:27729920

  1. Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products1[OPEN

    Science.gov (United States)

    Yuen, Macaire M.S.; Bohlmann, Jörg

    2016-01-01

    Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I–IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes. PMID:26936895

  2. Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products.

    Science.gov (United States)

    Geisler, Katrin; Jensen, Niels Berg; Yuen, Macaire M S; Madilao, Lina; Bohlmann, Jörg

    2016-05-01

    Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I-IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes.

  3. Auxin Biosynthesis: A Simple Two-Step Pathway Converts Tryptophan to Indole-3-Acetic Acid in Plants

    Institute of Scientific and Technical Information of China (English)

    Yunde Zhao

    2012-01-01

    Indole-3-acetic acid (IAA),the main naturally occurring auxin,is essential for almost every aspect of plant growth and development.However,only recently have studies finally established the first complete auxin biosynthesis pathway that converts tryptophan (Trp) to IAA in plants.Trp is first converted to indole-3-pyruvate (IPA) by the TAA family of amino transferases and subsequently IAA is produced from IPA by the YUC family of flavin monooxygenases.The two-step conversion of Trp to IAA is the main auxin biosynthesis pathway that plays an essential role in many developmental processes.

  4. Chlorogenic Acids Biosynthesis in Centella asiatica Cells Is not Stimulated by Salicylic Acid Manipulation.

    Science.gov (United States)

    Ncube, E N; Steenkamp, P A; Madala, N E; Dubery, I A

    2016-07-01

    Exogenous application of synthetic and natural elicitors of plant defence has been shown to result in mass production of secondary metabolites with nutraceuticals properties in cultured cells. In particular, salicylic acid (SA) treatment has been reported to induce the production of phenylpropanoids, including cinnamic acid derivatives bound to quinic acid (chlorogenic acids). Centella asiatica is an important medicinal plant with several therapeutic properties owing to its wide spectrum of secondary metabolites. We investigated the effect of SA on C. asiatica cells by monitoring perturbation of chlorogenic acids in particular. Different concentrations of SA were used to treat C. asiatica cells, and extracts from both treated and untreated cells were analysed using an optimised UHPLC-QTOF-MS/MS method. Semi-targeted multivariate data analyses with the aid of principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed a concentration-dependent metabolic response. Surprisingly, a range of chlorogenic acid derivatives were found to be downregulated as a consequence of SA treatment. Moreover, irbic acid (3,5-O-dicaffeoyl-4-O-malonilquinic acid) was found to be a dominant CGA in C. asiatica cells, although the SA treatment also had a negative effect on its concentration. Overall SA treatment was found to be an ineffective elicitor of CGA production in cultured C. asiatica cells.

  5. Redundant Systems of Phosphatidic Acid Biosynthesis via Acylation of Glycerol-3-Phosphate or Dihydroxyacetone Phosphate in the Yeast Saccharomyces cerevisiae

    OpenAIRE

    Athenstaedt, Karin; Weys, Sabine; Paltauf, Fritz; Daum, Günther

    1999-01-01

    In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611–7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p presen...

  6. The role of aromatic L-amino acid decarboxylase in bacillamide C biosynthesis by Bacillus atrophaeus C89

    OpenAIRE

    Lei Yuwen; Feng-Li Zhang; Qi-Hua Chen; Shuang-Jun Lin; Yi-Lei Zhao; Zhi-Yong Li

    2013-01-01

    For biosynthesis of bacillamide C by Bacillus atrophaeus C89 associated with South China sea sponge Dysidea avara, it is hypothesized that decarboxylation from L-tryptophan to tryptamine could be performed before amidation by the downstream aromatic L-amino acid decarboxylase (AADC) to the non-ribosomal peptide synthetases (NRPS) gene cluster for biosynthesizing bacillamide C. The structural analysis of decarboxylases' known substrates in KEGG database and alignment analysis of amino acid seq...

  7. Hardening with salicylic acid induces concentration-dependent changes in abscisic acid biosynthesis of tomato under salt stress.

    Science.gov (United States)

    Horváth, Edit; Csiszár, Jolán; Gallé, Ágnes; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2015-07-01

    The role of salicylic acid (SA) in the control of abscisic acid (ABA) biosynthesis is controversial although both plant growth regulators may accumulate in tissues under abiotic and biotic stress conditions. Hardening of tomato plants to salinity stress with 10(-4)M SA ("high SA") resulted in an up-regulation of ABA biosynthesis genes, zeaxanthin epoxidase (SlZEP1), 9-cis-epoxycarotenoid dioxygenase (SlNCED1) and aldehyde oxidases (SlAO1 and SlAO2) in the roots and led to ABA accumulation both in root and leaf tissues. In plants pre-treated with lower concentration of SA (10(-7)M, "low SA"), the up-regulation of SlNCED1 in the roots promoted ABA accumulation in the root tissues but the hormone concentration remained at control level in the leaves. Salt stress induced by 100mM NaCl reduced the transcript abundance of ABA biosynthetic genes and inhibited SlAO activity in plants hardened with "high SA", but the tissues maintained root ABA level over the untreated control. The combined effect of "high SA" and ABA under salt stress led to partially recovered photosynthetic activity, reduced ethylene production in root apices, and restored root growth, which is one of the main features of salt tolerance. Unlike "high SA", hardening with "low SA" had no influence on ethylene production, and led to reduced elongation of roots in plants exposed to 100mM NaCl. The up-regulation of carotenoid cleavage dioxygenases SlCCD1A and SlCCD1B by SA, which produce apocarotenoids, may open new pathways in SA sensing and signalling processes.

  8. Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid.

    Science.gov (United States)

    Zheng, Xiao-Yu; Zhou, Mian; Yoo, Heejin; Pruneda-Paz, Jose L; Spivey, Natalie Weaver; Kay, Steve A; Dong, Xinnian

    2015-07-28

    The plant hormone salicylic acid (SA) is essential for local defense and systemic acquired resistance (SAR). When plants, such as Arabidopsis, are challenged by different pathogens, an increase in SA biosynthesis generally occurs through transcriptional induction of the key synthetic enzyme isochorismate synthase 1 (ICS1). However, the regulatory mechanism for this induction is poorly understood. Using a yeast one-hybrid screen, we identified two transcription factors (TFs), NTM1-like 9 (NTL9) and CCA1 hiking expedition (CHE), as activators of ICS1 during specific immune responses. NTL9 is essential for inducing ICS1 and two other SA synthesis-related genes, phytoalexin-deficient 4 (PAD4) and enhanced disease susceptibility 1 (EDS1), in guard cells that form stomata. Stomata can quickly close upon challenge to block pathogen entry. This stomatal immunity requires ICS1 and the SA signaling pathway. In the ntl9 mutant, this response is defective and can be rescued by exogenous application of SA, indicating that NTL9-mediated SA synthesis is essential for stomatal immunity. CHE, the second identified TF, is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for the pathogen-induced SA synthesis in systemic tissues during SAR. CHE may also regulate ICS1 through the known transcription activators calmodulin binding protein 60g (CBP60g) and systemic acquired resistance deficient 1 (SARD1) because induction of these TF genes is compromised in the che-2 mutant. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner and therefore fills a gap in the signal transduction pathway between pathogen recognition and SA production.

  9. Plastoquinone and Ubiquinone in Plants: Biosynthesis, Physiological Function and Metabolic Engineering

    Science.gov (United States)

    Liu, Miaomiao; Lu, Shanfa

    2016-01-01

    Plastoquinone (PQ) and ubiquinone (UQ) are two important prenylquinones, functioning as electron transporters in the electron transport chain of oxygenic photosynthesis and the aerobic respiratory chain, respectively, and play indispensable roles in plant growth and development through participating in the biosynthesis and metabolism of important chemical compounds, acting as antioxidants, being involved in plant response to stress, and regulating gene expression and cell signal transduction. UQ, particularly UQ10, has also been widely used in people’s life. It is effective in treating cardiovascular diseases, chronic gingivitis and periodontitis, and shows favorable impact on cancer treatment and human reproductive health. PQ and UQ are made up of an active benzoquinone ring attached to a polyisoprenoid side chain. Biosynthesis of PQ and UQ is very complicated with more than thirty five enzymes involved. Their synthetic pathways can be generally divided into two stages. The first stage leads to the biosynthesis of precursors of benzene quinone ring and prenyl side chain. The benzene quinone ring for UQ is synthesized from tyrosine or phenylalanine, whereas the ring for PQ is derived from tyrosine. The prenyl side chains of PQ and UQ are derived from glyceraldehyde 3-phosphate and pyruvate through the 2-C-methyl-D-erythritol 4-phosphate pathway and/or acetyl-CoA and acetoacetyl-CoA through the mevalonate pathway. The second stage includes the condensation of ring and side chain and subsequent modification. Homogentisate solanesyltransferase, 4-hydroxybenzoate polyprenyl diphosphate transferase and a series of benzene quinone ring modification enzymes are involved in this stage. PQ exists in plants, while UQ widely presents in plants, animals and microbes. Many enzymes and their encoding genes involved in PQ and UQ biosynthesis have been intensively studied recently. Metabolic engineering of UQ10 in plants, such as rice and tobacco, has also been tested. In this

  10. The putative E3 ubiquitin ligase ECERIFERUM9 regulates abscisic acid biosynthesis and response during seed germination and postgermination growth in arabidopsis

    KAUST Repository

    Zhao, Huayan

    2014-05-08

    The ECERIFERUM9 (CER9) gene encodes a putative E3 ubiquitin ligase that functions in cuticle biosynthesis and the maintenance of plant water status. Here, we found that CER9 is also involved in abscisic acid (ABA) signaling in seeds and young seedlings of Arabidopsis (Arabidopsis thaliana). The germinated embryos of the mutants exhibited enhanced sensitivity to ABA during the transition from reversible dormancy to determinate seedling growth. Expression of the CER9 gene is closely related to ABA levels and displays a similar pattern to that of ABSCISIC ACID-INSENSITIVE5 (ABI5), which encodes a positive regulator of ABA responses in seeds. cer9 mutant seeds exhibited delayed germination that is independent of seed coat permeability. Quantitative proteomic analyses showed that cer9 seeds had a protein profile similar to that of the wild type treated with ABA. Transcriptomics analyses revealed that genes involved in ABA biosynthesis or signaling pathways were differentially regulated in cer9 seeds. Consistent with this, high levels of ABA were detected in dry seeds of cer9. Blocking ABA biosynthesis by fluridone treatment or by combining an ABA-deficient mutation with cer9 attenuated the phenotypes of cer9. Whereas introduction of the abi1-1, abi3-1, or abi4-103 mutation could completely eliminate the ABA hypersensitivity of cer9, introduction of abi5 resulted only in partial suppression. These results indicate that CER9 is a novel negative regulator of ABA biosynthesis and the ABA signaling pathway during seed germination. © 2014 American Society of Plant Biologists. All Rights Reserved.

  11. Positional distribution of fatty acids on the glycerol backbone during the biosynthesis of glycerolipids in Ectocarpus fasciculatus

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The biosynthesis of glycolipids in E. fasciculatus was studied by 14C label and chase. The fatty acids in sulphoquinovosyl diacylglycerol (SQDG) were almost 16-car- bon and 18-carbon ones. In addition to the two fatty acids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained 8.5 mol% and 31.0 mol% of eicosapentaenoic acid (20∶5), respectively, and this fatty acid was usually distributed in the sn-1 position of the glycerol backbone. When plants were incubated with [2-14C] acetate, differences existed in the positional distribution of the labeled fatty acids in sn-1 and sn-2 among the three glycerolipids. In SQDG, 14C-labeled fatty acids were distributed uniformly in the sn-1 and sn-2 positions. In DGDG, 14C-labeled fatty acids were mainly distributed in the sn-2 position. In MGDG, the radioactivity of fatty acids in sn-1 position was far greater than that in sn-2 position after a 30 min pulse label, and the difference in radioactivity between the two positions decreased rapidly. The above results indicated that differences in the positional distribution of 14C-labeled fatty acids between sn-1 and sn-2 positions might be related to 20∶5 and the biosynthesis of DGDG. Our results also suggested that E. fasciculatus had the same DGDG biosynthetic pathway as that in higher plants and galactosyl transferase was selective for MGDG.

  12. Salicylic Acid and its Function in Plant Immunity

    Institute of Scientific and Technical Information of China (English)

    Chuanfu An; Zhonglin Mou

    2011-01-01

    The small phenolic compound salicylic acid (SA) plays an important regulatory role in multiple physiological processes including plant immune response. Significant progress has been made during the past two decades in understanding the SA-mediated defense signaling network.Characterization of a number of genes functioning in SA biosynthesis,conjugation, accumulation, signaling, and crosstalk with other hormones such as jasmonic acid, ethylene, abscisic acid, auxin, gibberellic acid,cytokinin, brassinosteroid, and peptide hormones has sketched the finely tuned immune response network. Full understanding of the mechanism of plant immunity will need to take advantage of fast developing genomics tools and bioinformatics techniques. However, elucidating genetic components involved in these pathways by conventional genetics, biochemistry, and molecular biology approaches will continue to be a major task of the community. High-throughput method for SA quantification holds the potential for isolating additional mutants related to SA-mediated defense signaling.

  13. Molecular evolution and functional characterisation of haplotypes of an important rubber biosynthesis gene in Hevea brasiliensis.

    Science.gov (United States)

    Uthup, T K; Rajamani, A; Ravindran, M; Saha, T

    2016-07-01

    Hydroxy-methylglutaryl coenzyme-A synthase (HMGS) is a rate-limiting enzyme in the cytoplasmic isoprenoid biosynthesis pathway leading to natural rubber production in Hevea brasiliensis (rubber). Analysis of the structural variants of this gene is imperative to understand their functional significance in rubber biosynthesis so that they can be properly utilised for ongoing crop improvement programmes in Hevea. We report here allele richness and diversity of the HMGS gene in selected popular rubber clones. Haplotypes consisting of single nucleotide polymorphisms (SNPs) from the coding and non-coding regions with a high degree of heterozygosity were identified. Segregation and linkage disequilibrium analysis confirmed that recombination is the major contributor to the generation of allelic diversity, rather than point mutations. The evolutionarily conserved nature of some SNPs was identified by comparative DNA sequence analysis of HMGS orthologues from diverse taxa, demonstrating the molecular evolution of rubber biosynthesis genes in general. In silico three-dimensional structural studies highlighting the structural positioning of non-synonymous SNPs from different HMGS haplotypes revealed that the ligand-binding site on the enzyme remains impervious to the reported sequence variations. In contrast, gene expression results indicated the possibility of association between specific haplotypes and HMGS expression in Hevea clones, which may have a downstream impact up to the level of rubber production. Moreover, haplotype diversity of the HMGS gene and its putative association with gene expression can be the basis for further genetic association studies in rubber. Furthermore, the data also show the role of SNPs in the evolution of candidate genes coding for functional traits in plants.

  14. The response regulator YycF inhibits expression of the fatty acid biosynthesis repressor FabT in Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Maria Luz Mohedano

    2016-08-01

    Full Text Available The YycFG (also known as WalRK, VicRK, MicAB or TCS02 two-component system (TCS is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

  15. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae

    Science.gov (United States)

    Mohedano, Maria L.; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M.; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation. PMID:27610104

  16. Complex Binding of the FabR Repressor of Bacterial Unsaturated Fatty Acid Biosynthesis to its Cognate Promoters

    OpenAIRE

    Feng, Youjun; Cronan, John E.

    2011-01-01

    Two transcriptional regulators, the FadR activator and the FabR repressor control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was bloc...

  17. The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by C-13-labeling experiments

    DEFF Research Database (Denmark)

    Jensen, N.B.S.; Christensen, B.; Nielsen, Jette

    2002-01-01

    Uniformly C-13 labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with C-13 disclosing a substantial de novo...... biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium....

  18. COPI activity coupled with fatty acid biosynthesis is required for viral replication.

    Directory of Open Access Journals (Sweden)

    Sara Cherry

    2006-10-01

    Full Text Available During infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host-pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies.

  19. Dissociation of cephamycin C and clavulanic acid biosynthesis by 1,3-diaminopropane in Streptomyces clavuligerus.

    Science.gov (United States)

    Leite, Carla A; Cavallieri, André P; Baptista, Amanda S; Araujo, Maria L G C

    2016-01-01

    Streptomyces clavuligerus produces simultaneously cephamycin C (CephC) and clavulanic acid (CA). Adding 1,3-diaminopropane to culture medium stimulates production of beta-lactam antibiotics. However, there are no studies on the influence of this diamine on coordinated production of CephC and CA. This study indicates that 1,3-diaminopropane can dissociate CephC and CA productions. Results indicated that low diamine concentrations (below 1.25 g l(-1)) in culture medium increased CA production by 200%, but not that of CephC. Conversely, CephC production increased by 300% when 10 g l(-1) 1,3-diaminopropane was added to culture medium. Addition of just L-lysine (18.3 g l(-1)) to culture medium increased both biocompounds. On the other hand, while L-lysine plus 7.5 g l(-1) 1,3-diaminopropane increased volumetric production of CephC by 1100%, its impact on CA production was insignificant. The combined results suggest that extracellular concentration of 1,3-diaminopropane may trigger the dissociation of CephC and CA biosynthesis in S. clavuligerus.

  20. Co-culture engineering for microbial biosynthesis of 3-amino-benzoic acid in Escherichia coli.

    Science.gov (United States)

    Zhang, Haoran; Stephanopoulos, Gregory

    2016-07-01

    3-amino-benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli-E. coli co-culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co-culture system was found to improve 3AB production by 15 fold, compared to the mono-culture approach. Further engineering of the co-culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co-culture engineering can be a powerful new approach in the broad field of metabolic engineering.

  1. An extracytoplasmic function sigma factor, σ(25), differentially regulates avermectin and oligomycin biosynthesis in Streptomyces avermitilis.

    Science.gov (United States)

    Luo, Shuai; Sun, Di; Zhu, Jianya; Chen, Zhi; Wen, Ying; Li, Jilun

    2014-08-01

    σ(25) is an extracytoplasmic function (ECF) σ factor in the bacterium Streptomyces avermitilis that plays a differential regulatory role in avermectin and oligomycin biosynthesis. Gene deletion, complementation, and overexpression experiments showed that σ(25) inhibited avermectin production but promoted oligomycin production. σ(25) indirectly inhibited avermectin production by affecting the transcription of the pathway-specific activator gene aveR, whereas it directly activated oligomycin production by initiating transcription of the pathway-specific activator gene olmRI. The divergently transcribed genes smrAB are located upstream of sig25 and encode a putative two-component system (TCS). σ(25) was found to initiate its own transcription, and its expression was directly activated by SmrA. The precise SmrA-binding sites in the region upstream of sig25 were determined by DNase I footprinting assays and identified two direct repeat sequences CTGTGA-n5-CTGTGA, suggesting that SmrA regulates sig25 transcription by binding to these direct repeats. The deletion of smrAB had the similar effect on avermectin and oligomycin A production to the deletion of sig25, indicating that σ(25) and SmrAB function similarly in the regulation of antibiotic production. These findings helpfully clarify the regulation of antibiotic biosynthesis by an ECF σ factor-TCS signal transduction system in S. avermitilis.

  2. Biosynthesis of the Escherichia coli K1 group 2 polysialic acid capsule occurs within a protected cytoplasmic compartment.

    Science.gov (United States)

    Steenbergen, Susan M; Vimr, Eric R

    2008-06-01

    Capsular polysaccharides are important virulence determinants in a wide range of invasive infectious diseases. Although capsule synthesis has been extensively investigated, understanding polysaccharide export from the cytoplasm to the external environment has been more difficult. Here we present the results of a novel protection assay indicating that synthesis and export of the Escherichia coli K1 group 2 capsular polysialic acid (K1 antigen) occur within a protected subcellular compartment designated the sialisome. In addition to the polymerase encoded by neuS, localization and complementation analyses indicated that the sialisome includes the accessory membrane protein NeuE. The requirement for NeuE was suppressed by overproducing NeuS, suggesting that NeuE functions by stabilizing the polymerase or facilitating its assembly in the sialisome. Although an interaction between NeuE and NeuS could not be demonstrated with a bacterial two-hybrid system that reconstitutes an intracellular cell-signalling pathway, interactions between NeuS and KpsC as well as other sialisome components were detected. The combined results provide direct evidence for specific protein-protein interactions in the synthesis and export of group 2 capsular polysaccharides under in vivo conditions. The approaches developed here will facilitate further dissection of the sialisome, suggesting similar methodology for understanding the biosynthesis of other group 2 capsules.

  3. Abscisic acid induces biosynthesis of bisbibenzyls and tolerance to UV-C in the liverwort Marchantia polymorpha.

    Science.gov (United States)

    Kageyama, Akito; Ishizaki, Kimitsune; Kohchi, Takayuki; Matsuura, Hideyuki; Takahashi, Kosaku

    2015-09-01

    Environmental stresses are effective triggers for the biosynthesis of various secondary metabolites in plants, and phytohormones such as jasmonic acid and abscisic acid are known to mediate such responses in flowering plants. However, the detailed mechanism underlying the regulation of secondary metabolism in bryophytes remains unclear. In this study, the induction mechanism of secondary metabolites in the model liverwort Marchantia polymorpha was investigated. Abscisic acid (ABA) and ultraviolet irradiation (UV-C) were found to induce the biosynthesis of isoriccardin C, marchantin C, and riccardin F, which are categorized as bisbibenzyls, characteristic metabolites of liverworts. UV-C led to the significant accumulation of ABA. Overexpression of MpABI1, which encodes protein phosphatase 2C (PP2C) as a negative regulator of ABA signaling, suppressed accumulation of bisbibenzyls in response to ABA and UV-C irradiation and conferred susceptibility to UV-C irradiation. These data show that ABA plays a significant role in the induction of bisbibenzyl biosynthesis, which might confer tolerance against UV-C irradiation in M. polymorpha.

  4. Deuterium NMR used to indicate a common mechanism for the biosynthesis of ricinoleic acid by Ricinus communis and Claviceps purpurea.

    Science.gov (United States)

    Billault, Isabelle; Mantle, Peter G; Robins, Richard J

    2004-03-17

    Previous studies have shown that ricinoleic acid from castor bean oil of Ricinus communis is synthesized by the direct hydroxyl substitution of oleate, while it has been proposed that ricinoleate is formed by hydration of linoleate in the ergot fungus Claviceps purpurea. The mechanism of the enzymes specific to ricinoleate synthesis has not yet been established, but hydroxylation and desaturation of fatty acids in plants apparently involve closely related mechanisms. As mechanistic differences in the enzymes involved in the biosynthesis of natural products can lead to different isotopic distributions in the product, we could expect ricinoleate isolated from castor or ergot oil to show distinct (2)H distribution patterns. To obtain information concerning the substrate and isotope effects that occur during the biosynthesis of ricinoleate, the site-specific natural deuterium distributions in methyl ricinoleate isolated from castor oil and in methyl ricinoleate and methyl linoleate isolated from ergot oils have been measured by quantitative (2)H NMR. First, the deuterium profiles for methyl ricinoleate from the plant and fungus are equivalent. Second, the deuterium profile for methyl linoleate from ergot is incompatible with this chemical species being the precursor of methyl ricinoleate. Hence, it is apparent that 12-hydroxylation in C. purpurea is consistent with the biosynthetic mechanisms proposed for R. communis and is compatible with the general fundamental mechanistic similarities between hydroxylation and desaturation previously proposed for plant fatty acid biosynthesis.

  5. Differential expression of structural genes for the late phase of phytic acid biosynthesis in developing seeds of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Bhati, Kaushal Kumar; Aggarwal, Sipla; Sharma, Shivani; Mantri, Shrikant; Singh, Sudhir P; Bhalla, Sherry; Kaur, Jagdeep; Tiwari, Siddharth; Roy, Joy K; Tuli, Rakesh; Pandey, Ajay K

    2014-07-01

    In cereals, phytic acid (PA) or inositol hexakisphosphate (IP6) is a well-known phosphate storage compound as well as major chelator of important micronutrients (iron, zinc, calcium, etc.). Genes involved in the late phases of PA biosynthesis pathway are known in crops like maize, soybeans and barley but none have been reported from wheat. Our in silico analysis identified six wheat genes that might be involved in the biosynthesis of inositol phosphates. Four of the genes were inositol tetraphosphate kinases (TaITPK1, TaITPK2, TaITPK3, and TaITPK4), and the other two genes encode for inositol triphosphate kinase (TaIPK2) and inositol pentakisphosphate kinase (TaIPK1). Additionally, we identified a homolog of Zmlpa-1, an ABCC subclass multidrug resistance-associated transporter protein (TaMRP3) that is putatively involved in PA transport. Analyses of the mRNA expression levels of these seven genes showed that they are differentially expressed during seed development, and that some are preferentially expressed in aleurone tissue. These results suggest selective roles during PA biosynthesis, and that both lipid-independent and -dependent pathways are active in developing wheat grains. TaIPK1 and TaMRP3 were able to complement the yeast ScΔipk1 and ScΔycf1 mutants, respectively, providing evidence that the wheat genes have the expected biochemical functions. This is the first comprehensive study of the wheat genes involved in the late phase of PA biosynthesis. Knowledge generated from these studies could be utilized to develop strategies for generating low phyate wheat.

  6. Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Pittet, Martine; Conzelmann, Andreas

    2007-03-01

    Like most other eukaryotes, Saccharomyces cerevisiae harbors a GPI anchoring machinery and uses it to attach proteins to membranes. While a few GPI proteins reside permanently at the plasma membrane, a majority of them gets further processed and is integrated into the cell wall by a covalent attachment to cell wall glucans. The GPI biosynthetic pathway is necessary for growth and survival of yeast cells. The GPI lipids are synthesized in the ER and added onto proteins by a pathway comprising 12 steps, carried out by 23 gene products, 19 of which are essential. Some of the estimated 60 GPI proteins predicted from the genome sequence serve enzymatic functions required for the biosynthesis and the continuous shape adaptations of the cell wall, others seem to be structural elements of the cell wall and yet others mediate cell adhesion. Because of its genetic tractability S. cerevisiae is an attractive model organism not only for studying GPI biosynthesis in general, but equally for investigating the intracellular transport of GPI proteins and the peculiar role of GPI anchoring in the elaboration of fungal cell walls.

  7. Biosynthesis of 1-aminocyclopropane-1-carboxylic acid and ethylene from delta-aminolevulinic acid in ripening tomato fruits

    Energy Technology Data Exchange (ETDEWEB)

    El-Rayes, D.E.D.A.

    1987-01-01

    A new pathway for ethylene (C/sub 2/H/sub 4/) biosynthesis, which utilizes delta-aminolevulinic acid (ALA) as a precursor of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of C/sub 2/H/sub 4/, is presented. ALA enhanced ACC accumulation to 410% and C/sub 2/H/sub 4/ production to 232% of the control. The C/sub 2/H/sub 4/ production rate varied with the ALA concentration and the stage of tomato fruit development. As the ALA concentration increased from zero to 40 mM, the C/sub 2/H/sub 4/ production rate increased. Both treated and untreated pericarp discs from fruits at the pink stage of development yielded the largest C/sub 2/H/sub 4/ production rate. Radioactivity from (2,3-/sup 3/H)ALA was detected in both ACC and C/sub 2/H/sub 4/, and radioactivity from (4-/sup 14/C)ALA was detected in ACC and CO/sub 2/ but not in C/sub 2/H/sub 4/. However, radioactivity from (5-/sup 14/C)ALA was detected in CO/sub 2/, and its amount was greater than that obtained from (4-/sup 14/C)ALA. Neither ACC nor C/sub 2/H/sub 4/ showed any radioactivity when (5-/sup 14/C)ALA was supplied to the fruit discs. In addition, when (2,3-/sup 3/H)ALA or (4-/sup 14/C)ALA was supplied to the fruit discs, radioactivity was detected in other metabolites such as fumarate, succinate, malate, glutamate, glutamine, ..cap alpha..-ketoglutarate, and methionine, but the amount of radioactivity was insignificant as compared with the amount of radioactivity found in C/sub 2/H/sub 4/ and ACC.

  8. Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate

    Directory of Open Access Journals (Sweden)

    Schellenberg Barbara

    2009-10-01

    Full Text Available Abstract Background Syringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringae pv. syringae B728a with its host plant Phaseolus vulgaris (bean, was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames sylA-sylE allowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for. Results We have cloned a 22 kb genomic fragment containing the sylA-sylE gene cluster but no other complete gene into the broad host range cosmid pLAFR3. Transfer of the recombinant cosmid into Pseudomonas putida and P. syringae pv. syringae SM was sufficient to direct the biosynthesis of bona fide syringolin A in these heterologous organisms whose genomes do not contain homologous genes. NMR analysis of syringolin A isolated from cultures grown in the presence of NaH13CO3 revealed preferential 13C-labeling at the ureido carbonyl position. Conclusion The results show that no additional syringolin A-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the sylC gene product(s. A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.

  9. Biological functions of iduronic acid in chondroitin/dermatan sulfate.

    Science.gov (United States)

    Thelin, Martin A; Bartolini, Barbara; Axelsson, Jakob; Gustafsson, Renata; Tykesson, Emil; Pera, Edgar; Oldberg, Åke; Maccarana, Marco; Malmstrom, Anders

    2013-05-01

    The presence of iduronic acid in chondroitin/dermatan sulfate changes the properties of the polysaccharides because it generates a more flexible chain with increased binding potentials. Iduronic acid in chondroitin/dermatan sulfate influences multiple cellular properties, such as migration, proliferation, differentiation, angiogenesis and the regulation of cytokine/growth factor activities. Under pathological conditions such as wound healing, inflammation and cancer, iduronic acid has diverse regulatory functions. Iduronic acid is formed by two epimerases (i.e. dermatan sulfate epimerase 1 and 2) that have different tissue distribution and properties. The role of iduronic acid in chondroitin/dermatan sulfate is highlighted by the vast changes in connective tissue features in patients with a new type of Ehler-Danlos syndrome: adducted thumb-clubfoot syndrome. Future research aims to understand the roles of the two epimerases and their interplay with the sulfotransferases involved in chondroitin sulfate/dermatan sulfate biosynthesis. Furthermore, a better definition of chondroitin/dermatan sulfate functions using different knockout models is needed. In this review, we focus on the two enzymes responsible for iduronic acid formation, as well as the role of iduronic acid in health and disease.

  10. Overlapping functions of the starch synthases SSII and SSIII in amylopectin biosynthesis in Arabidopsis

    Directory of Open Access Journals (Sweden)

    D'Hulst Christophe

    2008-09-01

    Full Text Available Abstract Background The biochemical mechanisms that determine the molecular architecture of amylopectin are central in plant biology because they allow long-term storage of reduced carbon. Amylopectin structure imparts the ability to form semi-crystalline starch granules, which in turn provides its glucose storage function. The enzymatic steps of amylopectin biosynthesis resemble those of the soluble polymer glycogen, however, the reasons for amylopectin's architectural distinctions are not clearly understood. The multiplicity of starch biosynthetic enzymes conserved in plants likely is involved. For example, amylopectin chain elongation in plants involves five conserved classes of starch synthase (SS, whereas glycogen biosynthesis typically requires only one class of glycogen synthase. Results Null mutations were characterized in AtSS2, which codes for SSII, and mutant lines were compared to lines lacking SSIII and to an Atss2, Atss3 double mutant. Loss of SSII did not affect growth rate or starch quantity, but caused increased amylose/amylopectin ratio, increased total amylose, and deficiency in amylopectin chains with degree of polymerization (DP 12 to DP28. In contrast, loss of both SSII and SSIII caused slower plant growth and dramatically reduced starch content. Extreme deficiency in DP12 to DP28 chains occurred in the double mutant, far more severe than the summed changes in SSII- or SSIII-deficient plants lacking only one of the two enzymes. Conclusion SSII and SSIII have partially redundant functions in determination of amylopectin structure, and these roles cannot be substituted by any other conserved SS, specifically SSI, GBSSI, or SSIV. Even though SSIII is not required for the normal abundance of glucan chains of DP12 to DP18, the enzyme clearly is capable of functioning in production such chains. The role of SSIII in producing these chains cannot be detected simply by analysis of an individual mutation. Competition between

  11. Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels.

    Science.gov (United States)

    Mandal, Shantanu; Upadhyay, Shivangi; Wajid, Saima; Ram, Mauji; Jain, Dharam Chand; Singh, Ved Pal; Abdin, Malik Zainul; Kapoor, Rupam

    2015-07-01

    It is becoming increasingly evident that the formation of arbuscular mycorrhiza (AM) enhances secondary metabolite production in shoots. Despite mounting evidence, relatively little is known about the underlying mechanisms. This study suggests that increase in artemisinin concentration in Artemisia annua colonized by Rhizophagus intraradices is due to altered trichome density as well as transcriptional patterns that are mediated via enhanced jasmonic acid (JA) levels. Mycorrhizal (M) plants had higher JA levels in leaf tissue that may be due to induction of an allene oxidase synthase gene (AOS), encoding one of the key enzymes for JA production. Non-mycorrhizal (NM) plants were exogenously supplied with a range of methyl jasmonic acid concentrations. When leaves of NM and M plants with similar levels of endogenous JA were compared, these matched closely in terms of shoot trichome density, artemisinin concentration, and transcript profile of artemisinin biosynthesis genes. Mycorrhization increased artemisinin levels by increasing glandular trichome density and transcriptional activation of artemisinin biosynthesis genes. Transcriptional analysis of some rate-limiting enzymes of mevalonate and methyl erythritol phosphate (MEP) pathways revealed that AM increases isoprenoids by induction of the MEP pathway. A decline in artemisinin concentration in shoots of NM and M plants treated with ibuprofen (an inhibitor of JA biosynthesis) further confirmed the implication of JA in the mechanism of artemisinin production.

  12. The 5-lipoxygenase inhibitor, zileuton, suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages

    Science.gov (United States)

    Rossi, A; Pergola, C; Koeberle, A; Hoffmann, M; Dehm, F; Bramanti, P; Cuzzocrea, S; Werz, O; Sautebin, L

    2010-01-01

    BACKGROUND AND PURPOSE Zileuton is the only 5-lipoxygenase (5-LOX) inhibitor marketed as a treatment for asthma, and is often utilized as a selective tool to evaluate the role of 5-LOX and leukotrienes. The aim of this study was to investigate the effect of zileuton on prostaglandin (PG) production in vitro and in vivo. EXPERIMENTAL APPROACH Peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon γ (LPS/IFNγ), J774 macrophages and human whole blood stimulated with LPS were used as in vitro models and rat carrageenan-induced pleurisy as an in vivo model. KEY RESULTS Zileuton suppressed PG biosynthesis by interference with arachidonic acid (AA) release in macrophages. We found that zileuton significantly reduced PGE2 and 6-keto prostaglandin F1α (PGF1α) levels in activated mouse peritoneal macrophages and in J774 macrophages. This effect was not related to 5-LOX inhibition, because it was also observed in macrophages from 5-LOX knockout mice. Notably, zileuton inhibited PGE2 production in LPS-stimulated human whole blood and suppressed PGE2 and 6-keto PGF1α pleural levels in rat carrageenan-induced pleurisy. Interestingly, zileuton failed to inhibit the activity of microsomal PGE2 synthase1 and of cyclooxygenase (COX)-2 and did not affect COX-2 expression. However, zileuton significantly decreased AA release in macrophages accompanied by inhibition of phospholipase A2 translocation to cellular membranes. CONCLUSIONS AND IMPLICATION Zileuton inhibited PG production by interfering at the level of AA release. Its mechanism of action, as well as its use as a pharmacological tool, in experimental models of inflammation should be reassessed. PMID:20880396

  13. The NIFS protein can function as a selenide delivery protein in the biosynthesis of selenophosphate.

    Science.gov (United States)

    Lacourciere, G M; Stadtman, T C

    1998-11-20

    The NIFS protein from Azobacter vinelandii is a pyridoxal phosphate-containing homodimer that catalyzes the formation of equimolar amounts of elemental sulfur and L-alanine from the substrate L-cysteine (Zheng, L., White, R. H., Cash, V. L., Jack, R. F., and Dean, D. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2754-2758). A sulfur transfer role of NIFS in which the enzyme donates sulfur for iron sulfur center formation in nitrogenase was suggested. The fact that NIFS also can catalyze the decomposition of L-selenocysteine to elemental selenium and L-alanine suggested the possibility that this enzyme might serve as a selenide delivery protein for the in vitro biosynthesis of selenophosphate. In agreement with this hypothesis, we have shown that replacement of selenide with NIFS and L-selenocysteine in the in vitro selenophosphate synthetase assay results in an increased rate of formation of selenophosphate. These results thus support the view that a selenocysteine-specific enzyme similar to NIFS may be involved as an in vivo selenide delivery protein for selenophosphate biosynthesis. A kinetic characterization of the two NIFS catalyzed reactions carried out in the present study indicates that the enzyme favors L-cysteine as a substrate compared with its selenium analog. A specific activity for L-cysteine of 142 nmol/min/mg compared with 55 nmol/min/mg for L-selenocysteine was determined. This level of enzyme activity on the selenoamino acid substrate is adequate to deliver selenium to selenophosphate synthetase in the in vitro assay system described.

  14. Functional analysis of a cinnamyl alcohol dehydrogenase involved in lignin biosynthesis in wheat.

    Science.gov (United States)

    Ma, Qing-Hu

    2010-06-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step in the biosynthesis of monolignols. In the present study, a cDNA encoding a CAD was isolated from wheat, designated as TaCAD1. A genome-wide data mining in the wheat EST database revealed another 10 CAD-like homologues, namely TaCAD2 to TaCAD11. A phylogenetic analysis showed that TaCAD1 belonged to the bona fide CAD group involved in lignin synthesis. Two other putative CADs from the wheat genome (TaCAD2 and TaCAD4) also belonged to this group and were very close to TaCAD1, but lacked C-terminal domain, suggesting that they are pseudogenes. DNA gel blot analysis for the wheat genome showed two to three copies of CAD related to TaCAD1, but RNA gel blot analysis revealed only single band for TaCAD1, which was highly expressed in stem, with quite low expression in leaf and undetectable expression in root. The predicted three-dimension structure of TaCAD1 resembled that of AtCAD5, but two amino acid substitutions were identified in the substrate binding region. Recombinant TaCAD1 protein used coniferyl aldehyde as the most favoured substrate, also showed high efficiencies toward sinapyl and p-coumaryl aldehydes. TaCAD1 was an enzyme being pH-dependent and temperature-sensitive, and showing a typical random catalysing mechanism. At the milky stage of wheat, TaCAD1 mRNA abundance, protein level and enzyme activity in stem tissues were higher in a lodging-resistant cultivar (H4546) than in lodging-sensitive cultivar (C6001). These properties were correlated to the lignin contents and lodging indices of the two cultivars. These data suggest that TaCAD1 is the predominant CAD in wheat stem for lignin biosynthesis and is critical for lodging resistance.

  15. The p450 monooxygenase BcABA1 is essential for abscisic acid biosynthesis in Botrytis cinerea

    DEFF Research Database (Denmark)

    Siewers, V.; Smedsgaard, Jørn; Tudzynski, P.

    2004-01-01

    The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids...... but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this "direct" pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced...... the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential...

  16. Stereospecific synthesis of threo- and erythro-beta-hydroxyglutamic acid during kutzneride biosynthesis.

    Science.gov (United States)

    Strieker, Matthias; Nolan, Elizabeth M; Walsh, Christopher T; Marahiel, Mohamed A

    2009-09-23

    The antifungal and antimicrobial kutznerides, hexadepsipeptides composed of one alpha-hydroxy acid and five nonproteinogenic amino acids, are remarkable examples of the structural diversity found in nonribosomally produced natural products. They contain D-3-hydroxyglutamic acid, which is found in the threo and erythro isomers in mature kutznerides. In this study, two putative nonheme iron oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in vitro, and found to stereospecifically hydroxylate the beta-position of glutamic acid. KtzO generates threo-L-hydroxyglutamic acid and KtzP catalyzes the formation of the erythro-isomer bound to the peptidyl carrier protein of the third module of the nonribosomal peptide synthetase KtzH. This module has a truncated adenylation domain and is unable to activate and incorporate glutamic acid. The lack of a functional adenylation domain in the third KtzH module is compensated in trans by the stand-alone adenylation domain KtzN, which activates and transfers glutamic acid onto the carrier of KtzH in the presence of the truncated adenylation domain and either KtzO or KtzP. A method that employs nonhydrolyzable coenzyme A analogs was developed and used to determine the kinetic parameters for KtzO- and KtzP-catalyzed hydroxylation of glutamic acid bound to the carrier protein. A detailed mechanism for the in trans compensation of the truncated adenylation domain and the stereospecific hydroxyglutamic acid generation and incorporation is presented. These insights may guide the use of KtzO/KtzP and KtzN or other in trans modification/restoration tools in biocombinatorial engineering approaches.

  17. Gluconic acid-producing Pseudomonas sp. prevent γ-actinorhodin biosynthesis by Streptomyces coelicolor A3(2).

    Science.gov (United States)

    Galet, Justine; Deveau, Aurélie; Hôtel, Laurence; Leblond, Pierre; Frey-Klett, Pascale; Aigle, Bertrand

    2014-09-01

    Streptomyces are ubiquitous soil bacteria well known for their ability to produce a wide range of secondary metabolites including antibiotics. In their natural environments, they co-exist and interact with complex microbial communities and their natural products are assumed to play a major role in mediating these interactions. Reciprocally, their secondary metabolism can be influenced by the surrounding microbial communities. Little is known about these complex interactions and the underlying molecular mechanisms. During pairwise co-culture experiments, a fluorescent Pseudomonas, Pseudomonas fluorescens BBc6R8, was shown to prevent the production of the diffusible blue pigment antibiotic γ-actinorhodin by Streptomyces coelicolor A3(2) M145 without altering the biosynthesis of the intracellular actinorhodin. A mutant of the BBc6R8 strain defective in the production of gluconic acid from glucose and consequently unable to acidify the culture medium did not show any effect on the γ-actinorhodin biosynthesis in contrast to the wild-type strain and the mutant complemented with the wild-type allele. In addition, when glucose was substituted by mannitol in the culture medium, P. fluorescens BBc6R8 was unable to acidify the medium and to prevent the biosynthesis of the antibiotic. All together, the results show that P. fluorescens BBc6R8 impairs the biosynthesis of the lactone form of actinorhodin in S. coelicolor by acidifying the medium through the production of gluconic acid. Other fluorescent Pseudomonas and the opportunistic pathogen Pseudomonas aeruginosa PAO1 also prevented the γ-actinorhodin production in a similar way. We propose some hypotheses on the ecological significance of such interaction.

  18. PNPLA1 has a crucial role in skin barrier function by directing acylceramide biosynthesis

    Science.gov (United States)

    Hirabayashi, Tetsuya; Anjo, Tatsuki; Kaneko, Arisa; Senoo, Yuuya; Shibata, Akitaka; Takama, Hiroyuki; Yokoyama, Kohei; Nishito, Yasumasa; Ono, Tomio; Taya, Choji; Muramatsu, Kazuaki; Fukami, Kiyoko; Muñoz-Garcia, Agustí; Brash, Alan R.; Ikeda, Kazutaka; Arita, Makoto; Akiyama, Masashi; Murakami, Makoto

    2017-01-01

    Mutations in patatin-like phospholipase domain-containing 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, but the mechanism involved remains unclear. Here we show that PNPLA1, an enzyme expressed in differentiated keratinocytes, plays a crucial role in the biosynthesis of ω-O-acylceramide, a lipid component essential for skin barrier. Global or keratinocyte-specific Pnpla1-deficient neonates die due to epidermal permeability barrier defects with severe transepidermal water loss, decreased intercellular lipid lamellae in the stratum corneum, and aberrant keratinocyte differentiation. In Pnpla1−/− epidermis, unique linoleate-containing lipids including acylceramides, acylglucosylceramides and (O-acyl)-ω-hydroxy fatty acids are almost absent with reciprocal increases in their putative precursors, indicating that PNPLA1 catalyses the ω-O-esterification with linoleic acid to form acylceramides. Moreover, acylceramide supplementation partially rescues the altered differentiation of Pnpla1−/− keratinocytes. Our findings provide valuable insight into the skin barrier formation and ichthyosis development, and may contribute to novel therapeutic strategies for treatment of epidermal barrier defects. PMID:28248300

  19. Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing.

