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Sample records for acid biosynthetic pathway

  1. Substrate specificity of the sialic acid biosynthetic pathway

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    Jacobs, Christina L.; Goon, Scarlett; Yarema, Kevin J.; Hinderlich, Stephan; Hang, Howard C.; Chai, Diana H.; Bertozzi, Carolyn R.

    2001-07-18

    Unnatural analogs of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogs bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell-surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogs with ketone-containing N-acyl groups that varied in the lengthor steric bulk was chemically synthesized and tested for metabolic conversion to cell-surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

  2. Indole-3-acetic acid in Fusarium graminearum: Identification of biosynthetic pathways and characterization of physiological effects.

    Science.gov (United States)

    Luo, Kun; Rocheleau, Hélène; Qi, Peng-Fei; Zheng, You-Liang; Zhao, Hui-Yan; Ouellet, Thérèse

    2016-09-01

    Fusarium graminearum is a devastating pathogenic fungus causing fusarium head blight (FHB) of wheat. This fungus can produce indole-3-acetic acid (IAA) and a very large amount of IAA accumulates in wheat head tissues during the first few days of infection by F. graminearum. Using liquid culture conditions, we have determined that F. graminearum can use tryptamine (TAM) and indole-3-acetonitrile (IAN) as biosynthetic intermediates to produce IAA. It is the first time that F. graminearum is shown to use the l-tryptophan-dependent TAM and IAN pathways rather than the indole-3-acetamide or indole-3-pyruvic acid pathways to produce IAA. Our experiments also showed that exogenous IAA was metabolized by F. graminearum. Exogenous IAA, TAM, and IAN inhibited mycelial growth; IAA and IAN also affected the hyphae branching pattern and delayed macroconidium germination. IAA and TAM had a small positive effect on the production of the mycotoxin 15-ADON while IAN inhibited its production. Our results showed that IAA and biosynthetic intermediates had a significant effect on F. graminearum physiology and suggested a new area of exploration for fungicidal compounds. PMID:27567719

  3. Orthogonal Fatty Acid Biosynthetic Pathway Improves Fatty Acid Ethyl Ester Production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Eriksen, Dawn T; HamediRad, Mohammad; Yuan, Yongbo; Zhao, Huimin

    2015-07-17

    Fatty acid ethyl esters (FAEEs) are a form of biodiesel that can be microbially produced via a transesterification reaction of fatty acids with ethanol. The titer of microbially produced FAEEs can be greatly reduced by unbalanced metabolism and an insufficient supply of fatty acids, resulting in a commercially inviable process. Here, we report on a pathway engineering strategy in Saccharomyces cerevisiae for enhancing the titer of microbially produced FAEEs by providing the cells with an orthogonal route for fatty acid synthesis. The fatty acids generated from this heterologous pathway would supply the FAEE production, safeguarding endogenous fatty acids for cellular metabolism and growth. We investigated the heterologous expression of a Type-I fatty acid synthase (FAS) from Brevibacterium ammoniagenes coupled with WS/DGAT, the wax ester synthase/acyl-coenzyme that catalyzes the transesterification reaction with ethanol. Strains harboring the orthologous fatty acid synthesis yielded a 6.3-fold increase in FAEE titer compared to strains without the heterologous FAS. Variations in fatty acid chain length and degree of saturation can affect the quality of the biodiesel; therefore, we also investigated the diversity of the fatty acid production profile of FAS enzymes from other Actinomyces organisms. PMID:25594225

  4. Biosynthesis of monoterpenoids in higher plants. The biosynthetic pathway leading to the monoterpenoids from amino acids with a carbon-skeleton similar to mevalonic acid

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    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.

  5. Expression of genes associated with the biosynthetic pathways of abscisic acid, gibberellin, and ethylene during the germination of lettuce seeds.

    Science.gov (United States)

    Clemente, A C S; Guimarães, R M; Martins, D C; Gomes, L A A; Caixeta, F; Reis, R G E; Rosa, S D V F

    2015-01-01

    Seed germination and dormancy are complex phenomena that are controlled by many genes and environmental factors. Such genes are indicated by phytohormones that interact with each other, and may cause dormancy or promote seed germination. The objective of this study was to investigate gene expression associated with the biosynthetic pathways of abscisic acid (ABA), gibberellic acid (GA), and ethylene (ET) in dormant and germinated lettuce seeds. The expressions of LsNCED, LsGA3ox1, and ACO-B were evaluated in germinating and dormant seeds from the cultivars Everglades, Babá de Verão, Verônica, Salinas, Colorado, and Regina 71. The expressions of LsNCED, LsGA3ox1, and ACO-B were related to the biosynthesis of ABA, GA, and ET, respectively; therefore, the presence of these substances depends on genotype. LsNCED expression only occurred in dormant seeds, and was connected to dormancy. LsGA3ox1expression only occurred in germinated seeds, and was connected to germination. The ACO-B gene was involved in ET biosynthesis, and was expressed differently in germinated and dormant seeds, depending on the genotype, indicating different functions for different characteristics. Furthermore, sensitivity to phytohormones appeared to be more important than the expression levels of LsNCED, LsGA3ox1, or ACO-B.

  6. Cloning and characterization of the gene encoding β-amyrin synthase in the glycyrrhizic acid biosynthetic pathway in Glycyrrhiza uralensis

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    Honghao Chen

    2013-12-01

    Full Text Available Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic. Based on previous research, these effects are mediated by a number of active ingredients, especially glycyrrhizic acid (GA. In the present study, a gene encoding β-amyrin synthase (β-AS involved in GA biosynthesis in G. uralensis has been cloned and expressed in Saccharomyces cerevisiae. The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene into β-amyrin. In fact the β-AS gene is particularly important in the GA biosynthetic pathway in G. uralensis. The complete sequence of the enzyme was determined and a phylogenetic tree based on the β-AS gene of G. uralensis and 20 other species was created. This showed that Glycyrrhiza glabra had the closest kinship with G. uralensis. The results of this work will be useful in determining how to improve the efficacy of G. uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro.

  7. Evaluation of Biosynthetic Pathways of 2Н- and 13С-Labeled Amino Acids by an Obligate Methylotrophic Bacterium Methylobacillus Flagellatum and a Facultative Methylotrophic Bacterium Brevibacterium Methylicum

    Directory of Open Access Journals (Sweden)

    Oleg Mosin

    2016-06-01

    Full Text Available By the method of electron impact mass-spectrometry was studied the pathways of biosynthesis of 2H, 13C-labeled amino acids of a facultative methylotrophic bacterium Brevibacterium methylicum and an obligate methylotrophic bacterium Methylobacillus flagellatum obtained on growth media containing as a source of stable isotopes [2H]methanol, [13C]methanol and 2H2O. For mass-spectrometric analysis the multicomponential mixtures of 2H- and 13C-labeled amino acids, derived from cultural media and protein hydrolysates after hydrolysis in 6 M 2HСl (3 % phenol and 2 M Ва(OH2 were modified into N-benzyloxycarbonyl-derivatives of amino acids as well as into methyl esters of N-5-(dimethylaminonaphthalene-1-sulfonyl chloride (dansyl derivatives of [2H, 13С]amino acids, which were preparative separated using a method of reverse-phase HCLP. Biosynthetically obtained 2H- and 13C-labeled amino acids represented the mixtures differing in quantities of isotopes incorporated into molecule. The levels of 2H and 13С enrichment of secreted amino acids and amino acid resigues of protein were found to vary from 20,0 atom % to L-leucine/isoleucine up to 97,5 atom % for L-alanine depending on concentration of 2H- and 13C-labelled substrates.

  8. Mining and engineering natural-product biosynthetic pathways.

    Science.gov (United States)

    Wilkinson, Barrie; Micklefield, Jason

    2007-07-01

    Natural products continue to fulfill an important role in the development of therapeutic agents. In addition, with the advent of chemical genetics and high-throughput screening platforms, these molecules have become increasingly valuable as tools for interrogating fundamental aspects of biological systems. To access the vast portion of natural-product structural diversity that remains unexploited for these and other applications, genome mining and microbial metagenomic approaches are proving particularly powerful. When these are coupled with recombineering and related genetic tools, large biosynthetic gene clusters that remain intractable or cryptic in the native host can be more efficiently cloned and expressed in a suitable heterologous system. For lead optimization and the further structural diversification of natural-product libraries, combinatorial biosynthetic engineering has also become indispensable. However, our ability to rationally redesign biosynthetic pathways is often limited by our lack of understanding of the structure, dynamics and interplay between the many enzymes involved in complex biosynthetic pathways. Despite this, recent structures of fatty acid synthases should allow a more accurate prediction of the likely architecture of related polyketide synthase and nonribosomal peptide synthetase multienzymes. PMID:17576425

  9. Molecular cloning and promoter analysis of the specific salicylic acid biosynthetic pathway gene phenylalanine ammonia-lyase (AaPAL1) from Artemisia annua.

    Science.gov (United States)

    Zhang, Ying; Fu, Xueqing; Hao, Xiaolong; Zhang, Lida; Wang, Luyao; Qian, Hongmei; Zhao, Jingya

    2016-07-01

    Phenylalanine ammonia-lyase (PAL) is the key enzyme in the biosynthetic pathway of salicylic acid (SA). In this study, a full-length cDNA of PAL gene (named as AaPAL1) was cloned from Artemisia annua. The gene contains an open reading frame of 2,151 bps encoding 716 amino acids. Comparative and bioinformatics analysis revealed that the polypeptide protein of AaPAL1 was highly homologous to PALs from other plant species. Southern blot analysis revealed that it belonged to a gene family with three members. Quantitative RT-PCR analysis of various tissues of A. annua showed that AaPAL1 transcript levels were highest in the young leaves. A 1160-bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including W-box, TGACG-motif, and TC-rich repeats. Quantitative RT-PCR indicated that AaPAL1 was upregulated by salinity, drought, wounding, and SA stresses, which were corroborated positively with the identified cis-elements within the promoter region. AaPAL1 was successfully expressed in Escherichia. coli and the enzyme activity of the purified AaPAL1 was approximately 287.2 U/mg. These results substantiated the involvement of AaPAL1 in the phenylalanine pathway. PMID:26040426

  10. The biosynthetic pathway of vitamin C in higher plants.

    Science.gov (United States)

    Wheeler, G L; Jones, M A; Smirnoff, N

    1998-05-28

    Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

  11. Structural and Functional Analysis of Campylobacter jejuni PseG: a Udp-sugarhydrolase from the Pseudaminic Acid Biosynthetic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    E Rangarajan; A Proteau; Q Cui; S Logan; Z Potetinova; D Whitfield; E Purisima; M Cygler; A Matte; et al.

    2011-12-31

    Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-{beta}-l-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 {angstrom} resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His{sup 17}-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His{sup 17} functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C-O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.

  12. Biosynthetic pathway of terpenoid indole alkaloids in Catharanthus roseus.

    Science.gov (United States)

    Zhu, Xiaoxuan; Zeng, Xinyi; Sun, Chao; Chen, Shilin

    2014-09-01

    Catharanthus roseus is one of the most extensively investigated medicinal plants, which can produce more than 130 alkaloids, including the powerful antitumor drugs vinblastine and vincristine. Here we review the recent advances in the biosynthetic pathway of terpenoid indole alkaloids (TIAs) in C. roseus, and the identification and characterization of the corresponding enzymes involved in this pathway. Strictosidine is the central intermediate in the biosynthesis of different TIAs, which is formed by the condensation of secologanin and tryptamine. Secologanin is derived from terpenoid (isoprenoid) biosynthetic pathway, while tryptamine is derived from indole biosynthetic pathway. Then various specific end products are produced by different routes during downstream process. Although many genes and corresponding enzymes have been characterized in this pathway, our knowledge on the whole TIA biosynthetic pathway still remains largely unknown up to date. Full elucidation of TIA biosynthetic pathway is an important prerequisite to understand the regulation of the TIA biosynthesis in the medicinal plant and to produce valuable TIAs by synthetic biological technology. PMID:25159992

  13. Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms

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    Wang, Yan; Wang, Liuqing; Liu, Fei; Wang, Qi; Selvaraj, Jonathan Nimal; Xing, Fuguo; Zhao, Yueju; Liu, Yang

    2016-01-01

    Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes of the biosynthetic pathway. We discuss recent information on molecular studies of OTA biosynthesis despite insufficient genetic background in detail. Accordingly, genetic regulation has also been explored with regard to the interaction between the regulators and the environmental factors. In this review, we focus on three aspects of OTA: OTA-producing strains, OTA biosynthetic pathway and the regulation mechanisms of OTA production. This can pave the way to assist in protecting food and feed from OTA contamination by understanding OTA biosynthetic pathway and regulatory mechanisms. PMID:27007394

  14. THE CAROTENOID BIOSYNTHETIC PATHWAY: THINKING IN ALL DIMENSIONS

    OpenAIRE

    Shumskaya, Maria; Wurtzel, Eleanore T.

    2013-01-01

    The carotenoid biosynthetic pathway serves manifold roles in plants related to photosynthesis, photoprotection, development, stress hormones, and various volatiles and signalling apocarotenoids. The pathway also produces compounds that impact human nutrition and metabolic products that contribute to fragrance and flavour of food and non-food crops. It is no surprise that the pathway has been a target of metabolic engineering, most prominently in the case of Golden Rice. The future success and...

  15. The carotenoid biosynthetic pathway: thinking in all dimensions.

    Science.gov (United States)

    Shumskaya, Maria; Wurtzel, Eleanore T

    2013-07-01

    The carotenoid biosynthetic pathway serves manifold roles in plants related to photosynthesis, photoprotection, development, stress hormones, and various volatiles and signaling apocarotenoids. The pathway also produces compounds that impact human nutrition and metabolic products that contribute to fragrance and flavor of food and non-food crops. It is no surprise that the pathway has been a target of metabolic engineering, most prominently in the case of Golden Rice. The future success and predictability of metabolic engineering of carotenoids rests in the ability to target carotenoids for specific physiological purposes as well as to simultaneously modify carotenoids along with other desired traits. Here, we ask whether predictive metabolic engineering of the carotenoid pathway is indeed possible. Despite a long history of research on the pathway, at this point in time we can only describe the pathway as a parts list and have almost no knowledge of the location of the complete pathway, how it is assembled, and whether there exists any trafficking of the enzymes or the carotenoids themselves. We discuss the current state of knowledge regarding the "complete" pathway and make the argument that predictive metabolic engineering of the carotenoid pathway (and other pathways) will require investigation of the three dimensional state of the pathway as it may exist in plastids of different ultrastructures. Along with this message we point out the need to develop new types of visualization tools and resources that better reflect the dynamic nature of biosynthetic pathways. PMID:23683930

  16. Nutrient shortage triggers the hexosamine biosynthetic pathway via the GCN2-ATF4 signalling pathway.

    Science.gov (United States)

    Chaveroux, Cédric; Sarcinelli, Carmen; Barbet, Virginie; Belfeki, Sofiane; Barthelaix, Audrey; Ferraro-Peyret, Carole; Lebecque, Serge; Renno, Toufic; Bruhat, Alain; Fafournoux, Pierre; Manié, Serge N

    2016-06-03

    The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2α signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked β-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signalling.

  17. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids.

    Science.gov (United States)

    Kishimoto, Shinji; Sato, Michio; Tsunematsu, Yuta; Watanabe, Kenji

    2016-01-01

    Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin (tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details. PMID:27548127

  18. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

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    Shinji Kishimoto

    2016-08-01

    Full Text Available Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid, saframycin (tetrahydroisoquinoline alkaloid, strictosidine (monoterpene indole alkaloid, ergotamine (ergot alkaloid and opiates (benzylisoquinoline and morphinan alkaloid. This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.

  19. A kinetic model for the penicillin biosynthetic pathway in

    DEFF Research Database (Denmark)

    Nielsen, Jens; Jørgensen, Henrik

    1996-01-01

    A kinetic model for the first two steps in the penicillin biosynthetic pathway, i.e. the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS) is proposed. The model is based on Michaelis-Menten type kinetics with non-competitive inhibition of the ACVS by ACV, and competitive inhibition...... that there is a shift in the flux control from the ACVS to the IPNS during the cultivation....

  20. Evolutionary systems biology of amino acid biosynthetic cost in yeast.

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    Michael D Barton

    Full Text Available Every protein has a biosynthetic cost to the cell based on the synthesis of its constituent amino acids. In order to optimise growth and reproduction, natural selection is expected, where possible, to favour the use of proteins whose constituents are cheaper to produce, as reduced biosynthetic cost may confer a fitness advantage to the organism. Quantifying the cost of amino acid biosynthesis presents challenges, since energetic requirements may change across different cellular and environmental conditions. We developed a systems biology approach to estimate the cost of amino acid synthesis based on genome-scale metabolic models and investigated the effects of the cost of amino acid synthesis on Saccharomyces cerevisiae gene expression and protein evolution. First, we used our two new and six previously reported measures of amino acid cost in conjunction with codon usage bias, tRNA gene number and atomic composition to identify which of these factors best predict transcript and protein levels. Second, we compared amino acid cost with rates of amino acid substitution across four species in the genus Saccharomyces. Regardless of which cost measure is used, amino acid biosynthetic cost is weakly associated with transcript and protein levels. In contrast, we find that biosynthetic cost and amino acid substitution rates show a negative correlation, but for only a subset of cost measures. In the economy of the yeast cell, we find that the cost of amino acid synthesis plays a limited role in shaping transcript and protein expression levels compared to that of translational optimisation. Biosynthetic cost does, however, appear to affect rates of amino acid evolution in Saccharomyces, suggesting that expensive amino acids may only be used when they have specific structural or functional roles in protein sequences. However, as there appears to be no single currency to compute the cost of amino acid synthesis across all cellular and environmental

  1. Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through Combinatorial Pathway Reassembly in Engineered Yeast.

    Science.gov (United States)

    Tang, Man-Cheng; Lin, Hsiao-Ching; Li, Dehai; Zou, Yi; Li, Jian; Xu, Wei; Cacho, Ralph A; Hillenmeyer, Maureen E; Garg, Neil K; Tang, Yi

    2015-11-01

    The structural diversity and biological activities of fungal indole diterpenes (IDTs) are generated in large part by the IDT cyclases (IDTCs). Identifying different IDTCs from IDT biosynthetic pathways is therefore important toward understanding how these enzymes introduce chemical diversity from a common linear precursor. However, IDTCs involved in the cyclization of the well-known aflavinine subgroup of IDTs have not been discovered. Here, using Saccharomyces cerevisiae as a heterologous host and a phylogenetically guided enzyme mining approach, we combinatorially assembled IDT biosynthetic pathways using IDTCs homologues identified from different fungal hosts. We identified the genetically standalone IDTCs involved in the cyclization of aflavinine and anominine and produced new IDTs not previously isolated. The cyclization mechanisms of the new IDTCs were proposed based on the yeast reconstitution results. Our studies demonstrate heterologous pathway assembly is a useful tool in the reconstitution of unclustered biosynthetic pathways.

  2. Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

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    Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

    2015-09-30

    Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

  3. Evolution of tryptophan biosynthetic pathway in microbial genomes: a comparative genetic study.

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    Priya, V K; Sarkar, Susmita; Sinha, Somdatta

    2014-03-01

    Biosynthetic pathway evolution needs to consider the evolution of a group of genes that code for enzymes catalysing the multiple chemical reaction steps leading to the final end product. Tryptophan biosynthetic pathway has five chemical reaction steps that are highly conserved in diverse microbial genomes, though the genes of the pathway enzymes show considerable variations in arrangements, operon structure (gene fusion and splitting) and regulation. We use a combined bioinformatic and statistical analyses approach to address the question if the pathway genes from different microbial genomes, belonging to a wide range of groups, show similar evolutionary relationships within and between them. Our analyses involved detailed study of gene organization (fusion/splitting events), base composition, relative synonymous codon usage pattern of the genes, gene expressivity, amino acid usage, etc. to assess inter- and intra-genic variations, between and within the pathway genes, in diverse group of microorganisms. We describe these genetic and genomic variations in the tryptophan pathway genes in different microorganisms to show the similarities across organisms, and compare the same genes across different organisms to find the possible variability arising possibly due to horizontal gene transfers. Such studies form the basis for moving from single gene evolution to pathway evolutionary studies that are important steps towards understanding the systems biology of intracellular pathways. PMID:24592292

  4. Alanylclavam Biosynthetic Genes Are Clustered Together with One Group of Clavulanic Acid Biosynthetic Genes in Streptomyces clavuligerus▿ §

    Science.gov (United States)

    Zelyas, Nathan J.; Cai, Hui; Kwong, Thomas; Jensen, Susan E.

    2008-01-01

    Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway. PMID:18931110

  5. Elucidation of the Vanillin Biosynthetic Pathway in Vanilla planifolia

    DEFF Research Database (Denmark)

    Gallage, Nethaji Janeshawari

    harvested pods are processed by curing to stop the natural vegetative process and to initiate the enzymes that are responsible for the formation of the well-known aromatic flavour constituents. Vanillin (3-methoxy-4-hydroxybenzaldehyde) is the main flavour component of vanilla extract from cured vanilla...... pods. As vanillin is toxic to living organisms in high concentrations, vanilla plants store vanillin almost entirely as the glucose conjugated form, vanillin-β-D-glucoside. The highest concentration of vanillin glucoside is localized in the inner part of the pod including mesocarp and placenta 6 months...... after the pollination. Subcellular localization of vanillin and its glucoside was speculated to be in the vacuole. Despite the popularity of the flavour, the vanillin biosynthetic pathway has remained elusive, presumably due to lack of genetic and genomic resources during past few decades. The research...

  6. Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2008-07-01

    Full Text Available Abstract Background High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes. Results Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1 is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP increases the riboflavin accumulation significantly. Conclusion The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established.

  7. Narrow-spectrum inhibitors targeting an alternative menaquinone biosynthetic pathway of Helicobacter pylori.

    Science.gov (United States)

    Yamamoto, Tsuyoshi; Matsui, Hidenori; Yamaji, Kenzaburo; Takahashi, Tetsufumi; Øverby, Anders; Nakamura, Masahiko; Matsumoto, Atsuko; Nonaka, Kenichi; Sunazuka, Toshiaki; Ōmura, Satoshi; Nakano, Hirofumi

    2016-09-01

    We aimed to identify narrow-spectrum natural compounds that specifically inhibit an alternative menaquinone (MK; vitamin K2) biosynthetic pathway (the futalosine pathway) of Helicobacter pylori. Culture broth samples of 6183 microbes were examined using the paper disc method with different combinations of 2 of the following 3 indicator microorganisms: Bacillus halodurans C-125 and Kitasatospora setae KM-6054(T), which have only the futalosine pathway of MK biosynthesis, and Bacillus subtilis H17, which has only the canonical MK biosynthetic pathway. Most of the active compounds isolated from culture broth samples were from the families of polyunsaturated fatty acids (PUFAs). Only one compound isolated from the culture broth of Streptomyces sp. K12-1112, siamycin I (a 21-residue lasso peptide antibiotic), targeted the futalosine pathway. The inhibitory activities of representative PUFAs and siamycin I against the growth of B. halodurans or K. setae were abrogated by supplementation with MK. Thereafter, the growth of H. pylori strains SS1 and TN2GF4 in broth cultures was dose-dependently suppressed by eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or siamycin I, and these inhibitory effects were reduced by supplementation with MK. Daily administration of EPA (100 μM), DHA (100 μM), or siamycin I (2.5 μM) in drinking water reduced the H. pylori SS1 colonization in the gastric mucosa of C57BL/6 mice by 96%, 78%, and 68%, respectively. These data suggest that EPA, DHA, and siamycin I prevented H. pylori infection by inhibiting the futalosine pathway of MK biosynthesis. PMID:27346378

  8. Characterization of cyanobacterial hydrocarbon composition and distribution of biosynthetic pathways.

    Directory of Open Access Journals (Sweden)

    R Cameron Coates

    Full Text Available Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene and methyl group positions (3-, 4- and 5-methylheptadecane for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR and aldehyde deformylating oxygenase (ADO. The second involves a polyketide synthase (PKS pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS. Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both.

  9. Regulatory structure of the biosynthetic pathway for the aspartate family of amino acids in Lemna paucicostata Hegelm. 6746, with special reference to the role of aspartokinase

    International Nuclear Information System (INIS)

    Comprehensive studies were made with Lemna paucicostate Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme along being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [14C]threonine and [14C]homoserine in Lemna

  10. The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Marco Garavaglia

    Full Text Available Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.

  11. Apicoplast Biosynthetic Pathways as Possible Targetsfor Combination Therapy of Malaria

    Institute of Scientific and Technical Information of China (English)

    Solomon Tesfaye; Bhanu Prakash; Prati Pal Singh

    2015-01-01

    The emergence of malaria parasite strains resistant to practically all the antimalarial drugs in clinical use is now making itnecessary to discover and develop both new antimalarial drugs and treatments. Recent advances in molecular techniques along withthe availability of genome sequence ofPlasmodiumfalciparum may provide a wide range of novel targets in metabolic pathways likeisoprenoid biosynthesis, fatty acid biosynthesis and heme biosynthesis in the apicoplast of Plasmodiurn. On the other hand, thecombination therapy approach (currently used to retard the selection of parasite strains resistant to individual components of acombination of drugs) has proved to be a success in the combination of sulphadoxine and pyrimethamine, which targets two differentsteps in the folate pathway of malaria parasite. However, after the success of this therapeutic combination, the efficacy of othercombinations of drugs which target different enzymes in a particular metabolic pathway has, apparently, not been reported. Therefore,herein, we review various drug targets so far discovered in apicoplast-related anabolic pathways, especially, with a sharper focus onthe possibility to target more than one enzyme at a time in a particular metabolic pathway of malaria parasites.

  12. Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

    Energy Technology Data Exchange (ETDEWEB)

    Kyrpides, Nikos; Anderson, Iain; Rodriguez, Jason; Susanti, Dwi; Porat, Iris; Reich, Claudia; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Lykidis, Athanasios; Kim, Edwin; Thompson, Linda S.; Nolan, Matt; Land, Miriam; Copeland, Alex; Lapidus, Alla; Lucas, Susan; Detter, Chris; Zhulin, Igor B.; Olsen, Gary J.; Whitman, William; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching, hyperthermophilic member of the order Thermoproteales within the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein. Predicted highly expressed proteins do not include housekeeping genes, and instead include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins.

  13. The carnitine biosynthetic pathway in Arabidopsis thaliana shares similar features with the pathway of mammals and fungi.

    Science.gov (United States)

    Rippa, Sonia; Zhao, Yingjuan; Merlier, Franck; Charrier, Aurélie; Perrin, Yolande

    2012-11-01

    Carnitine is an essential quaternary ammonium amino acid that occurs in the microbial, plant and animal kingdoms. The role and synthesis of this compound are very well documented in bacteria, fungi and mammals. On the contrary, although the presence of carnitine in plant tissue has been reported four decades ago and information about its biological implication are available, nothing is known about its synthesis in plants. We designed experiments to determine if the carnitine biosynthetic pathway in Arabidopsis thaliana is similar to the pathway in mammals and in the fungi Neurospora crassa and Candida albicans. We first checked for the presence of trimetyllysine (TML) and γ-butyrobetaine (γ-BB), two precursors of carnitine in fungi and in mammals, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Both compounds were shown to be present in plant extracts at concentrations in the picomole range per mg of dry weight. We next synthesized deuterium-labeled TML and transferred A. thaliana seedlings on growth medium supplemented with 1 mM of the deuterated precursor. LC-ESI-MS/MS analysis of plant extracts clearly highlighted the synthesis of deuterium labeled γ-BB and labeled carnitine in deuterated-TML fed plants. The similarities between plant, fungal and mammalian pathways provide very useful information to search homologies between genomes. As a matter of fact the analysis of A. thaliana protein database provides homology for several enzymes responsible for carnitine synthesis in fungi and mammals. The study of mutants affected in the corresponding genes would be very useful to elucidate the plant carnitine biosynthetic pathway and to investigate further the role of carnitine in plant physiology.

  14. Biosynthetic Analysis of the Petrobactin Siderophore Pathway from Bacillus anthracis▿

    OpenAIRE

    Lee, Jung Yeop; Janes, Brian K.; Passalacqua, Karla D; Pfleger, Brian F.; Bergman, Nicholas H; Liu, Haichuan; Håkansson, Kristina; Somu, Ravindranadh V.; Aldrich, Courtney C.; Cendrowski, Stephen; Hanna, Philip C.; Sherman, David H.

    2006-01-01

    The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, a catecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. We initiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis of the asb operon, identification of accumulated intermediates, and addition of exogenous siderophores to asb mutant strains. As a starting point, in-frame deletions of each of...

  15. Computational genomic identification and functional reconstitution of plant natural product biosynthetic pathways

    NARCIS (Netherlands)

    Medema, Marnix H.; Osbourn, Anne

    2016-01-01

    Covering: 2003 to 2016 The last decade has seen the first major discoveries regarding the genomic basis of plant natural product biosynthetic pathways. Four key computationally driven strategies have been developed to identify such pathways, which make use of physical clustering, co-expression, e

  16. Diversity in biosynthetic pathways of galactolipids in the light of endosymbiotic origin of chloroplasts

    Directory of Open Access Journals (Sweden)

    Naoki eSato

    2016-02-01

    Full Text Available Cyanobacteria and chloroplasts perform oxygenic photosynthesis, and share a common origin. Galactolipids are present in the photosynthetic membranes of both cyanobacteria and chloroplasts, but the biosynthetic pathways of the galactolipids are significantly different in the two systems. In this minireview, we explain the history of the discovery of the cyanobacterial pathway, and present a probable scenario of the evolution of the two pathways.

  17. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk.

    Science.gov (United States)

    Tsunematsu, Yuta; Ishikawa, Noriyasu; Wakana, Daigo; Goda, Yukihiro; Noguchi, Hiroshi; Moriya, Hisao; Hotta, Kinya; Watanabe, Kenji

    2013-12-01

    Spirotryprostatins, an indole alkaloid class of nonribosomal peptides isolated from Aspergillus fumigatus, are known for their antimitotic activity in tumor cells. Because spirotryprostatins and many other chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, identifying and understanding the mechanism of spiro-carbon biosynthesis is of great interest. Here we report a detailed study of spiro-ring formation in spirotryprostatins from tryprostatins derived from the fumitremorgin biosynthetic pathway, using reactants and products prepared with engineered yeast and fungal strains. Unexpectedly, FqzB, an FAD-dependent monooxygenase from the unrelated fumiquinazoline biosynthetic pathway, catalyzed spiro-carbon formation in spirotryprostatin A via an epoxidation route. Furthermore, FtmG, a cytochrome P450 from the fumitremorgin biosynthetic pathway, was determined to catalyze the spiro-ring formation in spirotryprostatin B. Our results highlight the versatile role of oxygenating enzymes in the biosynthesis of structurally complex natural products and indicate that cross-talk of different biosynthetic pathways allows product diversification in natural product biosynthesis.

  18. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk.

    Science.gov (United States)

    Tsunematsu, Yuta; Ishikawa, Noriyasu; Wakana, Daigo; Goda, Yukihiro; Noguchi, Hiroshi; Moriya, Hisao; Hotta, Kinya; Watanabe, Kenji

    2013-12-01

    Spirotryprostatins, an indole alkaloid class of nonribosomal peptides isolated from Aspergillus fumigatus, are known for their antimitotic activity in tumor cells. Because spirotryprostatins and many other chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, identifying and understanding the mechanism of spiro-carbon biosynthesis is of great interest. Here we report a detailed study of spiro-ring formation in spirotryprostatins from tryprostatins derived from the fumitremorgin biosynthetic pathway, using reactants and products prepared with engineered yeast and fungal strains. Unexpectedly, FqzB, an FAD-dependent monooxygenase from the unrelated fumiquinazoline biosynthetic pathway, catalyzed spiro-carbon formation in spirotryprostatin A via an epoxidation route. Furthermore, FtmG, a cytochrome P450 from the fumitremorgin biosynthetic pathway, was determined to catalyze the spiro-ring formation in spirotryprostatin B. Our results highlight the versatile role of oxygenating enzymes in the biosynthesis of structurally complex natural products and indicate that cross-talk of different biosynthetic pathways allows product diversification in natural product biosynthesis. PMID:24121553

  19. Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway

    DEFF Research Database (Denmark)

    Liu, Qing; Manzano, David; Tanić, Nikola;

    2014-01-01

    Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew.......4μgg-1 parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding...

  20. Utility of the Biosynthetic Folate Pathway for Targets in Antimicrobial Discovery

    OpenAIRE

    Bourne, Christina R

    2014-01-01

    The need for new antimicrobials is great in face of a growing pool of resistant pathogenic organisms. This review will address the potential for antimicrobial therapy based on polypharmacological activities within the currently utilized bacterial biosynthetic folate pathway. The folate metabolic pathway leads to synthesis of required precursors for cellular function and contains a critical node, dihydrofolate reductase (DHFR), which is shared between prokaryotes and eukaryotes. The DHFR enz...

  1. A simple biosynthetic pathway for large product generation from small substrate amounts

    International Nuclear Information System (INIS)

    A recently emerging discipline of synthetic biology has the aim of constructing new biosynthetic pathways with useful biological functions. A major application of these pathways is generating a large amount of the desired product. However, toxicity due to the possible presence of toxic precursors is one of the main problems for such production. We consider here the problem of generating a large amount of product from a potentially toxic substrate. To address this, we propose a simple biosynthetic pathway, which can be induced in order to produce a large number of the product molecules, by keeping the substrate amount at low levels. Surprisingly, we show that the large product generation crucially depends on fast non-specific degradation of the substrate molecules. We derive an optimal induction strategy, which allows as much as three orders of magnitude increase in the product amount through biologically realistic parameter values. We point to a recently discovered bacterial immune system (CRISPR/Cas in E. coli) as a putative example of the pathway analysed here. We also argue that the scheme proposed here can be used not only as a stand-alone pathway, but also as a strategy to produce a large amount of the desired molecules with small perturbations of endogenous biosynthetic pathways. (paper)

  2. A simple biosynthetic pathway for large product generation from small substrate amounts

    Science.gov (United States)

    Djordjevic, Marko; Djordjevic, Magdalena

    2012-10-01

    A recently emerging discipline of synthetic biology has the aim of constructing new biosynthetic pathways with useful biological functions. A major application of these pathways is generating a large amount of the desired product. However, toxicity due to the possible presence of toxic precursors is one of the main problems for such production. We consider here the problem of generating a large amount of product from a potentially toxic substrate. To address this, we propose a simple biosynthetic pathway, which can be induced in order to produce a large number of the product molecules, by keeping the substrate amount at low levels. Surprisingly, we show that the large product generation crucially depends on fast non-specific degradation of the substrate molecules. We derive an optimal induction strategy, which allows as much as three orders of magnitude increase in the product amount through biologically realistic parameter values. We point to a recently discovered bacterial immune system (CRISPR/Cas in E. coli) as a putative example of the pathway analysed here. We also argue that the scheme proposed here can be used not only as a stand-alone pathway, but also as a strategy to produce a large amount of the desired molecules with small perturbations of endogenous biosynthetic pathways.

  3. Localization and interactions between Arabidopsis auxin biosynthetic enzymes in the TAA/YUC-dependent pathway.

    Science.gov (United States)

    Kriechbaumer, Verena; Botchway, Stanley W; Hawes, Chris

    2016-07-01

    The growth regulator auxin is involved in all key developmental processes in plants. A complex network of a multiplicity of potential biosynthetic pathways as well as transport, signalling plus conjugation and deconjugation lead to a complex and multifaceted system system for auxin function. This raises the question how such a system can be effectively organized and controlled. Here we report that a subset of auxin biosynthetic enzymes in the TAA/YUC route of auxin biosynthesis is localized to the endoplasmic reticulum (ER). ER microsomal fractions also contain a significant percentage of auxin biosynthetic activity. This could point toward a model of auxin function using ER membrane location and subcellular compartmentation for supplementary layers of regulation. Additionally we show specific protein-protein interactions between some of the enzymes in the TAA/YUC route of auxin biosynthesis. PMID:27208541

  4. Biosynthetic Pathway for the Epipolythiodioxopiperazine Acetylaranotin in Aspergillus terreus Revealed by Genome-based Deletion Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Chun-Jun; Yeh, Hsu-Hua; Chiang, Yi Ming; Sanchez, James F.; Chang, ShuLin; Bruno, Kenneth S.; Wang, Clay C.

    2013-04-15

    Abstract Epipolythiodioxopiperazines (ETPs) are a class of fungal secondary metabolites derived from cyclic peptides. Acetylaranotin belongs to one structural subgroup of ETPs characterized by the presence of a seven-membered dihydrooxepine ring. Defining the genes involved in acetylaranotin biosynthesis should provide a means to increase production of these compounds and facilitate the engineering of second-generation molecules. The filamentous fungus Aspergillus terreus produces acetylaranotin and related natural products. Using targeted gene deletions, we have identified a cluster of 9 genes including one nonribosomal peptide synthase gene, ataP, that is required for acetylaranotin biosynthesis. Chemical analysis of the wild type and mutant strains enabled us to isolate seventeen natural products that are either intermediates in the normal biosynthetic pathway or shunt products that are produced when the pathway is interrupted through mutation. Nine of the compounds identified in this study are novel natural products. Our data allow us to propose a complete biosynthetic pathway for acetylaranotin and related natural products.

  5. Reassembled biosynthetic pathway for large-scale carbohydrate synthesis: alpha-Gal epitope producing "superbug".

    Science.gov (United States)

    Chen, Xi; Liu, Ziye; Zhang, Jianbo; Zhang, Wei; Kowal, Przemyslaw; Wang, Peng George

    2002-01-01

    A metabolic pathway engineered Escherichia coli strain (superbug) containing one plasmid harboring an artificial gene cluster encoding all the five enzymes in the biosynthetic pathway of Galalpha l,3Lac through galactose metabolism has been developed. The plasmid contains a lambda promoter, a c1857 repressor gene, an ampicillin resistance gene, and a T7 terminator. Each gene was preceded by a Shine - Dalgarno sequence for ribosome binding. In a reaction catalyzed by the recombinant E. coli strain, Galalpha 1,3Lac trisaccharide accumulated at concentrations of 14.2 mM (7.2 gL(-1)) in a reaction mixture containing galactose, glucose, lactose, and a catalytic amount of uridine 5'-diphosphoglucose. This work demonstrates that large-scale synthesis of complex oligosaccharides can be achieved economically and efficiently through a single, biosynthetic pathway engineered microorganism. PMID:17590953

  6. Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Wei Sun

    Full Text Available Chalcone synthase (CHS catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1 encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

  7. Yellow flowers generated by expression of the aurone biosynthetic pathway.

    Science.gov (United States)

    Ono, Eiichiro; Fukuchi-Mizutani, Masako; Nakamura, Noriko; Fukui, Yuko; Yonekura-Sakakibara, Keiko; Yamaguchi, Masaatsu; Nakayama, Toru; Tanaka, Takaharu; Kusumi, Takaaki; Tanaka, Yoshikazu

    2006-07-18

    Flower color is most often conferred by colored flavonoid pigments. Aurone flavonoids confer a bright yellow color on flowers such as snapdragon (Antirrhinum majus) and dahlia (Dahlia variabilis). A. majus aureusidin synthase (AmAS1) was identified as the key enzyme that catalyzes aurone biosynthesis from chalcones, but transgenic flowers overexpressing AmAS1 gene failed to produce aurones. Here, we report that chalcone 4'-O-glucosyltransferase (4'CGT) is essential for aurone biosynthesis and yellow coloration in vivo. Coexpression of the Am4'CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). Furthermore, their coexpression combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi) resulted in yellow flowers. An Am4'CGT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. We therefore conclude that chalcones are 4'-O-glucosylated in the cytoplasm, their 4'-O-glucosides transported to the vacuole, and therein enzymatically converted to aurone 6-O-glucosides. This metabolic pathway is unique among the known examples of flavonoid, including anthocyanin biosynthesis because, for all other compounds, the carbon backbone is completed before transport to the vacuole. Our findings herein not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineering yellow flowers for major ornamental species lacking this color variant. PMID:16832053

  8. Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera

    DEFF Research Database (Denmark)

    Ono, Hajime; Rewitz, Kim; Shinoda, Tetsu;

    2006-01-01

    Ecdysteroids regulate many key developmental events in arthropods including molting and metamorphosis. Recently, members of the Drosophila Halloween group of genes, that are required for embryonic viability and cuticle deposition, have been shown to code for several cytochrome P450 enzymes that c...... Bombyx and Manduca that is expressed in both embryos and larva. These studies suggest an evolutionary split between Diptera and Lepidoptera in how the ecdysone biosynthetic pathway is regulated during development....

  9. Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera

    Science.gov (United States)

    Pandey, Akansha; Swarnkar, Vishakha; Pandey, Tushar; Srivastava, Piush; Kanojiya, Sanjeev; Mishra, Dipak Kumar; Tripathi, Vineeta

    2016-01-01

    Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG’s were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes. PMID:27703261

  10. A branched biosynthetic pathway is involved in production of roquefortine and related compounds in Penicillium chrysogenum.

    Directory of Open Access Journals (Sweden)

    Hazrat Ali

    Full Text Available Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD, dehydrohistidyltryptophanyldi-ketopiperazine (DHTD, roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN. It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.

  11. Divisions of labor in the thiamin biosynthetic pathway among tissues of maize

    Directory of Open Access Journals (Sweden)

    Jiahn Chou eGuan

    2014-08-01

    Full Text Available The B vitamin thiamin is essential for central metabolism in all cellular organisms including plants. While plants synthesize thiamin de novo, organs vary widely in their capacities for thiamin synthesis. We use a transcriptomics approach to appraise the distribution of de novo synthesis and thiamin salvage pathways among organs of maize. We identify at least six developmental contexts in which metabolically active, non-photosynthetic organs exhibit low expression of one or both branches of the de novo thiamin biosynthetic pathway indicating a dependence on inter-cellular transport of thiamin and/or thiamin precursors. Neither the thiazole (THI4 nor pyrimidine (THIC branches of the pathway are expressed in developing pollen implying a dependence on import of thiamin from surrounding floral and inflorescence organs. Consistent with that hypothesis, organs of the male inflorescence and flowers are shown to have high relative expression of the thiamin biosynthetic pathway and comparatively high thiamin contents. By contrast, divergent patterns of THIC and THI4 expression occur in the shoot apical meristem, embyro sac, embryo, endosperm, and root-tips suggesting that these sink organs acquire significant amounts of thiamin via salvage pathways. In the root and shoot meristems, expression of THIC in the absence of THI4 indicates a capacity for thiamin synthesis via salvage of thiazole, whereas the opposite pattern obtains in embryo and endosperm implying that seed storage organs are poised for pyrimidine salvage. Finally, stable isotope labeling experiments set an upper limit on the rate of de novo thiamin biosynthesis in maize leaf explants. Overall, the observed patterns of thiamin biosynthetic gene expression mirror the strategies for thiamin acquisition that have evolved in bacteria.

  12. The oxalic acid biosynthetic activity of Burkholderia mallei is encoded by a single locus

    Science.gov (United States)

    Although it is known that oxalic acid provides a selective advantage to the secreting microbe, our understanding of how this acid is biosynthesized remains incomplete. This study reports the identification, cloning, and partial characterization of the oxalic acid biosynthetic enzyme from the animal ...

  13. Involvement of the Octadecanoid Pathway and Protein Phosphorylation in Fungal Elicitor-Induced Expression of Terpenoid Indole Alkaloid Biosynthetic Genes in Catharanthus roseus

    Science.gov (United States)

    Menke, Frank L.H.; Parchmann, Stefanie; Mueller, Martin J.; Kijne, Jan W.; Memelink, Johan

    1999-01-01

    Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor α-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously . Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates. PMID:10198087

  14. Towards a palaeosalinity proxy: hydrogen isotopic fractionation between source water and lipids produced via different biosynthetic pathways in haptophyte algae

    Science.gov (United States)

    Chivall, David; M'Boule, Daniela; Heinzelmann, Sandra M.; Kasper, Sebastian; Sinke-Schoen, Daniëlle; Sininnghe-Damsté, Jaap S.; Schouten, Stefan; van der Meer, Marcel T. J.

    2014-05-01

    Palaeosalinity is one of the most important oceanographic parameters that cannot currently be quantified with reasonable accuracy from sedimentary records. Hydrogen isotopic fractionation between water and alkenones is dependent, amongst other factors, upon the salinity in which alkenone-producing haptophyte algae grow and is represented by the fractionation factor, α, increasing with salinity.1 As such, the hydrogen isotopic composition of alkenones is emerging as a palaeosalinity proxy. Understanding the mechanism behind the sensitivity of fractionation to salinity is important for the correct application of the proxy, however this mechanism is currently unknown. Here we present hydrogen isotopic compositions of lipids produced via different biosynthetic pathways from batch cultures of Emiliania huxleyi CCMP 1516 and Isochrysis galbana CCMP 1323 grown over a range of salinities and discuss the possible sources of the sensitivity of hydrogen isotope fractionation to salinity. α for C37 alkenones (produced via an unknown biosynthetic pathway but assumed to be acetogenic; e.g.2) and that for C14:0, C16:0, and C18:1 fatty acids (acetogenic) from exponential growth phase I. galbana show a similar sensitivity to salinity, increasing at 0.0013-0.0019 per salinity unit (S-1). Meanwhile, in exponential growth phase E. huxleyi, α for C37 alkenones and α for brassicasterol (mevalonate pathway) increase at 0.0015-0.0022 S-1, but α for phytol (methylerythritol pathway) shows no significant relationship with salinity. These results suggest that fractionation is sensitive to salinity for lipids formed both in the chloroplast and cytosol. They also suggest that the sensitivity may either originate in glyceralde-3-phosphate or pyruvate but is then lost through hydrogen exchange with cell water during sugar rearrangements in the methylerythritol pathway or sensitivity originates with the production and consumption of acetate. References Schouten, S., Ossebaar, J., Schreiber

  15. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway.

    Science.gov (United States)

    Umemoto, Naoyuki; Nakayasu, Masaru; Ohyama, Kiyoshi; Yotsu-Yamashita, Mari; Mizutani, Masaharu; Seki, Hikaru; Saito, Kazuki; Muranaka, Toshiya

    2016-08-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  16. A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery.

    Science.gov (United States)

    Karim, Ashty S; Jewett, Michael C

    2016-07-01

    Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms. PMID:26996382

  17. Hydroxycinnamic acid functional ingredients and their biosynthetic genes in tubers of Solanum tuberosum Group Phureja

    Directory of Open Access Journals (Sweden)

    Liyao Ji

    2016-12-01

    Full Text Available Potato is an ideal candidate for the delivery of functional ingredients due to its high worldwide consumption. The metabolites in cooked tubers of eight diploid potato genotypes from Colombia were explored. Potato tubers were harvested, cooked,lyophilized, and then stored at −80°C. Metabolites were extracted from flesh samples and analyzed using liquid chromatography and high-resolution mass spectrometry. A total of 294 metabolites were putatively identified, of which 87 metabolites were associated with health-benefiting roles for humans, such as anticancer and anti-inflammatory properties. Two metabolites, chlorogenic acid and N-Feruloyltyramine were detected in high abundance and were mapped on to the potato metabolic pathways to predict the related biosynthetic enzymes: hydroxycinnamoyl-CoA quinate transferase (HQT and tyramine hydroxycinnamoyl transferase (THT, respectively. The coding genes of these enzymes identified nonsynonymous single-nucleotide polymorphisms (nsSNPs in AC09, AC64, and Russet Burbank, with the highest enzyme stability found in AC09. This is consistent with the highest presence of hydroxycinnamic acids in the AC09 genotype. The metabolites detected at high fold change, their functional ingredient properties, and their enhancement through breeding to improve health of the indigenous communities’ of Colombia are discussed.

  18. Probing a Coral Genome for Components of the Photoprotective Scytonemin Biosynthetic Pathway and the 2-Aminoethylphosphonate Pathway

    Directory of Open Access Journals (Sweden)

    Nori Satoh

    2013-02-01

    Full Text Available Genome sequences of the reef-building coral, Acropora digitifera, have been decoded. Acropora inhabits an environment with intense ultraviolet exposure and hosts the photosynthetic endosymbiont, Symbiodinium. Acropora homologs of all four genes necessary for biosynthesis of the photoprotective cyanobacterial compound, shinorine, are present. Among metazoans, these genes are found only in anthozoans. To gain further evolutionary insights into biosynthesis of photoprotective compounds and associated coral proteins, we surveyed the Acropora genome for 18 clustered genes involved in cyanobacterial synthesis of the anti-UV compound, scytonemin, even though it had not previously been detected in corals. We identified candidates for only 6 of the 18 genes, including tyrP, scyA, and scyB. Therefore, it does not appear that Acropora digitifera can synthesize scytonemin independently. On the other hand, molecular phylogenetic analysis showed that one tyrosinase gene is an ortholog of vertebrate tyrosinase genes and that the coral homologs, scyA and scyB, are similar to bacterial metabolic genes, phosphonopyruvate (ppyr decarboxylase and glutamate dehydrogenase (GDH, respectively. Further genomic searches for ppyr gene-related biosynthetic components indicate that the coral possesses a metabolic pathway similar to the bacterial 2-aminoethylphosphonate (AEP biosynthetic pathway. The results suggest that de novo synthesis of carbon-phosphorus compounds is performed in corals.

  19. Trehalose Polyphleates Are Produced by a Glycolipid Biosynthetic Pathway Conserved across Phylogenetically Distant Mycobacteria.

    Science.gov (United States)

    Burbaud, Sophie; Laval, Françoise; Lemassu, Anne; Daffé, Mamadou; Guilhot, Christophe; Chalut, Christian

    2016-02-18

    Mycobacteria synthesize a variety of structurally related glycolipids with major biological functions. Common themes have emerged for the biosynthesis of these glycolipids, including several families of proteins. Genes encoding these proteins are usually clustered on bacterial chromosomal islets dedicated to the synthesis of one glycolipid family. Here, we investigated the function of a cluster of five genes widely distributed across non-tuberculous mycobacteria. Using defined mutant analysis and in-depth structural characterization of glycolipids from wild-type or mutant strains of Mycobacterium smegmatis and Mycobacterium abscessus, we established that they are involved in the formation of trehalose polyphleates (TPP), a family of compounds originally described in Mycobacterium phlei. Comparative genomics and lipid analysis of strains distributed along the mycobacterial phylogenetic tree revealed that TPP is synthesized by a large number of non-tuberculous mycobacteria. This work unravels a novel glycolipid biosynthetic pathway in mycobacteria and extends the spectrum of bacteria that produce TPP. PMID:27028886

  20. The biosynthetic pathway for myxol-2' fucoside (myxoxanthophyll) in the cyanobacterium Synechococcus sp. strain PCC 7002.

    Science.gov (United States)

    Graham, Joel E; Bryant, Donald A

    2009-05-01

    Synechococcus sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic beta-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2' fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2'-OH rather than on the 1'-OH position of the psi end of the molecule. In this study, the genes encoding two enzymes that modify the psi end of myxoxanthophyll in Synechococcus sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed cruF, encodes a 1',2'-hydroxylase [corrected] and that open reading frame SynPCC7002_A2031, renamed cruG, encodes a 2'-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when cruF was expressed in a lycopene-producing strain of Escherichia coli. Database searches showed that homologs of cruF and cruG occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain crtC homologs also contain cruF orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.

  1. Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target.

    Science.gov (United States)

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2016-06-01

    Malaria is a major cause of morbidity and mortality in humans. Artemisinins remain as the first-line treatment for Plasmodium falciparum (P. falciparum) malaria although drug resistance has already emerged and spread in Southeast Asia. Thus, to fight this disease, there is an urgent need to develop new antimalarial drugs for malaria chemotherapy. Unlike human host cells, P. falciparum cannot salvage preformed pyrimidine bases or nucleosides from the extracellular environment and relies solely on nucleotides synthesized through the de novo biosynthetic pathway. This review presents significant progress on understanding the de novo pyrimidine pathway and the functional enzymes in the human parasite P. falciparum. Current knowledge in genomics and metabolomics are described, particularly focusing on the parasite purine and pyrimidine nucleotide metabolism. These include gene annotation, characterization and molecular mechanism of the enzymes that are different from the human host pathway. Recent elucidation of the three-dimensional crystal structures and the catalytic reactions of three enzymes: dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase, as well as their inhibitors are reviewed in the context of their therapeutic potential against malaria.

  2. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli

    DEFF Research Database (Denmark)

    Stahlhut, Steen Gustav; Siedler, Solvej; Malla, Sailesh;

    2015-01-01

    -viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer׳s disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin...

  3. Biosynthetic studies on clavulanic acid: its biopathway and stereochemical course

    Energy Technology Data Exchange (ETDEWEB)

    Mao, S.S.

    1987-01-01

    A degradative analysis allowed determination of the stereochemistry at C-9 of clavulanic acid produced by Streptomyces clavuigerus. An over-all inversion of configuration from the C/sub 5/-unit precursor ornithine was observed. The diastereomeric (1R,2R)- and (1S,2R)-(1-/sup 3/H)-glycerols were separately synthesized and administered. Complementary results demonstrated an overall retention of configuration paralleling cysteine incorporation in the biosynthesis of penicillin. 3-Hydroxyornithine, a potential precursor to clavulanic acid, was prepared by a 1,3-dipolar addition of a nitrone and vinylglycine. However, 3-hydroxyornithine was not taken up by the organism and this possible intermediate could not be shown to be a specific precursor to clavulanic acid. (2-/sup 3/H)-L-Ornithine displays a preferential incorporation relative to D-ornithine. An epimerization by a one-base mechanism is suggested by the retention of half the tritium activity. ..beta..-Alanine, a potential precursor of the ..beta..-lactam segment was examined and shown not to play a direct role in the biosynthesis. Further, 3-hydroxypropionyl-ornithine, a parallel amide to the tripeptide intermediate in penicillin biosynthesis, was not incorporated into clavulanic acid. The role of 3-hydroxypropionate and glycerol were examined in both starch and triglyceride fermentation media.

  4. Utility of the Biosynthetic Folate Pathway for Targets in Antimicrobial Discovery

    Directory of Open Access Journals (Sweden)

    Christina R. Bourne

    2014-01-01

    Full Text Available The need for new antimicrobials is great in face of a growing pool of resistant pathogenic organisms. This review will address the potential for antimicrobial therapy based on polypharmacological activities within the currently utilized bacterial biosynthetic folate pathway. The folate metabolic pathway leads to synthesis of required precursors for cellular function and contains a critical node, dihydrofolate reductase (DHFR, which is shared between prokaryotes and eukaryotes. The DHFR enzyme is currently targeted by methotrexate in anti-cancer therapies, by trimethoprim for antibacterial uses, and by pyrimethamine for anti-protozoal applications. An additional anti-folate target is dihyropteroate synthase (DHPS, which is unique to prokaryotes as they cannot acquire folate through dietary means. It has been demonstrated as a primary target for the longest standing antibiotic class, the sulfonamides, which act synergistically with DHFR inhibitors. Investigations have revealed most DHPS enzymes possess the ability to utilize sulfa drugs metabolically, producing alternate products that presumably inhibit downstream enzymes requiring the produced dihydropteroate. Recent work has established an off-target effect of sulfonamide antibiotics on a eukaryotic enzyme, sepiapterin reductase, causing alterations in neurotransmitter synthesis. Given that inhibitors of both DHFR and DHPS are designed to mimic their cognate substrate, which contain shared substructures, it is reasonable to expect such “off-target” effects. These inhibitors are also likely to interact with the enzymatic neighbors in the folate pathway that bind products of the DHFR or DHPS enzymes and/or substrates of similar substructure. Computational studies designed to assess polypharmacology reiterate these conclusions. This leads to hypotheses exploring the vast utility of multiple members of the folate pathway for modulating cellular metabolism, and includes an appealing capacity for

  5. Utility of the Biosynthetic Folate Pathway for Targets in Antimicrobial Discovery.

    Science.gov (United States)

    Bourne, Christina R

    2014-01-01

    The need for new antimicrobials is great in face of a growing pool of resistant pathogenic organisms. This review will address the potential for antimicrobial therapy based on polypharmacological activities within the currently utilized bacterial biosynthetic folate pathway. The folate metabolic pathway leads to synthesis of required precursors for cellular function and contains a critical node, dihydrofolate reductase (DHFR), which is shared between prokaryotes and eukaryotes. The DHFR enzyme is currently targeted by methotrexate in anti-cancer therapies, by trimethoprim for antibacterial uses, and by pyrimethamine for anti-protozoal applications. An additional anti-folate target is dihyropteroate synthase (DHPS), which is unique to prokaryotes as they cannot acquire folate through dietary means. It has been demonstrated as a primary target for the longest standing antibiotic class, the sulfonamides, which act synergistically with DHFR inhibitors. Investigations have revealed most DHPS enzymes possess the ability to utilize sulfa drugs metabolically, producing alternate products that presumably inhibit downstream enzymes requiring the produced dihydropteroate. Recent work has established an off-target effect of sulfonamide antibiotics on a eukaryotic enzyme, sepiapterin reductase, causing alterations in neurotransmitter synthesis. Given that inhibitors of both DHFR and DHPS are designed to mimic their cognate substrate, which contain shared substructures, it is reasonable to expect such "off-target" effects. These inhibitors are also likely to interact with the enzymatic neighbors in the folate pathway that bind products of the DHFR or DHPS enzymes and/or substrates of similar substructure. Computational studies designed to assess polypharmacology reiterate these conclusions. This leads to hypotheses exploring the vast utility of multiple members of the folate pathway for modulating cellular metabolism, and includes an appealing capacity for prokaryotic

  6. Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

    NARCIS (Netherlands)

    Verdoes, J.C.; Sandmann, G.; Visser, H.; Diaz, M.; Mossel, van M.; Ooyen, van A.J.J.

    2003-01-01

    The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both si

  7. Biosynthetic pathways in Methanospirillum hungatei as determined by 13C nuclear magnetic resonance.

    OpenAIRE

    Ekiel, I; Smith, I C; Sprott, G D

    1983-01-01

    The main metabolic pathways in Methanospirillum hungatei GP1 were followed by using 13C nuclear magnetic resonance, with 13C-labeled acetate and CO2 as carbon sources. The labeling patterns found in carbohydrates, amino acids, lipids, and nucleosides were consistent with the formation of pyruvate from acetate and CO2 as the first step in biosynthesis. Carbohydrates are formed by the glucogenic pathway, and no scrambling of label was observed, indicating that the oxidative or reductive pentose...

  8. The heme biosynthetic pathway of the obligate Wolbachia endosymbiont of Brugia malayi as a potential anti-filarial drug target.

    Directory of Open Access Journals (Sweden)

    Bo Wu

    Full Text Available BACKGROUND: Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole can interrupt transmission predominantly by killing microfilariae (mf larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs. METHODS AND FINDINGS: Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP, which targets ferrochelatase (FC, the last step. Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a approximately 600-fold difference in drug sensitivities to succinyl acetone (SA between

  9. The Structure of L-Tyrosine 2,3-Aminomutase frmo the C-1027 Enediyne Antitumor Antibiotic Biosynthetic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Christianson,C.; Montavon, T.; Van Lanen, S.; Shen, B.; Bruner, S.

    2007-01-01

    The SgcC4 L-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-{beta}-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate L-tyrosine to generate (S)-{beta}-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the L-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.

  10. The structure of L-tyrosine 2,3-aminomutase from the C-1027 enediyne antitumor antibiotic biosynthetic pathway.

    Science.gov (United States)

    Christianson, Carl V; Montavon, Timothy J; Van Lanen, Steven G; Shen, Ben; Bruner, Steven D

    2007-06-19

    The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate l-tyrosine to generate (S)-beta-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the l-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations. PMID:17516659

  11. New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

    Energy Technology Data Exchange (ETDEWEB)

    Gallo, A.; Bruno, K. S.; Solfrizzo, M.; Perrone, G.; Mule, G.; Visconti, A.; Baker, S. E.

    2012-09-14

    Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been obtained in Penicillium species. In Aspergillus species only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase in OTA producing A. carbonarius ITEM 5010 has removed the ability of the fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in Aspergillus species. The absence of OTA and ochratoxin α-the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β- the dechloro analog of ochratoxin α- were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius, and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight in the biosynthetic pathway of the toxin.

  12. Comparative SNP diversity among four Eucalyptus species for genes from secondary metabolite biosynthetic pathways

    OpenAIRE

    Foley William J; Maintz Jens; Hui Yeoh Suat; Külheim Carsten; Moran Gavin F

    2009-01-01

    Abstract Background There is little information about the DNA sequence variation within and between closely related plant species. The combination of re-sequencing technologies, large-scale DNA pools and availability of reference gene sequences allowed the extensive characterisation of single nucleotide polymorphisms (SNPs) in genes of four biosynthetic pathways leading to the formation of ecologically relevant secondary metabolites in Eucalyptus. With this approach the occurrence and pattern...

  13. Depth-related distribution of a key gene of the tetraether lipid biosynthetic pathway in marine Thaumarchaeota

    OpenAIRE

    Villanueva, L.; Schouten, S; Sinninghe Damsté, J.S.

    2015-01-01

    The distribution of isoprenoid glycerol dialkyl glycerol tetraethers (GDGT) lipids synthesized by Thaumarchaeota has been shown to be temperature-dependent in world oceans. Depth-related differences in the ammonia monooxygenase (amoA) of Thaumarchaeota have led to the classification of ‘shallow’ and ‘deep water’ clusters, potentially affecting GDGT distributions. Here, we investigate if this classification is also reflected in a key gene of the thaumarchaeotal lipid biosynthetic pathway codin...

  14. Carotenoid Biosynthetic and Catabolic Pathways: Gene Expression and Carotenoid Content in Grains of Maize Landraces

    Directory of Open Access Journals (Sweden)

    Rafael da Silva Messias

    2014-01-01

    Full Text Available Plant carotenoids have been implicated in preventing several age-related diseases, and they also provide vitamin A precursors; therefore, increasing the content of carotenoids in maize grains is of great interest. It is not well understood, however, how the carotenoid biosynthetic pathway is regulated. Fortunately, the maize germplasm exhibits a high degree of genetic diversity that can be exploited for this purpose. Here, the accumulation of carotenoids and the expression of genes from carotenoid metabolic and catabolic pathways were investigated in several maize landraces. The carotenoid content in grains varied from 10.03, in the white variety MC5, to 61.50 μg·g−1, in the yellow-to-orange variety MC3, and the major carotenoids detected were lutein and zeaxanthin. PSY1 (phythoene synthase expression showed a positive correlation with the total carotenoid content. Additionally, the PSY1 and HYD3 (ferredoxin-dependent di-iron monooxygenase expression levels were positively correlated with β-cryptoxanthin and zeaxanthin, while CYP97C (cytochrome P450-type monooxygenase expression did not correlate with any of the carotenoids. In contrast, ZmCCD1 (carotenoid dioxygenase was more highly expressed at the beginning of grain development, as well as in the white variety, and its expression was inversely correlated with the accumulation of several carotenoids, suggesting that CCD1 is also an important enzyme to be considered when attempting to improve the carotenoid content in maize. The MC27 and MC1 varieties showed the highest HYD3/CYP97C ratios, suggesting that they are promising candidates for increasing the zeaxanthin content; in contrast, MC14 and MC7 showed low HYD3/CYP97C, suggesting that they may be useful in biofortification efforts aimed at promoting the accumulation of provitamin A. The results of this study demonstrate the use of maize germplasm to provide insight into the regulation of genes involved in the carotenoid pathway, which

  15. Comparative SNP diversity among four Eucalyptus species for genes from secondary metabolite biosynthetic pathways

    Directory of Open Access Journals (Sweden)

    Foley William J

    2009-09-01

    Full Text Available Abstract Background There is little information about the DNA sequence variation within and between closely related plant species. The combination of re-sequencing technologies, large-scale DNA pools and availability of reference gene sequences allowed the extensive characterisation of single nucleotide polymorphisms (SNPs in genes of four biosynthetic pathways leading to the formation of ecologically relevant secondary metabolites in Eucalyptus. With this approach the occurrence and patterns of SNP variation for a set of genes can be compared across different species from the same genus. Results In a single GS-FLX run, we sequenced over 103 Mbp and assembled them to approximately 50 kbp of reference sequences. An average sequencing depth of 315 reads per nucleotide site was achieved for all four eucalypt species, Eucalyptus globulus, E. nitens, E. camaldulensis and E. loxophleba. We sequenced 23 genes from 1,764 individuals and discovered 8,631 SNPs across the species, with about 1.5 times as many SNPs per kbp in the introns compared to exons. The exons of the two closely related species (E. globulus and E. nitens had similar numbers of SNPs at synonymous and non-synonymous sites. These species also had similar levels of SNP diversity, whereas E. camaldulensis and E. loxophleba had much higher SNP diversity. Neither the pathway nor the position in the pathway influenced gene diversity. The four species share between 20 and 43% of the SNPs in these genes. Conclusion By using conservative statistical detection methods, we were confident about the validity of each SNP. With numerous individuals sampled over the geographical range of each species, we discovered one SNP in every 33 bp for E. nitens and one in every 31 bp in E. globulus. In contrast, the more distantly related species contained more SNPs: one in every 16 bp for E. camaldulensis and one in 17 bp for E. loxophleba, which is, to the best of our knowledge, the highest frequency of SNPs

  16. Expression of fatty acid and lipid biosynthetic genes in developing endosperm of Jatropha curcas

    Directory of Open Access Journals (Sweden)

    Gu Keyu

    2012-07-01

    Full Text Available Abstract Background Temporal and spatial expression of fatty acid and lipid biosynthetic genes are associated with the accumulation of storage lipids in the seeds of oil plants. In jatropha (Jatropha curcas L., a potential biofuel plant, the storage lipids are mainly synthesized and accumulated in the endosperm of seeds. Although the fatty acid and lipid biosynthetic genes in jatropha have been identified, the expression of these genes at different developing stages of endosperm has not been systemically investigated. Results Transmission electron microscopy study revealed that the oil body formation in developing endosperm of jatropha seeds initially appeared at 28 days after fertilization (DAF, was actively developed at 42 DAF and reached to the maximum number and size at 56 DAF. Sixty-eight genes that encode enzymes, proteins or their subunits involved in fatty acid and lipid biosynthesis were identified from a normalized cDNA library of jatropha developing endosperm. Gene expression with quantitative reverse-transcription polymerase chain reaction analysis demonstrated that the 68 genes could be collectively grouped into five categories based on the patterns of relative expression of the genes during endosperm development. Category I has 47 genes and they displayed a bell-shaped expression pattern with the peak expression at 28 or 42 DAF, but low expression at 14 and 56 DAF. Category II contains 8 genes and expression of the 8 genes was constantly increased from 14 to 56 DAF. Category III comprises of 2 genes and both genes were constitutively expressed throughout endosperm development. Category IV has 9 genes and they showed a high expression at 14 and 28 DAF, but a decreased expression from 42 to 56 DAF. Category V consists of 2 genes and both genes showed a medium expression at 14 DAF, the lowest expression at 28 or 42 DAF, and the highest expression at 56 DAF. In addition, genes encoding enzymes or proteins with similar function were

  17. Differential expression of carotenoid biosynthetic pathway genes in two contrasting tomato genotypes for lycopene content

    Indian Academy of Sciences (India)

    Shilpa Pandurangaiah; Kundapura V Ravishankar; Kodthalu S Shivashankar; Avverahally T Sadashiva; Kavitha Pillakenchappa; Sunil Kumar Narayanan

    2016-06-01

    Tomato (Solanum lycopersicum L.) is one of the model plants to study the carotenoid biosynthesis. In the present study, the fruit carotenoid content were quantified at different developmental stages for two contrasting genotypes viz. IIHR-249-1and IIHR-2866 by UPLC. Lycopene content was high in IIHR-249-1(19.45 mg/100g fresh weight) compared to IIHR-2866 ((1.88 mg/100g fresh weight) at the ripe stage. qPCR was performed for genes that are involved in the carotenoid biosynthetic pathway to study the difference in lycopene content in fruits of both the genotypes. The expression of Phytoene Synthase (PSY) increased by 36 fold and Phytoene desaturase (PDS) increased by 14fold from immature green stage to ripe stage in IIHR-249-1. The expression of Chloroplast lycopene β cyclase (LCY-B) and Chromoplast lycopene β cyclase (CYC-B) decreased gradually from the initial stage to the ripe stage in IIHR-249-1. IIHR 249-1 showed 3 and 1.8 fold decrease in gene expression for Chloroplast lycopene β cyclase ((LCY-B) and Chromoplast lycopene β cyclase (CYC-B) .The F2 hybrids derived from IIHR-249-1 and IIHR-2866 were analyzed at the ripe stage for lycopene content. The gene expression of Chloroplast lycopene β cyclase (LCY-B) and Chromoplast lycopene β cyclase (CYC-B) in high and low lycopene lines from F2 progenies also showed the decrease in transcript levels of both the genes in high lycopene F2 lines. We wish to suggest that the differential expression of Lycopene β -cyclases can be used in marker assisted breeding.

  18. 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.

  19. Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440.

    Science.gov (United States)

    Molina-Henares, M Antonia; García-Salamanca, Adela; Molina-Henares, A Jesús; de la Torre, Jesús; Herrera, M Carmen; Ramos, Juan L; Duque, Estrella

    2009-01-01

    Pseudomonas putida KT2440 is a non-pathogenic prototrophic bacterium with high potential for biotechnological applications. Despite all that is known about this strain, the biosynthesis of essential chemicals has not been fully analysed and auxotroph mutants are scarce. We carried out massive mini-Tn5 random mutagenesis and screened for auxotrophs that require aromatic amino acids. The biosynthesis of aromatic amino acids was analysed in detail including physical and transcriptional organization of genes, complementation assays and feeding experiments to establish pathway intermediates. There is a single pathway from chorismate leading to the biosynthesis of tryptophan, whereas the biosynthesis of phenylalanine and tyrosine is achieved through multiple convergent pathways. Genes for tryptophan biosynthesis are grouped in unlinked regions with the trpBA and trpGDE genes organized as operons and the trpI, trpE and trpF genes organized as single transcriptional units. The pheA and tyrA gene-encoding multifunctional enzymes for phenylalanine and tyrosine biosynthesis are linked in the chromosome and form an operon with the serC gene involved in serine biosynthesis. The last step in the biosynthesis of these two amino acids requires an amino transferase activity for which multiple tyrB-like genes are present in the host chromosome. PMID:21261884

  20. Identification of a dTDP-rhamnose biosynthetic pathway that oscillates with the molting cycle in Caenorhabditis elegans

    Science.gov (United States)

    Feng, Likui; Shou, Qingyao; Butcher, Rebecca A.

    2016-01-01

    L-Rhamnose is a common component of cell-wall polysaccharides, glycoproteins and some natural products in bacteria and plants, but is rare in fungi and animals. In the present study, we identify and characterize a biosynthetic pathway for dTDP-rhamnose in Caenorhabditis elegans that is highly conserved across nematode species. We show that RML-1 activates glucose 1-phosphate (Glc-1-P) in the presence of either dTTP or UTP to yield dTDP-glucose or UDP-glucose, respectively. RML-2 is a dTDP-glucose 4,6-dehydratase, converting dTDP-glucose into dTDP-4-keto-6-deoxyglucose. Using mass spectrometry and NMR spectroscopy, we demonstrate that coincubation of dTDP-4-keto-6-deoxyglucose with RML-3 (3,5-epimerase) and RML-4 (4-keto-reductase) produces dTDP-rhamnose. RML-4 could only be expressed and purified in an active form through co-expression with a co-regulated protein, RML-5, which forms a complex with RML-4. Analysis of the sugar nucleotide pool in C. elegans established the presence of dTDP-rhamnose in vivo. Targeting the expression of the rhamnose biosynthetic genes by RNAi resulted in significant reductions in dTDP-rhamnose, but had no effect on the biosynthesis of a closely related sugar, ascarylose, found in the ascaroside pheromones. Therefore, the rhamnose and ascarylose biosynthetic pathways are distinct. We also show that transcriptional reporters for the rhamnose biosynthetic genes are expressed highly in the embryo, in the hypodermis during molting cycles and in the hypodermal seam cells specifically before the molt to the stress-resistant dauer larval stage. These expression patterns suggest that rhamnose biosynthesis may play an important role in hypodermal development or the production of the cuticle or surface coat during molting. PMID:27009306

  1. Structure-Based Design of Inhibitors of the Crucial Cysteine Biosynthetic Pathway Enzyme O-Acetyl Serine Sulfhydrylase.

    Science.gov (United States)

    Mazumder, Mohit; Gourinath, Samudrala

    2016-01-01

    The cysteine biosynthetic pathway is of fundamental importance for the growth, survival, and pathogenicity of the many pathogens. This pathway is present in many species but is absent in mammals. The ability of pathogens to counteract the oxidative defences of a host is critical for the survival of these pathogens during their long latent phases, especially in anaerobic pathogens such as Entamoeba histolytica, Leishmania donovani, Trichomonas vaginalis, and Salmonella typhimurium. All of these organisms rely on the de novo cysteine biosynthetic pathway to assimilate sulphur and maintain a ready supply of cysteine. The de novo cysteine biosynthetic pathway, on account of its being important for the survival of pathogens and at the same time being absent in mammals, is an important drug target for diseases such as amoebiasis, trichomoniasis & tuberculosis. Cysteine biosynthesis is catalysed by two enzymes: serine acetyl transferase (SAT) followed by O-acetylserine sulfhydrylase (OASS). OASS is well studied, and with the availability of crystal structures of this enzyme in different conformations, it is a suitable template for structure-based inhibitor development. Moreover, OASS is highly conserved, both structurally and sequence-wise, among the above-mentioned organisms. There have been several reports of inhibitor screening and development against this enzyme from different organisms such as Salmonella typhimurium, Mycobacterium tuberculosis and Entamoeba histolytica. All of these inhibitors have been reported to display micromolar to nanomolar binding affinities for the open conformation of the enzyme. In this review, we highlight the structural similarities of this enzyme in different organisms and the attempts for inhibitor development so far. We also propose that the intermediate state of the enzyme may be the ideal target for the design of effective highaffinity inhibitors. PMID:26303427

  2. Decoding Biosynthetic Pathways in Plants by Pulse-Chase Strategies Using 13CO2 as a Universal Tracer

    Directory of Open Access Journals (Sweden)

    Adelbert Bacher

    2016-07-01

    Full Text Available 13CO2 pulse-chase experiments monitored by high-resolution NMR spectroscopy and mass spectrometry can provide 13C-isotopologue compositions in biosynthetic products. Experiments with a variety of plant species have documented that the isotopologue profiles generated with 13CO2 pulse-chase labeling are directly comparable to those that can be generated by the application of [U-13C6]glucose to aseptically growing plants. However, the application of the 13CO2 labeling technology is not subject to the experimental limitations that one has to take into account for experiments with [U-13C6]glucose and can be applied to plants growing under physiological conditions, even in the field. In practical terms, the results of biosynthetic studies with 13CO2 consist of the detection of pairs, triples and occasionally quadruples of 13C atoms that have been jointly contributed to the target metabolite, at an abundance that is well above the stochastic occurrence of such multiples. Notably, the connectivities of jointly transferred 13C multiples can have undergone modification by skeletal rearrangements that can be diagnosed from the isotopologue data. As shown by the examples presented in this review article, the approach turns out to be powerful in decoding the carbon topology of even complex biosynthetic pathways.

  3. Auxin Biosynthesis: Are the Indole-3-Acetic Acid and Phenylacetic Acid Biosynthesis Pathways Mirror Images?

    Science.gov (United States)

    Cook, Sam D; Nichols, David S; Smith, Jason; Chourey, Prem S; McAdam, Erin L; Quittenden, Laura; Ross, John J

    2016-06-01

    The biosynthesis of the main auxin in plants (indole-3-acetic acid [IAA]) has been elucidated recently and is thought to involve the sequential conversion of Trp to indole-3-pyruvic acid to IAA However, the pathway leading to a less well studied auxin, phenylacetic acid (PAA), remains unclear. Here, we present evidence from metabolism experiments that PAA is synthesized from the amino acid Phe, via phenylpyruvate. In pea (Pisum sativum), the reverse reaction, phenylpyruvate to Phe, is also demonstrated. However, despite similarities between the pathways leading to IAA and PAA, evidence from mutants in pea and maize (Zea mays) indicate that IAA biosynthetic enzymes are not the main enzymes for PAA biosynthesis. Instead, we identified a putative aromatic aminotransferase (PsArAT) from pea that may function in the PAA synthesis pathway. PMID:27208245

  4. Dothistroma pini, a Forest Pathogen, Contains Homologs of Aflatoxin Biosynthetic Pathway Genes

    OpenAIRE

    Bradshaw, Rosie E.; Bhatnagar, Deepak; Ganley, Rebecca J.; Gillman, Carmel J.; Brendon J. Monahan; Seconi, Janet M.

    2002-01-01

    Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatox...

  5. Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

    Energy Technology Data Exchange (ETDEWEB)

    Geiger, Jim

    2013-11-30

    structure of ADP- Glucose pyrophosphorylase from potato in its inhibited conformation, and bound to both ATP and ADP-glucose. In addition, we have determined the first structure of glycogen synthase in its "closed", catalytically active conformation bound to ADP-glucose. We also determined the structure of glycogen synthase bound to malto-oligosaccharides, showing for the first time that an enzyme in the starch biosynthetic pathway recognizes glucans not just in its active site but on binding sites on the surface of the enzyme ten’s of Angstroms from the active site. In addition our structure of a glycogen branching enzyme bound to malto-oligosaccharides identified seven distinct binding sites distributed about the surface of the enzyme. We will now determine the function of these sites to get a molecular-level picture of exactly how these enzymes interact with their polymeric substrates and confer specificity leading to the complex structure of the starch granule. We will extend our studies to other isoforms of the enzymes, to understand how their structures give rise to their distinct function. Our goal is to understand what accounts for the various functional differences between SS and SBE isoforms at a molecular level.

  6. Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

    Energy Technology Data Exchange (ETDEWEB)

    Geiger, Jim

    2013-11-30

    structure of ADP- Glucose pyrophosphorylase from potato in its inhibited conformation, and bound to both ATP and ADP-glucose. In addition, we have determined the first structure of glycogen synthase in its "closed", catalytically active conformation bound to ADP-glucose. We also determined the structure of glycogen synthase bound to malto-oligosaccharides, showing for the first time that an enzyme in the starch biosynthetic pathway recognizes glucans not just in its active site but on binding sites on the surface of the enzyme ten’s of Angstroms from the active site. In addition our structure of a glycogen branching enzyme bound to malto-oligosaccharides identified seven distinct binding sites distributed about the surface of the enzyme. We will now determine the function of these sites to get a molecular-level picture of exactly how these enzymes interact with their polymeric substrates and confer specificity leading to the complex structure of the starch granule. We will extend our studies to other isoforms of the enzymes, to understand how their structures give rise to their distinct function. Our goal is to understand what accounts for the various functional differences between SS and SBE isoforms at a molecular level.

  7. ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Jensen, Michael Krogh; Lindemose, Søren; De Masi, Federico;

    2013-01-01

    ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT......[A,C,G]CGT as ATAF1 consensus binding sequences. Co-expression analysis across publicly available microarray experiments identified 25 genes co-expressed with ATAF1. The promoter regions of ATAF1 co-expressors were significantly enriched for ATAF1 binding sites, and TTGCGTA was identified in the promoter of the key...... abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis....

  8. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway1[OPEN

    Science.gov (United States)

    Nakayasu, Masaru; Ohyama, Kiyoshi; Saito, Kazuki

    2016-01-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  9. Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Nawrath, C.; Poirier, Y.; Somerville, C. (Carnegie Institution of Washington, Stanford, CA (United States))

    1994-12-20

    In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plans accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-[mu]m granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand. 20 refs., 6 figs.

  10. A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis.

    Science.gov (United States)

    Zhao, Qing; Zhang, Yang; Wang, Gang; Hill, Lionel; Weng, Jing-Ke; Chen, Xiao-Ya; Xue, Hongwei; Martin, Cathie

    2016-04-01

    Wogonin and baicalein are bioactive flavones in the popular Chinese herbal remedy Huang-Qin (Scutellaria baicalensis Georgi). These specialized flavones lack a 4'-hydroxyl group on the B ring (4'-deoxyflavones) and induce apoptosis in a wide spectrum of human tumor cells in vitro and inhibit tumor growth in vivo in different mouse tumor models. Root-specific flavones (RSFs) from Scutellaria have a variety of reported additional beneficial effects including antioxidant and antiviral properties. We describe the characterization of a new pathway for the synthesis of these compounds, in which pinocembrin (a 4'-deoxyflavanone) serves as a key intermediate. Although two genes encoding flavone synthase II (FNSII) are expressed in the roots of S. baicalensis, FNSII-1 has broad specificity for flavanones as substrates, whereas FNSII-2 is specific for pinocembrin. FNSII-2 is responsible for the synthesis of 4'-deoxyRSFs, such as chrysin and wogonin, wogonoside, baicalein, and baicalin, which are synthesized from chrysin. A gene encoding a cinnamic acid-specific coenzyme A ligase (SbCLL-7), which is highly expressed in roots, is required for the synthesis of RSFs by FNSII-2, as demonstrated by gene silencing. A specific isoform of chalcone synthase (SbCHS-2) that is highly expressed in roots producing RSFs is also required for the synthesis of chrysin. Our studies reveal a recently evolved pathway for biosynthesis of specific, bioactive 4'-deoxyflavones in the roots of S. baicalensis. PMID:27152350

  11. Aspergillus nidulans as a platform for discovery and characterization of complex biosynthetic pathways

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere

    as a model organism for a range of research disciplines and manygenetic engineering tools are available for working in this organism. This PhD study therefore employed A.nidulans as a model system to address the following on two aspects: 1) Developing A. nidulans as aplatform for pathway discovery...... of secondary metabolites and 2) Developing A. nidulans as a model systemfor protein production with human-like glycan structure.  The first part of this study resulted in the development of a method for the transfer and expression ofintact biosynthetic gene clusters to A. nidulans to facilitate pathway......Filamentous fungi produce a wide range of bioactive compounds, classified as secondary metabolites,which have the potential to be used as pharmaceuticals, insecticides, fungicides and food additives.Secondary metabolites also include mycotoxins, which are produced by fungi that contaminate food...

  12. Use of the valine biosynthetic pathway to convert glucose into isobutanol.

    Science.gov (United States)

    Savrasova, Ekaterina A; Kivero, Aleksander D; Shakulov, Rustem S; Stoynova, Nataliya V

    2011-09-01

    Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259-2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also--as has been demonstrated previously (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009)--used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD

  13. Tracing the biosynthetic source of essential amino acids in marine turtles using delta13C fingerprints.

    Science.gov (United States)

    Arthur, Karen E; Kelez, Shaleyla; Larsen, Thomas; Choy, C Anela; Popp, Brian N

    2014-05-01

    Plants, bacteria, and fungi produce essential amino acids (EAAs) with distinctive patterns of delta13C values that can be used as naturally occurring fingerprints of biosynthetic origin of EAAs in a food web. Because animals cannot synthesize EAAs and must obtain them from food, their tissues reflect delta13C(EAA) patterns found in diet, but it is not known how microbes responsible for hindgut fermentation in some herbivores influence the delta13C values of EAAs in their hosts' tissues. We examined whether distinctive delta13C fingerprints of hindgut flora are evident in the tissues of green turtles (Chelonia mydas), which are known to be facultative hindgut fermenters. We determined delta13C(EAA) values in tissues of green turtles foraging herbivorously in neritic habitats of Hawaii and compared them with those from green, olive ridley, and loggerhead turtles foraging carnivorously in oceanic environments of the central and southeast Pacific Ocean. Results of multivariate statistical analysis revealed two distinct groups that could be distinguished based on unique delta13C(EAA) patterns. A three-end-member predictive linear discriminant model indicated that delta13C(EAA) fingerprints existed in the tissues of carnivorous turtles that resembled patterns found in microalgae, which form the base of an oceanic food web, whereas herbivorous turtles derive EAAs from a bacterial or seagrass source. This study demonstrates the capacity for delta13C fingerprinting to establish the biosynthetic origin of EAAs in higher consumers, and that marine turtles foraging on macroalgal diets appear to receive nutritional supplementation from bacterial symbionts in their digestive system. PMID:25000760

  14. Tracing the biosynthetic source of essential amino acids in marine turtles using delta13C fingerprints.

    Science.gov (United States)

    Arthur, Karen E; Kelez, Shaleyla; Larsen, Thomas; Choy, C Anela; Popp, Brian N

    2014-05-01

    Plants, bacteria, and fungi produce essential amino acids (EAAs) with distinctive patterns of delta13C values that can be used as naturally occurring fingerprints of biosynthetic origin of EAAs in a food web. Because animals cannot synthesize EAAs and must obtain them from food, their tissues reflect delta13C(EAA) patterns found in diet, but it is not known how microbes responsible for hindgut fermentation in some herbivores influence the delta13C values of EAAs in their hosts' tissues. We examined whether distinctive delta13C fingerprints of hindgut flora are evident in the tissues of green turtles (Chelonia mydas), which are known to be facultative hindgut fermenters. We determined delta13C(EAA) values in tissues of green turtles foraging herbivorously in neritic habitats of Hawaii and compared them with those from green, olive ridley, and loggerhead turtles foraging carnivorously in oceanic environments of the central and southeast Pacific Ocean. Results of multivariate statistical analysis revealed two distinct groups that could be distinguished based on unique delta13C(EAA) patterns. A three-end-member predictive linear discriminant model indicated that delta13C(EAA) fingerprints existed in the tissues of carnivorous turtles that resembled patterns found in microalgae, which form the base of an oceanic food web, whereas herbivorous turtles derive EAAs from a bacterial or seagrass source. This study demonstrates the capacity for delta13C fingerprinting to establish the biosynthetic origin of EAAs in higher consumers, and that marine turtles foraging on macroalgal diets appear to receive nutritional supplementation from bacterial symbionts in their digestive system.

  15. Ketol-acid reductoisomerase enzymes and methods of use

    Energy Technology Data Exchange (ETDEWEB)

    Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O' Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

    2016-07-12

    Provided herein are polypeptides having ketol-acid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

  16. Yeast Extract and Silver Nitrate Induce the Expression of Phenylpropanoid Biosynthetic Genes and Induce the Accumulation of Rosmarinic Acid in Agastache rugosa Cell Culture.

    Science.gov (United States)

    Park, Woo Tae; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Yeo, Sun Kyung; Jeon, Jin; Park, Jong Seok; Lee, Sook Young; Park, Sang Un

    2016-01-01

    The present study aimed to investigate the role of yeast extract and silver nitrate on the enhancement of phenylpropanoid pathway genes and accumulation of rosmarinic acid in Agastache rugosa cell cultures. The treatment of cell cultures with yeast extract (500 mg/L) and silver nitrate (30 mg/L) for varying times enhanced the expression of genes in the phenylpropanoid pathway and the production of rosmarinic acid. The results indicated that the expression of RAS and HPPR was proportional to the amount of yeast extract and silver nitrate. The transcript levels of HPPR under yeast extract treatment were 1.84-, 1.97-, and 2.86-fold higher than the control treatments after 3, 6, and 12 h, respectively, whereas PAL expression under silver nitrate treatment was 52.31-fold higher than in the non-treated controls after 24 h of elicitation. The concentration of rosmarinic acid was directly proportional to the concentration of the applied elicitors. Yeast extract supplementation documented the highest amount of rosmarinic acid at 4.98 mg/g, whereas silver nitrate addition resulted in a comparatively lower amount of rosmarinic acid at 0.65 mg/g. In conclusion, addition of yeast extract to the cell cultures enhanced the accumulation of rosmarinic acid, which was evidenced by the expression levels of the phenylpropanoid biosynthetic pathway genes in A. rugosa.

  17. Yeast Extract and Silver Nitrate Induce the Expression of Phenylpropanoid Biosynthetic Genes and Induce the Accumulation of Rosmarinic Acid in Agastache rugosa Cell Culture

    Directory of Open Access Journals (Sweden)

    Woo Tae Park

    2016-03-01

    Full Text Available The present study aimed to investigate the role of yeast extract and silver nitrate on the enhancement of phenylpropanoid pathway genes and accumulation of rosmarinic acid in Agastache rugosa cell cultures. The treatment of cell cultures with yeast extract (500 mg/L and silver nitrate (30 mg/L for varying times enhanced the expression of genes in the phenylpropanoid pathway and the production of rosmarinic acid. The results indicated that the expression of RAS and HPPR was proportional to the amount of yeast extract and silver nitrate. The transcript levels of HPPR under yeast extract treatment were 1.84-, 1.97-, and 2.86-fold higher than the control treatments after 3, 6, and 12 h, respectively, whereas PAL expression under silver nitrate treatment was 52.31-fold higher than in the non-treated controls after 24 h of elicitation. The concentration of rosmarinic acid was directly proportional to the concentration of the applied elicitors. Yeast extract supplementation documented the highest amount of rosmarinic acid at 4.98 mg/g, whereas silver nitrate addition resulted in a comparatively lower amount of rosmarinic acid at 0.65 mg/g. In conclusion, addition of yeast extract to the cell cultures enhanced the accumulation of rosmarinic acid, which was evidenced by the expression levels of the phenylpropanoid biosynthetic pathway genes in A. rugosa.

  18. Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Qing Liu

    Full Text Available The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS and germacrene A oxidase (GAO, which together catalyse the biosynthesis of germacra-1(10,4,11(13-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS, CYP71BL3, can catalyse the oxidation of germacra-1(10,4,11(13-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS, chicory GAO (CiGAO, and chicory COS (CiCOS in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1 FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.

  19. Physiological and molecular responses of the isoprenoid biosynthetic pathway in a drought-resistant Mediterranean shrub, Cistus creticus exposed to water deficit.

    Science.gov (United States)

    Munné-Bosch, Sergi; Falara, Vasiliki; Pateraki, Irene; López-Carbonell, Marta; Cela, Jana; Kanellis, Angelos K

    2009-01-30

    The goal of the present research was to obtain new insights into the mechanisms underlying drought stress resistance in plants. Specifically, we evaluated changes in the expression of genes encoding enzymes involved in isoprenoid biosynthesis, together with the levels of the corresponding metabolites (chlorophylls, carotenoids, tocopherols and abscisic acid), in a drought-resistant Mediterranean shrub, Cistus creticus grown under Mediterranean field conditions. Summer drought led to reductions in the relative leaf water content (RWC) by 25%, but did not alter the maximum efficiency of PSII, indicating the absence of damage to the photosynthetic apparatus. While the expression of genes encoding C. creticus chlorophyll a oxygenase/chlorophyll b synthase (CAO) and phytoene synthase (PSY) were not affected by water deficit, the genes encoding homogentisate phytyl-transferase (HPT) and 9-cis-epoxycarotenoid dioxygenase (NCED) were induced in water-stressed (WS) plants. Drought-induced changes in gene expression were observed at early stages of drought and were strongly correlated with levels of the corresponding metabolites, with simultaneous increases in abscisic acid and alpha-tocopherol levels of up to 4-fold and 62%, respectively. Furthermore, alpha-tocopherol levels were strongly positively correlated with abscisic acid contents, but not with the levels of jasmonic acid and salicylic acid. We conclude that the abscisic acid and tocopherol biosynthetic pathway may be regulated at the transcript level in WS C. creticus plants, and that the genes encoding HPT and NCED may play a key role in the drought stress resistance of this Mediterranean shrub by modulating abscisic acid and tocopherol biosynthesis. PMID:18455260

  20. Construction of the astaxanthin biosynthetic pathway in a methanotrophic bacterium Methylomonas sp. strain 16a.

    Science.gov (United States)

    Ye, Rick W; Yao, Henry; Stead, Kristen; Wang, Tao; Tao, Luan; Cheng, Qiong; Sharpe, Pamela L; Suh, Wonchul; Nagel, Eva; Arcilla, Dennis; Dragotta, Dominic; Miller, Edward S

    2007-04-01

    Methylomonas sp. strain 16a is an obligate methanotrophic bacterium that uses methane or methanol as the sole carbon source. An effort was made to engineer this organism for astaxanthin production. Upon expressing the canthaxanthin gene cluster under the control of the native hps promoter in the chromosome, canthaxanthin was produced as the main carotenoid. Further conversion to astaxanthin was carried out by expressing different combinations of crtW and crtZ genes encoding the beta-carotenoid ketolase and hydroxylase. The carotenoid intermediate profile was influenced by the copy number of these two genes under the control of the hps promoter. Expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of the mono-ketolated product adonixanthin. On the other hand, expression of two copies of crtW and one copy of crtZ resulted in the presence of non-hydroxylated carotenoid canthaxanthin and the mono-hydroxylated adonirubin. Production of astaxanthin as the predominant carotenoid was obtained in a strain containing two complete sets of carotenoid biosynthetic genes. This strain had an astaxanthin titer ranging from 1 to 2.4 mg g(-1) of dry cell biomass depending on the growth conditions. More than 90% of the total carotenoid was astaxanthin, of which the majority was in the form of E-isomer. This result indicates that it is possible to produce astaxanthin with desirable properties in methanotrophs through genetic engineering.

  1. A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

    NARCIS (Netherlands)

    Ali, Hazrat; Ries, Marco I.; Nijland, Jeroen G.; Lankhorst, Peter P.; Hankemeier, Thomas; Bovenberg, Roel A. L.; Vreeken, Rob J.; Driessen, Arnold J. M.

    2013-01-01

    Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this

  2. The characterization of transgenic tomato overexpressing gibberellin 20-oxidase reveals induction of parthenocarpic fruit growth, higher yield, and alteration of the gibberellin biosynthetic pathway.

    Science.gov (United States)

    García-Hurtado, Noemí; Carrera, Esther; Ruiz-Rivero, Omar; López-Gresa, Maria Pilar; Hedden, Peter; Gong, Fan; García-Martínez, José Luis

    2012-10-01

    Fruit-set and growth in tomato depend on the action of gibberellins (GAs). To evaluate the role of the GA biosynthetic enzyme GA 20-oxidase (GA20ox) in that process, the citrus gene CcGA20ox1 was overexpressed in tomato (Solanum lycopersicum L.) cv Micro-Tom. The transformed plants were taller, had non-serrated leaves, and some flowers displayed a protruding stigma due to a longer style, thus preventing self-pollination, similar to GA(3)-treated plants. Flowering was delayed compared with wild-type (WT) plants. Both yield and number of fruits per plant, some of them seedless, were higher in the transgenic plants. The Brix index value of fruit juice was also higher due to elevated citric acid content, but not glucose or fructose content. When emasculated, 14-30% of ovaries from transgenic flowers developed parthenocarpically, whereas no parthenocarpy was found in emasculated WT flowers. The presence of early-13-hydroxylation and non-13-hydroxylation GA pathways was demonstrated in the shoot and fruit of Micro-Tom, as well as in two tall tomato cultivars (Ailsa Craig and UC-82). The transgenic plants had altered GA profiles containing higher concentrations of GA(4), from the non-13-hydroxylation pathway, which is generally a minor active GA in tomato. The effect of GA(4) application in enhancing stem growth and parthenocarpic fruit development was proportional to dose, with the same activity as GA(1). The results support the contention that GA20ox overexpression diverts GA metabolism from the early-13-hydroxylation pathway to the non-13-hydroxylation pathway. This led to enhanced GA(4) synthesis and higher yield, although the increase in GA(4) content in the ovary was not sufficient to induce full parthenocarpy. PMID:22945942

  3. Triterpenoid Saponin Biosynthetic Pathway Profiling and Candidate Gene Mining of the Ilex asprella Root Using RNA-Seq

    Directory of Open Access Journals (Sweden)

    Xiasheng Zheng

    2014-04-01

    Full Text Available Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield. According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins.

  4. Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro Biosynthetic Pathway in Streptomyces sp. US24 Strain

    Directory of Open Access Journals (Sweden)

    Samiha Sioud

    2007-01-01

    Full Text Available We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro diketopiperazine (DKP. To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS. The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide.

  5. Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant

    Science.gov (United States)

    Mohamed-Hussein, Zeti-Azura; Ng, Chyan Leong

    2016-01-01

    Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that’s highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity

  6. De novo transcriptome assembly and the putative biosynthetic pathway of steroidal sapogenins of Dioscorea composita.

    Science.gov (United States)

    Wang, Xia; Chen, Dijia; Wang, Yuqi; Xie, Jun

    2015-01-01

    The plant Dioscorea composita has important applications in the medical and energy industries, and can be used for the extraction of steroidal sapogenins (important raw materials for the synthesis of steroidal drugs) and bioethanol production. However, little is known at the genetic level about how sapogenins are biosynthesized in this plant. Using Illumina deep sequencing, 62,341 unigenes were obtained by assembling its transcriptome, and 27,720 unigenes were annotated. Of these, 8,022 unigenes were mapped to 243 specific pathways, and 531 unigenes were identified to be involved in 24 secondary metabolic pathways. 35 enzymes, which were encoded by 79 unigenes, were related to the biosynthesis of steroidal sapogenins in this transcriptome database, covering almost all the nodes in the steroidal pathway. The results of real-time PCR experiments on ten related transcripts (HMGR, MK, SQLE, FPPS, DXS, CAS, HMED, CYP51, DHCR7, and DHCR24) indicated that sapogenins were mainly biosynthesized by the mevalonate pathway. The expression of these ten transcripts in the tuber and leaves was found to be much higher than in the stem. Also, expression in the shoots was low. The nucleotide and protein sequences and conserved domains of four related genes (HMGR, CAS, SQS, and SMT1) were highly conserved between D. composita and D. zingiberensis; but expression of these four genes is greater in D. composita. However, there is no expression of these key enzymes in potato and no steroidal sapogenins are synthesized. PMID:25860891

  7. De novo transcriptome assembly and the putative biosynthetic pathway of steroidal sapogenins of Dioscorea composita.

    Directory of Open Access Journals (Sweden)

    Xia Wang

    Full Text Available The plant Dioscorea composita has important applications in the medical and energy industries, and can be used for the extraction of steroidal sapogenins (important raw materials for the synthesis of steroidal drugs and bioethanol production. However, little is known at the genetic level about how sapogenins are biosynthesized in this plant. Using Illumina deep sequencing, 62,341 unigenes were obtained by assembling its transcriptome, and 27,720 unigenes were annotated. Of these, 8,022 unigenes were mapped to 243 specific pathways, and 531 unigenes were identified to be involved in 24 secondary metabolic pathways. 35 enzymes, which were encoded by 79 unigenes, were related to the biosynthesis of steroidal sapogenins in this transcriptome database, covering almost all the nodes in the steroidal pathway. The results of real-time PCR experiments on ten related transcripts (HMGR, MK, SQLE, FPPS, DXS, CAS, HMED, CYP51, DHCR7, and DHCR24 indicated that sapogenins were mainly biosynthesized by the mevalonate pathway. The expression of these ten transcripts in the tuber and leaves was found to be much higher than in the stem. Also, expression in the shoots was low. The nucleotide and protein sequences and conserved domains of four related genes (HMGR, CAS, SQS, and SMT1 were highly conserved between D. composita and D. zingiberensis; but expression of these four genes is greater in D. composita. However, there is no expression of these key enzymes in potato and no steroidal sapogenins are synthesized.

  8. Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440

    OpenAIRE

    Molina‐Henares, M. Antonia; García‐Salamanca, Adela; Molina‐Henares, A. Jesús; De La Torre, Jesús; Herrera, M?? Carmen; Ramos, Juan L.; Duque, Estrella

    2008-01-01

    Summary Pseudomonas putida KT2440 is a non‐pathogenic prototrophic bacterium with high potential for biotechnological applications. Despite all that is known about this strain, the biosynthesis of essential chemicals has not been fully analysed and auxotroph mutants are scarce. We carried out massive mini‐Tn5 random mutagenesis and screened for auxotrophs that require aromatic amino acids. The biosynthesis of aromatic amino acids was analysed in detail including physical and transcriptional o...

  9. Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Mitra Partha P

    2009-08-01

    Full Text Available Abstract Background Natural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host. Results As an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5–7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology. Conclusion The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds

  10. Effective Antibiofilm Polyketides against Staphylococcus aureus from the Pyranonaphthoquinone Biosynthetic Pathways of Streptomyces Species

    OpenAIRE

    Oja, Terhi; San Martin Galindo, Paola; Taguchi, Takaaki; Manner, Suvi; Vuorela, Pia M.; Ichinose, Koji; Metsä-Ketelä, Mikko; Fallarero, Adyary

    2015-01-01

    Streptomyces bacteria are renowned for their ability to produce bioactive secondary metabolites. Recently, synthetic biology has enabled the production of intermediates and shunt products, which may have altered biological activities compared to the end products of the pathways. Here, we have evaluated the potential of recently isolated alnumycins and other closely related pyranonaphthoquinone (PNQ) polyketides against Staphylococcus aureus biofilms. The antimicrobial potency of the compounds...

  11. Comparative Analysis of the Biosynthetic Gene Clusters and Pathways for Three Structurally Related Antitumor Antibiotics Bleomycin, Tallysomycin and Zorbamycin†

    OpenAIRE

    Galm, Ute; Wendt-Pienkowski, Evelyn; Wang, Liyan; Huang, Sheng-Xiong; Unsin, Claudia; Tao, Meifeng; Coughlin, Jane M.; Shen, Ben

    2011-01-01

    The biosynthetic gene clusters for the glycopeptide antitumor antibiotics bleomycin (BLM), tallysomycin (TLM), and zorbamycin (ZBM) have been recently cloned and characterized from Streptomyces verticillus ATCC15003, Streptoalloteichus hindustanus E465-94 ATCC31158, and Streptomyces flavoviridis ATCC21892, respectively. The striking similarities and differences among the biosynthetic gene clusters for the three structurally related glycopeptide antitumor antibiotics prompted us to compare and...

  12. The biosynthetic pathway of curcuminoid in turmeric (Curcuma longa) as revealed by 13C-labeled precursors.

    Science.gov (United States)

    Kita, Tomoko; Imai, Shinsuke; Sawada, Hiroshi; Kumagai, Hidehiko; Seto, Haruo

    2008-07-01

    In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding (13)C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by (13)C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid > ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.

  13. Selectively improving nikkomycin Z production by blocking the imidazolone biosynthetic pathway of nikkomycin X and uracil feeding in Streptomyces ansochromogenes

    Directory of Open Access Journals (Sweden)

    Yang Haihua

    2009-11-01

    Full Text Available Abstract Background Nikkomycins are a group of peptidyl nucleoside antibiotics and act as potent inhibitors of chitin synthases in fungi and insects. Nikkomycin X and Z are the main components produced by Streptomyces ansochromogenes. Of them, nikkomycin Z is a promising antifungal agent with clinical significance. Since highly structural similarities between nikkomycin Z and X, separation of nikkomycin Z from the culture medium of S. ansochromogenes is difficult. Thus, generating a nikkomycin Z selectively producing strain is vital to scale up the nikkomycin Z yields for clinical trials. Results A nikkomycin Z producing strain (sanPDM was constructed by blocking the imidazolone biosynthetic pathway of nikkomycin X via genetic manipulation and yielded 300 mg/L nikkomycin Z and abolished the nikkomycin X production. To further increase the yield of nikkomycin Z, the effects of different precursors on its production were investigated. Precursors of nucleoside moiety (uracil or uridine had a stimulatory effect on nikkomycin Z production while precursors of peptidyl moiety (L-lysine and L-glutamate had no effect. sanPDM produced the maximum yields of nikkomycin Z (800 mg/L in the presence of uracil at the concentration of 2 g/L and it was approximately 2.6-fold higher than that of the parent strain. Conclusion A high nikkomycin Z selectively producing was obtained by genetic manipulation combined with precursors feeding. The strategy presented here might be applicable in other bacteria to selectively produce targeted antibiotics.

  14. Depth-related distribution of a key gene of the tetraether lipid biosynthetic pathway in marine Thaumarchaeota.

    Science.gov (United States)

    Villanueva, Laura; Schouten, Stefan; Sinninghe Damsté, Jaap S

    2015-10-01

    The distribution of isoprenoid glycerol dialkyl glycerol tetraethers (GDGT) lipids synthesized by Thaumarchaeota has been shown to be temperature-dependent in world oceans. Depth-related differences in the ammonia monooxygenase (amoA) of Thaumarchaeota have led to the classification of 'shallow' and 'deep water' clusters, potentially affecting GDGT distributions. Here, we investigate if this classification is also reflected in a key gene of the thaumarchaeotal lipid biosynthetic pathway coding for geranylgeranylglyceryl phosphate (GGGP) synthase. We investigated metagenomic databases, suspended particulate matter and surface sediment of the Arabian Sea oxygen minimum zone. These revealed significant differences in amoA and GGGP synthase between 'shallow' and 'deep water' Thaumarchaeota. Intriguingly, amoA and GGGP synthase sequences of benthic Thaumarchaeota clustered with the 'shallow water' rather than with 'deep water' Thaumarchaeota. This suggests that pressure and temperature are unlikely factors that drive the differentiation, and suggests an important role of ammonia concentration that is higher in benthic and 'shallow water' niches. Analysis of the relative abundance of GDGTs in the Arabian Sea and in globally distributed surface sediments showed differences in GDGT distributions from subsurface to deep waters that may be explained by differences in the GGGP synthase, suggesting a genetic control on GDGT distributions. PMID:24813867

  15. Flower color alteration in Lotus japonicus by modification of the carotenoid biosynthetic pathway.

    Science.gov (United States)

    Suzuki, Sakae; Nishihara, Masahiro; Nakatsuka, Takashi; Misawa, Norihiko; Ogiwara, Isao; Yamamura, Saburo

    2007-07-01

    To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding beta-carotene ketolase (4,4'-beta-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops. PMID:17265153

  16. Genome Engineering of the 2,3-Butanediol Biosynthetic Pathway for Tight Regulation in Cyanobacteria.

    Science.gov (United States)

    Nozzi, Nicole E; Atsumi, Shota

    2015-11-20

    Cyanobacteria have gained popularity among the metabolic engineering community as a tractable photosynthetic host for renewable chemical production. However, though a number of successfully engineered production systems have been reported, long-term genetic stability remains an issue for cyanobacterial systems. The genetic engineering toolbox for cyanobacteria is largely lacking inducible systems for expression control. The characterization of tight regulation systems for use in cyanobacteria may help to alleviate this problem. In this work we explore the function of the IPTG inducible promoter P(L)lacO1 in the model cyanobacterium Synechococcus elongatus PCC 7942 as well as the effect of gene order within an operon on pathway expression. According to our experiments, P(L)lacO1 functions well as an inducible promoter in S. elongatus. Additionally, we found that gene order within an operon can strongly influence control of expression of each gene.