    Science.gov (United States)

    Nagamatsu, Atsushi; Masuta, Chikara; Senda, Mineo; Matsuura, Hideyuki; Kasai, Atsushi; Hong, Jin-Sung; Kitamura, Keisuke; Abe, Jun; Kanazawa, Akira

    2007-11-01

    Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase (CHS) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase (F3'H) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.

  20. The biosynthesis of N-arachidonoyl dopamine (NADA), a putative endocannabinoid and endovanilloid, via conjugation of arachidonic acid with dopamine.

    Science.gov (United States)

    Hu, Sherry Shu-Jung; Bradshaw, Heather B; Benton, Valery M; Chen, Jay Shih-Chieh; Huang, Susan M; Minassi, Alberto; Bisogno, Tiziana; Masuda, Kim; Tan, Bo; Roskoski, Robert; Cravatt, Benjamin F; Di Marzo, Vincenzo; Walker, J Michael

    2009-10-01

    N-arachidonoyl dopamine (NADA) is an endogenous ligand that activates the cannabinoid type 1 receptor and the transient receptor potential vanilloid type 1 channel. Two potential biosynthetic pathways for NADA have been proposed, though no conclusive evidence exists for either. The first is the direct conjugation of arachidonic acid with dopamine and the other is via metabolism of a putative N-arachidonoyl tyrosine (NA-tyrosine). In the present study we investigated these biosynthetic mechanisms and report that NADA synthesis requires TH in dopaminergic terminals; however, NA-tyrosine, which we identify here as an endogenous lipid, is not an intermediate. We show that NADA biosynthesis primarily occurs through an enzyme-mediated conjugation of arachidonic acid with dopamine. While this conjugation likely involves a complex of enzymes, our data suggest a direct involvement of fatty acid amide hydrolase in NADA biosynthesis either as a rate-limiting enzyme that liberates arachidonic acid from AEA, or as a conjugation enzyme, or both.

  1. Methyl jasmonate stimulates biosynthesis of 2-phenylethylamine, phenylacetic acid and 2-phenylethanol in seedlings of common buckwheat.

    Science.gov (United States)

    Horbowicz, Marcin; Wiczkowski, Wiesław; Sawicki, Tomasz; Szawara-Nowak, Dorota; Sytykiewicz, Hubert; Mitrus, Joanna

    2015-01-01

    Methyl jasmonate has a strong effect on secondary metabolizm in plants, by stimulating the biosynthesis a number of phenolic compounds and alkaloids. Common buckwheat (Fagopyrum esculentum Moench) is an important source of biologically active compounds. This research focuses on the detection and quantification of 2-phenylethylamine and its possible metabolites in the cotyledons, hypocotyl and roots of common buckwheat seedlings treated with methyl jasmonate. In cotyledons of buckwheat sprouts, only traces of 2-phenylethylamine were found, while in the hypocotyl and roots its concentration was about 150 and 1000-times higher, respectively. Treatment with methyl jasmonate resulted in a 4-fold increase of the 2-phenylethylamine level in the cotyledons of 7-day buckwheat seedlings, and an 11-fold and 5-fold increase in hypocotyl and roots, respectively. Methyl jasmonate treatment led also to about 4-fold increase of phenylacetic acid content in all examined seedling organs, but did not affect the 2-phenylethanol level in cotyledons, and slightly enhanced in hypocotyl and roots. It has been suggested that 2-phenylethylamine is a substrate for the biosynthesis of phenylacetic acid and 2-phenylethanol, as well as cinnamoyl 2-phenethylamide. In organs of buckwheat seedling treated with methyl jasmonate, higher amounts of aromatic amino acid transaminase mRNA were found. The enzyme can be involved in the synthesis of phenylpyruvic acid, but the presence of this compound could not be confirmed in any of the examined organs of common buckwheat seedling.

  2. Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases1[W][OA

    Science.gov (United States)

    Hall, Dawn E.; Zerbe, Philipp; Jancsik, Sharon; Quesada, Alfonso Lara; Dullat, Harpreet; Madilao, Lina L.; Yuen, Macaire; Bohlmann, Jörg

    2013-01-01

    Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs. PMID:23370714

  3. Functional Characterization of 4′OMT and 7OMT Genes in BIA Biosynthesis

    Science.gov (United States)

    Gurkok, Tugba; Ozhuner, Esma; Parmaksiz, Iskender; Özcan, Sebahattin; Turktas, Mine; İpek, Arif; Demirtas, Ibrahim; Okay, Sezer; Unver, Turgay

    2016-01-01

    Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4′–methyltransferase (4′OMT) and reticuline 7-O-methyltransferase (7OMT) genes were subjected to manipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem, and capsule tissues accordingly. Suppression of 4′OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4′OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes (CNMT, TYDC, 6OMT, SAT, COR, 4′OMT, and 7OMT) were observed upon manipulation of 4′OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4′OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues. PMID:26909086

  4. Functional Characterization of 4´OMT and 7OMT Genes in BIA Biosynthesis

    Directory of Open Access Journals (Sweden)

    Tugba eGurkok

    2016-02-01

    Full Text Available Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L., the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4´-O-methyltransferase (4´OMT and reticuline 7-O-methyltransferase (7OMT genes were subjected tomanipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem and capsule tissues accordingly. Suppression of 4´OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4´OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes (CNMT, TYDC, 6OMT, SAT, COR, 4´OMT and 7OMT were observed upon manipulation of 4´OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4´OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues.

  5. Endurance exercise and conjugated linoleic acid (CLA) supplementation up-regulate CYP17A1 and stimulate testosterone biosynthesis.

    Science.gov (United States)

    Barone, Rosario; Macaluso, Filippo; Catanese, Patrizia; Marino Gammazza, Antonella; Rizzuto, Luigi; Marozzi, Paola; Lo Giudice, Giuseppe; Stampone, Tomaso; Cappello, Francesco; Morici, Giuseppe; Zummo, Giovanni; Farina, Felicia; Di Felice, Valentina

    2013-01-01

    A new role for fat supplements, in particular conjugated linoleic acid (CLA), has been delineated in steroidogenesis, although the underlying molecular mechanisms have not yet been elucidated. The aims of the present study were to identify the pathway stimulated by CLA supplementation using a cell culture model and to determine whether this same pathway is also stimulated in vivo by CLA supplementation associated with exercise. In vitro, Leydig tumour rat cells (R2C) supplemented with different concentrations of CLA exhibited increasing testosterone biosynthesis accompanied by increasing levels of CYP17A1 mRNA and protein. In vivo, trained mice showed an increase in free plasma testosterone and an up-regulation of CYP17A1 mRNA and protein. The effect of training on CYP17A1 expression and testosterone biosynthesis was significantly higher in the trained mice supplemented with CLA compared to the placebo. The results of the present study demonstrated that CLA stimulates testosterone biosynthesis via CYP17A1, and endurance training led to the synthesis of testosterone in vivo by inducing the overexpression of CYP17A1 mRNA and protein in the Leydig cells of the testis. This effect was enhanced by CLA supplementation. Therefore, CLA-associated physical activity may be used for its steroidogenic property in different fields, such as alimentary industry, human reproductive medicine, sport science, and anti-muscle wasting.

  6. Integrin-like Protein Is Involved in the Osmotic Stress-induced Abscisic Acid Biosynthesis in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Bing Lü; Feng Chen; Zhong-Hua Gong; Hong Xie; Jian-Sheng Liang

    2007-01-01

    We studied the perception of plant cells to osmotic stress that leads to the accumulation of abscisic acid (ABA) in stressed Arabidopsis thaliana L. cells. A significant difference was found between protoplasts and cells in terms of their responses to osmotic stress and ABA biosynthesis, implying that cell wall and/or cell wall-plasma membrane interaction are essential in identifying osmotic stress. Western blotting and immunofluorescence localization experiments, using polyclonal antibody against human integrin β1, revealed the existence of a protein similar to the integrin protein of animals in the suspension-cultured cells located in the plasma membrane fraction.Treatment with a synthetic pentapeptide, Gly-Arg-Gly-Asp-Ser (GRGDS), which contains an RGD domain and interacts specifically with integrin protein and thus blocks the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, but not in protoplasts. These results demonstrate that cell wall and/or cell wall-plasma membrane interaction mediated by integrin-like proteins played important roles in osmotic stress-induced ABA biosynthesis in Arabidopsis thaliana.

  7. Endurance exercise and conjugated linoleic acid (CLA supplementation up-regulate CYP17A1 and stimulate testosterone biosynthesis.

    Directory of Open Access Journals (Sweden)

    Rosario Barone

    Full Text Available A new role for fat supplements, in particular conjugated linoleic acid (CLA, has been delineated in steroidogenesis, although the underlying molecular mechanisms have not yet been elucidated. The aims of the present study were to identify the pathway stimulated by CLA supplementation using a cell culture model and to determine whether this same pathway is also stimulated in vivo by CLA supplementation associated with exercise. In vitro, Leydig tumour rat cells (R2C supplemented with different concentrations of CLA exhibited increasing testosterone biosynthesis accompanied by increasing levels of CYP17A1 mRNA and protein. In vivo, trained mice showed an increase in free plasma testosterone and an up-regulation of CYP17A1 mRNA and protein. The effect of training on CYP17A1 expression and testosterone biosynthesis was significantly higher in the trained mice supplemented with CLA compared to the placebo. The results of the present study demonstrated that CLA stimulates testosterone biosynthesis via CYP17A1, and endurance training led to the synthesis of testosterone in vivo by inducing the overexpression of CYP17A1 mRNA and protein in the Leydig cells of the testis. This effect was enhanced by CLA supplementation. Therefore, CLA-associated physical activity may be used for its steroidogenic property in different fields, such as alimentary industry, human reproductive medicine, sport science, and anti-muscle wasting.

  8. Identification and functional characterization of the putative polysaccharide biosynthesis protein (CapD) of Enterococcus faecium U0317.

    Science.gov (United States)

    Ali, Liaqat; Spiess, Meike; Wobser, Dominique; Rodriguez, Marta; Blum, Hubert E; Sakιnç, Türkân

    2016-01-01

    Most bacterial species produce capsular polysaccharides that contribute to disease pathogenesis through evasion of the host innate immune system and are also involved in inhibiting leukocyte killing. In the present study, we identified a gene in Enterococcus faecium U0317 with homologies to the polysaccharide biosynthesis protein CapD that is made up of 336 amino acids and putatively catalyzes N-linked glycosylation. A capD deletion mutant was constructed and complemented by homologous recombination that was confirmed by PCR and sequencing. The mutant revealed different growth behavior and morphological changes compared to wild-type by scanning electron microscopy, also the capD mutant showed a strong hydrophobicity and that was reversed in the reconstituted mutant. For further characterization and functional analyses, in-vitro cell culture and in-vivo a mouse infection models were used. Antibodies directed against alpha lipotechoic acid (αLTA) and the peptidyl-prolyl cis-trans isomerase (αPpiC), effectively mediated the opsonophagocytic killing in the capD knock-out mutant, while this activity was not observed in the wild-type and reconstituted mutant. By comparison more than 2-fold decrease was seen in mutant colonization and adherence to both T24 and Caco2 cells. However, a significant higher bacterial colonization was observed in capD mutant during bacteremia in the animal model, while virulence in a mouse UTI (urinary tract infection) model, there were no obvious differences. Further studies are needed to elucidate the function of capsular polysaccharide synthesis gene clusters and its involvement in the disease pathogenesis with the aim to develop targeted therapies to treat multidrug-resistant E. faecium infections.

  9. Nitric oxide mediates the fungal elicitor-induced puerarin biosynthesis in Pueraria thomsonii Benth. suspension cells through a salicylic acid (SA)-dependent and a jasmonic acid (JA)-dependent signal pathway

    Institute of Scientific and Technical Information of China (English)

    XU Maojun; DONG Jufang; ZHU Muyuan

    2006-01-01

    Nitric oxide (NO) has emerged as a key signaling molecule in plant secondary metabolite biosynthesis recently. In order to investigate the molecular basis of NO signaling in elicitor-induced secondary metabolite biosynthesis of plant cells, we determined the contents of NO, salicylic acid (SA), jasmonic acid (JA), and puerarin in Pueraria thomsonii Benth. suspension cells treated with the elicitors prepared from cell walls of Penicillium citrinum. The results showed that the fungal elicitor induced NO burst, SA accumulation and puerarin production of P. thomsonii Benth. cells. The elicitor-induced SA accumulation and puerarin production was suppressed by nitric oxide specific scavenger cPITO, indicating that NO was essential for elicitor-induced SA and puerarin biosynthesis in P. thomsonii Benth. cells. In transgenic NahG P. thomsonii Benth. cells, the fungal elicitor also induced puerarin biosynthesis, NO burst, and JA accumulation, though the SA biosynthesis was impaired. The elicitor-induced JA accumulation in transgenic cells was blocked by cPITO, which suggested that JA acted downstream of NO and its biosynthesis was controlled by NO. External application of NO via its donor sodium nitroprusside (SNP) enhanced puerarin biosynthesis in transgenic NahG P. thomsonii Benth. cells, and the NO-triggered puerarin biosynthesis was suppressed by JA inhibitors IBU and NDGA, which indicated that NO induced puerarin production through a JA-dependent signal pathway in the transgenic cells. Exogenous application of SA suppressed the elicitor-induced JA biosynthesis and reversed the inhibition of IBU and NDGA on elicitor-induced puerarin accumulation in transgenic cells, which indicated that SA inhibited JA biosynthesis in the cells and that SA might be used as a substitute for JA to mediate the elicitor- and NO-induced puerarin biosynthesis. It was, therefore, concluded that NO might mediate the elicitor-induced puerarin biosynthesis through SA- and JA-dependent signal

  10. Fatty acid biosynthesis. VIII. The fate of malonyl-CoA in fatty acid biosynthesis by purified enzymes from lactating-rabbit mammary gland

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Carey, E.M.; Dils, R.

    1971-01-01

    - 1. We have investigated the formation and utilization of malonyl-CoA in fatty acid synthesis catalysed by preparations of partially purified acetyl-CoA carboxylase and purified fatty acid synthetase from lactating-rabbit mammary gland. - 2. Carboxylation of [1-14C]acetyl-CoA was linked to fatty...

  11. Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways.

    Science.gov (United States)

    Willis, Lisa M; Whitfield, Chris

    2013-08-30

    Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis.

  12. Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Bulfer, Stacie L.; Scott, Erin M.; Couture, Jean-François; Pillus, Lorraine; Trievel, Raymond C.; (Michigan); (UCSD)

    2010-01-12

    Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed {alpha}/{beta} topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.

  13. Biosynthesis and biodegradation of wood components

    Energy Technology Data Exchange (ETDEWEB)

    Higuchi, T. (ed.)

    1985-01-01

    A textbook containing 22 chapters by various authors covers the structure of wood, the localization of polysaccharides and lignins in wood cell walls, metabolism and synthetic function of cambial tissue, cell organelles and their function in the biosynthesis of cell wall components, biosynthesis of plant cell wall polysaccharides, lignin, cutin, suberin and associated waxes, phenolic acids and monolignols, quinones, flavonoids, tannins, stilbenes and terpenoid wood extractives, the occurrence of extractives, the metabolism of phenolic acids, wood degradation by micro-organisms and fungi, and biodegradation of cellulose, hemicelluloses, lignin, and aromatic extractives of wood. An index is included.

  14. Biosynthesis of pseudoisoeugenols in tissue cultures of Pimpinella anisum. Phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase activities.

    Science.gov (United States)

    Reichling, J; Kemmerer, B; Sauer-Gürth, H

    1995-07-28

    The genus Pimpinella contains pseudoisoeugenols, phenylpropanoids with a rare 2,5-dioxy substitution pattern on the phenyl ring. To study the biosynthesis of these compounds, we set up a leaf-differentiating tissue culture of Pimpinella anisum. These cultures mainly produce epoxy-pseudoisoeugenol-(2-methylbutyrate). To corroborate the biosynthetic pathway of epoxy-pseudoisoeugenol-(2-methylbutyrate) as proposed on the basis of investigations with 13C/14C-labelled precursors, the key steps of the pathway were investigated at an enzyme level. Experiments with cell-free homogenates clearly revealed that L-phenylalanine is converted to (E)-cinnamic acid by phenylalanine ammonia lyase and that (E)-cinnamic acid is converted to p-coumaric acid by cinnamic acid 4-hydroxylase. L-2-aminooxy-3-phenylpropionic acid, an analogue of L-phenylalanine, inhibited the incorporation of L-[3'-13C]phenylalanine into epoxy-pseudoisoeugenol-(2-methylbutyrate). Up to 2% of the precursor DL-[3'-13C]phenyllactate was incorporated into epoxy-pseudoisoeugenol-(2-methylbutyrate). Inhibition experiments with oxalacetic acid clearly showed that cinnamic acid is not formed by dehydration of phenyllactic acid in this leaf-differentiating tissue culture of P. anisum.

  15. Activation of glycerol metabolic pathway by evolutionary engineering of Rhizopus oryzae to strengthen the fumaric acid biosynthesis from crude glycerol.

    Science.gov (United States)

    Huang, Di; Wang, Ru; Du, Wenjie; Wang, Guanyi; Xia, Menglei

    2015-11-01

    Rhizopus oryzae is strictly inhibited by biodiesel-based by-product crude glycerol, which results in low fumaric acid production. In this study, evolutionary engineering was employed to activate the glycerol utilization pathway for fumaric acid production. An evolved strain G80 was selected, which could tolerate and utilize high concentrations of crude glycerol to produce 14.9g/L fumaric acid with a yield of 0.248g/g glycerol. Key enzymes activity analysis revealed that the evolved strain displayed a significant upregulation in glycerol dissimilation, pyruvate consumption and reductive tricarboxylic acid pathways, compared with the parent strain. Subsequently, intracellular metabolic profiling analysis showed that amino acid biosynthesis, tricarboxylic acid cycle, fatty acid and stress response metabolites accounted for metabolic difference between two strains. Moreover, a glycerol fed-batch strategy was optimized to obtain the highest fumaric acid production of 25.5g/L, significantly increased by 20.9-fold than that of the parent strain of 1.2g/L.

  16. Amino Acids Attenuate Insulin Action on Gluconeogenesis and Promote Fatty Acid Biosynthesis via mTORC1 Signaling Pathway in trout Hepatocytes

    Directory of Open Access Journals (Sweden)

    Weiwei Dai

    2015-06-01

    Full Text Available Background/Aims: Carnivores exhibit poor utilization of dietary carbohydrates and glucose intolerant phenotypes, yet it remains unclear what are the causal factors and underlying mechanisms. We aimed to evaluate excessive amino acids (AAs-induced effects on insulin signaling, fatty acid biosynthesis and glucose metabolism in rainbow trout and determine the potential involvement of mTORC1 and p38 MAPK pathway. Methods: We stimulated trout primary hepatocytes with different AA levels and employed acute administration of rapamycin to inhibit mTORC1 activation. Results: Increased AA levels enhanced the phosphorylation of ribosomal protein S6 kinase (S6K1, S6, and insulin receptor substrate 1 (IRS-1 on Ser302 but suppressed Akt and p38 phosphorylation; up-regulated the expression of genes related to gluconeogenesis and fatty acid biosynthesis. mTORC1 inhibition not only inhibited the phosphorylation of mTORC1 downstream targets, but also blunted IRS-1 Ser302 phosphorylation and restored excessive AAs-suppressed Akt phosphorylation. Rapamycin also inhibited fatty acid biosynthetic and gluconeogenic gene expression. Conclusion: High levels of AAs up-regulate hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner, while attenuate insulin-mediated repression of gluconeogenesis through elevating IRS-1 Ser302 phosphorylation, which in turn impairs Akt activation and thereby weakening insulin action. We propose that p38 MAPK probably also involves in these AAs-induced metabolic changes.

  17. Effect of salinity on the biosynthesis of n-3 long-chain polyunsaturated fatty acids in silverside Chirostoma estor.

    Science.gov (United States)

    Fonseca-Madrigal, J; Pineda-Delgado, D; Martínez-Palacios, C; Rodríguez, C; Tocher, D R

    2012-08-01

    The genus Chirostoma (silversides) belongs to the family Atherinopsidae, which contains around 150 species, most of which are marine. However, Mexican silverside (Chirostoma estor) is one of the few representatives of freshwater atherinopsids and is only found in some lakes of the Mexican Central Plateau. However, studies have shown that C. estor has improved survival, growth, and development when cultured in water conditions with increased salinity. In addition, C. estor displays an unusual fatty acid composition for a freshwater fish with high docosahexaenoic acid (DHA)/ eicosapentaenoic acid (EPA) ratios. Freshwater and marine fish species display very different essential fatty acid metabolism and requirements, and so the present study investigated long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis to determine the capacity of C. estor for endogenous production of EPA and DHA, and the effect that salinity has on these pathways. Briefly, C. estor were maintained at three salinities (0, 5, and 15 ppt), and the metabolism of ¹⁴C-labeled 18:3n-3 was determined in isolated hepatocyte and enterocyte cells. The results showed that C. estor has the capacity for endogenous biosynthesis of LC-PUFA from 18-carbon fatty acid precursors, but that the pathway was essentially only active in saline conditions with virtually no activity in cells isolated from fish grown in freshwater. The activity of the LC-PUFA biosynthesis pathway was also higher in cells isolated from fish at 15 ppt compared with fish at 5 ppt. The activity was around fivefold higher in hepatocytes compared with enterocytes; although the majority of 18:3n-3 was converted to 18:4n-3 and 20:4n-3 in hepatocytes, the proportions of 18:3n-3 converted to EPA and DHA were higher in enterocytes. The data were consistent with the hypothesis that conversion of EPA to DHA could contribute, at least in part, to the generally high DHA/EPA ratios observed in the tissue lipids of C. estor.

  18. HpDTC1, a Stress-Inducible Bifunctional Diterpene Cyclase Involved in Momilactone Biosynthesis, Functions in Chemical Defence in the Moss Hypnum plumaeforme

    Science.gov (United States)

    Okada, Kazunori; Kawaide, Hiroshi; Miyamoto, Koji; Miyazaki, Sho; Kainuma, Ryosuke; Kimura, Honoka; Fujiwara, Kaoru; Natsume, Masahiro; Nojiri, Hideaki; Nakajima, Masatoshi; Yamane, Hisakazu; Hatano, Yuki; Nozaki, Hiroshi; Hayashi, Ken-ichiro

    2016-01-01

    Momilactones, which are diterpenoid phytoalexins with antimicrobial and allelopathic functions, have been found only in rice and the moss Hypnum plumaeforme. Although these two evolutionarily distinct plant species are thought to produce momilactones as a chemical defence, the momilactone biosynthetic pathway in H. plumaeforme has been unclear. Here, we identified a gene encoding syn-pimara-7,15-diene synthase (HpDTC1) responsible for the first step of momilactone biosynthesis in the moss. HpDTC1 is a bifunctional diterpene cyclase that catalyses a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene. HpDTC1 transcription was up-regulated in response to abiotic and biotic stress treatments. HpDTC1 promoter-GUS analysis in transgenic Physcomitrella patens showed similar transcriptional responses as H. plumaeforme to the stresses, suggesting that a common response system to stress exists in mosses. Jasmonic acid (JA), a potent signalling molecule for inducing plant defences, could not activate HpDTC1 expression. In contrast, 12-oxo-phytodienoic acid, an oxylipin precursor of JA in vascular plants, enhanced HpDTC1 expression and momilactone accumulation, implying that as-yet-unknown oxylipins could regulate momilactone biosynthesis in H. plumaeforme. These results demonstrate the existence of an evolutionarily conserved chemical defence system utilizing momilactones and suggest the molecular basis of the regulation for inductive production of momilactones in H. plumaeforme. PMID:27137939

  19. HpDTC1, a Stress-Inducible Bifunctional Diterpene Cyclase Involved in Momilactone Biosynthesis, Functions in Chemical Defence in the Moss Hypnum plumaeforme.

    Science.gov (United States)

    Okada, Kazunori; Kawaide, Hiroshi; Miyamoto, Koji; Miyazaki, Sho; Kainuma, Ryosuke; Kimura, Honoka; Fujiwara, Kaoru; Natsume, Masahiro; Nojiri, Hideaki; Nakajima, Masatoshi; Yamane, Hisakazu; Hatano, Yuki; Nozaki, Hiroshi; Hayashi, Ken-Ichiro

    2016-05-03

    Momilactones, which are diterpenoid phytoalexins with antimicrobial and allelopathic functions, have been found only in rice and the moss Hypnum plumaeforme. Although these two evolutionarily distinct plant species are thought to produce momilactones as a chemical defence, the momilactone biosynthetic pathway in H. plumaeforme has been unclear. Here, we identified a gene encoding syn-pimara-7,15-diene synthase (HpDTC1) responsible for the first step of momilactone biosynthesis in the moss. HpDTC1 is a bifunctional diterpene cyclase that catalyses a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene. HpDTC1 transcription was up-regulated in response to abiotic and biotic stress treatments. HpDTC1 promoter-GUS analysis in transgenic Physcomitrella patens showed similar transcriptional responses as H. plumaeforme to the stresses, suggesting that a common response system to stress exists in mosses. Jasmonic acid (JA), a potent signalling molecule for inducing plant defences, could not activate HpDTC1 expression. In contrast, 12-oxo-phytodienoic acid, an oxylipin precursor of JA in vascular plants, enhanced HpDTC1 expression and momilactone accumulation, implying that as-yet-unknown oxylipins could regulate momilactone biosynthesis in H. plumaeforme. These results demonstrate the existence of an evolutionarily conserved chemical defence system utilizing momilactones and suggest the molecular basis of the regulation for inductive production of momilactones in H. plumaeforme.

  20. Type III polyketide synthase is involved in the biosynthesis of protocatechuic acid in Aspergillus niger.

    Science.gov (United States)

    Lv, Yangyong; Xiao, Jing; Pan, Li

    2014-11-01

    Genomic studies have shown that not only plants but also filamentous fungi contain type III polyketide synthases. To study the function of type III polyketide synthase (AnPKSIII) in Aspergillus niger, a deletion strain (delAnPKSIII) and an overexpression strain (oeAnPKSIII) were constructed in A. niger MA169.4, a derivative of the wild-type (WT) A. niger ATCC 9029 that produces large quantities of gluconic acid. Alterations in the metabolites were analyzed by HPLC when the extract of the overexpression strain was compared with extracts of the WT and deletion strains. Protocatechuic acid (PCA; 3,4-dihydroxybenzoic acid, 3.2 mg/l) was isolated and identified as the main product of AnPKSIII when inductively expressed in A. niger MA169.4. The molecular weight of PCA was 154.1 (m/z 153.1 [M-H](-)), was detected by ESI-MS in the negative ionization mode, and (1)H and (13)C NMR data confirmed its structure.

  1. Functional analysis of gamma-carotene ketolase involved in the carotenoid biosynthesis of Deinococcus radiodurans.

    Science.gov (United States)

    Sun, Zongtao; Shen, Shaochuan; Tian, Bing; Wang, Hu; Xu, Zhenjian; Wang, Liangyan; Hua, Yuejin

    2009-11-01

    Deinococcus radiodurans strain R1 synthesizes a unique ketocarotenoid product named deinoxanthin. The detailed steps involved in the biosynthesis of deinoxanthin remain unresolved. A carotene ketolase homologue encoded by dr0093 was inactivated by gene mutation to verify its function in the native host D. radiodurans. Analysis of the carotenoids in the resultant mutant R1DeltacrtO demonstrated that dr0093 encodes gamma-carotene ketolase (CrtO) catalysing the introduction of one keto group into the C-4 position of gamma-carotene derivatives to form ketolated carotenoids. The mutant R1DeltacrtO became more sensitive to H(2)O(2) treatment than the wild-type strain R1, indicating that the C-4 keto group is important for the antioxidant activity of carotenoids in D. radiodurans. Carotenoid extracts from mutant R1DeltacrtO exhibited lower 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the wild-type strain R1. The enhanced antioxidant ability of ketocarotenoids in D. radiodurans might be attributed to its extended conjugated double bonds and relative stability by the C-4 keto group substitution.

  2. Functional characterization of CYP107W1 from Streptomyces avermitilis and biosynthesis of macrolide oligomycin A.

    Science.gov (United States)

    Han, Songhee; Pham, Tan-Viet; Kim, Joo-Hwan; Lim, Young-Ran; Park, Hyoung-Goo; Cha, Gun-Su; Yun, Chul-Ho; Chun, Young-Jin; Kang, Lin-Woo; Kim, Donghak

    2015-06-01

    Streptomyces avermitilis contains 33 cytochrome P450 genes in its genome, many of which play important roles in the biosynthesis process of antimicrobial agents. Here, we characterized the biochemical function and structure of CYP107W1 from S. avermitilis, which is responsible for the 12-hydroxylation reaction of oligomycin C. CYP107W1 was expressed and purified from Escherichia coli. Purified proteins exhibited the typical CO-binding spectrum of P450. Interaction of oligomycin C and oligomycin A (12-hydroxylated oligomycin C) with purified CYP107W1 resulted in a type I binding with Kd values of 14.4 ± 0.7 μM and 2.0 ± 0.1 μM, respectively. LC-mass spectrometry analysis showed that CYP107W1 produced oligomycin A by regioselectively hydroxylating C12 of oligomycin C. Steady-state kinetic analysis yielded a kcat value of 0.2 min(-1) and a Km value of 18 μM. The crystal structure of CYP107W1 was determined at 2.1 Å resolution. The overall P450 folding conformations are well conserved, and the open access binding pocket for the large macrolide oligomycin C was observed above the distal side of heme. This study of CYP107W1 can help a better understanding of clinically important P450 enzymes as well as their optimization and engineering for synthesizing novel antibacterial agents and other pharmaceutically important compounds.

  3. Biosynthesis and Functional Significance of Peripheral Node Addressin in Cancer-Associated TLO

    Directory of Open Access Journals (Sweden)

    Aliyah M. Weinstein

    2016-08-01

    Full Text Available Peripheral node addressin (PNAd marks high endothelial venules (HEV, which are crucial for the recruitment of lymphocytes into lymphoid organs in non-mucosal tissue sites. PNAd is a sulfated and fucosylated glycoprotein recognized by the prototypic monoclonal antibody MECA-79. PNAd is the ligand for L-selectin, which is expressed on the surface of naïve and central memory T cells, where it mediates leukocyte rolling on vascular endothelial surfaces. Although PNAd was first identified in the HEV of peripheral lymph nodes, recent work suggests a critical role for PNAd in the context of chronic inflammatory diseases, where it can be used as a marker for the formation of tertiary lymphoid organs (TLO. TLO form in tissues affected by sustained inflammation, such as the tumor microenvironment (TME, where they function as local sites of adaptive immune cell priming. This allows for specific B- and T-cell responses to be initiated or reactivated in inflamed tissues without dependency on secondary lymphoid organs (SLO. Recent studies of cancer in mice and humans have identified PNAd as a biomarker of improved disease prognosis, and blockade of PNAd or its ligand, L-selectin, abrogates protective anti-tumor immunity in murine models. This review examines pathways regulating PNAd biosynthesis by the endothelial cells integral to HEV and the formation and maintenance of lymphoid structures throughout the body, particularly in the setting of cancer.

  4. Transcriptome Profiling of Tomato Fruit Development Reveals Transcription Factors Associated with Ascorbic Acid, Carotenoid and Flavonoid Biosynthesis.

    Science.gov (United States)

    Ye, Jie; Hu, Tixu; Yang, Congmei; Li, Hanxia; Yang, Mingze; Ijaz, Raina; Ye, Zhibiao; Zhang, Yuyang

    2015-01-01

    Tomato (Solanum lycopersicum) serves as a research model for fruit development; however, while it is an important dietary source of antioxidant nutrients, the transcriptional regulation of genes that determine nutrient levels remains poorly understood. Here, the transcriptomes of fruit at seven developmental stages (7, 14, 21, 28, 35, 42 and 49 days after flowering) from two tomato cultivars (Ailsa Craig and HG6-61) were evaluated using the Illumina sequencing platform. A total of 26,397 genes, which were expressed in at least one developmental stage, were detected in the two cultivars, and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis. Gene Ontology term enrichment analysis indicated that genes involved in RNA regulation, secondary metabolism, hormone metabolism and cell wall metabolism were the most highly differentially expressed genes during fruit development and ripening. A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid, carotenoid and flavonoid biosynthesis. This transcriptional correlation was confirmed by agroinfiltration mediated transient expression, which showed that most of the enzymatic genes in the ascorbic acid biosynthesis were regulated by the overexpression of each of the three transcription factors that were tested. The metabolic dynamics of ascorbic acid, carotenoid and flavonoid were investigated during fruit development and ripening, and some selected transcription factors showed transcriptional correlation with the accumulation of ascorbic acid, carotenoid and flavonoid. This transcriptome study provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism.

  5. Transcriptome Profiling of Tomato Fruit Development Reveals Transcription Factors Associated with Ascorbic Acid, Carotenoid and Flavonoid Biosynthesis.

    Directory of Open Access Journals (Sweden)

    Jie Ye

    Full Text Available Tomato (Solanum lycopersicum serves as a research model for fruit development; however, while it is an important dietary source of antioxidant nutrients, the transcriptional regulation of genes that determine nutrient levels remains poorly understood. Here, the transcriptomes of fruit at seven developmental stages (7, 14, 21, 28, 35, 42 and 49 days after flowering from two tomato cultivars (Ailsa Craig and HG6-61 were evaluated using the Illumina sequencing platform. A total of 26,397 genes, which were expressed in at least one developmental stage, were detected in the two cultivars, and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis. Gene Ontology term enrichment analysis indicated that genes involved in RNA regulation, secondary metabolism, hormone metabolism and cell wall metabolism were the most highly differentially expressed genes during fruit development and ripening. A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid, carotenoid and flavonoid biosynthesis. This transcriptional correlation was confirmed by agroinfiltration mediated transient expression, which showed that most of the enzymatic genes in the ascorbic acid biosynthesis were regulated by the overexpression of each of the three transcription factors that were tested. The metabolic dynamics of ascorbic acid, carotenoid and flavonoid were investigated during fruit development and ripening, and some selected transcription factors showed transcriptional correlation with the accumulation of ascorbic acid, carotenoid and flavonoid. This transcriptome study provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism.

  6. Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance

    Science.gov (United States)

    Jenkins, Benjamin J.; Seyssel, Kevin; Chiu, Sally; Pan, Pin-Ho; Lin, Shih-Yi; Stanley, Elizabeth; Ament, Zsuzsanna; West, James A.; Summerhill, Keith; Griffin, Julian L.; Vetter, Walter; Autio, Kaija J.; Hiltunen, Kalervo; Hazebrouck, Stéphane; Stepankova, Renata; Chen, Chun-Jung; Alligier, Maud; Laville, Martine; Moore, Mary; Kraft, Guillaume; Cherrington, Alan; King, Sarah; Krauss, Ronald M.; de Schryver, Evelyn; Van Veldhoven, Paul P.; Ronis, Martin; Koulman, Albert

    2017-01-01

    Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1−/− mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1−/− only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease. PMID:28332596

  7. High levels of jasmonic acid antagonize the biosynthesis of gibberellins and inhibit the growth of Nicotiana attenuata stems.

    Science.gov (United States)

    Heinrich, Maria; Hettenhausen, Christian; Lange, Theo; Wünsche, Hendrik; Fang, Jingjing; Baldwin, Ian T; Wu, Jianqiang

    2013-02-01

    Hormones play pivotal roles in regulating plant development, growth, and stress responses, and cross-talk among different hormones fine-tunes various aspects of plant physiology. Jasmonic acid (JA) is important for plant defense against herbivores and necrotic fungi and also regulates flower development; in addition, Arabidopsis mutants over-producing JA usually have stunted stems and wound-induced jasmonates suppress Arabidopsis growth, suggesting that JA is also involved in stem elongation. Gibberellins (GAs) promote stem and leaf growth and modulate seed germination, flowering time, and the development of flowers, fruits, and seeds. However, little is known about the interaction between the JA and GA pathways. Two calcium-dependent protein kinases, CDPK4 and CDPK5, are important suppressors of JA accumulation in a wild tobacco species, Nicotiana attenuata. The stems of N. attenuata silenced in CDPK4 and CDPK5 (irCDPK4/5 plants) had dramatically increased levels of JA and exhibited stunted elongation and had very high contents of secondary metabolites. Genetic analysis indicated that the high JA levels in irCDPK4/5 stems accounted for the suppressed stem elongation and the accumulation of secondary metabolites. Supplementation of GA(3) to irCDPK4/5 plants largely restored normal stem growth to wild-type levels. Measures of GA levels indicated that over-accumulation of JA in irCDPK4/5 stems inhibited the biosynthesis of GAs. Finally, we show that JA antagonizes GA biosynthesis by strongly inhibiting the transcript accumulation of GA20ox and possibly GA13ox, the key genes in GA production, demonstrating that high JA levels antagonize GA biosynthesis in stems.

  8. Functional Expression of Enterobacterial O-Polysaccharide Biosynthesis Enzymes in Bacillus subtilis

    Science.gov (United States)

    Schäffer, Christina; Wugeditsch, Thomas; Messner, Paul; Whitfield, Chris

    2002-01-01

    The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes wbaP (formerly rfbP), wecA (formerly rfe), and wbbO (formerly rfbF) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in Bacillus subtilis. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and N-acetylglucosaminyl 1-phosphate from UDP-N-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional wbbO gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In B. subtilis hosts, the genes were effectively transcribed under the vegII promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the tet resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in Escherichia coli and B. subtilis, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-α-d-14C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in B. subtilis varied between 20 and 75% of those measured in E. coli. PMID:12324313

  9. Unique biological function of cathepsin L in secretory vesicles for biosynthesis of neuropeptides.

    Science.gov (United States)

    Funkelstein, Lydiane; Beinfeld, Margery; Minokadeh, Ardalan; Zadina, James; Hook, Vivian

    2010-12-01

    Neuropeptides are essential for cell-cell communication in the nervous and neuroendocrine systems. Production of active neuropeptides requires proteolytic processing of proneuropeptide precursors in secretory vesicles that produce, store, and release neuropeptides that regulate physiological functions. This review describes recent findings indicating the prominent role of cathepsin L in secretory vesicles for production of neuropeptides from their protein precursors. The role of cathepsin L in neuropeptide production was discovered using the strategy of activity-based probes for proenkephalin-cleaving activity for identification of the enzyme protein by mass spectrometry. The novel role of cathepsin L in secretory vesicles for neuropeptide production has been demonstrated in vivo by cathepsin L gene knockout studies, cathepsin L gene expression in neuroendocrine cells, and notably, cathepsin L localization in neuropeptide-containing secretory vesicles. Cathepsin L is involved in producing opioid neuropeptides consisting of enkephalin, β-endorphin, and dynorphin, as well as in generating the POMC-derived peptide hormones ACTH and α-MSH. In addition, NPY, CCK, and catestatin neuropeptides utilize cathepsin L for their biosynthesis. The neuropeptide-synthesizing functions of cathepsin L represent its unique activity in secretory vesicles, which contrasts with its role in lysosomes. Interesting evaluations of protease gene knockout studies in mice that lack cathepsin L compared to those lacking PC1/3 and PC2 (PC, prohormone convertase) indicate the key role of cathepsin L in neuropeptide production. Therefore, dual cathepsin L and prohormone convertase protease pathways participate in neuropeptide production. Significantly, the recent new findings indicate cathepsin L as a novel 'proprotein convertase' for production of neuropeptides that mediate cell-cell communication in health and disease.

  10. Effect of some saturated and unsaturated fatty acids on prostaglandin biosynthesis in washed human blood platelets from (1-/sup 14/ C)arachidonic acid

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, K.C.; Awasthi, K.K.; Lindegard, P.; Tiwari, K.P.

    1982-03-01

    The effects of some saturated (lauric, palmitic and stearic) an unsaturated (linoleic, gamma-linolenic, alpha-linolenic and oleic) fatty acids at 0.1. 0.25 and 0.5 mM concentrations on the in vitro metabolization of (1-14 C) arachidonic acid by washed human blood platelets have been studied. Effects of these fatty acids were studied with intact as well as lysed platelet preparations. With intact platelet preparations it was found that (i) all unsaturated fatty acids enhanced the biosynthesis of TxB2, PGE2, PGD2 and PGF2 alpha, (ii) unsaturated fatty acids reduced the formation of HHT and HETE with the exception of oleic acid which showed very little effect, (iii) unsaturated fatty acids reduced the formation of MDA, whereas palmitic and stearic acids increased its formation and (iv) all unsaturated fatty acids reduced the synthesis of prostaglandin endoperoxides. These results support our previous observations where effects of fatty acids were examined at higher concentrations (10). At 0.1 mM FA concentration, inconsistent results were obtained. With lysed platelet preparations all cyclooxygenase products were reduced in presence of unsaturated fatty acids, whereas HETE formation was reduced only in presence of linoleic and gamma-linolenic acids. Electron micrographs of washed platelet suspensions were obtained with untreated platelet preparations and platelet preparations treated with 0.25 and 0.5 mM linoleic acid concentrations. The results are discussed in the light of a possible soap-like effect of FA salt on platelets.

  11. Current Opinions on the Functions of Tocopherol Based on the Genetic Manipulation of Tocopherol Biosynthesis in Plants

    Institute of Scientific and Technical Information of China (English)

    Yin Li; Zinan Wang; Xiaofen Sun; Kexuan Tang

    2008-01-01

    As a member of an important group of lipid soluble antioxidants,tocopherols play a paramount role In the daily diet of humans and animals.Recently,genes required for tocochromanol biosynthesis pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms: Arabidopsis thaliana and Synechocystis sp.PCC 6803.At the basis of these foundations,genetic manipulation of tocochromanol biosynthesis pathway can give rise to strategies that enhance the level of tocochromanol content or convert the constitution of tocochromanol.In addition,genetic manipulations of the tocochromanol biosynthesis pathway provide help for the study of the function of tocopherol in plant systems.The present article summarizes recent advances and pays special attention to the functions of tocopherol in plants.The roles of tocopherol in the network of reactive oxygen species,antioxidants and phytohormones to maintain redox homeostasis and the functions of tocopherol as a signal molecule in chloroplast-to-nucleus signaling to regulate carbohydrate metabolism are also discussed.

  12. New tuberculostatic agents targeting nucleic acid biosynthesis: drug design using QSAR approaches.

    Science.gov (United States)

    Bueno, Renata V; Braga, Rodolpho C; Segretti, Natanael D; Ferreira, Elizabeth I; Trossini, Gustavo H G; Andrade, Carolina H

    2014-01-01

    Worldwide, tuberculosis (TB) is the leading cause of death among curable infectious diseases. The emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) TB is a growing global health concern and there is an urgent need for new anti-TB drugs. Enzymes involved in DNA and ATP biosynthesis are potential targets for tuberculostatic drug design, since these enzymes are essential for Mycobacterium tuberculosis growth. This review presents the current progress and applications of structure-activity relationship analysis for the discovery of innovative tuberculostatic agents as inhibitors of ribonucleotide reductase, DNA gyrase, ATP synthase, and thymidylate kinase enzymes, highlighting present challenges and new opportunities in TB drug design.