  17. Arctic mustard flower color polymorphism controlled by petal-specific downregulation at the threshold of the anthocyanin biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Cynthia A Dick

    Full Text Available Intra- and interspecific variation in flower color is a hallmark of angiosperm diversity. The evolutionary forces underlying the variety of flower colors can be nearly as diverse as the colors themselves. In addition to pollinator preferences, non-pollinator agents of selection can have a major influence on the evolution of flower color polymorphisms, especially when the pigments in question are also expressed in vegetative tissues. In such cases, identifying the target(s of selection starts with determining the biochemical and molecular basis for the flower color variation and examining any pleiotropic effects manifested in vegetative tissues. Herein, we describe a widespread purple-white flower color polymorphism in the mustard Parrya nudicaulis spanning Alaska. The frequency of white-flowered individuals increases with increasing growing-season temperature, consistent with the role of anthocyanin pigments in stress tolerance. White petals fail to produce the stress responsive flavonoid intermediates in the anthocyanin biosynthetic pathway (ABP, suggesting an early pathway blockage. Petal cDNA sequences did not reveal blockages in any of the eight enzyme-coding genes in white-flowered individuals, nor any color differentiating SNPs. A qRT-PCR analysis of white petals identified a 24-fold reduction in chalcone synthase (CHS at the threshold of the ABP, but no change in CHS expression in leaves and sepals. This arctic species has avoided the deleterious effects associated with the loss of flavonoid intermediates in vegetative tissues by decoupling CHS expression in petals and leaves, yet the correlation of flower color and climate suggests that the loss of flavonoids in the petals alone may affect the tolerance of white-flowered individuals to colder environments.

  18. Auxin Biosynthesis: Are the Indole-3-Acetic Acid and Phenylacetic Acid Biosynthesis Pathways Mirror Images?1[OPEN

    Science.gov (United States)

    Nichols, David S.; Smith, Jason; Chourey, Prem S.; McAdam, Erin L.; Quittenden, Laura

    2016-01-01

    The biosynthesis of the main auxin in plants (indole-3-acetic acid [IAA]) has been elucidated recently and is thought to involve the sequential conversion of Trp to indole-3-pyruvic acid to IAA. However, the pathway leading to a less well studied auxin, phenylacetic acid (PAA), remains unclear. Here, we present evidence from metabolism experiments that PAA is synthesized from the amino acid Phe, via phenylpyruvate. In pea (Pisum sativum), the reverse reaction, phenylpyruvate to Phe, is also demonstrated. However, despite similarities between the pathways leading to IAA and PAA, evidence from mutants in pea and maize (Zea mays) indicate that IAA biosynthetic enzymes are not the main enzymes for PAA biosynthesis. Instead, we identified a putative aromatic aminotransferase (PsArAT) from pea that may function in the PAA synthesis pathway. PMID:27208245

  19. Biosynthetic Pathways of Vibrio succinogenes growing with fumarate as terminal electron acceptor and sole carbon source.

    Science.gov (United States)

    Bronder, M; Mell, H; Stupperich, E; Kröger, A

    1982-05-01

    1. With fumarate as the terminal electron acceptor and either H2 or formate as donor, Vibrio succinogenes could grow anaerobically in a mineral medium using fumarate as the sole carbon source. Both the growth rate and the cell yield were increased when glutamate was also present in the medium. 2. Glutamate was incorporated only into the amino acids of the glutamate family (glutamate, glutamine, proline and arginine) of the protein. The residual cell constituents were synthesized from fumarate. 3. Pyruvate and phosphoenolpyruvate, as the central intermediates of most of the cell constituents, were formed through the action of malic enzyme and phosphoenolpyruvate synthetase. Fructose-1,6-bisphosphate aldolase was present in the bacterium suggesting that this enzyme is involved in carbohydrate synthesis. 4. In the absence of added glutamate the amino acids of the glutamate family were synthesized from fumarate via citrate. The enzymes involved in glutamate synthesis were present. 5. During growth in the presence of glutamate, net reducing equivalents were needed for cell synthesis. Glutamate and not H2 or formate was used as the source of these reducing equivalents. For this purpose part of the glutamate was oxidized to yield succinate and CO2. 6. The alpha-ketoglutarate dehydrogenase involved in this reaction was found to use ferredoxin as the electron acceptor. The ferredoxin of the bacterium was reoxidized by means of a NADP-ferredoxin oxidoreductase. Enzymes catalyzing the reduction of NAD, NADP or ferredoxin by H2 or formate were not detected in the bacterium. PMID:7103660

  20. The Glutathione Biosynthetic Pathway of Plasmodium Is Essential for Mosquito Transmission

    Science.gov (United States)

    Vega-Rodríguez, Joel; Janse, Chris J.; Pastrana-Mena, Rebecca; Waters, Andrew P.; Coppens, Isabelle; Rodríguez-Orengo, José F.; Jacobs-Lorena, Marcelo; Serrano, Adelfa E.

    2009-01-01

    Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that γ-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs− parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs− parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito. PMID:19229315

  1. 1. Improving the Yield of Biodiesel from Microalgae and Other Lipids. 2. Studies of the Wax Ester Biosynthetic Pathway and Potential Biotechnological Application

    OpenAIRE

    Wahlen, Bradley D.

    2012-01-01

    The production of biofuels and oleochemicals from renewable sources offers an opportunity to reduce our dependence on fossil fuels. The work contained in this dissertation has focused on developing and improving methods for the production of biodiesel from non-traditional feedstocks and understanding biosynthetic pathways that result in the production of oleochemicals and fuels. Pure vegetable oil can account for 70-80% of the total cost of biodiesel production. Many low-cost oils contain ...

  2. Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides.

    OpenAIRE

    Hunter, C N; Hundle, B S; Hearst, J E; Lang, H.P.; Gardiner, A.T.; Takaichi, S; Cogdell, R. J.

    1994-01-01

    Carotenoids have two major functions in bacterial photosynthesis, photoprotection and accessory light harvesting. The genes encoding many carotenoid biosynthetic pathways have now been mapped and cloned in several different species, and the availability of cloned genes which encode the biosynthesis of carotenoids not found in the photosynthetic genus Rhodobacter opens up the possibility of introducing a wider range of foreign carotenoids into the bacterial photosynthetic apparatus than would ...

  3. Exploring triacylglycerol biosynthetic pathway in developing seeds of Chia (Salvia hispanica L.): a transcriptomic approach.

    Science.gov (United States)

    R V, Sreedhar; Kumari, Priya; Rupwate, Sunny D; Rajasekharan, Ram; Srinivasan, Malathi

    2015-01-01

    Chia (Salvia hispanica L.), a member of the mint family (Lamiaceae), is a rediscovered crop with great importance in health and nutrition and is also the highest known terrestrial plant source of heart-healthy omega-3 fatty acid, alpha linolenic acid (ALA). At present, there is no public genomic information or database available for this crop, hindering research on its genetic improvement through genomics-assisted breeding programs. The first comprehensive analysis of the global transcriptome profile of developing Salvia hispanica L. seeds, with special reference to lipid biosynthesis is presented in this study. RNA from five different stages of seed development was extracted and sequenced separately using the Illumina GAIIx platform. De novo assembly of processed reads in the pooled transcriptome using Trinity yielded 76,014 transcripts. The total transcript length was 66,944,462 bases (66.9 Mb), with an average length of approximately 880 bases. In the molecular functions category of Gene Ontology (GO) terms, ATP binding and nucleotide binding were found to be the most abundant and in the biological processes category, the metabolic process and the regulation of transcription-DNA-dependent and oxidation-reduction process were abundant. From the EuKaryotic Orthologous Groups of proteins (KOG) classification, the major category was "Metabolism" (31.97%), of which the most prominent class was 'carbohydrate metabolism and transport' (5.81% of total KOG classifications) followed by 'secondary metabolite biosynthesis transport and catabolism' (5.34%) and 'lipid metabolism' (4.57%). A majority of the candidate genes involved in lipid biosynthesis and oil accumulation were identified. Furthermore, 5596 simple sequence repeats (SSRs) were identified. The transcriptome data was further validated through confirmative PCR and qRT-PCR for select lipid genes. Our study provides insight into the complex transcriptome and will contribute to further genome-wide research and

  4. Exploring triacylglycerol biosynthetic pathway in developing seeds of Chia (Salvia hispanica L.): a transcriptomic approach.

    Science.gov (United States)

    R V, Sreedhar; Kumari, Priya; Rupwate, Sunny D; Rajasekharan, Ram; Srinivasan, Malathi

    2015-01-01

    Chia (Salvia hispanica L.), a member of the mint family (Lamiaceae), is a rediscovered crop with great importance in health and nutrition and is also the highest known terrestrial plant source of heart-healthy omega-3 fatty acid, alpha linolenic acid (ALA). At present, there is no public genomic information or database available for this crop, hindering research on its genetic improvement through genomics-assisted breeding programs. The first comprehensive analysis of the global transcriptome profile of developing Salvia hispanica L. seeds, with special reference to lipid biosynthesis is presented in this study. RNA from five different stages of seed development was extracted and sequenced separately using the Illumina GAIIx platform. De novo assembly of processed reads in the pooled transcriptome using Trinity yielded 76,014 transcripts. The total transcript length was 66,944,462 bases (66.9 Mb), with an average length of approximately 880 bases. In the molecular functions category of Gene Ontology (GO) terms, ATP binding and nucleotide binding were found to be the most abundant and in the biological processes category, the metabolic process and the regulation of transcription-DNA-dependent and oxidation-reduction process were abundant. From the EuKaryotic Orthologous Groups of proteins (KOG) classification, the major category was "Metabolism" (31.97%), of which the most prominent class was 'carbohydrate metabolism and transport' (5.81% of total KOG classifications) followed by 'secondary metabolite biosynthesis transport and catabolism' (5.34%) and 'lipid metabolism' (4.57%). A majority of the candidate genes involved in lipid biosynthesis and oil accumulation were identified. Furthermore, 5596 simple sequence repeats (SSRs) were identified. The transcriptome data was further validated through confirmative PCR and qRT-PCR for select lipid genes. Our study provides insight into the complex transcriptome and will contribute to further genome-wide research and

  5. Exploring triacylglycerol biosynthetic pathway in developing seeds of Chia (Salvia hispanica L.: a transcriptomic approach.

    Directory of Open Access Journals (Sweden)

    Sreedhar R V

    Full Text Available Chia (Salvia hispanica L., a member of the mint family (Lamiaceae, is a rediscovered crop with great importance in health and nutrition and is also the highest known terrestrial plant source of heart-healthy omega-3 fatty acid, alpha linolenic acid (ALA. At present, there is no public genomic information or database available for this crop, hindering research on its genetic improvement through genomics-assisted breeding programs. The first comprehensive analysis of the global transcriptome profile of developing Salvia hispanica L. seeds, with special reference to lipid biosynthesis is presented in this study. RNA from five different stages of seed development was extracted and sequenced separately using the Illumina GAIIx platform. De novo assembly of processed reads in the pooled transcriptome using Trinity yielded 76,014 transcripts. The total transcript length was 66,944,462 bases (66.9 Mb, with an average length of approximately 880 bases. In the molecular functions category of Gene Ontology (GO terms, ATP binding and nucleotide binding were found to be the most abundant and in the biological processes category, the metabolic process and the regulation of transcription-DNA-dependent and oxidation-reduction process were abundant. From the EuKaryotic Orthologous Groups of proteins (KOG classification, the major category was "Metabolism" (31.97%, of which the most prominent class was 'carbohydrate metabolism and transport' (5.81% of total KOG classifications followed by 'secondary metabolite biosynthesis transport and catabolism' (5.34% and 'lipid metabolism' (4.57%. A majority of the candidate genes involved in lipid biosynthesis and oil accumulation were identified. Furthermore, 5596 simple sequence repeats (SSRs were identified. The transcriptome data was further validated through confirmative PCR and qRT-PCR for select lipid genes. Our study provides insight into the complex transcriptome and will contribute to further genome-wide research

  6. Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway.

    Science.gov (United States)

    Wei, Qiaoe; Ran, Tingting; Ma, Chencui; He, Jianhua; Xu, Dongqing; Wang, Weiwu

    2016-07-22

    Pyrroloquinoline quinone (PQQ) has received considerable attention due to its numerous important physiological functions. PqqA is a precursor peptide of PQQ with two conserved residues: glutamate and tyrosine. After linkage of the Cγ of glutamate and Cϵ of tyrosine by PqqE, these two residues are hypothesized to be cleaved from PqqA by PqqF. The linked glutamate and tyrosine residues are then used to synthesize PQQ. Here, we demonstrated that the pqqF gene is essential for PQQ biosynthesis as deletion of it eliminated the inhibition of prodigiosin production by glucose. We further determined the crystal structure of PqqF, which has a closed clamshell-like shape. The PqqF consists of two halves composed of an N- and a C-terminal lobe. The PqqF-N and PqqF-C lobes form a chamber with the volume of the cavity of ∼9400 Å(3) The PqqF structure conforms to the general structure of inverzincins. Compared with the most thoroughly characterized inverzincin insulin-degrading enzyme, the size of PqqF chamber is markedly smaller, which may define the specificity for its substrate PqqA. Furthermore, the 14-amino acid-residue-long tag formed by the N-terminal tag from expression vector precisely protrudes into the counterpart active site; this N-terminal tag occupies the active site and stabilizes the closed, inactive conformation. His-48, His-52, Glu-129 and His-14 from the N-terminal tag coordinate with the zinc ion. Glu-51 acts as a base catalyst. The observed histidine residue-mediated inhibition may be applicable for the design of a peptide for the inhibition of M16 metalloproteases.

  7. A new member of the 4-methylideneimidazole-5-one–containing aminomutase family from the enediyne kedarcidin biosynthetic pathway

    Science.gov (United States)

    Huang, Sheng-Xiong; Lohman, Jeremy R.; Huang, Tingting; Shen, Ben

    2013-01-01

    4-Methylideneimidazole-5-one (MIO)-containing aminomutases catalyze the conversion of l-α-amino acids to β-amino acids with either an (R) or an (S) configuration. l-Phenylalanine and l-tyrosine are the only two natural substrates identified to date. The enediyne chromophore of the chromoprotein antitumor antibiotic kedarcidin (KED) harbors an (R)-2-aza-3-chloro-β-tyrosine moiety reminiscent of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, the biosynthesis of which uncovered the first known MIO-containing aminomutase, SgcC4. Comparative analysis of the KED and C-1027 biosynthetic gene clusters inspired the proposal for (R)-2-aza-3-chloro-β-tyrosine biosynthesis starting from 2-aza-l-tyrosine, featuring KedY4 as a putative MIO-containing aminomutase. Here we report the biochemical characterization of KedY4, confirming its proposed role in KED biosynthesis. KedY4 is an MIO-containing aminomutase that stereospecifically catalyzes the conversion of 2-aza-l-tyrosine to (R)-2-aza-β-tyrosine, exhibiting no detectable activity toward 2-aza-l-phenylalanine or l-tyrosine as an alternative substrate. In contrast, SgcC4, which stereospecifically catalyzes the conversion of l-tyrosine to (S)-β-tyrosine in C-1027 biosynthesis, exhibits minimal activity with 2-aza-l-tyrosine as an alternative substrate but generating (S)-2-aza-β-tyrosine, a product with the opposite stereochemistry of KedY4. This report of KedY4 broadens the scope of known substrates for the MIO-containing aminomutase family, and comparative studies of KedY4 and SgcC4 provide an outstanding opportunity to examine how MIO-containing aminomutases control substrate specificity and product enantioselectivity. PMID:23633564

  8. Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Nara, Takeshi, E-mail: tnara@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Hashimoto, Muneaki; Hirawake, Hiroko [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Liao, Chien-Wei [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Parasitology, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan, ROC (China); Fukai, Yoshihisa; Suzuki, Shigeo; Tsubouchi, Akiko; Morales, Jorge; Takamiya, Shinzaburo [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Division of Proteomics and Biomolecular Science, Biomedical Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Fan, Chia-Kwung [Department of Parasitology, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan, ROC (China); Inaoka, Daniel Ken [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Inoue, Masayuki [Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Tanaka, Akiko [Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045 (Japan); Harada, Shigeharu [Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Kita, Kiyoshi [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); and others

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer An Escherichia coli strain co-expressing CPSII, ATC, and DHO of Trypanosoma cruzi was constructed. Black-Right-Pointing-Pointer Molecular interactions between CPSII, ATC, and DHO of T. cruzi were demonstrated. Black-Right-Pointing-Pointer CPSII bound with both ATC and DHO. Black-Right-Pointing-Pointer ATC bound with both CPSII and DHO. Black-Right-Pointing-Pointer A functional tri-enzyme complex might precede the establishment of the fused enzyme. -- Abstract: The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.

  9. The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways

    Science.gov (United States)

    Dugrand-Judek, Audray; Olry, Alexandre; Hehn, Alain; Costantino, Gilles; Ollitrault, Patrick; Froelicher, Yann; Bourgaud, Frédéric

    2015-01-01

    Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in

  10. Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

    International Nuclear Information System (INIS)

    Highlights: ► An Escherichia coli strain co-expressing CPSII, ATC, and DHO of Trypanosoma cruzi was constructed. ► Molecular interactions between CPSII, ATC, and DHO of T. cruzi were demonstrated. ► CPSII bound with both ATC and DHO. ► ATC bound with both CPSII and DHO. ► A functional tri-enzyme complex might precede the establishment of the fused enzyme. -- Abstract: The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded—and led to—gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.

  11. Influence of the dissolved oxygen concentration on the penicillin biosynthetic pathway in steady-state cultures of Penicillium chrysogenum

    DEFF Research Database (Denmark)

    Henriksen, Claus Maxel; Nielsen, Jens Bredal; Villadsen, John

    1997-01-01

    recovered to its maximum value when the dissolved oxygen concentration was reset to a value above 0.08 mM. The specific formation rates of a number of typical byproducts of the penicillin production, i.e., delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, isopenicillin N, 6-aminopenicillanic acid, 8......-hydroxypenillic acid and 6-oxopiperidine-2-carboxylic acid, the delta-lactam form of cl-aminoadipic acid, all increased with decreasing dissolved oxygen concentration. A simultaneous increase in the secretion of glutathione was observed, and a link between the secretion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine...... and glutathione is suggested. The intracellular pools of the pathway intermediates delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N increased respectively 2- and 3-fold when the dissolved oxygen concentration was lowered from 0.344 to 0.042 mM, whereas the intracellular pools of glutathione...

  12. Identification and functional characterization of genes encoding omega-3 polyunsaturated fatty acid biosynthetic activities from unicellular microalgae.

    Science.gov (United States)

    Vaezi, Royah; Napier, Johnathan A; Sayanova, Olga

    2013-12-01

    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. PMID:24351909

  13. 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.

  14. Biosynthetic Studies of 13-Desmethylspirolide C Produced by Alexandrium ostenfeldii (= A. peruvianum): Rationalization of the Biosynthetic Pathway Following Incorporation of (13)C-Labeled Methionine and Application of the Odd-Even Rule of Methylation.

    Science.gov (United States)

    Anttila, Matthew; Strangman, Wendy; York, Robert; Tomas, Carmelo; Wright, Jeffrey L C

    2016-03-25

    Understanding the biosynthesis of dinoflagellate polyketides presents many unique challenges. Because of the remaining hurdles to dinoflagellate genome sequencing, precursor labeling studies remain the only viable way to investigate dinoflagellate biosynthesis. However, prior studies have shown that polyketide chain assembly does not follow any of the established processes. Additionally, acetate, the common precursor for polyketides, is frequently scrambled, thus compromising interpretation. These factors are further compounded by low production yields of the compounds of interest. A recent report on the biosynthesis of spirolides, a group belonging to the growing class of toxic spiroimines, provided some insight into the polyketide assembly process based on acetate labeling studies, but many details were left uncertain. By feeding (13)C methyl-labeled methionine to cultures of Alexandrium ostenfeldii, the producing organism of 13-desmethylspirolide C, and application of the odd-even methylation rule, the complete biosynthetic pathway has been established.

  15. Primitive Extracellular Lipid Components on the Surface of the Charophytic Alga Klebsormidium flaccidum and Their Possible Biosynthetic Pathways as Deduced from the Genome Sequence

    Science.gov (United States)

    Kondo, Satoshi; Hori, Koichi; Sasaki-Sekimoto, Yuko; Kobayashi, Atsuko; Kato, Tsubasa; Yuno-Ohta, Naoko; Nobusawa, Takashi; Ohtaka, Kinuka; Shimojima, Mie; Ohta, Hiroyuki

    2016-01-01

    Klebsormidium flaccidum is a charophytic alga living in terrestrial and semiaquatic environments. K. flaccidum grows in various habitats, such as low-temperature areas and under desiccated conditions, because of its ability to tolerate harsh environments. Wax and cuticle polymers that contribute to the cuticle layer of plants are important for the survival of land plants, as they protect against those harsh environmental conditions and were probably critical for the transition from aquatic microorganism to land plants. Bryophytes, non-vascular land plants, have similar, but simpler, extracellular waxes and polyester backbones than those of vascular plants. The presence of waxes in terrestrial algae, especially in charophytes, which are the closest algae to land plants, could provide clues in elucidating the mechanism of land colonization by plants. Here, we compared genes involved in the lipid biosynthetic pathways of Arabidopsis thaliana to the K. flaccidum and the Chlamydomonas reinhardtii genomes, and identified wax-related genes in both algae. A simple and easy extraction method was developed for the recovery of the surface lipids from K. flaccidum and C. reinhardtii. Although these algae have wax components, their surface lipids were largely different from those of land plants. We also investigated aliphatic substances in the cell wall fraction of K. flaccidum and C. reinhardtii. Many of the fatty acids were determined to be lipophilic monomers in K. flaccidum, and a Fourier transform infrared spectroscopic analysis revealed that their possible binding mode was distinct from that of A. thaliana. Thus, we propose that K. flaccidum has a cuticle-like hydrophobic layer composed of lipids and glycoproteins, with a different composition from the cutin polymer typically found in land plant cuticles.

  16. Primitive Extracellular Lipid Components on the Surface of the Charophytic Alga Klebsormidium flaccidum and Their Possible Biosynthetic Pathways as Deduced from the Genome Sequence.

    Science.gov (United States)

    Kondo, Satoshi; Hori, Koichi; Sasaki-Sekimoto, Yuko; Kobayashi, Atsuko; Kato, Tsubasa; Yuno-Ohta, Naoko; Nobusawa, Takashi; Ohtaka, Kinuka; Shimojima, Mie; Ohta, Hiroyuki

    2016-01-01

    Klebsormidium flaccidum is a charophytic alga living in terrestrial and semiaquatic environments. K. flaccidum grows in various habitats, such as low-temperature areas and under desiccated conditions, because of its ability to tolerate harsh environments. Wax and cuticle polymers that contribute to the cuticle layer of plants are important for the survival of land plants, as they protect against those harsh environmental conditions and were probably critical for the transition from aquatic microorganism to land plants. Bryophytes, non-vascular land plants, have similar, but simpler, extracellular waxes and polyester backbones than those of vascular plants. The presence of waxes in terrestrial algae, especially in charophytes, which are the closest algae to land plants, could provide clues in elucidating the mechanism of land colonization by plants. Here, we compared genes involved in the lipid biosynthetic pathways of Arabidopsis thaliana to the K. flaccidum and the Chlamydomonas reinhardtii genomes, and identified wax-related genes in both algae. A simple and easy extraction method was developed for the recovery of the surface lipids from K. flaccidum and C. reinhardtii. Although these algae have wax components, their surface lipids were largely different from those of land plants. We also investigated aliphatic substances in the cell wall fraction of K. flaccidum and C. reinhardtii. Many of the fatty acids were determined to be lipophilic monomers in K. flaccidum, and a Fourier transform infrared spectroscopic analysis revealed that their possible binding mode was distinct from that of A. thaliana. Thus, we propose that K. flaccidum has a cuticle-like hydrophobic layer composed of lipids and glycoproteins, with a different composition from the cutin polymer typically found in land plant cuticles. PMID:27446179

  17. Primitive extracellular lipid components on the surface of the charophytic alga Klebsormidium flaccidum and their possible biosynthetic pathways as deduced from the genome sequence

    Directory of Open Access Journals (Sweden)

    Satoshi eKondo

    2016-06-01

    Full Text Available Klebsormidium flaccidum is a charophytic alga living in terrestrial and semiaquatic environments. K. flaccidum grows in various habitats, such as low-temperature areas and under desiccated conditions, because of its ability to tolerate harsh environments. Wax and cuticle polymers that contribute to the cuticle layer of plants are important for the survival of land plants, as they protect against those harsh environmental conditions and were probably critical for the transition from aquatic microorganism to land plants. Bryophytes, non-vascular land plants, have similar, but simpler, extracellular waxes and polyester backbones than those of vascular plants. The presence of waxes in terrestrial algae, especially in charophytes, which are the closest algae to land plants, could provide clues in elucidating the mechanism of land colonization by plants. Here, we compared genes involved in the lipid biosynthetic pathways of Arabidopsis thaliana to the K. flaccidum and the Chlamydomonas reinhardtii genomes, and identified wax-related genes in both algae. A simple and easy extraction method was developed for the recovery of the surface lipids from K. flaccidum and C. reinhardtii. Although these algae have wax components, their surface lipids were largely different from those of land plants. We also investigated aliphatic substances in the cell wall fraction of K. flaccidum and C. reinhardtii. Many of the fatty acids were determined to be lipophilic monomers in K. flaccidum, and a Fourier transform infrared spectroscopic analysis revealed that their possible binding mode was distinct from that of A. thaliana. Thus, we propose that K. flaccidum has a cuticle-like hydrophobic layer composed of lipids and glycoproteins, with a different composition from the cutin polymer typically found in land plant cuticles.

  18. Studying of Biosynthetic Pathways of 2H-labeled Purine Ribonucleoside Inosine in a Chemoheterotrophic Bacterium Bacillus subtilis B-3157 by FAB Mass-Spectrometry

    Directory of Open Access Journals (Sweden)

    Oleg Mosin

    2015-09-01

    Full Text Available This paper deals with studying biosynthetic pathways of 2H-labeled purine ribonucleoside inosine excreted into liquid microbial culture (LC by Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157 while growing of this bacterium on heavy water (HW medium with 2% (v/v hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2H-labeled growth substrates. Isolation of 2H-labeled inosine from LC was performed by adsorption/desorption on activated carbon with following extraction by 0,3 M ammonium–formate buffer (pH = 8,9, crystallization in 80% (v/v EtOH, and ion exchange chromatography (IEC on a column with AG50WX 4 cation exchange resin equilibrated with 0,3 M ammonium–formate buffer and 0,045 M NH4Cl. The investigation of deuterium incorporation into the inosine molecule by FAB method demonstrated incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment – 65,5 atom% 2H with 3 deuterium atoms being included into the ribose and 2 deuterium atoms – into the hypoxanthine residue of the molecule. Three non-exchangeable deuterium atoms were incorporated into the ribose residue owing to the preservation in this bacterium the minor pathways of de novo glucose biosynthesis in 2H2O-medium. These non-exchangeable deuterium atoms in the ribose residue were originated from HMP shunt reactions, while two other deuterium atoms at C2, C8-positions in the hypoxanthine residue were synthesized from [2H]amino acids, primarily glutamine and glycine, that originated from deuterated hydrolysate. A glycoside proton at -N9-glycosidic bond could be replaced with deuterium via the reaction of СО2 elimination at the stage of ribulose-5-monophosphate formation from 3-keto-6-phosphogluconic acid with subsequent proton (deuteron attachment at the С1-position of ribulose-5-monophosphate. Two other protons at C2(C3 and C4 positions in ribose residue could be

  19. Response differences between Ectocarpus siliculosus populations to copper stress involve cellular exclusion and induction of the phytochelatin biosynthetic pathway.

    Science.gov (United States)

    Roncarati, Francesca; Sáez, Claudio A; Greco, Maria; Gledhill, Martha; Bitonti, Maria B; Brown, Murray T

    2015-02-01

    Some populations of brown seaweed species inhabit metal-polluted environments and can develop tolerance to metal stress, but the mechanisms by which this is accomplished are still to be elucidated. To address this, the responses of two strains of the model brown alga Ectocarpus siliculosus isolated from sites with different histories of metal contamination exposed to total copper (CuT) concentrations ranging between 0 and 2.4 μM for 10 days were investigated. The synthesis of the metal-chelator phytochelatin (PCs) and relative levels of transcripts encoding the enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione synthase (GS) and phytochelatin synthase (PCS) that participate in the PC biosynthetic pathway were measured, along with the effects on growth, and adsorption and uptake of Cu. Growth of strain LIA, from a pristine site in Scotland, was inhibited to a greater extent, and at lower concentrations, than that of Es524, isolated from a Cu-contaminated site in Chile. Concentrations of intra-cellular Cu were higher and the exchangeable fraction was lower in LIA than Es524, especially at the highest exposure levels. Total glutathione concentrations increased in both strains with Cu exposure, whereas total PCs levels were higher in Es524 than LIA; PC2 and PC3 were detected in Es524 but PC2 only was found in LIA. The greater production and levels of polymerisation of PCs in Es524 can be explained by the up-regulation of genes encoding for key enzymes involved in the synthesis of PCs. In Es524 there was an increase in the transcripts of γ-GCS, GS and PCS, particularly under high Cu exposure, whereas in LIA4 transcripts of γ-GCS1 increased only slightly, γ-GCS2 and GS decreased and PCS did not change. The consequences of higher intra-cellular concentrations of Cu, lower production of PCs, and lower expression of enzymes involved in GSH-PCs synthesis may be contributing to an induced oxidative stress condition in LIA, which explains, at least in part, the

  20. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae (on linr)

    NARCIS (Netherlands)

    Wang, Kui-Lin; Bolitho, Karen; Grafton, Karryn; Kortstee, A.J.; Karunairetnam, Sakuntala; McGhie, T.K.; Espley, R.V.; Hellens, R.P.; Allan, A.C.

    2010-01-01

    Background - The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the

  1. Increased glycopeptide production after overexpression of shikimate pathway genes being part of the balhimycin biosynthetic gene cluster

    DEFF Research Database (Denmark)

    Thykær, Jette; Nielsen, Jens; Wohlleben, W.;

    2010-01-01

    Amycolatopsis balhimycina produces the vancomycin-analogue balhimycin. The strain therefore serves as a model strain for glycopeptide antibiotic production. Previous characterisation of the balhimycin biosynthetic cluster had shown that the border sequences contained both, a putative 3-deoxy-d-ar...

  2. Structure of ThiM from Vitamin B1 biosynthetic pathway of Staphylococcus aureus – Insights into a novel pro-drug approach addressing MRSA infections

    Science.gov (United States)

    Drebes, Julia; Künz, Madeleine; Windshügel, Björn; Kikhney, Alexey G.; Müller, Ingrid B.; Eberle, Raphael J.; Oberthür, Dominik; Cang, Huaixing; Svergun, Dmitri I.; Perbandt, Markus; Betzel, Christian; Wrenger, Carsten

    2016-03-01

    Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin). Thiamin in its activated form, thiamin pyrophosphate, is an essential co-factor for all organisms. Therefore, thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional co-factors by the bacterium can function as pro-drugs which thus block various co-factor dependent pathways. We characterized one of the key enzymes within the S. aureus vitamin B1 biosynthetic pathway, 5-(hydroxyethyl)-4-methylthiazole kinase (SaThiM; EC 2.7.1.50), a potential target for pro-drug compounds and analyzed the native structure of SaThiM and complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues.

  3. New insights into the regulation of plant immunity by amino acid metabolic pathways.

    Science.gov (United States)

    Zeier, Jürgen

    2013-12-01

    Besides defence pathways regulated by classical stress hormones, distinct amino acid metabolic pathways constitute integral parts of the plant immune system. Mutations in several genes involved in Asp-derived amino acid biosynthetic pathways can have profound impact on plant resistance to specific pathogen types. For instance, amino acid imbalances associated with homoserine or threonine accumulation elevate plant immunity to oomycete pathogens but not to pathogenic fungi or bacteria. The catabolism of Lys produces the immune signal pipecolic acid (Pip), a cyclic, non-protein amino acid. Pip amplifies plant defence responses and acts as a critical regulator of plant systemic acquired resistance, defence priming and local resistance to bacterial pathogens. Asp-derived pyridine nucleotides influence both pre- and post-invasion immunity, and the catabolism of branched chain amino acids appears to affect plant resistance to distinct pathogen classes by modulating crosstalk of salicylic acid- and jasmonic acid-regulated defence pathways. It also emerges that, besides polyamine oxidation and NADPH oxidase, Pro metabolism is involved in the oxidative burst and the hypersensitive response associated with avirulent pathogen recognition. Moreover, the acylation of amino acids can control plant resistance to pathogens and pests by the formation of protective plant metabolites or by the modulation of plant hormone activity.

  4. Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: heterologous expression and evidence for diverse and multiple catalytic functions of C(50) carotenoid cyclases.

    Science.gov (United States)

    Netzer, Roman; Stafsnes, Marit H; Andreassen, Trygve; Goksøyr, Audun; Bruheim, Per; Brautaset, Trygve

    2010-11-01

    We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids. PMID:20802040

  5. Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases▿ †

    Science.gov (United States)

    Netzer, Roman; Stafsnes, Marit H.; Andreassen, Trygve; Goksøyr, Audun; Bruheim, Per; Brautaset, Trygve

    2010-01-01

    We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C50 carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C40 lycopene, C45 nonaflavuxanthin, C50 flavuxanthin, and C50 sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C50 carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ɛ-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C50 carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ɛ-cyclic C50 carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ɛ- and γ-cyclic C50 carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C50 carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids. PMID:20802040

  6. 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

  7. Salicylic acid-independent plant defence pathways

    NARCIS (Netherlands)

    Pieterse, C.M.J.; Loon, L.C. van

    1999-01-01

    Salicylic acid is an important signalling molecule involved in both locally and systemically induced disease resistance responses. Recent advances in our understanding of plant defence signalling have revealed that plants employ a network of signal transduction pathways, some of which are independen

  8. A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum

    Science.gov (United States)

    Huang, Wenjun; Khaldun, A. B. M.; Chen, Jianjun; Zhang, Chanjuan; Lv, Haiyan; Yuan, Ling; Wang, Ying

    2016-01-01

    Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components (BCs) in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from Epimedium sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase) and EsFLS (flavonol synthase), but not the promoters of EsDFRs (dihydroflavonol 4-reductase) and EsANS (anthocyanidin synthase) in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase), NtCHI (chalcone isomerase), NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS) were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived BCs in E. sagittatum. Thus

  9. A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum.

    Science.gov (United States)

    Huang, Wenjun; Khaldun, A B M; Chen, Jianjun; Zhang, Chanjuan; Lv, Haiyan; Yuan, Ling; Wang, Ying

    2016-01-01

    Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components (BCs) in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from Epimedium sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase) and EsFLS (flavonol synthase), but not the promoters of EsDFRs (dihydroflavonol 4-reductase) and EsANS (anthocyanidin synthase) in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase), NtCHI (chalcone isomerase), NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS) were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived BCs in E. sagittatum. Thus

  10. A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional Chinese medicinal plant, Epimedium sagittatum

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    2016-07-01

    Full Text Available Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from E. sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase and EsFLS (flavonol synthase, but not the promoters of EsDFRs (dihydroflavonol 4-reductase and EsANS (anthocyanidin synthase in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase, NtCHI (chalcone isomerase, NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived bioactive components in E

  11. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Harashima, S; Hinnebusch, A G

    1986-11-01

    GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

  12. Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures.

    Science.gov (United States)

    Rodas-Junco, Beatriz A; Cab-Guillén, Yahaira; Muñoz-Sánchez, J Armando; Vázquez-Flota, Felipe; Monforte-González, Miriam; Hernández-Sotomayor, S M Teresa

    2013-10-01

    Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

  13. Inhibitory effect of eugenol on aflatoxin B1 production in Aspergillus parasiticus by downregulating the expression of major genes in the toxin biosynthetic pathway.

    Science.gov (United States)

    Jahanshiri, Zahra; Shams-Ghahfarokhi, Masoomeh; Allameh, Abdolamir; Razzaghi-Abyaneh, Mehdi

    2015-07-01

    Aflatoxin contamination of grains and agro-products is a serious food safety issue and a significant economic concern worldwide. In the present study, the effects of eugenol on Aspergillus parasiticus growth and aflatoxin production were studied in relation to the expression of some essential genes involved in aflatoxin biosynthetic pathway. The fungus was cultured in presence of serial two-fold concentrations of eugenol (15.62-500 μg mL(-1)) for 3 days at 28 °C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography. The expression of aflatoxin biosynthetic genes including ver-1, nor-1, pksA, omtA and aflR were evaluated by real-time PCR. Eugenol strongly inhibited A. parasiticus growth in the range of 19.16-95.83 % in a dose-dependent manner. Aflatoxin B1 production was also inhibited by the compound in the range of 15.07-98.0 %. The expressions of ver-1, nor-1, pksA, omtA and aflR genes were significantly suppressed by eugenol at concentrations of 62.5 and 125 μg mL(-1). These results indicate that eugenol may be considered as a good candidate to control toxigenic fungal growth and the subsequent contamination of food, feed and agricultural commodities by carcinogenic aflatoxins.

  14. Research Progress on Capsaicinoids Biosynthetic Pathway and Its Related Genes%辣椒素类物质生物合成途径及其相关基因研究进展

    Institute of Scientific and Technical Information of China (English)

    吴智明; 程蛟文; 唐鑫; 胡开林

    2012-01-01

    辣椒素类物质是辣椒果实胎座中产生的特异辣味代谢产物的总称.辣椒素类物质在辣椒果实中的生物合成主要有两条途径:以苯丙氨酸为前体的苯丙烷途径和以缬氨酸或亮氨酸为前体的支链脂肪酸途径.本文综述了近年来国内外学者在辣椒素类物质生物合成过程中的主要酶类基因的克隆、基因表达调控机制研究方面取得的最新进展.%Capsaicinoids are the substances responsible for the pungent sensation that synthesize and accumulate unique in fruits placental tissues of Capsicum species. Capsaicinoids are biosynthesized through 2 pathways: phenylpropanoid and branched-chain fatty acid pathways, which provide the precursors phenylalanine and valine or leucine, respectively. This paper reviewed the new research progress on studying the enzymes and genes participating in the biosynthetic pathway and the regulatory process that accounts for different accumulation levels of capsaicinoids in chili pepper fruits.

  15. Isolation and identification of dihydroartemisinic acid hydroperoxide from Artemisia annua : A novel biosynthetic precursor of artemisinin

    NARCIS (Netherlands)

    Wallaart, TE; Pras, N; Quax, WJ

    1999-01-01

    Dihydroartemisinic acid hydroperoxide (2) was isolated for the first time as a natural product from the plant Artemisia annua in a 29% yield. Its structure was identified by H-1 and C-13 NMR spectroscopy. Compound 2 is known as an intermediate of the photochemical oxidation of dihydroartemisinic aci

  16. New Role of Rosea1 in Regulating Anthocyanin Biosynthetic Pathway in Hairy Root of Snapdragon (Antirrhinum majus L.

    Directory of Open Access Journals (Sweden)

    An Zhang

    2013-09-01

    Full Text Available We investigated the transcriptional regulation of anthocyanin biosynthesis in hairy roots system by ectopically expressing Rosea1 and Delila and we found something different from previous research. The RT-PCR results revealed that Rosea1 could activate early and late biosynthetic genes tested, including CHS, DFR and ANS. Delila enhanced the expression of CHS weakly, but did not influence DFR or ANS. The two regulators, Rosea1 and Delila, failed to interplay each other. It was speculated that Delila would be ineffective in the absence of Rosea1, another MYB factor specifically controlling CHS may exist. This investigation provided a new way to increase anthocyanin content by over expressing a MYB factor, potentially to be used in the field of agriculture and food

  17. Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces

    OpenAIRE

    Brandl, M. T.; Quiñones, B.; Lindow, S E

    2001-01-01

    We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC. Nonpathogenic E. herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered fro...

  18. A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of Wolinella succinogenes

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Timothy H. [Cornell University, Ithaca, New York 14853-1301 (United States); Krishnamoorthy, Kalyanaraman; Begley, Tadhg P., E-mail: begley@tamu.edu [Texas A& M University, College Station, TX 77842 (United States); Ealick, Steven E., E-mail: begley@tamu.edu [Cornell University, Ithaca, New York 14853-1301 (United States)

    2011-10-01

    MetY is the first reported structure of an O-acetylhomoserine sulfhydrylase that utilizes a protein thiocarboxylate intermediate as the sulfur source in a novel methionine-biosynthetic pathway instead of catalyzing a direct sulfhydrylation reaction. O-Acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5′-phosphate (PLP) dependent sulfide-utilizing enzyme in the l-cysteine and l-methionine biosynthetic pathways of various enteric bacteria and fungi. OAHS catalyzes the conversion of O-acetylhomoserine to homocysteine using sulfide in a process known as direct sulfhydrylation. However, the source of the sulfur has not been identified and no structures of OAHS have been reported in the literature. Here, the crystal structure of Wolinella succinogenes OAHS (MetY) determined at 2.2 Å resolution is reported. MetY crystallized in space group C2 with two monomers in the asymmetric unit. Size-exclusion chromatography, dynamic light scattering and crystal packing indicate that the biological unit is a tetramer in solution. This is further supported by the crystal structure, in which a tetramer is formed using a combination of noncrystallographic and crystallographic twofold axes. A search for structurally homologous proteins revealed that MetY has the same fold as cystathionine γ-lyase and methionine γ-lyase. The active sites of these enzymes, which are also PLP-dependent, share a high degree of structural similarity, suggesting that MetY belongs to the γ-elimination subclass of the Cys/Met metabolism PLP-dependent family of enzymes. The structure of MetY, together with biochemical data, provides insight into the mechanism of sulfur transfer to a small molecule via a protein thiocarboxylate intermediate.

  19. The Biosynthetic Order of Amino Acid Addition to the Genetic Code

    CERN Document Server

    Davis, B K

    2002-01-01

    The previously formulated model for the evolution of the genetic code was shown to clarify why base triplets of some precursor amino acids differ by a single base from product amino acid codons, while others show less homology. First, the model indicated that the direction of code evolution changed on expansion from the N-fixers code (stage 2). Growth of the code from 16 codons in the NAN column (N, any standard nucleotide) proceeded by assignment of codons in the GNN, ANN, CNN and UNN rows. Expansion phase (stage 4 to 7) precursor/product pairs that spanned this shift included aspartate/threonine, aspartate/methionine and glutamate/proline. Both 5' and mid-base differ in the codons of each of these pairs. Second, post-expansion additions (stage 9 to 14) required codon reassignment, eliminating initial correlations. Codons for the post-expansion pair, aspartate (glutamate)/arginine, also differ at both 5' and mid-base sites. Third, the distribution of core structure groups among acceptors indicated that varia...

  20. Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the LLD-ACV:bisACV ratio on the flux control.

    Science.gov (United States)

    Theilgaard, H A; Nielsen, J

    1999-01-01

    An extended kinetic model for the first two steps of the penicillin biosynthetic pathway in Penicillium chrysogenum is set up. It includes the formation and reduction of the dimer bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) from the first pathway intermediate LLD-ACV and their parallel inhibition of the enzyme ACV synthetase (ACVS). The kinetic model is based on Michaelis-Menten type kinetics, with non-competitive inhibition of the ACVS by both LLD-ACV and bisACV, and competitive inhibition of the isopenicillin N synthetase (IPNS) by glutathione. The inhibition constant of LLD-ACV, KACV is determined to be 0.54 mm. With the kinetic model metabolic control analysis is performed to identify the distribution of rate-control in the pathway at all ratios of LLD-ACV:bisACV. It is concluded that the flux control totally resides at the IPNS. This is a result of the regulation of the ACVS by both the LLD-ACV and bisACV demanding a higher flux through the IPNS enzyme to alleviate their inhibition. The measurement of an intracellular ratio of LLD-ACV:bisACV to be in the range of 1-2 moles per moles emphasises the importance of a fast conversion of LLD-ACV to IPN, and accumulation of LLD-ACV above the K(m)-value of the IPNS should therefore be avoided.

  1. Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces.

    Science.gov (United States)

    Brandl, M T; Quiñones, B; Lindow, S E

    2001-03-13

    We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC. Nonpathogenic E. herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered from individual leaves 48 h after inoculation, and subjected to fluorescence in situ hybridization using a 16S rRNA oligonucleotide probe specific to strain 299R. Epifluorescence images captured through a rhodamine filter were used to distinguish the 5carboxytetramethylrhodamine-labeled cells of strain 299R from other leaf microflora. Quantification of the green fluorescence intensity of individual cells by analysis of digital images revealed that about 65% of the 299R cells recovered from bean leaves had higher ipdC expression than in culture. Additionally, 10% of the cells exhibited much higher levels of green fluorescence than the median fluorescence intensity, indicating that they are more heterogeneous with respect to ipdC expression on plants than in culture. Examination of 299R cells in situ on leaf surfaces by confocal laser scanning microscopy after fluorescence in situ hybridization of cells on leaf samples showed that even cells that were in close proximity exhibited dramatically different green fluorescence intensities, and thus, were in a physical or chemical microenvironment that induced differential expression of ipdC. PMID:11248099

  2. Marine omega-3 phospholipids suppress hepatic steatosis by a complex inhibition of biosynthetic pathways in dietary obese mice [Mus Musculus

    NARCIS (Netherlands)

    Schothorst, van E.M.; Keijer, J.

    2014-01-01

    Background & Aims: Non-alcoholic fatty liver disease accompanies obesity and is independently associated with cardiovascular disease. Omega-3 fatty acids, such as docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid, reduce the risk of cardiovascular disease due to their anti-inflammatory and hypol

  3. A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

    Directory of Open Access Journals (Sweden)

    Belshoff Alex C

    2011-05-01

    Full Text Available Abstract Background Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes. Results We utilized a stable isotope resolved metabolomics (SIRM approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits

  4. Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

    Science.gov (United States)

    Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

    2009-03-01

    The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts. PMID:19206228

  5. Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

    Science.gov (United States)

    Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

    2009-03-01

    The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

  6. Modification of the mannitol biosynthetic pathway in Aureobasidium pullulans to alter the structure of the polyol lipid liamocin

    Science.gov (United States)

    Aureobasidium pullulans is an important industrial organism that is utilized for the production of numerous bioproducts, such as pullulan, ß-glucan, aureobasidin, and polymalic acid. Our laboratory is also interested in production of the extracellular polyol lipids, called liamocins, produced by cer...

  7. Gene transcript profiles of the TIA biosynthetic pathway in response to ethylene and copper reveal their interactive role in modulating TIA biosynthesis in Catharanthus roseus.

    Science.gov (United States)

    Pan, Ya-Jie; Liu, Jia; Guo, Xiao-Rui; Zu, Yuan-Gang; Tang, Zhong-Hua

    2015-05-01

    Research on transcriptional regulation of terpenoid indole alkaloid (TIA) biosynthesis of the medicinal plant, Catharanthus roseus, has largely been focused on gene function and not clustering analysis of multiple genes at the transcript level. Here, more than ten key genes encoding key enzyme of alkaloid synthesis in TIA biosynthetic pathways were chosen to investigate the integrative responses to exogenous elicitor ethylene and copper (Cu) at both transcriptional and metabolic levels. The ethylene-induced gene transcripts in leaves and roots, respectively, were subjected to principal component analysis (PCA) and the results showed the overall expression of TIA pathway genes indicated as the Q value followed a standard normal distribution after ethylene treatments. Peak gene expression was at 15-30 μM of ethephon, and the pre-mature leaf had a higher Q value than the immature or mature leaf and root. Treatment with elicitor Cu found that Cu up-regulated overall TIA gene expression more in roots than in leaves. The combined effects of Cu and ethephon on TIA gene expression were stronger than their separate effects. It has been documented that TIA gene expression is tightly regulated by the transcriptional factor (TF) ethylene responsive factor (ERF) and mitogen-activated protein kinase (MAPK) cascade. The loading plot combination with correlation analysis for the genes of C. roseus showed that expression of the MPK gene correlated with strictosidine synthase (STR) and strictosidine b-D-glucosidase(SGD). In addition, ERF expression correlated with expression of secologanin synthase (SLS) and tryptophan decarboxylase (TDC), specifically in roots, whereas MPK and myelocytomatosis oncogene (MYC) correlated with STR and SGD genes. In conclusion, the ERF regulates the upstream pathway genes in response to heavy metal Cu mainly in C. roseus roots, while the MPK mainly participates in regulating the STR gene in response to ethylene in pre-mature leaf. Interestingly, the

  8. Inhibition of green tea and the catechins against 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    Science.gov (United States)

    Hui, Xian; Liu, Hui; Tian, Fang-Lin; Li, Fei-Fei; Li, Heng; Gao, Wen-Yun

    2016-09-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the MEP terpenoid biosynthetic pathway and also a validated antimicrobial target. Green tea which is rich in polyphenolic components such as the catechins, possesses a plenty of pharmacological activities, in particular an antibacterial effect. To uncover the antibacterial mechanism of green tea and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of green tea and its main antimicrobial catechins were investigated in this study. The results show that the raw extract of green tea and its ethyl acetate fraction are able to suppress DXR activity explicitly. Further determination of the DXR inhibitory capacity of eight catechin compounds demonstrates that the most active compound is gallocatechin gallate that is able to inhibit around 50% activity of DXR at 25μM. Based on these data, the primary structure-activity relationship of the catechins against DXR is discussed. This study would be very helpful to elucidate the antimicrobial mechanism of green tea and the catechins and also would be very useful to direct the rational utilization of them as food additives. PMID:27439219

  9. In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana.

    Science.gov (United States)

    Saifi, Monica; Nasrullah, Nazima; Ahmad, Malik Mobeen; Ali, Athar; Khan, Jawaid A; Abdin, M Z

    2015-09-01

    miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana.

  10. In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana.

    Science.gov (United States)

    Saifi, Monica; Nasrullah, Nazima; Ahmad, Malik Mobeen; Ali, Athar; Khan, Jawaid A; Abdin, M Z

    2015-09-01

    miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana. PMID:26042546

  11. Elucidation of the biosynthetic pathway for Okenone in Thiodictyon sp. CAD16 leads to the discovery of two novel carotene ketolases.

    Science.gov (United States)

    Vogl, Kajetan; Bryant, Donald A

    2011-11-01

    Okenone is a unique ketocarotenoid found in many purple sulfur bacteria; it is important because of its unique light absorption and photoprotection properties. Okenane, a compound formed by diagenetic reduction of okenone, is an important biomarker in geochemical analyses of sedimentary rocks. Despite its ecological and biogeochemical importance, the biochemical pathway for okenone synthesis has not yet been fully described. The genome sequence of an okenone-producing organism, Thiodictyon sp. strain CAD16, revealed four genes whose predicted proteins had strong sequence similarity to enzymes known to produce ψ-end group modifications of carotenoids in proteobacteria. These four genes encoded homologs of a 1,2-carotenoid hydratase (CrtC), an O-methyltransferase (CrtF), and two paralogs of carotenoid 3,4-desaturases (CrtD). Expression studies in lycopene- or neurosporene-producing strains of Escherichia coli confirmed the functions of crtC and crtF, but the crtD paralogs encoded enzymes with previously undescribed functions. One enzyme, CruS, was only distantly related to CrtD desaturases, was bifunctional, and performed a 3,4-desaturation and introduced a C-2 keto group into neurosporene derivatives in the presence of dioxygen. The enzyme encoded by the other crtD paralog also represents a new enzyme in carotenogenesis and was named cruO. CruO encodes the C-4/4' ketolase uniquely required for okenone biosynthesis. The identification of CruO and the demonstration of its biochemical activity complete the elucidation of the biosynthetic pathway for okenone and other related ketocarotenoids.

  12. Improvement of the enediyne antitumor antibiotic C-1027 production by manipulating its biosynthetic pathway regulation in Streptomyces globisporus.

    Science.gov (United States)

    Chen, Yihua; Yin, Min; Horsman, Geoff P; Shen, Ben

    2011-03-25

    The production of C-1027 in Streptomyces globisporus was previously increased 2- to 3-fold by manipulating three pathway-specific activators, SgcR1, SgcR2, and SgcR3. In this study, we have further characterized two putative C-1027 regulatory genes, sgcE1 and sgcR, by in vivo inactivation. The HxlR family DNA-binding protein SgcE1 was not essential for C-1027 biosynthesis, since inactivation of sgcE1 showed no effect on C-1027 production. In contrast, the proposed repressive role of the sgcR gene was confirmed by a 3-fold increase in C-1027 production in the ΔsgcR mutant S. globisporus SB1022 strain relative to the wild-type strain. Considering SgcR shows no significant similarity to any protein of known function, it may be representative of a new family of regulatory proteins. Finally, overexpression of the previously characterized activator sgcR1 in S. globisporus SB1022 increased the C-1027 yield to 37.5 ± 7.7 mg/L, which is about 7-fold higher than the wild-type strain. PMID:21250756

  13. Crystal Structure of Vancosaminyltransferase GtfD from the Vancomycin Biosynthetic Pathway: Interactions with Acceptor and Nucleotide Ligands

    Energy Technology Data Exchange (ETDEWEB)

    Mulichak, A.M.; Lu, W.; Losey, H.C.; Walsh, C.T.; Garavito, R.M. (Harvard-Med); (MSU)

    2010-03-08

    The TDP-vancosaminyltransferase GtfD catalyzes the attachment of L-vancosamine to a monoglucosylated heptapeptide intermediate during the final stage of vancomycin biosynthesis. Glycosyltransferases from this and similar antibiotic pathways are potential tools for the design of new compounds that are effective against vancomycin resistant bacterial strains. We have determined the X-ray crystal structure of GtfD as a complex with TDP and the natural glycopeptide substrate at 2.0 {angstrom} resolution. GtfD, a member of the bidomain GT-B glycosyltransferase superfamily, binds TDP in the interdomain cleft, while the aglycone acceptor binds in a deep crevice in the N-terminal domain. However, the two domains are more interdependent in terms of substrate binding and overall structure than was evident in the structures of closely related glycosyltransferases GtfA and GtfB. Structural and kinetic analyses support the identification of Asp13 as a catalytic general base, with a possible secondary role for Thr10. Several residues have also been identified as being involved in donor sugar binding and recognition.

  14. Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: strategies and applications.

    Science.gov (United States)

    Ludwig-Müller, Jutta; Jahn, Linda; Lippert, Annemarie; Püschel, Joachim; Walter, Antje

    2014-11-01

    A plethora of bioactive plant metabolites has been explored for pharmaceutical, food chemistry and agricultural applications. The chemical synthesis of these structures is often difficult, so plants are favorably used as producers. While whole plants can serve as a source for secondary metabolites and can be also improved by metabolic engineering, more often cell or organ cultures of relevant plant species are of interest. It should be noted that only in few cases the production for commercial application in such cultures has been achieved. Their genetic manipulation is sometimes faster and the production of a specific metabolite is more reliable, because of less environmental influences. In addition, upscaling in bioreactors is nowadays possible for many of these cultures, so some are already used in industry. There are approaches to alter the profile of metabolites not only by using plant genes, but also by using bacterial genes encoding modifying enzymes. Also, strategies to cope with unwanted or even toxic compounds are available. The need for metabolic engineering of plant secondary metabolite pathways is increasing with the rising demand for (novel) compounds with new bioactive properties. Here, we give some examples of recent developments for the metabolic engineering of plants and organ cultures, which can be used in the production of metabolites with interesting properties. PMID:24699436

  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. Biosynthetic control of the natural abundance of carbon 13 at specific positions within fatty acids in Escherichia coli. Evidence regarding the coupling of fatty acid and phospholipid synthesis

    International Nuclear Information System (INIS)

    Stable carbon isotope ratios (13C/12C) at natural abundance levels have been determined for individual carbon atoms in each of the major phospholipid fatty acids of Escherichia coli grown on glucose as the sole carbon source. Two models were constructed for the isotope effects and carbon flow pathways which must be responsible for the observed isotopic fractionations. Both models incorporate a branch in the carbon flow at which fatty acyl-acyl carrier protein (acyl-ACP) is utilized either for complex lipid synthesis or for elongation by fatty acid synthetase. Depletion of carbon 13 in the carboxyl groups of myristic and palmitoleic acids (relative to carbonyl groups in precursor acyl-ACP's) was observed to occur at this branching site. Only one of the models was consistent both with this observation and with the observation that exogenous fatty acids are incorporated into phospholipids but are not elongated. The successful model has free fatty acid as the intermediate product coupling fatty acid biosynthesis to phospholipid synthesis. Essential to this pathway are those reactions catalyzed by thioesterases I and II as well as acyl-ACP synthetase, enzymes whose roles have previously been unknown in vivo

  17. Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties.

    Science.gov (United States)

    Rama Reddy, Nagaraja Reddy; Mehta, Rucha Harishbhai; Soni, Palak Harendrabhai; Makasana, Jayanti; Gajbhiye, Narendra Athamaram; Ponnuchamy, Manivel; Kumar, Jitendra

    2015-01-01

    Senna (Cassia angustifolia Vahl.) is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090)', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various

  18. Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl., a Non-Model Plant with Potent Laxative Properties.

    Directory of Open Access Journals (Sweden)

    Nagaraja Reddy Rama Reddy

    Full Text Available Senna (Cassia angustifolia Vahl. is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG, Cluster of Orthologous Gene (COG and Gene Ontology (GO. Out of the total transcripts, 40138 (95.0% and 36349 (97.7% from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf and 32077 (mature leaf transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7% CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in

  19. 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.

  20. Formal modeling and analysis of the hexosamine biosynthetic pathway: role of O-linked N-acetylglucosamine transferase in oncogenesis and cancer progression

    Science.gov (United States)

    Saeed, Muhammad Tariq; Holowatyj, Andreana N.; Sheikh, Iftikhar A.; Zafar Paracha, Rehan; Shafi, Aamir; Siddiqa, Amnah; Bibi, Zurah; Khan, Mukaram

    2016-01-01

    The alteration of glucose metabolism, through increased uptake of glucose and glutamine addiction, is essential to cancer cell growth and invasion. Increased flux of glucose through the Hexosamine Biosynthetic Pathway (HBP) drives increased cellular O-GlcNAcylation (hyper-O-GlcNAcylation) and contributes to cancer progression by regulating key oncogenes. However, the association between hyper-O-GlcNAcylation and activation of these oncogenes remains poorly characterized. Here, we implement a qualitative modeling framework to analyze the role of the Biological Regulatory Network in HBP activation and its potential effects on key oncogenes. Experimental observations are encoded in a temporal language format and model checking is applied to infer the model parameters and qualitative model construction. Using this model, we discover step-wise genetic alterations that promote cancer development and invasion due to an increase in glycolytic flux, and reveal critical trajectories involved in cancer progression. We compute delay constraints to reveal important associations between the production and degradation rates of proteins. O-linked N-acetylglucosamine transferase (OGT), an enzyme used for addition of O-GlcNAc during O-GlcNAcylation, is identified as a key regulator to promote oncogenesis in a feedback mechanism through the stabilization of c-Myc. Silencing of the OGT and c-Myc loop decreases glycolytic flux and leads to programmed cell death. Results of network analyses also identify a significant cycle that highlights the role of p53-Mdm2 circuit oscillations in cancer recovery and homeostasis. Together, our findings suggest that the OGT and c-Myc feedback loop is critical in tumor progression, and targeting these mediators may provide a mechanism-based therapeutic approach to regulate hyper-O-GlcNAcylation in human cancer.

  1. Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes.

    Directory of Open Access Journals (Sweden)

    Fei Xia

    Full Text Available Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17 and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19 are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high

  2. Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

    Science.gov (United States)

    Zheng, Desong; Sun, Quanxi; Liu, Jiang; Li, Yaxiao; Hua, Jinping

    2016-01-01

    Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of

  3. Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes.

    Science.gov (United States)

    Xia, Fei; Li, Xueying; Li, Xinzheng; Zheng, Desong; Sun, Quanxi; Liu, Jiang; Li, Yaxiao; Hua, Jinping; Qi, Baoxiu

    2016-01-01

    Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of

  4. Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures

    Science.gov (United States)

    Rodas-Junco, Beatriz A; Cab-Guillen, Yahaira; Muñoz-Sanchez, J Armando; Vázquez-Flota, Felipe; Monforte-Gonzalez, Miriam; Hérnandez-Sotomayor, S M Teresa

    2013-01-01

    Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

  5. Genetic and metabolomic dissection of the ergothioneine and selenoneine biosynthetic pathway in the fission yeast, S. pombe, and construction of an overproduction system.

    Directory of Open Access Journals (Sweden)

    Tomáš Pluskal

    Full Text Available Ergothioneine is a small, sulfur-containing metabolite (229 Da synthesized by various species of bacteria and fungi, which can accumulate to millimolar levels in tissues or cells (e.g. erythrocytes of higher eukaryotes. It is commonly marketed as a dietary supplement due to its proposed protective and antioxidative functions. In this study we report the genes forming the two-step ergothioneine biosynthetic pathway in the fission yeast, Schizosaccharomyces pombe. We identified the first gene, egt1+ (SPBC1604.01, by sequence homology to previously published genes from Neurospora crassa and Mycobacterium smegmatis. We showed, using metabolomic analysis, that the Δegt1 deletion mutant completely lacked ergothioneine and its precursors (trimethyl histidine/hercynine and hercynylcysteine sulfoxide. Since the second step of ergothioneine biosynthesis has not been characterized in eukaryotes, we examined four putative homologs (Nfs1/SPBC21D10.11c, SPAC11D3.10, SPCC777.03c, and SPBC660.12c of the corresponding mycobacterial enzyme EgtE. Among deletion mutants of these genes, only one (ΔSPBC660.12c, designated Δegt2 showed a substantial decrease in ergothioneine, accompanied by accumulation of its immediate precursor, hercynylcysteine sulfoxide. Ergothioneine-deficient strains exhibited no phenotypic defects during vegetative growth or quiescence. To effectively study the role of ergothioneine, we constructed an egt1+ overexpression system by replacing its native promoter with the nmt1+ promoter, which is inducible in the absence of thiamine. We employed three versions of the nmt1 promoter with increasing strength of expression and confirmed corresponding accumulations of ergothioneine. We quantified the intracellular concentration of ergothioneine in S. pombe (0.3, 157.4, 41.6, and up to 1606.3 µM in vegetative, nitrogen-starved, glucose-starved, and egt1+-overexpressing cells, respectively and described its gradual accumulation under long

  6. DIFFERENTIAL EXPRESSION OF RETINOIC ACID BIOSYNTHETIC AND METABOLISM GENES IN LIVERS FROM MICE TREATED WITH HEPATOTUMORIGENIC AND NON-HEPATOTUMORIGENIC CONAZOLES

    Science.gov (United States)

    Conazoles are fungicides used in crop protection and as pharmaceuticals. Triadimefon and propiconazole are hepatotumorigenic in mice, while myclobutanil is not. Previous toxicogenomic studies suggest that alteration of the retinoic acid metabolism pathway may play a key event in ...

  7. Yeast Extract and Silver Nitrate Induce the Expression of Phenylpropanoid Biosynthetic Genes and Induce the Accumulation of Rosmarinic Acid in Agastache rugosa Cell Culture

    OpenAIRE

    Woo Tae Park; Mariadhas Valan Arasu; Naif Abdullah Al-Dhabi; Sun Kyung Yeo; Jin Jeon; Jong Seok Park; Sook Young Lee; Sang Un Park

    2016-01-01

    The present study aimed to investigate the role of yeast extract and silver nitrate on the enhancement of phenylpropanoid pathway genes and accumulation of rosmarinic acid in Agastache rugosa cell cultures. The treatment of cell cultures with yeast extract (500 mg/L) and silver nitrate (30 mg/L) for varying times enhanced the expression of genes in the phenylpropanoid pathway and the production of rosmarinic acid. The results indicated that the expression of RAS and HPPR was proportional to t...

  8. Exploring De Novo metabolic pathways from pyruvate to propionic acid.

    Science.gov (United States)

    Stine, Andrew; Zhang, Miaomin; Ro, Soo; Clendennen, Stephanie; Shelton, Michael C; Tyo, Keith E J; Broadbelt, Linda J

    2016-03-01

    Industrial biotechnology provides an efficient, sustainable solution for chemical production. However, designing biochemical pathways based solely on known reactions does not exploit its full potential. Enzymes are known to accept non-native substrates, which may allow novel, advantageous reactions. We have previously developed a computational program named Biological Network Integrated Computational Explorer (BNICE) to predict promiscuous enzyme activities and design synthetic pathways, using generalized reaction rules curated from biochemical reaction databases. Here, we use BNICE to design pathways synthesizing propionic acid from pyruvate. The currently known natural pathways produce undesirable by-products lactic acid and succinic acid, reducing their economic viability. BNICE predicted seven pathways containing four reaction steps or less, five of which avoid these by-products. Among the 16 biochemical reactions comprising these pathways, 44% were validated by literature references. More than 28% of these known reactions were not in the BNICE training dataset, showing that BNICE was able to predict novel enzyme substrates. Most of the pathways included the intermediate acrylic acid. As acrylic acid bioproduction has been well advanced, we focused on the critical step of reducing acrylic acid to propionic acid. We experimentally validated that Oye2p from Saccharomyces cerevisiae can catalyze this reaction at a slow turnover rate (10(-3) s(-1) ), which was unknown to occur with this enzyme, and is an important finding for further propionic acid metabolic engineering. These results validate BNICE as a pathway-searching tool that can predict previously unknown promiscuous enzyme activities and show that computational methods can elucidate novel biochemical pathways for industrial applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:303-311, 2016. PMID:26821575

  9. Minimum Information about a Biosynthetic Gene cluster

    NARCIS (Netherlands)

    Medema, M.H.; Kottmann, Renzo; Yilmaz, Pelin; Cummings, Matthew; Biggins, J.B.; Blin, Kai; Bruijn, De Irene; Chooi, Yit Heng; Claesen, Jan; Coates, R.C.; Cruz-Morales, Pablo; Duddela, Srikanth; Düsterhus, Stephanie; Edwards, Daniel J.; Fewer, David P.; Garg, Neha; Geiger, Christoph; Gomez-Escribano, Juan Pablo; Greule, Anja; Hadjithomas, Michalis; Haines, Anthony S.; Helfrich, Eric J.N.; Hillwig, Matthew L.; Ishida, Keishi; Jones, Adam C.; Jones, Carla S.; Jungmann, Katrin; Kegler, Carsten; Kim, Hyun Uk; Kötter, Peter; Krug, Daniel; Masschelein, Joleen; Melnik, Alexey V.; Mantovani, Simone M.; Monroe, Emily A.; Moore, Marcus; Moss, Nathan; Nützmann, Hans Wilhelm; Pan, Guohui; Pati, Amrita; Petras, Daniel; Reen, F.J.; Rosconi, Federico; Rui, Zhe; Tian, Zhenhua; Tobias, Nicholas J.; Tsunematsu, Yuta; Wiemann, Philipp; Wyckoff, Elizabeth; Yan, Xiaohui; Yim, Grace; Yu, Fengan; Xie, Yunchang; Aigle, Bertrand; Apel, Alexander K.; Balibar, Carl J.; Balskus, Emily P.; Barona-Gómez, Francisco; Bechthold, Andreas; Bode, Helge B.; Borriss, Rainer; Brady, Sean F.; Brakhage, Axel A.; Caffrey, Patrick; Cheng, Yi Qiang; Clardy, Jon; Cox, Russell J.; Mot, De René; Donadio, Stefano; Donia, Mohamed S.; Donk, Van Der Wilfred A.; Dorrestein, Pieter C.; Doyle, Sean; Driessen, Arnold J.M.; Ehling-Schulz, Monika; Entian, Karl Dieter; Fischbach, Michael A.; Gerwick, Lena; Gerwick, William H.; Gross, Harald; Gust, Bertolt; Hertweck, Christian; Höfte, Monica; Jensen, Susan E.; Ju, Jianhua; Katz, Leonard; Kaysser, Leonard; Klassen, Jonathan L.; Keller, Nancy P.; Kormanec, Jan; Kuipers, Oscar P.; Kuzuyama, Tomohisa; Kyrpides, Nikos C.; Kwon, Hyung Jin; Lautru, Sylvie; Lavigne, Rob; Lee, Chia Y.; Linquan, Bai; Liu, Xinyu; Liu, Wen; Luzhetskyy, Andriy; Mahmud, Taifo; Mast, Yvonne; Méndez, Carmen; Metsä-Ketelä, Mikko; Micklefield, Jason; Mitchell, Douglas A.; Moore, Bradley S.; Moreira, Leonilde M.; Müller, Rolf; Neilan, Brett A.; Nett, Markus; Nielsen, Jens; O'Gara, Fergal; Oikawa, Hideaki; Osbourn, Anne; Osburne, Marcia S.; Ostash, Bohdan; Payne, Shelley M.; Pernodet, Jean Luc; Petricek, Miroslav; Piel, Jörn; Ploux, Olivier; Raaijmakers, Jos M.; Salas, José A.; Schmitt, Esther K.; Scott, Barry; Seipke, Ryan F.; Shen, Ben; Sherman, David H.; Sivonen, Kaarina; Smanski, Michael J.; Sosio, Margherita; Stegmann, Evi; Süssmuth, Roderich D.; Tahlan, Kapil; Thomas, Christopher M.; Tang, Yi; Truman, Andrew W.; Viaud, Muriel; Walton, Jonathan D.; Walsh, Christopher T.; Weber, Tilmann; Wezel, Van Gilles P.; Wilkinson, Barrie; Willey, Joanne M.; Wohlleben, Wolfgang; Wright, Gerard D.; Ziemert, Nadine; Zhang, Changsheng; Zotchev, Sergey B.; Breitling, Rainer; Takano, Eriko; Glöckner, Frank Oliver

    2015-01-01

    A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploi

  10. Indole-3-acetic acid (IAA) induced changes in oil content, fatty acid profiles and expression of four fatty acid biosynthetic genes in Chlorella vulgaris at early stationary growth phase.

    Science.gov (United States)

    Jusoh, Malinna; Loh, Saw Hong; Chuah, Tse Seng; Aziz, Ahmad; Cha, Thye San

    2015-03-01

    Microalgae lipids and oils are potential candidates for renewable biodiesel. Many microalgae species accumulate a substantial amount of lipids and oils under environmental stresses. However, low growth rate under these adverse conditions account for the decrease in overall biomass productivity which directly influence the oil yield. This study was undertaken to investigate the effect of exogenously added auxin (indole-3-acetic acid; IAA) on the oil content, fatty acid compositions, and the expression of fatty acid biosynthetic genes in Chlorella vulgaris (UMT-M1). Auxin has been shown to regulate growth and metabolite production of several microalgae. Results showed that oil accumulation was highest on days after treatment (DAT)-2 with enriched levels of palmitic (C16:0) and stearic (C18:0) acids, while the linoleic (C18:2) and α-linolenic (C18:3n3) acids levels were markedly reduced by IAA. The elevated levels of saturated fatty acids (C16:0 and C18:0) were consistent with high expression of the β-ketoacyl ACP synthase I (KAS I) gene, while low expression of omega-6 fatty acid desaturase (ω-6 FAD) gene was consistent with low production of C18:2. However, the increment of stearoyl-ACP desaturase (SAD) gene expression upon IAA induction did not coincide with oleic acid (C18:1) production. The expression of omega-3 fatty acid desaturase (ω-3 FAD) gene showed a positive correlation with the synthesis of PUFA and C18:3n3.

  11. Functional characterization of a fatty acid double-bond hydratase from Lactobacillus plantarum and its interaction with biosynthetic membranes.

    Science.gov (United States)

    Ortega-Anaya, Joana; Hernández-Santoyo, Alejandra

    2015-12-01

    Hydrogenation of linoleic acid and other polyunsaturated fatty acids is a detoxification mechanism that is present in the Lactobacillus genus of lactic bacteria. The first stage in this multi-step process is hydration of the substrate with formation of 10-hydroxy-9-cis-octadecenoic acid due to fatty-acid hydratase activity that has been detected only in the membrane-associated cell fraction; however, its interaction with the cell membrane is unknown. To provide information in this respect we characterized the homotrimeric 64.7 kDa-native protein from Lactobacillus plantarum; afterwards, it was reconstituted in proteoliposomes and analyzed by confocal fluorescence microscopy. The results showed that hydratase is an extrinsic-membrane protein and hence, the enzymatic reaction occurs at the periphery of the cell. This location may be advantageous in the detoxifying process since the toxic linoleic acid molecule can be bound to hydratase and converted to non-toxic 10-hydroxy-9-cis-octadecenoic acid before it reaches cell membrane. Additionally, we propose that the interaction with membrane periphery occurs through electrostatic contacts. Finally, the structural model of L. plantarum hydratase was constructed based on the amino acid sequence and hence, the putative binding sites with linoleic acid were identified: site 1, located in an external hydrophobic pocket at the C-terminus of the protein and site 2, located at the core and in contact with a FAD molecule. Interestingly, it was found that the linoleic acid molecule arranges around a methionine residue in both sites (Met154 and Met81, respectively) that acts as a rigid pole, thus playing a key role in binding unsaturated fatty acids.

  12. Integrating nitric oxide into salicylic acid and jasmonic acid/ethylene plant defense pathways

    DEFF Research Database (Denmark)

    Mur, Luis A J; Prats, Elena; Pierre, Sandra;

    2013-01-01

    Plant defence against pests and pathogens is known to be conferred by either salicylic acid (SA) or jasmonic acid (JA)/ethylene (ET) pathways, depending on infection or herbivore-grazing strategy. It is well attested that SA and JA/ET pathways are mutually antagonistic allowing defence responses...

  13. Research Progress of Terpenoid Indole Alkaloids (TIAs) Biosynthetic Pathway of Catharanthus roseus%长春花萜类吲哚生物碱的生物合成途径

    Institute of Scientific and Technical Information of China (English)

    邢世海; 王荃; 潘琪芳; 赵静雅; 唐克轩

    2012-01-01

    There are more than 130 types of terpenoid indole alkaloids (TIAs) in medicinal plant Catharanthus roseus (L. ) G. Don. In this review, the upstream and downstream of the biosynthesis of these alkaloids in C. roseus, and related studies were summarized. During upstream pathway, tryptamine which came from indole biosynthetic pathway and secologamn which came from monoterpenoid pathway were synthesized into 3a(S)-Strictosidine which is the common precurors of different TIAs by the corresponding enzymatic catalysis. Then various TIAs were found from 3α(S)-Strictosidine by enzymes during downstream process. The TIAs biosynthetic pathway in C. roseus were outlined in the article in order to provide useful information for the researchers who are interested in terpenoid indole alkaloids biosynthetic pathway and metabolic regulation.%药用植物长春花含有130余种萜类吲哚生物碱,该文对近年来国内外有关长春花生物碱合成的上游和下游阶段及其相关研究进行详细的归纳总结.长春花上游合成途径中在相应的酶促作用下由吲哚途径产生的色胺和由类萜途径产生的裂环马钱子苷在异胡豆苷合成酶的催化作用下形成了所有长春花TIAs的共同前体物质3α-异胡豆苷,3α-异胡豆苷再由下游途径的各种酶促作用下生成种类各异的长春花TIAs.通过对长春花TIAs合成途径的阐述,为萜类吲哚生物碱合成及其代谢调控的相关研究提供参考.

  14. The Simultaneous Repression of CCR and CAD, Two Enzymes of the Lignin Biosynthetic Pathway, Results in Sterility and Dwarfism in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Johanne Thévenin; Brigitte Pollet; Bruno Letarnec; Luc Saulnier; Lionel Gissot; Alessandra Maia-Grondard; Catherine Lapierre; Lise Jouanina

    2011-01-01

    Cinnamoyl CoA reductase(CCR)and cinnamyl alcohol dehydrogenase(CAD)catalyze the last steps of monolignol biosynthesis.In Arabidopsis,one CCR gene(CCR1,At1g15950)and two CAD genes(CAD C At3g19450 and CAD D At4g34230)are involved in this pathway.A triple cad c cad d ccr1 mutant,named ccc,was obtained.This mutant displays a severe dwarf phenotype and male sterility.The lignin content in ccc mature stems is reduced to 50% of the wild-type level.In addition,stem lignin structure is severely affected,as shown by the dramatic enrichment in resistant inter-unit bonds and incorporation into the polymer of monolignol precursors such as coniferaldehyde,sinapaldehyde,and ferulic acid.Male sterility is due to the lack of lignification in the anther endothecium,which causes the failure of anther dehiscence and of pollen release.The ccc hypolignified stems accumulate higher amounts of flavonol glycosides,sinapoyl malate and feruloyl malate,which suggests a redirection of the phenolic pathway.Therefore,the absence of CAD and CCR,key enzymes of the monolignol pathway,has more severe consequences on the phenotype than the individual absence of each of them.Induction of another CCR(CCR2,At1g80820)and another CAD(CAD1,At4g39330)does not compensate the absence of the main CCR and CAD activities.This lack of CCR and CAD activities not only impacts lignification,but also severely affects the development of the plants.These consequences must be carefully considered when trying to reduce the lignin content of plants in order to facilitate the lignocellulose-to-bioethanol conversion process.

  15. Early lignin pathway enzymes and routes to chlorogenic acid in switchgrass (Panicum virgatum L.).

    Science.gov (United States)

    Escamilla-Treviño, Luis L; Shen, Hui; Hernandez, Timothy; Yin, Yanbin; Xu, Ying; Dixon, Richard A

    2014-03-01

    Studying lignin biosynthesis in Panicum virgatum (switchgrass) has provided a basis for generating plants with reduced lignin content and increased saccharification efficiency. Chlorogenic acid (CGA, caffeoyl quinate) is the major soluble phenolic compound in switchgrass, and the lignin and CGA biosynthetic pathways potentially share intermediates and enzymes. The enzyme hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) is responsible for CGA biosynthesis in tobacco, tomato and globe artichoke, but there are no close orthologs of HQT in switchgrass or in other monocotyledonous plants with complete genome sequences. We examined available transcriptomic databases for genes encoding enzymes potentially involved in CGA biosynthesis in switchgrass. The protein products of two hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) genes (PvHCT1a and PvHCT2a), closely related to lignin pathway HCTs from other species, were characterized biochemically and exhibited the expected HCT activity, preferring shikimic acid as acyl acceptor. We also characterized two switchgrass coumaroyl shikimate 3'-hydroxylase (C3'H) enzymes (PvC3'H1 and PvC3'H2); both of these cytochrome P450s had the capacity to hydroxylate 4-coumaroyl shikimate or 4-coumaroyl quinate to generate caffeoyl shikimate or CGA. Another switchgrass hydroxycinnamoyl transferase, PvHCT-Like1, is phylogenetically distant from HCTs or HQTs, but exhibits HQT activity, preferring quinic acid as acyl acceptor, and could therefore function in CGA biosynthesis. The biochemical features of the recombinant enzymes, the presence of the corresponding activities in plant protein extracts, and the expression patterns of the corresponding genes, suggest preferred routes to CGA in switchgrass.

  16. Hydrogen isotope analysis of amino acids and whole cells reflects biosynthetic processing of nutrient- and water-derived hydrogen

    Science.gov (United States)

    Griffin, P.; Newsome, S.; Steele, A.; Fogel, M. L.

    2011-12-01

    Hydrogen (H) isotopes serve as sensitive tracers of biochemical processes that can be exploited to answer critical questions in biogeochemistry, ecology, and microbiology. Despite this apparent utility, relatively little is known about the specific mechanisms of H isotope fractionation involved in biosynthesis. In order to understand how organisms incorporate hydrogen from their chemical milieu into biomass, we have cultured the model bacterium E. coli MG1655 in a variety of media composed of deuterium-labeled nutrients and waters. Isotopic analysis of bulk cell mass reveals that the H fractionation between media water and cell material varies as a function of the nutrient source, with commonly used organic food sources (glucose and tryptone) leading to far smaller fractionation signals than non-standard ones (such as formamide, adenine, and urea). In addition, we have completed compound specific isotope analysis of amino acids using combined GC-IRMS. Amino acids harvested from E. coli cultured on glucose in water of varied D/H composition posses an extraordinary range of isotopic compositions (400-600 %). Furthermore, these amino acids follow a systematic distribution of D/H where proline is always heaviest and glycine is always lightest. However, when the short-chain peptide tryptone is used in place of glucose, only the non-essential amino acids reflect media water D/H values, suggesting the direct incorporation of some media-borne amino acids into cellular protein. These observations provide a foundation for understanding the cellular routing of hydrogen obtained from food and water sources and indicate that D/H analysis can serve as a powerful probe of biological function.

  17. Deciphering ascorbic acid regulatory pathways in ripening tomato fruit using a weighted gene correlation network analysis approach.

    Science.gov (United States)

    Gao, Chao; Ju, Zheng; Li, Shan; Zuo, Jinhua; Fu, Daqi; Tian, Huiqin; Luo, Yunbo; Zhu, Benzhong

    2013-11-01

    Genotype is generally determined by the co-expression of diverse genes and multiple regulatory pathways in plants. Gene co-expression analysis combining with physiological trait data provides very important information about the gene function and regulatory mechanism. L-Ascorbic acid (AsA), which is an essential nutrient component for human health and plant metabolism, plays key roles in diverse biological processes such as cell cycle, cell expansion, stress resistance, hormone synthesis, and signaling. Here, we applied a weighted gene correlation network analysis approach based on gene expression values and AsA content data in ripening tomato (Solanum lycopersicum L.) fruit with different AsA content levels, which leads to identification of AsA relevant modules and vital genes in AsA regulatory pathways. Twenty-four modules were compartmentalized according to gene expression profiling. Among these modules, one negatively related module containing genes involved in redox processes and one positively related module enriched with genes involved in AsA biosynthetic and recycling pathways were further analyzed. The present work herein indicates that redox pathways as well as hormone-signal pathways are closely correlated with AsA accumulation in ripening tomato fruit, and allowed us to prioritize candidate genes for follow-up studies to dissect this interplay at the biochemical and molecular level.

  18. Deciphering Ascorbic Acid Regulatory Pathways in Ripening Tomato Fruit Using a Weighted Gene Correlation Network Analysis Approach

    Institute of Scientific and Technical Information of China (English)

    Chao Gao; Zheng Ju; Shan Li; Jinhua Zuo; Daqi Fu; Huiqin Tian; Yunbo Luo; Benzhong Zhu

    2013-01-01

    Genotype is generally determined by the co-expression of diverse genes and multiple regulatory pathways in plants. Gene co-expression analysis combining with physiological trait data provides very important information about the gene function and regulatory mechanism. L-Ascorbic acid (AsA), which is an essential nutrient component for human health and plant metabolism, plays key roles in diverse biological processes such as cell cycle, cell expansion, stress resistance, hormone synthesis, and signaling. Here, we applied a weighted gene correlation network analysis approach based on gene expression values and AsA content data in ripening tomato (Solanum lycopersicum L.) fruit with different AsA content levels, which leads to identification of AsA relevant modules and vital genes in AsA regulatory pathways. Twenty-four modules were compartmentalized according to gene expression profiling. Among these modules, one negatively related module containing genes involved in redox processes and one positively related module enriched with genes involved in AsA biosynthetic and recycling pathways were further analyzed. The present work herein indicates that redox pathways as well as hormone-signal pathways are closely correlated with AsA accumulation in ripening tomato fruit, and allowed us to prioritize candidate genes for follow-up studies to dissect this interplay at the biochemical and molecular level.

  19. Analysis of the transcriptome of Erigeron breviscapus uncovers putative scutellarin and chlorogenic acids biosynthetic genes and genetic markers.

    Directory of Open Access Journals (Sweden)

    Ni-Hao Jiang

    Full Text Available Erigeron breviscapus (Vant. Hand-Mazz. is a famous medicinal plant. Scutellarin and chlorogenic acids are the primary active components in this herb. However, the mechanisms of biosynthesis and regulation for scutellarin and chlorogenic acids in E. breviscapus are considerably unknown. In addition, genomic information of this herb is also unavailable.Using Illumina sequencing on GAIIx platform, a total of 64,605,972 raw sequencing reads were generated and assembled into 73,092 non-redundant unigenes. Among them, 44,855 unigenes (61.37% were annotated in the public databases Nr, Swiss-Prot, KEGG, and COG. The transcripts encoding the known enzymes involved in flavonoids and in chlorogenic acids biosynthesis were discovered in the Illumina dataset. Three candidate cytochrome P450 genes were discovered which might encode flavone 6-hydroase converting apigenin to scutellarein. Furthermore, 4 unigenes encoding the homologues of maize P1 (R2R3-MYB transcription factors were defined, which might regulate the biosynthesis of scutellarin. Additionally, a total of 11,077 simple sequence repeat (SSR were identified from 9,255 unigenes. Of SSRs, tri-nucleotide motifs were the most abundant motif. Thirty-six primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism. The result revealed that 34 (94.40% primer pairs were successfully amplified and 19 (52.78% primer pairs exhibited polymorphisms.Using next generation sequencing (NGS technology, this study firstly provides abundant genomic data for E. breviscapus. The candidate genes involved in the biosynthesis and transcriptional regulation of scutellarin and chlorogenic acids were obtained in this study. Additionally, a plenty of genetic makers were generated by identification of SSRs, which is a powerful tool for molecular breeding and genetics applications in this herb.

  20. Identification and Functional Characterization of Genes Encoding Omega-3 Polyunsaturated Fatty Acid Biosynthetic Activities from Unicellular Microalgae

    OpenAIRE

    Royah Vaezi; Napier, Johnathan A.; Olga Sayanova

    2013-01-01

    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...

  1. Diurnal variations of mouse plasma and hepatic bile acid concentrations as well as expression of biosynthetic enzymes and transporters.

    Directory of Open Access Journals (Sweden)

    Yu-Kun Jennifer Zhang

    Full Text Available BACKGROUND: Diurnal fluctuation of bile acid (BA concentrations in the enterohepatic system of mammals has been known for a long time. Recently, BAs have been recognized as signaling molecules beyond their well-established roles in dietary lipid absorption and cholesterol homeostasis. METHODS AND RESULTS: The current study depicted diurnal variations of individual BAs detected by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS in serum and livers collected from C57BL/6 mice fed a regular chow or a chow containing cholestyramine (resin. Circadian rhythms of mRNA of vital BA-related nuclear receptors, enzymes, and transporters in livers and ilea were determined in control- and resin-fed mice, as well as in farnesoid X receptor (FXR null mice. The circadian profiles of BAs showed enhanced bacterial dehydroxylation during the fasting phase and efficient hepatic reconjugation of BAs in the fed phase. The resin removed more than 90% of BAs with β-hydroxy groups, such as muricholic acids and ursodeoxycholic acid, from serum and livers, but did not exert as significant influence on CA and CDCA in both compartments. Both resin-fed and FXR-null mouse models indicate that BAs regulate their own biosynthesis through the FXR-regulated ileal fibroblast growth factor 15. BA flux also influences the daily mRNA levels of multiple BA transporters. CONCLUSION: BA concentration and composition exhibit circadian variations in mouse liver and serum, which influences the circadian rhythms of BA metabolizing genes in liver and ileum. The diurnal variations of BAs appear to serve as a signal that coordinates daily nutrient metabolism in mammals.

  2. A heteromeric membrane-bound prenyltransferase complex from hop catalyzes three sequential aromatic prenylations in the bitter acid pathway.

    Science.gov (United States)

    Li, Haoxun; Ban, Zhaonan; Qin, Hao; Ma, Liya; King, Andrew J; Wang, Guodong

    2015-03-01

    Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop. PMID:25564559

  3. AMPK-independent pathways regulate skeletal muscle fatty acid oxidation

    DEFF Research Database (Denmark)

    Dzamko, Nicolas; Schertzer, Jonathan D.; Ryall, James G.;

    2008-01-01

    with rates of fatty acid oxidation. To address this issue we have investigated the requirement for skeletal muscle AMPK in controlling aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside (AICAR) and contraction-stimulated fatty acid oxidation utilizing transgenic mice expressing a muscle-specific kinase...... beta (IKKbeta) and protein kinase D (PKD) may phosphorylate ACC2 at Ser-221 but during in vitro phosphorylation assays only AMPK phosphorylated ACC2. These data demonstrate that AMPK is not essential for the regulation of fatty acid oxidation by AICAR or muscle contraction.......The activation of AMP-activated protein kinase (AMPK) and phosphorylation/inhibition of acetyl-CoA carboxylase 2 (ACC2) is believed to be the principal pathway regulating fatty acid oxidation. However, during exercise AMPK activity and ACC Ser-221 phosphorylation does not always correlate...

  4. Expression, Crystallization and Preliminary X-ray Diffraction Analyses of Med-ORF10 in the Biosynthetic Pathway of an Antitumor Antibiotic Medermycin.

    Science.gov (United States)

    Liu, Yanli; Liu, Shasha; Yang, Tingting; Guo, Xiaoxia; Jiang, Yali; Zahid, Kashif Rafiq; Liu, Ke; Liu, Jinlin; Yang, Jihong; Zhao, Haobin; Yang, Yi; Li, Aiying; Qi, Chao

    2015-12-01

    Medermycin, as a prominent member of benzoisochromanequinones, possesses strong antitumor activity and is biosynthesized under the control of a 29-ORF-containing biosynthetic gene cluster. Most of ORFs in this gene cluster have not been characterized, including a small protein encoding gene med-ORF10, proposed to play a regulatory role in biosynthesis of medermycin in an unknown mode. In this study, we reported the expression, protein preparation, crystallization and preliminary X-ray diffraction analyses of Med-ORF10 of the wild type Streptomyces strain. Firstly, we cloned and overexpressed med-ORF10 in Escherichia coli and purified the protein with 98% purity and 3 mg/L yield. Then, we crystallized the protein at concentration of 20 mg/mL in condition 22% PEG 3350, 0.2 M magnesium formate and collected the data at 1.78 Å resolution. Finally, we detected the expression of Med-ORF10 in Streptomyces by western blotting. In conclusion, this study confirmed the expression of Med-ORF10 protein in the wild-type strain of Streptomyces AM-7161 and collected the X-ray diffraction data of Med-ORF10 crystal at 1.78 Å resolution. These studies provide evidences for the functional Med-ORF10 protein in Streptomyces strains and facilitate our further investigation.

  5. Conservation and Expression Patterns Divergence of Ascorbic Acid d-mannose/l-galactose Pathway Genes in Brassica rapa

    Science.gov (United States)

    Duan, Weike; Ren, Jun; Li, Yan; Liu, Tongkun; Song, Xiaoming; Chen, Zhongwen; Huang, Zhinan; Hou, Xilin; Li, Ying

    2016-01-01

    Ascorbic acid (AsA) participates in diverse biological processes, is regulated by multiple factors and is a potent antioxidant and cellular reductant. The D-Mannose/L-Galactose pathway is a major plant AsA biosynthetic pathway that is highly connected within biosynthetic networks, and generally conserved across plants. Previous work has shown that, although most genes of this pathway are expressed under standard growth conditions in Brassica rapa, some paralogs of these genes are not. We hypothesize that regulatory evolution in duplicate AsA pathway genes has occurred as an adaptation to environmental stressors, and that gene retention has been influenced by polyploidation events in Brassicas. To test these hypotheses, we explored the conservation of these genes in Brassicas and their expression patterns divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A genome), B. oleracea (C genome) and B. napus (AC genome). However, the number of genes that encode the same type of enzymes varied among the three plant species. With the exception of GMP, which has nine genes, there were one to four genes that encoded the other enzymes. Moreover, we found that expression patterns divergence widely exists among these genes. (i) VTC2 and VTC5 are paralogous genes, but only VTC5 is influenced by FLC. (ii) Under light treatment, PMI1 co-regulates the AsA pool size with other D-Man/L-Gal pathway genes, whereas PMI2 is regulated only by darkness. (iii) Under NaCl, Cu2+, MeJA and wounding stresses, most of the paralogs exhibit different expression patterns. Additionally, GME and GPP are the key regulatory enzymes that limit AsA biosynthesis in response to these treatments. In conclusion, our data support that the conservative and divergent expression patterns of D-Man/L-Gal pathway genes not only avoid AsA biosynthesis network instability but also allow B. rapa to better adapt to complex environments. PMID:27313597

  6. Conservation and Expression Patterns Divergence of Ascorbic Acid d-mannose/l-galactose Pathway Genes in Brassica rapa.

    Science.gov (United States)

    Duan, Weike; Ren, Jun; Li, Yan; Liu, Tongkun; Song, Xiaoming; Chen, Zhongwen; Huang, Zhinan; Hou, Xilin; Li, Ying

    2016-01-01

    Ascorbic acid (AsA) participates in diverse biological processes, is regulated by multiple factors and is a potent antioxidant and cellular reductant. The D-Mannose/L-Galactose pathway is a major plant AsA biosynthetic pathway that is highly connected within biosynthetic networks, and generally conserved across plants. Previous work has shown that, although most genes of this pathway are expressed under standard growth conditions in Brassica rapa, some paralogs of these genes are not. We hypothesize that regulatory evolution in duplicate AsA pathway genes has occurred as an adaptation to environmental stressors, and that gene retention has been influenced by polyploidation events in Brassicas. To test these hypotheses, we explored the conservation of these genes in Brassicas and their expression patterns divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A genome), B. oleracea (C genome) and B. napus (AC genome). However, the number of genes that encode the same type of enzymes varied among the three plant species. With the exception of GMP, which has nine genes, there were one to four genes that encoded the other enzymes. Moreover, we found that expression patterns divergence widely exists among these genes. (i) VTC2 and VTC5 are paralogous genes, but only VTC5 is influenced by FLC. (ii) Under light treatment, PMI1 co-regulates the AsA pool size with other D-Man/L-Gal pathway genes, whereas PMI2 is regulated only by darkness. (iii) Under NaCl, Cu(2+), MeJA and wounding stresses, most of the paralogs exhibit different expression patterns. Additionally, GME and GPP are the key regulatory enzymes that limit AsA biosynthesis in response to these treatments. In conclusion, our data support that the conservative and divergent expression patterns of D-Man/L-Gal pathway genes not only avoid AsA biosynthesis network instability but also allow B. rapa to better adapt to complex environments. PMID:27313597

  7. Conservation and expression patterns divergence of ascorbic acid D-mannose/L-galactose pathway genes in Brassica rapa

    Directory of Open Access Journals (Sweden)

    Weike eDuan

    2016-06-01

    Full Text Available Ascorbic acid (AsA participates in diverse biological processes, is regulated by multiple factors and is a potent antioxidant and cellular reductant. The D-mannose/L-galactose pathway is a major plant AsA biosynthetic pathway that is highly connected within biosynthetic networks, and generally conserved across plants. Previous work has shown that, although most genes of this pathway are expressed under standard growth conditions in Brassica rapa, some paralogs of these genes are not. We hypothesize that regulatory evolution in duplicate AsA pathway genes has occurred as an adaptation to environmental stressors, and that gene retention has been influenced by polyploidation events in Brassicas. To test these hypotheses, we explored the conservation of these genes in Brassicas and their expression patterns divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A genome, Brassica oleracea (C genome and Brassica napus (AC genome. However, the number of genes that encode the same type of enzymes varied among the three plant species. With the exception of GMP, which has nine genes, there were one to four genes that encoded the other enzymes. Moreover, we found that expression patterns divergence widely exists among these genes. i VTC2 and VTC5 are paralogous genes, but only VTC5 is influenced by FLC. ii Under light treatment, PMI1 co-regulates the AsA pool size with other D-Man/L-Gal pathway genes, whereas PMI2 is regulated only by darkness. iii Under NaCl, Cu2+, MeJA and wounding stresses, most of the paralogs exhibit different expression patterns. Additionally, GME and GPP are the key regulatory enzymes that limit AsA biosynthesis in response to these treatments. In conclusion, our data support that the conservative and divergent expression patterns of D-Man/L-Gal pathway genes not only avoid AsA biosynthesis network instability but also allow B. rapa to better adapt to complex environments.

  8. New Biosynthetic Step in the Melanin Pathway of Wangiella (Exophiala) dermatitidis: Evidence for 2-Acetyl-1,3,6,8-Tetrahydroxynaphthalene as a Novel Precursor

    Science.gov (United States)

    The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced as a pentaketide directly by an iter...

  9. Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis, phylogenetic analysis, and expression profiling

    OpenAIRE

    Li, Peirong; Zhang, Shujiang; Zhang, Shifan; Li, Fei; Zhang, Hui; Cheng, Feng; Wu, Jian; Wang, Xiaowu; Sun, Rifei

    2015-01-01

    Background Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa. Results We identified 67 carotenoid biosynthetic genes in B. rapa, which were ort...

  10. Concise Total Synthesis of (±)-Salinosporamide A, (±)-Cinnabaramide A, and Derivatives via a Bis-Cyclization Process: Implications for a Biosynthetic Pathway?

    OpenAIRE

    Ma, Gil; Nguyen, Henry; Romo, Daniel

    2007-01-01

    4-Alkylidene-β-lactones (hetero ketene dimers) and α-amino acids are useful precursors for total syntheses of the β-lactone containing proteasome inhibitors, salinosporamide A, cinnabaramide A, and derivatives. A key step is a nucleophile-promoted, bis-cyclization of keto acids that simultaneously generates the γ-lactam-and β-lactone of these natural products. This reaction sequence may have implications for the biosynthesis of these natural products.

  11. Anthocyanin biosynthetic genes in Brassica rapa

    OpenAIRE

    Guo, Ning; Cheng, Feng; Wu, Jian; Liu, Bo; Zheng, Shuning; Liang, Jianli; Wang, Xiaowu

    2014-01-01

    Background Anthocyanins are a group of flavonoid compounds. As a group of important secondary metabolites, they perform several key biological functions in plants. Anthocyanins also play beneficial health roles as potentially protective factors against cancer and heart disease. To elucidate the anthocyanin biosynthetic pathway in Brassica rapa, we conducted comparative genomic analyses between Arabidopsis thaliana and B. rapa on a genome-wide level. Results In total, we identified 73 genes in...

  12. Deciphering the Late Biosynthetic Steps of Antimalarial Compound FR-900098

    OpenAIRE

    Johannes, Tyler W.; DeSieno, Matthew A.; Griffin, Benjamin M.; Thomas, Paul M.; Kelleher, Neil L.; Metcalf, William W.; Zhao, Huimin

    2010-01-01

    FR-900098 is a potent chemotherapeutic agent for the treatment of malaria. Here we report the heterologous production of this compound in E. coli by re-constructing the entire biosynthetic pathway using a three plasmid system. Based on this system, whole cell feeding assays in combination with in vitro enzymatic activity assays reveal an unprecedented functional role of nucleotide conjugation and lead to the complete elucidation of the previously unassigned late biosynthetic steps. These stud...

  13. Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits.

    Science.gov (United States)

    Smita, Shuchi; Rajwanshi, Ravi; Lenka, Sangram Keshari; Katiyar, Amit; Chinnusamy, Viswanathan; Bansal, Kailash Chander

    2013-12-01

    Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the beta-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype-Pusa Rohini. We found that expression of phytoene synthase and beta-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.

  14. Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits

    Indian Academy of Sciences (India)

    Shuchi Smita; Ravi Rajwanshi; Sangram Keshari Lenka; Amit Katiyar; Viswanathan Chinnusamy; Kailash Chander Bansal

    2013-12-01

    Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the -carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype—Pusa Rohini. We found that expression of phytoene synthase and -carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.

  15. Structural Insights Into the Evolutionary Paths of Oxylipin Biosynthetic Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Lee, D.-S.; Nioche, P.; Hamberg, M.; Raman, C.S.

    2009-05-20

    The oxylipin pathway generates not only prostaglandin-like jasmonates but also green leaf volatiles (GLVs), which confer characteristic aromas to fruits and vegetables. Although allene oxide synthase (AOS) and hydroperoxide lyase are atypical cytochrome P450 family members involved in the synthesis of jasmonates and GLVs, respectively, it is unknown how these enzymes rearrange their hydroperoxide substrates into different products. Here we present the crystal structures of Arabidopsis thaliana AOS, free and in complex with substrate or intermediate analogues. The structures reveal an unusual active site poised to control the reactivity of an epoxyallylic radical and its cation by means of interactions with an aromatic {pi}-system. Replacing the amino acid involved in these steps by a non-polar residue markedly reduces AOS activity and, unexpectedly, is both necessary and sufficient for converting AOS into a GLV biosynthetic enzyme. Furthermore, by combining our structural data with bioinformatic and biochemical analyses, we have discovered previously unknown hydroperoxide lyase in plant growth-promoting rhizobacteria, AOS in coral, and epoxyalcohol synthase in amphioxus. These results indicate that oxylipin biosynthetic genes were present in the last common ancestor of plants and animals, but were subsequently lost in all metazoan lineages except Placozoa, Cnidaria and Cephalochordata.

  16. Evolution in biosynthetic pathways: two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region.

    OpenAIRE

    Belfaiza, J.; Parsot, C; Martel, A.; de la Tour, C B; Margarita, D; Cohen, G. N.; Saint-Girons, I

    1986-01-01

    The metC gene of Escherichia coli K-12 was cloned and the nucleotide sequence of the metC gene and its flanking regions was determined. The translation initiation codon was identified by sequencing the NH2-terminal part of beta-cystathionase, the MetC gene product. The metC gene (1185 nucleotides) encodes a protein having 395 amino acid residues. The 5' noncoding region was found to contain a "Met box" homologous to sequences suggestive of operator structures upstream from other methionine ge...

  17. Minimum Information about a Biosynthetic Gene cluster : commentary

    NARCIS (Netherlands)

    Medema, Marnix H; Kottmann, Renzo; Yilmaz, Pelin; Cummings, Matthew; Biggins, John B; Blin, Kai; de Bruijn, Irene; Chooi, Yit Heng; Claesen, Jan; Coates, R Cameron; Cruz-Morales, Pablo; Duddela, Srikanth; Dusterhus, Stephanie; Edwards, Daniel J; Fewer, David P; Garg, Neha; Geiger, Christoph; Gomez-Escribano, Juan Pablo; Greule, Anja; Hadjithomas, Michalis; Haines, Anthony S; Helfrich, Eric J N; Hillwig, Matthew L; Ishida, Keishi; Jones, Adam C; Jones, Carla S; Jungmann, Katrin; Kegler, Carsten; Kim, Hyun Uk; Kotter, Peter; Krug, Daniel; Masschelein, Joleen; Melnik, Alexey V; Mantovani, Simone M; Monroe, Emily A; Moore, Marcus; Moss, Nathan; Nutzmann, Hans-Wilhelm; Pan, Guohui; Pati, Amrita; Petras, Daniel; Reen, F Jerry; Rosconi, Federico; Rui, Zhe; Tian, Zhenhua; Tobias, Nicholas J; Tsunematsu, Yuta; Wiemann, Philipp; Wyckoff, Elizabeth; Yan, Xiaohui; Yim, Grace; Yu, Fengan; Xie, Yunchang; Aigle, Bertrand; Apel, Alexander K; Balibar, Carl J; Balskus, Emily P; Barona-Gomez, Francisco; Bechthold, Andreas; Bode, Helge B; Borriss, Rainer; Brady, Sean F; Brakhage, Axel A; Caffrey, Patrick; Cheng, Yi-Qiang; Clardy, Jon; Cox, Russell J; De Mot, Rene; Donadio, Stefano; Donia, Mohamed S; van der Donk, Wilfred A; Dorrestein, Pieter C; Doyle, Sean; Driessen, Arnold J M; Ehling-Schulz, Monika; Entian, Karl-Dieter; Fischbach, Michael A; Gerwick, Lena; Gerwick, William H; Gross, Harald; Gust, Bertolt; Hertweck, Christian; Hofte, Monica; Jensen, Susan E; Ju, Jianhua; Katz, Leonard; Kaysser, Leonard; Klassen, Jonathan L; Keller, Nancy P; Kormanec, Jan; Kuipers, Oscar P; Kuzuyama, Tomohisa; Kyrpides, Nikos C; Kwon, Hyung-Jin; Lautru, Sylvie; Lavigne, Rob; Lee, Chia Y; Linquan, Bai; Liu, Xinyu; Liu, Wen; Luzhetskyy, Andriy; Mahmud, Taifo; Mast, Yvonne; Mendez, Carmen; Metsa-Ketela, Mikko; Micklefield, Jason; Mitchell, Douglas A; Moore, Bradley S; Moreira, Leonilde M; Muller, Rolf; Neilan, Brett A; Nett, Markus; Nielsen, Jens; O'Gara, Fergal; Oikawa, Hideaki; Osbourn, Anne; Osburne, Marcia S; Ostash, Bohdan; Payne, Shelley M; Pernodet, Jean-Luc; Petricek, Miroslav; Piel, Jorn; Ploux, Olivier; Raaijmakers, Jos M; Salas, Jose A; Schmitt, Esther K; Scott, Barry; Seipke, Ryan F; Shen, Ben; Sherman, David H; Sivonen, Kaarina; Smanski, Michael J; Sosio, Margherita; Stegmann, Evi; Sussmuth, Roderich D; Tahlan, Kapil; Thomas, Christopher M; Tang, Yi; Truman, Andrew W; Viaud, Muriel; Walton, Jonathan D; Walsh, Christopher T; Weber, Tilmann; van Wezel, Gilles P; Wilkinson, Barrie; Willey, Joanne M; Wohlleben, Wolfgang; Wright, Gerard D; Ziemert, Nadine; Zhang, Changsheng; Zotchev, Sergey B; Breitling, Rainer; Takano, Eriko; Glockner, Frank Oliver

    2015-01-01

    A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit.

  18. Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

    DEFF Research Database (Denmark)

    Brinch-Pedersen, Henrik; Galili, G; Knudsen, S;

    1996-01-01

    In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the...

  19. Fatty acid hydroperoxides pathways in plants. A review.

    Directory of Open Access Journals (Sweden)

    Fauconnier, M. L.

    1997-02-01

    Full Text Available The present paper focusses on the fatty acid hydroperoxides pathways, mainly hydroperoxide lyase and hydroperoxide dehydrase. For each enzyme, the definition, occurrence and subcellular localization is presented. Particular attention is given to reaction mecanisms and to substrate specificity. Physiological roles of reaction products are also discussed.

    El presente artículo se centra en las rutas de los hidroperóxidos de ácidos grasos, principalmente la hidroperóxido liasa y la hidroperóxido dehidrasa. Se presenta para cada enzima, la definición, distribución y localización subcelular. Se da atención particular a los mecanismos de reacción y a la especificidad de sustrato. También se discuten los papeles fisiológicos de los productos de reacción.

  20. The floral transcriptome of ylang ylang (Cananga odorata var. fruticosa) uncovers biosynthetic pathways for volatile organic compounds and a multifunctional and novel sesquiterpene synthase.

    Science.gov (United States)

    Jin, Jingjing; Kim, Mi Jung; Dhandapani, Savitha; Tjhang, Jessica Gambino; Yin, Jun-Lin; Wong, Limsoon; Sarojam, Rajani; Chua, Nam-Hai; Jang, In-Cheol

    2015-07-01

    The pleasant fragrance of ylang ylang varieties (Cananga odorata) is mainly due to volatile organic compounds (VOCs) produced by the flowers. Floral scents are a key factor in plant-insect interactions and are vital for successful pollination. C. odorata var. fruticosa, or dwarf ylang ylang, is a variety of ylang ylang that is popularly grown in Southeast Asia as a small shrub with aromatic flowers. Here, we describe the combined use of bioinformatics and chemical analysis to discover genes for the VOC biosynthesis pathways and related genes. The scented flowers of C. odorata var. fruticosa were analysed by gas chromatography/mass spectrometry and a total of 49 VOCs were identified at four different stages of flower development. The bulk of these VOCs were terpenes, mainly sesquiterpenes. To identify the various terpene synthases (TPSs) involved in the production of these essential oils, we performed RNA sequencing on mature flowers. From the RNA sequencing data, four full-length TPSs were functionally characterized. In vitro assays showed that two of these TPSs were mono-TPSs. CoTPS1 synthesized four products corresponding to β-thujene, sabinene, β-pinene, and α-terpinene from geranyl pyrophosphate and CoTPS4 produced geraniol from geranyl pyrophosphate. The other two TPSs were identified as sesqui-TPSs. CoTPS3 catalysed the conversion of farnesyl pyrophosphate to α-bergamotene, whereas CoTPS2 was found to be a multifunctional and novel TPS that could catalyse the synthesis of three sesquiterpenes, β-ylangene, β-copaene, and β-cubebene. Additionally, the activities of the two sesqui-TPSs were confirmed in planta by transient expression of these TPS genes in Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. PMID:25956881

  1. Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway.