  13. Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

    Science.gov (United States)

    Kalscheuer, Rainer; Stöveken, Tim; Luftmann, Heinrich; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

    2006-02-01

    Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

  14. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    Science.gov (United States)

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  15. Deletion of the carboxyl-terminal region of 1-aminocyclopropane-1-carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme.

    Science.gov (United States)

    Li, N; Mattoo, A K

    1994-03-04

    1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is a key enzyme regulating biosynthesis of the plant hormone ethylene. The expression of an enzymatically active, wound-inducible tomato (Lycopersicon esculentum L. cv Pik-Red) ACC synthase (485 amino acids long) in a heterologous Escherichia coli system allowed us to study the importance of hypervariable COOH terminus in enzymatic activity and protein conformation. We constructed several deletion mutants of the gene, expressed these in E. coli, purified the protein products to apparent homogeneity, and analyzed both conformation and enzyme kinetic parameters of the wild-type and truncated ACC syntheses. Deletion of the COOH terminus through Arg429 results in complete inactivation of the enzyme. Deletion of 46-52 amino acids from the COOH terminus results in an enzyme that has nine times higher affinity for the substrate S-adenosylmethionine than the wild-type enzyme. The highly efficient, truncated ACC synthase was found to be a monomer of 52 +/- 1.8 kDa as determined by gel filtration, whereas the wild-type ACC synthase, analyzed under similar conditions, is a dimer. These results demonstrate that the non-conserved COOH terminus of ACC synthase affects its enzymatic function as well as dimerization.

  16. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba

    Directory of Open Access Journals (Sweden)

    Qiangwen Chen

    2017-01-01

    Full Text Available Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs, are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT and mevalonate kinase (MVK as core enzymes. In this study, two full-length (cDNAs encoding AACT (GbAACT, GenBank Accession No. KX904942 and MVK (GbMVK, GenBank Accession No. KX904944 were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  17. Identification and functional analysis of gene cluster involvement in biosynthesis of the cyclic lipopeptide antibiotic pelgipeptin produced by Paenibacillus elgii

    Directory of Open Access Journals (Sweden)

    Qian Chao-Dong

    2012-09-01

    Full Text Available Abstract Background Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a β-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway. Results A potential pelgipeptin synthetase gene cluster (plp was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPSs, with one, seven, and one module(s, respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1 provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis. Conclusions In this study, a gene cluster (plp responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.

  18. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Messing, Simon A.J.; Gabelli, Sandra B.; Echeverria, Ignacia; Vogel, Jonathan T.; Guan, Jiahn Chou; Tan, Bao Cai; Klee, Harry J.; McCarty, Donald R.; Amzel, L. Mario (JHU); (Florida)

    2011-09-06

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  19. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid W

    Energy Technology Data Exchange (ETDEWEB)

    Messing, S.; Gabelli, S; Echeverria, I; Vogel, J; Guan, J; Tan, B; Klee, H; McCarty, D; Amzela, M

    2010-01-01

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  20. Differential control of cholesterol and fatty acid biosynthesis in sensitive and multidrug-resistant LoVo tumor cells.

    Science.gov (United States)

    Santini, Maria Teresa; Napolitano, Mariarosaria; Ferrante, Antonella; Rainaldi, Gabriella; Arancia, Giuseppe; Bravo, Elena

    2003-01-01

    Multidrug resistance (MDR) describes the decrease in sensitivity of tumor cells to a wide variety of cytotoxic compounds. Although a central role has been ascribed to the P-glycoprotein (Pgp) pump in MDR, lipids also appear to be extremely important. However, their precise role in MDR is not yet fully understood. It was the aim of the present paper to gain a deeper understanding of intracellular lipid equilibrium in both sensitive and MDR tumor cells. In particular, intracellular cholesterol biosynthesis and cholesterol esterification were examined in LoVo-sensitive and Pgp-overexpressing resistant cells. The data presented seem to suggest that the higher synthesis of cholesteryl ester and triglyceride observed in resistant with respect to wild-type cells is due to a greater production of fatty acids in these cells. The results are discussed in view of the possible roles of sterol regulatory element-binding proteins and Pgp in these phenomena.

  1. Retinoic acid treatment enhances lipid oxidation and inhibits lipid biosynthesis capacities in the liver of mice.

    Science.gov (United States)

    Amengual, Jaume; Ribot, Joan; Bonet, M Luisa; Palou, Andreu

    2010-01-01

    Vitamin A, mainly as retinoic acid (RA), is known to affect the development and function of adipose tissues. Treatment with RA reduces body weight and adiposity independent of changes in food intake in mice. Lipid metabolism in liver can have a major impact on whole body adiposity. The aim of this work was to investigate the effects of an in vivo treatment with RA on hepatic lipid metabolism in mice. Adult, standard diet-fed mice were treated with different doses of all-trans RA or vehicle (subcutaneous injection) for 4 days before sacrifice. Food intake and body weight changes during treatment were determined, as well as adiposity, liver composition, levels of circulating metabolites and lipoproteins and expression levels of key mRNA species in liver following sacrifice. RA treatment resulted in reduced body weight and adiposity, as expected. In the liver, RA treatment triggered an increase in the mRNA expression levels of peroxisome proliferator-activated receptor alpha, retinoid X receptor alpha, uncoupling protein 2, liver-type carnitine palmitoyltransferase 1, and carnitine/acylcarnitine carrier, and a reduction in the mRNA expression levels of sterol regulatory element binding protein 1c and fatty acid synthase. Consistent with the changes in gene expression, hepatic triacylglycerol content and circulating VLDL fraction were reduced and levels of circulating ketone bodies increased after RA treatment. These results point to a capacity of active vitamin A forms to shift liver lipid metabolism in vivo towards increased catabolism and reduced lipogenesis. These effects might contribute to the reduction of adiposity brought about by RA treatment.

  2. PROTEIN METABOLISM IN REGENERATING WOUND TISSUE: FUNCTION OF THE SULFUR AMINO ACIDS.

    Science.gov (United States)

    PROTEINS, *TISSUES(BIOLOGY), METABOLISM, TISSUES(BIOLOGY), REGENERATION(ENGINEERING), WOUNDS AND INJURIES, TISSUES(BIOLOGY), TRACER STUDIES, METHIONINE, COLLAGEN, TYROSINE, BIOSYNTHESIS, AMINO ACIDS .

  3. Natural product inhibitors of fatty acid biosynthesis: synthesis of the marine microbial metabolites pseudopyronines A and B and evaluation of their anti-infective activities

    DEFF Research Database (Denmark)

    Giddens, Anna C.; Nielsen, Lone; Boshoff, Helena I.;

    2007-01-01

    of pathogenic microorganisms and were found to exhibit good potency (IC50≥0.46 μg/mL) and selectivity towards Leishmania donovani. Several of the compounds inhibited recombinant fatty acid biosynthesis enzymes from both Plasmodium falciparum and Mycobacterium tuberculosis, validating these targets in the search...

  4. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth

    DEFF Research Database (Denmark)

    Jakočiūnė, Dzuiga; Herrero-Fresno, Ana; Jelsbak, Lotte;

    2016-01-01

    RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis...

  5. Antisense-mediated suppression of C-hordein biosynthesis in the barley grain results in correlated changes in the transcriptome, protein profile, and amino acid composition

    DEFF Research Database (Denmark)

    Hansen, Mette; Lange, Marianne; Friis, Carsten;

    2007-01-01

    Antisense- or RNAi-mediated suppression of the biosynthesis of nutritionally inferior storage proteins is a promising strategy for improving the amino acid profile of seeds. However, the potential pleiotropic effects of this on interconnected pathways and the agronomic quality traits need...

  6. [Effect of organic acids on the biosynthesis of carotenes by an Actinomyces chrysomallus strain].

    Science.gov (United States)

    Nefelova, M V; Sverdlova, A N; Alekseeva, L N

    1978-01-01

    Synthesis of carotenes by Actinomyces chrysomallus var. carotenoides was stimulated by citric, acetic, oxalacetic, fumaric, succinic, malic, alpha-ketoglutaric, tartaric, pyruvic, and propionic acids. Acetic acid acts as a precursor of carotene synthesis and also has another stimulating mechanism of action on carotenogenesis of the actinomycete. Acetic, furmaric, malic, succinic, and alpha-ketoglutaric acids stimulate cyclization of lycopene yielding beta-carotene.

  7. Extracellular biosynthesis of functionalized silver nanoparticles by strains of Cladosporium cladosporioides fungus

    NARCIS (Netherlands)

    Balaji, D. S.; Basavaraja, S.; Deshpande, R.; Mahesh, D. Bedre; Prabhakar, B. K.; Venkataraman, A.

    2009-01-01

    In the present investigation, we report the extracellular biosynthesis of silver nanoparticles (AgNP) employing the fungus Cladosporium cladosporioides. The extracellular solution of C. cladosporioides was used for the reduction of AgNO(3) solution to AgNP. The present study includes time dependent

  8. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Marta Spodenkiewicz

    2016-10-01

    Full Text Available Glutamine synthetase (GS is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS can cause an ultra-rare recessive inborn error of metabolism—congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition.

  9. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis.

    Science.gov (United States)

    Spodenkiewicz, Marta; Diez-Fernandez, Carmen; Rüfenacht, Véronique; Gemperle-Britschgi, Corinne; Häberle, Johannes

    2016-10-19

    Glutamine synthetase (GS) is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS) can cause an ultra-rare recessive inborn error of metabolism-congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i) this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii) early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition.

  10. Phosphatidic acid: biosynthesis, pharmacokinetics, mechanisms of action and effect on strength and body composition in resistance-trained individuals.

    Science.gov (United States)

    Bond, Peter

    2017-01-01

    The mechanistic target of rapamycin complex 1 (mTORC1) has received much attention in the field of exercise physiology as a master regulator of skeletal muscle hypertrophy. The multiprotein complex is regulated by various signals such as growth factors, energy status, amino acids and mechanical stimuli. Importantly, the glycerophospholipid phosphatidic acid (PA) appears to play an important role in mTORC1 activation by mechanical stimulation. PA has been shown to modulate mTOR activity by direct binding to its FKBP12-rapamycin binding domain. Additionally, it has been suggested that exogenous PA activates mTORC1 via extracellular conversion to lysophosphatidic acid and subsequent binding to endothelial differentiation gene receptors on the cell surface. Recent trials have therefore evaluated the effects of PA supplementation in resistance-trained individuals on strength and body composition. As research in this field is rapidly evolving, this review attempts to provide a comprehensive overview of its biosynthesis, pharmacokinetics, mechanisms of action and effect on strength and body composition in resistance-trained individuals.

  11. Biosynthesis of poly(4-hydroxybutyrate) in recombinant Escherichia coli grown on glycerol is stimulated by propionic acid.

    Science.gov (United States)

    Kämpf, Michael M; Thöny-Meyer, Linda; Ren, Qun

    2014-11-01

    One of the most promising polyhydroxyalkanoates (PHAs) for medical applications is poly(4-hydroxybutyrate) (P4HB) due to its biodegradability, biocompatibility and mechanical properties. Currently, the major hurdle for expanding P4HB applications is the production and recovery cost. In this study, we investigated the stimulating factors for P4HB biosynthesis with the ultimate goal of reducing production cost. We found that addition of propionic acid to the culture medium stimulates the P4HB accumulation in recombinant Escherichia coli JM109 grown on glycerol. This stimulating effect was significantly weakened by addition of exogenous methionine, whereas it was not influenced by addition of cysteine. These results suggest that propionic acid enhances P4HB synthesis by reducing the intracellular methionine pool. Utilizing these findings for P4HB production in batch cultures on glycerol, the volumetric yield of P4HB could be improved 4 fold from 0.9g/L to 3.7g/L by adding 2g/L propionic acid into the medium.

  12. Acid monolayer functionalized iron oxide nanoparticle catalysts

    Science.gov (United States)

    Ikenberry, Myles

    Superparamagnetic iron oxide nanoparticle functionalization is an area of intensely active research, with applications across disciplines such as biomedical science and heterogeneous catalysis. This work demonstrates the functionalization of iron oxide nanoparticles with a quasi-monolayer of 11-sulfoundecanoic acid, 10-phosphono-1-decanesulfonic acid, and 11-aminoundecanoic acid. The carboxylic and phosphonic moieties form bonds to the iron oxide particle core, while the sulfonic acid groups face outward where they are available for catalysis. The particles were characterized by thermogravimetric analysis (TGA), transmission electron microscopy (TEM), potentiometric titration, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), inductively coupled plasma optical emission spectrometry (ICP-OES), X-ray photoelectron spectrometry (XPS), and dynamic light scattering (DLS). The sulfonic acid functionalized particles were used to catalyze the hydrolysis of sucrose at 80° and starch at 130°, showing a higher activity per acid site than the traditional solid acid catalyst Amberlyst-15, and comparing well against results reported in the literature for sulfonic acid functionalized mesoporous silicas. In sucrose catalysis reactions, the phosphonic-sulfonic nanoparticles (PSNPs) were seen to be incompletely recovered by an external magnetic field, while the carboxylic-sulfonic nanoparticles (CSNPs) showed a trend of increasing activity over the first four recycle runs. Between the two sulfonic ligands, the phosphonates produced a more tightly packed monolayer, which corresponded to a higher sulfonic acid loading, lower agglomeration, lower recoverability through application of an external magnetic field, and higher activity per acid site for the hydrolysis of starch. Functionalizations with 11-aminoundecanoic acid resulted in some amine groups binding to the surfaces of iron oxide nanoparticles. This amine binding is commonly ignored in iron oxide

  13. Stereospecific Synthesis of threo- and erythro-β-Hydroxyglutamic Acid During Kutzneride Biosynthesis

    OpenAIRE

    Strieker, Matthias; Nolan, Elizabeth M.; Walsh, Christopher T.; Marahiel, Mohamed A. (Prof. Dr.)

    2009-01-01

    The antifungal and antimicrobial kutznerides, hexadepsipeptides comprised of one α-hydroxy acid and five non-proteinogenic amino acids, are remarkable examples of the structural diversity found in nonribosomally-produced natural products. They contain D-3-hydroxyglutamic acid, which is found in the threo and erythro isomers in mature kutznerides. In this study, two putative non-heme iron oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in vitro, and ...

  14. Salicylic Acid Induction of Flavonoid Biosynthesis Pathways in Wheat Varies by Treatment.

    Science.gov (United States)

    Gondor, Orsolya K; Janda, Tibor; Soós, Vilmos; Pál, Magda; Majláth, Imre; Adak, Malay K; Balázs, Ervin; Szalai, Gabriella

    2016-01-01

    Salicylic acid is a promising compound for the reduction of stress sensitivity in plants. Although several biochemical and physiological changes have been described in plants treated with salicylic acid, the mode of action of the various treatments has not yet been clarified. The present work reports a detailed comparative study on the effects of different modes of salicylic acid application at the physiological, metabolomic, and transcriptomic levels. Seed soaking and hydroponic treatments were found to induce various changes in the protective mechanisms of wheat plants. The possible involvement of the flavonoid metabolism in salicylic acid-related stress signaling was also demonstrated. Different salicylic acid treatments were shown to induce different physiological and biochemical processes, with varying responses in the leaves and roots. Hydroponic treatment enhanced the level of oxidative stress, the expression of genes involved in the flavonoid metabolism and the amount of non-enzymatic antioxidant compounds, namely ortho-hydroxycinnamic acid and the flavonol quercetin in the leaves, while it decreased the ortho-hydroxycinnamic acid and flavonol contents and enhanced ascorbate peroxidase activity in the roots. In contrast, seed soaking only elevated the gene expression level of phenylalanine ammonia lyase in the roots and caused a slight increase in the amount of flavonols. These results draw attention to the fact that the effects of exogenous salicylic acid application cannot be generalized in different experimental systems and that the flavonoid metabolism may be an important part of the action mechanisms induced by salicylic acid.

  15. Salicylic Acid Induction of Flavonoid Biosynthesis Pathways in Wheat Varies by Treatment

    Science.gov (United States)

    Gondor, Orsolya K.; Janda, Tibor; Soós, Vilmos; Pál, Magda; Majláth, Imre; Adak, Malay K.; Balázs, Ervin; Szalai, Gabriella

    2016-01-01

    Salicylic acid is a promising compound for the reduction of stress sensitivity in plants. Although several biochemical and physiological changes have been described in plants treated with salicylic acid, the mode of action of the various treatments has not yet been clarified. The present work reports a detailed comparative study on the effects of different modes of salicylic acid application at the physiological, metabolomic, and transcriptomic levels. Seed soaking and hydroponic treatments were found to induce various changes in the protective mechanisms of wheat plants. The possible involvement of the flavonoid metabolism in salicylic acid-related stress signaling was also demonstrated. Different salicylic acid treatments were shown to induce different physiological and biochemical processes, with varying responses in the leaves and roots. Hydroponic treatment enhanced the level of oxidative stress, the expression of genes involved in the flavonoid metabolism and the amount of non-enzymatic antioxidant compounds, namely ortho-hydroxycinnamic acid and the flavonol quercetin in the leaves, while it decreased the ortho-hydroxycinnamic acid and flavonol contents and enhanced ascorbate peroxidase activity in the roots. In contrast, seed soaking only elevated the gene expression level of phenylalanine ammonia lyase in the roots and caused a slight increase in the amount of flavonols. These results draw attention to the fact that the effects of exogenous salicylic acid application cannot be generalized in different experimental systems and that the flavonoid metabolism may be an important part of the action mechanisms induced by salicylic acid.

  16. Biosynthetic mechanism for L-Gulose in main polar lipids of Thermoplasma acidophilum and possible resemblance to plant ascorbic acid biosynthesis.

    Science.gov (United States)

    Yamauchi, Noriaki; Nakayama, Yusuke

    2013-01-01

    L-Gulose is a very rare sugar, but appears as a sugar component of the main polar lipids characteristic in such a thermophilic archaeon as Thermoplasma acidophilum that lives without cell walls in a highly acidic environment. The biosynthesis of L-gulose in this thermophilic organism was investigated with deuterium-labeling experiments. L-Gulose was found to be biosynthesized from D-glucose via stepwise stereochemical inversion at C-2 and C-5. The involvement of an epimerase related to GDP-mannose 3,5-epimerase, the key enzyme of plant ascorbate biosynthesis, was also suggested in this C-5 inversion. The resemblance of L-gulose biosynthesis in archaea and plants might be suggested from these results.

  17. Structure-activity relationships of the nonredox-type non-competitive leukotriene biosynthesis inhibitor acetyl-11-keto-β-boswellic acid.

    Science.gov (United States)

    Sailer, E R; Hoernlein, R F; Ammon, H P; Safayhi, H

    1996-05-01

    Acetyl-11-keto-β-boswellic acid (AKBA) from Boswellia serrata Roxb. and italics Boswellia carterii Birdw. is the first selective, direct, non-competitive and non-redox-type inhibitor of 5-lipoxygenase, the key enzyme for leukotriene biosynthesis (Safayhi et al., 1992). Previously, we showed that AKBA interacts with the 5-lipoxygenase via a pentacyclic triterpene selective effector site (Safayhi et al., 1995). In order to study the impact of AKBA's functional groups on enzyme inhibition, natural and synthetic analogues of this boswellic acid were tested for 5-lipoxygenase inhibition in intact rat neutrophils (Sailer et al., 1996 a). The results reveal that the carboxylic group of AKBA combined with the 11-keto-group is essential for enzyme inhibition, whereas the acetoxy-group on position C-3 α increases the affinity of AKBA to its effector site. Furthermore, other experiments demonstrated that minor structural modifications could cause a total loss of binding affinity and/or inhibitory activity of these compounds.

  18. Transcriptional regulation of chlorogenic acid biosynthesis in carrot root slices exposed to UV-B light

    Science.gov (United States)

    Orange carrots are well known for their nutritional value as producers of ß-carotene, a Vitamin A precursor. Lesser known, is their ability to accumulate antioxidants such as chlorogenic acid. Chlorogenic acid is produced through the same biosynthetic pathway that produces lignins, anthocyanins, f...

  19. Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes.

    Science.gov (United States)

    Yuan, Yuan; Wang, Zhouyong; Jiang, Chao; Wang, Xumin; Huang, Luqi

    2014-01-25

    Chlorogenic acids (CGAs) and luteolin are active compounds in Lonicera japonica, a plant of high medicinal value in traditional Chinese medicine. This study provides a comprehensive overview of gene families involved in chlorogenic acid and luteolin biosynthesis in L. japonica, as well as its substitutes Lonicera hypoglauca and Lonicera macranthoides. The gene sequence feature and gene expression patterns in various tissues and buds of the species were characterized. Bioinformatics analysis revealed that 14 chlorogenic acid and luteolin biosynthesis-related genes were identified from the L. japonica transcriptome assembly. Phylogenetic analyses suggested that the function of individual gene could be differentiation and induce active compound diversity. Their orthologous genes were also recognized in L. hypoglauca and L. macranthoides genomic datasets, except for LHCHS1 and LMC4H2. The expression patterns of these genes are different in the tissues of L. japonica, L. hypoglauca and L. macranthoides. Results also showed that CGAs were controlled in the first step of biosynthesis, whereas both steps controlled luteolin in the bud of L. japonica. The expression of LJFNS2 exhibited positive correlation with luteolin levels in L. japonica. This study provides significant information for understanding the functional diversity of gene families involved in chlorogenic acid and the luteolin biosynthesis, active compound diversity of L. japonica and its substitutes, and the different usages of the three species.

  20. Heteroconium chaetospira induces resistance to clubroot via upregulation of host genes involved in jasmonic acid, ethylene, and auxin biosynthesis.

    Directory of Open Access Journals (Sweden)

    Rachid Lahlali

    Full Text Available An endophytic fungus, Heteroconium chaetospira isolate BC2HB1 (Hc, suppressed clubroot (Plasmodiophora brassicae -Pb on canola in growth-cabinet trials. Confocal microscopy demonstrated that Hc penetrated canola roots and colonized cortical tissues. Based on qPCR analysis, the amount of Hc DNA found in canola roots at 14 days after treatment was negatively correlated (r = 0.92, P<0.001 with the severity of clubroot at 5 weeks after treatment at a low (2×10(5 spores pot(-1 but not high (2×10(5 spores pot(-1 dose of pathogen inoculum. Transcript levels of nine B. napus (Bn genes in roots treated with Hc plus Pb, Pb alone and a nontreated control were analyzed using qPCR supplemented with biochemical analysis for the activity of phenylalanine ammonia lyases (PAL. These genes encode enzymes involved in several biosynthetic pathways related potentially to plant defence. Hc plus Pb increased the activity of PAL but not that of the other two genes (BnCCR and BnOPCL involved also in phenylpropanoid biosynthesis, relative to Pb inoculation alone. In contrast, expression of several genes involved in the jasmonic acid (BnOPR2, ethylene (BnACO, auxin (BnAAO1, and PR-2 protein (BnPR-2 biosynthesis were upregulated by 63, 48, 3, and 3 fold, respectively, by Hc plus Pb over Pb alone. This indicates that these genes may be involved in inducing resistance in canola by Hc against clubroot. The upregulation of BnAAO1 appears to be related to both pathogenesis of clubroot and induced defence mechanisms in canola roots. This is the first report on regulation of specific host genes involved in induced plant resistance by a non-mycorrhizal endophyte.

  1. Functional Characterization of 4′OMT and 7OMT Genes in BIA Biosynthesis

    OpenAIRE

    Gurkok, Tugba; Ozhuner, Esma; Parmaksiz, Iskender; Özcan, Sebahattin; Turktas, Mine; İPEK, Arif; Demirtas, Ibrahim; Okay, Sezer; Unver, Turgay

    2016-01-01

    Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes...

  2. Functional Characterization of 4´OMT and 7OMT Genes in BIA Biosynthesis

    OpenAIRE

    Tugba eGurkok; Esma eOzhuner; Iskender eParmaksiz; Sebahattin eÖzcan; Mine eTurktas; Arif eIpek; Sezer eOkay; Turgay eUNVER

    2016-01-01

    Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes...

  3. Identification and Characterization of Late Pathway Enzymes in Phytic Acid Biosynthesis in Glycine max

    OpenAIRE

    Stiles, Amanda Rose

    2007-01-01

    Phytic acid, also known as myo-inositol hexakisphosphate or Ins(1,2,3,4,5,6)P6, is the major storage form of phosphorus in plant seeds. Phytic acid is poorly digested by non-ruminant animals such as swine and poultry, and it chelates mineral cations including calcium, iron, zinc, and potassium, classifying it as an anti-nutrient. The excretion of unutilized phytic acid in manure translates to an excess amount of phosphorus runoff that can lead to eutrophication of lakes and ponds. Understand...

  4. Molecular Structure of WlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Thoden, James B.; Holden, Hazel M. (UW)

    2010-09-08

    The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 {angstrom} resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimeric quaternary structure and belongs to the L{beta}H superfamily of N-acyltransferases. Each subunit contains 27 {beta}-strands, 23 of which form the canonical left-handed {beta}-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O{sup {delta}1} of Asn 84 and the sugar C-3{prime} amino group and the second between the backbone amide group of Arg 94 and the sugar C-5{prime} carboxylate. The sugar C-3{prime} amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.

  5. Ascorbic Acid Biosynthesis and Brackish Water Acclimation in the Euryhaline Freshwater White-Rimmed Stingray, Himantura signifer.

    Science.gov (United States)

    Wong, Samuel Z H; Ching, Biyun; Chng, You R; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2013-01-01

    L-gulono-γ-lactone oxidase (Gulo) catalyzes the last step of ascorbic acid biosynthesis, which occurs in the kidney of elasmobranchs. This study aimed to clone and sequence gulonolactone oxidase (gulo) from the kidney of the euryhaline freshwater stingray, Himantura signifer, and to determine the effects of acclimation from freshwater to brackish water (salinity 20) on its renal gulo mRNA expression and Gulo activity. We also examined the effects of brackish water acclimation on concentrations of ascorbate, dehydroascorbate and ascorbate + dehydroascorbate in the kidney, brain and gill. The complete cDNA coding sequence of gulo from the kidney of H. signifer contained 1323 bp coding for 440 amino acids. The expression of gulo was kidney-specific, and renal gulo expression decreased significantly by 67% and 50% in fish acclimated to brackish water for 1 day and 6 days, respectively. There was also a significant decrease in renal Gulo activity after 6 days of acclimation to brackish water. Hence, brackish water acclimation led to a decrease in the ascorbic acid synthetic capacity in the kidney of H. signifer. However, there were significant increases in concentrations of ascorbate and ascorbate + dehydroascorbate in the gills (after 1 or 6 days), and a significant increase in the concentration of ascorbate and a significant decrease in the concentration of dehydroascorbate in the brain (after 1 day) of fish acclimated to brackish water. Taken together, our results indicate that H. signifer might experience greater salinity-induced oxidative stress in freshwater than in brackish water, possibly related to its short history of freshwater invasion. These results also suggest for the first time a possible relationship between the successful invasion of the freshwater environment by some euryhaline marine elasmobranchs and the ability of these elasmobranchs to increase the capacity of ascorbic acid synthesis in response to hyposalinity stress.

  6. Ascorbic Acid Biosynthesis and Brackish Water Acclimation in the Euryhaline Freshwater White-Rimmed Stingray, Himantura signifer.

    Directory of Open Access Journals (Sweden)

    Samuel Z H Wong

    Full Text Available L-gulono-γ-lactone oxidase (Gulo catalyzes the last step of ascorbic acid biosynthesis, which occurs in the kidney of elasmobranchs. This study aimed to clone and sequence gulonolactone oxidase (gulo from the kidney of the euryhaline freshwater stingray, Himantura signifer, and to determine the effects of acclimation from freshwater to brackish water (salinity 20 on its renal gulo mRNA expression and Gulo activity. We also examined the effects of brackish water acclimation on concentrations of ascorbate, dehydroascorbate and ascorbate + dehydroascorbate in the kidney, brain and gill. The complete cDNA coding sequence of gulo from the kidney of H. signifer contained 1323 bp coding for 440 amino acids. The expression of gulo was kidney-specific, and renal gulo expression decreased significantly by 67% and 50% in fish acclimated to brackish water for 1 day and 6 days, respectively. There was also a significant decrease in renal Gulo activity after 6 days of acclimation to brackish water. Hence, brackish water acclimation led to a decrease in the ascorbic acid synthetic capacity in the kidney of H. signifer. However, there were significant increases in concentrations of ascorbate and ascorbate + dehydroascorbate in the gills (after 1 or 6 days, and a significant increase in the concentration of ascorbate and a significant decrease in the concentration of dehydroascorbate in the brain (after 1 day of fish acclimated to brackish water. Taken together, our results indicate that H. signifer might experience greater salinity-induced oxidative stress in freshwater than in brackish water, possibly related to its short history of freshwater invasion. These results also suggest for the first time a possible relationship between the successful invasion of the freshwater environment by some euryhaline marine elasmobranchs and the ability of these elasmobranchs to increase the capacity of ascorbic acid synthesis in response to hyposalinity stress.

  7. Biosynthesis of Unsaturated Fatty Acids via Mortierella Isabellina Cultivated in a Medium Containing Butanol

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Mortierella isabellina was found to accumulate large amounts of unsaturated fatty acids when it was grown in a medium containing butanol(5 g/L) and yeast extract(10 g/L) and cultivated at 25 ℃ for 5 d during which additional feeding butanol of 2 g/L was fed after being cultivated for 48 h. The resultant mycelial lipids accounted for 40.1% of the dry mycelia, while about 34% of butanol in the medium was converted. The mycelial lipids contained four kinds of unsaturated fatty acids, i.e., palmitoleic(4.9%), oleic(54.1%), linoleic(10.4%) and linolenic(5.4%) acids. Those accounted for 74.8% of the total fatty acids. The effects of the culture conditions, such as cultivation temperature, initial pH of the medium and additional feeding butanol in the course of cultivation, on the production of mycelial lipids by M. isabellina were studied.

  8. The ability of Clostridium bifermentans strains to lactic acid biosynthesis in various environmental conditions

    OpenAIRE

    Leja, Katarzyna; Myszka, Kamila; Czaczyk, Katarzyna

    2013-01-01

    Clostridium bifermentans strains, isolated from a manure, were examinated for their ability to produce lactic acid from PY medium with glycerol under different pH conditions and when PY medium was supplemented with saccharides such as fructose, sorbitol, glucose, mannose, mannitol, maltose, xylose, raffinose, and arabinose. In the last test performed, the ability of investigated strains to produce lactic acid from mixed carbon source (glycerol plus saccharide) was checked. The strains of Cl. ...

  9. Bile acid signaling and biliary functions

    Directory of Open Access Journals (Sweden)

    Hannah Jones

    2015-03-01

    Full Text Available This review focuses on various components of bile acid signaling in relation to cholangiocytes. Their roles as targets for potential therapies for cholangiopathies are also explored. While many factors are involved in these complex signaling pathways, this review emphasizes the roles of transmembrane G protein coupled receptor (TGR5, farnesoid X receptor (FXR, ursodeoxycholic acid (UDCA and the bicarbonate umbrella. Following a general background on cholangiocytes and bile acids, we will expand the review and include sections that are most recently known (within 5–7 years regarding the field of bile acid signaling and cholangiocyte function. These findings all demonstrate that bile acids influence biliary functions which can, in turn, regulate the cholangiocyte response during pathological events.

  10. Effect of fatty acids on leukocyte function

    Directory of Open Access Journals (Sweden)

    Pompéia C.

    2000-01-01

    Full Text Available Fatty acids have various effects on immune and inflammatory responses, acting as intracellular and intercellular mediators. Polyunsaturated fatty acids (PUFAs of the omega-3 family have overall suppressive effects, inhibiting lymphocyte proliferation, antibody and cytokine production, adhesion molecule expression, natural killer cell activity and triggering cell death. The omega-6 PUFAs have both inhibitory and stimulatory effects. The most studied of these is arachidonic acid that can be oxidized to eicosanoids, such as prostaglandins, leukotrienes and thromboxanes, all of which are potent mediators of inflammation. Nevertheless, it has been found that many of the effects of PUFA on immune and inflammatory responses are not dependent on eicosanoid generation. Fatty acids have also been found to modulate phagocytosis, reactive oxygen species production, cytokine production and leukocyte migration, also interfering with antigen presentation by macrophages. The importance of fatty acids in immune function has been corroborated by many clinical trials in which patients show improvement when submitted to fatty acid supplementation. Several mechanisms have been proposed to explain fatty acid modulation of immune response, such as changes in membrane fluidity and signal transduction pathways, regulation of gene transcription, protein acylation, and calcium release. In this review, evidence is presented to support the proposition that changes in cell metabolism also play an important role in the effect of fatty acids on leukocyte functioning, as fatty acids regulate glucose and glutamine metabolism and mitochondrial depolarization.

  11. Cloning,sequencing and function of sanA,a gene involved in nikkomycin biosynthesis of Streptomyces ansochromogenes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Several genetically stable mutants blocked in nikkomycin biosynthesis were obtained after the slightly germinated spores of Streptomyces ansochromogenes,a nikkomycin producer,were treated with ultra violet radiation.One of the mutants is the same in morpholotical differentiation as the wild type strain and is designated as NBB19.A DNA library was constructed using plasmid pIJ702 as cloning vector,NBB19 as cloning recipient.A 6 kb DNA fragment which can genetically complement NBB19 was cloned when screening the library for antifungal activity.Sequence analysis showed that the 3 kb Bgl II-Sal I fragment contains one complete ORF (ORF1) and one partial ORF (ORF2).ORF1 is designated as sanA.sanA is 1 365 bp,encoding a protein consisting of 454 amino acid residues.Database searching indicated that sanA is homologous to the hypothetical methyltransferase in Pyrococcus horikoshii with 25% identities and 41% positives.Disruptant of sanA lost the ability to synthesize nikkomycin.It indicated that sanA is a novel gene which is essential for nikkomycin biosynthesis.

  12. Biochemical and Genetic Engineering of Diatoms for Polyunsaturated Fatty Acid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Hong-Ye Li

    2014-01-01

    Full Text Available The role of diatoms as a source of bioactive compounds has been recently explored. Diatom cells store a high amount of fatty acids, especially certain polyunsaturated fatty acids (PUFAs. However, many aspects of diatom metabolism and the production of PUFAs remain unclear. This review describes a number of technical strategies, such as modulation of environmental factors (temperature, light, chemical composition of culture medium and culture methods, to influence the content of PUFAs in diatoms. Genetic engineering, a newly emerging field, also plays an important role in controlling the synthesis of fatty acids in marine microalgae. Several key points in the biosynthetic pathway of PUFAs in diatoms as well as recent progresses are also a critical part and are summarized here.

  13. Iron-sulphur clusters, their biosynthesis, and biological functions in protozoan parasites.

    Science.gov (United States)

    Ali, Vahab; Nozaki, Tomoyoshi

    2013-01-01

    Fe-S clusters are ensembles of sulphide-linked di-, tri-, and tetra-iron centres of a variety of metalloproteins that play important roles in reduction and oxidation of mitochondrial electron transport, energy metabolism, regulation of gene expression, cell survival, nitrogen fixation, and numerous other metabolic pathways. The Fe-S clusters are assembled by one of four distinct systems: NIF, SUF, ISC, and CIA machineries. The ISC machinery is a house-keeping system conserved widely from prokaryotes to higher eukaryotes, while the other systems are present in a limited range of organisms and play supplementary roles under certain conditions such as stress. Fe-S cluster-containing proteins and the components required for Fe-S cluster biosynthesis are modulated under stress conditions, drug resistance, and developmental stages. It is also known that a defect in Fe-S proteins and Fe-S cluster biogenesis leads to many genetic disorders in humans, which indicates the importance of the systems. In this review, we describe the biological and physiological significance of Fe-S cluster-containing proteins and their biosynthesis in parasitic protozoa including Plasmodium, Trypanosoma, Leishmania, Giardia, Trichomonas, Entamoeba, Cryptosporidium, Blastocystis, and microsporidia. We also discuss the roles of Fe-S cluster biosynthesis in proliferation, differentiation, and stress response in protozoan parasites. The heterogeneity of the systems and the compartmentalization of Fe-S cluster biogenesis in the protozoan parasites likely reflect divergent evolution under highly diverse environmental niches, and influence their parasitic lifestyle and pathogenesis. Finally, both Fe-S cluster-containing proteins and their biosynthetic machinery in protozoan parasites are remarkably different from those in their mammalian hosts. Thus, they represent a rational target for the development of novel chemotherapeutic and prophylactic agents against protozoan infections.

  14. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    Science.gov (United States)

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  15. Regulation of indole-3-acetic acid biosynthesis by branched-chain amino acids in Enterobacter cloacae UW5.

    Science.gov (United States)

    Parsons, Cassandra V; Harris, Danielle M M; Patten, Cheryl L

    2015-09-01

    The soil bacterium Enterobacter cloacae UW5 produces the rhizosphere signaling molecule indole-3-acetic acid (IAA) via the indolepyruvate pathway. Expression of indolepyruvate decarboxylase, a key pathway enzyme encoded by ipdC, is upregulated by the transcription factor TyrR in response to aromatic amino acids. Some members of the TyrR regulon may also be controlled by branched-chain amino acids and here we show that expression from the ipdC promoter and production of IAA are downregulated by valine, leucine and isoleucine. Regulation of the IAA synthesis pathway by both aromatic and branched-chain amino acids suggests a broader role for this pathway in bacterial physiology, beyond plant interactions.

  16. Biosynthesis of terephthalic acid, isophthalic acid and their derivatives from the corresponding dinitriles by tetrachloroterephthalonitrile-induced Rhodococcus sp.

    Science.gov (United States)

    He, Yu-Cai; Wu, Ya-Dong; Pan, Xue-He; Ma, Cui-Luan

    2014-02-01

    The nitrilase from Rhodococcus sp. CCZU10-1 catalyses the hydrolysis of dinitriles to acids without the formation of amides and cyanocarboxylic acids. It was induced by benzonitrile and its analogues (tetrachloroterephthalonitrile > ε-caprolactam > benzonitrile > phenylacetonitrile), and had activity towards aromatic nitriles (terephthalonitrile > tetrachloroterephthalonitrile > isophthalonitrile > tetrachloroisophthalonitrile > tetrafluoroterephthalonitrile > benzonitrile). After the optimization, the highest nitrilase induction [311 U/(g DCW)] was achieved with tetrachloroterephthalonitrile (1 mM) in the medium after 24 h at 30 °C after optimum enzyme activity was at pH 6.8 and at 30 °C. Efficient biocatalyst recycling was achieved by cell immobilization in calcium alginate, with a product-to-biocatalyst ratios of 776 g terephthalic acid/g DCW and 630 g isophthalic acid/g DCW.

  17. Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C.; Jacobs, Jr, William R.; Kremer, Laurent (CNRS-UMR); (Einstein); (TAM)

    2011-08-24

    The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA{_}T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.

  18. Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis

    Science.gov (United States)

    Caenorhabditis elegans secretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, perception of which enables worms to gauge depletion of food or a high worm population density. As a result, worms enter the dauer state, a specific developmental stage capable of surviv...

  19. Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis

    NARCIS (Netherlands)

    Degu, A.; Hatew, B.; Nunes-Nesi, A.; Shlizerman, L.; Zur, N.; Fernie, A.R.; Blumwald, E.; Sadka, A.

    2011-01-01

    Citrate, a major determinant of citrus fruit quality, accumulates early in fruit development and declines towards maturation. The isomerization of citrate to isocitrate, catalyzed by aconitase is a key step in acid metabolism. Inhibition of mitochondrial aconitase activity early in fruit development

  20. Conjugated linoleic acid or omega 3 fatty acids increase mitochondrial biosynthesis and metabolism in skeletal muscle cells

    OpenAIRE

    Vaughan Roger A; Garcia-Smith Randi; Bisoffi Marco; Conn Carole A; Trujillo Kristina A

    2012-01-01

    Abstract Background Polyunsaturated fatty acids are popular dietary supplements advertised to contribute to weight loss by increasing fat metabolism in liver, but the effects on overall muscle metabolism are less established. We evaluated the effects of conjugated linoleic acid (CLA) or combination omega 3 on metabolic characteristics in muscle cells. Methods Human rhabdomyosarcoma cells were treated with either DMSO control, or CLA or combination omega 3 for 24 or 48 hours. RNA was determine...

  1. Fatty acid biosynthesis VII. Substrate control of chain-length of products synthesised by rat liver fatty acid synthetase

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Carey, E.M.; Dils, R.

    1970-01-01

    - 1. Gas-liquid and paper chromatography have been used to determine the chain-lengths of fatty acids synthesised by purified rat liver fatty acid synthetase from [1-14C]acetyl-CoA, [1,3-14C2]malonyl-CoA and from [1-14C]acetyl-CoA plus partially purified rat liver acetyl-CoA carboxylase. - 2...

  2. Microbial biosynthesis of alkanes.

    Science.gov (United States)

    Schirmer, Andreas; Rude, Mathew A; Li, Xuezhi; Popova, Emanuela; del Cardayre, Stephen B

    2010-07-30

    Alkanes, the major constituents of gasoline, diesel, and jet fuel, are naturally produced by diverse species; however, the genetics and biochemistry behind this biology have remained elusive. Here we describe the discovery of an alkane biosynthesis pathway from cyanobacteria. The pathway consists of an acyl-acyl carrier protein reductase and an aldehyde decarbonylase, which together convert intermediates of fatty acid metabolism to alkanes and alkenes. The aldehyde decarbonylase is related to the broadly functional nonheme diiron enzymes. Heterologous expression of the alkane operon in Escherichia coli leads to the production and secretion of C13 to C17 mixtures of alkanes and alkenes. These genes and enzymes can now be leveraged for the simple and direct conversion of renewable raw materials to fungible hydrocarbon fuels.

  3. GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes.

    Science.gov (United States)

    Kriel, Allison; Brinsmade, Shaun R; Tse, Jessica L; Tehranchi, Ashley K; Bittner, Alycia N; Sonenshein, Abraham L; Wang, Jue D

    2014-01-01

    The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp(0) cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

  4. Expression of Tropodithietic Acid Biosynthesis Is Controlled by a Novel Autoinducer▿ †

    OpenAIRE

    Geng, Haifeng; Belas, Robert

    2010-01-01

    The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tr...

  5. Evidence for a universal pathway of abscisic acid biosynthesis in higher plants from sup 18 O incorporation patterns

    Energy Technology Data Exchange (ETDEWEB)

    Zeevaart, J.A.D.; Heath, T.G.; Gage, D.A. (Michigan State University, East Lansing (USA))

    1989-12-01

    Previous labeling studies of abscisic acid (ABA) with {sup 18}O{sub 2} have been mainly conducted with water-stressed leaves. In this study, {sup 18}O incorporation into ABA of stressed leaves of various species was compared with {sup 18}O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), {sup 18}O was most abundant in the carboxyl group, whereas incorporation of a second and third {sup 18}O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in {sup 18}O{sub 2}. ABA from turgid bean leaves showed significant {sup 18}O incorporation, again with highest {sup 18}O enrichment in the carboxyl group. On the basis of {sup 18}O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precusor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.

  6. Mutation in the key enzyme of sialic acid biosynthesis causes severe glomerular proteinuria and is rescued by N-acetylmannosamine.

    Science.gov (United States)

    Galeano, Belinda; Klootwijk, Riko; Manoli, Irini; Sun, MaoSen; Ciccone, Carla; Darvish, Daniel; Starost, Matthew F; Zerfas, Patricia M; Hoffmann, Victoria J; Hoogstraten-Miller, Shelley; Krasnewich, Donna M; Gahl, William A; Huizing, Marjan

    2007-06-01

    Mutations in the key enzyme of sialic acid biosynthesis, uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK), result in hereditary inclusion body myopathy (HIBM), an adult-onset, progressive neuromuscular disorder. We created knockin mice harboring the M712T Gne/Mnk mutation. Homozygous mutant (Gne(M712T/M712T)) mice did not survive beyond P3. At P2, significantly decreased Gne-epimerase activity was observed in Gne(M712T/M712T) muscle, but no myopathic features were apparent. Rather, homozygous mutant mice had glomerular hematuria, proteinuria, and podocytopathy. Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration yielded survival beyond P3 in 43% of the Gne(M712T/M712T) pups. Survivors exhibited improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gne-epimerase activities. These findings establish this Gne(M712T/M712T) knockin mouse as what we believe to be the first genetic model of podocyte injury and segmental glomerular basement membrane splitting due to hyposialylation. The results also support evaluation of ManNAc as a treatment not only for HIBM but also for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane.

  7. Genetic engineering activates biosynthesis of aromatic fumaric acid amides in the human pathogen Aspergillus fumigatus.

    Science.gov (United States)

    Kalb, Daniel; Heinekamp, Thorsten; Lackner, Gerald; Scharf, Daniel H; Dahse, Hans-Martin; Brakhage, Axel A; Hoffmeister, Dirk

    2015-03-01

    The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds.

  8. Brassinosteroids Improve Quality of Summer Tea (Camellia sinensis L.) by Balancing Biosynthesis of Polyphenols and Amino Acids

    Science.gov (United States)

    Li, Xin; Ahammed, Golam J.; Li, Zhi-Xin; Zhang, Lan; Wei, Ji-Peng; Shen, Chen; Yan, Peng; Zhang, Li-Ping; Han, Wen-Yan

    2016-01-01

    Summer grown green tea is less popular due to bitterness and high astringency, which are attributed to high levels of tea polyphenols (TP) and low levels of amino acids (AA) in tea leaves (Camellia sinensis L.). Brassinosteroids (BRs), a group of steroidal plant hormones can regulate primary and secondary metabolism in a range of plant species under both normal and stress conditions. However, specific effects of BRs on the photosynthesis of tea plants and the quality of summer green tea are largely unknown. Here we show that 24-epibrassinolide (EBR), a bioactive BR, promoted photosynthesis in tea plants in a concentration-dependent manner. Stimulation in photosynthesis by EBR resulted in an increased summer tea yield. Although all tested concentrations (0.01, 0.05, 0.1, 0.5, and 1.0 ppm) of EBR increased concentrations of TP and AA, a moderate concentration (0.5 ppm) caused the highest decrease in TP to AA ratio, an important feature of quality tea. Time-course analysis using 0.5 ppm EBR as foliar spray revealed that TP or AA concentration increased as early as 3 h after EBR application, reaching the highest peak at 24 h and that remained more or less stable. Importantly, such changes in TP and AA concentration by EBR resulted in a remarkably decreased but stable TP to AA ratio at 24 h and onward. Furthermore, concentrations of catechins and theanine increased, while that of caffeine remained unaltered following treatment with EBR. EBR improved activity of phenylalanine ammonia-lyase (PAL) and glutamine: 2-oxoglutarate aminotransferase (GOGAT) enzymes involved in catechins and theanine biosynthesis, respectively. Transcript analysis revealed that transcript levels of CsPAL and CsGS peaked as early as 6 h, while that of CsGOGAT peaked at 12 h following application of EBR, implying that EBR increased the concentration of TP and AA by inducing their biosynthesis. These results suggest a positive role of BR in enhancing green tea quality, which might have potential

  9. Brassinosteroids Improve Quality of Summer Tea by Balancing Biosynthesis of Polyphenols and Amino Acids in Camellia sinensis L.

    Directory of Open Access Journals (Sweden)

    Xin Li

    2016-08-01

    Full Text Available Summer grown green tea is less popular due to bitterness and high astringency that are attributed to high levels of tea polyphenol (TP and low levels of amino acids (AA in tea leaves (Camellia sinensis L.. Brassinosteroids (BRs, a group of steroidal plant hormones can regulate primary and secondary metabolism in a range of plant species under both normal and stress conditions. However, specific effects of BRs on the photosynthesis of tea plants and the quality of summer green tea are largely unknown. Here we show that 24-epibrassinolide (EBR, a bioactive BR, promoted photosynthesis in tea plants in a concentration-dependent manner. Stimulation in photosynthesis by EBR resulted in an increased summer tea yield. Although all tested concentrations (0.01, 0.05, 0.1, 0.5 and 1.0 ppm increased concentrations of TP and AA, a moderate concentration of EBR (0.5 ppm caused the highest decrease in TP to AA ratio, an important feature of quality tea. Time-course analysis using 0.5 ppm EBR as foliar spray revealed that TP or AA concentration increased as early as 3 h after EBR application, reaching the highest peak at 24 h and that remained more or less stable. Importantly, such changes in TP and AA concentration by EBR resulted in a remarkably decreased but stable TP to AA ratio at 24 h and onward. Furthermore, concentrations of catechins and theanine increased, while that of caffeine remained unaltered following treatment with EBR. EBR improved activity of phenylalanine ammonia-lyase (PAL and glutamine: 2-oxoglutarate (GOGAT enzymes involved in catechins and theanine biosynthesis, respectively. Transcript analysis revealed that transcript levels of CsPAL and CsGS peaked as early as 6 h, while that of CsGOGAT peaked at 12 h following application of EBR, implying that EBR increased the concentration of TP and AA by inducing their biosynthesis. These results suggest a positive role of BR in enhancing green tea quality, which might have potential implication

  10. In silico analysis of amino acid biosynthesis and proteolysis in Lactobacillus delbrueckii subsp. bulgaricus 2038 and the implications for bovine milk fermentation.

    Science.gov (United States)

    Zheng, Huajun; Liu, Enuo; Hao, Pei; Konno, Tomonobu; Oda, Munehiro; Ji, Zai-Si

    2012-08-01

    The amino acid biosynthesis pathway and proteolytic system of Lactobacillus delbrueckii subsp. bulgaricus 2038 (L. bulgaricus 2038), a mainstay of large-scale yogurt production, were modeled based on its genomic sequence. L. bulgaricus 2038 retains more potential for amino acid synthesis and a more powerful proteolytic system than other L. bulgaricus strains, but favors amino acid uptake over de novo synthesis. Free amino acids and peptides in bovine milk provide the main nitrogen sources; whey is more important than casein for L. bulgaricus during fermentation. Free amino acids are imported by amino acid permeases and by ABC-type transport systems whereas exogenous oligopeptides are imported by ABC-type proteins only. Histidine is neither synthesized nor imported singly, which might explain why L. bulgaricus cannot grow in synthetic media.

  11. Amino acid metabolism of Astacus leptodactylus (Esch.)—II. Biosynthesis of the non-essential amino acids

    NARCIS (Netherlands)

    Marrewijk, Willibrordus J.A.; Zandee, Daniel I.

    1975-01-01

    1. 1. Incubation of Astacus leptodactylus with U-14C-glucose or 1-14C-acetate induced labelling of α- and β-alanine, aspartic and glutamic acids, glutamine, glycine, proline and serine. No radioactivity was incorporated into arginine, asparagine, histidine, isoleucine, leucine, lysine, ornithine, ph

  12. Compartmentation of hepatic fatty-acid-binding protein in liver cells and its effect on microsomal phosphatidic acid biosynthesis.

    Science.gov (United States)

    Bordewick, U; Heese, M; Börchers, T; Robenek, H; Spener, F

    1989-03-01

    Fatty-acid-binding proteins are known to occur in the cytosol of mammalian cells and to bind fatty acids and their CoA-esters. Application of the postembedding protein A-gold labeling method with antibody against the hepatic type fatty-acid-binding protein (hFABP) to cross-sections of liver cells and a newly developed gel-chromatographic immunofluorescence assay established qualitatively (1) that hFABP in mitochondria was confined to outer mitochondrial membranes, (2) the presence of this protein in microsomes and (3) that nuclei were also filled with hFABP. Quantitative data elaborated with a non-competitive ELISA confirmed these results. A significant difference to the distribution of cardiac FABP in heart muscle cells, where this type of protein was found in cytosol, matrix and nuclei, was observed (Börchers et al. (1989) Biochim. Biophys. Acta, in the press). hFABP-containing rat liver microsomes were incubated with long-chain acyl-CoAs in the presence of hFABP (isolated from rat liver cytosol) in a study on the acylation of sn-glycerol-3-phosphate and lysophosphatidic acid. Both acyltransferases were stimulated by addition of hFABP to the incubation medium. The morphological, immunochemical as well as kinetic data infer a direct interaction of hFABP with microsomal membranes in liver cells.

  13. Biosynthesis and actions of 5-oxoeicosatetraenoic acid (5-oxo-ETE) on feline granulocytes1

    OpenAIRE

    Cossette, Chantal; Gravel, Sylvie; Reddy, Chintam Nagendra; Gore, Vivek; Chourey, Shishir; Ye, Qiuji; Snyder, Nathaniel W.; Mesaros, Clementina A.; Ian A Blair; Lavoie, Jean-Pierre; Carol R Reinero; Rokach, Joshua; Powell, William S.

    2015-01-01

    The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most powerful human eosinophil chemoattractant among lipid mediators and could play a major pathophysiological role in eosinophilic diseases such as asthma. Its actions are mediated by the OXE receptor, orthologs of which are found in many species from humans to fish, but not rodents. The unavailability of rodent models to examine the pathophysiological roles of 5-oxo-ETE and the OXE receptor has substantially...

  14. Biosynthesis of Dictyostelium Discoideum Differentation-Inducing Factor by a Hybrid Type I Fatty Acid A-Type III polyketide synthase

    Energy Technology Data Exchange (ETDEWEB)

    Austin,M.; Saito, T.; Bowman, M.; Haydock, S.; Kato, A.; Moore, B.; Kay, R.; Noel, J.

    2006-01-01

    Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two {approx}3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.

  15. Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots.

    Science.gov (United States)

    Armendáriz, O; Gil-Monreal, M; Zulet, A; Zabalza, A; Royuela, M

    2016-05-01

    The objective of this work was to ascertain whether there is a general pattern of carbon allocation and utilisation in plants following herbicide supply, independent of the site of application: sprayed on leaves or supplied to nutrient solution. The herbicides studied were the amino acid biosynthesis-inhibiting herbicides (ABIH): glyphosate, an inhibitor of aromatic amino acid biosynthesis, and imazamox, an inhibitor of branched-chain amino acid biosynthesis. All treated plants showed impaired carbon metabolism; carbohydrate accumulation was detected in both leaves and roots of the treated plants. The accumulation in roots was due to lack of use of available sugars as growth was arrested, which elicited soluble carbohydrate accumulation in the leaves due to a decrease in sink strength. Under aerobic conditions, ethanol fermentative metabolism was enhanced in roots of the treated plants. This fermentative response was not related to a change in total respiration rates or cytochrome respiratory capacity, but an increase in alternative oxidase capacity was detected. Pyruvate accumulation was detected after most of the herbicide treatments. These results demonstrate that both ABIH induce the less-efficient, ATP-producing pathways, namely fermentation and alternative respiration, by increasing the key metabolite, pyruvate. The plant response was similar not only for the two ABIH but also after foliar or residual application.

  16. Conformational classification of functional nucleic acids.

    Science.gov (United States)

    Fujii, S

    1999-01-01

    The kink parameters would provide the tolerant aspect for irregular helical structure of nucleic acid. Using these kink parameters, the classification of conformation space was carried for the functional nucleic acid molecules. The kink parameters could afford us the simple structural aspects about the constructive parts of functional molecules. Local elastic kink phenomena can be classified by rod like models with the combination of kink parameters. The constructive parts, such as the stable tetra nucleotides loop, U-turn conformation and adenosine platform, were selected and the statistical analyses were carried on the parameters calculated by program BIOCON.

  17. Association with AflR in Endosomes Reveals New Functions for AflJ in Aflatoxin Biosynthesis

    Directory of Open Access Journals (Sweden)

    John E. Linz

    2012-12-01

    Full Text Available Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus ΔaflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export.

  18. Transcriptomic insight into terpenoid and carbazole alkaloid biosynthesis, and functional characterization of two terpene synthases in curry tree (Murraya koenigii)

    Science.gov (United States)

    Meena, Seema; Rajeev Kumar, Sarma; Dwivedi, Varun; Kumar Singh, Anup; Chanotiya, Chandan S.; Akhtar, Md. Qussen; Kumar, Krishna; Kumar Shasany, Ajit; Nagegowda, Dinesh A.

    2017-01-01

    Curry tree (Murraya koenigii L.) is a rich source of aromatic terpenes and pharmacologically important carbazole alkaloids. Here, M. koenigii leaf transcriptome was generated to gain insight into terpenoid and alkaloid biosynthesis. Analysis of de novo assembled contigs yielded genes for terpene backbone biosynthesis and terpene synthases. Also, gene families possibly involved in carbazole alkaloid formation were identified that included polyketide synthases, prenyltransferases, methyltransferases and cytochrome P450s. Further, two genes encoding terpene synthases (MkTPS1 and MkTPS2) with highest in silico transcript abundance were cloned and functionally characterized to determine their involvement in leaf volatile formation. Subcellular localization using GFP fusions revealed the plastidial and cytosolic localization of MkTPS1 and MkTPS2, respectively. Enzymatic characterization demonstrated the monoterpene synthase activity of recombinant MkTPS1, which produced primarily (−)-sabinene from geranyl diphosphate (GPP). Recombinant MkTPS2 exhibited sesquiterpene synthase activity and formed (E,E)-α-farnesene as the major product from farnesyl diphosphate (FPP). Moreover, mRNA expression and leaf volatile analyses indicated that MkTPS1 accounts for (−)-sabinene emitted by M. koenigii leaves. Overall, the transcriptome data generated in this study will be a great resource and the start point for characterizing genes involved in the biosynthetic pathway of medicinally important carbazole alkaloids. PMID:28272514

  19. Fermentation and alternative oxidase contribute to the action of amino acid biosynthesis-inhibiting herbicides.

    Science.gov (United States)

    Zulet, Amaia; Gil-Monreal, Miriam; Zabalza, Ana; van Dongen, Joost T; Royuela, Mercedes

    2015-03-01

    Acetolactate synthase inhibitors (ALS-inhibitors) and glyphosate (GLP) are two classes of herbicide that act by the specific inhibition of an enzyme in the biosynthetic pathway of branched-chain or aromatic amino acids, respectively. The physiological effects that are detected after application of these two classes of herbicides are not fully understood in relation to the primary biochemical target inhibition, although they have been well documented. Interestingly, the two herbicides' toxicity includes some common physiological effects suggesting that they kill the treated plants by a similar pattern despite targeting different enzymes. The induction of aerobic ethanol fermentation and alternative oxidase (AOX) are two examples of these common effects. The objective of this work was to gain further insight into the role of fermentation and AOX induction in the toxic consequences of ALS-inhibitors and GLP. For this, Arabidopsis T-DNA knockout mutants of alcohol dehydrogenase (ADH) 1 and AOX1a were used. The results found in wild-type indicate that both GLP and ALS-inhibitors reduce ATP production by inducing fermentation and alternative respiration. The main physiological effects in the process of herbicide activity upon treated plants were accumulation of carbohydrates and total free amino acids. The effects of the herbicides on these parameters were less pronounced in mutants compared to wild-type plants. The role of fermentation and AOX regarding pyruvate availability is also discussed.

  20. Microorganism mediated biosynthesis of metal chalcogenides; a powerful tool to transform toxic effluents into functional nanomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Vena, M. Paula; Jobbágy, Matías; Bilmes, Sara A., E-mail: sarabil@qi.fcen.uba.ar

    2016-09-15

    Cadmium contained in soil and water can be taken up by certain crops and aquatic organisms and accumulate in the food-chain, thus removal of Cd from mining or industrial effluents – i.e. Ni-Cd batteries, electroplating, pigments, fertilizers – becomes mandatory for human health. In parallel, there is an increased interest in the production of luminescent Q-dots for applications in bioimaging, sensors and electronic devices, even the present synthesis methods are economic and environmentally costly. An alternative green pathway for producing Metal chalcogenides (MC: CdS, CdSe, CdTe) nanocrystals is based on the metabolic activity of living organisms. Intracellular and extracellular biosynthesis of can be achieved within a biomimetic approach feeding living organisms with Cd precursors providing new routes for combining bioremediation with green routes for producing MC nanoparticles. In this mini-review we present the state-of-the-art of biosynthesis of MC nanoparticles with a critical discussion of parameters involved and protocols. Few existing examples of scaling-up are also discussed. A modular reactor based on microorganisms entrapped in biocompatible mineral matrices – already proven for bioremediation of dissolved dyes – is proposed for combining both Cd-depletion and MC nanoparticle's production. - Highlights: • Removal of heavy metals by living matter is feasible trough biosorption and bioaccumulation • Algae, fungi, bacteria and yeasts can synthesize CdS, CdSe and CdTe Q-dots • Encapsulation of microorganisms in mineral gels provides building blocks for reactor design. • Depletion of Cd with production of Q-dots can be achieved with modular bioreactors with entrapped cells.

  1. Caffeoylquinic Acids Biosynthesis and Accumulation in Cynara cardunculus: State of the Art

    NARCIS (Netherlands)

    Moglia, A.; Comino, C.; Menin, B.; Portis, E.; Aquadro, A.; Beekwilder, M.J.; Hehn, A.; Bourgaud, F.; Lanteri, S.

    2013-01-01

    Plant secondary metabolites are highly evolved compounds performing different functions, and have been widely exploited from food to medicine. A constant supply of phenols, a class of secondary metabolites, provides preventive and defensive mechanisms to reduce the risk of chronic diseases in human

  2. Cell wall structure and function in lactic acid bacteria.

    Science.gov (United States)

    Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

    2014-08-29

    The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.

  3. Suppression of Aflatoxin Biosynthesis in Aspergillus flavus by 2-Phenylethanol Is Associated with Stimulated Growth and Decreased Degradation of Branched-Chain Amino Acids.

    Science.gov (United States)

    Chang, Perng-Kuang; Hua, Sui Sheng T; Sarreal, Siov Bouy L; Li, Robert W

    2015-09-24

    The saprophytic soil fungus Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE), is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 μL/mL) using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO) analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids (valine, leucine and isoleucine) were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids OPEN ACCESS Toxins 2015, 7 3888 were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in

  4. Suppression of Aflatoxin Biosynthesis in Aspergillus flavus by 2-Phenylethanol Is Associated with Stimulated Growth and Decreased Degradation of Branched-Chain Amino Acids

    Directory of Open Access Journals (Sweden)

    Perng-Kuang Chang

    2015-09-01

    Full Text Available The saprophytic soil fungus Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE, is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 μL/mL using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids (valine, leucine and isoleucine were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in branched-chain amino

  5. Identification of a Δ5-like fatty acyl desaturase from the cephalopod Octopus vulgaris (Cuvier 1797) involved in the biosynthesis of essential fatty acids.

    Science.gov (United States)

    Monroig, Oscar; Navarro, Juan C; Dick, James R; Alemany, Frederic; Tocher, Douglas R

    2012-08-01

    Long-chain polyunsaturated fatty acids (LC-PUFA) have been identified as essential compounds for common octopus (Octopus vulgaris), but precise dietary requirements have not been determined due, in part, to the inherent difficulties of performing feeding trials on paralarvae. Our objective is to establish the essential fatty acid (EFA) requirements for paralarval stages of the common octopus through characterisation of the enzymes of endogenous LC-PUFA biosynthetic pathways. In this study, we isolated a cDNA with high homology to fatty acyl desaturases (Fad). Functional characterisation in recombinant yeast showed that the octopus Fad exhibited Δ5-desaturation activity towards saturated and polyunsaturated fatty acyl substrates. Thus, it efficiently converted the yeast's endogenous 16:0 and 18:0 to 16:1n-11 and 18:1n-13, respectively, and desaturated exogenously added PUFA substrates 20:4n-3 and 20:3n-6 to 20:5n-3 (EPA) and 20:4n-6 (ARA), respectively. Although the Δ5 Fad enables common octopus to produce EPA and ARA, the low availability of its adequate substrates 20:4n-3 and 20:3n-6, either in the diet or by limited endogenous synthesis from C(18) PUFA, might indicate that EPA and ARA are indeed EFA for this species. Interestingly, the octopus Δ5 Fad can also participate in the biosynthesis of non-methylene-interrupted FA, PUFA that are generally uncommon in vertebrates but have been found previously in marine invertebrates, including molluscs, and now also confirmed to be present in specific tissues of common octopus.

  6. An Acidic pH is a determinant factor for TRI genes expression and trichothecenes B biosynthesis in Fusarium graminearum

    OpenAIRE

    2010-01-01

    Abstract Reducing production of trichothecene B by Fusarium graminearum on cereals is necessary to avoid contamination leading to yields reduction and having harmful impacts on human and animal health. Understanding how trichothecenes biosynthesis is induced is essential. Effect of ambient pH on fungal growth, toxin biosynthesis and TRI genes expression was studied during in vitro liquid culture of F. graminearum on minimal medium. Fungal development stopped at day 3 after a sharp ...

  7. Multifunctional oxidosqualene cyclases and cytochrome P450involved in the biosynthesis of apple fruit triterpenic acids

    OpenAIRE

    Andre, Christelle; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; M.Cooney, Janine; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; A.Laing, William

    2016-01-01

    Summary Apple (Malus × domestica) accumulates bioactive ursane‐, oleanane‐, and lupane‐type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology‐based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with co...

  8. Biosynthesis and actions of 5-oxoeicosatetraenoic acid (5-oxo-ETE) on feline granulocytes.

    Science.gov (United States)

    Cossette, Chantal; Gravel, Sylvie; Reddy, Chintam Nagendra; Gore, Vivek; Chourey, Shishir; Ye, Qiuji; Snyder, Nathaniel W; Mesaros, Clementina A; Blair, Ian A; Lavoie, Jean-Pierre; Reinero, Carol R; Rokach, Joshua; Powell, William S

    2015-08-01

    The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most powerful human eosinophil chemoattractant among lipid mediators and could play a major pathophysiological role in eosinophilic diseases such as asthma. Its actions are mediated by the OXE receptor, orthologs of which are found in many species from humans to fish, but not rodents. The unavailability of rodent models to examine the pathophysiological roles of 5-oxo-ETE and the OXE receptor has substantially hampered progress in this area. As an alternative, we have explored the possibility that the cat could serve as an appropriate animal model to investigate the role of 5-oxo-ETE. We found that feline peripheral blood leukocytes synthesize 5-oxo-ETE and that physiologically relevant levels of 5-oxo-ETE are present in bronchoalveolar lavage fluid from cats with experimentally induced asthma. 5-Oxo-ETE (EC50, 0.7nM) is a much more potent activator of actin polymerization in feline eosinophils than various other eicosanoids, including leukotriene (LT) B4 and prostaglandin D2. 5-Oxo-ETE and LTB4 induce feline leukocyte migration to similar extents at low concentrations (1nM), but at higher concentrations the response to 5-oxo-ETE is much greater. Although high concentrations of selective human OXE receptor antagonists blocked 5-oxo-ETE-induced actin polymerization in feline granulocytes, their potencies were about 200 times lower than for human granulocytes. We conclude that feline leukocytes synthesize and respond to 5-oxo-ETE, which could potentially play an important role in feline asthma, a common condition in this species. The cat could serve as a useful animal model to investigate the pathophysiological role of 5-oxo-ETE.

  9. Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer.

    Science.gov (United States)

    Geng, Haifeng; Belas, Robert

    2010-09-01

    The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda(-) mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda(-) mutants-tdaA and tdaH failed to respond-by placing wild-type (Tda(+)) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

  10. Expression of Tropodithietic Acid Biosynthesis Is Controlled by a Novel Autoinducer▿ †

    Science.gov (United States)

    Geng, Haifeng; Belas, Robert

    2010-01-01

    The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda− mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda− mutants—tdaA and tdaH failed to respond—by placing wild-type (Tda+) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal. PMID:20601479

  11. Observation of an Acryloyl–Thiamin Diphosphate Adduct in the First Step of Clavulanic Acid Biosynthesis

    Science.gov (United States)

    Merski, Matthew

    2011-01-01

    The first committed biosynthetic step toward clavulanic acid, the clinically-important β-lactamase inhibitor, is catalyzed by the thiamin diphosphate (ThDP)-dependent enzyme N2-(2-carboxyethyl)arginine synthase (CEAS). This protein carries out a unique reaction among ThDP-dependent processes in which a C–N bond is formed, and an electrophilic acryloyl–thiazolium intermediate of ThDP is proposed to be involved, unlike the nucleophilic enamine species typically generated by this class of enzymes. Here we present evidence for the existence of the putative acryloyl adduct, and report the unexpected observation of a long-wavelength chromophore (λ = 433 nm), which we attribute to this enzyme bound species. Chemical models were synthesized that both confirm its expected absorption (λ = 310–320 nm), and exclude self-condensation and intramolecular imine formation with the cofactor as its cause. Circular dichroism experiments and others discount charge transfer as a likely explanation for the ~120 nm red shift of the chromophore (~25 kcal). Examples are well-known of charged molecules that exhibit significantly red-shifted UV-visible spectra compared to their neutral forms as, for example, polyene cations and dyes such as indigo and the cyanines. Rhodopsin is the classic biochemical example where the protein (opsin)-bound protonated Schiff base of retinal displays a remarkable range of red-shifted absorptions modulated by the protein environment. Similar tuning of the chromophoric behavior of the enzyme-bound CEAS acryloyl•ThDP species may be occurring. PMID:18052280

  12. Systems biology of lignin biosynthesis in Populus trichocarpa: heteromeric 4-coumaric acid:coenzyme A ligase protein complex formation, regulation, and numerical modeling.

    Science.gov (United States)

    Chen, Hsi-Chuan; Song, Jina; Wang, Jack P; Lin, Ying-Chung; Ducoste, Joel; Shuford, Christopher M; Liu, Jie; Li, Quanzi; Shi, Rui; Nepomuceno, Angelito; Isik, Fikret; Muddiman, David C; Williams, Cranos; Sederoff, Ronald R; Chiang, Vincent L

    2014-03-01

    As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein-protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.

  13. Chronic Over-expression of Fibroblast Growth Factor 21 Increases Bile Acid Biosynthesis by Opposing FGF15/19 Action

    Directory of Open Access Journals (Sweden)

    Jun Zhang

    2017-02-01

    Full Text Available Pharmacological doses of fibroblast growth factor (FGF 21 effectively normalize glucose, lipid and energy homeostasis in multiple animal models with many benefits translating to obese humans with type 2 diabetes. However, a role for FGF21 in the regulation of bile acid metabolism has not been reported. Herein, we demonstrate AAV-mediated FGF21 overexpression in mice increases liver expression of the key bile acid producing enzyme, Cyp7a1, resulting in an increased bile acid pool. Furthermore, in cholecystectomized mice, FGF21-mediated bile acid pool increase led to increased transit of bile acids into colon. We elucidate that the mechanism of FGF21 induced bile acid changes is mainly through antagonizing FGF15/19 function on liver βKlotho/FGFR4 receptor complex; thus inhibiting FGF15/19-mediated suppression of Cyp7a1 expression. In conclusion, these data reveal a previously unidentified role for FGF21 on bile acid metabolism and may be relevant to understand the effects of FGF21 analogs in clinical studies.

  14. NOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis and Rubisco formation in rice.

    Directory of Open Access Journals (Sweden)

    Qiaosong Yang

    Full Text Available NITRIC OXIDE-ASSOCIATED1 (NOA1 encodes a circularly permuted GTPase (cGTPase known to be essential for ribosome assembly in plants. While the reduced chlorophyll and Rubisco phenotypes were formerly noticed in both NOA1-suppressed rice and Arabidopsis, a detailed insight is still necessary. In this study, by using RNAi transgenic rice, we further demonstrate that NOA1 functions in a temperature-dependent manner to regulate chlorophyll and Rubisco levels. When plants were grown at 30°C, the chlorophyll and Rubisco levels in OsNOA1-silenced plants were only slightly lower than those in WT. However, at 22°C, the silenced plants accumulated far less chlorophyll and Rubisco than WT. It was further revealed that the regulation of chlorophyll and Rubisco occurs at the anabolic level. Etiolated WT seedlings restored chlorophyll and Rubisco accumulations readily once returned to light, at either 30°C or 15°C. Etiolated OsNOA1-silenced plants accumulated chlorophyll and Rubisco to normal levels only at 30°C, and lost this ability at low temperature. On the other hand, de-etiolated OsNOA1-silenced seedlings maintained similar levels of chlorophyll and Rubisco as WT, even after being shifted to 15°C for various times. Further expression analyses identified several candidate genes, including OsPorA (NADPH: protochlorophyllide oxidoreductase A, OsrbcL (Rubisco large subunit, OsRALyase (Ribosomal RNA apurinic site specific lyase and OsPuf4 (RNA-binding protein of the Puf family, which may be involved in OsNOA1-regulated chlorophyll biosynthesis and Rubisco formation. Overall, our results suggest OsNOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis, Rubisco formation and plastid development in rice.

  15. Members of the Penicillium chrysogenum velvet complex play functionally opposing roles in the regulation of penicillin biosynthesis and conidiation.

    Science.gov (United States)

    Kopke, Katarina; Hoff, Birgit; Bloemendal, Sandra; Katschorowski, Alexandra; Kamerewerd, Jens; Kück, Ulrich

    2013-02-01

    A velvet multisubunit complex was recently detected in the filamentous fungus Penicillium chrysogenum, the major industrial producer of the β-lactam antibiotic penicillin. Core components of this complex are P. chrysogenum VelA (PcVelA) and PcLaeA, which regulate secondary metabolite production, hyphal morphology, conidiation, and pellet formation. Here we describe the characterization of PcVelB, PcVelC, and PcVosA as novel subunits of this velvet complex. Using yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC), we demonstrate that all velvet proteins are part of an interaction network. Functional analyses using single- and double-knockout strains clearly indicate that velvet subunits have opposing roles in the regulation of penicillin biosynthesis and light-dependent conidiation. PcVelC, together with PcVelA and PcLaeA, activates penicillin biosynthesis, while PcVelB represses this process. In contrast, PcVelB and PcVosA promote conidiation, while PcVelC has an inhibitory effect. Our genetic analyses further show that light-dependent spore formation depends not only on PcVelA but also on PcVelB and PcVosA. The results provided here contribute to our fundamental understanding of the function of velvet subunits as part of a regulatory network mediating signals responsible for morphology and secondary metabolism and will be instrumental in generating mutants with newly derived properties that are relevant to strain improvement programs.

  16. Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions.

    Science.gov (United States)

    Luo, Chuping; Liu, Xuehui; Zhou, Huafei; Wang, Xiaoyu; Chen, Zhiyi

    2015-01-01

    Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.

  17. Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites: hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis.

    Science.gov (United States)

    Takamatsu, Satoshi; Xu, Lian-Hua; Fushinobu, Shinya; Shoun, Hirofumi; Komatsu, Mamoru; Cane, David E; Ikeda, Haruo

    2011-01-01

    Pentalenic acid (1) has been isolated from many Streptomyces sp. as a co-metabolite of the sesquiterpenoid antibiotic pentalenolactone and related natural products. We have previously reported the identification of a 13.4-kb gene cluster in the genome of Streptomyces avermitilis implicated in the biosynthesis of the pentalenolactone family of metabolites consisting of 13 open reading frames. Detailed molecular genetic and biochemical studies have revealed that at least seven genes are involved in the biosynthesis of the newly discovered metabolites, neopentalenoketolactone, but no gene specifically dedicated to the formation of pentalenic acid (1) was evident in the same gene cluster. The wild-type strain of S. avermitilis, as well as its derivatives, mainly produce pentalenic acid (1), together with neopentalenoketolactone (9). Disruption of the sav7469 gene encoding a cytochrome P450 (CYP105D7), members of which class are associated with the hydroxylation of many structurally different compounds, abolished the production of pentalenic acid (1). The sav7469-deletion mutant derived from SUKA11 carrying pKU462∷ptl-clusterΔptlH accumulated 1-deoxypentalenic acid (5), but not pentalenic acid (1). Reintroduction of an extra-copy of the sav7469 gene to SUKA11 Δsav7469 carrying pKU462∷ptl-clusterΔptlH restored the production of pentalenic acid (1). Recombinant CYP105D7 prepared from Escherichia coli catalyzed the oxidative conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1) in the presence of the electron-transport partners, ferredoxin (Fdx) and Fdx reductase, both in vivo and in vitro. These results unambiguously demonstrate that CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1), a shunt product in the biosynthesis of the pentalenolactone family of metabolites.

  18. Biosynthesis of the 22nd Genetically Encoded Amino Acid Pyrrolysine: Structure and Reaction Mechanism of PylC at 1.5Å Resolution

    KAUST Repository

    Quitterer, Felix

    2012-12-01

    The second step in the biosynthesis of the 22nd genetically encoded amino acid pyrrolysine (Pyl) is catalyzed by PylC that forms the pseudopeptide l-lysine-Nε-3R-methyl-d-ornithine. Here, we present six crystal structures of the monomeric active ligase in complex with substrates, reaction intermediates, and products including ATP, the non-hydrolyzable ATP analogue 5′-adenylyl-β-γ-imidodiphosphate, ADP, d-ornithine (d-Orn), l-lysine (Lys), phosphorylated d-Orn, l-lysine-Nε-d-ornithine, inorganic phosphate, carbonate, and Mg2 +. The overall structure of PylC reveals similarities to the superfamily of ATP-grasp enzymes; however, there exist unique structural and functional features for a topological control of successive substrate entry and product release. Furthermore, the presented high-resolution structures provide detailed insights into the reaction mechanism of isopeptide bond formation starting with phosphorylation of d-Orn by transfer of a phosphate moiety from activated ATP. The binding of Lys to the enzyme complex is then followed by an SN2 reaction resulting in l-lysine-Nε-d-ornithine and inorganic phosphate. Surprisingly, PylC harbors two adenine nucleotides bound at the active site, what has not been observed in any ATP-grasp protein analyzed to date. Whereas one ATP molecule is involved in catalysis, the second adenine nucleotide functions as a selective anchor for the C- and N-terminus of the Lys substrate and is responsible for protein stability as shown by mutagenesis. © 2012 Elsevier Ltd.

  19. Enzymology of the carnitine biosynthesis pathway.

    Science.gov (United States)

    Strijbis, Karin; Vaz, Frédéric M; Distel, Ben

    2010-05-01

    The water-soluble zwitterion carnitine is an essential metabolite in eukaryotes required for fatty acid oxidation as it functions as a carrier during transfer of activated acyl and acetyl groups across intracellular membranes. Most eukaryotes are able to synthesize carnitine endogenously, besides their capacity to take up carnitine from the diet or extracellular medium through plasma membrane transporters. This review discusses the current knowledge on carnitine homeostasis with special emphasis on the enzymology of the four steps of the carnitine biosynthesis pathway.

  20. The role of beta-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower.

    Science.gov (United States)

    González-Mellado, Damián; von Wettstein-Knowles, Penny; Garcés, Rafael; Martínez-Force, Enrique

    2010-05-01

    The beta-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.

  1. Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis.

    Science.gov (United States)

    Herrero, Joaquín; Fernández-Pérez, Francisco; Yebra, Tatiana; Novo-Uzal, Esther; Pomar, Federico; Pedreño, Ma Ángeles; Cuello, Juan; Guéra, Alfredo; Esteban-Carrasco, Alberto; Zapata, José Miguel

    2013-06-01

    Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in ΦPSII and q L in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.

  2. Aspergillus niger Enhance Bioactive Compounds Biosynthesis As Well As Expression of Functional Genes in Adventitious Roots of Glycyrrhiza uralensis Fisch.

    Science.gov (United States)

    Li, Jing; Wang, Juan; Li, Jinxin; Liu, Dahui; Li, Hongfa; Gao, Wenyuan; Li, Jianli; Liu, Shujie

    2016-02-01

    In the present study, the culture conditions for the accumulation of Glycyrrhiza uralensis adventitious root metabolites in balloon-type bubble bioreactors (BTBBs) have been optimized. The results of the culture showed that the best culture conditions were a cone angle of 90° bioreactor and 0.4-0.6-0.4-vvm aeration volume. Aspergillus niger can be used as a fungal elicitor to enhance the production of defense compounds in plants. With the addition of a fungal elicitor (derived from Aspergillus niger), the maximum accumulation of total flavonoids (16.12 mg g(-1)) and glycyrrhetinic acid (0.18 mg g(-1)) occurred at a dose of 400 mg L(-1) of Aspergillus niger resulting in a 3.47-fold and 1.8-fold increase over control roots. However, the highest concentration of polysaccharide (106.06 mg g(-1)) was achieved with a mixture of elicitors (Aspergillus niger and salicylic acid) added to the medium, resulting in a 1.09-fold increase over Aspergillus niger treatment alone. Electrospray ionization tandem mass spectrometry (ESI-MS(n)) analysis was performed, showing that seven compounds were present after treatment with the elicitors, including uralsaponin B, licorice saponin B2, liquiritin, and (3R)-vestitol, only identified in the mixed elicitor treatment group. It has also been found that elicitors (Aspergillus niger and salicylic acid) significantly upregulated the expression of the cinnamate 4-hydroxylase (C4H), β-amyrin synthase (β-AS), squalene epoxidase (SE) and a cytochrome P450 monooxygenase (CYP72A154) genes, which are involved in the biosynthesis of bioactive compounds, and increased superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity.

  3. Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica.

    Science.gov (United States)

    Chen, Jia-Wen; Liu, Wan-Jun; Hu, Dong-Xiong; Wang, Xiang; Balamurugan, Srinivasan; Alimujiang, Adili; Yang, Wei-Dong; Liu, Jie-Sheng; Li, Hong-Ye

    2016-08-30

    Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to lack of suitable industrial strain with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT-overexpressing N. oceanica showed higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. GC-MS analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of EPA (C20:5) increased by 8%, which is a polyunsaturated fatty acid necessary for animal nutrition. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential. This article is protected by copyright. All rights reserved.

  4. Imaging Functional Nucleic Acid Delivery to Skin.

    Science.gov (United States)

    Kaspar, Roger L; Hickerson, Robyn P; González-González, Emilio; Flores, Manuel A; Speaker, Tycho P; Rogers, Faye A; Milstone, Leonard M; Contag, Christopher H

    2016-01-01

    Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A wide range of technologies for traversing these barriers has been described and moderate success has been reported for several approaches. The lessons learned from these studies include the need for combinations of approaches to facilitate nucleic acid delivery across these skin barriers and then functional delivery across the cellular and nuclear membranes for expression (e.g., reporter genes, DNA oligonucleotides or shRNA) or into the cytoplasm for regulation (e.g., siRNA, miRNA, antisense oligos). The tools for topical delivery that have been evaluated include chemical, physical and electrical methods, and the development and testing of each of these approaches has been greatly enabled by imaging tools. These techniques allow delivery and real time monitoring of reporter genes, therapeutic nucleic acids and also triplex nucleic acids for gene editing. Optical imaging is comprised of a number of modalities based on properties of light-tissue interaction (e.g., scattering, autofluorescence, and reflectance), the interaction of light with specific molecules (e.g., absorbtion, fluorescence), or enzymatic reactions that produce light (bioluminescence). Optical imaging technologies operate over a range of scales from macroscopic to microscopic and if necessary, nanoscopic, and thus can be used to assess nucleic acid delivery to organs, regions, cells

  5. The ornibactin biosynthesis and transport genes of Burkholderia cenocepacia are regulated by an extracytoplasmic function sigma factor which is a part of the Fur regulon.

    Science.gov (United States)

    Agnoli, Kirsty; Lowe, Carolyn A; Farmer, Kate L; Husnain, Seyyed I; Thomas, Mark S

    2006-05-01

    Burkholderia cenocepacia mutants that fail to produce the siderophore ornibactin were obtained following mutagenesis with mini-Tn5Tp. These mutants were shown to be growth restricted under conditions of iron depletion. In eight of the mutants, the transposon had integrated into one of two genes, orbI and orbJ, encoding nonribosomal peptide synthetases. In the other mutant, the transposon had inserted into an open reading frame, orbS, located upstream from orbI. The polypeptide product of orbS exhibits a high degree of similarity to the Pseudomonas aeruginosa extracytoplasmic function (ECF) sigma factor PvdS but possesses an N-terminal extension of approximately 29 amino acids that is not present in PvdS. Three predicted OrbS-dependent promoters were identified within the ornibactin gene cluster, based on their similarity to PvdS-dependent promoters. The iron-regulated activity of these promoters was shown to require OrbS. Transcription of the orbS gene was found to be under the control of an iron-regulated sigma(70)-dependent promoter. This promoter, but not the OrbS-dependent promoters, was shown to be a target for repression by the global regulator Fur. Our results demonstrate that production of ornibactin by B. cenocepacia in response to iron starvation requires transcription of an operon that is dependent on the Fur-regulated ECF sigma factor gene orbS. A mechanism is also proposed for the biosynthesis of ornibactin.

  6. VisG is essential for biosynthesis of virginiamycin S, a streptogramin type B antibiotic, as a provider of the nonproteinogenic amino acid phenylglycine.

    Science.gov (United States)

    Ningsih, Fitria; Kitani, Shigeru; Fukushima, Eriko; Nihira, Takuya

    2011-11-01

    A streptogramin type B antibiotic, virginiamycin S (VS), is produced by Streptomyces virginiae, together with a streptogramin type A antibiotic, virginiamycin M1 (VM), as its synergistic counterpart. VS is a cyclic hexadepsipeptide containing a nonproteinogenic amino acid, Lphenylglycine (L-pheGly), in its core structure. We have identified, in the left-hand extremity of the virginiamycin supercluster, two genes that direct VS biosynthesis with L-pheGly incorporation. Transcriptional analysis revealed that visF, encoding a nonribosomal peptide synthetase, and visG, encoding a protein with homology to a hydroxyphenylacetyl-CoA dioxygenase, are under the transcriptional regulation of virginiae butanolide (VB), a small diffusing signalling molecule that governs virginiamycin production. Gene deletion of visG resulted in complete loss of VS production without any changes in VM production, suggesting that visG is required for VS biosynthesis. The abolished VS production in the visG disruptant was fully recovered either by the external addition of pheGly or by gene complementation, which indicates that VisG is involved in VS biosynthesis as the provider of an L-pheGly molecule. A feeding experiment with L-pheGly analogues suggested that VisF, which is responsible for the last condensation step, has high substrate specificity toward L-pheGly.

  7. Cloning, sequencing and function of sanA, a gene involved in nikkomycin biosynthesis of Streptomyces ansochromogenes

    Institute of Scientific and Technical Information of China (English)

    贾君永[1; 李文利[2; 陈蔚[3; 聂丽平[4; 谭华荣[5

    2000-01-01

    Several genetically stable mutants blocked in nikkomycin biosynthesis were obtained after the slightly germinated spores of Streptomyces ansochromogenes, a nikkomycin producer, were treated with ultra violet radiation. One of the mutants is the same in morpholotical differentiation as the wild type strain and is designated as NBB19. A DMA library was constructed using plasmid plJ702 as cloning vector, NBB19 as cloning recipient. A 6 kb DNA fragment which can genetically complement NBB19 was cloned when screening the library for antifungal activity. Sequence analysis showed that the 3 kb Bgl II-Sal I fragment contains one complete ORF (ORF1) and one partial ORF (ORF2). ORF1 is designated as sanA. sanA is 1 365 bp, encoding a protein consisting of 454 amino acid residues. Database searching indicated that sanA is homologous to the hypothetical methyltransferase in Pyrococcus horikoshli with 25% identities and 41% positives. Disruptant of sanA lost the ability to synthesize nikkomycin. It indicated that sa

  8. The role of ß-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

    DEFF Research Database (Denmark)

    González-Mellado, Damián; von Wettstein, Penelope Margaret; Garcés, Rafael;

    2010-01-01

    a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains....... proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons...