    Science.gov (United States)

    Jawed, Kamran; Mattam, Anu Jose; Fatma, Zia; Wajid, Saima; Abdin, Malik Z; Yazdani, Syed Shams

    2016-01-01

    Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product. PMID:27466817

  2. Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway

    Science.gov (United States)

    Jawed, Kamran; Mattam, Anu Jose; Fatma, Zia; Wajid, Saima; Abdin, Malik Z.; Yazdani, Syed Shams

    2016-01-01

    Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product. PMID:27466817

  3. Effects of overexpressing individual lignin biosynthetic enzymes on feeding and growth of corn earworms and fall armyworms

    Science.gov (United States)

    Lignin is an important insect resistance component of plants. Enhancing or disrupting the lignin biosynthetic pathway for different bioenergy uses may alter pest resistance. The lignin biosynthetic pathway is complex, and a number of pathway compounds are also involved in the biosynthesis of simpler...

  4. Hormonal Regulation and Expression Profiles of Wheat Genes Involved during Phytic Acid Biosynthesis Pathway

    OpenAIRE

    Sipla Aggarwal; Vishnu Shukla; Kaushal Kumar Bhati; Mandeep Kaur; Shivani Sharma; Anuradha Singh; Shrikant Mantri; Ajay Kumar Pandey

    2015-01-01

    Phytic acid (PA) biosynthesis pathway genes were reported from multiple crop species. PA accumulation was enhanced during grain filling and at that time, hormones like Abscisic acid (ABA) and Gibberellic acid (GA3) interplay to control the process of seed development. Regulation of wheat PA pathway genes has not yet been reported in seeds. In an attempt to find the clues for the regulation by hormones, the promoter region of wheat PA pathway genes was analyzed for the presence of cis-elements...

  5. Subcloning of the enterobactin biosynthetic gene entB: Expression, purification, characterization, and substrate specificity of isochorismatase

    International Nuclear Information System (INIS)

    The Escherichia coli entB, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 μM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway

  6. HPLC-MS/MS analyses show that the near-Starchless aps1 and pgm leaves accumulate wild type levels of ADPglucose: further evidence for the occurrence of important ADPglucose biosynthetic pathway(s alternative to the pPGI-pPGM-AGP pathway.

    Directory of Open Access Journals (Sweden)

    Abdellatif Bahaji

    Full Text Available In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a involves plastidic phosphoglucomutase (pPGM, ADPglucose (ADPG pyrophosphorylase (AGP and starch synthase (SS, and (b is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI. This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b pPGM and AGP are not major determinants of intracellular ADPG content, and (c the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.

  7. Directed evolution of the CpcA biosynthetic pathway and optimization of conditions for CpcA production and its properties.

    Science.gov (United States)

    Dong, Dalu; Pan, Hangtao; Yu, Ping

    2014-06-01

    To improve the production of phycocyanin holo-α-subunit (CpcA) from Spirulina maxima, five genes and their spacer region sequences involved in its biosynthesis were subject to the directed evolution by error-prone PCR using the plasmid pETDuet-6 as the template. Mutants were screened by determining the CpcA yield in 96-well plates directly. A mutant strain CPCA713 with the highest CpcA yield of 17.36 mg/l in 96-well plates was obtained, and this yield was 29.7 % higher than that from the control strain ZJGSU09 containing pETDuet-6 (13.38 mg/l). Sequence alignments indicated that 10 nucleotides and 5 amino acids were mutated. Glycerol and beef extract were found to be the best carbon and nitrogen sources for accumulating CpcA in the screened CPCA713 strain, respectively. The concentrations of the key factors that affected the CpcA yield were optimized by response surface methodology with a Box-Behnken design and were as follows: glycerol, 16.0 g/l; yeast extract, 18.2 g/l; and beef extract, 4.8 g/l. Under the optimal conditions, the CpcA yield was up to 71.21 mg/l in the shake flask. Time-course of the CpcA production before and after optimization were performed and compared. After being purified by a Hi-Trap metal chelating affinity column loaded with 100 mM nickel sulfate, CpcA presented a single protein band with an estimated molecular weight of 29 kDa in the sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The purified CpcA had the maximal absorptive and fluorescent emission wavelengths at 623 and 650.8 nm, respectively, and was stable at temperatures of 40 °C below and pHs of 5.5-8.0, and in the dark or in the dim light. It had also a strong scavenging ability to three free radicals ·OH, ·O2 (-), and di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH). The IC50 values of ·OH, ·O2 (-), and DPPH free radicals by purified CpcA were 0.08, 0.46, and 0.48 mg/ml, respectively. This study lays a good foundation for the industrial

  8. Retrobiosynthetic study of salicylic acid in Catharanthus roseus cell suspension cultures

    OpenAIRE

    Mustafa, Natali Rianika

    2007-01-01

    Salicylic acid (SA) is an important signal compound in systemic acquired resistance in plants. The level of this C6C1 compound in plants increases after a pathogenic attack. There are two biosynthetic pathways of SA, the phenylalanine pathway, which is thought to occur in plants, and the isochorismate pathway, which is found in microorganisms. The biosynthetic pathway of SA in plant is still a matter of debate and some studies showed that the presence of the microbial pathway in plants is als...

  9. Crosstalk between cystine and glutathione is critical for the regulation of amino acid signaling pathways and ferroptosis.

    Science.gov (United States)

    Yu, Xinlei; Long, Yun Chau

    2016-01-01

    Although essential amino acids regulate mechanistic target of rapamycin complex 1 (mTORC1) and the integrated stress response (ISR), the role of cysteine is unknown. We found that in hepatoma HepG2 cells, cystine (oxidized form of cysteine) activated mTORC1 and suppressed the ISR. Cystine deprivation induced GSH efflux and extracellular degradation, which aimed to restore cellular cysteine. Inhibition of γ-glutamyl transpeptidase (GGT) impaired the ability of GSH or cell-permeable GSH to restore mTORC1 signaling and the ISR, suggesting that the capacity of GSH to release cysteine, but not GSH per se, regulated the signaling networks. Inhibition of protein translation restored both mTORC1 signaling and the ISR during cystine starvation, suggesting the bulk of cellular cysteine was committed to the biosynthetic process. Cellular cysteine and GSH displayed overlapping protective roles in the suppression of ferroptosis, further supporting their cooperation in the regulation of cell signaling. Thus, cellular cysteine and its derivative GSH cooperate to regulate mTORC1 pathway, the ISR and ferroptosis. PMID:27425006

  10. Biosynthetic engineering of nonribosomal peptide synthetases.

    Science.gov (United States)

    Kries, Hajo

    2016-09-01

    From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27465074

  11. Retinoic acid activates two pathways required for meiosis in mice.

    Directory of Open Access Journals (Sweden)

    Jana Koubova

    2014-08-01

    Full Text Available In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA, the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function.

  12. In vitro reconstitution of an abscisic acid signalling pathway

    KAUST Repository

    Fujii, Hiroaki

    2009-11-18

    The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants; it has critical functions in stress resistance and in growth and development. Several proteins have been reported to function as ABA receptors, and many more are known to be involved in ABA signalling. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear. Here we show that by combining the recently identified ABA receptor PYR1 with the type 2C protein phosphatase (PP2C) ABI1, the serine/threonine protein kinase SnRK2.6/OST1 and the transcription factor ABF2/AREB1, we can reconstitute ABA-triggered phosphorylation of the transcription factor in vitro. Introduction of these four components into plant protoplasts results in ABA-responsive gene expression. Protoplast and test-tube reconstitution assays were used to test the function of various members of the receptor, protein phosphatase and kinase families. Our results suggest that the default state of the SnRK2 kinases is an autophosphorylated, active state and that the SnRK2 kinases are kept inactive by the PP2Cs through physical interaction and dephosphorylation. We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway. © 2009 Macmillan Publishers Limited. All rights reserved.

  13. The biosynthetic products of chinese insect medicine, Aspongopus chinensis

    Science.gov (United States)

    Luo, Xiao-Hong; Wang, Xiao-Zheng; Jiang, Hai-Long; Yang, Jun-Li; Crews, Phillip; Valeriote, Frederick A.; Wu, Quan-Xiang

    2012-01-01

    A new oxazole (1) was obtained from chinese insect medicine Aspongopus chinensis, along with three known N-acetyldopamine derivatives (2–4). Their structures were determined on the basis of NMR and ESI-MS analyses. The possible biosynthetic pathways of the isolated compounds are discussed. Cytotoxicities of those compounds against 10 selected cancer cells were measured in vitro. PMID:22430116

  14. Genome mining of the hitachimycin biosynthetic gene cluster: involvement of a phenylalanine-2,3-aminomutase in biosynthesis.

    Science.gov (United States)

    Kudo, Fumitaka; Kawamura, Koichi; Uchino, Asuka; Miyanaga, Akimasa; Numakura, Mario; Takayanagi, Ryuichi; Eguchi, Tadashi

    2015-04-13

    Hitachimycin is a macrolactam antibiotic with (S)-β-phenylalanine (β-Phe) at the starter position of its polyketide skeleton. To understand the incorporation mechanism of β-Phe and the modification mechanism of the unique polyketide skeleton, the biosynthetic gene cluster for hitachimycin in Streptomyces scabrisporus was identified by genome mining. The identified gene cluster contains a putative phenylalanine-2,3-aminomutase (PAM), five polyketide synthases, four β-amino-acid-carrying enzymes, and a characteristic amidohydrolase. A hitA knockout mutant showed no hitachimycin production, but antibiotic production was restored by feeding with (S)-β-Phe. We also confirmed the enzymatic activity of the HitA PAM. The results suggest that the identified gene cluster is responsible for the biosynthesis of hitachimycin. A plausible biosynthetic pathway for hitachimycin, including a unique polyketide skeletal transformation mechanism, is proposed.

  15. Retinoic acid suppresses the canonical Wnt signaling pathway in embryonic stem cells and activates the noncanonical Wnt signaling pathway

    Science.gov (United States)

    Osei-Sarfo, Kwame; Gudas, Lorraine J.

    2014-01-01

    Embryonic stem cells (ESCs) have both the ability to self-renew and to differentiate into various cell lineages. Retinoic acid (RA), a metabolite of Vitamin A, has a critical function in initiating lineage differentiation of ESCs through binding to the retinoic acid receptors (RARs). Additionally, the Wnt signaling pathway plays a role in pluripotency and differentiation, depending on the activation status of the canonical and noncanonical pathways. The activation of the canonical Wnt signaling pathway, which requires the nuclear accumulation of β-catenin and its interaction with Tcf1/Lef at Wnt response elements, is involved in ESC stemness maintenance. The noncanonical Wnt signaling pathway, through actions of Tcf3, can antagonize the canonical pathway. We show that RA activates the noncanonical Wnt signaling pathway, while concomitantly inhibiting the canonical pathway. RA increases the expression of ligands and receptors of the noncanonical Wnt pathway (Wnt 5a, 7a, Fzd2 and Fzd6), downstream signaling, and Tcf3 expression. RA reduces the phosphorylated β-catenin level by 4-fold, though total β-catenin levels don't change. We show that RA signaling increases the dissociation of Tcf1 and the association of Tcf3 at promoters of genes that regulate stemness (e.g. NR5A2,Lrh-1) or differentiation (eg. Cyr61, Zic5). Knockdown of Tcf3 increases Lrh-1 transcript levels in mESCs and prevents the RA-associated, ∼4-fold increase in Zic5, indicating that RA requires Tcf3 to effect changes in Zic5 levels. We demonstrate a novel role for RA in altering the activation of these two Wnt signaling pathways and show that Tcf3 mediates some actions of RA during differentiation. PMID:24648413

  16. Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues.

    Directory of Open Access Journals (Sweden)

    Valerio Mori

    Full Text Available NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the "amidated" and "deamidated" routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT, which in mammals comprises three distinct isozymes, and NAD synthetase (NADS. First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide. In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.

  17. Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

    DEFF Research Database (Denmark)

    Liu, Chengwei; Tagami, Koichi; Minami, Atsushi;

    2015-01-01

    KULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products....

  18. Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network.

    Science.gov (United States)

    Widhalm, Joshua R; Gutensohn, Michael; Yoo, Heejin; Adebesin, Funmilayo; Qian, Yichun; Guo, Longyun; Jaini, Rohit; Lynch, Joseph H; McCoy, Rachel M; Shreve, Jacob T; Thimmapuram, Jyothi; Rhodes, David; Morgan, John A; Dudareva, Natalia

    2015-09-10

    In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles, as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network.

  19. Reconstruction of Pathways Associated with Amino Acid Metabolism in Human Mitochondria

    Institute of Scientific and Technical Information of China (English)

    Purnima Guda; Chittibabu Guda; Shankar Subramaniam

    2007-01-01

    We have used a bioinformatics approach for the identification and reconstruction of metabolic pathways associated with amino acid metabolism in human mitochon- dria. Human mitochondrial proteins determined by experimental and computa- tional methods have been superposed on the reference pathways from the KEGG database to identify mitochondrial pathways. Enzymes at the entry and exit points for each reconstructed pathway were identified, and mitochondrial solute carrier proteins were determined where applicable. Intermediate enzymes in the mito- chondrial pathways were identified based on the annotations available from public databases, evidence in current literature, or our MITOPRED program, which pre- dicts the mitochondrial localization of proteins. Through integration of the data derived from experimental, bibliographical, and computational sources, we recon- structed the amino acid metabolic pathways in human mitochondria, which could help better understand the mitochondrial metabolism and its role in human health.

  20. Retrobiosynthetic study of salicylic acid in Catharanthus roseus cell suspension cultures

    NARCIS (Netherlands)

    Mustafa, Natali Rianika

    2007-01-01

    Salicylic acid (SA) is an important signal compound in systemic acquired resistance in plants. The level of this C6C1 compound in plants increases after a pathogenic attack. There are two biosynthetic pathways of SA, the phenylalanine pathway, which is thought to occur in plants, and the isochorisma

  1. Horizontal gene transfer and redundancy of tryptophan biosynthetic enzymes in dinotoms.

    Science.gov (United States)

    Imanian, Behzad; Keeling, Patrick J

    2014-02-01

    A tertiary endosymbiosis between a dinoflagellate host and diatom endosymbiont gave rise to "dinotoms," cells with a unique nuclear and mitochondrial redundancy derived from two evolutionarily distinct eukaryotic lineages. To examine how this unique redundancy might have affected the evolution of metabolic systems, we investigated the transcription of genes involved in biosynthesis of the amino acid tryptophan in three species, Durinskia baltica, Kryptoperidinium foliaceum, and Glenodinium foliaceum. From transcriptome sequence data, we recovered two distinct sets of protein-coding transcripts covering the entire tryptophan biosynthetic pathway. Phylogenetic analyses suggest a diatom origin for one set of the proteins, which we infer to be expressed in the endosymbiont, and that the other arose from multiple horizontal gene transfer events to the dinoflagellate ancestor of the host lineage. This is the first indication that these cells retain redundant sets of transcripts and likely metabolic pathways for the biosynthesis of small molecules and extend their redundancy to their two distinct nuclear genomes. PMID:24448981

  2. Sorbic acid stress activates the Candida glabrata high osmolarity glycerol MAP kinase pathway

    Directory of Open Access Journals (Sweden)

    Zeljkica eJandric

    2013-11-01

    Full Text Available Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in Candida glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologues CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species, and required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.

  3. Selective deoxygenation of stearic acid via an anhydride pathway

    NARCIS (Netherlands)

    Hollak, S.A.W.; Bitter, W.; Haveren, van J.; Es, van D.S.

    2012-01-01

    Stearic anhydride is proposed as reactive intermediate in the hydrogen free decarbonylation and ketonization of stearic acid over Pd/Al2O3 at 523 K. This information is crucial towards developing of a selective low temperature decarbonylation process of fatty acids towards olefins.

  4. Arginine-dependent acid-resistance pathway in Shigella boydii

    Science.gov (United States)

    Ability to survive the low pH of the human stomach is considered be an important virulent determinant. Acid tolerance of Shigella boydii 18 CDPH, the strain implicated in an outbreak may have played an important role in surviving the acidic food (bean salad). The strain was capable of inducing arg...

  5. Dual responsive physical networks from asymmetric biosynthetic triblock copolymers

    NARCIS (Netherlands)

    Pham, T.H.T.

    2013-01-01

      The aim of the project is to develop biosynthetically produced amino acid polymers which are composed of three distinct blocks A-C-B, each with a separate function. A is a first self-assembling block capable of ‘recognizing’ (upon a trigger) other A blocks; C is an inert, random

  6. Opposing effects of bile acids deoxycholic acid and ursodeoxycholic acid on signal transduction pathways in oesophageal cancer cells.

    Science.gov (United States)

    Abdel-Latif, Mohamed M; Inoue, Hiroyasu; Reynolds, John V

    2016-09-01

    Ursodeoxycholic acid (UDCA) was reported to reduce bile acid toxicity, but the mechanisms underlying its cytoprotective effects are not fully understood. The aim of the present study was to examine the effects of UDCA on the modulation of deoxycholic acid (DCA)-induced signal transduction in oesophageal cancer cells. Nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activity was assessed using a gel shift assay. NF-κB activation and translocation was performed using an ELISA-based assay and immunofluorescence analysis. COX-2 expression was analysed by western blotting and COX-2 promoter activity was assessed by luciferase assay. DCA induced NF-κB and AP-1 DNA-binding activities in SKGT-4 and OE33 cells. UDCA pretreatment inhibited DCA-induced NF-κB and AP-1 activation and NF-κB translocation. This inhibitory effect was coupled with a blockade of IκB-α degradation and inhibition of phosphorylation of IKK-α/β and ERK1/2. Moreover, UDCA pretreatment inhibited COX-2 upregulation. Using transient transfection of the COX-2 promoter, UDCA pretreatment abrogated DCA-induced COX-2 promoter activation. In addition, UDCA protected oesophageal cells from the apoptotic effects of deoxycholate. Our findings indicate that UDCA inhibits DCA-induced signalling pathways in oesophageal cancer cells. These data indicate a possible mechanistic role for the chemopreventive actions of UDCA in oesophageal carcinogenesis.

  7. 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

    Establishing efficient synthetic pathways for microbial production of biochemicals is often hampered by competing pathways and/or insufficient precursor supply. Compartmentalization in cellular organelles can isolate synthetic pathways from competing pathways, and provide a compact and suitable...... 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...

  8. 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...

  9. 酵母UDP-葡萄糖合成途径的优化及黄酮葡萄糖苷的合成%Optimization of biosynthetic pathway of uridine diphosphate glucose in yeast for production of flavonoid glucosides

    Institute of Scientific and Technical Information of China (English)

    王惠敏; 杨燕; 田雷瑜; 周文龙; 王伟

    2016-01-01

    目的:研究在酿酒酵母细胞内高表达大肠杆菌 UDP-葡萄糖合成途径的2个关键酶对工程酵母细胞的葡萄糖苷化反应活性的影响。方法利用 PCR方法获得大肠杆菌磷酸葡萄糖变位酶(PGM)和 UDP-葡萄糖焦磷酸化酶(GalU)基因,构建整合型表达载体,转化酵母;同时共表达具有催化黄酮葡萄糖苷化的 UDP-葡萄糖转移酶,研究摇瓶培养条件下PGM和 GalU 基因的表达对工程细胞生物催化黄酮类化合物葡萄糖苷化的影响。结果在酵母细胞内表达大肠杆菌 UDP-葡萄糖合成途径的2个关键酶 PGM和 GalU,提高了组合表达 UDP-葡萄糖转移酶的工程菌生物催化合成野黄芩素-7-O-葡萄糖苷的效率,表明PGM和GalU基因的高表达能促进 UDP-葡萄糖的合成;所构建的工程菌也能催化木犀草素、柚皮素、白杨素、汉黄芩素、木蝴蝶素 A、黄芩素、槲皮素以及大豆黄酮等黄酮类化合物的葡萄糖苷化。结论组合表达大肠杆菌 PGM 和 GalU 基因能促进酵母细胞内 UDP-葡萄糖的合成,进而提高了工程细胞催化合成黄酮葡萄糖苷的效率。%Objective To investigate the effect of expression of phosphoglucomutase and UTP-glucose-1-phosphate uridylyltransferase [two key enzymes in the pathway of synthesizing uracil diphosphate glucose (UDP-glucose)] fromEscherichia coli on glucosylation capacity of engineering yeast. Methods In order to up-regulate the biosynthetic pathway of UDP-glucose ofSaccharomyces cerevisiae, the two genes encoding phosphoglucomutase (PGM) and UTP-glucose-1-phosphate uridylyltransferase (GalU) ofE. coli were amplified by PCR and then inserted into the yeast integration vectors. The resulting plasmids were linearized by restriction enzymeNot I and transformed into the engineering strain. The integration expression plasmid pδGAPg-SbGT34 with a UDP-glucose flavonoid glucosyltransferase gene cloned fromScutellaria baicalensis

  10. 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.

  11. Transcriptomic Analysis and Systematic Mining of Genes Involved in Biosynthetic Pathway of Triterpenoid Saponins in Ilex Asprella%岗梅转录组及其乌索烷型三萜皂苷生物合成相关酶基因的发掘

    Institute of Scientific and Technical Information of China (English)

    郑夏生; 罗秀秀; 徐晖; 詹若挺; 陈蔚文

    2014-01-01

    This study was aimed to screen candidate genes involved in the triterpenoid saponins biosynthetic pathway of the Ilex asprella root. The Illumina platform was applied to perform transcriptomic sequencing of I. asprella root, followed by a series of bioinformatics analysis. The results showed that a total of 272 candidate unigenes were anno-tated to be involved in the biosynthetic pathway of terpenoid in the transcriptome of I. asprella root, including 72 u-nigenes for the upstream pathway and 26 unigenes for cyclization, oxidation and glycosylation in the downstream pathway. Phylogenetic analysis was carried out to further analyze the evolution relationship of some candidate uni-genes and their homologous genes. Two genes IaA S1 and IaA S2 were proved to be mixed amyrin synthases in yeast expression system. Moreover, IaA S1 was identified to one of the rare ASs with α-amyrin as the major product. It was concluded that a series of candidate genes, which might be involved in the biosynthetic pathway of triterpenoid saponins, were screened out from the transcriptome of I. asprella root. Further investigation of these candidate genes will provide insight into their actual functions in the triterpenoid saponins biosynthetic pathway in I. asprella.%目的:发掘与岗梅乌索烷型三萜皂苷生物合成相关的酶基因。方法:利用Illumina测序平台对岗梅根进行转录组测序,通过基因注释、同源分析、系统发生分析等生物信息学手段,发掘可能参与合成的单基因簇(Unigenes)。结果:在岗梅根转录组中发现了272条与萜类生物合成相关的Unigenes。这其中包括72条可能参与萜类生物合成上游途径的Unigenes,以及26条可能与三萜合成途径下游母核合成、氧化和糖基化相关的Unigenes。对其中部分Unigenes进行系统发生分析,进一步揭示了这些Unigenes与同源基因间的进化关系。利用酵母表达系统,鉴定了两个香树酯醇合酶IaAS1

  12. Carnosic acid biosynthesis elucidated by a synthetic biology platform.

    Science.gov (United States)

    Ignea, Codruta; Athanasakoglou, Anastasia; Ioannou, Efstathia; Georgantea, Panagiota; Trikka, Fotini A; Loupassaki, Sofia; Roussis, Vassilios; Makris, Antonios M; Kampranis, Sotirios C

    2016-03-29

    Synthetic biology approaches achieving the reconstruction of specific plant natural product biosynthetic pathways in dedicated microbial "chassis" have provided access to important industrial compounds (e.g., artemisinin, resveratrol, vanillin). However, the potential of such production systems to facilitate elucidation of plant biosynthetic pathways has been underexplored. Here we report on the application of a modular terpene production platform in the characterization of the biosynthetic pathway leading to the potent antioxidant carnosic acid and related diterpenes in Salvia pomifera and Rosmarinus officinalis.Four cytochrome P450 enzymes are identified (CYP76AH24, CYP71BE52, CYP76AK6, and CYP76AK8), the combined activities of which account for all of the oxidation events leading to the biosynthesis of the major diterpenes produced in these plants. This approach develops yeast as an efficient tool to harness the biotechnological potential of the numerous sequencing datasets that are increasingly becoming available through transcriptomic or genomic studies. PMID:26976595

  13. Parallel proton transfer pathways in aqueous acid-base reactions

    OpenAIRE

    Cox, M. J.; Bakker, H.J.

    2008-01-01

    We study the mechanism of proton transfer (PT) between the photoacid 8-hydroxy-1,3, 6-pyrenetrisulfonic acid (HPTS) and the base chloroacetate in aqueous solution. We investigate both proton and deuteron transfer reactions in solutions with base concentrations ranging from 0.25M to 4M. Using femtosecond midinfrared spectroscopy, we probe the vibrational responses of HPTS, its conjugate photobase, the hydrated proton/deuteron, and chloroacetate. The measurement of these four resonances allows ...

  14. Biocontrol of Potato Common Scab is Associated with High Pseudomonas fluorescens LBUM223 Populations and Phenazine-1-Carboxylic Acid Biosynthetic Transcript Accumulation in the Potato Geocaulosphere.

    Science.gov (United States)

    Arseneault, Tanya; Goyer, Claudia; Filion, Martin

    2016-09-01

    Pseudomonads are often used as biocontrol agents because they display a broad range of mechanisms to control diseases. Common scab of potato, caused by Streptomyces scabies, was previously reported to be controlled by Pseudomonas fluorescens LBUM223 through phenazine-1-carboxylic acid (PCA) production. In this study, we aimed at characterizing the population dynamics of LBUM223 and the expression of phzC, a key gene involved in the biosynthesis of PCA, in the rhizosphere and geocaulosphere of potato plants grown under controlled and field conditions. Results obtained from controlled experiments showed that soil populations of LBUM223 significantly declined over a 15-week period. However, at week 15, the presence of S. scabies in the geocaulosphere was associated with significantly higher populations of LBUM223 than when the pathogen was absent. It also led to the detection of significantly higher phzC gene transcript numbers. Under field conditions, soil populations of LBUM223 followed a similar decline in time when a single inoculation was applied in spring but remained stable when reinoculated biweekly, which also led to greater phzC gene transcripts accumulation. Taken together, our findings suggest that LBUM223 must colonize the potato geocaulosphere at high levels (10(7) bacteria/g of soil) in order to achieve biocontrol of common scab through increased PCA production. PMID:27088392

  15. The role of MAPK signalling pathways in acetic acid-induced cell death of Saccharomyces cerevisiae

    OpenAIRE

    Azevedo, Flávio Humberto Torres Dias Feio de

    2011-01-01

    Dissertação de mestrado em Genética Molecular Mitogenic Activated Protein Kinase (MAPK) cascades are important signalling pathways that allow yeast cells to swiftly adapt to changing environmental conditions. Previous studies suggested that the High Osmolarity Glycerol (HOG) MAPK pathway and ceramide production are involved in acetic-acid induced apoptosis in yeast. Evidence that changes in the levels of endogenous ceramides can affect yeast cell fate has also been put forth...

  16. Permanganate oxidation of α-amino acids: kinetic correlations for the nonautocatalytic and autocatalytic reaction pathways.

    Science.gov (United States)

    Perez-Benito, Joaquin F

    2011-09-01

    The reactions of permanganate ion with seven α-amino acids in aqueous KH(2)PO(4)/K(2)HPO(4) buffers have been followed spectrophotometrically at two different wavelengths: 526 nm (decay of MnO(4)(-)) and 418 nm (formation of colloidal MnO(2)). All of the reactions studied were autocatalyzed by colloidal MnO(2), with the contribution of the autocatalytic reaction pathway decreasing in the order glycine > l-threonine > l-alanine > l-glutamic acid > l-leucine > l-isoleucine > l-valine. The rate constants corresponding to the nonautocatalytic and autocatalytic pathways were obtained by means of either a differential rate law or an integrated one, the latter requiring the use of an iterative method for its implementation. The activation parameters for the two pathways were determined and analyzed to obtain statistically significant correlations for the series of reactions studied. The activation enthalpy of the nonautocatalytic pathway showed a strong, positive dependence on the standard Gibbs energy for the dissociation of the protonated amino group of the α-amino acid. Linear enthalpy-entropy correlations were found for both pathways, leading to isokinetic temperatures of 370 ± 21 K (nonautocatalytic) and 364 ± 28 K (autocatalytic). Mechanisms in agreement with the experimental data are proposed for the two reaction pathways.

  17. Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis.

    Science.gov (United States)

    Yoon, Mee-Sup; Chen, Jie

    2013-12-01

    Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner. Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.

  18. Engineering the fatty acid metabolic pathway in Saccharomyces cerevisiae for advanced biofuel production

    Directory of Open Access Journals (Sweden)

    Xiaoling Tang

    2015-12-01

    Full Text Available Fatty acid-derived fuels and chemicals have attracted a great deal of attention in recent decades, due to their following properties of high compatibility to gasoline-based fuels and existing infrastructure for their direct utilization, storage and distribution. The yeast Saccharomyces cerevisiae is the ideal biofuel producing candidate, based on the wealth of available genetic information and versatile tools designed to manipulate its metabolic pathways. Engineering the fatty acid metabolic pathways in S. cerevisiae is an effective strategy to increase its fatty acid biosynthesis and provide more pathway precursors for production of targeted products. This review summarizes the recent progress in metabolic engineering of yeast cells for fatty acids and fatty acid derivatives production, including the regulation of acetyl-CoA biosynthesis, NADPH production, fatty acid elongation, and the accumulation of activated precursors of fatty acids for converting enzymes. By introducing specific enzymes in the engineered strains, a powerful platform with a scalable, controllable and economic route for advanced biofuel production has been established.

  19. Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system

    OpenAIRE

    Nah, Hee-Ju; Woo, Min-Woo; Choi, Si-Sun; Kim, Eung-Soo

    2015-01-01

    Background Direct cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-si...

  20. Omega-3 fatty acid deficiency disrupts endocytosis, neuritogenesis, and mitochondrial protein pathways in the mouse hippocampus.

    Directory of Open Access Journals (Sweden)

    Jane A English

    2013-10-01

    Full Text Available Omega-3 fatty acid (n-3 FA deficiency is an environmental risk factor for schizophrenia, yet characterisation of the consequences of deficiency at the protein level in the brain is limited. We aimed to identify the protein pathways disrupted as a consequence of chronic n-3 deficiency in the hippocampus of mice. Fatty acid analysis of the hippocampus following chronic dietary deficiency revealed a 3-fold decrease (p

  1. Development of Fatty Acid-Producing Corynebacterium glutamicum Strains

    OpenAIRE

    Takeno, Seiki; Takasaki, Manami; Urabayashi, Akinobu; Mimura, Akinori; Muramatsu, Tetsuhiro; Mitsuhashi, Satoshi; Ikeda, Masato

    2013-01-01

    To date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway of Corynebacterium glutamicum. To develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. This was followed by genome analysis to characterize its genetic background. The selection of spontaneous mutants resistant to the palmitic acid ester s...

  2. Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis.

    OpenAIRE

    Duester, G; Shean, M L; McBride, M S; Stewart, M J

    1991-01-01

    Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between...

  3. 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

    the amino acids valine, isoleucine, and leucine, and required the short branched chain acids 2-methylbutanoic acid or 2-methylpropanoic acid for growth in a defined medium. The isoleucine related metabolites, alpha-keto-beta-methylvaleric acid and 2-methylbutanal also served as growth factors. Growth...... was observed. Despite the deficiency in IlvE activity, the mutant strain was still able to produce the short chain carboxylic acids, 3-methylbutanoic acid and 2-methylpropanoic acid when cultivated in rich medium. Supplementation experiments employing deuterated glucose induced the valine biosynthetic pathway...

  4. Ketol-acid reductoisomerase enzymes and methods of use

    Science.gov (United States)

    Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O'Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

    2015-10-27

    Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

  5. Enzymology of the branched-chain amino acid oxidation disorders: the valine pathway

    OpenAIRE

    Wanders, Ronald J.A.; Duran, Marinus; Loupatty, Ference J.

    2010-01-01

    Valine is one of the three branched-chain amino acids which undergoes oxidation within mitochondria. In this paper, we describe the current state of knowledge with respect to the enzymology of the valine oxidation pathway and the different disorders affecting oxidation.

  6. Enzymology of the branched-chain amino acid oxidation disorders: the valine pathway.

    Science.gov (United States)

    Wanders, Ronald J A; Duran, Marinus; Loupatty, Ference J

    2012-01-01

    Valine is one of the three branched-chain amino acids which undergoes oxidation within mitochondria. In this paper, we describe the current state of knowledge with respect to the enzymology of the valine oxidation pathway and the different disorders affecting oxidation.

  7. Biosynthesis of indole-3-acetic acid via the indole-3-acetamide pathway in Streptomyces spp.

    Science.gov (United States)

    Manulis, S; Shafrir, H; Epstein, E; Lichter, A; Barash, I

    1994-05-01

    Various Streptomyces spp. including S. violaceus, S. scabies, S. griseus, S. exfoliatus, S. coelicolor and S. lividans secrete indole-3-acetic acid (IAA) when fed with L-tryptophan (Trp). Production of IAA was detected in Streptomyces strains causing potato scab as well as in non-pathogenic strains. The pathways for IAA synthesis from Trp were investigated in S. violaceus and S. exfoliatus. Indole-3-acetamide (IAM), indole-3-lactic acid (ILA), indole-3-ethanol (IEt) and IAA were identified by HPLC and GC-MS. Streptomyces cells were capable of catabolizing IAM, ILA, IEt and indole-3-acetaldehyde (IAAId) into IAA. Incorporation of radioactivity into IAM, IAA and ILA but not IEt was detected when cells were fed with L-[3-14C]tryptophan. Results indicate the presence of the IAM pathway (Trp-->IAM-->IAA) and the possible presence of additional pathways for IAA biosynthesis in Streptomyces. PMID:8025670

  8. Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway.

    Science.gov (United States)

    Sorigué, Damien; Légeret, Bertrand; Cuiné, Stéphan; Morales, Pablo; Mirabella, Boris; Guédeney, Geneviève; Li-Beisson, Yonghua; Jetter, Reinhard; Peltier, Gilles; Beisson, Fred

    2016-08-01

    Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway. PMID:27288359

  9. Molecular Cloning and Characterization of a DFR from Developing Seeds of Blue-grained Wheat in Anthocyanin Biosynthetic Pathway%蓝粒小麦花青素生物合成途径中的二氢黄酮醇4-还原酶基因的分子克隆

    Institute of Scientific and Technical Information of China (English)

    杨国华; 赵学强; 李滨; 刘建中; 郑琪; 童依平; 李振声

    2003-01-01

    Blue-grained wheat derived from the hybrid Triticum aestivum L. x Thinopyrum ponticum (Podp.) Barkworth et D. R. Dewey (Agropyron elongatum (Host) P. Beauv., 2n=70). The molecular biological mechanism of the biosynthetic pathway of blue pigments in the blue grain remains unclear yet. Dihydroflavonol 4-reductase (DFR) is one of the key enzymes controlling flavonoid synthesis in anthocyanin biosynthetic pathway, and may directly participate in the formation of blue pigment in the aleurone layer of blue-grained wheat. Here we cloned a DFRcDNA (TaDFR) from the developing seeds of blue-grained wheat, and four DFR genomic DNAs from Th. ponticum (ThpDFR.t), blue-grained wheat (TaDFR.bg), white-grained offspring of light blue-grained wheat (TaDFR.wg) and Chinese Spring (2n=42) ( TaDFR. csg), respectively. TaDFRcDNA encodes a 354 amino-acids polypeptide with high identity to DFR from Hordeum vulgare L. (94%), Oryza sativa L. (83%), Zea mays L.(84%). The result of cluster analysis showed that TaDFR cDNA nucleotide sequence has 100% identity with that of TaDFR.csg. The four DFRgenomic DNAs have extraordinary high homology and each has three introns. The differences of the four DFR genomic DNAs mainly exist in introns. Southern blotting analysis showed that there are at least 3-5 DFR copies in wheat, the copy numbers in different color grain wheats are not significantly different. The hybridization band patterns were the same, but different from that of Th. ponticum. DFR in blue-grained wheat belongs to a DFR supeffamily. Northern blotting analysis indicated that the DFRexpressed in the developing seeds of both blue- and white-grained wheat at 15 d after flowering (DAF), the mRNA levels of DFRreached the highest at 18 DAF, then declined quickly and disappeared at 33 DAF. But the expression levels in blue-grained seeds were higher than that in white grain at the same seed developing stages. DFRtranscripts accumulated in young leaves, and leaf sheaths of blue- and white

  10. Bile acid promotes liver regeneration via farnesoid X receptor signaling pathways in rats.

    Science.gov (United States)

    Ding, Long; Yang, Yu; Qu, Yikun; Yang, Ting; Wang, Kaifeng; Liu, Weixin; Xia, Weibin

    2015-06-01

    Bile acids, which are synthesized from cholesterol in the hepatocytes of the liver, are amphipathic molecules with a steroid backbone. Studies have shown that bile acid exhibits important effects on liver regeneration. However, the mechanism underlying these effects remains unclear. The aim of the present study was to investigate the effect of bile acid and the farnesoid X receptor (FXR) on hepatic regeneration and lipid metabolism. Rats were fed with 0.2% bile acid or glucose for 7 days and then subjected to a 50 or 70% hepatectomy. Hepatic regeneration rate, serum and liver levels of bile acid, and expression of FXR and Caveolin‑1, were detected at 24, 48 or 72 h following hepatectomy. The expression of proliferating cell nuclear antigen (PCNA) in the liver was measured using immunohistochemistry at the end of the study. Hepatocytes isolated from rats were treated with bile acid, glucose, FXR agonist and FXR antagonist, separately or in combination. Lipid metabolism, the expression of members of the FXR signaling pathway and energy metabolism‑related factors were measured using ELISA kits or western blotting. Bile acid significantly increased the hepatic regeneration rate and the expression of FXR, Caveolin‑1 and PCNA. Levels of total cholesterol and high density lipoprotein were increased in bile acid‑ or FXR agonist‑treated hepatocytes in vitro. Levels of triglyceride, low density lipoprotein and free fatty acid were decreased. In addition, bile acid and FXR agonists increased the expression of bile salt export pump and small heterodimer partner, and downregulated the expression of apical sodium‑dependent bile acid transporter, Na+/taurocholate cotransporting polypeptide and cholesterol 7α‑hydroxylase. These results suggested that physiological concentrations of bile acid may promote liver regeneration via FXR signaling pathways, and may be associated with energy metabolism. PMID:25634785

  11. Phenylalanine ammonia lyase catalyzed synthesis of amino acids by an MIO-cofactor independent pathway.

    Science.gov (United States)

    Lovelock, Sarah L; Lloyd, Richard C; Turner, Nicholas J

    2014-04-25

    Phenylalanine ammonia lyases (PALs) belong to a family of 4-methylideneimidazole-5-one (MIO) cofactor dependent enzymes which are responsible for the conversion of L-phenylalanine into trans-cinnamic acid in eukaryotic and prokaryotic organisms. Under conditions of high ammonia concentration, this deamination reaction is reversible and hence there is considerable interest in the development of PALs as biocatalysts for the enantioselective synthesis of non-natural amino acids. Herein the discovery of a previously unobserved competing MIO-independent reaction pathway, which proceeds in a non-stereoselective manner and results in the generation of both L- and D-phenylalanine derivatives, is described. The mechanism of the MIO-independent pathway is explored through isotopic-labeling studies and mutagenesis of key active-site residues. The results obtained are consistent with amino acid deamination occurring by a stepwise E1 cB elimination mechanism.

  12. Uncarilic Acid and Secouncarilic Acid, Two New Triterpenoids from Uucaria sessilifructus

    Directory of Open Access Journals (Sweden)

    Yan Li

    2013-08-01

    Full Text Available Two new compounds, the 6-oxo oleanane-type triterpenoid uncarilic acid, and its 5,6-secotriterpenoid derivative, secouncarilic acid, were isolated from the hooks and stems of Uucaria sessilifructus together with seven known ursane-type triterpenoids. Uncarilic acid is the second 6-oxo oleanane-type triterpenoid ever reported, while secouncarilic acid is the first oleanane-type 5,6-secotriterpenoid. A plausible biosynthetic pathway from uncarilic acid to secouncarilic acid was also postulated. The inhibitory activities of all the nine compounds against LPS-induced nitric oxide production in RAW264.7 macrophages were evaluated.

  13. 钝齿棒杆菌argR基因缺失株构建及其缺失对精氨酸生物合成途径相关基因转录水平的影响%Construction of Corynebacterium crenatum AS 1.542△argR and analysis of transcriptional levels of the related genes of arginine biosynthetic pathway

    Institute of Scientific and Technical Information of China (English)

    陈雪岚; 汤立; 焦海涛; 徐峰; 熊勇华

    2013-01-01

    [Objective] ArgR, coded by the argR gene from Corynebacterium crenatum AS 1. 542, acts as a negative regulator in arginine biosynthetic pathway. However, the effect of argR on transcriptional levels of the related biosynthetic genes has not been reported. Here, we constructed a deletion mutant of argR gene; C. Crenatum AS 1.542 △argR using marker-less knockout technology, and compared the changes of transcriptional levels of the arginine biosynthetic genes between the mutant strain and the wild-type strain. [Methods] We used marker-less knockout technology to construct C. Crenatum AS 1. 542△argR and analyzed the changes of the relate genes at the transcriptional level using real-time fluorescence quantitative PCR. [Results] C. Crenatum AS 1. 542△argR was successfully obtained and the transcriptional level of arginine biosynthetic genes in this mutant increased significantly with an average of about 162. 1 folds. [Conclusion] The arginine biosynthetic genes in C. Crenatum are clearly controlled by the negative regulator ArgR. However, the deletion of this regulator does not result in a clear change in arginine production in the bacteria.%[目的]钝齿棒杆菌AS 1.542中argR基因编码的蛋白ArgR在精氨酸生物合成途径中扮演负调控的角色,但其对相关基因在转录水平的影响还未见报道.因此,本课题组构建了钝齿棒杆菌argR基因缺失株,并在转录水平上比较野生株与缺失株精氨酸生物合成途径相关基因的变化.[方法]采用无痕敲除的方法构建了钝齿棒杆菌argR基因缺失株,并采用荧光定量PCR方法分析缺失株和野生株精氨酸生物合成途径相关基因在转录水平的变化.[结果]利用pK18mobsacB质粒中蔗糖致死基因sacB反向筛选标记及PCR方法成功筛选到钝齿棒杆菌argR基因缺失株;荧光定量PCR结果表明,argR基因缺失株精氨酸生物合成途径中相关基因在转录水平获得大量提高,平均约上调162.13倍.[结论]

  14. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    OpenAIRE

    Mie Bech Lukassen; Wagma Saei; Teis Esben Sondergaard; Anu Tamminen; Abhishek Kumar; Frank Kempken; Wiebe, Marilyn G.; Jens Laurids Sørensen

    2015-01-01

    Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus n...

  15. Accessing natural product biosynthetic processes by mass spectrometry.

    Science.gov (United States)

    Bumpus, Stefanie B; Kelleher, Neil L

    2008-10-01

    Two important classes of natural products are made by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). With most biosynthetic intermediates covalently tethered during biogenesis, protein mass spectrometry (MS) has proven invaluable for their interrogation. New mass spectrometric assay formats (such as selective cofactor ejection and proteomics style LC-MS) are showcased here in the context of functional insights into new breeds of NRPS/PKS enzymes, including the first characterization of an 'iterative' PKS, the biosynthesis of the enediyne antitumor antibiotics, the study of a new strategy for PKS initiation via a GNAT-like mechanism, and the analysis of branching strategies in the so-called 'AT-less' NRPS/PKS hybrid systems. The future of MS analysis of NRPS and PKS biosynthetic pathways lies in adoption and development of methods that continue bridging enzymology with proteomics as both fields continue their post-genomic acceleration. PMID:18706516

  16. Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis.

    Science.gov (United States)

    Yoon, Mee-Sup; Chen, Jie

    2013-12-01

    Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner. Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling. PMID:24068326

  17. Molecular cloning of a novel glucuronokinase/putative pyrophosphorylase from zebrafish acting in an UDP-glucuronic acid salvage pathway.

    Directory of Open Access Journals (Sweden)

    Roman Gangl

    Full Text Available In animals, the main precursor for glycosaminoglycan and furthermore proteoglycan biosynthesis, like hyaluronic acid, is UDP-glucuronic acid, which is synthesized via the nucleotide sugar oxidation pathway. Mutations in this pathway cause severe developmental defects (deficiency in the initiation of heart valve formation. In plants, UDP-glucuronic acid is synthesized via two independent pathways. Beside the nucleotide sugar oxidation pathway, a second minor route to UDP-glucuronic acid exist termed the myo-inositol oxygenation pathway. Within this myo-inositol is ring cleaved into glucuronic acid, which is subsequently converted to UDP-glucuronic acid by glucuronokinase and UDP-sugar pyrophosphorylase. Here we report on a similar, but bifunctional enzyme from zebrafish (Danio rerio which has glucuronokinase/putative pyrophosphorylase activity. The enzyme can convert glucuronic acid into UDP-glucuronic acid, required for completion of the alternative pathway to UDP-glucuronic acid via myo-inositol and thus establishes a so far unknown second route to UDP-glucuronic acid in animals. Glucuronokinase from zebrafish is a member of the GHMP-kinase superfamily having unique substrate specificity for glucuronic acid with a Km of 31 ± 8 µM and accepting ATP as the only phosphate donor (Km: 59 ± 9 µM. UDP-glucuronic acid pyrophosphorylase from zebrafish has homology to bacterial nucleotidyltransferases and requires UTP as nucleosid diphosphate donor. Genes for bifunctional glucuronokinase and putative UDP-glucuronic acid pyrophosphorylase are conserved among some groups of lower animals, including fishes, frogs, tunicates, and polychaeta, but are absent from mammals. The existence of a second pathway for UDP-glucuronic acid biosynthesis in zebrafish likely explains some previous contradictory finding in jekyll/ugdh zebrafish developmental mutants, which showed residual glycosaminoglycans and proteoglycans in knockout mutants of UDP

  18. 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.

  19. Common biosynthetic origins for polycyclic tetramate macrolactams from phylogenetically diverse bacteria.

    Science.gov (United States)

    Blodgett, Joshua A V; Oh, Dong-Chan; Cao, Shugeng; Currie, Cameron R; Kolter, Roberto; Clardy, Jon

    2010-06-29

    A combination of small molecule chemistry, biosynthetic analysis, and genome mining has revealed the unexpected conservation of polycyclic tetramate macrolactam biosynthetic loci in diverse bacteria. Initially our chemical analysis of a Streptomyces strain associated with the southern pine beetle led to the discovery of frontalamides A and B, two previously undescribed members of this antibiotic family. Genome analyses and genetic manipulation of the producing organism led to the identification of the frontalamide biosynthetic gene cluster and several biosynthetic intermediates. The biosynthetic locus for the frontalamides' mixed polyketide/amino acid structure encodes a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), which resembles iterative enzymes known in fungi. No such mixed iterative PKS-NRPS enzymes have been characterized in bacteria. Genome-mining efforts revealed strikingly conserved frontalamide-like biosynthetic clusters in the genomes of phylogenetically diverse bacteria ranging from proteobacteria to actinomycetes. Screens for environmental actinomycete isolates carrying frontalamide-like biosynthetic loci led to the isolation of a number of positive strains, the majority of which produced candidate frontalamide-like compounds under suitable growth conditions. These results establish the prevalence of frontalamide-like gene clusters in diverse bacterial types, with medicinally important Streptomyces species being particularly enriched. PMID:20547882

  20. The tryptophan pathway genes of the Sargasso Sea metagenome: new operon structures and the prevalence of non-operon organization

    OpenAIRE

    Kagan, Juliana; Sharon, Itai; Beja, Oded; Kuhn, Jonathan C

    2008-01-01

    Background The enormous database of microbial DNA generated from the Sargasso Sea metagenome provides a unique opportunity to locate genes participating in different biosynthetic pathways and to attempt to understand the relationship and evolution of those genes. In this article, an analysis of the Sargasso Sea metagenome is made with respect to the seven genes of the tryptophan pathway. Results At least 5% of all the genes that are related to amino acid biosynthesis are tryptophan (trp) gene...

  1. Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis [v1; ref status: indexed, http://f1000r.es/2tz

    Directory of Open Access Journals (Sweden)

    Yaya Wang

    2014-02-01

    Full Text Available Fluorination has been widely used in chemical synthesis, but is rare in nature. The only known biological fluorination scope is represented by the fl pathway from Streptomyces cattleya that produces fluoroacetate (FAc and 4-fluorothreonine (4-FT. Here we report the identification of a novel pathway for FAc and 4-FT biosynthesis from the actinomycetoma-causing pathogen Nocardia brasiliensis ATCC 700358. The new pathway shares overall conservation with the fl pathway in S. cattleya. Biochemical characterization of the conserved domains revealed a novel fluorinase NobA that can biosynthesize 5’-fluoro-5’-deoxyadenosine (5’-FDA from inorganic fluoride and S-adenosyl-l-methionine (SAM. The NobA shows similar halide specificity and characteristics to the fluorination enzyme FlA of the fl pathway. Kinetic parameters for fluoride (Km 4153 μM, kcat 0.073 min-1 and SAM (Km 416 μM, kcat 0.139 min-1 have been determined, revealing that NobA is slightly (2.3 fold slower than FlA. Upon sequence comparison, we finally identified a distinct loop region in the fluorinases that probably accounts for the disparity of fluorination activity.

  2. Biochemical and Structural Characterization of a Ureidoglycine Aminotransferase in the Klebsiella pneumoniae Uric Acid Catabolic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    French, Jarrod B.; Ealick, Steven E. (Cornell)

    2010-09-03

    Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an {alpha}-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

  3. Enhanced fatty acid accumulation in Isochrysis galbana by inhibition of the mitochondrial alternative oxidase pathway under nitrogen deprivation.

    Science.gov (United States)

    Zhang, Litao; Liu, Jianguo

    2016-07-01

    The purpose of this study was to clarify the interrelation between the mitochondrial alternative oxidase (AOX) pathway and fatty acid accumulation in marine microalga Isochrysis galbana. Under normal conditions, the activity of the AOX pathway was maintained at a low level in I. galbana. Compared with the normal condition, nitrogen deprivation significantly increased the AOX pathway activity and fatty acid accumulation. Under nitrogen deprivation, the inhibition of the AOX pathway by salicylhydroxamic acid caused the accumulation of reducing equivalents and the over-reduction of chloroplasts in I. galbana cells, leading to a decrease in the photosynthetic O2 evolution rate. The over-production of reducing equivalents due to the inhibition of the AOX pathway under nitrogen deprivation further enhanced the accumulation of fatty acids in I. galbana cells.

  4. Biosynthetic porphyrins and the origin of photosynthesis

    Science.gov (United States)

    Mauzerall, D.; Ley, A.; Mercer-Smith, J. A.

    1986-01-01

    Since the prebiotic atmosphere was anaerobic, if not reducing, a useful function of primordial photosynthesis would have been to photooxidize reduced substrates such as Fe(+2), S(-2) or reduced organic molecules and to emit hydrogen. Experiments have shown that the early biogenic pigments uroporphyrin and coproporphyrin do photooxidize organic compounds and emit hydrogen in the presence of a platinum catalyst. These experiments were carried out in dilute aqueous solution near neutral pH under anaerobic atmosphere, and quantum yields near 10-2 were obtained. Thus relevant prebiotic conditions were maintained. Rather then to further optimize conditions, attempts were made to replace the platinum catalyst by a more prebiotically suitable catalyst. Trials with an Fe4S4(SR)4 cluster, in analogy to the present hydrogenase and nitrogenase, were not successful. However, experiments using cobalt complexes to catalyze the formation of hydrogen are promising. In analogy with biological photosynthetic systems which group pigments, electron transfer molecules and enzymes in clusters for efficiency, it was found that binding the biogenic porphyrins to the polyvinyl alcohol used to support the platinum catalyst did increase the quantum yield of the reaction. It was also found that ultraviolet light can serve to photo-oxidize porphyrinogens to porphyrins under anaerobic conditions. Thus the formation of the colorless porphyriogens by the extraordinarily simple biosynthetic pathway would not be a problem because of the prevalence of UV light in the prebiotic, anoxic atmosphere.

  5. Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Recent studies have shown that polyunsaturated fatty acids (PUFA) regulated the functions of membrane receptors in T cells and suppressed T cell -mediated immune responses. But the molecular mechanisms of immune regulation are not yet elucidated. Lipid rafts are plasma membrane microdomains, in which many receptors localized. The purpose of this study was to investigate the effect of DHA on IL-2R signaling pathway in lipid rafts. We isolated lipid rafts by discontinuous sucrose density gradient ultracentrifugation, and found that DHA could change the composition of lipid rafts and alter the distribution of key molecules of IL-2R signaling pathway, which transferred from lipid rafts to detergent-soluble membrane fractions. These results revealed that DHA treatment increased the proportion of polyunsaturated fatty acids especially n(3 polyunsaturated fatty acids in lipid rafts and changed the lipid environment of membrane microdomains in T cells. Compared with controls, DHA changed the localization of IL-2R, STAT5a and STAT5b in lipid rafts and suppressed the expression of JAK1, JAK3 and tyrosine phosphotyrosine in soluble membrane fractions. Summarily, this study concluded the effects of DHA on IL-2R signaling pathway in lipid rafts and explained the regulation of PUFAs in T cell-mediated immune responses.