  9. Evidence that the Essential Response Regulator YycF in Streptococcus pneumoniae Modulates Expression of Fatty Acid Biosynthesis Genes and Alters Membrane Composition†

    Science.gov (United States)

    Mohedano, M. Luz; Overweg, Karin; de la Fuente, Alicia; Reuter, Mark; Altabe, Silvia; Mulholland, Francis; de Mendoza, Diego; López, Paloma; Wells, Jerry M.

    2005-01-01

    The YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism. PMID:15774879

  10. Transcription factors FabR and FadR regulate both aerobic and anaerobic pathways for unsaturated fatty acid biosynthesis in Shewanella oneidensis

    Directory of Open Access Journals (Sweden)

    Qixia eLuo

    2014-12-01

    Full Text Available As genes for type II fatty acid synthesis are essential to the growth of Escherichia coli, its sole (anaerobic pathway has significant potential as a target for novel antibacterial drug, and has been extensively studied. Despite this, we still know surprisingly little about fatty acid synthesis in bacteria because this anaerobic pathway in fact is not widely distributed. In this study, we show a novel model of unsaturated fatty acid (UFA synthesis in Shewanella, emerging human pathogens in addition to well-known metal reducers. We identify both anaerobic and aerobic UFA biosynthesis pathways in the representative species, S. oneidensis. Uniquely, the bacterium also contains two regulators FabR and FadR, whose counterparts in other bacteria control the anaerobic pathway. However, we show that in S. oneidensis these two regulators are involved in regulation of both pathways, in either direct or indirect manner. Overall, our results indicate that the UFA biosynthesis and its regulation are far more complex than previously expected, and S. oneidensis serves as a good research model for further work.

  11. COPPER AMINE OXIDASE1 (CuA01)of Arabidopsis thaliana Contributes to Abscisic Acid-and Polyamine-Induced Nitric Oxide Biosynthesis and Abscisic Acid Signal Transduction

    Institute of Scientific and Technical Information of China (English)

    Rinukshi Wimalasekera; Corina Villar; Tahmina Begum; Günther F. E. Scherer

    2011-01-01

    Polyamines (PA), polyamine oxidases, copper amine oxidases, and nitric oxide (NO)play important roles in physiology and stress responses in plants. NO biosynthesis as a result of catabolism of PA by polyamine oxidases and copper amine oxidases may explain in part PA-mediated responses. Involvement of a copper amine oxidase gene, COPPER AMINE OXIDASE1 (CuA01), of Arabidopsis was tested for its role in stress responses using the knockouts cuaol.1 and cuaol-2. PA-induced and ABA-induced NO production investigated by fluorometry and fluorescence microscopy showed that the cuaol-1 and cuaol-2 are impaired in NO production, suggesting a function of CuAO1 in PA and ABA-mediated NO production. Furthermore, we found a PA-dependent increase in protein S-nitrosylation. The addition of PA and ABA also resulted in HO increases, cuaol-1 and cuaol-2 showed less sensitivity to exogenous ABA supplementation during ger-mination, seedling establishment, and root growth inhibition as compared to wild-type. In response to ABA treatment,expression levels of the stress-responsive genes RD29A and ADH1 were significantly lower in the knockouts. These obser-vations characterize cuaol-1 and cuaol-2 as ABA-insensitive mutants. Taken together, our findings extend the ABA signal transduction network to include CuAO1 as one potential contributor to enhanced NO production by ABA.

  12. Calcium ionophore (A-23187 induced peritoneal eicosanoid biosynthesis: a rapid method to evaluate inhibitors of arachidonic acid metabolism in vivo

    Directory of Open Access Journals (Sweden)

    T. S. Rao

    1993-01-01

    Full Text Available The present investigation characterizes calcium ionophore (A-23187 induced peritoneal eicosanoid biosynthesis in the rat. Intraperitoneal injection of A-23187 (20 μg/rat stimulated marked biosynthesis of 6-keto-PGF1α (6-KPA, TxB2, LTC4 and LTB4, with no detectable changes on levels of PGE2. Levels of all eicosanoids decreased rapidly after a peak which was seen as early as 5 min. Enzyme markers of cellular contents of neutrophils and mononuclear cells, MPO and NAG respectively, decreased rapidly after ionophore injection; this was followed by increases after 60 min. Indomethacin, a selective cyclooxygenase inhibitor, and zileuton and ICI D-2138, two selective 5-lipoxygenase inhibitors attenuated prostaglandin and leukotriene pathways respectively. Oral administration of zileuton (20 mg/kg, p.o. inhibited LTB4 biosynthesis for up to 6 h suggesting a long duration of pharmacological activity in the rats consistent with its longer half-life. The rapid onset and the magnitude of increases in levels of eicosanoids render the ionophore induced peritoneal eicosanoid biosynthesis a useful model to evaluate pharmacological profiles of inhibitors of eicosanoid pathways in vivo.

  13. Peptides in the nervous systems of cnidarians: structure, function, and biosynthesis

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Leviev, I; Carstensen, Kathrine

    1996-01-01

    molecule. In addition to well-known, "classical" processing enzymes, novel prohormone processing enzymes must be present in cnidarian neurons. These include a processing enzyme hydrolyzing at the C-terminal sides of acidic (Asp and Glu) residues and a dipeptidyl aminopeptidase digesting at the C......-terminal sides of N-terminally located X-Pro and X-Ala sequences. All this shows that the primitive nervous systems of cnidarians are already quite complex, and that neuropeptides play a central role in the physiology of these animals....

  14. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.

    Science.gov (United States)

    Neshat, Armin; Mentz, Almut; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts.

  15. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.

    Directory of Open Access Journals (Sweden)

    Jie Chen

    Full Text Available BACKGROUND: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1 and membrane-bound (Tre-2 trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. PRINCIPAL FINDINGS: The membrane-bound trehalase of Spodoptera exigua (SeTre-2 was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1 and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. CONCLUSIONS: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2

  16. Influence of dietary docosahexaenoic acid in combination with other long-chain polyunsaturated fatty acids on expression of biosynthesis genes and phospholipid fatty acid compositions in tissues of post-smolt Atlantic salmon (Salmo salar).

    Science.gov (United States)

    Betancor, Mónica B; Howarth, Fraser J E; Glencross, Brett D; Tocher, Douglas R

    2014-01-01

    To investigate interactions of dietary LC-PUFA, a dose-response study with a range of docosahexaenoic acid (DHA; 22:6n-3) levels (1 g kg(-1), 5 g kg(-1), 10 g kg(-1), 15 g kg(-1) and 20 g kg(-1)) was performed with post-smolts (111 ± 2.6g; mean ± S.D.) over a nine-week feeding period. Additional diets included 10 g kg(-1) DHA in combination with 10 g kg(-1) of either eicosapentaenoic acid (EPA; 20:5n-3) or arachidonic acid (ARA; 20:4n-6), and a diet containing 5 g kg(-1) each of DHA and EPA. The liver, brain, head kidney and gill were collected at the conclusion of the trial, and lipid and fatty acid compositions were determined as well as expression of genes of LC-PUFA biosynthesis. Total lipid content and class composition were largely unaffected by changes in dietary LC-PUFA. However, phospholipid (PL) fatty acid compositions generally reflected that of the diet, although the response varied between tissues. The liver most strongly reflected diet, followed by the head kidney. In both tissues increasing dietary DHA led to significantly increased DHA in PL and inclusion of EPA or ARA led to higher levels of these fatty acids. The brain showed the most conserved composition and gene expression profile, with increased dietary LC-PUFA resulting in only minor changes in PL fatty acids. Dietary LC-PUFA significantly affected the expression of Δ6 and Δ5 desaturases, Elovl 2, 4 and 5, and SREBPs although this varied between tissues with greatest effects observed in the liver followed by the head kidney, similar to PL fatty acid compositions.

  17. Acylphloroglucinol Biosynthesis in Strawberry Fruit.

    Science.gov (United States)

    Song, Chuankui; Ring, Ludwig; Hoffmann, Thomas; Huang, Fong-Chin; Slovin, Janet; Schwab, Wilfried

    2015-11-01

    Phenolics have health-promoting properties and are a major group of metabolites in fruit crops. Through reverse genetic analysis of the functions of four ripening-related genes in the octoploid strawberry (Fragaria × ananassa), we discovered four acylphloroglucinol (APG)-glucosides as native Fragaria spp. fruit metabolites whose levels were differently regulated in the transgenic fruits. The biosynthesis of the APG aglycones was investigated by examination of the enzymatic properties of three recombinant Fragaria vesca chalcone synthase (FvCHS) proteins. CHS is involved in anthocyanin biosynthesis during ripening. The F. vesca enzymes readily catalyzed the condensation of two intermediates in branched-chain amino acid metabolism, isovaleryl-Coenzyme A (CoA) and isobutyryl-CoA, with three molecules of malonyl-CoA to form phlorisovalerophenone and phlorisobutyrophenone, respectively, and formed naringenin chalcone when 4-coumaroyl-CoA was used as starter molecule. Isovaleryl-CoA was the preferred starter substrate of FvCHS2-1. Suppression of CHS activity in both transient and stable CHS-silenced fruit resulted in a substantial decrease of APG glucosides and anthocyanins and enhanced levels of volatiles derived from branched-chain amino acids. The proposed APG pathway was confirmed by feeding isotopically labeled amino acids. Thus, Fragaria spp. plants have the capacity to synthesize pharmaceutically important APGs using dual functional CHS/(phloriso)valerophenone synthases that are expressed during fruit ripening. Duplication and adaptive evolution of CHS is the most probable scenario and might be generally applicable to other plants. The results highlight that important promiscuous gene function may be missed when annotation relies solely on in silico analysis.

  18. Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond.

    Science.gov (United States)

    Niwa, Ryusuke; Niwa, Yuko S

    2014-01-01

    Steroid hormones are responsible for the coordinated regulation of many aspects of biological processes in multicellular organisms. Since the last century, many studies have identified and characterized steroidogenic enzymes in vertebrates, including mammals. However, much less is known about invertebrate steroidogenic enzymes. In the last 15 years, a number of steroidogenic enzymes and their functions have been characterized in ecdysozoan animals, especially in the fruit fly Drosophila melanogaster. In this review, we summarize the latest knowledge of enzymes crucial for synthesizing ecdysteroids, the principal insect steroid hormones. We also discuss the functional conservation and diversity of ecdysteroidogenic enzymes in other insects and even non-insect species, such as nematodes, vertebrates, and lower eukaryotes.

  19. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.

    OpenAIRE

    Berlemont, Renaud; Delsaute, Maud; Pipers, Delphine; D'Amico, Salvino; Feller, Georges; Galleni, Moreno; Power, Pablo

    2009-01-01

    In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carb...

  20. Biosynthesis of biphenyls and benzophenones--evolution of benzoic acid-specific type III polyketide synthases in plants.

    Science.gov (United States)

    Beerhues, Ludger; Liu, Benye

    2009-01-01

    Type III polyketide synthases (PKSs) generate a diverse array of secondary metabolites by varying the starter substrate, the number of condensation reactions, and the mechanism of ring closure. Among the starter substrates used, benzoyl-CoA is a rare starter molecule. Biphenyl synthase (BIS) and benzophenone synthase (BPS) catalyze the formation of identical linear tetraketide intermediates from benzoyl-CoA and three molecules of malonyl-CoA but use alternative intramolecular cyclization reactions to form 3,5-dihydroxybiphenyl and 2,4,6-trihydroxybenzophenone, respectively. In a phylogenetic tree, BIS and BPS group together closely, indicating that they arise from a relatively recent functional diversification of a common ancestral gene. The functionally diverse PKSs, which include BIS and BPS, and the ubiquitously distributed chalcone synthases (CHSs) form separate clusters, which originate from a gene duplication event prior to the speciation of the angiosperms. BIS is the key enzyme of biphenyl metabolism. Biphenyls and the related dibenzofurans are the phytoalexins of the Maloideae. This subfamily of the Rosaceae includes a number of economically important fruit trees, such as apple and pear. When incubated with ortho-hydroxybenzoyl (salicoyl)-CoA, BIS catalyzes a single decarboxylative condensation with malonyl-CoA to form 4-hydroxycoumarin. A well-known anticoagulant derivative of this enzymatic product is dicoumarol. Elicitor-treated cell cultures of Sorbus aucuparia also formed 4-hydroxycoumarin when fed with the N-acetylcysteamine thioester of salicylic acid (salicoyl-NAC). BPS is the key enzyme of benzophenone metabolism. Polyprenylated benzophenone derivatives with bridged polycyclic skeletons are widely distributed in the Clusiaceae (Guttiferae). Xanthones are regioselectively cyclized benzophenone derivatives. BPS was converted into a functional phenylpyrone synthase (PPS) by a single amino acid substitution in the initiation/elongation cavity. The

  1. Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L. ) cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Kuehnl, T.K.; Koch, U.; Heller, W.; Wellmann, E.

    1987-10-01

    Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsible for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

  2. Aflatoxin biosynthesis: current frontiers.

    Science.gov (United States)

    Roze, Ludmila V; Hong, Sung-Yong; Linz, John E

    2013-01-01

    Aflatoxins are among the principal mycotoxins that contaminate economically important food and feed crops. Aflatoxin B1 is the most potent naturally occurring carcinogen known and is also an immunosuppressant. Occurrence of aflatoxins in crops has vast economic and human health impacts worldwide. Thus, the study of aflatoxin biosynthesis has become a focal point in attempts to reduce human exposure to aflatoxins. This review highlights recent advances in the field of aflatoxin biosynthesis and explores the functional connection between aflatoxin biosynthesis, endomembrane trafficking, and response to oxidative stress. Dissection of the regulatory mechanisms involves a complete comprehension of the aflatoxin biosynthetic process and the dynamic network of transcription factors that orchestrates coordinated expression of the target genes. Despite advancements in the field, development of a safe and effective multifaceted approach to solve the aflatoxin food contamination problem is still required.

  3. The function and catalysis of 2-oxoglutarate-dependent oxygenases involved in plant flavonoid biosynthesis.

    Science.gov (United States)

    Cheng, Ai-Xia; Han, Xiao-Juan; Wu, Yi-Feng; Lou, Hong-Xiang

    2014-01-15

    Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism. They fulfil a variety of functions in plants and have health benefits for humans. During the synthesis of the tricyclic flavonoid natural products in plants, oxidative modifications to the central C ring are catalyzed by four of FeII and 2-oxoglutarate dependent (2-ODD) oxygenases, namely flavone synthase I (FNS I), flavonol synthase (FLS), anthocyanidin synthase (ANS) and flavanone 3β-hydroxylase (FHT). FNS I, FLS and ANS are involved in desaturation of C2-C3 of flavonoids and FHT in hydroxylation of C3. FNS I, which is restricted to the Apiaceae species and in rice, is predicted to have evolved from FHT by duplication. Due to their sequence similarity and substrate specificity, FLS and ANS, which interact with the α surface of the substrate, belong to a group of dioxygenases having a broad substrate specificity, while FNS I and FHT are more selective, and interact with the naringenin β surface. Here, we summarize recent findings regarding the function of the four 2-ODD oxygenases and the relationship between their catalytic activity, their polypeptide sequence and their tertiary structure.

  4. The Function and Catalysis of 2-Oxoglutarate-Dependent Oxygenases Involved in Plant Flavonoid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Ai-Xia Cheng

    2014-01-01

    Full Text Available Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism. They fulfil a variety of functions in plants and have health benefits for humans. During the synthesis of the tricyclic flavonoid natural products in plants, oxidative modifications to the central C ring are catalyzed by four of FeII and 2-oxoglutarate dependent (2-ODD oxygenases, namely flavone synthase I (FNS I, flavonol synthase (FLS, anthocyanidin synthase (ANS and flavanone 3β-hydroxylase (FHT. FNS I, FLS and ANS are involved in desaturation of C2–C3 of flavonoids and FHT in hydroxylation of C3. FNS I, which is restricted to the Apiaceae species and in rice, is predicted to have evolved from FHT by duplication. Due to their sequence similarity and substrate specificity, FLS and ANS, which interact with the α surface of the substrate, belong to a group of dioxygenases having a broad substrate specificity, while FNS I and FHT are more selective, and interact with the naringenin β surface. Here, we summarize recent findings regarding the function of the four 2-ODD oxygenases and the relationship between their catalytic activity, their polypeptide sequence and their tertiary structure.

  5. Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans

    Science.gov (United States)

    Blanchette, Cassandra R.; Thackeray, Andrea; Perrat, Paola N.; Hekimi, Siegfried; Bénard, Claire Y.

    2017-01-01

    The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development. PMID:28068429

  6. Amino acids: metabolism, functions, and nutrition.

    Science.gov (United States)

    Wu, Guoyao

    2009-05-01

    Recent years have witnessed the discovery that amino acids (AA) are not only cell signaling molecules but are also regulators of gene expression and the protein phosphorylation cascade. Additionally, AA are key precursors for syntheses of hormones and low-molecular weight nitrogenous substances with each having enormous biological importance. Physiological concentrations of AA and their metabolites (e.g., nitric oxide, polyamines, glutathione, taurine, thyroid hormones, and serotonin) are required for the functions. However, elevated levels of AA and their products (e.g., ammonia, homocysteine, and asymmetric dimethylarginine) are pathogenic factors for neurological disorders, oxidative stress, and cardiovascular disease. Thus, an optimal balance among AA in the diet and circulation is crucial for whole body homeostasis. There is growing recognition that besides their role as building blocks of proteins and polypeptides, some AA regulate key metabolic pathways that are necessary for maintenance, growth, reproduction, and immunity. They are called functional AA, which include arginine, cysteine, glutamine, leucine, proline, and tryptophan. Dietary supplementation with one or a mixture of these AA may be beneficial for (1) ameliorating health problems at various stages of the life cycle (e.g., fetal growth restriction, neonatal morbidity and mortality, weaning-associated intestinal dysfunction and wasting syndrome, obesity, diabetes, cardiovascular disease, the metabolic syndrome, and infertility); (2) optimizing efficiency of metabolic transformations to enhance muscle growth, milk production, egg and meat quality and athletic performance, while preventing excess fat deposition and reducing adiposity. Thus, AA have important functions in both nutrition and health.

  7. Functional analysis of metabolic channeling and regulation in lignin biosynthesis: a computational approach.

    Directory of Open Access Journals (Sweden)

    Yun Lee

    Full Text Available Lignin is a polymer in secondary cell walls of plants that is known to have negative impacts on forage digestibility, pulping efficiency, and sugar release from cellulosic biomass. While targeted modifications of different lignin biosynthetic enzymes have permitted the generation of transgenic plants with desirable traits, such as improved digestibility or reduced recalcitrance to saccharification, some of the engineered plants exhibit monomer compositions that are clearly at odds with the expected outcomes when the biosynthetic pathway is perturbed. In Medicago, such discrepancies were partly reconciled by the recent finding that certain biosynthetic enzymes may be spatially organized into two independent channels for the synthesis of guaiacyl (G and syringyl (S lignin monomers. Nevertheless, the mechanistic details, as well as the biological function of these interactions, remain unclear. To decipher the working principles of this and similar control mechanisms, we propose and employ here a novel computational approach that permits an expedient and exhaustive assessment of hundreds of minimal designs that could arise in vivo. Interestingly, this comparative analysis not only helps distinguish two most parsimonious mechanisms of crosstalk between the two channels by formulating a targeted and readily testable hypothesis, but also suggests that the G lignin-specific channel is more important for proper functioning than the S lignin-specific channel. While the proposed strategy of analysis in this article is tightly focused on lignin synthesis, it is likely to be of similar utility in extracting unbiased information in a variety of situations, where the spatial organization of molecular components is critical for coordinating the flow of cellular information, and where initially various control designs seem equally valid.

  8. Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L.

    Science.gov (United States)

    Du, L; Lykkesfeldt, J; Olsen, C E; Halkier, B A

    1995-01-01

    An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combined use of an improved isolation medium and jasmonic acid-induced etiolated seedlings of Sinapis alba L. An 8-fold induction of de novo biosynthesis of the L-tyrosine-derived p-hydroxybenzylglucosinolate was obtained in etiolated S. alba seedlings upon treatment with jasmonic acid. Formation of inhibitory glucosinolate degradation products upon tissue homogenization was prevented by inactivation of myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. The biosynthetically active microsomal enzyme system converted L-tyrosine into p-hydroxyphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The Km and Vmax values of the enzyme system were 346 microM and 538 pmol per mg of protein per h, respectively. The nature of the enzyme catalyzing the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 by photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous cytochrome P450 inhibitors confirms the involvement of cytochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glycosides. Images Fig. 1 Fig. 2 Fig. 4 PMID:8618930

  9. ABA biosynthesis defective mutants reduce some free amino acids accumulation under drought stress in tomato leaves in comparison with Arabidopsis plants tissues

    Directory of Open Access Journals (Sweden)

    Adnan Ali Al.Asbahi

    2012-05-01

    Full Text Available The ability of plants to tolerate drought conditions is crucial for plant survival and crop production worldwide. The present data confirm previous findings reported existence of a strong relation between abscisic acid (ABA content and amino acid accumulation as response water stress which is one of the most important defense mechanism activated during water stress in many plant species. Therefore, free amino acids were measured to determine any changes in the metabolite pool in relation to ABA content. The ABA defective mutants of Arabidopsis plants were subjected to leaf dehydration for Arabidopsis on Whatman 3 mm filter paper at room temperature while, tomato mutant plants were subjected to drought stresses for tomato plants by withholding water. To understand the signal transduction mechanisms underlying osmotic stress-regulating gene induction and activation of osmoprotectant free amino acid synthesizing genes, we carried out a genetic screen to isolate Arabidopsis mutants defective in ABA biosynthesis under drought stress conditions. The present results revealed an accumulation of specific free amino acid in water stressed tissues in which majority of free amino acids are increased especially those playing an osmoprotectant role such as proline and glycine. Drought stress related Amino acids contents are significantly reduced in the mutants under water stress condition while they are increased significantly in the wild types plants. The exhibited higher accumulation of other amino acids under stressed condition in the mutant plants suggest that, their expressions are regulated in an ABA independent pathways. In addition, free amino acids content changes during water stress condition suggest their contribution in drought toleration as common compatible osmolytes.

  10. A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon.

    Science.gov (United States)

    Goh, Yong Jun; Klaenhammer, Todd R

    2013-09-01

    Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log-phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.

  11. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.

    Science.gov (United States)

    Berlemont, Renaud; Delsaute, Maud; Pipers, Delphine; D'Amico, Salvino; Feller, Georges; Galleni, Moreno; Power, Pablo

    2009-09-01

    In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.

  12. Effects of heavy metals and light levels on the biosynthesis of carotenoids and fatty acids in the macroalgae Gracilaria tenuistipitata (var. liui Zhang & Xia

    Directory of Open Access Journals (Sweden)

    Ernani Pinto

    2011-04-01

    Full Text Available We present here the effect of heavy metals and of different light intensities on the biosynthesis of fatty acids and pigments in the macroalga Gracilaria tenuistipitata (var. liui Zhang & Xia. In order to verify the fatty acid content, gas chromatography with flame ionization detection (GC-FID was employed. Pigments (major carotenoids and chlorophyl-a were monitored by liquid chromatography with diode array detection (HPLC-DAD. Cultures of G. tenuistipitata were exposed to cadmium (Cd2+, 200 ppb and copper (Cu2+, 200 ppb, as well as to different light conditions (low light: 100 µmol.photons.m-2.s-1, or high light: 1000 µmol.photons.m-2.s-1. Cd2+ and Cu2+ increased the saturated and monounsaturated fatty acid content [14:0, 16:0, 18:0, 18:1 (n-7 and 18:1 (n-9] and all major pigments (violaxanthin, antheraxanthin, lutein, zeaxanthin, chlorophyll-a and β-carotene. Both heavy metals decreased the levels of polyunsaturated fatty acids (PUFA [18:2 (n-6, 18:3 (n-6, 18:5 (n-4, 20:4 (n-6, 20:5 (n-3, 22:6 (n-3]. G. tenuistipitata cultures were exposed to high light intensity for five days and no statistically significant differences were observed in the content of fatty acids. On the other hand, the levels of pigments rose markedly for chlorophyll-a and all of the carotenoids studied.

  13. Functional characterization of enzymes involved in cysteine biosynthesis and H(2)S production in Trypanosoma cruzi.

    Science.gov (United States)

    Marciano, Daniela; Santana, Marianela; Nowicki, Cristina

    2012-10-01

    Trypanosoma cruzi is expected to synthetize de novo cysteine by different routes, among which the two-step pathway involving serine acetyltransferase and cysteine synthase (CS) is comprised. Also, cystathionine β synthase (CBS) might contribute to the de novo generation of cysteine in addition to catalyze the first step of the reverse transsulfuration route producing cystathionine. However, neither the functionality of CS nor that of cystathionine γ lyase (CGL) has been assessed. Our results show that T. cruzi CS could participate notably more actively than CBS in the de novo synthesis of cysteine. Interestingly, at the protein level T. cruzi CS is more abundant in amastigotes than in epimastigotes. Unlike the mammalian homologues, T. cruzi CGL specifically cleaves cystathionine into cysteine and is unable to produce H(2)S. The expression pattern of T. cruzi CGL parallels that of CBS, which unexpectedly suggests that in addition to the de novo synthesis of cysteine, the reverse transsulfuration pathway could be operative in the mammalian and insect stages. Besides, T. cruzi CBS produces H(2)S by decomposing cysteine or via condensation of cysteine with homocysteine. The latter reaction leads to cystathionine production, and is catalyzed remarkably more efficiently than the breakdown of cysteine. In T. cruzi like in other organisms, H(2)S could exert regulatory effects on varied metabolic processes. Notably, T. cruzi seems to count on stage-specific routes involved in cysteine production, the multiple cysteine-processing alternatives could presumably reflect this parasite's high needs of reducing power for detoxification of reactive oxygen species.

  14. Biosynthesis of gold and silver nanoparticles by natural precursor clove and their functionalization with amine group

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Ashwani Kumar; Talat, Mahe [Banaras Hindu University, Nanoscience and Nanotechnology Unit, Department of Physics (India); Singh, D. P. [Southern Illinois University Carbondale, Department of Physics (United States); Srivastava, O. N., E-mail: hepons@yahoo.co [Banaras Hindu University, Nanoscience and Nanotechnology Unit, Department of Physics (India)

    2010-06-15

    We report a simple and cost effective way for synthesis of metallic nanoparticles (Au and Ag) using natural precursor clove. Au and Ag nanoparticles have been synthesized by reducing the aqueous solution of AuCl{sub 4} and AgNO{sub 3} with clove extract. One interesting aspect here is that reduction time is quite small (few minutes instead of hours as compared to other natural precursors). We synthesized gold and silver nanoparticles of different shape and size by varying the ratio of AuCl{sub 4} and AgNO{sub 3} with respect to clove extract, where the dominant component is eugenol. The evolution of Au and Ag nanoparticles from the reduction of different ratios of AuCl{sub 4} and AgNO{sub 3} with optimised concentration of the clove extract has been evaluated through monitoring of surface plasmon behaviour as a function of time. The reduction of AuCl{sub 4} and AgNO{sub 3} by eugenol is because of the inductive effect of methoxy and allyl groups which are present at ortho and para positions of proton releasing -OH group as two electrons are released from one molecule of eugenol. This is followed by the formation of resonating structure of the anionic form of eugenol. The presence of methoxy and allyl groups has been confirmed by FTIR. To the best of our knowledge, use of clove as reducing agent, the consequent very short time (minutes instead of hours and without any scavenger) and the elucidation of mechanism of reduction based on FTIR analysis has not been attempted earlier.

  15. A wheat R2R3-MYB protein PURPLE PLANT1 (TaPL1) functions as a positive regulator of anthocyanin biosynthesis.

    Science.gov (United States)

    Shin, Dong Ho; Choi, Myoung-Goo; Kang, Chon-Sik; Park, Chul-Soo; Choi, Sang-Bong; Park, Youn-Il

    2016-01-15

    Transcriptional activation of anthocyanin biosynthesis genes in vegetative tissues of monocotyledonous plants is mediated by cooperative activity of one component from each of the following two transcription factor families: MYB encoded by PURPLE PLANT1/COLORED ALEURONE1 (PL1/C1), and basic helix-loop-helix (bHLH) encoded by RED/BOOSTER (R1/B1). In the present study, putative PL cDNA was cloned from the wheat (Triticum aestivum) cultivar Iksan370, which preferentially expresses anthocyanins in coleoptiles. Phylogenetic tree analysis of deduced amino acid sequences showed that a putative TaPL1 is highly homologous to barley (Hordeum vulgare) HvPL1, but is distinct from wheat TaC1. Transgenic Arabidopsis thaliana stably expressing putative TaPL1 accumulated anthocyanin pigments in leaves and up-regulated structural genes involved in both early and late anthocyanin biosynthesis steps. TaPL1 transcript levels in Iksan370 were more prominent in vegetative tissues such as young coleoptiles than in reproductive tissues such as spikelets. TaPL1 expression was significantly up-regulated by environmental stresses including cold, salt, and light, which are known to induce anthocyanin accumulation. These combined results suggest that TaPL1 is an active positive regulator of anthocyanin biosynthesis in wheat coleoptiles.

  16. Functional identiifcation of phenazine biosynthesis genes in plant pathogenic bacteriaPseudomonas syringae pv.tomato and Xanthomonas oryzaepv.oryzae

    Institute of Scientific and Technical Information of China (English)

    LI Wen; XU You-ping; Jean-Pierre Munyampundu; XU Xin; QI Xian-fei; GU Yuan; CAI Xin-zhong

    2016-01-01

    Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens. In this study, we identiifed phenazine biosynthesis (phz) genes in two genome-completed plant pathogenic bacteriaPseudomonas syringae pv.tomato(Pst) DC3000 andXanthomonas oryzaepv.oryzae(Xoo) PXO99A. Unlike the phz genes in typical phenazine-producing pseudomonads,phz homologs inPst DC3000 andXoo PXO99A consisted of phzC/D/E/F/G andphzC/E1/E2/F/G, respectively, and the both were not organized into an operon. Detection experiments demonstrated that phenazine-1-carboxylic acid (PCA) ofPst DC3000 accumulated to 13.4 μg L–1, while that ofXoo PXO99A was almost undetectable. Moreover,Pst DC3000 was resistant to 1 mg mL–1 PCA, whileXoo PXO99A was sensitive to 50 μg mL–1 PCA. Furthermore, mutation ofphzF blocked the PCA production and signiifcantly reduced the pathogenicity ofPst DC3000 in tomato, while the complementary strains restored these phenotypes. These results revealed thatPst DC3000 produces low level of and is resistant to phenazines and thus is unable to be biologicaly controled by phenazines. Additionaly,phz-mediated PCA production is required for ful pathogenicity ofPst DC3000. To our knowledge, this is the ifrst report of PCA production and its function in pathogenicity of a plant pathogenicP. syringaestrain.

  17. Supplementation with linoleic acid-rich soybean oil stimulates macrophage foam cell formation via increased oxidative stress and diacylglycerol acyltransferase1-mediated triglyceride biosynthesis.

    Science.gov (United States)

    Rom, Oren; Jeries, Helana; Hayek, Tony; Aviram, Michael

    2017-01-02

    During the last decades there has been a staggering rise in human consumption of soybean oil (SO) and its major polyunsaturated fatty acid linoleic acid (LA). The role of SO or LA in cardiovascular diseases is highly controversial, and their impact on macrophage foam cell formation, the hallmark of early atherogenesis, is unclear. To investigate the effects of high SO or LA intake on macrophage lipid metabolism and the related mechanisms of action, C57BL/6 mice were orally supplemented with increasing levels of SO-based emulsion or equivalent levels of purified LA for 1 month, followed by analyses of lipid accumulation and peroxidation in aortas, serum and in peritoneal macrophages (MPM) of the mice. Lipid peroxidation and triglyceride mass in aortas from SO or LA supplemented mice were dose-dependently and significantly increased. In MPM from SO or LA supplemented mice, lipid peroxides were significantly increased and a marked accumulation of cellular triglycerides was found in accordance with enhanced triglyceride biosynthesis rate and overexpression of diacylglycerol acyltransferase1 (DGAT1), the key enzyme in triglyceride biosynthesis. In cultured J774A.1 macrophages treated with SO or LA, triglyceride accumulated via increased oxidative stress and a p38 mitogen-activated protein kinase (MAPK)-mediated overexpression of DGAT1. Accordingly, anti-oxidants (pomegranate polyphenols), inhibition of p38 MAPK (by SB202190) or DGAT1 (by oleanolic acid), all significantly attenuated SO or LA-induced macrophage triglyceride accumulation. These findings reveal novel mechanisms by which supplementation with SO or LA stimulate macrophage foam cell formation, suggesting a pro-atherogenic role for overconsumption of SO or LA. © 2016 BioFactors, 43(1):100-116, 2017.

  18. Acid-functionalized polyolefin materials and their use in acid-promoted chemical reactions

    Energy Technology Data Exchange (ETDEWEB)

    Oyola, Yatsandra; Tian, Chengcheng; Bauer, John Christopher; Dai, Sheng

    2016-06-07

    An acid-functionalized polyolefin material that can be used as an acid catalyst in a wide range of acid-promoted chemical reactions, wherein the acid-functionalized polyolefin material includes a polyolefin backbone on which acid groups are appended. Also described is a method for the preparation of the acid catalyst in which a precursor polyolefin is subjected to ionizing radiation (e.g., electron beam irradiation) of sufficient power and the irradiated precursor polyolefin reacted with at least one vinyl monomer having an acid group thereon. Further described is a method for conducting an acid-promoted chemical reaction, wherein an acid-reactive organic precursor is contacted in liquid form with a solid heterogeneous acid catalyst comprising a polyolefin backbone of at least 1 micron in one dimension and having carboxylic acid groups and either sulfonic acid or phosphoric acid groups appended thereto.

  19. Functional amino acids in nutrition and health.

    Science.gov (United States)

    Wu, Guoyao

    2013-09-01

    The recent years have witnessed growing interest in biochemistry, physiology and nutrition of amino acids (AA) in growth, health and disease of humans and other animals. This results from the discoveries of AA in cell signaling involving protein kinases, G protein-coupled receptors, and gaseous molecules (i.e., NO, CO and H2S). In addition, nutritional studies have shown that dietary supplementation with several AA (e.g., arginine, glutamine, glutamate, leucine, and proline) modulates gene expression, enhances growth of the small intestine and skeletal muscle, or reduces excessive body fat. These seminal findings led to the new concept of functional AA, which are defined as those AA that participate in and regulate key metabolic pathways to improve health, survival, growth, development, lactation, and reproduction of the organisms. Functional AA hold great promise in prevention and treatment of metabolic diseases (e.g., obesity, diabetes, and cardiovascular disorders), intrauterine growth restriction, infertility, intestinal and neurological dysfunction, and infectious disease (including viral infections).

  20. Current topics in the biotechnological production of essential amino acids, functional amino acids, and dipeptides.

    Science.gov (United States)

    Mitsuhashi, Satoshi

    2014-04-01

    Amino acids play important roles in both human and animal nutrition and in the maintenance of health. Here, amino acids are classified into three groups: first, essential amino acids, which are essential to nutrition; second, functional amino acids, recently found to be important in the promotion of physiological functions; and third, dipeptides, which are used to resolve problematic features of specific free amino acids, such as their instability or insolubility. This review focusses on recent researches concerning the microbial production of essential amino acids (lysine and methionine), functional amino acids (histidine and ornithine), and a dipeptide (L-alanyl-L-glutamine).

  1. Metabolic engineering of Rhizopus oryzae: Effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose

    Science.gov (United States)

    Fumaric acid, a dicarboxylic acid used as a food acidulant and in manufacturing synthetic resins, can be produced from glucose in fermentation by Rhizopus oryzae. However, the fumaric acid yield is limited by the co-production of ethanol and other byproducts. To increase fumaric acid production, ove...

  2. Inducing Effect of Dihydroartemisinic Acid in the Biosynthesis of Artemisinins with Cultured Cells of Artemisia annua by Enhancing the Expression of Genes

    Directory of Open Access Journals (Sweden)

    Jianhua Zhu

    2014-01-01

    Full Text Available Artemisinin has been used in the production of “artemisinin combination therapies” for the treatment of malaria. Feeding of precursors has been proven to be one of the most effective methods to enhance artemisinin production in plant cultured cells. At the current paper, the biosynthesis of artemisinin (ART and its four analogs from dihydroartemisinic acid (DHAA in suspension-cultured cells of Artemisia annua were investigated. ARTs were detected by HPLC/GC-MS and isolated by various chromatography methods. The structures of four DHAA metabolites, namely, dihydro-epi-deoxyarteannuin B, arteannuin I, arteannuin K, and 3-β-hydroxy-dihydro-epi-deoxyarteannuin B, were elucidated by physicochemical and spectroscopic analyses. The correlation between gene expression and ART content was investigated. The results of RT-PCR showed that DHAA could up-regulate expression of amorpha-4,11-diene synthase gene (ADS, amorpha-4,11-diene C-12 oxidase gene (CYP71AV1, and farnesyl diphosphate synthase gene (FPS (3.19-, 7.21-, and 2.04-fold higher than those of control group, resp., which indicated that biosynthesis processes from DHAA to ART were enzyme-mediated.

  3. De novo sequencing and transcriptome analysis of Pinellia ternata identify the candidate genes involved in the biosynthesis of benzoic acid and ephedrine

    Directory of Open Access Journals (Sweden)

    Zhang Guang Hui

    2016-08-01

    Full Text Available Background: The medicinal herb, Pinellia ternate, is purported to be an anti-emetic with analgesic and sedative effects. Alkaloids are the main biologically active compounds in P. ternata, especially ephedrine that is a phenylpropylamino alkaloid specifically produced by Ephedra and Catha edulis. However, how ephedrine is synthesized in plants is uncertain. Only the phenylalanine ammonia lyase (PAL and relevant genes in this pathway have been characterized. Genomic information of P. ternata is also unavailable. Results: We analyzed the transcriptome of the tuber of P. ternata with the Illumina HiSeqTM 2000 sequencing platform. 66,813,052 high-quality reads were generated, and these reads were assembled de novo into 89,068 unigenes. Most known genes involved in benzoic acid biosynthesis were identified in the unigene dataset of P. ternate, and the expression patterns of some ephedrine biosynthesis-related genes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR. Also, 14,468 simple sequence repeats (SSRs were identified from 12,000 unigenes. Twenty primer pairs for SSRs were randomly selected for the validation of their amplification effect. Conclusion: RNA-seq data was firstly used to provide a comprehensive gene information on P. ternata at the transcriptional level. These data will advance molecular genetics in this valuable medicinal plant.

  4. Transcriptomic analysis of genes involved in the biosynthesis, recycling and degradation of L-ascorbic acid in pepper fruits (Capsicum annuum L.).

    Science.gov (United States)

    Alós, Enriqueta; Rodrigo, María J; Zacarías, Lorenzo

    2013-06-01

    Sweet pepper (Capsicum annuum L.) is widely recognized among the vegetables with high content of ascorbic acid (AsA). However, the metabolic pathways involved in the biosynthesis, recycling and degradation of AsA and their relative contribution to the concentration of AsA have not been established yet. In the present work, the expression levels of selected genes involved in the AsA biosynthesis, degradation and recycling pathways were analyzed during development and ripening of pepper fruit cv. Palermo and in mature fruit of four cultivars (Lipari, C-116, Surrentino and Italverde) with different AsA concentrations. An inverse correlation was found between the expression of the biosynthetic genes and AsA concentrations, which could indicate that a feedback mechanism regulates AsA homeostasis in pepper fruits. Interestingly, analysis of mRNA levels of ascorbate oxidase, involved in the degradation of AsA, suggests that this enzyme plays a critical role in the regulation of the AsA pool during fruit development and ripening.

  5. Progress on Abscisic Acid Biosynthesis and Signal Transduction in Higher Plants%高等植物脱落酸生物合成及其信号转导研究进展

    Institute of Scientific and Technical Information of China (English)

    杨洪强; 接玉玲

    2001-01-01

    绍介了近年来高等植物体脱落酸(ABA)生物合成缺陷型及反应敏感性突变体、逆境胁迫下ABA的合成、ABA生物合成途径以及ABA信号的细胞识别与转导等几方面的研究进展。%Abscisic acid(ABA) plays a major role in plant growth and development, as well as in adaptation to environmental stress. The research of abscisic acid biosynthesis and signal transduction is advancing rapidly in recent years. In this article, it is reviewed that the ABA-biosynthetic mutants and insensitive or ypersensitivive mutants, ABA biosynthesis under stress, the pathway of ABA biosynthesis, ABA signal perception and transduction.

  6. Indole-3-acetic acid (IAA) biosynthesis in the smut fungus Ustilago maydis and its relevance for increased IAA levels in infected tissue and host tumour formation.

    Science.gov (United States)

    Reineke, Gavin; Heinze, Bernadette; Schirawski, Jan; Buettner, Hermann; Kahmann, Regine; Basse, Christoph W

    2008-05-01

    Infection of maize (Zea mays) plants with the smut fungus Ustilago maydis is characterized by excessive host tumour formation. U. maydis is able to produce indole-3-acetic acid (IAA) efficiently from tryptophan. To assess a possible connection to the induction of host tumours, we investigated the pathways leading to fungal IAA biosynthesis. Besides the previously identified iad1 gene, we identified a second indole-3-acetaldehyde dehydrogenase gene, iad2. Deltaiad1Deltaiad2 mutants were blocked in the conversion of both indole-3-acetaldehyde and tryptamine to IAA, although the reduction in IAA formation from tryptophan was not significantly different from Deltaiad1 mutants. To assess an influence of indole-3-pyruvic acid on IAA formation, we deleted the aromatic amino acid aminotransferase genes tam1 and tam2 in Deltaiad1Deltaiad2 mutants. This revealed a further reduction in IAA levels by five- and tenfold in mutant strains harbouring theDeltatam1 andDeltatam1Deltatam2 deletions, respectively. This illustrates that indole-3-pyruvic acid serves as an efficient precursor for IAA formation in U. maydis. Interestingly, the rise in host IAA levels upon U. maydis infection was significantly reduced in tissue infected with Deltaiad1Deltaiad2Deltatam1 orDeltaiad1Deltaiad2Deltatam1Deltatam2 mutants, whereas induction of tumours was not compromised. Together, these results indicate that fungal IAA production critically contributes to IAA levels in infected tissue, but this is apparently not important for triggering host tumour formation.

  7. A functional gene cluster for toxoflavin biosynthesis in the genome of the soil bacterium Pseudomonas protegens Pf-5

    Science.gov (United States)

    Toxoflavin is a broad-spectrum toxin best known for its role in virulence of Burkholderia glumae, which causes panicle blight of rice. A gene cluster containing homologs of toxoflavin biosynthesis genes (toxA-E) of B. glumae is present in the genome of Pseudomonas protegens Pf-5, a biological contr...

  8. In vitro selection of functional nucleic acids

    Science.gov (United States)

    Wilson, D. S.; Szostak, J. W.

    1999-01-01

    In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

  9. The Arabidopsis male-sterile mutant dde2-2 is defective in the ALLENE OXIDE SYNTHASE gene encoding one of the key enzymes of the jasmonic acid biosynthesis pathway

    DEFF Research Database (Denmark)

    von Malek, Bernadette; van der Graaff, Eric; Schneitz, Kay;

    2002-01-01

    exhibits a male-sterile phenotype. The dde2-2 phenotype can be rescued by application of methyl jasmonate, indicating that the mutant is affected in jasmonic acid biosynthesis. The combination of genetic mapping and a candidate-gene approach identified a frameshift mutation in the ALLENE OXIDE SYNTHASE...