  6. Biosynthetic gene clusters for relevant secondary metabolites produced by Penicillium roqueforti in blue cheeses.

    Science.gov (United States)

    García-Estrada, Carlos; Martín, Juan-Francisco

    2016-10-01

    Ripening of blue-veined cheeses, such as the French Bleu and Roquefort, the Italian Gorgonzola, the English Stilton, the Danish Danablu or the Spanish Cabrales, Picón Bejes-Tresviso, and Valdeón, requires the growth and enzymatic activity of the mold Penicillium roqueforti, which is responsible for the characteristic texture, blue-green spots, and aroma of these types of cheeses. This filamentous fungus is able to synthesize different secondary metabolites, including andrastins, mycophenolic acid, and several mycotoxins, such as roquefortines C and D, PR-toxin and eremofortins, isofumigaclavines A and B, and festuclavine. This review provides a detailed description of the main secondary metabolites produced by P. roqueforti in blue cheese, giving a special emphasis to roquefortine, PR-toxin and mycophenolic acid, and their biosynthetic gene clusters and pathways. The knowledge of these clusters and secondary metabolism pathways, together with the ability of P. roqueforti to produce beneficial secondary metabolites, is of interest for commercial purposes. PMID:27554495

  7. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis.

    Science.gov (United States)

    Lukassen, Mie Bech; Saei, Wagma; Sondergaard, Teis Esben; Tamminen, Anu; Kumar, Abhishek; Kempken, Frank; Wiebe, Marilyn G; Sørensen, Jens Laurids

    2015-07-01

    Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster. PMID:26184239

  8. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    Directory of Open Access Journals (Sweden)

    Mie Bech Lukassen

    2015-07-01

    Full Text Available Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine. Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1, a polyketide synthase (PKS2, a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster.

  9. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    Science.gov (United States)

    Lukassen, Mie Bech; Saei, Wagma; Sondergaard, Teis Esben; Tamminen, Anu; Kumar, Abhishek; Kempken, Frank; Wiebe, Marilyn G.; Sørensen, Jens Laurids

    2015-01-01

    Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster. PMID:26184239

  10. c9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway[S

    Science.gov (United States)

    Mollica, Maria Pina; Trinchese, Giovanna; Cavaliere, Gina; De Filippo, Chiara; Cocca, Ennio; Gaita, Marcello; Della-Gatta, Antonio; Marano, Angela; Mazzarella, Giuseppe; Bergamo, Paolo

    2014-01-01

    Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis. PMID:24634500

  11. Advanced biofuel-oriented engineering of fatty acid pathway:a review%先进生物燃料导向的脂肪酸途径合成生物学改造

    Institute of Scientific and Technical Information of China (English)

    周雍进; 赵宗保

    2011-01-01

    化石能源日益枯竭,迫切需要寻找新型燃料.脂肪族生物燃料由于其热值高、性能好而受到广泛重视.微生物脂肪酸代谢途径是生产先进生物燃料的重要途径.文中综述了近几年基于合成生物学理念改造脂肪酸途径的进展,介绍了合成生物学在微生物柴油、中长链脂肪醇、长链烃类化合物生物合成中的应用,并展望了脂肪族生物燃料的发展方向.%Biofuel is in high demand as an alternative energy source for petroleum and diesel. Fatty acid-based biofuel has higher energy density and better compatibility with existing infrastructures. Microbial fatty acid biosynthetic pathway is important to develop biofuel. In this article, recent progresses on the modification and reconstruction of fatty acid metabolism for the production of biofuel were reviewed, with a focus on micro-diesel, long chain fatty alcohol and alkane. Problems, solutions and directions for further development of fatty acid-based biofuel were also discussed in the respect of synthetic biology.

  12. Identification of the phytosphingosine metabolic pathway leading to odd-numbered fatty acids.

    Science.gov (United States)

    Kondo, Natsuki; Ohno, Yusuke; Yamagata, Maki; Obara, Takashi; Seki, Naoya; Kitamura, Takuya; Naganuma, Tatsuro; Kihara, Akio

    2014-01-01

    The long-chain base phytosphingosine is a component of sphingolipids and exists in yeast, plants and some mammalian tissues. Phytosphingosine is unique in that it possesses an additional hydroxyl group compared with other long-chain bases. However, its metabolism is unknown. Here we show that phytosphingosine is metabolized to odd-numbered fatty acids and is incorporated into glycerophospholipids both in yeast and mammalian cells. Disruption of the yeast gene encoding long-chain base 1-phosphate lyase, which catalyzes the committed step in the metabolism of phytosphingosine to glycerophospholipids, causes an ~40% reduction in the level of phosphatidylcholines that contain a C15 fatty acid. We also find that 2-hydroxypalmitic acid is an intermediate of the phytosphingosine metabolic pathway. Furthermore, we show that the yeast MPO1 gene, whose product belongs to a large, conserved protein family of unknown function, is involved in phytosphingosine metabolism. Our findings provide insights into fatty acid diversity and identify a pathway by which hydroxyl group-containing lipids are metabolized. PMID:25345524

  13. 烟草绿原酸合成关键基因NtHQT1的克隆及表达分析%Cloning and Expression Analysis of Chlorogenic Acid Biosynthetic Gene NtHQT1 from Nicotiana tabacum

    Institute of Scientific and Technical Information of China (English)

    武明珠; 许亚龙; 李锋; 魏攀; 王中; 罗朝鹏; 王燃; 张剑锋; 林福呈

    2015-01-01

    Chlorogenic acid is the dominant polyphenol in leaf tobacco. Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) is the key enzyme in the metabolic pathway of chlorogenic acid in plant. In order to study the function mechanism of HQT gene in the synthesis of chlorogenic acid in tobacco, a new HQT gene was cloned from Nicotiana tabacum by homologous cloning technology and named as NtHQT1. The results of bioinformatics analysis and expression pattern analysis of genes treated by different hormones showed that NtHQT1 cDNA was 1 305 bp in length and encoded 435 amino acids; it was a hydrophobic protein and located in cytoplasm, its secondary structure was mainly of helix and random coil. The expression of NtHQT1 gene was obviously up-regulated by methyl jasmonate (MeJA), auxin (3-Indoleacetic acid, IAA), strigolactone (GR24), cytokinin (6-BA) and gibberellin acid (GA), while not obviously affected by abscisic acid (ABA). It suggested that NtHQT1 might play an important role in the course of growth, development and disease resistance of tobacco.%绿原酸(Chlorogenic acid,CGA)是烟叶中含量最高的多酚类物质,羟基肉桂酰辅酶A奎尼羟基肉桂转移酶(Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase,HQT)是植物绿原酸合成代谢途径中的关键酶.为研究HQT基因在烟草绿原酸合成中的作用机制,通过同源克隆技术从烟草中克隆到一个新的HQT基因,命名为NtHQT1.生物信息学和激素处理后基因表达模式分析结果表明:NtHQT1 cDNA全长1 305 bp,编码435个氨基酸,NtHQT1定位在细胞质中,属于疏水性蛋白,其二级结构主要是螺旋和无规则卷曲;茉莉酸甲酯(Methyl jasmonate,MeJA)、生长素(3-Indoleacetic acid,IAA)、独角金内酯类似物(GR24)、细胞分裂素(6-Benzylaminopurin,6-BA)和赤霉素(Gibberellic acid,GA)能明显上调NtHQT1基因的表达,脱落酸(Abscisic acid,ABA)对基因表达没有明显作用,预示NtHQT1基因在烟草生长发育和抗

  14. A mitochondrial pathway for biosynthesis of lipid mediators

    Science.gov (United States)

    Tyurina, Yulia Y.; Poloyac, Samuel M.; Tyurin, Vladimir A.; Kapralov, Alexander A.; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I.; Vikulina, Anna S.; Jung, Mi-Yeon; Epperly, Michael W.; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C.; Kochanek, Patrick M.; Pitt, Bruce R.; Greenberger, Joel S.; Vladimirov, Yury A.; Bayır, Hülya; Kagan, Valerian E.

    2014-06-01

    The central role of mitochondria in metabolic pathways and in cell-death mechanisms requires sophisticated signalling systems. Essential in this signalling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin (CL), is oxidized by the intermembrane-space haemoprotein, cytochrome c. We show that a number of oxygenated CL species undergo phospholipase A2-catalysed hydrolysis and thus generate multiple oxygenated fatty acids, including well-known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway, which includes the oxidation of polyunsaturated CLs and accumulation of their hydrolysis products (oxygenated linoleic, arachidonic acids and monolysocardiolipins), is activated in vivo after acute tissue injury.

  15. Caffeic Acid Inhibits NFkappaB Activation of Osteoclastogenesis Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Ferry Sandra

    2011-12-01

    Full Text Available BACKGROUND: Caffeic acid (3,4-dihydroxycinnamic acids is involved in various green plants. Based on our previous report, a major component of sweet potato extracts, possibly caffeic acid, was shown as a promising inhibitor of osteoclastogenesis. However, the effect of caffeic acid in inhibiting osteoclastogenesis needs to be confirmed. The underlying mechanism needs to be disclosed as well. METHODS: Caffeic acid in various concentrations was added to in vitro osteoclastogenesis of receptor activator nuclear factor kB ligand (RANKL-tumor necrosis factor alpha (TNF-α-macrophage colony stimulating factor (M-CSF-induced bone marrow-derived monocyte/macrophage precursor cells (BMMs and RANKL-TNF-α-induced RAW264 cells D-Clone (RAW-D cells. Tartrate resistant acid phosphatase (TRAP staining was performed and TRAP-positive polynucleated cells (PNCs were counted. For apoptosis analysis, caffeic acid-treated BMMs, RAW-D cells and osteoclast-like PNCs were subjected to Sub-G1 Apoptosis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assays. To measure NFkB activity, RAW-D cells were transfected with pNFkB-TA-Luc and subjected to Dual Luciferase Reporter Assay System. RESULTS: Caffeic acid inhibited osteoclastogenesis of RANKL-TNF-α-M-CSF-induced BMMs as well as RANKL-TNF-α-induced RAW-D cells in a dose dependent manner. Caffeic acid did not induce apoptosis in BMMs, RAW-D cells and osteoclast-like PNCs. RANKL-TNF-α-induced NFkB activity in RAW-D was diminished by caffeic acid in a dose dependent manner. Significant NFkB activity inhibtion was observed starting from 1µg/mL caffeic acid. CONCLUSIONS: Caffeic acid could be a potent osteoclastogenesis inhibitor through inhibition of NFkB activity. Our present study should be further followed up to disclose caffeic acid's possible overlying signaling pathways in inhibiting osteoclastogenesis. KEYWORDS: caffeic acid, osteoclastogenesis, NFkB, RANKL, TNF-α.

  16. Identification of diacylglycerol and triacylglycerol containing 11,12,13-trihydroxy-9,14-octadecadienoic acid in castor oil.

    Science.gov (United States)

    Lin, Jiann-Tsyh; Chen, Grace Q

    2011-02-28

    Castor oil has many industrial uses. Molecular species of acylglycerols containing monohydroxy, dihydroxy and trihydroxy fatty acids in castor oil have been reported. We report here the identification of acylglycerols containing a triOH18:2 fatty acid in castor oil. The structure of this novel fatty acid was proposed as 11,12,13-trihydroxy-9,14-octadecadienoic acid by the mass spectrometry of the lithiated adducts of acylglycerols in the HPLC fractions of castor oil. The fragmentation pathways of the lithiated adduct of 11,12,13-trihydroxy-9,14-octadecadienoic acid were proposed. We also proposed the biosynthetic pathways of polyhydroxy fatty acids in castor.

  17. N2O production pathways in the subtropical acid forest soils in China

    International Nuclear Information System (INIS)

    To date, N2O production pathways are poorly understood in the humid subtropical and tropical forest soils. A 15N-tracing experiment was carried out under controlled laboratory conditions to investigate the processes responsible for N2O production in four subtropical acid forest soils (pH2O emission in the subtropical acid forest soils, being responsible for 56.1%, 53.5%, 54.4%, and 55.2% of N2O production, in the GC, GS, GB, and TC soils, respectively, under aerobic conditions (40%-52%WFPS). The heterotrophic nitrification (recalcitrant organic N oxidation) accounted for 27.3%-41.8% of N2O production, while the contribution of autotrophic nitrification was little in the studied subtropical acid forest soils. The ratios of N2O-N emission from total nitrification (heterotrophic+autotrophic nitrification) were higher than those in most previous references. The soil with the lowest pH and highest organic-C content (GB) had the highest ratio (1.63%), suggesting that soil pH-organic matter interactions may exist and affect N2O product ratios from nitrification. The ratio of N2O-N emission from heterotrophic nitrification varied from 0.02% to 25.4% due to soil pH and organic matter. Results are valuable in the accurate modeling of N2O production in the subtropical acid forest soils and global budget. - Highlights: → We studied N2O production pathways in subtropical acid forest soil under aerobic conditions. → Denitrification was the main source of N2O production in subtropical acid forest soils. → Heterotrophic nitrification accounted for 27.3%-41.8% of N2O production. → While, contribution of autotrophic nitrification to N2O production was little. → Ratios of N2O-N emission from nitrification were higher than those in most previous references.

  18. Identification of genes and pathways involved in the synthesis of Mead acid (20:3n-9), an indicator of essential fatty acid deficiency.

    Science.gov (United States)

    Ichi, Ikuyo; Kono, Nozomu; Arita, Yuka; Haga, Shizuka; Arisawa, Kotoko; Yamano, Misato; Nagase, Mana; Fujiwara, Yoko; Arai, Hiroyuki

    2014-01-01

    In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n-9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1-6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n-9, 20:1n-9 and 20:2n-9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n-9→(Fads2)→18:2n-9→(Elovl5)→20:2n-9→(Fads1)→20:3n-9 and pathway 2) 18:1n-9→(Elovl5)→20:1n-9→(Fads2)→20:2n-9→(Fads1)→20:3n-9. PMID:24184513

  19. The effect pathway of retinoic acid through regulation of retinoic acid receptor in gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Su Liu; Qiao Wu; Zheng-Ming Chen; Wen-Jin Su

    2001-01-01

    AIM To evaluate the role of RARa gene in mediating the growth inhibitory effect of ail-trans retinoic acid (ATRA)on gastric cancer cells.``METHODS The expression levels of retinoic acid receptors (RARs) in gastric cancer cells were detected by Northern blot. Transient transfection and chlorophenicol acetyl transferase (CAT) assay were used to show the transcriptional activity of β retinoic acid response element (βRARE) and AP-l activity. Cell growth inhibition was determined by MTT assay and anchorage-independent growth assay, respectively. Stable transfection was performed by the method of Lipofectamine, and the cells were screened by G418.``RESULTS ATRA could induce expression level of RARα in MGC80-3, BGCC8823 and SGC-7901 cells obviously,resulting in growth inhibition of these cell lines. After sense RARa gene was transfected into MKN-45 cells that expressed rather Iow level of RARα and could not be induced by ATRA, the cell growth was inhibited by ATRA markedly. In contrast, when antisense RARα gene was transfected into BGC-825 cells, a little inhibitory effect by ATRA was seen, compared with the parallel BGC-823cells. In transient transfection assay, ATRA effectively induced transcriptional activity of βRARE in MGC80-3,BGC.823, SGC-7902 and MKN/RARa cell lines, but not in MKN-45 and BGC/aRARa cell lines. Similar results were observed in measuring anti-AP-l activity by ATRA in these cancer cell lines.``CONCLUSION ATRA inhibits the growth of gastric cancer cells by up-regulating the level of RARa; RARa is the major mediator of ATRA action in gastric cancer cells; and adequate level of RAPa is required for ATRA effect on gastric cancer cells.``

  20. Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Wang, Zheng; Chen, Yun;

    2015-01-01

    biomass into the products of interest. We engineered Saccharomyces cerevisiae for production of 3-hydroxypropionic acid (3HP), a potential building block for acrylates, from glucose and xylose. We introduced the 3HP biosynthetic pathways via malonyl-CoA or β-alanine intermediates into a xylose...

  1. SELECTIVE SEPARATION OF BIOSYNTHETIC PRODUCTS BY PERTRACTION - CHALLENGE FOR THE “WHITE BIOTECHNOLOGY”

    OpenAIRE

    Dan Cascaval; Anca-Irina Galaction

    2010-01-01

    This review presents our original results on selective separation of some biosynthetic products (antibiotics, carboxylic acids, amino acids) by free or facilitated pertraction (extraction and transport through liquid membranes). Selecting the optimum conditions, for all studied cases these pertraction technique simplify the technologies applied at industrial scale for separation and purification, allows to reaching high selectivity and reducing the overall cost of the products.

  2. Changes in actin dynamics are involved in salicylic acid signaling pathway.

    Science.gov (United States)

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented.

  3. Distinct pathways of ERK1/2 activation by hydroxy-carboxylic acid receptor-1.

    Directory of Open Access Journals (Sweden)

    Guo Li

    Full Text Available Mechanistic investigations have shown that, upon agonist activation, hydroxy-carboxylic acid receptor-1(HCA1 couples to a Gi protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for HCA1 signaling remain largely unknown. Using CHO-K1 cells stably expressing HCA1, and L6 cells, which endogenously express rat HCA1 receptors, we found that activation of ERK1/2 by HCA1 was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that HCA1 induced ERK1/2 activation via the extracellular Ca2+, PKC and IGF-I receptor transactivation-dependent pathways. In addition, we observed that pretreated the cells with M119K, an inhibitor of Gβγ subunit-dependent signaling, effectively attenuated the ERK1/2 activation triggered by HCA1, suggesting a critical role for βγ-subunits in HCA1-activated ERK1/2 phosphorylation. Furthermore, the present results also indicated that the arrestin2/3 were not required for ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to agonist, HCA1 receptors initially activate Gi, leading to dissociation of the Gβγ subunit from activated Gi, and subsequently induce ERK1/2 activation via two distinct pathways: one PKC-dependent pathway and the other IGF-IR transactivation-dependent pathway. Our results provide the first in-depth evidence that defines the molecular mechanism of HCA1-mediated ERK1/2 activation.

  4. Eicosapentaenoic Acid Protects against Palmitic Acid-Induced Endothelial Dysfunction via Activation of the AMPK/eNOS Pathway

    Directory of Open Access Journals (Sweden)

    Che-Hsin Lee

    2014-06-01

    Full Text Available Recent studies have shown that free fatty acids are associated with chronic inflammation, which may be involved in vascular injury. The intake of eicosapentaenoic acid (EPA can decrease cardiovascular disease risks, but the protective mechanisms of EPA on endothelial cells remain unclear. In this study, primary human umbilical vein endothelial cells (HUVECs treated with palmitic acid (PA were used to explore the protective effects of EPA. The results revealed that EPA attenuated PA-induced cell death and activation of apoptosis-related proteins, such as caspase-3, p53 and Bax. Additionally, EPA reduced the PA-induced increase in the generation of reactive oxygen species, the activation of NADPH oxidase, and the upregulation of inducible nitric oxide synthase (iNOS. EPA also restored the PA-mediated reduction of endothelial nitric oxide synthase (eNOS and AMP-activated protein kinase (AMPK phosphorylation. Using AMPK siRNA and the specific inhibitor compound C, we found that EPA restored the PA-mediated inhibitions of eNOS and AKT activities via activation of AMPK. Furthermore, the NF-κB signals that are mediated by p38 mitogen-activated protein kinase (MAPK were involved in protective effects of EPA. In summary, these results provide new insight into the possible molecular mechanisms by which EPA protects against atherogenesis via the AMPK/eNOS-related pathway.

  5. Aqueous phototransformation of bisphenol S: the competitive radical-attack pathway to p-hydroxybenzenesulfonic acid.

    Science.gov (United States)

    Wang, Xiaowen; Ma, Jiahai; Wang, Zhenpeng; Guo, Rongrong; Hu, Xuefeng

    2014-01-01

    The kinetics, environmental influencing factors, products and reaction mechanism of aqueous phototransformation of bisphenol S (BPS), as an alternative to bisphenol A, which is of environmental concern, were investigated. p-Hydroxybenzenesulfonic acid, as the major transformation product was confirmed by gas chromatography - mass spectrometry, electrospray ionization, ¹H nuclear magnetic resonance and fluorescence spectrum analysis. A reaction pathway was proposed based on the reactive oxygen species related results by electron paramagnetic resonance and radical traps. The competition of the excited state of BPS between transferring electron to O₂ to •O₂(-) and directly oxidizing H₂O to •OH was revealed.

  6. 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.

  7. 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. PMID:26020634

  8. Origin of saxitoxin biosynthetic genes in cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Ahmed Moustafa

    Full Text Available BACKGROUND: Paralytic shellfish poisoning (PSP is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX. STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway. METHODOLOGY/PRINCIPAL FINDINGS: We generated a draft genome assembly of the saxitoxin-producing (STX+ cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxin-genes (named sxtA to sxtZ that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non-toxic (STX- sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX- strains among Anabaena

  9. The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans.

    Science.gov (United States)

    Do, Eunsoo; Park, Minji; Hu, Guanggan; Caza, Mélissa; Kronstad, James W; Jung, Won Hee

    2016-09-01

    The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisiae. In this study, we identified the ortholog of LYS4 in the human fungal pathogen, Cryptococcus neoformans, and found that LYS4 expression is regulated by iron levels and by the iron-related transcription factors Hap3 and HapX. Deletion of the LYS4 gene resulted in lysine auxotrophy suggesting that Lys4 is essential for lysine biosynthesis. Our study also revealed that lysine uptake was mediated by two amino acid permeases, Aap2 and Aap3, and influenced by nitrogen catabolite repression (NCR). Furthermore, the lys4 mutant showed increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs. We showed that these phenotypes were due in part to impaired mitochondrial function as a result of LYS4 deletion, which we propose disrupts iron homeostasis in the organelle. The combination of defects are consistent with our observation that the lys4 mutant was attenuated virulence in a mouse inhalation model of cryptococcosis. PMID:27353379

  10. Defining and Modeling Known Adverse Outcome Pathways: Domoic Acid and Neuronal Signaling as a Case Study

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Karen H.; Andersen, Melvin E.; Basu, Nil; Carvan, Michael J.; Crofton, Kevin M.; King, Kerensa A.; Sunol, Cristina; Tiffany-Castiglioni, Evelyn; Schultz, Irvin R.

    2011-01-01

    An adverse outcome pathway (AOP) is a sequence of key events from a molecular-level initiating event and an ensuing cascade of steps to an adverse outcome with population level significance. To implement a predictive strategy for ecotoxicology, the multiscale nature of an AOP requires computational models to link salient processes (e.g., in chemical uptake, toxicokinetics, toxicodynamics, and population dynamics). A case study with domoic acid was used to demonstrate strategies and enable generic recommendations for developing computational models in an effort to move toward a toxicity testing paradigm focused on toxicity pathway perturbations applicable to ecological risk assessment. Domoic acid, an algal toxin with adverse effects on both wildlife and humans, is a potent agonist for kainate receptors (ionotropic glutamate receptors whose activation leads to the influx of Na+ and Ca2+). Increased Ca2+ concentrations result in neuronal excitotoxicity and cell death primarily in the hippocampus, which produces seizures, impairs learning and memory, and alters behavior in some species. Altered neuronal Ca2+ is a key process in domoic acid toxicity which can be evaluated in vitro. Further, results of these assays would be amenable to mechanistic modeling for identifying domoic acid concentrations and Ca2+ perturbations that are normal, adaptive, or clearly toxic. In vitro assays with outputs amenable to measurement in exposed populations can link in vitro to in vivo conditions, and toxicokinetic information will aid in linking in vitro results to the individual organism. Development of an AOP required an iterative process with three important outcomes: (1) a critically reviewed, stressor-specific AOP; (2) identification of key processes suitable for evaluation with in vitro assays; and (3) strategies for model development.

  11. Pt-Pd nanoelectrocatalyst of ultralow Pt content for the oxidation of formic acid: Towards tuning the reaction pathway

    Indian Academy of Sciences (India)

    Sourov Ghosh; C Retna Raj

    2015-05-01

    Synthesis of highly efficient functional electrocatalyst that favours the electrochemical oxidation of formic acid via CO-free dehydrogenation pathway is required for direct formic acid fuel cells. Traditional catalysts favour the dehydration pathway involving the generation of poisonous CO. Herein we demonstrate the superior electrocatalytic performance of Pt-Pd bimetallic nanoelectrocatalyst of ultralow Pt content and tuning the reaction pathway by controlling the Pt content. Bimetallic nanoparticles of Pt4Pd96, Pt7Pd93 and Pt47Pd53 compositions are synthesized by electrochemical co-deposition method in aqueous solution. The nanoparticles of ultralow Pt content, Pt4Pd96, favour the CO-free dehydrogenation pathway for formic acid oxidation with an onset potential of 0 V (SHE) whereas the Pt47Pd53 nanoparticles favour the dehydration pathway involving the formation of CO at high positive potential. The Pt content of the bimetallic nanoparticles actually controls the oxidation peak potential and catalytic activity. Significant negative shift (∼350 mV) in the oxidation peak potential and remarkable enhancement in the current density (2.6 times) are observed for Pt4Pd96 nanoparticles with respect to Pt47Pd53. The absence of three adjacent Pt and Pd atoms could be the reason for the suppression of CO pathway. The electrochemical impedance measurements indirectly support the CO-free pathway for the formic acid oxidation on Pt4Pd96 nanoparticles.

  12. White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways

    Directory of Open Access Journals (Sweden)

    V. Barquissau

    2016-05-01

    Conclusions: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.

  13. Dual responsive physical networks from asymmetric biosynthetic triblock copolymers

    OpenAIRE

    T.H.T Pham

    2013-01-01

      The aim of the project is to develop biosynthetically produced amino acid polymers which are composed of three distinct blocks A-C-B, each with a separate function. A is a first self-assembling block capable of ‘recognizing’ (upon a trigger) other A blocks; C is an inert, random coil-like connector, and B is a second self-assembling block. A and B have to be chosen such that they do not cross-assemble. With these molecules it should be possible to fabricate hydrogels in whi...

  14. Targeting sialic acid dependent and independent pathways of invasion in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Rosalynn Louise Ord

    Full Text Available The pathology of malaria is a consequence of the parasitaemia which develops through the cyclical asexual replication of parasites in a patient's red blood cells. Multiple parasite ligand-erythrocyte receptor interactions must occur for successful Plasmodium invasion of the human red cell. Two major malaria ligand families have been implicated in these variable ligand-receptor interactions used by Plasmodium falciparum to invade human red cells: the micronemal proteins from the Erythrocyte Binding Ligands (EBL family and the rhoptry proteins from the Reticulocyte binding Homolog (PfRH family. Ligands from the EBL family largely govern the sialic acid (SA dependent pathways of invasion and the RH family ligands (except for RH1 mediate SA independent invasion. In an attempt to dissect out the invasion inhibitory effects of antibodies against ligands from both pathways, we have used EBA-175 and RH5 as model members of each pathway. Mice were immunized with either region II of EBA-175 produced in Pichia pastoris or full-length RH5 produced by the wheat germ cell-free system, or a combination of the two antigens to look for synergistic inhibitory effects of the induced antibodies. Sera obtained from these immunizations were tested for native antigen recognition and for efficacy in invasion inhibition assays. Results obtained show promise for the potential use of such hybrid vaccines to induce antibodies that can block multiple parasite ligand-red cell receptor interactions and thus inhibit parasite invasion.

  15. 柠檬酸铵对紫杉醇生物合成途径的诱导作用研究%Elicitation of taxol biosynthetic pathway by ammonium citrate

    Institute of Scientific and Technical Information of China (English)

    未作君; 苗志奇; 元英进

    2001-01-01

    目的确定柠檬酸铵在紫杉醇生物代谢途径的作用位点。方法分别在0,0.1,1,10,20μmol/L的柠檬酸铵诱导下,观察紫杉烷类物质产量的变化,并通过生物代谢途径进行定性分析。结果紫杉醇的产量提高近3倍,而10-DAB(10-deactylbaccatin Ⅲ)与baccatin Ⅲ浓度下降。结论柠檬酸铵提高了baccatin Ⅲ与紫杉醇间的反应速度,即其作用位点位于baccatin Ⅲ与紫杉醇之间。%Object To study the eliciting effec t of ammonium citrate (AC) onthe yield of taxol. Methods The study was carried out on plant cell cultures with different concentrations of ad ded AC. Results Yield of taxol was increased by 3 times under 10 μmol/L AC elicitation, while the concentrations of 10-deacetyl baccatin Ⅲ and baccatin Ⅲ were simultaneously decreased. Conclusion By a nalyzing the changing characters between substrate and enzyme in the simplified taxol synthetic pathway. AC was considered to play the role in increasing the reaction rate from baccatin Ⅲ to taxol.

  16. Chitosan oligosaccharide and salicylic acid up-regulate gene expression differently in relation to the biosynthesis of artemisinin in Artemisia annua L

    DEFF Research Database (Denmark)

    Yin, Heng; Kjær, Anders; Fretté, Xavier;

    2012-01-01

    oligosaccharide (COS) and salicylic acid (SA) on both artemisinin production and gene expression related to the biosynthetic pathway of artemisinin. COS up-regulated the transcriptional levels of the genes ADS and TTG1 2.5 fold and 1.8 fold after 48 h individually, whereas SA only up-regulated ADS 2.0 fold after...

  17. N{sub 2}O production pathways in the subtropical acid forest soils in China

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Jinbo [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China); Cai Zucong, E-mail: zccai@mail.issas.ac.cn [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China); Zhu Tongbin [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China)

    2011-07-15

    To date, N{sub 2}O production pathways are poorly understood in the humid subtropical and tropical forest soils. A {sup 15}N-tracing experiment was carried out under controlled laboratory conditions to investigate the processes responsible for N{sub 2}O production in four subtropical acid forest soils (pH<4.5) in China. The results showed that denitrification was the main source of N{sub 2}O emission in the subtropical acid forest soils, being responsible for 56.1%, 53.5%, 54.4%, and 55.2% of N{sub 2}O production, in the GC, GS, GB, and TC soils, respectively, under aerobic conditions (40%-52%WFPS). The heterotrophic nitrification (recalcitrant organic N oxidation) accounted for 27.3%-41.8% of N{sub 2}O production, while the contribution of autotrophic nitrification was little in the studied subtropical acid forest soils. The ratios of N{sub 2}O-N emission from total nitrification (heterotrophic+autotrophic nitrification) were higher than those in most previous references. The soil with the lowest pH and highest organic-C content (GB) had the highest ratio (1.63%), suggesting that soil pH-organic matter interactions may exist and affect N{sub 2}O product ratios from nitrification. The ratio of N{sub 2}O-N emission from heterotrophic nitrification varied from 0.02% to 25.4% due to soil pH and organic matter. Results are valuable in the accurate modeling of N2O production in the subtropical acid forest soils and global budget. - Highlights: {yields} We studied N{sub 2}O production pathways in subtropical acid forest soil under aerobic conditions. {yields} Denitrification was the main source of N{sub 2}O production in subtropical acid forest soils. {yields} Heterotrophic nitrification accounted for 27.3%-41.8% of N{sub 2}O production. {yields} While, contribution of autotrophic nitrification to N{sub 2}O production was little. {yields} Ratios of N{sub 2}O-N emission from nitrification were higher than those in most previous references.

  18. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

    KAUST Repository

    Ross, Avena C.

    2013-01-23

    Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus. © 2012 American Chemical Society.

  19. Study on Erythritol Biosynthetic Pathway and The Optimization Strategy Abroad%国外对赤藓糖醇生物合成途径及优化策略的研究

    Institute of Scientific and Technical Information of China (English)

    李雨; 秦海青; 邱学良; 王成福; 高永旭; 李毅; 吴庆国

    2013-01-01

    赤藓糖醇是一种四碳醇,是应用于食品和医药工业的天然甜味剂。由于赤藓糖醇具有特殊的营养特性,它作为功能糖被应用于食品中,给糖尿病人和肥胖患者带来了福音。在国外,工业上一般采用Aureobasidium sp.和Pseudozyma tsukubaensis等突变的高渗酵母生产赤藓糖醇。由于生产效率高,成本适中,它还被用作生产其他糖的原料。本文主要综述了赤藓糖醇的理化性质、生物合成途径以及提高微生物赤藓糖醇生产力的策略。%Erythritol, a four-carbon polyol, is a biological sweetener with applications in food and pharmaceutical industries .It is also used as a functional sugar substitute in special foods for people with diabetes and obesity because of its unique nutritional properties .In abroad , Erythritol is produced by microbial methods using mostly osmophilic yeasts and has been produced commercially using mutant strains of Aureobasidium sp.and Pseudozyma tsukubaensis.Due to the high yield and productivity in the industrial scale of production , erythritol serves as an inexpensive starting material for the production of other sugars .This review focuses on the physicochemical properties of erythritol , pathway of erythritol biosynthesis and strategies used to enhance erythritol productivity in microbes .

  20. Retinoic acid triggers meiosis initiation via stra8-dependent pathway in Southern catfish, Silurus meridionalis.

    Science.gov (United States)

    Li, Minghui; Feng, Ruijuan; Ma, He; Dong, Ranran; Liu, Zhilong; Jiang, Wentao; Tao, Wenjing; Wang, Deshou

    2016-06-01

    Existing studies demonstrated that retinoic acid (RA) regulates meiotic initiation via stra8-independent pathway in teleosts which lack stra8 in their genomes. However, stra8 was recently identified from several fish species including Southern catfish (Silurus meridionalis). To explore the existence of stra8-dependent pathway in RA mediated meiotic initiation in fishes, in the present study, the genes encoding RA synthase aldh1a2 and catabolic enzyme cyp26a1 and cyp26b1 were cloned from the Southern catfish. By immunohistochemistry, Aldh1a2 signal was observed in gonads of both sexes during the meiotic initiation period. By real-time PCR, differentially expressed gene was observed for cyp26a1, but not for cyp26b1, in gonads during the meiotic initiation. Administration of exogenous RA or inhibition of endogenous RA degradation by either KET (RA catabolic enzyme inhibitor) or cyp26a1 knockdown using CRISPR/Cas9 induced advanced meiotic initiation in the ovaries as demonstrated by increased Stra8/stra8 expression and appearance of oocytes. In contrast, treatment with RA synthase inhibitor DEAB resulted in delayed meiotic initiation and Stra8/stra8 expression in the ovaries, which was rescued by exogenous RA administration. These results indicated that (1) RA triggers the onset of meiosis via stra8-dependent pathway in stra8 existing teleosts, as it does in tetrapods; (2) exogenous RA can rescue the endogenous RA deficiency; (3) Cyp26a1, instead of Cyp26b1, is the key catabolic enzyme involved in meiosis initiation in teleosts. Taken together, RA might trigger meiotic initiation via stra8-dependent and -independent pathway in different teleosts. PMID:26764212

  1. Effects of Retinoic Acid on the β-catenin/TCF Pathway in Cultured Porcine Tracheobronchial Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    李媛; 吴人亮; 王曦; 陈文书

    2004-01-01

    The effects of retinoic acid on the β-catenin/TCF pathway in cultured porcine tracheobronchial epithelial cells (TBEC) were investigated. After TBEC were treated with retinoic acid at various concentrations, mRNA and protein changes of β-catenin in cytoplasm, nucleus and whole cell of the TBEC were observed by immunocytochemical stain, RT-PCR and Western blotting. And the changes of the target gene cyclinD1 of β-catenin/TCF pathway were also observed. It was found that there was no significant difference in β-cat mRNA level after retinoic acid treatment. However,the expression of β-catenin in the whole cell and cytoplasm was elevated with the increase of retinoic acid concentration (P<0.01). The nuclear protein β-catenin and target gene cyclinD1 of β-catenin/TCF pathway was decreased (P<0.05). It was indicated that retinoic acid could increase β-catenin level of the whole cell protein and decrease nuclear β-catenin, downregulating β-cat/TCF signaling activity and reducing target gene cyclinD1 protein level. As a result, retinoic acid can downregulate β-catenin/TCF pathway in porcine tracheobronchial epithelial cell, suggesting that retinoic acid can inhibit the proliferation and accelerate differentiation of tracheobronchial epithelial cells.

  2. Characteristics and biosynthetic pathway of melanin in Ascochyta anemones%白头翁叶斑病菌黑色素的理化性质及其生物合成途径初步研究

    Institute of Scientific and Technical Information of China (English)

    苏丹; 吕国忠; 周如军; 杨红; 傅俊范

    2014-01-01

    对白头翁叶斑病菌胞壁结合黑色素和胞外黑色素进行了理化性质和红外光谱扫描测定,结果表明两者具有相似的理化性质,均易溶于KOH、H2 O2和NaClO,不溶于水、乙醇和丙酮。红外光谱分析表明,白头翁叶斑病菌YS-24菌株的胞壁结合黑色素与胞外黑色素为同一种类型的黑色素。DHN黑色素的特异性抑制剂—三环唑,对白头翁叶斑病菌黑色素的产生有明显的抑制作用;以白头翁叶斑病菌基因组DNA为模板,通过PCR扩增,得到了聚酮体合成酶基因的同源片段AaPKS,初步推断白头翁叶斑病菌黑色素合成属于DHN途径。%The characteristics of melanin,extracted from cell wall and fermented filtrate of Ascochyta anemones, were determined by diagnostic test and infrared spectroscopy. The results indicated that they have similar physical and chemical properties,and they are soluble in KOH,H2 O2 and NaClO,but not in water,ethanol and acetone. Infrared spectrum analysis showed that the wall-bound melanin and extracellular melanin belonged to the same type. Tricyclazole,a specific inhibitor of DHN melanin synthesis,could inhibit the fungal melanin’s biosynthesis of A.anemones. The homologous fragment of polyketide synthase gene (AaPKS)was amplified by PCR using A.anemones genomic DNA as a template. The results suggested that the melanin in A.anemones might be synthe-sized from DHN pathway.

  3. Drosophila fatty acid taste signals through the PLC pathway in sugar-sensing neurons.

    Directory of Open Access Journals (Sweden)

    Pavel Masek

    Full Text Available Taste is the primary sensory system for detecting food quality and palatability. Drosophila detects five distinct taste modalities that include sweet, bitter, salt, water, and the taste of carbonation. Of these, sweet-sensing neurons appear to have utility for the detection of nutritionally rich food while bitter-sensing neurons signal toxicity and confer repulsion. Growing evidence in mammals suggests that taste for fatty acids (FAs signals the presence of dietary lipids and promotes feeding. While flies appear to be attracted to fatty acids, the neural basis for fatty acid detection and attraction are unclear. Here, we demonstrate that a range of FAs are detected by the fly gustatory system and elicit a robust feeding response. Flies lacking olfactory organs respond robustly to FAs, confirming that FA attraction is mediated through the gustatory system. Furthermore, flies detect FAs independent of pH, suggesting the molecular basis for FA taste is not due to acidity. We show that low and medium concentrations of FAs serve as an appetitive signal and they are detected exclusively through the same subset of neurons that sense appetitive sweet substances, including most sugars. In mammals, taste perception of sweet and bitter substances is dependent on phospholipase C (PLC signaling in specialized taste buds. We find that flies mutant for norpA, a Drosophila ortholog of PLC, fail to respond to FAs. Intriguingly, norpA mutants respond normally to other tastants, including sucrose and yeast. The defect of norpA mutants can be rescued by selectively restoring norpA expression in sweet-sensing neurons, corroborating that FAs signal through sweet-sensing neurons, and suggesting PLC signaling in the gustatory system is specifically involved in FA taste. Taken together, these findings reveal that PLC function in Drosophila sweet-sensing neurons is a conserved molecular signaling pathway that confers attraction to fatty acids.

  4. Drosophila fatty acid taste signals through the PLC pathway in sugar-sensing neurons.

    Science.gov (United States)

    Masek, Pavel; Keene, Alex C

    2013-01-01

    Taste is the primary sensory system for detecting food quality and palatability. Drosophila detects five distinct taste modalities that include sweet, bitter, salt, water, and the taste of carbonation. Of these, sweet-sensing neurons appear to have utility for the detection of nutritionally rich food while bitter-sensing neurons signal toxicity and confer repulsion. Growing evidence in mammals suggests that taste for fatty acids (FAs) signals the presence of dietary lipids and promotes feeding. While flies appear to be attracted to fatty acids, the neural basis for fatty acid detection and attraction are unclear. Here, we demonstrate that a range of FAs are detected by the fly gustatory system and elicit a robust feeding response. Flies lacking olfactory organs respond robustly to FAs, confirming that FA attraction is mediated through the gustatory system. Furthermore, flies detect FAs independent of pH, suggesting the molecular basis for FA taste is not due to acidity. We show that low and medium concentrations of FAs serve as an appetitive signal and they are detected exclusively through the same subset of neurons that sense appetitive sweet substances, including most sugars. In mammals, taste perception of sweet and bitter substances is dependent on phospholipase C (PLC) signaling in specialized taste buds. We find that flies mutant for norpA, a Drosophila ortholog of PLC, fail to respond to FAs. Intriguingly, norpA mutants respond normally to other tastants, including sucrose and yeast. The defect of norpA mutants can be rescued by selectively restoring norpA expression in sweet-sensing neurons, corroborating that FAs signal through sweet-sensing neurons, and suggesting PLC signaling in the gustatory system is specifically involved in FA taste. Taken together, these findings reveal that PLC function in Drosophila sweet-sensing neurons is a conserved molecular signaling pathway that confers attraction to fatty acids.

  5. Myogenic and metabolic feedback in cerebral autoregulation: Putative involvement of arachidonic acid-dependent pathways.

    Science.gov (United States)

    Berg, Ronan M G

    2016-07-01

    The present paper presents a mechanistic model of cerebral autoregulation, in which the dual effects of the arachidonic acid metabolites 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) on vascular smooth muscle mediate the cerebrovascular adjustments to a change in cerebral perfusion pressure (CPP). 20-HETE signalling in vascular smooth muscle mediates myogenic feedback to changes in vessel wall stretch, which may be modulated by metabolic feedback through EETs released from astrocytes and endothelial cells in response to changes in brain tissue oxygen tension. The metabolic feedback pathway is much faster than 20-HETE-dependent myogenic feedback, and the former thus initiates the cerebral autoregulatory response, while myogenic feedback comprises a relatively slower mechanism that functions to set the basal cerebrovascular tone. Therefore, assessments of dynamic cerebral autoregulation, which may provide information on the response time of the cerebrovasculature, may specifically be used to yield information on metabolic feedback mechanisms, while data based on assessments of static cerebral autoregulation represent the integrated functionality of myogenic and metabolic feedback. PMID:27241246

  6. Clay minerals on Mars: Riotinto mining district (Huelva, Spain) as Earth analogue for acidic alteration pathways

    Science.gov (United States)

    Mavris, C.; Cuadros, J.; Bishop, J. L.; Nieto, J. M.; Michalski, J. R.

    2015-12-01

    Combined satellite and in-situ measurements of Mars surface have detected mineral assemblages indicating processes for which Earth analogues exist. Among them, aluminous clay-sulfate assemblages have been observed, which suggest alteration by acidic fluids. The Riotinto mining district (SW Spain) provides an Earth analogue site for such Martian processes. The parent rocks belong to an Upper Palaeozoic (Late Famennian-Tournaisian) volcano-sedimentary complex including siliciclastic sediments and mafic and felsic volcanics, all of which underwent hydrothermal alteration. The oxidation of an extensive pyrite-rich orebody provided mild to extreme acidic fluxes that leached the surrounding rocks for over 20 million years. The mineral assemblages are strongly dependent on their acidic alteration intensity. The observed mineralogical parageneses and leaching conditions for our sites at Riotinto are consistent with three alteration sequences: i) Mild: containing a range of clay minerals from vermiculite to kaolinite, with a wide variety of crystal order and mixed-layering; ii) Intermediate: containing smectite to kaolinite with jarosite-group phases; iii) Advanced: containing kaolinite, jarosite-group phases, and iron oxides. Our findings suggest that, even within this general scheme, the specific alteration pathways can be different.

  7. Palmitic acid interferes with energy metabolism balance by adversely switching the SIRT1-CD36-fatty acid pathway to the PKC zeta-GLUT4-glucose pathway in cardiomyoblasts.

    Science.gov (United States)

    Chen, Yeh-Peng; Tsai, Chia-Wen; Shen, Chia-Yao; Day, Cecilia-Hsuan; Yeh, Yu-Lan; Chen, Ray-Jade; Ho, Tsung-Jung; Padma, V Vijaya; Kuo, Wei-Wen; Huang, Chih-Yang

    2016-05-01

    Metabolic regulation is inextricably linked with cardiac function. Fatty acid metabolism is a significant mechanism for creating energy for the heart. However, cardiomyocytes are able to switch the fatty acids or glucose, depending on different situations, such as ischemia or anoxia. Lipotoxicity in obesity causes impairments in energy metabolism and apoptosis in cardiomyocytes. We utilized the treatment of H9c2 cardiomyoblast cells palmitic acid (PA) as a model for hyperlipidemia to investigate the signaling mechanisms involved in these processes. Our results show PA induces time- and dose-dependent lipotoxicity in H9c2 cells. Moreover, PA enhances cluster of differentiation 36 (CD36) and reduces glucose transporter type 4 (GLUT4) pathway protein levels following a short period of treatment, but cells switch from CD36 back to the GLUT4 pathway after during long-term exposure to PA. As sirtuin 1 (SIRT1) and protein kinase Cζ (PKCζ) play important roles in CD36 and GLUT4 translocation, we used the SIRT1 activator resveratrol and si-PKCζ to identify the switches in metabolism. Although PA reduced CD36 and increased GLUT4 metabolic pathway proteins, when we pretreated cells with resveratrol to activate SIRT1 or transfected si-PKCζ, both were able to significantly increase CD36 metabolic pathway proteins and reduce GLUT4 pathway proteins. High-fat diets affect energy metabolism pathways in both normal and aging rats and involve switching the energy source from the CD36 pathway to GLUT4. In conclusion, PA and high-fat diets cause lipotoxicity in vivo and in vitro and adversely switch the energy source from the CD36 pathway to the GLUT4 pathway. PMID:27133433

  8. All-trans retinoic acid modulates mitogen-activated protein kinase pathway activation in human scleral fibroblasts through retinoic acid receptor beta

    OpenAIRE

    Huo, Lijun; Cui, Dongmei; Yang, Xiao; Gao, Zhenya; Trier, Klaus; Zeng, Junwen

    2013-01-01

    Purpose All-trans retinoic acid (ATRA) is known to inhibit the proliferation of human scleral fibroblasts (HSFs) and to modulate the scleral intercellular matrix composition, and may therefore serve as a mediator for controlling eye growth. Cell proliferation is regulated by the mitogen-activated protein kinase (MAPK) pathway. The aim of the current study was to investigate whether changed activation of the MAPK pathway could be involved in the response of HSFs exposed to ATRA. Methods HSFs w...

  9. From carrot to clinic: an overview of the retinoic acid signaling pathway.

    Science.gov (United States)

    Theodosiou, Maria; Laudet, Vincent; Schubert, Michael

    2010-05-01

    Vitamin A is essential for the formation and maintenance of many body tissues. It is also important for embryonic growth and development and can act as a teratogen at critical periods of development. Retinoic acid (RA) is the biologically active form of vitamin A and its signaling is mediated by the RA and retinoid X receptors. In addition to its role as an important molecule during development, RA has also been implicated in clinical applications, both as a potential anti-tumor agent as well as for the treatment of skin diseases. This review presents an overview of how dietary retinoids are converted to RA, hence presenting the major players in RA metabolism and signaling, and highlights examples of treatment applications of retinoids. Moreover, we discuss the origin and diversification of the retinoid pathway, which are important factors for understanding the evolution of ligand-specificity among retinoid receptors. PMID:20140749

  10. Producing the Ethylene Signal: Regulation and Diversification of Ethylene Biosynthetic Enzymes.

    Science.gov (United States)

    Booker, Matthew A; DeLong, Alison

    2015-09-01

    Strictly controlled production of ethylene gas lies upstream of the signaling activities of this crucial regulator throughout the plant life cycle. Although the biosynthetic pathway is enzymatically simple, the regulatory circuits that modulate signal production are fine tuned to allow integration of responses to environmental and intrinsic cues. Recently identified posttranslational mechanisms that control ethylene production converge on one family of biosynthetic enzymes and overlay several independent reversible phosphorylation events and distinct mediators of ubiquitin-dependent protein degradation. Although the core pathway is conserved throughout seed plants, these posttranslational regulatory mechanisms may represent evolutionarily recent innovations. The evolutionary origins of the pathway and its regulators are not yet clear; outside the seed plants, numerous biochemical and phylogenetic questions remain to be addressed.

  11. Wnt/β-Catenin and Retinoic Acid Receptor Signaling Pathways Interact to Regulate Chondrocyte Function and Matrix Turnover*

    OpenAIRE

    Yasuhara, Rika; Yuasa, Takahito; Williams, Julie A.; Byers, Stephen W.; Shah, Salim; Pacifici, Maurizio; Iwamoto, Masahiro; Enomoto-Iwamoto, Motomi

    2009-01-01

    Activation of the Wnt/β-catenin and retinoid signaling pathways is known to tilt cartilage matrix homeostasis toward catabolism. Here, we investigated possible interactions between these pathways. We found that all-trans-retinoic acid (RA) treatment of mouse epiphyseal chondrocytes in culture did increase Wnt/β-catenin signaling in the absence or presence of exogenous Wnt3a, as revealed by lymphoid enhancer factor/T-cell factor/β-catenin reporter activity and β-catenin nuclear accumulation. T...

  12. Bardoxolone methyl (BARD) ameliorates aristolochic acid (AA)-induced acute kidney injury through Nrf2 pathway

    International Nuclear Information System (INIS)

    Bardoxolone methyl (BARD) is an antioxidant modulator that acts through induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. This study aimed to investigate the role of BARD in protecting kidneys from aristolochic acid (AA)-induced acute kidney injury (AKI). Male C57BL/6 mice received intraperitoneal (i.p.) injections of aristolochic acid I (AAI) (5 mg/kg/day) for 5 days to produce acute AA nephropathy (AAN) model. BARD (10 mg/kg/day, i.p.) was applied for 7 consecutive days, starting 2 days prior to AAI administration. The mice in the AA group showed AKI as evidenced by worsening kidney function evaluated by blood urea nitrogen (BUN) and serum creatinine (SCr) levels, and severe tubulointerstitial injury marked by massive tubule necrosis in kidney tissues. BARD significantly reduced BUN and SCr levels which were elevated by AAI. Additionally, AAI-induced histopathological renal damage was ameliorated by BARD. Furthermore, the expression of Nrf2 was reduced, and its repressor Kelch-like ECH-associated protein 1 (Keap1) was increased significantly, whereas heme oxygenase-1 (HO-1) was upregulated and NAD(P)H quinone oxidoreductase-1 (NQO1) was barely increased in the cytoplasm of tubules in kidneys after treatment with AAI. BARD significantly upregulated renal Nrf2, NQO1 and HO-1 expression and downregulated Keap1 expression compared with those in the AA group. Moreover, it was found that Nrf2 was expressed both in the cytoplasm and nuclear of glomeruli and tubules, whereas NQO1 and HO-1 were localized in the cytoplasm of tubules only. In conclusion, AA-induced acute renal injury was associated with impaired Nrf2 activation and expression of its downstream target genes in renal tissues. BARD prevented renal damage induced by AAI, and this renoprotective effect may be exerted by activating the Nrf2 signaling pathway and increasing expression of the downstream target genes

  13. 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.

  14. Hyaluronic acid fragments evoke Kupffer cells via TLR4 signaling pathway

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Kupffer cells, expressing toll-like receptor 4 (TLR4), play a central role in hepatic ischemia/reperfusion (I/R) injury. Hyaluronic acid (HA) fragments, degradative products of high-molecular-weight HA (HMW-HA), acquire the ability to activate immune cells under inflammatory conditions. Here we inves- tigated whether HA fragments could activate Kupffer cells and analyzed the underlying mechanism. Kupffer cells were isolated from wild-type mice (WT, C3H/HeN) and TLR4 mutant mice (C3H/HeJ) and HA fragments were produced by the methods of enzyme digestion and chromatography. Then Kupffer cells were stimulated by HA fragments or other control stimuli. The activation of Kupffer cells was estimated as the release of pro-inflammatory cytokines. The activation of p38 MAPK pathway of Kupffer cells was checked and blocking experiments were done as well. The results indicated that HA fragments acquired the ability to activate Kupffer cells in vitro, which was TLR4 dependent and not due to contamination of lipopolysaccharide. Experiments of p38 MAPK kinase inhibition by SB-203580 verified p38 MAPK was required in HA fragments induced Kupffer cells activation. This suggests that HA fragments, degradative products of one of the major glycosaminoglycans of the extracellular matrix, play critical roles in Kupffer cell activation mediated by TLR4 signaling pathway, which is, at least partially, de- pendent on p38 MAPK activation.

  15. Salvianolic Acid B Attenuates Experimental Pulmonary Fibrosis through Inhibition of the TGF-β Signaling Pathway.

    Science.gov (United States)

    Liu, Qingmei; Chu, Haiyan; Ma, Yanyun; Wu, Ting; Qian, Feng; Ren, Xian; Tu, Wenzhen; Zhou, Xiaodong; Jin, Li; Wu, Wenyu; Wang, Jiucun

    2016-01-01

    Pulmonary fibrosis is a progressive and fatal disorder. In our previous study, we found that the Yiqihuoxue formula (YQHX), a prescription of Traditional Chinese Medicine, had a curative effect on scleroderma, a typical fibrotic disease. The aim of this study was to determine the key ingredient mediating the therapeutic effects of YQHX and to examine its effect on pulmonary fibrosis, including its mechanism. Luciferase reporter assays showed that the most important anti-fibrotic component of the YQHX was Salviae miltiorrhiza (SM). Experiments performed using a bleomycin-instilled mouse model of pulmonary fibrosis showed that Salvianolic acid B (SAB), the major ingredient of SM, had strong anti-inflammatory and anti-fibrotic effects through its inhibition of inflammatory cell infiltration, alveolar structure disruption, and collagen deposition. Furthermore, SAB suppressed TGF-β-induced myofibroblastic differentiation of MRC-5 fibroblasts and TGF-β-mediated epithelial-to-mesenchymal transition of A549 cells by inhibiting both Smad-dependent signaling and the Smad-independent MAPK pathway. Taken together, our results suggest that SM is the key anti-fibrotic component of the YQHX and that SAB, the major ingredient of SM, alleviates experimental pulmonary fibrosis both in vivo and in vitro by inhibiting the TGF-β signaling pathway. Together, these results suggest that SAB potently inhibits pulmonary fibrosis. PMID:27278104

  16. Salvianolic Acid B Attenuates Experimental Pulmonary Fibrosis through Inhibition of the TGF-β Signaling Pathway

    Science.gov (United States)

    Liu, Qingmei; Chu, Haiyan; Ma, Yanyun; Wu, Ting; Qian, Feng; Ren, Xian; Tu, Wenzhen; Zhou, Xiaodong; Jin, Li; Wu, Wenyu; Wang, Jiucun

    2016-01-01

    Pulmonary fibrosis is a progressive and fatal disorder. In our previous study, we found that the Yiqihuoxue formula (YQHX), a prescription of Traditional Chinese Medicine, had a curative effect on scleroderma, a typical fibrotic disease. The aim of this study was to determine the key ingredient mediating the therapeutic effects of YQHX and to examine its effect on pulmonary fibrosis, including its mechanism. Luciferase reporter assays showed that the most important anti-fibrotic component of the YQHX was Salviae miltiorrhiza (SM). Experiments performed using a bleomycin-instilled mouse model of pulmonary fibrosis showed that Salvianolic acid B (SAB), the major ingredient of SM, had strong anti-inflammatory and anti-fibrotic effects through its inhibition of inflammatory cell infiltration, alveolar structure disruption, and collagen deposition. Furthermore, SAB suppressed TGF-β-induced myofibroblastic differentiation of MRC-5 fibroblasts and TGF-β-mediated epithelial-to-mesenchymal transition of A549 cells by inhibiting both Smad-dependent signaling and the Smad-independent MAPK pathway. Taken together, our results suggest that SM is the key anti-fibrotic component of the YQHX and that SAB, the major ingredient of SM, alleviates experimental pulmonary fibrosis both in vivo and in vitro by inhibiting the TGF-β signaling pathway. Together, these results suggest that SAB potently inhibits pulmonary fibrosis. PMID:27278104

  17. 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.

  18. Amino acids attenuate insulin action on gluconeogenesis and promote fatty acid biosynthesis via mTORC1 signaling pathway in trout hepatocytes

    OpenAIRE

    Dai, Wei Wei; Panserat, Stephane; Plagnes- Juan, Elisabeth; Seiliez, Iban; Skiba-Cassy, Sandrine

    2015-01-01

    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 emplo...

  19. Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens.

    Science.gov (United States)

    Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin; Simonsen, Henrik Toft; Hamberger, Björn

    2014-01-01

    Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has already been widely recognized as a viable alternative for industrial-scale production of biopharmaceuticals. For expression of terpenoid biosynthetic genes, and reconstruction of heterologous pathways, Physcomitrella has unique attributes that makes it a very promising biotechnological host. These features include a high native tolerance to terpenoids, a simple endogenous terpenoid profile, convenient genome editing using homologous recombination, and cultivation techniques that allow up-scaling from single cells in microtiter plates to industrial photo-bioreactors. Beyond its use for functional characterization of terpenoid biosynthetic genes, engineered Physcomitrella can be a green biotechnological platform for production of terpenoids. Here, we describe two complementary and simple procedures for stable nuclear transformation of Physcomitrella with terpenoid biosynthetic genes, selection and cultivation of transgenic lines, and metabolite analysis of terpenoids produced in transgenic moss lines. We also provide tools for metabolic engineering through genome editing using homologous recombination. PMID:24777804

  20. Circadian and Dopaminergic Regulation of Fatty Acid Oxidation Pathway Genes in Retina and Photoreceptor Cells

    Science.gov (United States)

    Vancura, Patrick; Wolloscheck, Tanja; Baba, Kenkichi; Tosini, Gianluca; Iuvone, P. Michael; Spessert, Rainer

    2016-01-01

    The energy metabolism of the retina might comply with daily changes in energy demand and is impaired in diabetic retinopathy—one of the most common causes of blindness in Europe and the USA. The aim of this study was to investigate putative adaptation of energy metabolism in healthy and diabetic retina. Hence expression analysis of metabolic pathway genes was performed using quantitative polymerase chain reaction, semi-quantitative western blot and immunohistochemistry. Transcriptional profiling of key enzymes of energy metabolism identified transcripts of mitochondrial fatty acid β-oxidation enzymes, i.e. carnitine palmitoyltransferase-1α (Cpt-1α) and medium chain acyl-CoA dehydrogenase (Acadm) to display daily rhythms with peak values during daytime in preparations of the whole retina and microdissected photoreceptors. The cycling of both enzymes persisted in constant darkness, was dampened in mice deficient for dopamine D4 (D4) receptors and was altered in db/db mice—a model of diabetic retinopathy. The data of the present study are consistent with circadian clock-dependent and dopaminergic regulation of fatty acid oxidation in retina and its putative disturbance in diabetic retina. PMID:27727308

  1. Ginkgolic acid suppresses the development of pancreatic cancer by inhibiting pathways driving lipogenesis.

    Science.gov (United States)

    Ma, Jiguang; Duan, Wanxing; Han, Suxia; Lei, Jianjun; Xu, Qinhong; Chen, Xin; Jiang, Zhengdong; Nan, Ligang; Li, Jiahui; Chen, Ke; Han, Liang; Wang, Zheng; Li, Xuqi; Wu, Erxi; Huo, Xiongwei

    2015-08-28

    Ginkgolic acid (GA) is a botanical drug extracted from the seed coat of Ginkgo biloba L. with a wide range of bioactive properties, including anti-tumor effect. However, whether GA has antitumor effect on pancreatic cancer cells and the underlying mechanisms have yet to be investigated. In this study, we show that GA suppressed the viability of cancer cells but has little toxicity on normal cells, e.g, HUVEC cells. Furthermore, treatment of GA resulted in impaired colony formation, migration, and invasion ability and increased apoptosis of cancer cells. In addition, GA inhibited the de novo lipogenesis of cancer cells through inducing activation of AMP-activated protein kinase (AMPK) signaling and downregulated the expression of key enzymes (e.g. acetyl-CoA carboxylase [ACC], fatty acid synthase [FASN]) involved in lipogenesis. Moreover, the in vivo experiment showed that GA reduced the expression of the key enzymes involved in lipogenesis and restrained the tumor growth. Taken together, our results suggest that GA may serve as a new candidate against tumor growth of pancreatic cancer partially through targeting pathway driving lipogenesis. PMID:25895130

  2. Investigating sources and pathways of perfluoroalkyl acids (PFAAs) in aquifers in Tokyo using multiple tracers

    International Nuclear Information System (INIS)

    We employed a multi-tracer approach to investigate sources and pathways of perfluoroalkyl acids (PFAAs) in urban groundwater, based on 53 groundwater samples taken from confined aquifers and unconfined aquifers in Tokyo. While the median concentrations of groundwater PFAAs were several ng/L, the maximum concentrations of perfluorooctane sulfonate (PFOS, 990 ng/L), perfluorooctanoate (PFOA, 1800 ng/L) and perfluorononanoate (PFNA, 620 ng/L) in groundwater were several times higher than those of wastewater and street runoff reported in the literature. PFAAs were more frequently detected than sewage tracers (carbamazepine and crotamiton), presumably owing to the higher persistence of PFAAs, the multiple sources of PFAAs beyond sewage (e.g., surface runoff, point sources) and the formation of PFAAs from their precursors. Use of multiple methods of source apportionment including principal component analysis–multiple linear regression (PCA–MLR) and perfluoroalkyl carboxylic acid ratio analysis highlighted sewage and point sources as the primary sources of PFAAs in the most severely polluted groundwater samples, with street runoff being a minor source (44.6% sewage, 45.7% point sources and 9.7% street runoff, by PCA–MLR). Tritium analysis indicated that, while young groundwater (recharged during or after the 1970s, when PFAAs were already in commercial use) in shallow aquifers (< 50 m depth) was naturally highly vulnerable to PFAA pollution, PFAAs were also found in old groundwater (recharged before the 1950s, when PFAAs were not in use) in deep aquifers (50–500 m depth). This study demonstrated the utility of multiple uses of tracers (pharmaceuticals and personal care products; PPCPs, tritium) and source apportionment methods in investigating sources and pathways of PFAAs in multiple aquifer systems. - Highlights: • Aquifers in Tokyo had high levels of perfluoroalkyl acids (up to 1800 ng/L). • PFAAs were more frequently detected than sewage

  3. Investigating sources and pathways of perfluoroalkyl acids (PFAAs) in aquifers in Tokyo using multiple tracers

    Energy Technology Data Exchange (ETDEWEB)

    Kuroda, Keisuke, E-mail: keisukekr@gmail.com [Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656 (Japan); Murakami, Michio [Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8505 (Japan); Oguma, Kumiko [Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656 (Japan); Takada, Hideshige [Laboratory of Organic Geochemistry (LOG), Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509 (Japan); Takizawa, Satoshi [Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656 (Japan)

    2014-08-01

    We employed a multi-tracer approach to investigate sources and pathways of perfluoroalkyl acids (PFAAs) in urban groundwater, based on 53 groundwater samples taken from confined aquifers and unconfined aquifers in Tokyo. While the median concentrations of groundwater PFAAs were several ng/L, the maximum concentrations of perfluorooctane sulfonate (PFOS, 990 ng/L), perfluorooctanoate (PFOA, 1800 ng/L) and perfluorononanoate (PFNA, 620 ng/L) in groundwater were several times higher than those of wastewater and street runoff reported in the literature. PFAAs were more frequently detected than sewage tracers (carbamazepine and crotamiton), presumably owing to the higher persistence of PFAAs, the multiple sources of PFAAs beyond sewage (e.g., surface runoff, point sources) and the formation of PFAAs from their precursors. Use of multiple methods of source apportionment including principal component analysis–multiple linear regression (PCA–MLR) and perfluoroalkyl carboxylic acid ratio analysis highlighted sewage and point sources as the primary sources of PFAAs in the most severely polluted groundwater samples, with street runoff being a minor source (44.6% sewage, 45.7% point sources and 9.7% street runoff, by PCA–MLR). Tritium analysis indicated that, while young groundwater (recharged during or after the 1970s, when PFAAs were already in commercial use) in shallow aquifers (< 50 m depth) was naturally highly vulnerable to PFAA pollution, PFAAs were also found in old groundwater (recharged before the 1950s, when PFAAs were not in use) in deep aquifers (50–500 m depth). This study demonstrated the utility of multiple uses of tracers (pharmaceuticals and personal care products; PPCPs, tritium) and source apportionment methods in investigating sources and pathways of PFAAs in multiple aquifer systems. - Highlights: • Aquifers in Tokyo had high levels of perfluoroalkyl acids (up to 1800 ng/L). • PFAAs were more frequently detected than sewage

  4. Dietary omega-3 fatty acids modulate the eicosanoid profile in man primarily via the CYP-epoxygenase pathway[S

    OpenAIRE

    Fischer, Robert; Konkel, Anne; Mehling, Heidrun; Blossey, Katrin; Gapelyuk, Andrej; Wessel, Niels; von Schacky, Clemens; Dechend, Ralf; Muller, Dominik N.; Rothe, Michael; Luft, Friedrich C.; Weylandt, Karsten; Schunck, Wolf-Hagen

    2014-01-01

    Cytochrome P450 (CYP)-dependent metabolites of arachidonic acid (AA) contribute to the regulation of cardiovascular function. CYP enzymes also accept EPA and DHA to yield more potent vasodilatory and potentially anti-arrhythmic metabolites, suggesting that the endogenous CYP-eicosanoid profile can be favorably shifted by dietary omega-3 fatty acids. To test this hypothesis, 20 healthy volunteers were treated with an EPA/DHA supplement and analyzed for concomitant changes in the circulatory an...

  5. Aromatic amino acid activation of signaling pathways in bone marrow mesenchymal stem cells depends on oxygen tension.

    Directory of Open Access Journals (Sweden)

    Mona El Refaey

    Full Text Available The physiologic oxygen pressures inside the bone marrow environment are much lower than what is present in the peripheral circulation, ranging from 1-7%, compared to values as high as 10-13% in the arteries, lungs and liver. Thus, experiments done with bone marrow mesenchymal stem cells (BMMSCs using standard culture conditions may not accurately reflect the true hypoxic bone marrow microenvironment. However, since aging is associated with an increased generation of reactive oxygen species, experiments done under 21%O2 conditions may actually more closely resemble that of the aging bone marrow environment. Aromatic amino acids are known to be natural anti-oxidants. We have previously reported that aromatic amino acids are potent agonists for stimulating increases in intracellular calcium and phospho-c-Raf and in promoting BMMSC differentiation down the osteogenic pathway. Our previous experiments were performed under normoxic conditions. Thus, we next decided to compare a normoxic (21% O2 vs. a hypoxic environment (3% O2 alone or after treatment with aromatic amino acids. Reverse-phase protein arrays showed that 3% O2 itself up-regulated proliferative pathways. Aromatic amino acids had no additional effect on signaling pathways under these conditions. However, under 21%O2 conditions, aromatic amino acids could now significantly increase these proliferative pathways over this "normoxic" baseline. Pharmacologic studies are consistent with the aromatic amino acids activating the extracellular calcium-sensing receptor. The effects of aromatic amino acids on BMMSC function in the 21% O2 environment is consistent with a potential role for these amino acids in an aging environment as functional anti oxidants.

  6. Metabolic pathways regulated by γ-aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass (Agrostis stolonifera).

    Science.gov (United States)

    Li, Zhou; Yu, Jingjin; Peng, Yan; Huang, Bingru

    2016-07-26

    γ-Aminobutyric acid is a non-protein amino acid involved in various metabolic processes. The objectives of this study were to examine whether increased GABA could improve heat tolerance in cool-season creeping bentgrass through physiological analysis, and to determine major metabolic pathways regulated by GABA through metabolic profiling. Plants were pretreated with 0.5 mM GABA or water before exposed to non-stressed condition (21/19 °C) or heat stress (35/30 °C) in controlled growth chambers for 35 d. The growth and physiological analysis demonstrated that exogenous GABA application significantly improved heat tolerance of creeping bentgrass. Metabolic profiling found that exogenous application of GABA led to increases in accumulations of amino acids (glutamic acid, aspartic acid, alanine, threonine, serine, and valine), organic acids (aconitic acid, malic acid, succinic acid, oxalic acid, and threonic acid), sugars (sucrose, fructose, glucose, galactose, and maltose), and sugar alcohols (mannitol and myo-inositol). These findings suggest that GABA-induced heat tolerance in creeping bentgrass could involve the enhancement of photosynthesis and ascorbate-glutathione cycle, the maintenance of osmotic adjustment, and the increase in GABA shunt. The increased GABA shunt could be the supply of intermediates to feed the tricarboxylic acid cycle of respiration metabolism during a long-term heat stress, thereby maintaining metabolic homeostasis.

  7. Metabolic pathways regulated by γ-aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass (Agrostis stolonifera).

    Science.gov (United States)

    Li, Zhou; Yu, Jingjin; Peng, Yan; Huang, Bingru

    2016-01-01

    γ-Aminobutyric acid is a non-protein amino acid involved in various metabolic processes. The objectives of this study were to examine whether increased GABA could improve heat tolerance in cool-season creeping bentgrass through physiological analysis, and to determine major metabolic pathways regulated by GABA through metabolic profiling. Plants were pretreated with 0.5 mM GABA or water before exposed to non-stressed condition (21/19 °C) or heat stress (35/30 °C) in controlled growth chambers for 35 d. The growth and physiological analysis demonstrated that exogenous GABA application significantly improved heat tolerance of creeping bentgrass. Metabolic profiling found that exogenous application of GABA led to increases in accumulations of amino acids (glutamic acid, aspartic acid, alanine, threonine, serine, and valine), organic acids (aconitic acid, malic acid, succinic acid, oxalic acid, and threonic acid), sugars (sucrose, fructose, glucose, galactose, and maltose), and sugar alcohols (mannitol and myo-inositol). These findings suggest that GABA-induced heat tolerance in creeping bentgrass could involve the enhancement of photosynthesis and ascorbate-glutathione cycle, the maintenance of osmotic adjustment, and the increase in GABA shunt. The increased GABA shunt could be the supply of intermediates to feed the tricarboxylic acid cycle of respiration metabolism during a long-term heat stress, thereby maintaining metabolic homeostasis. PMID:27455877

  8. In silico tools for the analysis of antibiotic biosynthetic pathways

    DEFF Research Database (Denmark)

    Weber, Tilmann

    2014-01-01

    Natural products of bacteria and fungi are the most important source for antimicrobial drug leads. For decades, such compounds were exclusively found by chemical/bioactivity-guided screening approaches. The rapid progress in sequencing technologies only recently allowed the development of novel s...... and tools are crucial for genome mining. In this review, a comprehensive overview is given on programs and databases for the identification and analysis of antibiotic biosynthesis gene clusters in genomic data....

  9. Fusarium Mycotoxins: Biosynthetic Pathways and Role in Virulence

    Science.gov (United States)

    Fusarium head blight (FHB) of wheat and barley is a devastating disease that has reached global proportions. Not only does this disease result in lower yields, but the mycotoxins produced by the fungus affect the quality of the grain. Fusarium sp. can produce a number of mycotoxins, including tric...

  10. Up-regulation of abscisic acid signaling pathway facilitates aphid xylem absorption and osmoregulation under drought stress.

    Science.gov (United States)

    Guo, Huijuan; Sun, Yucheng; Peng, Xinhong; Wang, Qinyang; Harris, Marvin; Ge, Feng

    2016-02-01

    The activation of the abscisic acid (ABA) signaling pathway reduces water loss from plants challenged by drought stress. The effect of drought-induced ABA signaling on the defense and nutrition allocation of plants is largely unknown. We postulated that these changes can affect herbivorous insects. We studied the effects of drought on different feeding stages of pea aphids in the wild-type A17 of Medicago truncatula and ABA signaling pathway mutant sta-1. We examined the impact of drought on plant water status, induced plant defense signaling via the abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) pathways, and on the host nutritional quality in terms of leaf free amino acid content. During the penetration phase of aphid feeding, drought decreased epidermis/mesophyll resistance but increased mesophyll/phloem resistance of A17 but not sta-1 plants. Quantification of transcripts associated with ABA, JA and SA signaling indicated that the drought-induced up-regulation of ABA signaling decreased the SA-dependent defense but increased the JA-dependent defense in A17 plants. During the phloem-feeding phase, drought had little effect on the amino acid concentrations and the associated aphid phloem-feeding parameters in both plant genotypes. In the xylem absorption stage, drought decreased xylem absorption time of aphids in both genotypes because of decreased water potential. Nevertheless, the activation of the ABA signaling pathway increased water-use efficiency of A17 plants by decreasing the stomatal aperture and transpiration rate. In contrast, the water potential of sta-1 plants (unable to close stomata) was too low to support xylem absorption activity of aphids; the aphids on sta-1 plants had the highest hemolymph osmolarity and lowest abundance under drought conditions. Taken together this study illustrates the significance of cross-talk between biotic-abiotic signaling pathways in plant-aphid interaction, and reveals the mechanisms leading to alter

  11. Involvement of a Natural Fusion of a Cytochrome P450 and a Hydrolase in Mycophenolic Acid Biosynthesis

    OpenAIRE

    Hansen, Bjarne Gram; Mnich, Ewelina; Nielsen, Kristian Fog; Nielsen, Jakob Blæsbjerg; Nielsen, Morten Thrane; Mortensen, Uffe Hasbro; Larsen, Thomas Ostenfeld; Patil, Kiran Raosaheb

    2012-01-01

    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 MpaC, 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 bioi...

  12. Identification of the Fucose Synthetase Gene in the Colanic Acid Gene Cluster of Escherichia coli K-12

    OpenAIRE

    Andrianopoulos, Kanella; WANG Lei; Reeves, Peter R.

    1998-01-01

    GDP–l-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP–d-mannose. l-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains. We previously sequenced the E. coli colanic acid gene cluster and identified one of the GDP–l-fucose biosynthetic pathway genes, gmd. We report here the identification ...

  13. Cloning and Expression Analysis of a Brassinosteroid Biosynthetic Enzyme Gene, GhDWF1, from Cotton (Gossypium hirsuturm L.)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Brassinosteroids (BRs) are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. To determine the effects of BRs on the development of cotton fibers, through screening cotton fiber EST database and contigging the candidate ESTs, a key gene (GhDWF1) involved in the upstream biosynthetic pathway of BRs was cloned from developing fibers of upland cotton (Gossypium hirsutum L.) cv. Xuzhou 142. The full length of the cloned cDNA is 1 849 bp, including a 37 bp 5'-untranslated region, an ORF of 1692 bp, and a 120 bp 3'-untranslated region.The cDNA encodes a polypeptide of 563 amino acid residues with a predicted molecular mass of 65 kD. The deduced amino acid sequence has high homology with the BR biosynthetic enzyme, DWARF1/DIMINUTO, from rice, maize, pea,tomato, and Arabidopsis. Furthermore, the typical conserved structures, such as the transmembrane domain, the FAD-dependent oxidase domain, and the FAD-binding site, are present in the GhDWF1 protein. The Southern blot indicated that the GhDWF1 gene is a single copy in upland cotton genome. RT-PCR analysis revealed that the highest level of GhDWF1 expression was detected in 0 DPA (day post anthesis) ovule (with fibers) while the lowest level was observed in cotyledon. The GhDWF1 gene presents high expression levels in root, young stem, and fiber, especially, at the fiber developmental stage of secondary cell wall accumulation. Moreover, the expression level was higher in ovules (with fibers) of wildtype (Xuzhou 142) than in ovules of fuzzless-lintless mutant at the same developmental stages (0 and 4 DPA). The results suggest that the GhDWF1 gene plays a crucial role in fiber development.

  14. Biosynthesis of resorcylic acid lactone lasiodiplodin in Lasiodiplodia theobromae.

    Science.gov (United States)

    Kashima, Takasumi; Takahashi, Kosaku; Matsuura, Hideyuki; Nabeta, Kensuke

    2009-05-01

    The biosynthesis of lasiodiplodin (1) and its (5S)-5-hydroxylated derivative (2) were investigated by the administration of (13)C-labeled acetates to Lasiodiplodia theobromae. The labeling patterns of biosynthetically (13)C-labeled 1 and 2 were determined by (13)C-NMR and INADEQUATE spectra, demonstrating the octaketide origins of 1 and 2. Taking into account the biosynthetic study of resorcylic acid lactones, the involvement of highly reduced acyl intermediates in the biosynthesis of lasiodiplodins was presumed; thus, we synthesized (2)H-labeled hypothetical acyl intermediates of 1, 9-hydroxydecanoic acid (4) and its N-acetylcysteamine thioester (SNAC, 5). When L. theobromae was incubated with 5 mM of a (2)H-labeled intermediate, the (2)H-label from the intermediate was incorporated at the expected position of 1. These incorporation studies revealed that 1 was produced via a pathway which closely resembles that of resorcylic acid lactone biosynthesis. PMID:19420710

  15. CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes.

    Science.gov (United States)

    Li, Chun Yao; Leopold, Alex L; Sander, Guy W; Shanks, Jacqueline V; Zhao, Le; Gibson, Susan I

    2015-01-01

    Terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Toward this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression of CrBPF1 causes increases in the transcript levels for 11 out of 13 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a "fine-tune" regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression. PMID:26483828

  16. CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes

    Directory of Open Access Journals (Sweden)

    Chun Yao eLi

    2015-10-01

    Full Text Available Terpenoid indole alkaloid (TIA biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Towards this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression of CrBPF1 causes increases in the transcript levels for 11 out of 13 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a fine-tune regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression.

  17. The influence of alternative pathways of respiration that utilize branched-chain amino acids following water shortage in Arabidopsis.

    Science.gov (United States)

    Pires, Marcel V; Pereira Júnior, Adilson A; Medeiros, David B; Daloso, Danilo M; Pham, Phuong Anh; Barros, Kallyne A; Engqvist, Martin K M; Florian, Alexandra; Krahnert, Ina; Maurino, Veronica G; Araújo, Wagner L; Fernie, Alisdair R

    2016-06-01

    During dark-induced senescence isovaleryl-CoA dehydrogenase (IVDH) and D-2-hydroxyglutarate dehydrogenase (D-2HGDH) act as alternate electron donors to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) pathway. However, the role of this pathway in response to other stresses still remains unclear. Here, we demonstrated that this alternative pathway is associated with tolerance to drought in Arabidopsis. In comparison with wild type (WT) and lines overexpressing D-2GHDH, loss-of-function etfqo-1, d2hgdh-2 and ivdh-1 mutants displayed compromised respiration rates and were more sensitive to drought. Our results demonstrated that an operational ETF/ETFQO pathway is associated with plants' ability to withstand drought and to recover growth once water becomes replete. Drought-induced metabolic reprogramming resulted in an increase in tricarboxylic acid (TCA) cycle intermediates and total amino acid levels, as well as decreases in protein, starch and nitrate contents. The enhanced levels of the branched-chain amino acids in loss-of-function mutants appear to be related to their increased utilization as substrates for the TCA cycle under water stress. Our results thus show that mitochondrial metabolism is highly active during drought stress responses and provide support for a role of alternative respiratory pathways within this response.

  18. Identification and analysis of the paulomycin biosynthetic gene cluster and titer improvement of the paulomycins in Streptomyces paulus NRRL 8115.

    Directory of Open Access Journals (Sweden)

    Jine Li

    Full Text Available The paulomycins are a group of glycosylated compounds featuring a unique paulic acid moiety. To locate their biosynthetic gene clusters, the genomes of two paulomycin producers, Streptomyces paulus NRRL 8115 and Streptomyces sp. YN86, were sequenced. The paulomycin biosynthetic gene clusters were defined by comparative analyses of the two genomes together with the genome of the third paulomycin producer Streptomyces albus J1074. Subsequently, the identity of the paulomycin biosynthetic gene cluster was confirmed by inactivation of two genes involved in biosynthesis of the paulomycose branched chain (pau11 and the ring A moiety (pau18 in Streptomyces paulus NRRL 8115. After determining the gene cluster boundaries, a convergent biosynthetic model was proposed for paulomycin based on the deduced functions of the pau genes. Finally, a paulomycin high-producing strain was constructed by expressing an activator-encoding gene (pau13 in S. paulus, setting the stage for future investigations.

  19. Excretion pathways and ruminal disappearance of glyphosate and its degradation product aminomethylphosphonic acid in dairy cows.

    Science.gov (United States)

    von Soosten, D; Meyer, U; Hüther, L; Dänicke, S; Lahrssen-Wiederholt, M; Schafft, H; Spolders, M; Breves, G

    2016-07-01

    From 6 balance experiments with total collection of feces and urine, samples were obtained to investigate the excretion pathways of glyphosate (GLY) in lactating dairy cows. Each experiment lasted for 26d. The first 21d served for adaptation to the diet, and during the remaining 5d collection of total feces and urine was conducted. Dry matter intake and milk yield were recorded daily and milk and feed samples were taken during the sampling periods. In 2 of the 6 experiments, at the sampling period for feces and urine, duodenal contents were collected for 5d. Cows were equipped with cannulas at the dorsal sac of the rumen and the proximal duodenum. Duodenal contents were collected every 2h over 5 consecutive days. The daily duodenal dry matter flow was measured by using chromium oxide as a volume marker. All samples (feed, feces, urine, milk and duodenal contents were analyzed for GLY and aminomethylphosphonic acid (AMPA). Overall, across the 6 experiments (n=32) the range of GLY intake was 0.08 to 6.67mg/d. The main proportion (61±11%; ±SD) of consumed GLY was excreted with feces; whereas excretion by urine was 8±3% of GLY intake. Elimination via milk was negligible. The GLY concentrations above the limit of quantification were not detected in any of the milk samples. A potential ruminal degradation of GLY to AMPA was derived from daily duodenal GLY flow. The apparent ruminal disappearance of GLY intake was 36 and 6%. In conclusion, the results of the present study indicate that the gastrointestinal absorption of GLY is of minor importance and fecal excretion represents the major excretion pathway. A degradation of GLY to AMPA by rumen microbes or a possible retention in the body has to be taken into account. PMID:27108173

  20. Amino Acid Deprivation Promotes Tumor Angiogenesis through the GCN2/ATF4 Pathway

    Directory of Open Access Journals (Sweden)

    Yugang Wang

    2013-08-01

    Full Text Available As tumors continue to grow and exceed their blood supply, nutrients become limited leading to deficiencies in amino acids (AAD, glucose (GD, and oxygen (hypoxia. These alterations result in significant changes in gene expression. While tumors have been shown to overcome the stress associated with GD or hypoxia by stimulating vascular endothelial growth factor (VEGF-mediated angiogenesis, the role of AAD in tumor angiogenesis remains to be elucidated. We found that in human tumors, the expression of the general control non-derepressible 2 (GCN2, an AAD sensor kinase is elevated at both protein and mRNA levels. In vitro studies revealed that VEGF expression is universally induced by AAD treatment in all five cell lines tested (five of five. This is in contrast to two other angiogenesis mediators interleukin-6 (two of five and fibroblast growth factor 2 (two of five that have a more restricted expression. Suppressing GCN2 expression significantly decreased AAD-induced VEGF expression. Silencing activating transcription factor 4 (ATF4, a downstream transcription factor of the GCN2 signaling pathway, is also associated with strong inhibition of AAD-induced VEGF expression. PKR-like kinase, the key player in GD-induced unfolded protein response is not involved in this process. In vivo xenograft tumor studies in nonobese diabetic/severe combined immunodeficient mice confirmed that knockdown of GCN2 in tumor cells retards tumor growth and decreases tumor blood vessel density. Our results reveal that the GCN2/ATF4 pathway promotes tumor growth and angiogenesis through AAD-mediated VEGF expression and, thus, is a potential target in cancer therapy.

  1. Polymorphisms in the genes involved in the arachidonic acid-pathway, fish consumption and the risk of colorectal cancer.

    NARCIS (Netherlands)

    Siezen, Christine L E; Bueno-de-Mesquita, H Bas; Peeters, Petra H M; Kram, Nicolien R; Doeselaar, Marina van; Kranen, Henk J van

    2006-01-01

    The objective of this study on colorectal cancer was to investigate the associations between SNPs in the genes involved in the arachidonic acid (AA)-pathway, their haplotypes and colorectal cancer. Moreover, interactions between SNPs and fish consumption were considered. In this study, a total of 50

  2. Anodic coupling of carboxylic acids to electron-rich double bonds: A surprising non-Kolbe pathway to lactones

    Directory of Open Access Journals (Sweden)

    Robert J. Perkins

    2013-08-01

    Full Text Available Carboxylic acids have been electro-oxidatively coupled to electron-rich olefins to form lactones. Kolbe decarboxylation does not appear to be a significant competing pathway. Experimental results indicate that oxidation occurs at the olefin and that the reaction proceeds through a radical cation intermediate.

  3. Colorectal adenoma risk is modified by the interplay between polymorphisms in arachidonic acid pathway genes and fish consumption.

    NARCIS (Netherlands)

    Siezen, C.L.; Leeuwen, A.I. van; Kram, N.R.; Luken, M.E.; Kranen, H.J. van; Kampman, E.

    2005-01-01

    Associations between polymorphisms in genes (SNPs) involved in the arachidonic acid (AA) pathway and colorectal adenomas have been investigated in a Dutch case control study including 384 cases and 403 polyp-free controls. Twenty-one polymorphisms in seven candidate genes were studied and a potentia

  4. Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

    Directory of Open Access Journals (Sweden)

    Jungsuwadee Paiboon

    2011-02-01

    Full Text Available Abstract Background Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. Results A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC and Adenosine Triphosphate Binding Cassette (ABC superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175. Conclusions We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and

  5. Biochemical and Structural Studies of NADH-Dependent FabG Used To Increase the Bacterial Production of Fatty Acids under Anaerobic Conditions

    OpenAIRE

    Javidpour, Pouya; Pereira, Jose H.; Goh, Ee-Been; McAndrew, Ryan P.; Ma, Suzanne M.; Friedland, Gregory D.; Keasling, Jay D.; Chhabra, Swapnil R; Adams, Paul D.; Beller, Harry R.

    2014-01-01

    Major efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the propo...

  6. Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway1[OPEN

    Science.gov (United States)

    Légeret, Bertrand; Mirabella, Boris; Guédeney, Geneviève; Jetter, Reinhard; Peltier, Gilles

    2016-01-01

    Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway. PMID:27288359

  7. Engineering the Saccharomyces cerevisiae β-oxidation pathway to increase medium chain fatty acid production as potential biofuel.

    Directory of Open Access Journals (Sweden)

    Liwei Chen

    Full Text Available Fatty acid-derived biofuels and biochemicals can be produced in microbes using β-oxidation pathway engineering. In this study, the β-oxidation pathway of Saccharomyces cerevisiae was engineered to accumulate a higher ratio of medium chain fatty acids (MCFAs when cells were grown on fatty acid-rich feedstock. For this purpose, the haploid deletion strain Δpox1 was obtained, in which the sole acyl-CoA oxidase encoded by POX1 was deleted. Next, the POX2 gene from Yarrowia lipolytica, which encodes an acyl-CoA oxidase with a preference for long chain acyl-CoAs, was expressed in the Δpox1 strain. The resulting Δpox1 [pox2+] strain exhibited a growth defect because the β-oxidation pathway was blocked in peroxisomes. To unblock the β-oxidation pathway, the gene CROT, which encodes carnitine O-octanoyltransferase, was expressed in the Δpox1 [pox2+] strain to transport the accumulated medium chain acyl-coAs out of the peroxisomes. The obtained Δpox1 [pox2+, crot+] strain grew at a normal rate. The effect of these genetic modifications on fatty acid accumulation and profile was investigated when the strains were grown on oleic acids-containing medium. It was determined that the engineered strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] had increased fatty acid accumulation and an increased ratio of MCFAs. Compared to the wild-type (WT strain, the total fatty acid production of the strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] were increased 29.5% and 15.6%, respectively. The intracellular level of MCFAs in Δpox1 [pox2+] and Δpox1 [pox2+, crot+] increased 2.26- and 1.87-fold compared to the WT strain, respectively. In addition, MCFAs in the culture medium increased 3.29-fold and 3.34-fold compared to the WT strain. These results suggested that fatty acids with an increased MCFAs ratio accumulate in the engineered strains with a modified β-oxidation pathway. Our approach exhibits great potential for transforming low value fatty acid

  8. SELECTIVE SEPARATION OF BIOSYNTHETIC PRODUCTS BY PERTRACTION - CHALLENGE FOR THE “WHITE BIOTECHNOLOGY”

    Directory of Open Access Journals (Sweden)

    Dan Cascaval

    2010-04-01

    Full Text Available This review presents our original results on selective separation of some biosynthetic products (antibiotics, carboxylic acids, amino acids by free or facilitated pertraction (extraction and transport through liquid membranes. Selecting the optimum conditions, for all studied cases these pertraction technique simplify the technologies applied at industrial scale for separation and purification, allows to reaching high selectivity and reducing the overall cost of the products.

  9. E-2-hexenal promotes susceptibility to Pseudomonas syringae by activating jasmonic acid pathways in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Alessandra eScala

    2013-04-01

    Full Text Available Green Leaf Volatiles (GLVs are C6-molecules - alcohols, aldehydes and esters - produced by plants upon herbivory or during pathogen infection. Exposure to this blend of volatiles induces defence-related responses in neighboring undamaged plants, thus assigning a role to GLVs in regulating plant defences. Here we compared Arabidopsis thaliana ecotype Ler with a hydroperoxide lyase line, hpl1, unable to synthesize GLVs, for susceptibility to Pseudomonas syringae pv. tomato (DC3000. We found that the growth of DC3000 was significantly reduced in the hpl1 mutant. This phenomenon correlated with lower jasmonic acid (JA levels and higher salicylic acid (SA levels in the hpl1 mutant. Furthermore, upon infection, the JA-responsive genes VSP2 and LEC were only slightly or not induced, respectively, in hpl1. This suggests that the reduced growth of DC3000 in hpl1 plants is due to the constraint of JA-dependent responses. Treatment of hpl1 plants with E-2-hexenal, one of the more reactive GLVs, prior to infection with DC3000, resulted in increased growth of DC3000 in hpl1, thus complementing this mutant. Interestingly, the growth of DC3000 also increased in Ler plants treated with E-2-hexenal. This stronger growth was not dependent on the JA-signaling component MYC2, but on ORA59, an integrator of JA and ethylene signaling pathways, and on the production of coronatine by DC3000. GLVs may have multiple effects on plant-pathogen interactions, in this case reducing resistance to P. syringae via JA and ORA59.

  10. From ether to acid: A plausible degradation pathway of glycerol dialkyl glycerol tetraethers

    Science.gov (United States)

    Liu, Xiao-Lei; Birgel, Daniel; Elling, Felix J.; Sutton, Paul A.; Lipp, Julius S.; Zhu, Rong; Zhang, Chuanlun; Könneke, Martin; Peckmann, Jörn; Rowland, Steven J.; Summons, Roger E.; Hinrichs, Kai-Uwe

    2016-06-01

    Glycerol dialkyl glycerol tetraethers (GDGTs) are ubiquitous microbial lipids with extensive demonstrated and potential roles as paleoenvironmental proxies. Despite the great attention they receive, comparatively little is known regarding their diagenetic fate. Putative degradation products of GDGTs, identified as hydroxyl and carboxyl derivatives, were detected in lipid extracts of marine sediment, seep carbonate, hot spring sediment and cells of the marine thaumarchaeon Nitrosopumilus maritimus. The distribution of GDGT degradation products in environmental samples suggests that both biotic and abiotic processes act as sinks for GDGTs. More than a hundred newly recognized degradation products afford a view of the stepwise degradation of GDGT via (1) ether bond hydrolysis yielding hydroxyl isoprenoids, namely, GDGTol (glycerol dialkyl glycerol triether alcohol), GMGD (glycerol monobiphytanyl glycerol diether), GDD (glycerol dibiphytanol diether), GMM (glycerol monobiphytanol monoether) and bpdiol (biphytanic diol); (2) oxidation of isoprenoidal alcohols into corresponding carboxyl derivatives and (3) chain shortening to yield C39 and smaller isoprenoids. This plausible GDGT degradation pathway from glycerol ethers to isoprenoidal fatty acids provides the link to commonly detected head-to-head linked long chain isoprenoidal hydrocarbons in petroleum and sediment samples. The problematic C80 to C82 tetraacids that cause naphthenate deposits in some oil production facilities can be generated from H-shaped glycerol monoalkyl glycerol tetraethers (GMGTs) following the same process, as indicated by the distribution of related derivatives in hydrothermally influenced sediments.

  11. Priming by Hexanoic acid induce activation of mevalonic and linolenic pathways and promotes the emission of plant volatiles.

    Directory of Open Access Journals (Sweden)

    Eugenio eLlorens

    2016-04-01

    Full Text Available Hexanoic acid is a short natural monocarboxylic acid present in some fruits and plants. Previous studies reported that soil drench application of this acid induces effective resistance in tomato plants against Botrytis cinerea and Pseudomonas syringae and in citrus against Alternaria alternata and Xanthomonas citri. In this work, we performed an in deep study of the metabolic changes produced in citrus by the application of hexanoic acid in response to the challenge pathogen Alternaria alternata, focusing on the response of the plant. Moreover, we used 13C labeled hexanoic to analyze its behavior inside the plants. Finally, we studied the volatile emission of the treated plants after the challenge inoculation. Drench application of 13C labeled hexanoic demonstrated that this molecule stays in the roots and is not mobilized to the leaves, suggesting long distance induction of resistance. Moreover, the study of the metabolic profile showed an alteration of more than two hundred molecules differentially induced by the application of the compound and the inoculation with the fungus. Bioinformatics analysis of data showed that most of these altered molecules could be related with the mevalonic and linolenic pathways suggesting the implication of these pathways in the induced resistance mediated by hexanoic acid. Finally, the application of this compound showed an enhancement of the emission of 17 volatile metabolites. Taken together, this study indicates that after the application of hexanoic acid this compound remains in the roots, provoking molecular changes that may trigger the defensive response in the rest of the plant mediated by changes in the mevalonic and linolenic pathways and enhancing the emission of volatile compounds, suggesting for the first time the implication of mevalonic pathway in response to hexanoic application.

  12. c9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway[S

    OpenAIRE

    Mollica, Maria Pina; Trinchese, Giovanna; Cavaliere, Gina; De Filippo, Chiara; Cocca, Ennio; Gaita, Marcello; Della-Gatta, Antonio; Marano, Angela; Mazzarella, Giuseppe; Bergamo, Paolo

    2014-01-01

    Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA iso...

  13. Nucleotide Metabolism and its Control in Lactic Acid Bacteria

    DEFF Research Database (Denmark)

    Kilstrup, Mogens; Hammer, Karin; Jensen, Peter Ruhdal;

    2005-01-01

    the interconversion pathways, the formation of deoxyribonucleotides and the salvage pathways for use of exogenous precursors. The data for the enzymatic and the genetic regulation of these pathways are reviewed, as well as the gene organizations in different lactic acid bacteria. Mutant phenotypes and methods...... for manipulation of nucleotide pools are also discussed. Our aim is to provide an overview of the physiology and genetics of nucleotide metabolism and its regulation that will facilitate the interpretation of data arising from genetics, metabolomics, proteomics, and transcriptomics in lactic acid bacteria.......Most metabolic reactions are connected through either their utilization of nucleotides or their utilization of nucleotides or their regulation by these metabolites. In this review the biosynthetic pathways for pyrimidine and purine metabolism in lactic acid bacteria are described including...

  14. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues.

    Science.gov (United States)

    Myers, Jennifer S; von Lersner, Ariana K; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  15. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues

    Science.gov (United States)

    Myers, Jennifer S.; von Lersner, Ariana K.; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  16. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    Energy Technology Data Exchange (ETDEWEB)

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G., E-mail: deborah.murdock@vanderbilt.edu

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  17. The fatty acid beta-oxidation pathway is important for decidualization of endometrial stromal cells in both humans and mice.

    Science.gov (United States)

    Tsai, Jui-He; Chi, Maggie M-Y; Schulte, Maureen B; Moley, Kelle H

    2014-02-01

    Embryo implantation and development requires the endometrial stromal cells (ESCs) to undergo decidualization. This differentiation process requires glucose utilization, and blockade of the pentose phosphate pathway inhibits decidualization of ESCs both in vitro and in vivo. Glucose and fatty acids are energy substrates for many cell types, and fatty acid beta-oxidation is critical for embryo implantation. Here, we investigated whether beta-oxidation is required for decidualization of ESCs. As assessed by marker gene expression, decidualization of human primary ESCs was blocked by reducing activity of carnitine calmitoyltransferase I, the rate-limiting enzyme in beta-oxidation, either by short hairpin RNA-mediated silencing or by treatment with the inhibitor etomoxir. Ranolazine (RAN), a partial beta-oxidation inhibitor, blocked early decidualization of a human ESC line. However, decidualization resumed after several days, most likely due to a compensatory up-regulation of GLUT1 expression and an increase in glucose metabolism. Simultaneous inhibition of the beta-oxidation pathway with RAN and the pentose phosphate pathway with glucosamine (GlcN) impaired in vitro decidualization of human ESCs more strongly than inhibition of either pathway alone. These findings were confirmed in murine ESCs in vitro, and exposure to RAN plus GlcN inhibited decidualization in vivo in a deciduoma model. Finally, intrauterine implantation of time-release RAN and GlcN pellets reduced pup number. Importantly, pup number returned to normal after the end of the pellet-active period. This work indicates that both fatty acids and glucose metabolism pathways are important for ESC decidualization, and suggests novel pathways to target for the design of future nonhormonal contraceptives.

  18. Genomics of pyrrolnitrin biosynthetic loci : evidence for conservation and whole-operon mobility within Gram-negative bacteria

    NARCIS (Netherlands)

    Costa, Rodrigo; van Aarle, Ingrid M.; Mendes, Rodrigo; van Elsas, Jan Dirk

    2009-01-01

    Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of Gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway tha

  19. Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development

    DEFF Research Database (Denmark)

    Rabatel, Andréane; Febvay, Gérard; Gaget, Karen;

    2013-01-01

    embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes...... encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data...... are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together...

  20. Polymorphism of Kynurenine Pathway-Related Genes, Kynurenic Acid, and Psychopathological Symptoms in HIV.

    Science.gov (United States)

    Douet, Vanessa; Tanizaki, Naomi; Franke, Adrian; Li, Xingnan; Chang, Linda

    2016-09-01

    HIV-infection is associated with neuroinflammation and greater psychopathological symptoms, which may be mediated by imbalances in the kynurenic pathway (KP). Two key KP enzymes that catabolize kynurenine include kynurenine-aminotransferase II (KATII), which yields antioxidative kynurenine acid [KYNA] in astrocytes, and kynurenine-3-monooxygenase (KMO), which produces neurotoxic metabolites in microglia. The relationships between polymorphisms in KMO and KATII, psychopathological symptoms, and cerebrospinal fluid (CSF) [KYNA] were evaluated in subjects with and without HIV-infection. Seventy-two HIV-positive and 72-seronegative (SN) participants were genotyped for KATII-rs1480544 and KMO-rs1053230. Although our participants were not currently diagnosed with depression or anxiety, they were assessed for psychopathological distress with Center for Epidemiologic Studies-Depression scale and Symptom Checklist-90-Revised. CSF-[KYNA] was also measured in 100 subjects (49 HIV/51 SN). HIV-participants had more psychopathological distress than SN, especially for anxiety. KATII-by-HIV interactions were found on anxiety, interpersonal sensitivity and obsessive compulsivity; KATII-C-carriers had lower scores than TT-carriers in SN but not in HIV. In contrast, the KMO-polymorphism had no influence on psychopathological symptoms in both groups. Overall, CSF-[KYNA] increased with age independently of HIV-serostatus, except KATII-TT-carriers tended to show no age-dependent variations. Therefore, the C-allele in KATII-rs1480544 appears to be protective against psychopathological distress in SN but not in HIV individuals, who had more psychopathological symptoms and likely greater neuroinflammation. The age-dependent increase in CSF-[KYNA] may reflect a compensatory response to age-related inflammation, which may be deficient in KATII-TT-carriers. Targeted treatments that decrease neuroinflammation and increase KYNA in at risk KATII-TT-carriers may reduce psychopathological symptoms

  1. Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPARβ/δ in HepG2 cells

    International Nuclear Information System (INIS)

    Highlights: • Palmitic acid significantly inhibited APOM gene expression in HepG2 cells. • Palmitic acid could obviously increase PPARB/D mRNA levels in HepG2 cells. • PPARβ/δ antagonist, GSK3787, had no effect on APOM expression. • GSK3787 could reverse the palmitic acid-induced down-regulation of APOM expression. • Palmitic acid induced suppression of APOM expression is mediated via the PPARβ/δ pathway. - Abstract: It has been demonstrated that apolipoprotein M (APOM) is a vasculoprotective constituent of high density lipoprotein (HDL), which could be related to the anti-atherosclerotic property of HDL. Investigation of regulation of APOM expression is of important for further exploring its pathophysiological function in vivo. Our previous studies indicated that expression of APOM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF), leptin, hyperglycemia and etc., in vivo and/or in vitro. In the present study, we demonstrated that palmitic acid could significantly inhibit APOM gene expression in HepG2 cells. Further study indicated neither PI-3 kinase (PI3K) inhibitor LY294002 nor protein kinase C (PKC) inhibitor GFX could abolish palmitic acid induced down-regulation of APOM expression. In contrast, the peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) antagonist GSK3787 could totally reverse the palmitic acid-induced down-regulation of APOM expression, which clearly demonstrates that down-regulation of APOM expression induced by palmitic acid is mediated via the PPARβ/δ pathway

  2. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production.

    Science.gov (United States)

    Man, Zaiwei; Xu, Meijuan; Rao, Zhiming; Guo, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L(-1) L-arginine with yield up to 0.431 g L-arginine g(-1) glucose in fed-batch fermentation. PMID:27338253

  3. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production

    Science.gov (United States)

    Man, Zaiwei; Xu, Meijuan; Rao, Zhiming; Guo, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L−1 L-arginine with yield up to 0.431 g L-arginine g−1 glucose in fed-batch fermentation. PMID:27338253

  4. Metabolic pathways of the wheat (Triticum aestivum endosperm amyloplast revealed by proteomics

    Directory of Open Access Journals (Sweden)

    Dupont Frances M

    2008-04-01

    Full Text Available Abstract Background By definition, amyloplasts are plastids specialized for starch production. However, a proteomic study of amyloplasts isolated from wheat (Triticum aestivum Butte 86 endosperm at 10 days after anthesis (DPA detected enzymes from many other metabolic and biosynthetic pathways. To better understand the role of amyloplasts in food production, the data from that study were evaluated in detail and an amyloplast metabolic map was outlined. Results Analysis of 288 proteins detected in an amyloplast preparation predicted that 178 were amyloplast proteins. Criteria included homology with known plastid proteins, prediction of a plastid transit peptide for the wheat gene product or a close homolog, known plastid location of the pathway, and predicted plastid location for other members of the same pathway. Of these, 135 enzymes were arranged into 18 pathways for carbohydrate, lipid, amino acid, nucleic acid and other biosynthetic processes that are critical for grain-fill. Functions of the other proteins are also discussed. Conclusion The pathways outlined in this paper suggest that amyloplasts play a central role in endosperm metabolism. The interacting effects of genetics and environment on starch and protein production may be mediated in part by regulatory mechanisms within this organelle.

  5. 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.

  6. Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein

    OpenAIRE

    Longo, Liam M.; Lee, Jihun; Blaber, Michael

    2013-01-01

    A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 “prebiotic” α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a “foldable set”—that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides?...

  7. A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Fan, J.; Xu, C.; Andre, C.

    2011-06-23

    Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.

  8. Tyrosol degradation via the homogentisic acid pathway in a newly isolated Halomonas strain from olive processing effluents

    OpenAIRE

    Liebgott, Pierre-Pol; Labat, Marc; Amouric, Agnès; Tholozan, Jean-Luc; LORQUIN, Jean

    2008-01-01

    To isolate a new Halomonas sp. strain capable of degrading tyrosol, a toxic compound present in olive mill wastewater, through the homogentisic acid (HGA) pathway. A moderately halophilic Gram-negative bacterium belonging to the Halomonas genus and designated strain TYRC17 was isolated from olive processing effluents. This strain was able to completely degrade tyrosol (2-(p-hydroxyphenyl)-ethanol), a toxic compound found in such effluent. Tyrosol degradation begins by an oxidation to 4-hydrox...

  9. Probing phosphorylation by non-mammalian isoprenoid biosynthetic enzymes using 1H–31P–31P correlation NMR spectroscopy†‡

    OpenAIRE

    Majumdar, Ananya; Shah, Meha H.; Bitok, J. Kipchirchir; Hassis-LeBeau, Maria E.; Freel Meyers, Caren L.

    2009-01-01

    The biogenesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) is accomplished by the methylerythritol phosphate (MEP) pathway in plants, bacteria and parasites, making it a potential target for the development of anti-infective agents and herbicides. The biosynthetic enzymes comprising this pathway catalyze intriguing chemical transformations on diphosphate scaffolds, offering an opportunity to generate novel analogs in this synthetically challenging compound class...

  10. The shikimate pathway: review of amino acid sequence, function and three-dimensional structures of the enzymes.

    Science.gov (United States)

    Mir, Rafia; Jallu, Shais; Singh, T P

    2015-06-01

    The aromatic compounds such as aromatic amino acids, vitamin K and ubiquinone are important prerequisites for the metabolism of an organism. All organisms can synthesize these aromatic metabolites through shikimate pathway, except for mammals which are dependent on their diet for these compounds. The pathway converts phosphoenolpyruvate and erythrose 4-phosphate to chorismate through seven enzymatically catalyzed steps and chorismate serves as a precursor for the synthesis of variety of aromatic compounds. These enzymes have shown to play a vital role for the viability of microorganisms and thus are suggested to present attractive molecular targets for the design of novel antimicrobial drugs. This review focuses on the seven enzymes of the shikimate pathway, highlighting their primary sequences, functions and three-dimensional structures. The understanding of their active site amino acid maps, functions and three-dimensional structures will provide a framework on which the rational design of antimicrobial drugs would be based. Comparing the full length amino acid sequences and the X-ray crystal structures of these enzymes from bacteria, fungi and plant sources would contribute in designing a specific drug and/or in developing broad-spectrum compounds with efficacy against a variety of pathogens.

  11. Phenylbutyric acid induces the cellular senescence through an Akt/p21{sup WAF1} signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hag Dong [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Jang, Chang-Young [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Choe, Jeong Min [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Sohn, Jeongwon, E-mail: biojs@korea.ac.kr [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Kim, Joon, E-mail: joonkim@korea.ac.kr [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Phenylbutyric acid induces cellular senescence. Black-Right-Pointing-Pointer Phenylbutyric acid activates Akt kinase. Black-Right-Pointing-Pointer The knockdown of PERK also can induce cellular senescence. Black-Right-Pointing-Pointer Akt/p21{sup WAF1} pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21{sup WAF1} induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21{sup WAF1} pathway by PERK inhibition.