  10. Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use.

    Science.gov (United States)

    Yoshida, Kiyohito; Hashimoto, Mikako; Hori, Ryuji; Adachi, Takumi; Okuyama, Hidetoshi; Orikasa, Yoshitake; Nagamine, Tadashi; Shimizu, Satoru; Ueno, Akio; Morita, Naoki

    2016-05-12

    The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

  11. Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use

    Directory of Open Access Journals (Sweden)

    Kiyohito Yoshida

    2016-05-01

    Full Text Available The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase, the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

  12. First discovery of two polyketide synthase genes for mitorubrinic acid and mitorubrinol yellow pigment biosynthesis and implications in virulence of Penicillium marneffei.

    Directory of Open Access Journals (Sweden)

    Patrick C Y Woo

    Full Text Available BACKGROUND: The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes. METHODOLOGY/PRINCIPAL FINDINGS: All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05. There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05. CONCLUSIONS/SIGNIFICANCE: The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid

  13. SIFT: predicting amino acid changes that affect protein function

    OpenAIRE

    Ng, Pauline C.; Henikoff, Steven

    2003-01-01

    Single nucleotide polymorphism (SNP) studies and random mutagenesis projects identify amino acid substitutions in protein-coding regions. Each substitution has the potential to affect protein function. SIFT (Sorting Intolerant From Tolerant) is a program that predicts whether an amino acid substitution affects protein function so that users can prioritize substitutions for further study. We have shown that SIFT can distinguish between functionally neutral and deleterious amino acid changes in...

  14. Inhibition of retinoic acid biosynthesis by the bisdichloroacetyldiamine WIN 18,446 markedly suppresses spermatogenesis and alters retinoid metabolism in mice.

    Science.gov (United States)

    Paik, Jisun; Haenisch, Michael; Muller, Charles H; Goldstein, Alex S; Arnold, Samuel; Isoherranen, Nina; Brabb, Thea; Treuting, Piper M; Amory, John K

    2014-05-23

    Knowledge of the regulation of testicular retinoic acid synthesis is crucial for understanding its role in spermatogenesis. Bisdichloroacetyldiamines strongly inhibit spermatogenesis. We reported previously that one of these compounds, WIN 18,446, potently inhibited spermatogenesis in rabbits by inhibiting retinoic acid synthesis. To understand how WIN 18,446 inhibits retinoic acid synthesis, we characterized its effects on human retinal dehydrogenase ALDH1A2 in vitro as well as its effects on retinoid metabolism in vivo using mice. WIN 18,446 strongly and irreversibly inhibited ALDH1A2 in vitro. In vivo, WIN 18,446 treatment completely abolished spermatogenesis after 4 weeks of treatment and modestly reduced adiposity in mice fed a chow diet. Effects of WIN 18,446 on retinoid concentrations were tissue-dependent. Although lung and liver retinyl ester concentrations were lower in WIN 18,446-treated animals, adipose retinyl ester levels were increased following the treatment. Interestingly, animals treated with WIN 18,446 had significantly higher circulating retinol concentrations compared with control mice. The effect on spermatogenesis by WIN 18,446 was not prevented by simultaneous treatment with retinoic acid, whereas effects on other tissues were partially or completely reversed. Cessation of WIN 18,446 treatment for 4 weeks reversed most retinoid-related phenotypes except for inhibition of spermatogenesis. Our data suggest that WIN 18,446 may be a useful model of systemic acquired retinoic acid deficiency. Given the effects observed in our study, inhibition of retinoic acid biosynthesis may have relevance for the treatment of obesity and in the development of novel male contraceptives.

  15. The occurrence and biosynthesis of gamma-linolenic acid in a blue-green alga,Spirulina platensis.

    Science.gov (United States)

    Nichols, B W; Wood, B J

    1968-01-01

    The acyl-lipid and fatty acid composition of six blue-green algae, namely,Spirulina platensis, Myxosarcina chroococcoides, Chlorogloea fritschii, Anabaena cylindrica, Anabaena flos-aquae, and Mastigocladus laminosus is reported.All contain major proportions of mono-and digalactosyl diglyceride, sulfoquinovosyl diglyceride, and phosphatidyl glycerol, but none possess lecithin, phophatidyl ethanolamine, or phosphatidyl inositol. Trans-3-hexadecenoic acid was absent from all extracts.The analyses provide further evidence that there is no general chemical or physical requirement for any specific fatty acid in photosynthesis. S. platensis is unique among photoautotrophic organisms so far studied, containing major quantities of gamma-linolenic acid (6,9,12-octadecatrienoic acid). This acid is synthesized by the alga by direct desaturation of linoleic acid and is primarily located in the mono- and digalactosyl diglyceride fractions.The possible phylogenetic relationship betweenS. platensis and other plant forms is discussed.

  16. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis

    Directory of Open Access Journals (Sweden)

    Wen-Bin Xue

    2016-07-01

    Full Text Available The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA. To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively.

  17. Functional characterization of Dihydroflavonol-4-reductase in anthocyanin biosynthesis of purple sweet potato underlies the direct evidence of anthocyanins function against abiotic stresses.

    Science.gov (United States)

    Wang, Hongxia; Fan, Weijuan; Li, Hong; Yang, Jun; Huang, Jirong; Zhang, Peng

    2013-01-01

    Dihydroflavonol-4-reductase (DFR) is a key enzyme in the catalysis of the stereospecific reduction of dihydroflavonols to leucoanthocyanidins in anthocyanin biosynthesis. In the purple sweet potato (Ipomoea batatas Lam.) cv. Ayamurasaki, expression of the IbDFR gene was strongly associated with anthocyanin accumulation in leaves, stems and roots. Overexpression of the IbDFR in Arabidopsis tt3 mutants fully complemented the pigmentation phenotype of the seed coat, cotyledon and hypocotyl. Downregulation of IbDFR expression in transgenic sweet potato (DFRi) using an RNAi approach dramatically reduced anthocyanin accumulation in young leaves, stems and storage roots. In contrast, the increase of flavonols quercetin-3-O-hexose-hexoside and quercetin-3-O-glucoside in the leaves and roots of DFRi plants is significant. Therefore, the metabolic pathway channeled greater flavonol influx in the DFRi plants when their anthocyanin and proanthocyanidin accumulation were decreased. These plants also displayed reduced antioxidant capacity compared to the wild type. After 24 h of cold treatment and 2 h recovery, the wild-type plants were almost fully restored to the initial phenotype compared to the slower recovery of DFRi plants, in which the levels of electrolyte leakage and hydrogen peroxide accumulation were dramatically increased. These results provide direct evidence of anthocyanins function in the protection against oxidative stress in the sweet potato. The molecular characterization of the IbDFR gene in the sweet potato not only confirms its important roles in flavonoid metabolism but also supports the protective function of anthocyanins of enhanced scavenging of reactive oxygen radicals in plants under stressful conditions.

  18. BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS.

    Science.gov (United States)

    Creelman, Robert A.; Mullet, John E.

    1997-06-01

    Jasmonic acid and its derivatives can modulate aspects of fruit ripening, production of viable pollen, root growth, tendril coiling, and plant resistance to insects and pathogens. Jasmonate activates genes involved in pathogen and insect resistance, and genes encoding vegetative storage proteins, but represses genes encoding proteins involved in photosynthesis. Jasmonic acid is derived from linolenic acid, and most of the enzymes in the biosynthetic pathway have been extensively characterized. Modulation of lipoxygenase and allene oxide synthase gene expression in transgenic plants raises new questions about the compartmentation of the biosynthetic pathway and its regulation. The activation of jasmonic acid biosynthesis by cell wall elicitors, the peptide systemin, and other compounds will be related to the function of jasmonates in plants. Jasmonate modulates gene expression at the level of translation, RNA processing, and transcription. Promoter elements that mediate responses to jasmonate have been isolated. This review covers recent advances in our understanding of how jasmonate biosynthesis is regulated and relates this information to knowledge of jasmonate modulated gene expression.

  19. Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine.

    Science.gov (United States)

    Bontpart, Thibaut; Marlin, Thérèse; Vialet, Sandrine; Guiraud, Jean-Luc; Pinasseau, Lucie; Meudec, Emmanuelle; Sommerer, Nicolas; Cheynier, Véronique; Terrier, Nancy

    2016-05-01

    In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, β-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites.

  20. Effect of gibberellic acid and calliterpenone on plant growth attributes, trichomes, essential oil biosynthesis and pathway gene expression in differential manner in Mentha arvensis L.

    Science.gov (United States)

    Bose, Subir K; Yadav, Ritesh Kumar; Mishra, Smrati; Sangwan, Rajender S; Singh, A K; Mishra, B; Srivastava, A K; Sangwan, Neelam S

    2013-05-01

    Extensive research is going on throughout the world to find out new molecules from natural sources to be used as plant growth promoter. Mentha arvensis L. is the main source of menthol rich essential oil used commercially in various food, pharmaceutical and other preparations. Experiments were conducted on field grown plants for understanding the effect of calliterpenone (CA), a stereo-isomer of abbeokutone, in comparison to gibberellic acid (GA3) on growth attributes, trichomes, essential oil biosynthesis and expression of some oil biosynthetic pathway genes. The exogenous application of CA (1 μM, 10 μM and 100 μM) was found to be better in improving plant biomass and stolon yield, leaf area, branching and leaf stem ratio than with counterpart GA3 at the same concentrations. CA treated plants showed higher glandular trichome number, density and diameter and also correlated with enhanced oil biogenetic capacity as revealed by feeding labeled (14)C-sucrose for 72 h to excised shoots. Semi-quantitative PCR analysis of key pathway genes revealed differential up regulation under CA treatments. Transcript level of menthol dehydrogenase/menthone reductase was found highly up regulated in CA treated plants with increased content of menthone and menthol in oil. These findings demonstrate that CA positively regulated the yields by enhanced branching and higher density of trichomes resulting into higher accumulation of essential oil. The results suggest CA as a novel plant derived diterpenoid with growth promoting action and opens up new possibilities for improving the crop yields and essential oil biosynthesis in qualitative and quantitative manner.

  1. The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase.

    Science.gov (United States)

    Ishikawa, Takahiro; Nishikawa, Hitoshi; Gao, Youngshun; Sawa, Yoshihiro; Shibata, Hitoshi; Yabuta, Yukinori; Maruta, Takanori; Shigeoka, Shigeru

    2008-11-07

    We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.

  2. Omega-3 fatty acids and cognitive function in women

    OpenAIRE

    Robinson, Jennifer G.; Ijioma, Nkechinyere; Harris, William

    2010-01-01

    Omega-3 fatty acids (FAs) could play an important role in maintaining cognitive function in aging individuals. The omega-3 FA docosahexaenoic acid is a major constituent of neuronal membranes and, along with the other long-chain omega-3 FAs from fish such as eicosapentaentoic acid, has been shown to have a wide variety of beneficial effects on neuronal functioning, inflammation, oxidation and cell death, as well as on the development of the characteristic pathology of Alzheimer’s disease. Ome...

  3. Caffeic Acid Expands Anti-Tumor Effect of Metformin in Human Metastatic Cervical Carcinoma HTB-34 Cells: Implications of AMPK Activation and Impairment of Fatty Acids De Novo Biosynthesis

    Science.gov (United States)

    Tyszka-Czochara, Malgorzata; Konieczny, Pawel; Majka, Marcin

    2017-01-01

    The efficacy of cancer treatments is often limited and associated with substantial toxicity. Appropriate combination of drug targeting specific mechanisms may regulate metabolism of tumor cells to reduce cancer cell growth and to improve survival. Therefore, we investigated the effects of anti-diabetic drug Metformin (Met) and a natural compound caffeic acid (trans-3,4-dihydroxycinnamic acid, CA) alone and in combination to treat an aggressive metastatic human cervical HTB-34 (ATCC CRL­1550) cancer cell line. CA at concentration of 100 µM, unlike Met at 10 mM, activated 5'-adenosine monophosphate-activated protein kinase (AMPK). What is more, CA contributed to the fueling of mitochondrial tricarboxylic acids (TCA) cycle with pyruvate by increasing Pyruvate Dehydrogenase Complex (PDH) activity, while Met promoted glucose catabolism to lactate. Met downregulated expression of enzymes of fatty acid de novo synthesis, such as ATP Citrate Lyase (ACLY), Fatty Acid Synthase (FAS), Fatty Acyl-CoA Elongase 6 (ELOVL6), and Stearoyl-CoA Desaturase-1 (SCD1) in cancer cells. In conclusion, CA mediated reprogramming of glucose processing through TCA cycle via oxidative decarboxylation. The increased oxidative stress, as a result of CA treatment, sensitized cancer cells and, acting on cell biosynthesis and bioenergetics, made HTB-34 cells more susceptible to Met and successfully inhibited neoplastic cells. The combination of Metformin and caffeic acid to suppress cervical carcinoma cells by two independent mechanisms may provide a promising approach to cancer treatment. PMID:28230778

  4. Amino Acid Catabolism in Plants.

    Science.gov (United States)

    Hildebrandt, Tatjana M; Nunes Nesi, Adriano; Araújo, Wagner L; Braun, Hans-Peter

    2015-11-02

    Amino acids have various prominent functions in plants. Besides their usage during protein biosynthesis, they also represent building blocks for several other biosynthesis pathways and play pivotal roles during signaling processes as well as in plant stress response. In general, pool sizes of the 20 amino acids differ strongly and change dynamically depending on the developmental and physiological state of the plant cell. Besides amino acid biosynthesis, which has already been investigated in great detail, the catabolism of amino acids is of central importance for adjusting their pool sizes but so far has drawn much less attention. The degradation of amino acids can also contribute substantially to the energy state of plant cells under certain physiological conditions, e.g. carbon starvation. In this review, we discuss the biological role of amino acid catabolism and summarize current knowledge on amino acid degradation pathways and their regulation in the context of plant cell physiology.

  5. Cyclic Comonomers for the Synthesis of Carboxylic Acid and Amine Functionalized Poly(l-Lactic Acid

    Directory of Open Access Journals (Sweden)

    Markus Heiny

    2015-03-01

    Full Text Available Degradable aliphatic polyesters such as poly(lactic acid are widely used in biomedical applications, however, they lack functional moieties along the polymer backbone that are amenable for functionalization reactions or could be the basis for interactions with biological systems. Here we present a straightforward route for the synthesis of functional α-ω epoxyesters as comonomers for lactide polymerization. Salient features of these highly functionalized epoxides are versatility in functionality and a short synthetic route of less than four steps. The α-ω epoxyesters presented serve as a means to introduce carboxylic acid and amine functional groups into poly(lactic acid polymers via ring-opening copolymerization.

  6. Dairy Streptococcus thermophilus improves cell viability of Lactobacillus brevis NPS-QW-145 and its γ-aminobutyric acid biosynthesis ability in milk.

    Science.gov (United States)

    Wu, Qinglong; Law, Yee-Song; Shah, Nagendra P

    2015-08-06

    Most high γ-aminobutyric acid (GABA) producers are Lactobacillus brevis of plant origin, which may be not able to ferment milk well due to its poor proteolytic nature as evidenced by the absence of genes encoding extracellular proteinases in its genome. In the present study, two glutamic acid decarboxylase (GAD) genes, gadA and gadB, were found in high GABA-producing L. brevis NPS-QW-145. Co-culturing of this organism with conventional dairy starters was carried out to manufacture GABA-rich fermented milk. It was observed that all the selected strains of Streptococcus thermophilus, but not Lactobacillus delbrueckii subsp. bulgaricus, improved the viability of L. brevis NPS-QW-145 in milk. Only certain strains of S. thermophilus improved the gadA mRNA level in L. brevis NPS-QW-145, thus enhanced GABA biosynthesis by the latter. These results suggest that certain S. thermophilus strains are highly recommended to co-culture with high GABA producer for manufacturing GABA-rich fermented milk.

  7. L-Ascorbic Acid: A Multifunctional Molecule Supporting Plant Growth and Development

    OpenAIRE

    2013-01-01

    L-Ascorbic acid (vitamin C) is as essential to plants as it is to animals. Ascorbic acid functions as a major redox buffer and as a cofactor for enzymes involved in regulating photosynthesis, hormone biosynthesis, and regenerating other antioxidants. Ascorbic acid regulates cell division and growth and is involved in signal transduction. In contrast to the single pathway responsible for ascorbic acid biosynthesis in animals, plants use multiple pathways to synthesize ascorbic acid, perhaps re...

  8. Transcellular biosynthesis of eicosanoid lipid mediators.

    Science.gov (United States)

    Capra, Valérie; Rovati, G Enrico; Mangano, Paolo; Buccellati, Carola; Murphy, Robert C; Sala, Angelo

    2015-04-01

    The synthesis of oxygenated eicosanoids is the result of the coordinated action of several enzymatic activities, from phospholipase A2 that releases the polyunsaturated fatty acids from membrane phospholipids, to primary oxidative enzymes, such as cyclooxygenases and lipoxygenases, to isomerases, synthases and hydrolases that carry out the final synthesis of the biologically active metabolites. Cells possessing the entire enzymatic machinery have been studied as sources of bioactive eicosanoids, but early on evidence proved that biosynthetic intermediates, albeit unstable, could move from one cell type to another. The biosynthesis of bioactive compounds could therefore be the result of a coordinated effort by multiple cell types that has been named transcellular biosynthesis of the eicosanoids. In several cases cells not capable of carrying out the complete biosynthetic process, due to the lack of key enzymes, have been shown to efficiently contribute to the final production of prostaglandins, leukotrienes and lipoxins. We will review in vitro studies, complex functional models, and in vivo evidences of the transcellular biosynthesis of eicosanoids and the biological relevance of the metabolites resulting from this unique biosynthetic pathway. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

  9. Tyramine biosynthesis is transcriptionally induced at low pH and improves the fitness of Enterococcus faecalis in acidic environments.

    Science.gov (United States)

    Perez, Marta; Calles-Enríquez, Marina; Nes, Ingolf; Martin, Maria Cruz; Fernandez, Maria; Ladero, Victor; Alvarez, Miguel A

    2015-04-01

    Enterococcus faecalis is a commensal bacterium of the human gut that requires the ability to pass through the stomach and therefore cope with low pH. E. faecalis has also been identified as one of the major tyramine producers in fermented food products, where they also encounter acidic environments. In the present work, we have constructed a non-tyramine-producing mutant to study the role of the tyramine biosynthetic pathway, which converts tyrosine to tyramine via amino acid decarboxylation. Wild-type strain showed higher survival in a system that mimics gastrointestinal stress, indicating that the tyramine biosynthetic pathway has a role in acid resistance. Transcriptional analyses of the E. faecalis V583 tyrosine decarboxylase cluster showed that an acidic pH, together with substrate availability, induces its expression and therefore the production of tyramine. The protective role of the tyramine pathway under acidic conditions appears to be exerted through the maintenance of the cytosolic pH. Tyramine production should be considered important in the adaptability of E. faecalis to acidic environments, such as fermented dairy foods, and to survive passage through the human gastrointestinal tract.

  10. Biosynthesis of Citric Acid from Glycerol by Acetate Mutants of Yarrowia lipolytica in Fed-Batch Fermentation

    Directory of Open Access Journals (Sweden)

    Anita Rywińska

    2009-01-01

    Full Text Available Pure and crude glycerol from biodiesel production have been used as substrates for citric acid production by acetate-negative mutants of Yarrowia lipolytica in fed-batch fermentation. Both the final concentration and the yield of the product were the highest when Y. lipolytica Wratislavia AWG7 strain was used in the culture with pure or crude glycerol. With a medium containing 200 g/L of glycerol, production reached a maximum of citric acid of 139 g/L after 120 h. This high yield of the product (up to 0.69 g of citric acid per gram of glycerol consumed was achieved with both pure and crude glycerol. Lower yield of citric acid in the culture with Y. lipolytica Wratislavia K1 strain (about 0.45 g/g resulted from increased erythritol concentrations (up to 40 g/L, accumulated simultaneously with the citric acid. The concentration of isocitric acid, a by-product in this fermentation, was very low, in the range from 2.6 to 4.6 g/L.

  11. 极大螺旋微藻(分节螺旋属)在一六面体光合生物反应器中生物合成13C标识氨基酸和糖%Biosynthesis of 13C-Labeled Amino Acids and Sugars by Spirulina (Arthrospira) Maxima in a Parallelepiped Photobioreactor

    Institute of Scientific and Technical Information of China (English)

    夏金兰; 聂珍媛

    2002-01-01

    This paper presents the investigation on biosynthesis of high-value-added amino acids and sugars labeleduniformly with stable isotope 13C by microalga Spirulina (Arthrospira) maxima in a parallelepiped photobioreactor.The kinetic data of both batch and continuous cultures with characterization of the amino acids and sugars areshown. The continuous culture without nutrients deficiency is for biosynthesis of amino acids, with tyrosine as oneof the principal constituents, and the batch culture with deficiency in nitrogen is for biosynthesis of labeled glucosethat is up to 64% versus dry mass of cells.

  12. Biosynthesis of high yield fatty acids from Chlorella vulgaris NIES-227 under nitrogen starvation stress during heterotrophic cultivation.

    Science.gov (United States)

    Shen, Xiao-Fei; Chu, Fei-Fei; Lam, Paul K S; Zeng, Raymond J

    2015-09-15

    In this study the heterotrophic cultivation of Chlorella vulgaris NIES-227 fed with glucose was investigated systematically using six media types; combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion. It was found that a high yield of fatty acids (0.88 of fed glucose-COD) and a high content of fatty acid methyl esters (FAMEs) (89% of dry weight) were obtained under nitrogen starved conditions. To our knowledge it is the first report on such high COD conversion yield and FAME content in microalgae. The dominant fatty acid (>50%) was methyl oleate (C18:1), a desirable component for biodiesel synthesis. FAME content under nitrogen starved conditions was significantly higher than under nitrogen sufficient conditions, while phosphorus had no significant influence, indicating that nitrogen starvation was the real "fatty acids trigger" in heterotrophic cultivation. These findings could simplify the downstream extraction process, such as the extrusion of oil from soybeans, and could reduce operating costs by improving the fatty acid yield from waste COD.

  13. Involvement of a Natural Fusion of a Cytochrome P450 and a Hydrolase in Mycophenolic Acid Biosynthesis

    DEFF Research Database (Denmark)

    Hansen, Bjarne Gram; Mnich, Ewelina; Nielsen, Kristian Fog;

    2012-01-01

    C, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway......Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by Mpa...... that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme...

  14. Repression by H-NS of genes required for the biosynthesis of the Vibrio cholerae biofilm matrix is modulated by the second messenger cyclic diguanylic acid.

    Science.gov (United States)

    Ayala, Julio C; Wang, Hongxia; Silva, Anisia J; Benitez, Jorge A

    2015-08-01

    Expression of Vibrio cholerae genes required for the biosynthesis of exopolysacchide (vps) and protein (rbm) components of the biofilm matrix is enhanced by cyclic diguanylate (c-di-GMP). In a previous study, we reported that the histone-like nucleoid structuring (H-NS) protein represses the transcription of vpsA, vpsL and vpsT. Here we demonstrate that the regulator VpsT can disrupt repressive H-NS nucleoprotein complexes at the vpsA and vpsL promoters in the presence of c-di-GMP, while H-NS could disrupt the VpsT-promoter complexes in the absence of c-di-GMP. Chromatin immunoprecipitation-Seq showed a remarkable trend for H-NS to cluster at loci involved in biofilm development such as the rbmABCDEF genes. We show that the antagonistic relationship between VpsT and H-NS regulates the expression of the rbmABCDEF cluster. Epistasis analysis demonstrated that VpsT functions as an antirepressor at the rbmA/F, vpsU and vpsA/L promoters. Deletion of vpsT increased H-NS occupancy at these promoters while increasing the c-di-GMP pool had the opposite effect and included the vpsT promoter. The negative effect of c-di-GMP on H-NS occupancy at the vpsT promoter required the regulator VpsR. These results demonstrate that c-di-GMP activates the transcription of genes required for the biosynthesis of the biofilm matrix by triggering a coordinated VpsR- and VpsT-dependent H-NS antirepression cascade.

  15. Hybride magnetic nanostructure based on amino acids functionalized polypyrrole

    Science.gov (United States)

    Nan, Alexandrina; Bunge, Alexander; Turcu, Rodica

    2015-12-01

    Conducting polypyrrole is especially promising for many commercial applications because of its unique optical, electric, thermal and mechanical properties. We report the synthesis and characterization of novel pyrrole functionalized monomers and core-shell hybrid nanostructures, consisting of a conjugated polymer layer (amino acids functionalized pyrrole copolymers) and a magnetic nanoparticle core. For functionalization of the pyrrole monomer we used several amino acids: tryptophan, leucine, phenylalanine, serine and tyrosine. These amino acids were linked via different types of hydrophobic linkers to the nitrogen atom of the pyrrole monomer. The magnetic core-shell hybrid nanostructures are characterized by various methods such as FTIR spectroscopy, transmission electron microscopy (TEM) and magnetic measurements.

  16. SIFT: Predicting amino acid changes that affect protein function.

    Science.gov (United States)

    Ng, Pauline C; Henikoff, Steven

    2003-07-01

    Single nucleotide polymorphism (SNP) studies and random mutagenesis projects identify amino acid substitutions in protein-coding regions. Each substitution has the potential to affect protein function. SIFT (Sorting Intolerant From Tolerant) is a program that predicts whether an amino acid substitution affects protein function so that users can prioritize substitutions for further study. We have shown that SIFT can distinguish between functionally neutral and deleterious amino acid changes in mutagenesis studies and on human polymorphisms. SIFT is available at http://blocks.fhcrc.org/sift/SIFT.html.

  17. Hybride magnetic nanostructure based on amino acids functionalized polypyrrole

    Energy Technology Data Exchange (ETDEWEB)

    Nan, Alexandrina, E-mail: alexandrina.nan@itim-cj.ro; Bunge, Alexander; Turcu, Rodica [National Institute for Research and Development of Isotopic and Molecular Technologies, 67-103 Donat, 400293 Cluj-Napoca (Romania)

    2015-12-23

    Conducting polypyrrole is especially promising for many commercial applications because of its unique optical, electric, thermal and mechanical properties. We report the synthesis and characterization of novel pyrrole functionalized monomers and core-shell hybrid nanostructures, consisting of a conjugated polymer layer (amino acids functionalized pyrrole copolymers) and a magnetic nanoparticle core. For functionalization of the pyrrole monomer we used several amino acids: tryptophan, leucine, phenylalanine, serine and tyrosine. These amino acids were linked via different types of hydrophobic linkers to the nitrogen atom of the pyrrole monomer. The magnetic core-shell hybrid nanostructures are characterized by various methods such as FTIR spectroscopy, transmission electron microscopy (TEM) and magnetic measurements.

  18. Production of novel antioxidative phenolic amides through heterologous expression of the plant’s chlorogenic acid biosynthesis genes in yeast

    NARCIS (Netherlands)

    Moglia, A.; Comino, C.; Lanteri, S.; Vos, de C.H.; Waard, de P.; Beek, van T.A.; Goitre, L.; Retta, S.F.; Beekwilder, M.J.

    2010-01-01

    Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker’s yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified a

  19. Characterization of a citrus R2R3-MYB transcription factor that regulates the flavonol and hydroxycinnamic acid biosynthesis

    Science.gov (United States)

    Flavonols and hydroxycinnamic acids are important phenylpropanoid metabolites in plants. In this study, we isolated and characterized a citrus R2R3-MYB transcription factor CsMYBF1, encoding a protein belonging to the flavonol-specific MYB subgroup. Ectopic expression of CsMYBF1 in tomato led to an ...

  20. Auxin biosynthesis and storage forms.

    Science.gov (United States)

    Korasick, David A; Enders, Tara A; Strader, Lucia C

    2013-06-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development.

  1. Effect of an Oxadiazoline and a Lignan on Mycolic Acid Biosynthesis and Ultrastructural Changes of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Eduard Baquero

    2011-01-01

    Full Text Available Tuberculosis (TB is an important disease that causes thousands of deaths around the world. Resistance against antitubercular available drugs has been reported; so, research on new effective antimycobacterial molecules is needed. Antimycobacterial activity of three lignans and two synthetic hydrazones was assessed against Mycobacterium tuberculosis H37Rv by antimycobacterial microdilution assay (TEMA. An oxadiazoline (AC451 and a lignan (ethoxycubebin were the most active compounds (MIC 6.09 and 62.4 μM, resp.. Several changes in mycolic acid profile of treated bacteria were detected with both compounds by mass spectrometry analysis. Additionally, the level of reduction of mycolic acids in ethoxycubebin treatment was correlated to disruption in bacterial morphology.

  2. Detection of phytohormones in temperate forest fungi predicts consistent abscisic acid production and a common pathway for cytokinin biosynthesis.

    Science.gov (United States)

    Morrison, Erin N; Knowles, Sarah; Hayward, Allison; Thorn, R Greg; Saville, Barry J; Emery, R J N

    2015-01-01

    The phytohormones, abscisic acid and cytokinin, once were thought to be present uniquely in plants, but increasing evidence suggests that these hormones are present in a wide variety of organisms. Few studies have examined fungi for the presence of these "plant" hormones or addressed whether their levels differ based on the nutrition mode of the fungus. This study examined 20 temperate forest fungi of differing nutritional modes (ectomycorrhizal, wood-rotting, saprotrophic). Abscisic acid and cytokinin were present in all fungi sampled; this indicated that the sampled fungi have the capacity to synthesize these two classes of phytohormones. Of the 27 cytokinins analyzed by HPLC-ESI MS/MS, seven were present in all fungi sampled. This suggested the existence of a common cytokinin metabolic pathway in fungi that does not vary among different nutritional modes. Predictions regarding the source of isopentenyl, cis-zeatin and methylthiol CK production stemming from the tRNA degradation pathway among fungi are discussed.

  3. Tyramine biosynthesis is transcriptionally induced at low pH and improves the fitness of Enterococcus faecalis in acidic environments

    OpenAIRE

    Pérez, Marta M.; Calles-Enríquez, Marina; Nes, Ingolf F.; Martín, M. Cruz; Fernández García, María; Ladero Losada, Víctor Manuel; Álvarez González, Miguel Ángel

    2015-01-01

    © 2014, Springer-Verlag Berlin Heidelberg. Enterococcus faecalis is a commensal bacterium of the human gut that requires the ability to pass through the stomach and therefore cope with low pH. E. faecalis has also been identified as one of the major tyramine producers in fermented food products, where they also encounter acidic environments. In the present work, we have constructed a non-tyramine-producing mutant to study the role of the tyramine biosynthetic pathway, which converts tyrosine ...

  4. Arabidopsis cytochrome P450s that catalyze the first step of tryptophan-dependent indole-3-acetic acid biosynthesis

    OpenAIRE

    Hull, Anna K.; Vij, Rekha; Celenza, John L.

    2000-01-01

    Plants synthesize numerous secondary metabolites that are used as developmental signals or as defense against pathogens. Tryptophan (Trp)-derived secondary metabolites include camalexin, indole glucosinolates, and indole-3-acetic acid (IAA); however, the steps in their synthesis from Trp or its precursors remain unclear. We have identified two Arabidopsis cytochrome P450s (CYP79B2 and CYP79B3) that can convert Trp to indole-3-acetaldoxime (IAOx), a precursor to IAA and indole glucosinolates.

  5. Biochemical and Structural Characterization of WlbA from Bordetella pertussis and Chromobacterium violaceum: Enzymes Required for the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Thoden, James B.; Holden, Hazel M. (UW)

    2011-12-22

    The unusual sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, or ManNAc3NAcA, has been observed in the lipopolysaccharides of both pathogenic and nonpathogenic Gram-negative bacteria. It is added to the lipopolysaccharides of these organisms by glycosyltransferases that use as substrates UDP-ManNAc3NAcA. Five enzymes are ultimately required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetylglucosamine. The second enzyme in the pathway, encoded by the wlba gene and referred to as WlbA, catalyzes the NAD-dependent oxidation of the C-3' hydroxyl group of the UDP-linked sugar. Here we describe a combined structural and functional investigation of the WlbA enzymes from Bordetella pertussis and Chromobacterium violaceum. For this investigation, ternary structures were determined in the presence of NAD(H) and substrate to 2.13 and 1.5 {angstrom} resolution, respectively. Both of the enzymes display octameric quaternary structures with their active sites positioned far apart. The octamers can be envisioned as tetramers of dimers. Kinetic studies demonstrate that the reaction mechanisms for these enzymes are sequential and that they do not require {alpha}-ketoglutarate for activity. These results are in sharp contrast to those recently reported for the WlbA enzymes from Pseudomonas aeruginosa and Thermus thermophilus, which function via ping-pong mechanisms that involve {alpha}-ketoglutarate. Taken together, the results reported here demonstrate that there are two distinct families of WlbA enzymes, which differ with respect to amino acid sequences, quaternary structures, active site architectures, and kinetic mechanisms.

  6. Overexpression of EsMcsu1 from the halophytic plant Eutrema salsugineum promotes abscisic acid biosynthesis and increases drought resistance in alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Zhou, C; Ma, Z Y; Zhu, L; Guo, J S; Zhu, J; Wang, J F

    2015-12-17

    The stress phytohormone abscisic acid (ABA) plays pivotal roles in plants' adaptive responses to adverse environments. Molybdenum cofactor sulfurases influence aldehyde oxidase activity and ABA biosynthesis. In this study, we isolated a novel EsMcsu1 gene encoding a molybdenum cofactor sulfurase from Eutrema salsugineum. EsMcus1 transcriptional patterns varied between organs, and its expression was significantly upregulated by abiotic stress or ABA treatment. Alfalfa plants that overexpressed EsMcsu1 had a higher ABA content than wild-type (WT) plants under drought stress conditions. Furthermore, levels of reactive oxygen species (ROS), ion leakage, and malondialdehyde were lower in the transgenic plants than in the WT plants after drought treatment, suggesting that the transgenic plants experienced less ROS-mediated damage. However, the expression of several stress-responsive genes, antioxidant enzyme activity, and osmolyte (proline and total soluble sugar) levels in the transgenic plants were higher than those in the WT plants after drought treatment. Therefore, EsMcsu1 overexpression improved drought tolerance in alfalfa plants by activating a series of ABA-mediated stress responses.

  7. Glyphosate’s Suppression of Cytochrome P450 Enzymes and Amino Acid Biosynthesis by the Gut Microbiome: Pathways to Modern Diseases

    Directory of Open Access Journals (Sweden)

    Anthony Samsel

    2013-04-01

    Full Text Available Glyphosate, the active ingredient in Roundup®, is the most popular herbicide used worldwide. The industry asserts it is minimally toxic to humans, but here we argue otherwise. Residues are found in the main foods of the Western diet, comprised primarily of sugar, corn, soy and wheat. Glyphosate's inhibition of cytochrome P450 (CYP enzymes is an overlooked component of its toxicity to mammals. CYP enzymes play crucial roles in biology, one of which is to detoxify xenobiotics. Thus, glyphosate enhances the damaging effects of other food borne chemical residues and environmental toxins. Negative impact on the body is insidious and manifests slowly over time as inflammation damages cellular systems throughout the body. Here, we show how interference with CYP enzymes acts synergistically with disruption of the biosynthesis of aromatic amino acids by gut bacteria, as well as impairment in serum sulfate transport. Consequences are most of the diseases and conditions associated with a Western diet, which include gastrointestinal disorders, obesity, diabetes, heart disease, depression, autism, infertility, cancer and Alzheimer’s disease. We explain the documented effects of glyphosate and its ability to induce disease, and we show that glyphosate is the “textbook example” of exogenous semiotic entropy: the disruption of homeostasis by environmental toxins.

  8. Functional characterization of the Bombyx mori fatty acid transport protein (BmFATP) within the silkmoth pheromone gland.

    Science.gov (United States)

    Ohnishi, Atsushi; Hashimoto, Kana; Imai, Kiyohiro; Matsumoto, Shogo

    2009-02-20

    Fatty acid transport protein (FATP) is an evolutionarily conserved membrane-bound protein that facilitates the uptake of extracellular long chain fatty acids. In humans and mice, six FATP isoforms have been identified and their tissue-specific distributions suggest that each plays a discrete role in lipid metabolism in association with fatty acid uptake. While the presence of FATP homologs in insects has been demonstrated, their functional role remains to be characterized. Pheromonogenesis is defined as the dynamic period in which all machinery required for sex pheromone biosynthesis is generated and organized within the pheromone gland (PG) cells. By exploiting this unique system in the PG of the silkmoth, Bombyx mori, we found that BmFATP is predominantly expressed in the PG and undergoes up-regulation 1 day prior to eclosion. Before eclosion, B. mori PG cells accumulate cytoplasmic lipid droplets (LDs), which play a role in storing the pheromone (bombykol) precursor fatty acid in the form of triacylglycerol. RNAi-mediated gene silencing of BmFATP in vivo significantly suppressed LD accumulation by preventing the synthesis of triacylglycerols and resulted in a significant reduction in bombykol production. These results, in conjunction with the findings that BmFATP stimulates the uptake of extracellular long-chain fatty acids and BmFATP knockdown reduces cellular long-chain acyl-CoA synthetase activity, suggest that BmFATP plays an essential role in bombykol biosynthesis by stimulating both LD accumulation and triacylglycerol synthesis via a process called vectorial acylation that couples the uptake of extracellular fatty acids with activation to CoA thioesters during pheromonogenesis.

  9. Co2 chemosorption by functionalized amino acid derivatives

    DEFF Research Database (Denmark)

    2015-01-01

    The absorption and desorption behaviour of carbon dioxide (CO2) using a composition comprising an ionic compound comprising a cation [A+] and an anion [B-] is described, wherein the anion [B-] is a mono-amine functionalized amino acid.......The absorption and desorption behaviour of carbon dioxide (CO2) using a composition comprising an ionic compound comprising a cation [A+] and an anion [B-] is described, wherein the anion [B-] is a mono-amine functionalized amino acid....

  10. Functional loss of pAMT results in biosynthesis of capsinoids, capsaicinoid analogs, in Capsicum annuum cv. CH-19 Sweet.

    Science.gov (United States)

    Lang, Yaqin; Kisaka, Hiroaki; Sugiyama, Ryuji; Nomura, Kenzo; Morita, Akihito; Watanabe, Tatsuo; Tanaka, Yoshiyuki; Yazawa, Susumu; Miwa, Tetsuya

    2009-09-01

    Capsaicinoids are responsible for the spicy flavor of pungent peppers (Capsicum). The cultivar CH-19 Sweet is a non-pungent pepper mutant derived from a pungent pepper strain, Capsicum annuum CH-19. CH-19 Sweet biosynthesizes capsaicinoid analogs, capsinoids. We determined the genetic and metabolic mechanisms of capsinoid biosynthesis in this cultivar. We analyzed the putative aminotransferase (pAMT) that is thought to catalyze the formation of vanillylamine from vanillin in the capsaicinoid biosynthetic pathway. Enzyme assays revealed that pAMT activity catalyzing vanillylamine formation was completely lost in CH-19 Sweet placenta tissue. RT-PCR analysis showed normal mRNA transcription of the pAMT gene; however, SNP analysis of the cDNA sequence showed a T nucleotide insertion at 1291 bp in the pAMT gene of CH-19 Sweet. This insertion formed a new stop codon, TGA, that prevented normal translation of the gene, and the pAMT protein did not accumulate in CH-19 Sweet as determined using Western blot analysis. We developed a dCAPS marker based on the T insertion in the pAMT gene of CH-19 Sweet, and showed that the pAMT genotype co-segregated with the capsinoid or capsaicinoid fruit phenotype in the F(2) population. The T insertion was not found in other pungent and non-pungent Capsicum lines, suggesting that it is specific to CH-19 Sweet. CH-19 Sweet's pAMT gene mutation is an example of a nonsense mutation in a single gene that alters a secondary metabolite biosynthetic pathway, resulting in the biosynthesis of analogs. The dCAPS marker will be useful in selecting lines with capsinoid-containing fruits in pepper-breeding programs.

  11. Cerato-platanin induces resistance in Arabidopsis leaves through stomatal perception, overexpression of salicylic acid- and ethylene-signalling genes and camalexin biosynthesis.

    Science.gov (United States)

    Baccelli, Ivan; Lombardi, Lara; Luti, Simone; Bernardi, Rodolfo; Picciarelli, Piero; Scala, Aniello; Pazzagli, Luigia

    2014-01-01

    Microbe-associated molecular patterns (MAMPs) lead to the activation of the first line of plant defence. Few fungal molecules are universally qualified as MAMPs, and proteins belonging to the cerato-platanin protein (CPP) family seem to possess these features. Cerato-platanin (CP) is the name-giving protein of the CPP family and is produced by Ceratocystis platani, the causal agent of the canker stain disease of plane trees (Platanus spp.). On plane tree leaves, the biological activity of CP has been widely studied. Once applied on the leaf surface, CP acts as an elicitor of defence responses. The molecular mechanism by which CP elicits leaves is still unknown, and the protective effect of CP against virulent pathogens has not been clearly demonstrated. In the present study, we tried to address these questions in the model plant Arabidopsis thaliana. Our results suggest that stomata rapidly sense CP since they responded to the treatment with ROS signalling and stomatal closure, and that CP triggers salicylic acid (SA)- and ethylene (ET)-signalling pathways, but not the jasmonic acid (JA)-signalling pathway, as revealed by the expression pattern of 20 marker genes. Among these, EDS1, PAD4, NPR1, GRX480, WRKY70, ACS6, ERF1a/b, COI1, MYC2, PDF1.2a and the pathogenesis-related (PR) genes 1-5. CP rapidly induced MAPK phosphorylation and induced the biosynthesis of camalexin within 12 hours following treatment. The induction of localised resistance was shown by a reduced susceptibility of the leaves to the infection with Botrytis cinerea and Pseudomonas syringae pv. tomato. These results contribute to elucidate the key steps of the signalling process underlying the resistance induction in plants by CP and point out the central role played by the stomata in this process.

  12. The Biosynthesis of Deuterium Labeled Amino Acids Using a Strain of Facultative Methylotrophic Bacterium Вrevibacterium Methylicum 5662 With RuMP Cycle of Carbon Assimilation

    Directory of Open Access Journals (Sweden)

    Oleg Mosin

    2015-03-01

    Full Text Available We used Gram-positive aerobic facultative methylotrophic bacterium, Brevibacterium methylicum, L-phenylalanine producer with ribulose-5-monophosphate (RuMP cycle for carbon assimilation for microbiological preparation of [2H]phenylalanine via conversion of low molecular weight substrates ([U-2H]MeOH and 2H2O. For this purpose, the cells of the methylotroph with improved growth characteristics were used on minimal salt media M9 supplemented with 2 % (v/v [U-2H]MeOH and increasing gradient of 2Н2O concentration from 0; 24,5; 49,0; 73,5 up to 98 % (v/v 2Н2O. L-phenylalanine was isolated from the growth medium after adding 5 M 2HCl (in 2Н2О, pH = 2,0 by extraction with isopropanol and subsequent crystallization in ethanol (output 0,65 g/l. Alanine, valine, and leucine/isoleucine were produced and accumulated exogenously in amounts of 5–6 mol in addition to the main product of biosynthesis. The method allows to obtain [2Н]amino acids with different levels of deuterium enrichment, depending on 2Н2O concentration in growth media, from 17 atom% 2Н (2 deuterium atoms (on the growth medium with 24,5 % (v/v 2Н2О up to 75 atom% 2Н (6 deuterium atoms (on the growth medium with 98 % (v/v 2Н2О with introduction of deuterium to benzyl С6Н5СН2-fragment of molecule that is confirmed with the data of electron impact (EI mass spectrometry analysis of methyl ethers of N-5-dimethylamino(naphthalene-1-sulfochloride [2H]amino acids after the separation by reverse-phase HPLC.

  13. Cerato-platanin induces resistance in Arabidopsis leaves through stomatal perception, overexpression of salicylic acid- and ethylene-signalling genes and camalexin biosynthesis.

    Directory of Open Access Journals (Sweden)

    Ivan Baccelli

    Full Text Available Microbe-associated molecular patterns (MAMPs lead to the activation of the first line of plant defence. Few fungal molecules are universally qualified as MAMPs, and proteins belonging to the cerato-platanin protein (CPP family seem to possess these features. Cerato-platanin (CP is the name-giving protein of the CPP family and is produced by Ceratocystis platani, the causal agent of the canker stain disease of plane trees (Platanus spp.. On plane tree leaves, the biological activity of CP has been widely studied. Once applied on the leaf surface, CP acts as an elicitor of defence responses. The molecular mechanism by which CP elicits leaves is still unknown, and the protective effect of CP against virulent pathogens has not been clearly demonstrated. In the present study, we tried to address these questions in the model plant Arabidopsis thaliana. Our results suggest that stomata rapidly sense CP since they responded to the treatment with ROS signalling and stomatal closure, and that CP triggers salicylic acid (SA- and ethylene (ET-signalling pathways, but not the jasmonic acid (JA-signalling pathway, as revealed by the expression pattern of 20 marker genes. Among these, EDS1, PAD4, NPR1, GRX480, WRKY70, ACS6, ERF1a/b, COI1, MYC2, PDF1.2a and the pathogenesis-related (PR genes 1-5. CP rapidly induced MAPK phosphorylation and induced the biosynthesis of camalexin within 12 hours following treatment. The induction of localised resistance was shown by a reduced susceptibility of the leaves to the infection with Botrytis cinerea and Pseudomonas syringae pv. tomato. These results contribute to elucidate the key steps of the signalling process underlying the resistance induction in plants by CP and point out the central role played by the stomata in this process.

  14. Isolation of 14{sub C} labelled amino acids by biosynthesis in maize plants (Zea mais L.); Obtencin de aminoacidos marcados con 14{sub C} por biosintesis en plantulas de maiz (Zea mais L)

    Energy Technology Data Exchange (ETDEWEB)

    Carreras, N.; Mazon, M. P.

    1983-07-01

    A method of obtaining 14{sub C} labelled amino acids by biosynthesis in maize plants which had assimilated 14CO{sub 2}, has been assayed. The plants were labelled for 60 minutes with 14{sub C}O2 produced from Ba 14{sub C}O3 (specific activity of 148 KBq/{mu}mol). An extract of the soluble compounds was obtained with 80% ethanol and the amino acids were separated from the rest of the soluble compounds by ion exchange chromatography on column of Dowex 50-X8 resin. Finally, seventeen amino acids were isolated and identified from the purified extract. The acid amino acids were separated in anionic column (Dowex 1-X8) and the neutral and basic amino acids in cationic column (Dowex 50-X4). (Author) 56 refs.

  15. Effect of precipitation, geographical location and biosynthesis on New Zealand milk powder bulk and fatty acids D/H ratios

    Science.gov (United States)

    Frew, R.; Emad Ehtesham, R.; Van Hale, R.; Hayman, A.; Baisden, T.

    2012-04-01

    D/H ratio measurements provide useful information for the investigation of biogeochemical influences on natural and agricultural produce, particularly with application to food traceability and authentication. Numerous studies have shown that variation of a product's D/H ratio is influenced by both environmental factors and biological processes. This study investigates the D/H ratio of New Zealand milk powder and individual fatty acids, and causal determinants of isotopic variation. One of the key environmental factors is precipitation, and the D/H ratio "isoscaping" of NZ has been undertaken. New Zealand provides a unique geography for these kinds of study in terms of proximity to the ocean and natural geographical variability from sea level to elevations as high as 3700 m. Milk powder samples were collected from different geographical regions from milk processing units, which were supplied by producers in the immediate region. H/D ratios of bulk milk powder and of individual fatty acids were determined. Initial comparison of the precipitation and milk powder bulk D/H data show a very good differentiation from north to southernmost parts of New Zealand and a relation between rain and milk bulk D/H abundance ratio. Almost 98% of milk FAs are in the form of triglycerides that have been extracted and hydrolysed to free FAs. Free FAs were esterified and analyzed with GC-IRMS. Individual FAs show variation in D/H ratio, and all values are depleted relative to the precipitation data. The difference in D/H ratio amongst individual FAs reflects the geographical environment and biological processes i.e. micro-organisms activity in the rumen of the cow. Short chain FAs (less than 8 carbons), particularly C4 (Butyric acid), appear to be key determinants. The variation in the data can be rationalized using statistical multivariate analysis.

  16. Identification and Functional Characterization of Genes Encoding Omega-3 Polyunsaturated Fatty Acid Biosynthetic Activities from Unicellular Microalgae

    Directory of Open Access Journals (Sweden)

    Royah Vaezi

    2013-12-01

    Full Text Available In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative “front-end” desaturases (Δ6 and Δ4 from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of putative open reading frames (ORFs in yeast revealed that the encoded enzyme activities efficiently convert their respective substrates: 54.1% conversion of α-linolenic acid for Δ6-desaturase, 15.1% conversion of 22:5n-3 for Δ4-desaturase and 38.1% conversion of γ-linolenic acid for Δ6-elongase. The Δ6-desaturase from Ostreococcus RCC809 displays a very strong substrate preference resulting in the predominant synthesis of stearidonic acid (C18:4Δ6,9,12,15. These data confirm the functional characterization of omega-3 long chain polyunsaturated fatty acid biosynthetic genes from these two species which have until now not been investigated for such activities. The identification of these new genes will also serve to expand the repertoire of activities available for metabolically engineering the omega-3 trait in heterologous hosts as well as providing better insights into the synthesis of eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA in marine microalgae.

  17. Phosphate Starvation Responses and Gibberellic Acid Biosynthesis Are Regulated by the MYB62 Transcription Factor in Arabiclopsis

    Institute of Scientific and Technical Information of China (English)

    Ballachanda N. Devaiah; Ramaiah Madhuvanthi; Athikkattuvalasu S. Karthikeyan; Kashchandra G. Raghothama

    2009-01-01

    The limited availability of phosphate (Pi) in most soils results in the manifestation of Pi starvation responses in plants. To dissect the transcriptional regulation of Pi stress-response mechanisms, we have characterized the biological role of MYB62, an R2R3-type MYB transcription factor that is induced in response to Pi deficiency. The induction of MYB62 is a specific response in the leaves during Pi deprivation. The MYB62 protein localizes to the nucleus. The overexpression of MYB62 resulted in altered root architecture, Pi uptake, and acid phosphatase activity, leading to decreased total Pi content in the shoots. The expression of several Pi starvation-induced (PSI) genes was also suppressed in the MYB62 overexpressing plants. Overexpression of MYB62 resulted in a characteristic gibberellic acid (GA)-deficient phenotype that could be partially reversed by exogenous application of GA. In addition, the expression of SOC1 and SUPERMAN, molecular regulators of flowering, was suppressed in the MYB62 overexpressing plants. Interestingly, the expression of these genes was also reduced during Pi deprivation in wild-type plants, suggesting a role for GA biosynthetic and floral regulatory genes in Pi starvation responses. Thus, this study highlights the role of MYB62 in the regulation of phosphate starvation responses via changes in GA metabolism and signaling. Such cross-talk between Pi homeostasis and GA might have broader implications on flowering, root development and adaptive mechanisms during nutrient stress.

  18. Biosynthesis of monoterpenoids in higher plants. The biosynthetic pathway leading to the monoterpenoids from amino acids with a carbon-skeleton similar to mevalonic acid

    Energy Technology Data Exchange (ETDEWEB)

    Tange, K. (Hiroshima Univ. (Japan). Faculty of Science)

    1981-09-01

    Radioisotopically labeled L-valine, DL-alanine, sodium acetate, and DL-mevalonic acid were incorporated into linalool by the intact plant of Cinnamomum camphora Sieb. var. linalooliferum Fujita and into geraniol and citronellol by that of Pelargonium roseum Bourbon. The uptake of leucine and valine resulted in the preferential location of the radioactivity on the 3,3-dimethylallyl pyrophosphate-derived moiety of these acyclic monoterpenoids, whereas the uptake of alanine resulted in the preferential location on the isopentenyl pyrophosphate-derived moiety, much as in the cases of mevalonic acid and sodium acetate. A biosynthetic pathway leading to the monoterpenoids from the amino acids is discussed.

  19. Campylobacter jejuni fatty acid synthase II: Structural and functional analysis of [beta]-hydroxyacyl-ACP dehydratase (FabZ)

    Energy Technology Data Exchange (ETDEWEB)

    Kirkpatrick, Andrew S.; Yokoyama, Takeshi; Choi, Kyoung-Jae; Yeo, Hye-Jeong; (Houston)

    2009-08-14

    Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The {beta}-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide a framework for investigation on roles of FAS II in C. jejuni virulence

  20. Fatty acids as modulators of neutrophil recruitment, function and survival.

    Science.gov (United States)

    Rodrigues, Hosana G; Takeo Sato, Fabio; Curi, Rui; Vinolo, Marco A R

    2016-08-15

    Neutrophils are well-known to act in the destruction of invading microorganisms. They have also been implicated in the activation of other immune cells including B- and T-lymphocytes and in the resolution of inflammation and tissue regeneration. Neutrophils are produced in the bone marrow and released into the circulation from where they migrate to tissues to perform their effector functions. Neutrophils are in constant contact with fatty acids that can modulate their function, activation and fate (survival or cell death) through different mechanisms. In this review, the effects of fatty acids pertaining to five classes, namely, long-chain saturated fatty acids (LCSFAs), short-chain fatty acids (SCFAs), and omega-3 (n-3), omega-6 (n-6) and omega-9 (n-9) unsaturated fatty acids, on neutrophils and the relevance of these effects for disease development are discussed.

  1. 分枝杆菌枝菌酸合成及其调控%Biosynthesis and regulation of mycolic acids in mycobacterium tuberculosis-A review

    Institute of Scientific and Technical Information of China (English)

    罗红丽; 庞蕾; 谢建平

    2012-01-01

    Tuberculosis ( TB ) is one of the world's deadliest diseases. Approximately eight million individuals develop active tuberculosis annually, and two million die of tuberculosis. The emergence of multi-drug resistance strains, HIV co-infection , and an increasing aging population further worsen this scenario. Mycolic acids ( Mas, also mycolate) are integral cell wall components of Mycobacterium tuberculosis, other mycobacterium and most actinomycetes, engaging in the remarkable survival ability of Mycobacterium tuberculosis within infected hosts, virulence and evasion of immunity. The biosynthesis and regulation of mycolic acids are rife with anti-tuberculosis drug targets. First-line tuberculosis drugs such as isoniazid and ethambutol target this pathway. In-depth investigation of this aspect will provide more opportunities to find better measures to combat tuberculosis. To this end, we reviewed the structures, classification, biosynthesis pathway, regulation factors in pathway of mycolic acid, as well as promising drug targets.%结核病是危害人类健康的重要传染病,每年200多万人死于结核病.耐(多)药菌株的出现、与HIV共感染以及人口老龄化等原因与全球结核病的卷土重来密切相关.枝菌酸是存在于结核分枝杆菌、其他分枝杆菌和许多放线菌的细胞壁中的关键组分,与结核分枝杆菌的致病、毒力和免疫逃避都有关系.枝菌酸在抗结核研究中有着极其重要的地位.结核分枝杆菌枝菌酸的生物合成途径一直是很重要的抗结核药物靶标,异烟肼、乙胺丁醇等抗结核药物都是以此为靶标.深入研究枝菌酸的合成、调控有助于发现更多的药物靶标,为开发结核病控制新措施提供基础.本文综述了结核分枝杆菌枝菌酸的结构与分类、生物合成途径及其调控、作为抗结核药物靶标的前景与应用,以期对枝菌酸有更深入的了解并为新型抗结核药物靶标的发现提供基础.

  2. Abscisic acid enhances tolerance of wheat seedlings to drought and regulates transcript levels of genes encoding ascorbate-glutathione biosynthesis

    Directory of Open Access Journals (Sweden)

    Wei eLiting

    2015-06-01

    Full Text Available Glutathione (GSH and ascorbate (ASA are associated with the abscisic acid (ABA-induced abiotic tolerance in higher plant, however, its molecular mechanism remains obscure. In this study, exogenous application (10 μM of ABA significantly increased the tolerance of seedlings of common wheat (Triticum aestivum L. suffering from 5 days of 15% polyethylene glycol (PEG-stimulated drought stress, as demonstrated by increased shoot lengths and shoot and root dry weights, while showing decreased contents of hydrogen peroxide (H2O2 and malondialdehyde (MDA. Under drought stress conditions, ABA markedly increased contents of GSH and ASA in both leaves and roots of ABA-treated plants. Temporal and spatial expression patterns of eight genes encoding ASA and GSH synthesis-related enzymes were measured using quantitative real-time reverse transcription polymerase chain reaction (qPCR. The results showed that ABA temporally regulated the transcript levels of genes encoding ASA-GSH cycle enzymes. Moreover, these genes exhibited differential expression patterns between the root and leaf organs of ABA-treated wheat seedlings during drought stress. These results implied that exogenous ABA increased the levels of GSH and ASA in drought-stressed wheat seedlings in time- and organ-specific manners. Moreover, the transcriptional profiles of ASA-GSH synthesis-related enzyme genes in the leaf tissue were compared between ABA- and salicylic acid (SA-treated wheat seedlings under PEG-stimulated drought stress, suggesting that they increased the contents of ASA and GSH by differentially regulating expression levels of ASA-GSH synthesis enzyme genes. Our results increase our understanding of the molecular mechanism of ABA-induced drought tolerance in higher plants

  3. Arabinogalactan biosynthesis

    DEFF Research Database (Denmark)

    Poulsen, Christian Peter; Dilokpimol, Adiphol; Geshi, Naomi

    2015-01-01

    not only to the Golgi cisternae but also to smaller compartments, which may be a part of the unconventional protein secretory pathway in plants. In Poulsen et al.,1 we have demonstrated increased targeting of AtGALT29A to small compartments when Y144 is substituted with another amino acid, and we...

  4. Density functional theory study of the oligomerization of carboxylic acids.

    Science.gov (United States)

    Di Tommaso, Devis; Watson, Ken L

    2014-11-20

    We present a density functional theory [M06-2X/6-31+G(d,p)] study of the structures and free energies of formation of oligomers of four carboxylic acids (formic acid, acetic acid, tetrolic acid, and benzoic acid) in water, chloroform, and carbon tetrachloride. Solvation effects were treated using the SMD continuum solvation model. The low-lying energy structures of molecular complexes were located by adopting an efficient search procedure to probe the potential energy surfaces of the oligomers of carboxylic acids (CA)n (n = 2-6). The free energies of the isomers of (CA)n in solution were determined as the sum of the electronic energy, vibrational-rotational-translational gas-phase contribution, and solvation free energy. The assessment of the computational protocol adopted in this study with respect to the dimerization of acetic acid, (AA)2, and formic acid, (FA)2, located new isomers of (AA)2 and (FA)2 and gave dimerization constants in good agreement with the experimental values. The calculation of the self-association of acetic acid, tetrolic acid, and benzoic acid shows the following: (i) Classic carboxylic dimers are the most stable isomer of (CA)2 in both the gas phase and solution. (ii) Trimers of carboxylic acid are stable in apolar aprotic solvents. (iii) Molecular clusters consisting of two interacting classic carboxylic dimers (CA)4,(D+D) are the most stable type of tetramers, but their formation from the self-association of classic carboxylic dimers is highly unfavorable. (iv) For acetic acid and tetrolic acid the reactions (CA)2 + 2CA → (CA)4,(D+D) and (CA)3 + CA → (CA)4,(D+D) are exoergonic, but these aggregation pathways go through unstable clusters that could hinder the formation of tetrameric species. (v) For tetrolic acid the prenucleation species that are more likely to form in solution are dimeric and trimeric structures that have encoded structural motifs resembling the α and β solid forms of tetrolic acid. (vi) Stable tetramers of

  5. The antimalarial drug quinine interferes with serotonin biosynthesis and action.

    Science.gov (United States)

    Islahudin, Farida; Tindall, Sarah M; Mellor, Ian R; Swift, Karen; Christensen, Hans E M; Fone, Kevin C F; Pleass, Richard J; Ting, Kang-Nee; Avery, Simon V

    2014-01-01

    The major antimalarial drug quinine perturbs uptake of the essential amino acid tryptophan, and patients with low plasma tryptophan are predisposed to adverse quinine reactions; symptoms of which are similar to indications of tryptophan depletion. As tryptophan is a precursor of the neurotransmitter serotonin (5-HT), here we test the hypothesis that quinine disrupts serotonin function. Quinine inhibited serotonin-induced proliferation of yeast as well as human (SHSY5Y) cells. One possible cause of this effect is through inhibition of 5-HT receptor activation by quinine, as we observed here. Furthermore, cells exhibited marked decreases in serotonin production during incubation with quinine. By assaying activity and kinetics of the rate-limiting enzyme for serotonin biosynthesis, tryptophan hydroxylase (TPH2), we showed that quinine competitively inhibits TPH2 in the presence of the substrate tryptophan. The study shows that quinine disrupts both serotonin biosynthesis and function, giving important new insight to the action of quinine on mammalian cells.

  6. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

    Directory of Open Access Journals (Sweden)

    Smrati Mishra

    Full Text Available Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.

  7. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

    Science.gov (United States)

    Mishra, Smrati; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh; Asha; Jadaun, Jyoti Singh; Sangwan, Neelam S

    2016-01-01

    Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.

  8. Role of Acid Functionality and Placement on Morphological Evolution and Strengthening of Acid Copolymers

    Science.gov (United States)

    Middleton, Luri Robert; Schwartz, Eric; Winey, Karen

    Functional polymers with specific interactions produce hierarchical morphologies that directly impact mechanical properties. We recently reported that the formation of acid-rich layered morphologies in precise poly(ethylene-co-acrylic acid) copolymers improves tensile strength. We now explore the generality of this phenomenon through variations in pendant acid chemistries, acid content and precision in placement of acid groups in polyethylene-based copolymers. In situ X-ray scattering measurements during tensile deformation reveal that the precision in acid group placement is critical to forming well-defined layered morphologies. This phenomenon was observed in both semi-crystalline and amorphous precise acid copolymers with varied acid chemistries (acrylic, geminal acrylic and phosphonic acids). Compositionally identical polymers but with pseudo random acid placement do not form layered morphologies. Acid chemistry and acid content influence morphological evolution predominately though modification of the copolymer Tg and crystallinity. Our results indicate that hierarchical layered structures, commensurate with improved mechanical properties, form in the presence of uniformity in chemical structure and sufficient chain mobility to strongly align during deformation.

  9. Covalently functionalized graphene sheets with biocompatible natural amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Mallakpour, Shadpour, E-mail: mallak@cc.iut.ac.ir [Organic Polymer Chemistry Research Laboratory, Department of Chemistry, Isfahan University of Technology, Isfahan, 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of); Nanotechnology and Advanced Materials Institute, Isfahan University of Technology, Isfahan, 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of); Abdolmaleki, Amir, E-mail: abdolmaleki@cc.iut.ac.ir [Organic Polymer Chemistry Research Laboratory, Department of Chemistry, Isfahan University of Technology, Isfahan, 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of); Nanotechnology and Advanced Materials Institute, Isfahan University of Technology, Isfahan, 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of); Borandeh, Sedigheh [Organic Polymer Chemistry Research Laboratory, Department of Chemistry, Isfahan University of Technology, Isfahan, 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of)

    2014-07-01

    Graphene sheets were covalently functionalized with aromatic–aliphatic amino acids (phenylalanine and tyrosine) and aliphatic amino acids (alanine, isoleucine, leucine, methionine and valine) by simple and green procedure. For this aim, at first natural graphite was converted into graphene oxide (GO) through strong oxidation procedure; then, based on the surface-exposed epoxy and carboxylic acid groups in GO solid, its surface modification with naturally occurring amino acids, occurred easily throughout the corresponding nucleophilic substitution and condensation reactions. Amino acid functionalized graphene demonstrates stable dispersion in water and common organic solvents. Fourier transform infrared, Raman and X-ray photoelectron spectroscopies, X-ray diffraction, field emission scanning electron microscopy and transmission electron microscopy were used to investigate the nanostructures and properties of prepared materials. Each amino acid has different considerable effects on the structure and morphology of the pure graphite, from increasing the layer spacing to layer scrolling, based on their structures, functional groups and chain length. In addition, therogravimetric analysis was used for demonstrating a successful grafting of amino acid molecules to the surface of graphene.

  10. Fatty acids, lipid mediators, and T-cell function.

    Science.gov (United States)

    de Jong, Anja J; Kloppenburg, Margreet; Toes, René E M; Ioan-Facsinay, Andreea

    2014-01-01

    Research toward the mechanisms underlying obesity-linked complications has intensified during the last years. As a consequence, it has become clear that metabolism and immunity are intimately linked. Free fatty acids and other lipids acquired in excess by current feeding patterns have been proposed to mediate this link due to their immune modulatory capacity. The functional differences between saturated and unsaturated fatty acids, in combination with their dietary intake are believed to modulate the outcome of immune responses. Moreover, unsaturated fatty acids can be oxidized in a tightly regulated and specific manner to generate either potent pro-inflammatory or pro-resolving lipid mediators. These oxidative derivatives of fatty acids have received detailed attention during the last years, as they have proven to have strong immune modulatory capacity, even in pM ranges. Both fatty acids and oxidized fatty acids have been studied especially in relation to macrophage and T-cells functions. In this review, we propose to focus on the effect of fatty acids and their oxidative derivatives on T-cells, as it is an active area of research during the past 5 years. The effect of fatty acids and their derivatives on activation and proliferation of T-cells, as well as the delicate balance between stimulation and lipotoxicity will be discussed. Moreover, the receptors involved in the interaction between free fatty acids and their derivatives with T-cells will be summarized. Finally, the mechanisms involved in modulation of T-cells by fatty acids will be addressed, including cellular signaling and metabolism of T-cells. The in vitro results will be placed in context of in vivo studies both in humans and mice. In this review, we summarize the latest findings on the immune modulatory function of lipids on T-cells and will point out novel directions for future research.

  11. Indole-3-acetic acid biosynthesis is deficient in Gluconacetobacter diazotrophicus strains with mutations in cytochrome c biogenesis genes.

    Science.gov (United States)

    Lee, Sunhee; Flores-Encarnación, M; Contreras-Zentella, M; Garcia-Flores, L; Escamilla, J E; Kennedy, Christina

    2004-08-01

    Gluconacetobacter diazotrophicus is an endophyte of sugarcane frequently found in plants grown in agricultural areas where nitrogen fertilizer input is low. Recent results from this laboratory, using mutant strains of G. diazotrophicus unable to fix nitrogen, suggested that there are two beneficial effects of G. diazotrophicus on sugarcane growth: one dependent and one not dependent on nitrogen fixation. A plant growth-promoting substance, such as indole-3-acetic acid (IAA), known to be produced by G. diazotrophicus, could be a nitrogen fixation-independent factor. One strain, MAd10, isolated by screening a library of Tn5 mutants, released only approximately 6% of the amount of IAA excreted by the parent strain in liquid culture. The mutation causing the IAA(-) phenotype was not linked to Tn5. A pLAFR3 cosmid clone that complemented the IAA deficiency was isolated. Sequence analysis of a complementing subclone indicated the presence of genes involved in cytochrome c biogenesis (ccm, for cytochrome c maturation). The G. diazotrophicus ccm operon was sequenced; the individual ccm gene products were 37 to 52% identical to ccm gene products of Escherichia coli and equivalent cyc genes of Bradyrhizobium japonicum. Although several ccm mutant phenotypes have been described in the literature, there are no reports of ccm gene products being involved in IAA production. Spectral analysis, heme-associated peroxidase activities, and respiratory activities of the cell membranes revealed that the ccm genes of G. diazotrophicus are involved in cytochrome c biogenesis.

  12. A fermentative approach towards optimizing directed biosynthesis of fumaric acid by Rhizopus oryzae 1526 utilizing apple industry waste biomass.

    Science.gov (United States)

    Das, Ratul Kumar; Brar, Satinder Kaur; Verma, Mausam

    2015-12-01

    The present research account deals with the bioproduction of fumaric acid (FA) from apple pomace ultrafiltration sludge (APUS) and apple pomace (AP) through fermentation. The filamentous fungus Rhizopus oryzae 1526 was used as a biocatalyst and its morphological impact on FA production was analysed in detail. For submerged fermentation, 40 g L(-1) of total solids concentration of APUS, pH 6.0, 30 °C, 200 rpm flask shaking speed and 72 h of incubation were found to be optimum for FA production (25.2 ± 1.0 g L(-1), 0.350 g (L(-1) h(-1))). Broth viscosity (cP), residual reducing sugar (g L(-1)) and ethanol (g L(-1)) produced as by-product, were also analysed. Plastic trays were used for solid state fermentation and at optimized level of moisture and incubation period, 52 ± 2.67 g FA per kg dry weight of AP was obtained. Changes in the total phenolic content (mg g(-1) dry weight of AP) were monitored at regular intervals. Utilization of APUS and AP for the directed synthesis of the high-value platform chemical FA by the fungal strain R. oryzae 1526 was an excellent display of fungal physiological and morphological control over a fermentative product.

  13. Improvement of poly-γ-glutamic acid biosynthesis in a moving bed biofilm reactor by Bacillus subtilis NX-2.

    Science.gov (United States)

    Jiang, Yongxiang; Tang, Bao; Xu, Zongqi; Liu, Kun; Xu, Zheng; Feng, Xiaohai; Xu, Hong

    2016-10-01

    The production of poly-γ-glutamic acid (γ-PGA) by Bacillus subtilis NX-2 using a moving bed biofilm reactor (MBBR) system was tested for the first time in this study. Polypropylene TL-2 was chosen as a suitable carrier, and γ-PGA concentration of 42.7±0.86g/L and productivity of 0.59±0.06g/(Lh) were obtained in batch fermentation. After application of the strategy of dissolved oxygen (DO)-stat feeding, higher γ-PGA concentration and productivity were achieved than with glucose feedback feeding. Finally, the repeated fed-batch cultures implemented in the MBBR system showed high stability, and the maximal γ-PGA concentration and productivity of 74.2g/L and 1.24g/(Lh) were achieved, respectively. In addition, the promotion of oxygen transfer by an MBBR carrier was well explained by a computational fluid dynamics (CFD) simulation. These results suggest that an MBBR system could be applied to large-scale γ-PGA production.

  14. FADS2 function loss at the cancer hotspot 11q13 locus diverts lipid signaling precursor synthesis to unusual eicosanoid fatty acids.

    Directory of Open Access Journals (Sweden)

    Woo Jung Park

    Full Text Available BACKGROUND: Genes coding for the fatty acid desaturases (FADS1, 2, 3 localized at the cancer genomic hotspot 11q13 locus are required for the biosynthesis of 20 carbon polyunsaturated fatty acids (PUFA that are direct eicosanoid precursors. In several cancer cell lines, FADS2 encoded Δ6 and Δ8 desaturation is not functional. METHODOLOGY/PRINCIPAL FINDINGS: Analyzing MCF7 cell fatty acids with detailed structural mass spectrometry, we show that in the absence of FADS2 activity, the FADS1 product Δ5-desaturase operates to produce 5,11,14-20∶3 and 5,11,14,17-20∶4. These PUFA are missing the 8-9 double bond of the eicosanoid signaling precursors arachidonic acid (5,8,11,14-20∶4 and eicosapentaenoic acid (5,8,11,14,17-20∶5. Heterologous expression of FADS2 restores Δ6 and Δ8-desaturase activity and normal eicosanoid precursor synthesis. CONCLUSIONS/SIGNIFICANCE: The loss of FADS2-encoded activities in cancer cells shuts down normal PUFA biosynthesis, deleting the endogenous supply of eicosanoid and downstream docosanoid precursors, and replacing them with unusual butylene-interrupted fatty acids. If recapitulated in vivo, the normal eicosanoid and docosanoid cell signaling milieu would be depleted and altered due to reduction and substitution of normal substrates with unusual substrates, with unpredictable consequences for cellular communication.

  15. Retinoid X Receptor Agonists Upregulate Genes Responsible for the Biosynthesis of All-Trans-Retinoic Acid in Human Epidermis.

    Directory of Open Access Journals (Sweden)

    Lizhi Wu

    Full Text Available UAB30 is an RXR selective agonist that has been shown to have potential cancer chemopreventive properties. Due to high efficacy and low toxicity, it is currently being evaluated in human Phase I clinical trials by the National Cancer Institute. While UAB30 shows promise as a low toxicity chemopreventive drug, the mechanism of its action is not well understood. In this study, we investigated the effects of UAB30 on gene expression in human organotypic skin raft cultures and mouse epidermis. The results of this study indicate that treatment with UAB30 results in upregulation of genes responsible for the uptake and metabolism of all-trans-retinol to all-trans-retinoic acid (ATRA, the natural agonist of RAR nuclear receptors. Consistent with the increased expression of these genes, the steady-state levels of ATRA are elevated in human skin rafts. In ultraviolet B (UVB irradiated mouse skin, the expression of ATRA target genes is found to be reduced. A reduced expression of ATRA sensitive genes is also observed in epidermis of mouse models of UVB-induced squamous cell carcinoma and basal cell carcinomas. However, treatment of mouse skin with UAB30 prior to UVB irradiation prevents the UVB-induced decrease in expression of some of the ATRA-responsive genes. Considering its positive effects on ATRA signaling in the epidermis and its low toxicity, UAB30 could be used as a chemoprophylactic agent in the treatment of non-melanoma skin cancer, particularly in organ transplant recipients and other high risk populations.

  16. Retinoid X Receptor Agonists Upregulate Genes Responsible for the Biosynthesis of All-Trans-Retinoic Acid in Human Epidermis.

    Science.gov (United States)

    Wu, Lizhi; Chaudhary, Sandeep C; Atigadda, Venkatram R; Belyaeva, Olga V; Harville, Steven R; Elmets, Craig A; Muccio, Donald D; Athar, Mohammad; Kedishvili, Natalia Y

    2016-01-01

    UAB30 is an RXR selective agonist that has been shown to have potential cancer chemopreventive properties. Due to high efficacy and low toxicity, it is currently being evaluated in human Phase I clinical trials by the National Cancer Institute. While UAB30 shows promise as a low toxicity chemopreventive drug, the mechanism of its action is not well understood. In this study, we investigated the effects of UAB30 on gene expression in human organotypic skin raft cultures and mouse epidermis. The results of this study indicate that treatment with UAB30 results in upregulation of genes responsible for the uptake and metabolism of all-trans-retinol to all-trans-retinoic acid (ATRA), the natural agonist of RAR nuclear receptors. Consistent with the increased expression of these genes, the steady-state levels of ATRA are elevated in human skin rafts. In ultraviolet B (UVB) irradiated mouse skin, the expression of ATRA target genes is found to be reduced. A reduced expression of ATRA sensitive genes is also observed in epidermis of mouse models of UVB-induced squamous cell carcinoma and basal cell carcinomas. However, treatment of mouse skin with UAB30 prior to UVB irradiation prevents the UVB-induced decrease in expression of some of the ATRA-responsive genes. Considering its positive effects on ATRA signaling in the epidermis and its low toxicity, UAB30 could be used as a chemoprophylactic agent in the treatment of non-melanoma skin cancer, particularly in organ transplant recipients and other high risk populations.

  17. DIETARY FOOD FORTIFIED WITH OROTIC ACID AND LIVER FUNCTION

    Directory of Open Access Journals (Sweden)

    Yohanes Buang

    2011-11-01

    Full Text Available The effects of dietary food fortified with orotic acid (1.0% on liver function were studied in rats. The rats fed withorotic acid promoted liver triglyceride content markedly, that was 5-fold higher than that of the control. The livermalondialdehyde (MDA content increased by 10%, but the gluthation peroxidase (GSH-Px activity decreased by 50%.The serum aspartate aminotransferase (AST and alanine aminotransferase (ALT activities increased by 25% and 30%,respectively. Therefore, the decreased GSH-Px activity was associated with the promotions of AST, ALT, and the liverMDA levels. In conclusion: dietary orotic acid promotes lipid peroxidation but reduces the rate of the antioxidantenzyme. Therefore, dietary food fortified with orotic acid attenuates the liver function.

  18. Biosynthesis and transport of terpenes

    NARCIS (Netherlands)

    Ting, H.M.

    2014-01-01

    Terpenoids are the largest class of natural product that are produced by plants, with functions that range from a role in plant development to direct defence against pathogens and indirect defence against insects through the attraction of natural enemies. While terpene biosynthesis genes have been w

  19. Essential fatty acids as functional components of foods- a review.

    Science.gov (United States)

    Kaur, Narinder; Chugh, Vishal; Gupta, Anil K

    2014-10-01

    During the recent decades, awareness towards the role of essential fatty acids in human health and disease prevention has been unremittingly increasing among people. Fish, fish oils and some vegetable oils are rich sources of essential fatty acids. Many studies have positively correlated essential fatty acids with reduction of cardiovascular morbidity and mortality, infant development, cancer prevention, optimal brain and vision functioning, arthritis, hypertension, diabetes mellitus and neurological/neuropsychiatric disorders. Beneficial effects may be mediated through several different mechanisms, including alteration in cell membrane composition, gene expression or eicosanoid production. However, the mechanisms whereby essential fatty acids affect gene expression are complex and involve multiple processes. Further understanding of the molecular aspects of essential fatty acids will be the key to devising novel approaches to the treatment and prevention of many diseases.

  20. Functional divergence of dafachronic acid pathways in the control of C. elegans development and lifespan.

    Science.gov (United States)

    Dumas, Kathleen J; Guo, Chunfang; Wang, Xi; Burkhart, Kirk B; Adams, Elizabeth J; Alam, Hena; Hu, Patrick J

    2010-04-15

    Steroid hormone and insulin/insulin-like growth factor signaling (IIS) pathways control development and lifespan in the nematode Caenorhabditis elegans by regulating the activity of the nuclear receptor DAF-12 and the FoxO transcription factor DAF-16, respectively. The DAF-12 ligands Delta(4)- and Delta(7)-dafachronic acid (DA) promote bypass of the dauer diapause and proper gonadal migration during larval development; in adults, DAs influence lifespan. Whether Delta(4)- and Delta(7)-DA have unique biological functions is not known. We identified the 3-beta-hydroxysteroid dehydrogenase (3betaHSD) family member HSD-1, which participates in Delta(4)-DA biosynthesis, as an inhibitor of DAF-16/FoxO activity. Whereas IIS promotes the cytoplasmic sequestration of DAF-16/FoxO, HSD-1 inhibits nuclear DAF-16/FoxO activity without affecting DAF-16/FoxO subcellular localization. Thus, HSD-1 and IIS inhibit DAF-16/FoxO activity via distinct and complementary mechanisms. In adults, HSD-1 was required for full lifespan extension in IIS mutants, indicating that HSD-1 interactions with IIS are context-dependent. In contrast to the Delta(7)-DA biosynthetic enzyme DAF-36, HSD-1 is dispensable for proper gonadal migration and lifespan extension induced by germline ablation. These findings provide insights into the molecular interface between DA and IIS pathways and suggest that Delta(4)- and Delta(7)-DA pathways have unique as well as overlapping biological functions in the control of development and lifespan.

  1. Retinoic acid synthesis and functions in early embryonic development

    Directory of Open Access Journals (Sweden)

    Kam Richard Kin Ting

    2012-03-01

    Full Text Available Abstract Retinoic acid (RA is a morphogen derived from retinol (vitamin A that plays important roles in cell growth, differentiation, and organogenesis. The production of RA from retinol requires two consecutive enzymatic reactions catalyzed by different sets of dehydrogenases. The retinol is first oxidized into retinal, which is then oxidized into RA. The RA interacts with retinoic acid receptor (RAR and retinoic acid X receptor (RXR which then regulate the target gene expression. In this review, we have discussed the metabolism of RA and the important components of RA signaling pathway, and highlighted current understanding of the functions of RA during early embryonic development.

  2. Chemical functionalization of hyaluronic acid for drug delivery applications

    Energy Technology Data Exchange (ETDEWEB)

    Vasi, Ana-Maria [“Gheorghe Asachi” Technical University of Iasi, Faculty of Chemical Engineering and Environmental Protection, 73 Prof. dr. docent Dimitrie Mangeron Street, 700050 Iasi (Romania); Popa, Marcel Ionel, E-mail: mipopa@ch.tuiasi.ro [“Gheorghe Asachi” Technical University of Iasi, Faculty of Chemical Engineering and Environmental Protection, 73 Prof. dr. docent Dimitrie Mangeron Street, 700050 Iasi (Romania); Butnaru, Maria [“Grigore T. Popa” University of Medicine Pharmacy, Faculty of Medical Bioengineering, 9-13 Kogalniceanu Street, 700454 Iasi (Romania); Dodi, Gianina [“Gheorghe Asachi” Technical University of Iasi, Faculty of Chemical Engineering and Environmental Protection, 73 Prof. dr. docent Dimitrie Mangeron Street, 700050 Iasi (Romania); SCIENT — Research Center for Instrumental Analysis, S.C. CROMATEC PLUS, 18 Sos. Cotroceni, 060114 Bucharest (Romania); Verestiuc, Liliana [“Grigore T. Popa” University of Medicine Pharmacy, Faculty of Medical Bioengineering, 9-13 Kogalniceanu Street, 700454 Iasi (Romania)

    2014-05-01

    Functionalized hyaluronic acid (HA) derivatives were obtained by ring opening mechanism of maleic anhydride (MA). FTIR and H{sup 1} NMR spectroscopy were used to confirm the chemical linkage of MA on the hyaluronic acid chains. Thermal analysis (TG-DTG and DSC) and GPC data for the new products revealed the formation of new functional groups, without significant changes in molecular weight and thermal stability. New gels based on hyaluronic acid modified derivatives were obtained by acrylic acid copolymerization in the presence of a redox initiation system. The resulted circular and interconnected pores of the gels were visualized by SEM. The release profiles of an ophthalmic model drug, pilocarpine from tested gels were studied in simulated media. Evaluation of the cytotoxicity and cell proliferation properties indicates the potential of the new systems to be used in contact with biological media in drug delivery applications. - Highlights: • New functionalized hyaluronic acid was prepared by ring opening of maleic anhydride. • Gels with circular pores based on acrylic acid copolymerization were formulated. • In vitro drug loading/release profile was evaluated in simulated ophthalmic media. • The cytotoxicity indicates the potential of derivatives to be used in vivo.

  3. Iron-sulfur cluster biosynthesis: functional characterization of the N- and C-terminal domains of human NFU.

    Science.gov (United States)

    Liu, Yushi; Qi, Wenbin; Cowan, J A

    2009-02-10

    Human NFU (also known as HIRIP5) has been implicated in cellular iron-sulfur cluster biosynthesis. Bacterial and yeast forms are smaller than the human protein and are homologous to the C-terminal domain of the latter. This C-terminal domain contains a pair of redox active cysteines and demonstrates thioredoxin-like activity by mediating persulfide bond cleavage of sulfur-loaded NifS (an IscS-type protein), the sulfide donor for [2Fe-2S] cluster assembly on ISU-type scaffold proteins. Herein, the affinity of full-length human NFU and the individual N- and C-terminal domains for sulfide donor and cluster scaffold proteins is assessed. The influence of the N-terminal domain on C-terminal NFU binding to NifS and persulfide reductase activity is also examined. Only the C-terminal domain is required for persulfide reductase activity, while complex formation of NifS with full-length NFU is similar to that of the C-terminal domain alone (K(D) approximately 9.7 +/- 0.7 and 10.1 +/- 0.6 microM, respectively). There is negligible affinity between the isolated C- and N-terminal domains, while the N-terminal domain has negligible affinity for either sulfide donor or cluster scaffold proteins. The temperature dependence of the binding enthalpy for formation of the complex between NifS and the C-terminal domain of NFU yields a change in molar heat capacity (DeltaC(p) approximately 138 cal mol(-1) K(-1)) that suggests bonding at the protein-protein interface is dominated by electrostatic interactions. This is consistent with electrostatic potential maps for bacterial homologues of the N- and C-terminal domains of human NFU, which most likely reflect the structural characteristics expected for full-length human NFU.

  4. Gibberellin biosynthesis in Gibberlla fujikuroi

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, S.W.; Coolbaugh, R.C. (Iowa State Univ., Ames (USA))

    1989-04-01

    Gibberellins (GAs) are a group of plant growth hormones which were first isolated from the fungus Gibberella fujikuori. We have examined the biosynthesis of GAs in this fungus in liquid cultures using HPLC followed by GC-MS. Furthermore we have used cell-free enzyme extracts with {sup 14}C-labeled intermediates to examine the regulation of specific parts of the biosynthetic pathway. GA{sub 3} is the predominant GA in well aerated cultures. GA{sub 4} and GA{sub 7}, intermediates in GA{sub 3} biosynthesis, accumulate in cultures with low levels of dissolved oxygen, but are not detectable in more aerated cultures. Light stimulates GA production in G. fujikuroi cultures grown from young stock. Cell-free enzyme studies indicate that light has no effect on incorporation of mevalonic acid into kaurene, but does significantly stimulate the oxidation of kaurenoic acid.

  5. The cloning, characterization, and functional analysis of a gene encoding an isopentenyl diphosphate isomerase involved in triterpene biosynthesis in the Lingzhi or reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes).

    Science.gov (United States)

    Wu, Feng-Li; Shi, Liang; Yao, Jian; Ren, Ang; Zhou, Chao; Mu, Da-Shuai; Zhao, Ming-Wen

    2013-01-01

    An isopentenyl diphosphate isomerase (IDI) gene, GlIDI, was isolated from Ganoderma lucidum, which produces triterpenes through the mevalonate pathway. The open reading frame of GlIDI encodes a 252 amino acid polypeptide with a theoretical molecular mass of 28.71 kDa and a theoretical isoelectric point of 5.36. GlIDI is highly homologous to other fungal IDIs and contains conserved active residues and nudix motifs shared by the IDI protein family. The color complementation assay indicated that GlIDI can accelerate the accumulation of β-carotene and confirmed that the cloned complementary DNA encoded a functional GlIDI protein. Gene expression analysis showed that the GlIDI transcription level was relatively low in the mycelia and reached a relatively high level in the mushroom primordia. In addition, its expression level could be up-regulated by 254 µM methyl jasmonate. Our results suggest that this enzyme may play an important role in triterpene biosynthesis.

  6. Characterization of multiple SPS knockout mutants reveals redundant functions of the four Arabidopsis sucrose phosphate synthase isoforms in plant viability, and strongly indicates that enhanced respiration and accelerated starch turnover can alleviate the blockage of sucrose biosynthesis.

    Science.gov (United States)

    Bahaji, Abdellatif; Baroja-Fernández, Edurne; Ricarte-Bermejo, Adriana; Sánchez-López, Ángela María; Muñoz, Francisco José; Romero, Jose M; Ruiz, María Teresa; Baslam, Marouane; Almagro, Goizeder; Sesma, María Teresa; Pozueta-Romero, Javier

    2015-09-01

    We characterized multiple knock-out mutants of the four Arabidopsis sucrose phosphate synthase (SPSA1, SPSA2, SPSB and SPSC) isoforms. Despite their reduced SPS activity, spsa1/spsa2, spsa1/spsb, spsa2/spsb, spsa2/spsc, spsb/spsc, spsa1/spsa2/spsb and spsa2/spsb/spsc mutants displayed wild type (WT) vegetative and reproductive morphology, and showed WT photosynthetic capacity and respiration. In contrast, growth of rosettes, flowers and siliques of the spsa1/spsc and spsa1/spsa2/spsc mutants was reduced compared with WT plants. Furthermore, these plants displayed a high dark respiration phenotype. spsa1/spsb/spsc and spsa1/spsa2/spsb/spsc seeds poorly germinated and produced aberrant and sterile plants. Leaves of all viable sps mutants, except spsa1/spsc and spsa1/spsa2/spsc, accumulated WT levels of nonstructural carbohydrates. spsa1/spsc leaves possessed high levels of metabolic intermediates and activities of enzymes of the glycolytic and tricarboxylic acid cycle pathways, and accumulated high levels of metabolic intermediates of the nocturnal starch-to-sucrose conversion process, even under continuous light conditions. Results presented in this work show that SPS is essential for plant viability, reveal redundant functions of the four SPS isoforms in processes that are important for plant growth and nonstructural carbohydrate metabolism, and strongly indicate that accelerated starch turnover and enhanced respiration can alleviate the blockage of sucrose biosynthesis in spsa1/spsc leaves.

  7. Carboxylic acid-functionalized SBA-15 nanorods for gemcitabine delivery

    Science.gov (United States)

    Bahrami, Zohreh; Badiei, Alireza; Ziarani, Ghodsi Mohammadi

    2015-03-01

    The present study deals with the functionalization of mesoporous silica nanoparticles as drug delivery systems. Mono, di, and tri amino-functionalized SBA-15 nanorods were synthesized by post-grafting method using (3-aminopropyl) triethoxysilane, N-(2-aminoethyl-)3- aminopropyltrimethoxysilane, and 3-[2-(2-aminoethylamino) ethylamino] propyl trimethoxysilane, respectively. The carboxylic acid derivatives of the amino-functionalized samples were obtained using succinic anhydride. Tminopropyltrimethoxysilanehe obtained modified materials were investigated as matrixes for the anticancer drug (gemcitabine) delivery. The prepared samples were characterized by SAXS, N2 adsorption/desorption, SEM, transmission electron microscopy, thermogravimetric analysis, and FTIR and UV spectroscopies. The adsorption and release properties of all samples were studied. It was revealed that the adsorption capacity and release behavior of gemcitabine were highly dependent on the type of the introduced functional groups. The carboxylic acid-modified samples have higher loading content, due to the strong interaction with gemcitabine. The maximum content of deposited drug in the modified SBA-15 nanorods is close to 40 wt%. It was found that the surface functionalization leads toward significant decrease of the drug release rate. The carboxylic acid-functionalized samples have slower release rate in contrast with the amino-functionalized samples.

  8. Carboxylic acid-functionalized SBA-15 nanorods for gemcitabine delivery

    Energy Technology Data Exchange (ETDEWEB)

    Bahrami, Zohreh; Badiei, Alireza, E-mail: abadiei@khayam.ut.ac.ir [University of Tehran, School of Chemistry, College of Science (Iran, Islamic Republic of); Ziarani, Ghodsi Mohammadi [Alzahra University, Research Laboratory of Pharmaceutical (Iran, Islamic Republic of)

    2015-03-15

    The present study deals with the functionalization of mesoporous silica nanoparticles as drug delivery systems. Mono, di, and tri amino-functionalized SBA-15 nanorods were synthesized by post-grafting method using (3-aminopropyl) triethoxysilane, N-(2-aminoethyl-)3- aminopropyltrimethoxysilane, and 3-[2-(2-aminoethylamino) ethylamino] propyl trimethoxysilane, respectively. The carboxylic acid derivatives of the amino-functionalized samples were obtained using succinic anhydride. Tminopropyltrimethoxysilanehe obtained modified materials were investigated as matrixes for the anticancer drug (gemcitabine) delivery. The prepared samples were characterized by SAXS, N{sub 2} adsorption/desorption, SEM, transmission electron microscopy, thermogravimetric analysis, and FTIR and UV spectroscopies. The adsorption and release properties of all samples were studied. It was revealed that the adsorption capacity and release behavior of gemcitabine were highly dependent on the type of the introduced functional groups. The carboxylic acid-modified samples have higher loading content, due to the strong interaction with gemcitabine. The maximum content of deposited drug in the modified SBA-15 nanorods is close to 40 wt%. It was found that the surface functionalization leads toward significant decrease of the drug release rate. The carboxylic acid-functionalized samples have slower release rate in contrast with the amino-functionalized samples.

  9. Engineering of Glucosinolate Biosynthesis

    DEFF Research Database (Denmark)

    Møldrup, Morten Emil; Salomonsen, Bo; Halkier, Barbara Ann

    2012-01-01

    -efficient methods for identification and validation of candidate genes are needed. This chapter covers the methodology we are using for gene discovery in glucosinolate engineering, namely, guilt-by-association-based in silico methods and fast proof-of-function screens by transient expression in Nicotiana...... of glucosinolate biosynthesis, although in planta validation of candidate gene function often is hampered by time-consuming generation of knockout and overexpression lines in Arabidopsis. To better exploit the increasing amount of data available from genomic sequencing, microarray database and RNAseq, time...... benthamiana. Moreover,the lessons learned in the rapid, transient tobacco system are readily translated to our robust, versatile yeast expression platform, where additional genes critical for large-scale microbial production of glucosinolates can be identified. We anticipate that the methodology presented...

  10. Fruit color mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis

    Science.gov (United States)

    Isoprenoids are a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, IPP is synthesized in the cytoplasm from mevalonic acid via the “MVA pathway” a...

  11. Structural and functional characterization of CalS11, a TDP-rhamnose 3′-O-methyltransferase involved in calicheamicin biosynthesis

    Science.gov (United States)

    Singh, Shanteri; Chang, Aram; Helmich, Kate E.; Bingman, Craig A.; Wrobel, Russel L.; Beebe, Emily T.; Makino, Shin-Ichi; Aceti, David J.; Dyer, Kevin; Hura, Greg L.; Sunkara, Manjula; Morris, Andrew J.; Phillips, George N.; Thorson, Jon S.

    2013-01-01

    Sugar methyltransferases (MTs) are an important class of tailoring enzymes which catalyze the transfer of a methyl group from S-adenosyl-L-methionine to sugar-based N-, C- and O- nucleophiles. While sugar N- and C-MTs involved in natural product biosynthesis have been found to act on sugar nucleotide substrates prior to a subsequent glycosyltransferase reaction, corresponding sugar O-methylation reactions studied thus far occur after the glycosyltransfer reaction. Herein we report the first in vitro characterization using 1H-13C-gHSQC with isotopically-labeled substrates and the X-ray structure determination at 1.55 Å resolution of the TDP-3′-O-rhamnose-methyltransferase CalS11 from Micromonospora echinospora. This study highlights a unique NMR-based methyltransferase assay, implicates CalS11 to be a metal and general acid/base-dependent O-methyltransferase and, as a first crystal structure for a TDP-hexose-O-methyltransferase, presents a new template for mechanistic studies and/or engineering. PMID:23662776

  12. Structural and functional characterization of CalS11, a TDP-rhamnose 3'-O-methyltransferase involved in calicheamicin biosynthesis.

    Science.gov (United States)

    Singh, Shanteri; Chang, Aram; Helmich, Kate E; Bingman, Craig A; Wrobel, Russell L; Beebe, Emily T; Makino, Shin-Ichi; Aceti, David J; Dyer, Kevin; Hura, Greg L; Sunkara, Manjula; Morris, Andrew J; Phillips, George N; Thorson, Jon S

    2013-07-19

    Sugar methyltransferases (MTs) are an important class of tailoring enzymes that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to sugar-based N-, C- and O-nucleophiles. While sugar N- and C-MTs involved in natural product biosynthesis have been found to act on sugar nucleotide substrates prior to a subsequent glycosyltransferase reaction, corresponding sugar O-methylation reactions studied thus far occur after the glycosyltransfer reaction. Herein we report the first in vitro characterization using (1)H-(13)C-gHSQC with isotopically labeled substrates and the X-ray structure determination at 1.55 Å resolution of the TDP-3'-O-rhamnose-methyltransferase CalS11 from Micromonospora echinospora. This study highlights a unique NMR-based methyltransferase assay, implicates CalS11 to be a metal- and general acid/base-dependent O-methyltransferase, and as a first crystal structure for a TDP-hexose-O-methyltransferase, presents a new template for mechanistic studies and/or engineering.

  13. Ferulic acid 5-hydroxylase 1 is essential for expression of anthocyanin biosynthesis-associated genes and anthocyanin accumulation under photooxidative stress in Arabidopsis.

    Science.gov (United States)

    Maruta, Takanori; Noshi, Masahiro; Nakamura, Maki; Matsuda, Shun; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

    2014-04-01

    Anthocyanins are important for preventing photoinhibition and photodamage. By comprehensive reverse genetic analysis of chloroplast-produced H2O2-responsive genes, we isolated here an anthocyanin-deficient mutant under photooxidative stress, which lacked ferulate 5-hydroxylase 1 (FAH1) involved in the phenylpropanoid pathway. Interestingly, the expression of anthocyanin biosynthesis-associated genes was also inhibited in this mutant. These findings suggest that FAH1 is essential for expression of anthocyanin biosynthesis-associated genes and anthocyanin accumulation under photooxidative stress in Arabidopsis. Furthermore, we found that estrogen-inducible silencing of thylakoid membrane-bound ascorbate peroxidase, which is a major H2O2-scavenging enzyme in chloroplasts, enhances the expression of FAH1 and anthocyanin biosynthesis-associated genes and accumulation of anthocyanin without any application of stress. Thus, it is likely that chloroplastic H2O2 activates FAH1 expression to induce anthocyanin accumulation for protecting cells from photooxidative stress.

  14. Functionalization of carbon nanotube yarn by acid treatment

    Directory of Open Access Journals (Sweden)

    H.E. Misak

    2014-01-01

    Full Text Available Carbon nanotube (CNT yarn was functionalized using sulfuric and nitric acid solutions in 3:1 volumetric ratio. Successful functionalization of CNT yarn with carboxyl and hydroxyl groups (e.g., COOH, COO–, OH, etc. was confirmed by attenuated total reflectance spectroscopy. X-ray diffraction revealed no significant change to the atomic in-plane alignment in the CNTs; however, the coherent length along the diameter was significantly reduced during functionalization. A morphology change of wavy extensions protruding from the surface was observed after the functionalization treatment. The force required to fracture the yarn remained the same after the functionalization process; however, the linear density was increased (310%. The increase in linear density after functionalization reduced the tenacity. However, the resistivity density product of the CNT yarn was reduced significantly (234% after functionalization.

  15. 4-mercaptophenylboronic acid functionalized gold nanoparticles for colorimetric sialic acid detection.

    Science.gov (United States)

    Sankoh, Supannee; Thammakhet, Chongdee; Numnuam, Apon; Limbut, Warakorn; Kanatharana, Proespichaya; Thavarungkul, Panote

    2016-11-15

    A simple and selective colorimetric sensor for sialic acid detection, based on the aggregation of 4-mercaptophenylboronic acid functionalized gold nanoparticles (4-MPBA-AuNPs) was developed. The color of the solution changed from wine-red to blue after binding with sialic acid. The colorimetric sensor provided good analytical performances with a linear dynamic range of 80µM to 2.00mM and a 68±2µM limit of detection without any effect from possible interferences and sample matrix. In addition, the quantitative results were obtained within only 10min. This developed sensor was used to detect sialic acid in blood serum samples and the results were in good agreement with those from the current periodate-resorcinol method (P>0.05) thus indicating that this developed colorimetric sensor can be used as an alternative method for sialic acid detection with a shorter analysis time and a high accuracy.

  16. 乳酸菌胞外多糖的生物合成及其遗传调控%Biosynthesis and Genetic Regulation of Exopolysaccharides from Lactic Acid Bacteria

    Institute of Scientific and Technical Information of China (English)

    姚晶; 任婧; 吴正钧; 王荫榆; 郭本恒

    2011-01-01

    Exopolysaccharides is produced by Lactic acid bacteria which is General Recognized As Safe status (GRAS). As its excellent functions, it is becoming a research hotspot recently. However, its industrial application is limited by the low production level. Therefore, it is significant for studying the genetics to improve the production.This paper mainly concerns about the classification and structure of exopolysaccharides, biosynthesis of homopolysaccharides and heteropolysaccharides, besides the discrepancies between these two exopolysaccharides. Genetic regulation and some effective regulation measures are closely discussed as well. This paper should provide a theoretical reference for further research.%作为食品级的乳酸菌分泌的胞外多糖,由于其具有很多优良的功能特性,成为了近年来的研究热点.胞外多糖产量偏低限制了其工业化应用,因此从基因水平上探索增加乳酸菌胞外多糖产量的途径是大有裨益的.文中详细阐述了胞外多糖的分类、结构,同型多糖和异性多糖生物合成过程及其差异,以及在合成过程中的遗传调控,并介绍了一些增加产量的有效调控手段,为深入的研究提供理论参考.

  17. A functional genomics approach to understand the control and regulation of storage protein biosynthesis in barley grain

    DEFF Research Database (Denmark)

    Vincze, É; Hansen, M; Bowra, S;

    2008-01-01

    The aim of the study was to obtain an insight into amino acid and storage protein metabolism in the developing barley grain at the molecular level. Our strategy was to analyse the transcriptome of relevant pathways in developing grains of field grown barley using a grain specific microarray...... reference to the temporal expression levels of a range of genes involved in the photosynthesis process, amino acid and storage protein metabolism. We conclude that the grain specific microarray is a reliable and cost-effective tool for monitoring temporal changes in the transcriptome of the major metabolic...... pathways in the barley grain. The study described here could provide a strong complement to existing knowledge assisting further  understanding of seed development and thereby provide a foundation for plant breeding towards storage protein with improved nutritional quality....

  18. Functional genomics of lactic acid bacteria: from food to health.

    Science.gov (United States)

    Douillard, François P; de Vos, Willem M

    2014-08-29

    Genome analysis using next generation sequencing technologies has revolutionized the characterization of lactic acid bacteria and complete genomes of all major groups are now available. Comparative genomics has provided new insights into the natural and laboratory evolution of lactic acid bacteria and their environmental interactions. Moreover, functional genomics approaches have been used to understand the response of lactic acid bacteria to their environment. The results have been instrumental in understanding the adaptation of lactic acid bacteria in artisanal and industrial food fermentations as well as their interactions with the human host. Collectively, this has led to a detailed analysis of genes involved in colonization, persistence, interaction and signaling towards to the human host and its health. Finally, massive parallel genome re-sequencing has provided new opportunities in applied genomics, specifically in the characterization of novel non-GMO strains that have potential to be used in the food industry. Here, we provide an overview of the state of the art of these functional genomics approaches and their impact in understanding, applying and designing lactic acid bacteria for food and health.

  19. Analysis of the pmsCEAB Gene Cluster Involved in Biosynthesis of Salicylic Acid and the Siderophore Pseudomonine in the Biocontrol Strain Pseudomonas fluorescens WCS374

    NARCIS (Netherlands)

    Mercado-Blanco, J.; Drift, K.M.G.M. van der; Olsson, P.E.; Thomas-Oates, J.E.; Loon, L.C. van; Bakker, P.A.H.M.

    2001-01-01

    Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the bios

  20. Transcription factors in alkaloid biosynthesis.

    Science.gov (United States)

    Yamada, Yasuyuki; Sato, Fumihiko

    2013-01-01

    Higher plants produce a large variety of low-molecular weight secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used pharmaceutically. Whereas alkaloid chemistry has been intensively investigated, alkaloid biosynthesis, including the relevant biosynthetic enzymes, genes and their regulation, and especially transcription factors, is largely unknown, as only a limited number of plant species produce certain types of alkaloids and they are difficult to study. Recently, however, several groups have succeeded in isolating the transcription factors that are involved in the biosynthesis of several types of alkaloids, including bHLH, ERF, and WRKY. Most of them show Jasmonate (JA) responsiveness, which suggests that the JA signaling cascade plays an important role in alkaloid biosynthesis. Here, we summarize the types and functions of transcription factors that have been isolated in alkaloid biosynthesis, and characterize their similarities and differences compared to those in other secondary metabolite pathways, such as phenylpropanoid and terpenoid biosyntheses. The evolution of this biosynthetic pathway and regulatory network, as well as the application of these transcription factors to metabolic engineering, is discussed.

  1. Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L. and their relevance for perfume manufacture

    Directory of Open Access Journals (Sweden)

    Caniard Anne

    2012-07-01

    Full Text Available Abstract Background Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Results Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S-copalyl diphosphate. A class I diTPS (SsSS then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae. Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. Conclusions SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid

  2. Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture

    Science.gov (United States)

    2012-01-01

    Background Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Results Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. Conclusions SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol

  3. From heterochromatin islands to the NAD World: a hierarchical view of aging through the functions of mammalian Sirt1 and systemic NAD biosynthesis.

    Science.gov (United States)

    Imai, Shin-ichiro

    2009-10-01

    For the past couple of decades, aging science has been rapidly evolving, and powerful genetic tools have identified a variety of evolutionarily conserved regulators and signaling pathways for the control of aging and longevity in model organisms. Nonetheless, a big challenge still remains to construct a comprehensive concept that could integrate many distinct layers of biological events into a systemic, hierarchical view of aging. The "heterochromatin island" hypothesis was originally proposed 10 years ago to explain deterministic and stochastic aspects of cellular and organismal aging, which drove the author to the study of evolutionarily conserved Sir2 proteins. Since a surprising discovery of their NAD-dependent deacetylase activity, Sir2 proteins, now called "sirtuins," have been emerging as a critical epigenetic regulator for aging. In this review, I will follow the process of conceptual development from the heterochromatin island hypothesis to a novel, comprehensive concept of a systemic regulatory network for mammalian aging, named "NAD World," summarizing recent studies on the mammalian NAD-dependent deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis. This new concept of the NAD World provides critical insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of aging in mammals.

  4. Molecular Cloning and Functional Analysis of Gene Clusters for the Biosynthesis of Indole-Diterpenes in Penicillium crustosum and P. janthinellum.

    Science.gov (United States)

    Nicholson, Matthew J; Eaton, Carla J; Stärkel, Cornelia; Tapper, Brian A; Cox, Murray P; Scott, Barry

    2015-07-23

    The penitremane and janthitremane families of indole-diterpenes are abundant natural products synthesized by Penicillium crustosum and P. janthinellum. Using a combination of PCR, cosmid library screening, and Illumina sequencing we have identified gene clusters encoding enzymes for the synthesis of these compounds. Targeted deletion of penP in P. crustosum abolished the synthesis of penitrems A, B, D, E, and F, and led to accumulation of paspaline, a key intermediate for paxilline biosynthesis in P. paxilli. Similarly, deletion of janP and janD in P. janthinellum abolished the synthesis of prenyl-elaborated indole-diterpenes, and led to accumulation in the latter of 13-desoxypaxilline, a key intermediate for the synthesis of the structurally related aflatremanes synthesized by Aspergillus flavus. This study helps resolve the genetic basis for the complexity of indole-diterpene natural products found within the Penicillium and Aspergillus species. All indole-diterpene gene clusters identified to date have a core set of genes for the synthesis of paspaline and a suite of genes encoding multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases, and prenyl transferases that catalyse various regio- and stereo- specific oxidations that give rise to the diversity of indole-diterpene products synthesized by this group of fungi.

  5. Molecular Cloning and Functional Analysis of Gene Clusters for the Biosynthesis of Indole-Diterpenes in Penicillium crustosum and P. janthinellum

    Directory of Open Access Journals (Sweden)

    Matthew J. Nicholson

    2015-07-01

    Full Text Available The penitremane and janthitremane families of indole-diterpenes are abundant natural products synthesized by Penicillium crustosum and P. janthinellum. Using a combination of PCR, cosmid library screening, and Illumina sequencing we have identified gene clusters encoding enzymes for the synthesis of these compounds. Targeted deletion of penP in P. crustosum abolished the synthesis of penitrems A, B, D, E, and F, and led to accumulation of paspaline, a key intermediate for paxilline biosynthesis in P. paxilli. Similarly, deletion of janP and janD in P. janthinellum abolished the synthesis of prenyl-elaborated indole-diterpenes, and led to accumulation in the latter of 13-desoxypaxilline, a key intermediate for the synthesis of the structurally related aflatremanes synthesized by Aspergillus flavus. This study helps resolve the genetic basis for the complexity of indole-diterpene natural products found within the Penicillium and Aspergillus species. All indole-diterpene gene clusters identified to date have a core set of genes for the synthesis of paspaline and a suite of genes encoding multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases, and prenyl transferases that catalyse various regio- and stereo- specific oxidations that give rise to the diversity of indole-diterpene products synthesized by this group of fungi.

  6. Cloning and bioinformatic analysis of lovastatin biosynthesis regulatory gene lovE

    Institute of Scientific and Technical Information of China (English)

    HUANG Xin; LI Hao-ming

    2009-01-01

    Background Lovastatin is an effective drug for treatment of hyperlipidemia.This study aimed to clone Iovastatin biosynthesis regulatory gene lovE and analyze the structure and function of its encoding protein.Methods According to the Iovastatin synthase gene sequence from genebank,primers were designed to amplify and clone the Iovastatin biosynthesis regulatory gene lovE from Aspergillus terms genomic DNA.Bioinformatic analysis of lovE and its encoding animo acid sequence was performed through intemet resources and software like DNAMAN.Results Target fragment lovE,almost 1500 bp in length,was amplified from Aspergillus terms genomic DNA and the secondary and three-dimensional structures of LovE protein were predicted.Conclusion In the Iovastatin biosynthesis process lovE is a regulatory gene and LovE protein is a GAL4-1ike transcriptional factor.

  7. Application of carboxyphenylboronic acid-functionalized magnetic nanoparticles for extracting nucleic acid from seeds.

    Science.gov (United States)

    Sun, Ning; Deng, Congliang; Ge, Guanglu; Xia, Qiang

    2015-01-01

    Magnetic iron oxide nanoparticles functionalized with 4-carboxyphenylboronic acid (CPBA-MNPs) were developed for extracting genomic DNA, total RNA and nucleic acids from seeds. The seed samples were genetically-modified maize seeds and unmodified soybean seeds infected by bean pod mottle virus and tobacco ringspot virus. The total nucleic acids, genomic DNA, and RNA could be separately extracted from these seeds with high qualities using CPBA-MNPs under different conditions. Furthermore, the results of real-time quantitative qPCR and real-time reverse transcription (RT)-PCR indicated that the nucleic acids extracted from these seeds using CPBA-MNPs were suitable for the detection of genetically-modified seeds and seed-borne viruses.

  8. Functional fermented whey-based beverage using lactic acid bacteria.

    Science.gov (United States)

    Pescuma, Micaela; Hébert, Elvira María; Mozzi, Fernanda; de Valdez, Graciela Font

    2010-06-30

    Whey protein concentrate (WPC) is employed as functional food ingredient because of its nutritional value and emulsifying properties. However, the major whey protein beta-lactoglobulin (BLG) is the main cause of milk allergy. The aim of this study was to formulate a fermented whey beverage using selected lactic acid bacteria and WPC35 (WPC containing 35% of proteins) to obtain a fermented product with low lactose and BLG contents and high essential amino acid concentration. Cell viability, lactose consumption, lactic acid production, proteolytic activity, amino acid release and BLG degradation by the selected strains Lactobacillus acidophilus CRL 636, Lactobacillus delbrueckii subsp. bulgaricus CRL 656 and Streptococcus thermophilus CRL 804, as single or mixed (SLaB) cultures were evaluated in WPC35 (10%, w/v) incubated at 37 degrees C for 24h. Then, the fermented WPC35 was mixed with peach juice and calcium lactate (2%, w/v) and stored at 10 degrees C for 28 days. During fermentation, single cultures grew 1.7-3.1 log CFU/ml and produced 25.1-95.0 mmol/l of lactic acid as consequence of lactose consumption (14.0-41.8 mmol/l) after 12h fermentation. L. delbrueckii subsp. bulgaricus CRL 656 was the most proteolytic strain (626 microg/ml Leu) and released the branched-chain essential amino acids Leu (16 microg/ml), Ile (27 microg/ml) and Val (43 microg/ml). All strains were able to degrade BLG in a range of 41-85% after 12h incubation. The starter culture SLaB grew 3.0 log CFU/ml, showed marked pH reduction, produced 122.0 mmol/l of lactic acid, displayed high proteolytic activity (484 microg/ml Leu) releasing Leu (13 microg/ml), Ile (18 microg/ml) and Val (35 microg/ml), and hydrolyzed 92% of BLG. The addition of calcium lactate to WPC35 maintained the drink pH stable during shelf life; no contamination was detected during this period. After 28 days, a decrease in cell viability of all strains was observed being more pronounced for L. delbrueckii subsp. bulgaricus

  9. Fatty Acid Signaling: The New Function of Intracellular Lipases

    Directory of Open Access Journals (Sweden)

    Zuzana Papackova

    2015-02-01

    Full Text Available Until recently, intracellular triacylglycerols (TAG stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed.

  10. Amino acid rejection behaviour as a function of concentration.

    Science.gov (United States)

    Shirley, Jason; Mandale, Stephen; Williams, Paul M

    2011-05-11

    The solute rejection versus concentration behaviour of five different amino acids has been investigated using a Nitto Denko NTR7450 nanofiltration membrane. The experimental data for amino acid rejection was also compared against a combined steric and charge rejection model. At its isoelectric point, lysine was effectively neutral and its behaviour was well described by the model incorporating a steric function only. For phenylalanine, the combined model was found to fit the data well. In contrast there was poor agreement between the model and rejection data for glutamine, glutamic acid and glycine whose rejection values at first increased with concentration. This result implied that another governing process was in operation. Dimerisation as an explanation for the observed phenomena was also investigated. Size analysis of amino acid molecules as a function of the prevailing concentration using dynamic light scattering was limited but showed no evidence of dimerisation. This data was supported by osmotic pressure measurements which demonstrated no evidence of non-linearity in the relation between osmotic pressure and concentration.

  11. Functional properties and fatty acids profile of different beans varieties.

    Science.gov (United States)

    Lo Turco, Vincenzo; Potortì, Angela Giorgia; Rando, Rossana; Ravenda, Pietro; Dugo, Giacomo; Di Bella, Giuseppa

    2016-10-01

    Dried seeds of four varieties of Phaseolus vulgaris, three of Vigna unguiculata ssp. unguiculata and two of Vigna angularis grown and marketed in Italy, Mexico, India, Japan, Ghana and Ivory Coast were analysed for fatty acids content. In oils from seeds of P. vulgaris, the main fatty acids were linolenic (34.7-41.5%) and linoleic (30.7-40.3%), followed by palmitic (10.7-16.8%). The first three aforementioned fatty acids in the lipid fraction of V. unguiculata varieties were 28.4, 28.7 and 26.2%, respectively; while in V. angularis varieties, main fatty acids were linoleic (36.4-39.1%) and palmitic (26.9-33.3%), followed by linolenic (17.9-22.2%). Statistical analyses indicate that botanical species play a rule in bean fatty acids distribution, while the same was not verified for geographical origin. Furthermore, the atherogenic index (AI) and the thrombogenic index (TI) were investigated for health and nutritional information. The results showed that these wide spread legumes have functional features to human health.

  12. NAMPT-mediated NAD+ biosynthesis in adipocytes regulates adipose tissue function and multi-organ insulin sensitivity in mice

    Science.gov (United States)

    Stromsdorfer, Kelly L.; Yamaguchi, Shintaro; Yoon, Myeong Jin; Moseley, Anna C.; Franczyk, Michael P.; Kelly, Shannon C.; Qi, Nathan; Imai, Shin-ichiro; Yoshino, Jun

    2016-01-01

    SUMMARY Obesity is associated with adipose tissue dysfunction and multi-organ insulin resistance. However, the mechanisms of such obesity-associated systemic metabolic complications are not clear. Here, we characterized mice with adipocyte-specific deletion of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting NAD+ biosynthetic enzyme known to decrease in adipose tissue of obese and aged rodents and people. We found that adipocyte-specific Nampt knockout mice had severe insulin resistance in adipose tissue, liver, and skeletal muscle, and adipose tissue dysfunction, manifested by increased plasma free fatty acids concentrations and decreased plasma concentrations of a major insulin-sensitizing adipokine, adiponectin. Loss of Nampt increased phosphorylation of CDK5 and PPARγ (serine-273) and decreased gene expression of obesity-linked phosphorylated PPARγ targets in adipose tissue. Remarkably, these deleterious alterations were normalized by administering rosiglitazone or a key NAD+ intermediate, nicotinamide mononucleotide (NMN). Collectively, our results provide important mechanistic and therapeutic insights into obesity-associated systemic metabolic derangements, particularly multi-organ insulin resistance. PMID:27498863

  13. De novo biosynthesis of linoleic acid and its conversion to the hydrocarbon (Z,Z)-6,9-heptadecadiene in the astigmatid mite, Carpoglyphus lactis: incorporation experiments with 13C-labeled glucose.

    Science.gov (United States)

    Shimizu, Nobuhiro; Naito, Michiya; Mori, Naoki; Kuwahara, Yasumasa

    2014-02-01

    De novo biosynthesis of linoleic acid (LA) and its conversion to (Z,Z)-6,9-heptadecadiene were examined in Carpoglyphus lactis (Acarina, Carpoglyphidae). Experiments involving (13)C-administration using [1-(13)C]-d-glucose revealed that (13)C atoms were incorporated into LA of total lipid extracted from the mite, resulting in labeling of all even-numbered carbons. This result demonstrated that LA was produced from (13)C-labeled acetyl-CoA, which is indicative of direct de novo biosynthesis. In these feeding experiments involving [1-(13)C]-D-glucose, (13)C atoms were also incorporated into (Z,Z)-6,9-heptadecadiene, which is one of the major secretory components in the mite. The labeling pattern of (Z,Z)-6,9-heptadecadiene at odd-numbered carbons agreed well with that of LA after loss of the carboxyl carbon. It was concluded that the mites could stably convert LA into (Z,Z)-6,9-heptadecadiene without the dietary requirement of this essential fatty acid.

  14. Cytochrome P450 family member CYP704B2 catalyzes the {omega}-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice.

    Science.gov (United States)

    Li, Hui; Pinot, Franck; Sauveplane, Vincent; Werck-Reichhart, Danièle; Diehl, Patrik; Schreiber, Lukas; Franke, Rochus; Zhang, Ping; Chen, Liang; Gao, Yawei; Liang, Wanqi; Zhang, Dabing

    2010-01-01

    The anther cuticle and microspore exine act as protective barriers for the male gametophyte and pollen grain, but relatively little is known about the mechanisms underlying the biosynthesis of the monomers of which they are composed. We report here the isolation and characterization of a rice (Oryza sativa) male sterile mutant, cyp704B2, which exhibits a swollen sporophytic tapetal layer, aborted pollen grains without detectable exine, and undeveloped anther cuticle. In addition, chemical composition analysis indicated that cutin monomers were hardly detectable in the cyp704B2 anthers. These defects are caused by a mutation in a cytochrome P450 family gene, CYP704B2. The CYP704B2 transcript is specifically detected in the tapetum and the microspore from stage 8 of anther development to stage 10. Heterologous expression of CYP704B2 in yeast demonstrated that CYP704B2 catalyzes the production of omega -hydroxylated fatty acids with 16 and 18 carbon chains. Our results provide insights into the biosynthesis of the two biopolymers sporopollenin and cutin. Specifically, our study indicates that the omega -hydroxylation pathway of fatty acids relying on this ancient CYP704B family, conserved from moss to angiosperms, is essential for the formation of both cuticle and exine during plant male reproductive and spore development.

  15. (-)-Menthol biosynthesis and molecular genetics

    Science.gov (United States)

    Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

    2005-12-01

    (-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

  16. A UDP-glucosyltransferase functions in both acylphloroglucinol glucoside and anthocyanin biosynthesis in strawberry (Fragaria × ananassa).

    Science.gov (United States)

    Song, Chuankui; Zhao, Shuai; Hong, Xiaotong; Liu, Jingyi; Schulenburg, Katja; Schwab, Wilfried

    2016-03-01

    Physiologically active acylphloroglucinol (APG) glucosides were recently found in strawberry (Fragaria sp.) fruit. Although the formation of the APG aglycones has been clarified, little is known about APG glycosylation in plants. In this study we functionally characterized ripening-related glucosyltransferase genes in Fragaria by comprehensive biochemical analyses of the encoded proteins and by a RNA interference (RNAi) approach in vivo. The allelic proteins UGT71K3a/b catalyzed the glucosylation of diverse hydroxycoumarins, naphthols and flavonoids as well as phloroglucinols, enzymatically synthesized APG aglycones and pelargonidin. Total enzymatic synthesis of APG glucosides was achieved by co-incubation of recombinant dual functional chalcone/valerophenone synthase and UGT71K3 proteins with essential coenzyme A esters and UDP-glucose. An APG glucoside was identified in strawberry fruit which has not yet been reported in other plants. Suppression of UGT71K3 activity in transient RNAi-silenced fruits led to a loss of pigmentation and a substantial decrease of the levels of various APG glucosides and an anthocyanin. Metabolite analyses of transgenic fruits confirmed UGT71K3 as a UDP-glucose:APG glucosyltransferase in planta. These results provide the foundation for the breeding of fruits with improved health benefits and for the biotechnological production of bioactive natural products.

  17. Characterization of an Arabidopsis-Phytophthora pathosystem: resistance requires a functional PAD2 gene and is independent of salicylic acid, ethylene and jasmonic acid signalling.

    Science.gov (United States)

    Roetschi, A; Si-Ammour, A; Belbahri, L; Mauch, F; Mauch-Mani, B

    2001-11-01

    Arabidopsis accessions were screened with isolates of Phytophthora porri originally isolated from other crucifer species. The described Arabidopsis-Phytophthora pathosystem shows the characteristics of a facultative biotrophic interaction similar to that seen in agronomically important diseases caused by Phytophthora species. In susceptible accessions, extensive colonization of the host tissue occurred and sexual and asexual spores were formed. In incompatible combinations, the plants reacted with a hypersensitive response (HR) and the formation of papillae at the sites of attempted penetration. Defence pathway mutants such as jar1 (jasmonic acid-insensitive), etr1 (ethylene receptor mutant) and ein2 (ethylene-insensitive) remained resistant towards P. porri. However, pad2, a mutant with reduced production of the phytoalexin camalexin, was hyper-susceptible. The accumulation of salicylic acid (SA) and PR1 protein was strongly reduced in pad2. Surprisingly, this lack of SA accumulation does not appear to be the cause of the hyper-susceptibility because interference with SA signalling in nahG plants or sid2 or npr1 mutants had only a minor effect on resistance. In addition, the functional SA analogue benzothiadiazol (BTH) did not induce resistance in susceptible plants including pad2. Similarly, the complete blockage of camalexin biosynthesis in pad3 did not cause susceptibility. Resistance of Arabidopsis against P. porri appears to depend on unknown defence mechanisms that are under the control of PAD2.

  18. Functional conservation of the capacity for ent-kaurene biosynthesis and an associated operon in certain rhizobia.

    Science.gov (United States)

    Hershey, David M; Lu, Xuan; Zi, Jiachen; Peters, Reuben J

    2014-01-01

    Bacterial interactions with plants are accompanied by complex signal exchange processes. Previously, the nitrogen-fixing symbiotic (rhizo)bacterium Bradyrhizobium japonicum was found to carry adjacent genes encoding two sequentially acting diterpene cyclases that together transform geranylgeranyl diphosphate to ent-kaurene, the olefin precursor to the gibberellin plant hormones. Species from the three other major genera of rhizobia were found to have homologous terpene synthase genes. Cloning and functional characterization of a representative set of these enzymes confirmed the capacity of each genus to produce ent-kaurene. Moreover, comparison of their genomic context revealed that these diterpene synthases are found in a conserved operon which includes an adjacent isoprenyl diphosphate synthase, shown here to produce the geranylgeranyl diphosphate precursor, providing a critical link to central metabolism. In addition, the rest of the operon consists of enzymatic genes that presumably lead to a more elaborated diterpenoid, although the production of gibberellins was not observed. Nevertheless, it has previously been shown that the operon is selectively expressed during nodulation, and the scattered distribution of the operon via independent horizontal gene transfer within the symbiotic plasmid or genomic island shown here suggests that such diterpenoid production may modulate the interaction of these particular symbionts with their host plants.

  19. Chiroptical Properties of Amino Acids: A Density Functional Theory Study

    Directory of Open Access Journals (Sweden)

    Martine Adrian-Scotto

    2010-04-01

    Full Text Available Amino acids are involved in many scientific theories elucidating possible origins of life on Earth. One of the challenges when discussing the evolutionary origin of biopolymers such as proteins and oligonucleotides in living organisms is the phenomenon that these polymers implement monomers of exclusively one handedness, a feature called biomolecular homochirality. Many attempts have been made to understand this process of racemic symmetry breaking. Assuming an extraterrestrial origin of the molecular building blocks of living organisms, their susceptibility to asymmetric photolysis by the absorption of circularly polarized electromagnetic radiation in interstellar space was proposed. In order to predict whether the interaction of circularly polarized light with various racemic amino acids can induce an enantiomeric excess, we investigated the electronic and chiroptical properties of the amino acids valine and isovaline by a molecular modelling approach based on quantum chemistry (Density Functional Theory. The average spectra of both L-valine and L-isovaline have been produced on the basis of Boltzmann population analysis using computed spectra for the various conformations of each amino acid.

  20. The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts.

    Science.gov (United States)

    Kowalska, Ewa; Kozik, Andrzej

    2008-01-01

    Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge.

  1. An extra-cytoplasmic function sigma factor and anti-sigma factor control carotenoid biosynthesis in Azospirillum brasilense.

    Science.gov (United States)

    Thirunavukkarasu, Nagarajan; Mishra, Mukti Nath; Spaepen, Stijn; Vanderleyden, Jos; Gross, Carol A; Tripathi, Anil K

    2008-07-01

    Strains Sp7 and Cd of Azospirillum brasilense, a plant growth-promoting rhizobacterium, differ in synthesis of carotenoids. While colonies of strain Sp7 have a white-cream colour on plates, colonies of strain Cd are orange-pink coloured because of the synthesis of carotenoids. Screening of a mini-Tn5 mutant library of A. brasilense Sp7 revealed two orange-pink-coloured mutants that produced carotenoids. Cloning and sequencing of the Tn5 flanking region in both the carotenoid-producing mutants of Sp7 revealed insertion of Tn5 in an ORF encoding anti-sigma factor, a ChrR-like protein. The upstream region of the Tn5-mutated ORF contained another ORF that encoded an extra-cytoplasmic function (ECF)-class sigma factor (sigma(E), RpoE). When the nucleotide sequences of the corresponding ORFs from the carotenoid-producing strain Cd were analysed, the sequence of the Cd sigma(E) was identical to that of the carotenoid non-producing strain Sp7, but the Cd anti-sigma(E) ORF had a deletion that caused frame shifting and creation of a stop codon. This resulted in the premature termination of the protein, which was about 7 kDa smaller than the Sp7 anti-sigma(E). Cloning of Sp7 anti-sigma(E) in a broad-host-range expression vector and expression in A. brasilense Cd and in the anti-sigma(E) knockout mutant of A. brasilense Sp7 resulted in the inhibition of carotenoid synthesis. Similarly, cloning and overexpression of A. brasilense Sp7 sigma(E) in A. brasilense Sp7 resulted in the production of carotenoids. These observations clearly indicate that carotenoid synthesis in A. brasilense is controlled by sigma(E) with its cognate anti-sigma(E).

  2. The upregulation of thiamine (vitamin B1 biosynthesis in Arabidopsis thaliana seedlings under salt and osmotic stress conditions is mediated by abscisic acid at the early stages of this stress response

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    Rapala-Kozik Maria

    2012-01-01

    Full Text Available Abstract Background Recent reports suggest that vitamin B1 (thiamine participates in the processes underlying plant adaptations to certain types of abiotic and biotic stress, mainly oxidative stress. Most of the genes coding for enzymes involved in thiamine biosynthesis in Arabidopsis thaliana have been identified. In our present study, we examined the expression of thiamine biosynthetic genes, of genes encoding thiamine diphosphate-dependent enzymes and the levels of thiamine compounds during the early (sensing and late (adaptation responses of Arabidopsis seedlings to oxidative, salinity and osmotic stress. The possible roles of plant hormones in the regulation of the thiamine contribution to stress responses were also explored. Results The expression of Arabidopsis genes involved in the thiamine diphosphate biosynthesis pathway, including that of THI1, THIC, TH1 and TPK, was analyzed for 48 h in seedlings subjected to NaCl or sorbitol treatment. These genes were found to be predominantly up-regulated in the early phase (2-6 h of the stress response. The changes in these gene transcript levels were further found to correlate with increases in thiamine and its diphosphate ester content in seedlings, as well as with the enhancement of gene expression for enzymes which require thiamine diphosphate as a cofactor, mainly α-ketoglutarate dehydrogenase, pyruvate dehydrogenase and transketolase. In the case of the phytohormones including the salicylic, jasmonic and abscisic acids which are known to be involved in plant stress responses, only abscisic acid was found to significantly influence the expression of thiamine biosynthetic genes, the thiamine diphosphate levels, as well as the expression of genes coding for main thiamine diphosphate-dependent enzymes. Using Arabidopsis mutant plants defective in abscisic acid production, we demonstrate that this phytohormone is important in the regulation of THI1 and THIC gene expression during salt stress

  3. Regulatory cross-talks and cascades in rice hormone biosynthesis pathways contribute to stress signaling

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    Arindam Deb

    2016-08-01

    Full Text Available Crosstalk among different hormone signaling pathways play an important role in modulating plant response to both biotic and abiotic stress. Hormone activity is controlled by its bio-availability, which is again influenced by its biosynthesis. Thus independent hormone biosynthesis pathways must be regulated and co-ordinated to mount an integrated response. One of the possibilities is to use cis-regulatory elements to orchestrate expression of hormone biosynthesis genes. Analysis of CREs, associated with differentially expressed hormone biosynthesis related genes in rice leaf under Magnaporthe oryzae attack and drought stress enabled us to obtain insights about cross-talk among hormone biosynthesis pathways at the transcriptional level. We identified some master transcription regulators that co-ordinate different hormone biosynthesis pathways under stress. We found that Abscisic acid and Brassinosteroid regulate Cytokinin conjugation; conversely Brassinosteroid biosynthesis is affected by both Abscisic acid and Cytokinin. Jasmonic acid and Ethylene biosynthesis may be modulated by Abscisic acid through DREB transcription factors. Jasmonic acid or Salicylic acid biosynthesis pathways are co-regulated but they are unlikely to influence each other’s production directly. Thus multiple hormones may modulate hormone biosynthesis pathways through a complex regulatory network, where biosynthesis of one hormone is affected by several other contributing hormones.

  4. Regulatory Cross-Talks and Cascades in Rice Hormone Biosynthesis Pathways Contribute to Stress Signaling.

    Science.gov (United States)

    Deb, Arindam; Grewal, Rumdeep K; Kundu, Sudip

    2016-01-01

    Crosstalk among different hormone signaling pathways play an important role in modulating plant response to both biotic and abiotic stress. Hormone activity is controlled by its bio-availability, which is again influenced by its biosynthesis. Thus, independent hormone biosynthesis pathways must be regulated and co-ordinated to mount an integrated response. One of the possibilities is to use cis-regulatory elements to orchestrate expression of hormone biosynthesis genes. Analysis of CREs, associated with differentially expressed hormone biosynthesis related genes in rice leaf under Magnaporthe oryzae attack and drought stress enabled us to obtain insights about cross-talk among hormone biosynthesis pathways at the transcriptional level. We identified some master transcription regulators that co-ordinate different hormone biosynthesis pathways under stress. We found that Abscisic acid and Brass