  12. Extending the biosynthetic repertoires of cyanobacteria and chloroplasts.

    Science.gov (United States)

    Nielsen, Agnieszka Zygadlo; Mellor, Silas Busck; Vavitsas, Konstantinos; Wlodarczyk, Artur Jacek; Gnanasekaran, Thiyagarajan; Perestrello Ramos H de Jesus, Maria; King, Brian Christopher; Bakowski, Kamil; Jensen, Poul Erik

    2016-07-01

    Chloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts. PMID:27005523

  13. Sugars as the optimal biosynthetic carbon substrate of aqueous life throughout the universe

    Science.gov (United States)

    Weber, A. L.

    2000-01-01

    Our previous analysis of the energetics of metabolism showed that both the biosynthesis of amino acids and lipids from sugars, and the fermentation of organic substrates, were energetically driven by electron transfer reactions resulting in carbon redox disproportionation (Weber, 1997). Redox disproportionation--the spontaneous (energetically favorable) direction of carbon group transformation in biosynthesis--is brought about and driven by the energetically downhill transfer of electron pairs from more oxidized carbon groups (with lower half-cell reduction potentials) to more reduced carbon groups (with higher half-cell reduction potentials). In this report, we compare the redox and kinetic properties of carbon groups in order to evaluate the relative biosynthetic capability of organic substrates, and to identify the optimal biosubstrate. This analysis revealed that sugars (monocarbonyl alditols) are the optimal biosynthetic substrate because they contain the maximum number of biosynthetically useful high energy electrons/carbon atom while still containing a single carbonyl group needed to kinetically facilitate their conversion to useful biosynthetic intermediates. This conclusion applies to aqueous life throughout the Universe because it is based on invariant aqueous carbon chemistry--primarily, the universal reduction potentials of carbon groups.

  14. Emergent biosynthetic capacity in simple microbial communities.

    Directory of Open Access Journals (Sweden)

    Hsuan-Chao Chiu

    2014-07-01

    Full Text Available Microbes have an astonishing capacity to transform their environments. Yet, the metabolic capacity of a single species is limited and the vast majority of microorganisms form complex communities and join forces to exhibit capabilities far exceeding those achieved by any single species. Such enhanced metabolic capacities represent a promising route to many medical, environmental, and industrial applications and call for the development of a predictive, systems-level understanding of synergistic microbial capacity. Here we present a comprehensive computational framework, integrating high-quality metabolic models of multiple species, temporal dynamics, and flux variability analysis, to study the metabolic capacity and dynamics of simple two-species microbial ecosystems. We specifically focus on detecting emergent biosynthetic capacity--instances in which a community growing on some medium produces and secretes metabolites that are not secreted by any member species when growing in isolation on that same medium. Using this framework to model a large collection of two-species communities on multiple media, we demonstrate that emergent biosynthetic capacity is highly prevalent. We identify commonly observed emergent metabolites and metabolic reprogramming patterns, characterizing typical mechanisms of emergent capacity. We further find that emergent secretion tends to occur in two waves, the first as soon as the two organisms are introduced, and the second when the medium is depleted and nutrients become limited. Finally, aiming to identify global community determinants of emergent capacity, we find a marked association between the level of emergent biosynthetic capacity and the functional/phylogenetic distance between community members. Specifically, we demonstrate a "Goldilocks" principle, where high levels of emergent capacity are observed when the species comprising the community are functionally neither too close, nor too distant. Taken together

  15. Jasmonic acid is involved in the signaling pathway for fungal endophyte-induced volatile oil accumulation of Atractylodes lancea plantlets

    Directory of Open Access Journals (Sweden)

    Ren Cheng-Gang

    2012-08-01

    Full Text Available Abstract Background Jasmonic acid (JA is a well-characterized signaling molecule in plant defense responses. However, its relationships with other signal molecules in secondary metabolite production induced by endophytic fungus are largely unknown. Atractylodes lancea (Asteraceae is a traditional Chinese medicinal plant that produces antimicrobial volatiles oils. We incubated plantlets of A. lancea with the fungus Gilmaniella sp. AL12. to research how JA interacted with other signal molecules in volatile oil production. Results Fungal inoculation increased JA generation and volatile oil accumulation. To investigate whether JA is required for volatile oil production, plantlets were treated with JA inhibitors ibuprofen (IBU and nordihydroguaiaretic acid. The inhibitors suppressed both JA and volatile oil production, but fungal inoculation could still induce volatile oils. Plantlets were further treated with the nitric oxide (NO-specific scavenger 2-(4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO, the H2O2 inhibitors diphenylene iodonium (DPI and catalase (CAT, and the salicylic acid (SA biosynthesis inhibitors paclobutrazol and 2-aminoindan-2-phosphonic acid. With fungal inoculation, IBU did not inhibit NO production, and JA generation was significantly suppressed by cPTIO, showing that JA may act as a downstream signal of the NO pathway. Exogenous H2O2 could reverse the inhibitory effects of cPTIO on JA generation, indicating that NO mediates JA induction by the fungus through H2O2-dependent pathways. With fungal inoculation, the H2O2 scavenger DPI/CAT could inhibit JA generation, but IBU could not inhibit H2O2 production, implying that H2O2 directly mediated JA generation. Finally, JA generation was enhanced when SA production was suppressed, and vice versa. Conclusions Jasmonic acid acts as a downstream signaling molecule in NO- and H2O2-mediated volatile oil accumulation induced by endophytic fungus and has

  16. Folic Acid Is Able to Polarize the Inflammatory Response in LPS Activated Microglia by Regulating Multiple Signaling Pathways

    Science.gov (United States)

    Salvatore, Rosaria; Porro, Chiara; Trotta, Teresa

    2016-01-01

    We investigated the ability of folic acid to modulate the inflammatory responses of LPS activated BV-2 microglia cells and the signal transduction pathways involved. To this aim, the BV-2 cell line was exposed to LPS as a proinflammatory response inducer, in presence or absence of various concentrations of folic acid. The production of nitric oxide (NO) was determined by the Griess test. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 were determined by ELISA. Inducible NO synthase (iNOS), nuclear transcription factor-kappa B (NF-κB) p65, MAPKs protein, and suppressors of cytokine signaling (SOCS)1 and SOCS3 were analyzed by western blotting. TNF-α and IL-1β, as well as iNOS dependent NO production, resulted significantly inhibited by folic acid pretreatment in LPS-activated BV-2 cells. We also observed that folic acid dose-dependently upregulated both SOCS1 and SOCS3 expression in BV-2 cells, leading to an increased expression of the anti-inflammatory cytokine IL-10. Finally, p-IκBα, which indirectly reflects NF-κB complex activation, and JNK phosphorylation resulted dose-dependently downregulated by folic acid pretreatment of LPS-activated cells, whereas p38 MAPK phosphorylation resulted significantly upregulated by folic acid treatment. Overall, these results demonstrated that folic acid was able to modulate the inflammatory response in microglia cells, shifting proinflammatory versus anti-inflammatory responses through regulating multiple signaling pathways.

  17. A genomics based discovery of secondary metabolite biosynthetic gene clusters in Aspergillus ustus.

    Directory of Open Access Journals (Sweden)

    Borui Pi

    Full Text Available Secondary metabolites (SMs produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

  18. Chlorogenic acid improves late diabetes through adiponectin receptor signaling pathways in db/db mice.

    Directory of Open Access Journals (Sweden)

    Shasha Jin

    Full Text Available The aim of this study was to examine the effects of chlorogenic acid (CGA on glucose and lipid metabolism in late diabetic db/db mice, as well as on adiponectin receptors and their signaling molecules, to provide evidence for CGA in the prevention of type 2 diabetes. We randomly divided 16 female db/db mice into db/db-CGA and db/db-control (CON groups equally; db/m mice were used as control mice. The mice in both the db/db-CGA and db/m-CGA groups were administered 80 mg/kg/d CGA by lavage for 12 weeks, whereas the mice in both CON groups were given equal volumes of phosphate-buffered saline (PBS by lavage. At the end of the intervention, we assessed body fat and the parameters of glucose and lipid metabolism in the plasma, liver and skeletal muscle tissues as well as the levels of aldose reductase (AR and transforming growth factor-β1 (TGF-β1 in the kidneys and measured adiponectin receptors and the protein expression of their signaling molecules in liver and muscle tissues. After 12 weeks of intervention, compared with the db/db-CON group, the percentage of body fat, fasting plasma glucose (FPG and glycosylated hemoglobin (HbA1c in the db/db-CGA group were all significantly decreased; TGF-β1 protein expression and AR activity in the kidney were both decreased; and the adiponectin level in visceral adipose was increased. The protein expression of adiponectin receptors (ADPNRs, the phosphorylation of AMP-activated protein kinase (AMPK in the liver and muscle, and the mRNA and protein levels of peroxisome proliferator-activated receptor alpha (PPAR-α in the liver were all significantly greater. CGA could lower the levels of fasting plasma glucose and HbA1c during late diabetes and improve kidney fibrosis to some extent through the modulation of adiponectin receptor signaling pathways in db/db mice.

  19. DMPD: Nucleic acid-sensing TLRs as modifiers of autoimmunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17082566 Nucleic acid-sensing TLRs as modifiers of autoimmunity. Deane JA, Bolland ...S. J Immunol. 2006 Nov 15;177(10):6573-8. (.png) (.svg) (.html) (.csml) Show Nucleic acid-sensing TLRs as mo...difiers of autoimmunity. PubmedID 17082566 Title Nucleic acid-sensing TLRs as modifiers of autoimmunity. Aut

  20. Biosynthetic Studies on Water-Soluble Derivative 5c (DTX5c

    Directory of Open Access Journals (Sweden)

    José J. Fernández

    2012-10-01

    Full Text Available The dinoflagellate Prorocentrum belizeanum is responsible for the production of several toxins involved in the red tide phenomenon known as Diarrhetic Shellfish Poisoning (DSP. In this paper we report on the biosynthetic origin of an okadaic acid water-soluble ester derivative, DTX5c, on the basis of the spectroscopical analysis of 13C enriched samples obtained by addition of labelled sodium [l-13C], [2-13C] acetate to artificial cultures of this dinoflagellate.

  1. The preparation of nucleotides uniformly labelled with carbon-14 by biosynthetic methods. Isolation of adenylic, uridylic, cytidylic,and guanylic acids, from the alkaline hydrolysate of escherichia coli RNA

    International Nuclear Information System (INIS)

    A method is described for the preparation and analysis of adenylic, uri dilic, cytidi- 11c and guanylic acids, labelled with 14C. Escherichia coli cells have been labelled by growing them in a medi dia containing glucose-14C as their only source of carbon. RNA is isolated from the cells, and after hydrolysis of the molecule the resulting nucleotides are separated by gel filtration and exchange chromatography. Chemical and radiochemical purity of the Isolated nucleotides is determined, and also its specific radioactivity. (Author) 30 refs

  2. Biosynthetic Machinery Involved in Aberrant Glycosylation: Promising Targets for Development Drugs Against Cancer

    Directory of Open Access Journals (Sweden)

    Andreia eVasconcelos-dos-Santos

    2015-06-01

    Full Text Available Cancer cells depend on altered metabolism and nutrient uptake to generate and keep the malignant phenotype. The hexosamine biosynthetic pathway (HBP is a branch of glucose metabolism that produces UDP-GlcNAc, and its derivatives, UDP-GalNAc and CMP-Neu5Ac, donor substrates used in the production of glycoproteins and glycolipids. Growing evidence demonstrates that alteration of the pool of activated substrates might lead to different glycosylation and cell signaling. It is already well established that aberrant glycosylation can modulate tumor growth and malignant transformation in different cancer types. Therefore, biosynthetic machinery involved in the assembly of aberrant glycans are becoming prominent targets for anti-tumor drugs. This review describes three classes of glycosylation, O-GlcNAcylation, N-linked and mucin type O-linked glycosylation, involved in tumor progression, their biosynthesis and highlights the available inhibitors as potential anti-tumor drugs.

  3. PPARα signal pathway gene expression is associated with fatty acid content in yak and cattle longissimus dorsi muscle.

    Science.gov (United States)

    Qin, W; Liang, C N; Guo, X; Chu, M; Pei, J; Bao, P J; Wu, X Y; Li, T K; Yan, P

    2015-11-19

    Intramuscular fatty acid (FA) is related to meat qualities such as juiciness, tenderness, palatability, and shear force. PPARα plays an important role in lipid metabolism in the liver and skeletal muscle. This study investigated FA composition in yaks and cattle, in order to ascertain whether a correlation between PPARα signal pathway genes as candidate genes and meat FA composition in yaks and cattle exists. Statistical analyses revealed that levels of monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) in yaks were significantly higher than those in cattle (P cattle (P cattle. However, LPL expression in yaks was significantly higher than that in cattle (P cattle, the mRNA level of PLTP was positively correlated with SFA (P meat quality.

  4. Gaseous 3-pentanol primes plant immunity against a bacterial speck pathogen, Pseudomonas syringae pv. tomato via salicylic acid and jasmonic acid-dependent signaling pathways in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Geun Cheol eSong

    2015-10-01

    Full Text Available 3-Pentanol is an active organic compound produced by plants and is a component of emitted insect sex pheromones. A previous study reported that drench application of 3-pentanol elicited plant immunity against microbial pathogens and an insect pest in crop plants. Here, we evaluated whether 3-pentanol and the derivatives 1-pentanol and 2-pentanol induced plant systemic resistance using the in vitro I-plate system. Exposure of Arabidopsis seedlings to 10 M and 100 nM 3-pentanol evaporate elicited an immune response to Pseudomonas syringae pv. tomato DC3000. We performed quantitative real-time PCR to investigate the 3-pentanol-mediated Arabidopsis immune responses by determining Pathogenesis-Related (PR gene expression levels associated with defense signaling through SA, JA, and ethylene signaling pathways. The results show that exposure to 3-pentanol and subsequent pathogen challenge upregulated PDF1.2 and PR1 expression. Selected Arabidopsis mutants confirmed that the 3-pentanol-mediated immune response involved salicylic acid (SA and jasmonic acid (JA signaling pathways and the NPR1 gene. Taken together, this study indicates that gaseous 3-pentanol triggers induced resistance in Arabidopsis by priming SA and JA signaling pathways. To our knowledge, this is the first report that a volatile compound of an insect sex pheromone triggers plant systemic resistance against a bacterial pathogen.

  5. Metabolic engineering of the fungal D-galacturonate pathway for L-ascorbic acid production

    OpenAIRE

    Kuivanen, Joosu

    2015-01-01

    Industrial biotechnology is one of the enabling technologies for biorefineries, where biomass is converted into value-added products. In addition to biofuels, several platform and fine chemicals can be produced from biomass using biotechnological routes taking advantage of metabolic pathways in the cell. Some of these metabolic pathways exist naturally in the cells that are used as production hosts. However, many of the desired chemical products are not naturally produced by the cellular meta...

  6. Alpha-picolinic Acid Activates Diverse Defense Responses of Salicylic Acid-, Jasmonic Acid/Ethylene- and Ca2 -dependent Pathways in Arabidopsis and Rice Suspension Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANGHai-Kuo; ZHANGXin; LIQun; HEZu-Hua

    2004-01-01

    Alpha-picolinic acid (PA) is an apoptosis inducer in animal cells, and could elicit hypersensitiv eresponse (HR) in rice, a monocotyledonous model plant. Here we report that PA is an HR inducer in plants. It induced HR in Arabidopsis, a dicotyledonous model plant, including the oxidative burst and cell death. We investigated defense signal transduction activated by PA through marker genes of particular defense pathways in Arabidopsis. The result indicated that both the salicylic acid-dependent and jasmonic acid/ethylene-dependent pathways were activated by PA, in which the marker defense genes PR-1, PR-2 and PDF 1.2 were all induced in dose-dependent and time-course manners. We also observed that the PAinduced reactive oxygen species (ROS) production in rice suspension cells was Ca2+-dependent. Together with our previous studies of PA-induced defense activation in rice, we conclude that PA acts as a nonspecific elicitor in plant defense and has a potential utilization in cellular model establishment of systemicac quired resistance (SAR) activation.

  7. Activation of the salicylic acid signaling pathway enhances Clover yellow vein virus virulence in susceptible pea cultivars.

    Science.gov (United States)

    Atsumi, Go; Kagaya, Uiko; Kitazawa, Hiroaki; Nakahara, Kenji Suto; Uyeda, Ichiro

    2009-02-01

    The wild-type strain (Cl-WT) of Clover yellow vein virus (ClYVV) systemically induces cell death in pea cv. Plant introduction (PI) 118501 but not in PI 226564. A single incompletely dominant gene, Cyn1, controls systemic cell death in PI 118501. Here, we show that activation of the salicylic acid (SA) signaling pathway enhances ClYVV virulence in susceptible pea cultivars. The kinetics of virus accumulation was not significantly different between PI 118501 (Cyn1) and PI 226564 (cyn1); however, the SA-responsive chitinase gene (SA-CHI) and the hypersensitive response (HR)-related gene homologous to tobacco HSR203J were induced only in PI 118501 (Cyn1). Two mutant viruses with mutations in P1/HCPro, which is an RNA-silencing suppressor, reduced the ability to induce cell death and SA-CHI expression. The application of SA and of its analog benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) partially complemented the reduced virulence of mutant viruses. These results suggest that high activation of the SA signaling pathway is required for ClYVV virulence. Interestingly, BTH could enhance Cl-WT symptoms in PI 226564 (cyn1). However, it could not enhance symptoms induced by White clover mosaic virus and Bean yellow mosaic virus. Our report suggests that the SA signaling pathway has opposing functions in compatible interactions, depending on the virus-host combination. PMID:19132869

  8. Hidden Brønsted acid catalysis: pathways of accidental or deliberate generation of triflic acid from metal triflates.

    Science.gov (United States)

    Dang, Tuan Thanh; Boeck, Florian; Hintermann, Lukas

    2011-11-18

    The generation of a hidden Brønsted acid as a true catalytic species in hydroalkoxylation reactions from metal precatalysts has been clarified in case studies. The mechanism of triflic acid (CF(3)SO(3)H or HOTf) generation starting either from AgOTf in 1,2-dichloroethane (DCE) or from a Cp*RuCl(2)/AgOTf/phosphane combination in toluene has been elucidated. The deliberate and controlled generation of HOTf from AgOTf and cocatalytic amounts of tert-butyl chloride in the cold or from AgOTf in DCE at elevated temperatures results in a hidden Brønsted acid catalyst useful for mechanistic control experiments or for synthetic applications. PMID:22010906

  9. Loss of Nuclear Receptor SHP Impairs but Does Not Eliminate Negative Feedback Regulation of Bile Acid Synthesis

    OpenAIRE

    Kerr, Thomas A.; Saeki, Shigeru; Schneider, Manfred; Schaefer, Karen; Berdy, Sara; Redder, Thadd; Shan, Bei; Russell, David W.; Schwarz, Margrit

    2002-01-01

    The in vivo role of the nuclear receptor SHP in feedback regulation of bile acid synthesis was examined. Loss of SHP in mice caused abnormal accumulation and increased synthesis of bile acids due to derepression of rate-limiting CYP7A1 and CYP8B1 hydroxylase enzymes in the biosynthetic pathway. Dietary bile acids induced liver damage and restored feedback regulation. A synthetic agonist of the nuclear receptor FXR was not hepatotoxic and had no regulatory effects. Reduction of the bile acid p...

  10. Biosynthesis of Akaeolide and Lorneic Acids and Annotation of Type I Polyketide Synthase Gene Clusters in the Genome of Streptomyces sp. NPS554

    Directory of Open Access Journals (Sweden)

    Tao Zhou

    2015-01-01

    Full Text Available The incorporation pattern of biosynthetic precursors into two structurally unique polyketides, akaeolide and lorneic acid A, was elucidated by feeding experiments with 13C-labeled precursors. In addition, the draft genome sequence of the producer, Streptomyces sp. NPS554, was performed and the biosynthetic gene clusters for these polyketides were identified. The putative gene clusters contain all the polyketide synthase (PKS domains necessary for assembly of the carbon skeletons. Combined with the 13C-labeling results, gene function prediction enabled us to propose biosynthetic pathways involving unusual carbon-carbon bond formation reactions. Genome analysis also indicated the presence of at least ten orphan type I PKS gene clusters that might be responsible for the production of new polyketides.

  11. Retinol oxidation to retinoic acid in human thyroid glandular cells.

    Science.gov (United States)

    Taibi, Gennaro; Gueli, Maria Concetta; Nicotra, Concetta M A; Cocciadiferro, Letizia; Carruba, Giuseppe

    2014-12-01

    Abstract Retinoic acid is regarded as the retinol metabolite that controls proliferation and differentiation of epithelial cells. In the present study, we investigated the potential role of xanthine dehydrogenase (XDH) in retinoic acid biosynthesis in human thyroid glandular cells (HTGC). In particular, we observed that cellular retinoids binding proteins (CRBPs) are also implicated in the biosynthetic pathway leading to retinoic acid formation in primary cultures of HTGC, as we have already reported for human mammary epithelial cells (HMEC). After partial protein purification, the enzyme responsible for retinoic acid biosynthesis was identified and quantified as XDH by immunoassay, by its ability to oxidize xanthine to uric acid and its sensitivity to the inhibitory effect of oxypurinol. The evidence of XDH-driven formation of retinoic acid in HTGC cultures further corroborates the potential role of XDH in retinoic acid biosynthesis in the epithelia. PMID:24506204

  12. 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.

  13. The Controversial Role of Retinoic Acid in Fibrotic Diseases: Analysis of Involved Signaling Pathways

    OpenAIRE

    Yuan-Han Qin; Drummen, Gregor P. C.; Tian-Biao Zhou

    2012-01-01

    Fibrotic diseases, such as liver, pulmonary and renal fibrosis, are common end-stage conditions and represent a major global health problem. Furthermore, effective therapeutic measures are presently unavailable. Extracellular matrix accumulation is the most prominent characteristic in the pathogenesis of fibrotic disease. Retinoic acid, including all-trans retinoic acid, 9-cis and 13-cis retinoic acid, play important roles in various physiological processes, such as in embryonic development, ...

  14. Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase-mediated abscisic acid pathway.

    Science.gov (United States)

    Yin, Cui-Cui; Ma, Biao; Collinge, Derek Phillip; Pogson, Barry James; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Chen, Hui; Yang, Chao; Lu, Xiang; Wang, Yi-Qin; Zhang, Wan-Ke; Chu, Cheng-Cai; Sun, Xiao-Hong; Fang, Shuang; Chu, Jin-Fang; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-04-01

    Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.

  15. A natural protecting group strategy to carry an amino acid starter unit in the biosynthesis of macrolactam polyketide antibiotics.

    Science.gov (United States)

    Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2011-11-16

    Macrolactam antibiotics are an important class of macrocyclic polyketides that contain a unique nitrogen-containing starter unit. In the present study, a set of starter biosynthetic enzymes in the macrolactam antibiotic vicenistatin was characterized. We found that the protection-deprotection strategy of the aminoacyl-ACP intermediate was critical in this system. On the basis of bioinformatics, the described pathway is also proposed as a common method for carrying amino acids in the biosynthesis of other macrolactam antibiotics. PMID:22010945

  16. 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.

  17. Fe2+ and Cu2+ increase the production of hyaluronic acid by lactobacilli via affecting different stages of the pentose phosphate pathway.

    Science.gov (United States)

    Choi, Sy-Bing; Lew, Lee-Ching; Hor, Kok-Chiu; Liong, Min-Tze

    2014-05-01

    This study aimed at optimizing the production of hyaluronic acid by Lactobacillus acidophilus FTDC 1231 using response surface methodology and evaluating the effects of divalent metal ions along the production pathway using molecular docking. Among different divalent metal ions that were screened, only iron (II) sulphate and copper (II) sulphate significantly (P pentose phosphate pathway.

  18. An adverse outcome pathway framework for neural tube and axial defects mediated by modulation of retinoic acid homeostasis.

    Science.gov (United States)

    Tonk, Elisa C M; Pennings, Jeroen L A; Piersma, Aldert H

    2015-08-01

    Developmental toxicity can be caused through a multitude of mechanisms and can therefore not be captured through a single simple mechanistic paradigm. However, it may be possible to define a selected group of overarching mechanisms that might allow detection of the vast majority of developmental toxicants. Against this background, we have explored the usefulness of retinoic acid mediated regulation of neural tube and axial patterning as a general mechanism that, when perturbed, may result in manifestations of developmental toxicity that may cover a large part of malformations known to occur in experimental animals and in man. Through a literature survey, we have identified key genes in the regulation of retinoic acid homeostasis, as well as marker genes of neural tube and axial patterning, that may be used to detect developmental toxicants in in vitro systems. A retinoic acid-neural tube/axial patterning adverse outcome pathway (RA-NTA AOP) framework was designed. The framework was tested against existing data of flusilazole exposure in the rat whole embryo culture, the zebrafish embryotoxicity test, and the embryonic stem cell test. Flusilazole is known to interact with retinoic acid homeostasis, and induced common and unique NTA marker gene changes in the three test systems. Flusilazole-induced changes were similar in directionality to gene expression responses after retinoic acid exposure. It is suggested that the RA-NTA framework may provide a general tool to define mechanistic pathways and biomarkers of developmental toxicity that may be used in alternative in vitro assays for the detection of embryotoxic compounds.

  19. The cockroach Blattella germanica obtains nitrogen from uric acid through a metabolic pathway shared with its bacterial endosymbiont.

    Science.gov (United States)

    Patiño-Navarrete, Rafael; Piulachs, Maria-Dolors; Belles, Xavier; Moya, Andrés; Latorre, Amparo; Peretó, Juli

    2014-07-01

    Uric acid stored in the fat body of cockroaches is a nitrogen reservoir mobilized in times of scarcity. The discovery of urease in Blattabacterium cuenoti, the primary endosymbiont of cockroaches, suggests that the endosymbiont may participate in cockroach nitrogen economy. However, bacterial urease may only be one piece in the entire nitrogen recycling process from insect uric acid. Thus, in addition to the uricolytic pathway to urea, there must be glutamine synthetase assimilating the released ammonia by the urease reaction to enable the stored nitrogen to be metabolically usable. None of the Blattabacterium genomes sequenced to date possess genes encoding for those enzymes. To test the host's contribution to the process, we have sequenced and analysed Blattella germanica transcriptomes from the fat body. We identified transcripts corresponding to all genes necessary for the synthesis of uric acid and its catabolism to urea, as well as for the synthesis of glutamine, asparagine, proline and glycine, i.e. the amino acids required by the endosymbiont. We also explored the changes in gene expression with different dietary protein levels. It appears that the ability to use uric acid as a nitrogen reservoir emerged in cockroaches after its age-old symbiotic association with bacteria.

  20. Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli.

    Science.gov (United States)

    Menendez-Bravo, Simón; Comba, Santiago; Sabatini, Martín; Arabolaza, Ana; Gramajo, Hugo

    2014-07-01

    Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate.

  1. Genetic variation in genes of the fatty acid synthesis pathway and breast cancer risk

    NARCIS (Netherlands)

    Campa, Daniele; McKay, James; Sinilnikova, Olga; Huesing, Anika; Vogel, Ulla; Hansen, Rikke Dalgaard; Overvad, Kim; Witt, Petra Mariann; Clavel-Chapelon, Francoise; Boutron-Ruault, Marie-Christine; Chajes, Veronique; Rohrmann, Sabine; Chang-Claude, Jenny; Boeing, Heiner; Fisher, Eva; Trichopoulou, Antonia; Trichopoulos, Dimitrios; Palli, Domenico; Villarini, Anna; Sacerdote, Carlotta; Mattiello, Amalia; Tumino, Rosario; Peeters, Petra H. M.; van Gils, Carla H.; Bueno-de-Mesquita, H. Bas; Lund, Eiliv; Dolores Chirlaque, Maria; Sala, Nuria; Rodriguez Suarez, Laudina; Barricarte, Aurelio; Dorronsoro, Miren; Sanchez, Maria-Jose; Lenner, Per; Hallmans, Goeran; Tsilidis, Kostas; Bingham, Sheila; Khaw, Kay-Tee; Gallo, Valentina; Norat, Teresa; Riboli, Elio; Rinaldi, Sabina; Lenoir, Gilbert; Tavtigian, Sean V.; Canzian, Federico; Kaaks, Rudolf

    2009-01-01

    Fatty acid synthase (FAS) is the major enzyme of lipogenesis. It catalyzes the NADPH-dependent condensation of acetyl-CoA and malonyl-CoA to produce palmitic acid. Transcription of the FAS gene is controlled synergistically by the transcription factors ChREBP (carbohydrate response element-binding p

  2. Genetic variation in genes of the fatty acid synthesis pathway and breast cancer risk

    DEFF Research Database (Denmark)

    Campa, Daniele; McKay, James; Sinilnikova, Olga;

    2009-01-01

    Fatty acid synthase (FAS) is the major enzyme of lipogenesis. It catalyzes the NADPH-dependent condensation of acetyl-CoA and malonyl-CoA to produce palmitic acid. Transcription of the FAS gene is controlled synergistically by the transcription factors ChREBP (carbohydrate response element-bindin...

  3. A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes

    DEFF Research Database (Denmark)

    Andersen, Gorm; Bjornberg, Olof; Polakova, Silvia;

    2008-01-01

    Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces...... kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil. Six loci, named URC1-6 (for uracil catabolism), are involved in the novel catabolic pathway. Four of them, URC3,5, URC6, and URC2 encode urea amidolyase, uracil phosphoribosyltransferase, and a putative transcription factor...

  4. Elucidating the Pseudomonas aeruginosa Fatty Acid Degradation Pathway: Identification of Additional Fatty Acyl-CoA Synthetase Homologues

    OpenAIRE

    Zarzycki-Siek, Jan; Norris, Michael H.; Kang, Yun (Kenneth); Sun, Zhenxin; Bluhm, Andrew P.; McMillan, Ian A.; Hoang, Tung T.

    2013-01-01

    The fatty acid (FA) degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. T...

  5. UV-induced photoreaction pathways of salicylic acid: Identification of the fourth stable conformer and ketoketene-water complex

    Science.gov (United States)

    Miyagawa, Masaya; Akai, Nobuyuki; Nakata, Munetaka

    2014-05-01

    Photoreaction pathways of salicylic acid have been investigated by the low-temperature matrix-isolation infrared spectroscopy with an aid of the density-functional-theory calculations. Only the most stable E form existed in an argon matrix. The second stable R form and the fourth stable H form were produced by UV irradiation (λ > 290 nm). The third stable O form was assumed to return immediately to E form even if it was produced. Ketoketene-water complex was yielded from R form by dissociation of the H and OH parts interacting through intramolecular hydrogen bonding upon prolonged UV irradiation.

  6. Evidence of salicylic acid pathway with EDS1 and PAD4 proteins by molecular dynamics simulation for grape improvement.

    Science.gov (United States)

    Tandon, Gitanjali; Jaiswal, Sarika; Iquebal, M A; Kumar, Sunil; Kaur, Sukhdeep; Rai, Anil; Kumar, Dinesh

    2015-01-01

    Biotic stress is a major cause of heavy loss in grape productivity. In order to develop biotic stress-resistant grape varieties, the key defense genes along with its pathway have to be deciphered. In angiosperm plants, lipase-like protein phytoalexin deficient 4 (PAD4) is well known to be essential for systemic resistance against biotic stress. PAD4 functions together with its interacting partner protein enhanced disease susceptibility 1 (EDS1) to promote salicylic acid (SA)-dependent and SA-independent defense pathway. Existence and structure of key protein of systemic resistance EDS1 and PAD4 are not known in grapes. Before SA pathway studies are taken in grape, molecular evidence of EDS1: PAD4 complex is to be established. To establish this, EDS1 protein sequence was retrieved from NCBI and homologous PAD4 protein was generated using Arabidopsis thaliana as template and conserved domains were confirmed. In this study, computational methods were used to model EDS1 and PAD4 and simulated the interactions of EDS1 and PAD4. Since no structural details of the proteins were available, homology modeling was employed to construct three-dimensional structures. Further, molecular dynamic simulations were performed to study the dynamic behavior of the EDS1 and PAD4. The modeled proteins were validated and subjected to molecular docking analysis. Molecular evidence of stable complex of EDS1:PAD4 in grape supporting SA defense pathway in response to biotic stress is reported in this study. If SA defense pathway genes are explored, then markers of genes involved can play pivotal role in grape variety development especially against biotic stress leading to higher productivity.

  7. The Heparan and Heparin Metabolism Pathway is Involved in Regulation of Fatty Acid Composition

    Science.gov (United States)

    Six genes involved in the heparan sulfate and heparin metabolism pathway, DSEL (dermatan sulfate epimerase-like), EXTL1 (exostoses (multiple)-like 1), HS6ST1 (heparan sulfate 6-O-sulfotransferase 1), HS6ST3 (heparan sulfate 6-O-sulfotransferase 3), NDST3 (N-deacetylase/N-sulfotransferase (heparan gl...

  8. 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...

  9. Burdock fructooligosaccharide induces fungal resistance in postharvest Kyoho grapes by activating the salicylic acid-dependent pathway and inhibiting browning.

    Science.gov (United States)

    Sun, Fei; Zhang, Pengying; Guo, Moran; Yu, Wenqian; Chen, Kaoshan

    2013-05-01

    Burdock fructooligosaccharide (BFO) is a natural elicitor from Arcitum lappa. The effects of BFO in controlling postharvest disease in grape, apple, banana, kiwi, citrus, strawberry, and pear were investigated. The disease index, decay percentage, and area under the disease progress curve indicated that BFO has general control effects on postharvest disease of fruits. Kyoho grapes were studied to elucidate the mechanism of BFO in boosting the resistance of grapes to Botrytis cinerea infection. BFO treatment induced upregulation of the npr1, pr1, pal, and sts genes, and inhibited the total phenol content decrease, which activated chitinase and β-1,3-glucanase. These results indicated that the salicylic acid-dependent signalling pathway was induced. The delayed colour change and peroxidase and polyphenoloxidase activity suggested that BFO delayed grape browning. The reduced respiration rate, weight loss, and titratable acidity prolonged the shelf life of postharvest grapes. BFO is a promising elicitor in postharvest disease control.

  10. Studies on Fragmentation Pathways of N-Ethoxy(phenyl) phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The positive and negative ESI-MS/MS spectra of N-ethoxy(phenyl) phosphoryl amino acids(EPP-AA) were investigated by electrospray ionization(ESI) ion trap mass spectrometry. The fragmentation pathways of [M + Na]+ and [M-H]- ions areproposed and rationalized. The observation may have some potential applications in the interpretation of the MS/MS spectra of novel N-phosphoryl compounds. The complexity of MS/MS spectra of EPP-AA [M + Na] + ions is decreased compared with that of N-dialkyloxyphosphoryl amino acid. Therefore, the new phosphonamidate method may be considered one of the superior methods that can be used in sequencing peptides and proteins extensively.

  11. Production of tartrates by cyanide-mediated dimerization of glyoxylate: a potential abiotic pathway to the citric acid cycle.

    Science.gov (United States)

    Butch, Christopher; Cope, Elizabeth D; Pollet, Pamela; Gelbaum, Leslie; Krishnamurthy, Ramanarayanan; Liotta, Charles L

    2013-09-11

    An abiotic formation of meso- and DL-tartrates in 80% yield via the cyanide-catalyzed dimerization of glyoxylate under alkaline conditions is demonstrated. A detailed mechanism for this conversion is proposed, supported by NMR evidence and (13)C-labeled reactions. Simple dehydration of tartrates to oxaloacetate and an ensuing decarboxylation to form pyruvate are known processes that provide a ready feedstock for entry into the citric acid cycle. While glyoxylate and high hydroxide concentration are atypical in the prebiotic literature, there is evidence for natural, abiotic availability of each. It is proposed that this availability, coupled with the remarkable efficiency of tartrate production from glyoxylate, merits consideration of an alternative prebiotic pathway for providing constituents of the citric acid cycle.

  12. 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

  13. 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

  14. Anti-inflammatory potential of 2-styrylchromones regarding their interference with arachidonic acid metabolic pathways

    OpenAIRE

    Gomes, Ana; Fernandes, Eduarda; Silva, Artur; Santos, Clementina M.M.; Pinto, Diana; Cavaleiro, José; Lima, José Costa

    2009-01-01

    Abstract Cyclooxygenases (COXs) are the key enzymes in the biosynthesis of prostanoids. COX-1 is a constitutive enzyme while the expression of COX-2 is highly stimulated in the event of inflammatory processes, leading to the production of large amounts of prostaglandins (PGs), in particular PGE2 and PGI2, which are pro-inflammatory mediators. Lipoxygenases (LOXs) are enzymes that produce hydroxy acids and leukotrienes (LTs). 5-LOX metabolizes arachidonic acid to yield, a...

  15. Both the Jasmonic Acid and the Salicylic Acid Pathways Contribute to Resistance to the Biotrophic Clubroot Agent Plasmodiophora brassicae in Arabidopsis.

    Science.gov (United States)

    Lemarié, Séverine; Robert-Seilaniantz, Alexandre; Lariagon, Christine; Lemoine, Jocelyne; Marnet, Nathalie; Jubault, Mélanie; Manzanares-Dauleux, Maria J; Gravot, Antoine

    2015-11-01

    The role of salicylic acid (SA) and jasmonic acid (JA) signaling in resistance to root pathogens has been poorly documented. We assessed the contribution of SA and JA to basal and partial resistance of Arabidopsis to the biotrophic clubroot agent Plasmodiophora brassicae. SA and JA levels as well as the expression of the SA-responsive genes PR2 and PR5 and the JA-responsive genes ARGAH2 and THI2.1 were monitored in infected roots of the accessions Col-0 (susceptible) and Bur-0 (partially resistant). SA signaling was activated in Bur-0 but not in Col-0. The JA pathway was weakly activated in Bur-0 but was strongly induced in Col-0. The contribution of both pathways to clubroot resistance was then assessed using exogenous phytohormone application and mutants affected in SA or JA signaling. Exogenous SA treatment decreased clubroot symptoms in the two Arabidopsis accessions, whereas JA treatment reduced clubroot symptoms only in Col-0. The cpr5-2 mutant, in which SA responses are constitutively induced, was more resistant to clubroot than the corresponding wild type, and the JA signaling-deficient mutant jar1 was more susceptible. Finally, we showed that the JA-mediated induction of NATA1 drove N(δ)-acetylornithine biosynthesis in infected Col-0 roots. The 35S::NATA1 and nata1 lines displayed reduced or enhanced clubroot symptoms, respectively, thus suggesting that in Col-0 this pathway was involved in the JA-mediated basal clubroot resistance. Overall, our data support the idea that, depending on the Arabidopsis accession, both SA and JA signaling can play a role in partial inhibition of clubroot development in compatible interactions with P. brassicae.

  16. Saturated Fatty Acid Induces Insulin Resistance Partially Through Nucleotide-binding Oligomerization Domain 1 Signaling Pathway in Adipocytes

    Institute of Scientific and Technical Information of China (English)

    Yi-jun Zhou; Yin-si Tang; Yu-ling Song; Ai Li; Hui Zhou; Yan Li

    2013-01-01

    Objective To investigate the potential role of nucleotide-binding oligomerization domain 1 (NOD1), a component of the innate immune system, in mediating lipid-induced insulin resistance in adipocytes. Methods Adipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments. The effect of oleate/palmitate mixture on nuclear factor-κB (NF-κB) activation was analyzed by reporter plasmid assay. The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[3H] glucose uptake assay. Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1 upon fatty acids treatment were analyzed. Results Oleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4, and these effects were blocked by siRNA targeting NOD1. Furthermore, saturated fatty acids decreased the ability of insulin-stimulated glucose uptake. Importantly, siRNA targeting NOD1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake. Conclusion NOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes, suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.

  17. Salicylic acid receptors activate jasmonic acid signalling through a non-canonical pathway to promote effector-triggered immunity

    Science.gov (United States)

    Liu, Lijing; Sonbol, Fathi-Mohamed; Huot, Bethany; Gu, Yangnan; Withers, John; Mwimba, Musoki; Yao, Jian; He, Sheng Yang; Dong, Xinnian

    2016-01-01

    It is an apparent conundrum how plants evolved effector-triggered immunity (ETI), involving programmed cell death (PCD), as a major defence mechanism against biotrophic pathogens, because ETI-associated PCD could leave them vulnerable to necrotrophic pathogens that thrive on dead host cells. Interestingly, during ETI, the normally antagonistic defence hormones, salicylic acid (SA) and jasmonic acid (JA) associated with defence against biotrophs and necrotrophs respectively, both accumulate to high levels. In this study, we made the surprising finding that JA is a positive regulator of RPS2-mediated ETI. Early induction of JA-responsive genes and de novo JA synthesis following SA accumulation is activated through the SA receptors NPR3 and NPR4, instead of the JA receptor COI1. We provide evidence that NPR3 and NPR4 may mediate this effect by promoting degradation of the JA transcriptional repressor JAZs. This unique interplay between SA and JA offers a possible explanation of how plants can mount defence against a biotrophic pathogen without becoming vulnerable to necrotrophic pathogens. PMID:27725643

  18. Hormone Signaling Pathways in Plants: The Role of Jasmonic Acid in Plant Cell Signaling

    OpenAIRE

    TİRYAKİ, İskender

    2004-01-01

    Plant growth and metabolism are affected by various biotic and abiotic stimuli including microorganisms and insects attack as well as light and environmental stresses. Such a diverse plant response requires a communication system that uses a group of chemical messengers called hormones. Hormones promote, inhibit, or qualitatively modify plant growth and development. This complex process requires a signal transduction that defines a specific information pathway within a cell that translat...

  19. Influence of ellagic acid on prostate cancer cell proliferation:A caspase-dependent pathway

    Institute of Scientific and Technical Information of China (English)

    Arshi Malik; Sarah Afaq; Mohammad Shahid; Kafil Akhtar; Abdullah Assiri

    2011-01-01

    Objective:To evaluate the effect of allagic acid treatment on the cell viability of human prostate cancer cells.Methods: Ellagic acid (10-100mol/L) treatment (48 h) of human prostate carcinomaPC3 cells was found to result in a dose-dependent inhibition of cell growth and apoptosis ofPC3 cells as assessed by MTTassay, western blotting, flow cytometry and confocal microscopy.Results: We observed that ellagic acid treatment ofPC3 cells resulted in a dose dependent inhibition of cell growth/cell viability. This ellagic acid caused cell growth inhibition was found to be accompanied by induction of apoptosis, as assessed by the cleavage of poly (ADP-ribose) polymerase(PARP) and morphological changes. Further, induction of apoptosis accompanied a decrease in the levels of antiapoptotic protein Bcl-2 and increase in proapoptotic protein Bax, thus shifting the Bax: Bcl-2 ratio in favor of apoptosis. Ellagic acid treatment of PC3 cells was also found to result in significant activation of caspases, as shown by the dose dependent decrease in the protein expression of procaspase-3, -6, -8 and-9. This ellagic acid-mediated induction of apoptosis was significantly (80%-90%) inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone(Z-VAD-FMK). Thus these data suggested an essential role of caspases in ellagic acid-mediated apoptosis ofPC3 cells.Conclusions:It is tempting to suggest that consumption of tropical pigmented fruits and vegetables could be an effective strategy to combat prostate cancer.

  20. 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. PMID:26853081

  1. All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways

    Directory of Open Access Journals (Sweden)

    Fang Bo

    2009-07-01

    Full Text Available Abstract Background All-trans retinoic acid (RA is one of the most important morphogens with pleiotropic actions. Its embryonic distribution correlates with neural differentiation in the developing central nervous system. To explore the precise effects of RA on neural differentiation of mouse embryonic stem cells (ESCs, we detected expression of RA nuclear receptors and RA-metabolizing enzymes in mouse ESCs and investigated the roles of RA in adherent monolayer culture. Results Upon addition of RA, cell differentiation was directed rapidly and exclusively into the neural lineage. Conversely, pharmacological interference with RA signaling suppressed this neural differentiation. Inhibition of fibroblast growth factor (FGF signaling did not suppress significantly neural differentiation in RA-treated cultures. Pharmacological interference with extracellular signal-regulated kinase (ERK pathway or activation of Wnt pathway effectively blocked the RA-promoted neural specification. ERK phosphorylation was enhanced in RA-treated cultures at the early stage of differentiation. Conclusion RA can promote neural lineage entry by ESCs in adherent monolayer culture systems. This effect depends on RA signaling and its crosstalk with the ERK and Wnt pathways.

  2. Effect of methacrylic acid beads on the sonic hedgehog signaling pathway and macrophage polarization in a subcutaneous injection mouse model.

    Science.gov (United States)

    Lisovsky, Alexandra; Zhang, David K Y; Sefton, Michael V

    2016-08-01

    Poly(methacrylic acid-co-methyl methacrylate) (MAA) beads promote a vascular regenerative response when used in diabetic wound healing. Previous studies reported that MAA beads modulated the expression of sonic hedgehog (Shh) and inflammation related genes in diabetic wounds. The aim of this work was to follow up on these observations in a subcutaneous injection model to study the host response in the absence of the confounding factors of diabetic wound healing. In this model, MAA beads improved vascularization in healthy mice of both sexes compared to control poly(methyl methacrylate) (MM) beads, with a stronger effect seen in males than females. MAA-induced vessels were perfusable, as evidenced from the CLARITY-processed images. In Shh-Cre-eGFP/Ptch1-LacZ non-diabetic transgenic mice, the increased vessel formation was accompanied by a higher density of cells expressing GFP (Shh) and β-Gal (patched 1, Ptch1) suggesting MAA enhanced the activation of the Shh pathway. Ptch1 is the Shh receptor and a target of the pathway. MAA beads also modulated the inflammatory cell infiltrate in CD1 mice: more neutrophils and more macrophages were noted with MAA relative to MM beads at days 1 and 7, respectively. In addition, MAA beads biased macrophages towards a MHCII-CD206+ ("M2") polarization state. This study suggests that the Shh pathway and an altered inflammatory response are two elements of the complex mechanism whereby MAA-based biomaterials effect vascular regeneration. PMID:27264502

  3. The effects of centrally injected arachidonic acid on respiratory system: Involvement of cyclooxygenase to thromboxane signaling pathway.

    Science.gov (United States)

    Erkan, Leman Gizem; Guvenc, Gokcen; Altinbas, Burcin; Niaz, Nasir; Yalcin, Murat

    2016-05-01

    Arachidonic acid (AA) is a polyunsaturated fatty acid that is present in the phospholipids of the cell membranes of the body and is abundant in the brain. Exogenously administered AA has been shown to affect brain metabolism and to exhibit cardiovascular and neuroendocrine actions. However, little is known regarding its respiratory actions and/or central mechanism of its respiratory effects. Therefore, the present study was designed to investigate the possible effects of centrally injected AA on respiratory system and the mediation of the central cyclooxygenase (COX) to thromboxane A2 (TXA2) signaling pathway on AA-induced respiratory effects in anaesthetized rats. Intracerebroventricular (i.c.v.) administration of AA induced dose- and time-dependent increase in tidal volume, respiratory rates and respiratory minute ventilation and also caused an increase in partial oxygen pressure (pO2) and decrease in partial carbon dioxide pressure (pCO2) in male anaesthetized Spraque Dawley rats. I.c.v. pretreatment with ibuprofen, a non-selective COX inhibitor, completely blocked the hyperventilation and blood gases changes induced by AA. In addition, central pretreatment with different doses of furegrelate, a TXA2 synthesis inhibitor, also partially prevented AA-evoked hyperventilation and blood gases effects. These data explicitly show that centrally administered AA induces hyperventilation with increasing pO2 and decreasing pCO2 levels which are mediated by the activation of central COX to TXA2 signaling pathway. PMID:26767978

  4. Clinical observation of thioctic acid combined with Isophane Protamine Biosynthetic Human Insulin Injection in treatment of type 2 diabetes%硫辛酸联合精蛋白生物合成人胰岛素注射液治疗2型糖尿病的疗效观察

    Institute of Scientific and Technical Information of China (English)

    胡开宇; 程晓芸; 汤春红

    2016-01-01

    目的:研究硫辛酸联合精蛋白生物合成人胰岛素注射液治疗2型糖尿病的临床疗效。方法选取2012年8月—2015年12月上海市闵行区龙柏社区卫生服务中心收治的2型糖尿病患者300例,随机分为对照组和治疗组,每组各150例。对照组患者皮下注射精蛋白生物合成人胰岛素注射液,初次为0.4 U/kg,2次/d 分别是每日早晚餐前,以后根据患者的治疗状况确定注射剂量的增减。治疗组在对照组的基础上静脉注射硫辛酸注射液,0.6 g 加入生理盐水250 mL,1次/d。两组均连续治疗1个月。观察两组胰岛素用量情况,比较治疗前后两组空腹血糖(FBG)、餐后2 h 血糖(2 h PBG)、糖化血红蛋白(HbA1c)、生活质量评分的变化。结果治疗组的胰岛素注射剂量在早、晚餐前,全天用量均显著低于对照组,两组比较差异具有统计学意义(P<0.05)。治疗后,两组患者 FBG、2 h PBG、HbA1c 均较治疗前显著降低,同组治疗前后差异具有统计学意义(P<0.05);治疗后治疗组这些观察指标的改善程度优于对照组,两组比较差异具有统计学意义(P<0.05)。治疗后,两组患者承受压力评分、社会关系评分、营养状况评分、运动状况评分均较治疗前显著增加,同组治疗前后差异具有统计学意义(P<0.05);治疗后治疗组这些观察指标的改善程度优于对照组,两组比较差异具有统计学意义(P<0.05)。结论硫辛酸联合精蛋白生物合成人胰岛素注射液治疗2型糖尿病具有较好的临床疗效,可减少患者注射胰岛素的剂量,能够更好地控制空腹血糖和餐后2 h 血糖,提高患者的生活质量,具有一定的临床推广应用价值。%Objective To investigate the clinical effect of thioctic acid combined with Isophane Protamine Biosynthetic Human Insulin Injection in treatment of type 2 diabetes. Methods Patients

  5. Expanding the diversity of unnatural cell surface sialic acids

    Energy Technology Data Exchange (ETDEWEB)

    Luchansky, Sarah J.; Goon, Scarlett; Bertozzi, Carolyn R.

    2003-10-30

    Novel chemical reactivity can be introduced onto cell surfaces through metabolic oligosaccharide engineering. This technique exploits the substrate promiscuity of cellular biosynthetic enzymes to deliver unnatural monosaccharides bearing bioorthogonal functional groups into cellular glycans. For example, derivatives of N-acetylmannosamine (ManNAc) are converted by the cellular biosynthetic machinery into the corresponding sialic acids and subsequently delivered to the cell surface in the form of sialoglycoconjugates. Analogs of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) are also metabolized and incorporated into cell surface glycans, likely through the sialic acid and GalNAc salvage pathways, respectively. Furthermore, GlcNAc analogs can be incorporated into nucleocytoplasmic proteins in place of {beta}-O-GlcNAc residues. These pathways have been exploited to integrate unique electrophiles such as ketones and azides into the target glycoconjugate class. These functional groups can be further elaborated in a chemoselective fashion by condensation with hydrazides and by Staudinger ligation, respectively, thereby introducing detectable probes onto the cell. In conclusion, sialic acid derivatives are efficient vehicles for delivery of bulky functional groups to cell surfaces and masking of their hydroxyl groups improves their cellular uptake and utilization. Furthermore, the successful introduction of photoactivatable aryl azides into cell surface glycans opens up new avenues for studying sialic acid-binding proteins and elucidating the role of sialic acid in essential processes such as signaling and cell adhesion.

  6. All-trans retinoic acid modulates mitogen-activated protein kinase pathway activation in human scleral fibroblasts through retinoic acid receptor beta

    Science.gov (United States)

    Huo, Lijun; Cui, Dongmei; Yang, Xiao; Gao, Zhenya; Trier, Klaus

    2013-01-01

    Purpose All-trans retinoic acid (ATRA) is known to inhibit the proliferation of human scleral fibroblasts (HSFs) and to modulate the scleral intercellular matrix composition, and may therefore serve as a mediator for controlling eye growth. Cell proliferation is regulated by the mitogen-activated protein kinase (MAPK) pathway. The aim of the current study was to investigate whether changed activation of the MAPK pathway could be involved in the response of HSFs exposed to ATRA. Methods HSFs were cultured in Dulbecco Modified Eagle's Medium/F12 (DMEM/F12) and exposed to 1 μmol/l ATRA for 10 min, 30 min, 1 h, 8 h, or 24 h. The activation of extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun N-terminal kinase (JNK) in HSFs was assessed with western blot analysis and immunocytofluorescence. Results After exposure to ATRA for 24 h, the HSFs appeared shrunken and thinner than the control cells. The intercellular spaces were wider, and the HSFs appeared less numerous than in the control culture. Western blot showed decreased activation of ERK 1/2 in the HSFs from 30 min (p=0.01) to 24 h (p<0.01) after the start of exposure to ATRA, and increased activation of the JNK protein from 10 to 30 min (p<0.01) after the start of exposure to ATRA. Indirect immunofluorescence confirmed changes in activation of ERK 1/2 and JNK in HSFs exposed to ATRA. No change in activation of p38 in HSFs was observed after exposure to ATRA. Pretreatment of the HSFs with LE135, an antagonist of retinoic acid receptor beta (RARβ), abolished the ATRA-induced changes inactivation of ERK 1/2 and JNK. Conclusions ATRA inhibits HSF proliferation by a mechanism associated with modulation of ERK 1/2 and JNK activation and depends on stimulation of retinoic acid receptor beta. PMID:23946634

  7. 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.

  8. Distinctive effects of eicosapentaenoic and docosahexaenoic acids in regulating neural stem cell fate are mediated via endocannabinoid signalling pathways.

    Science.gov (United States)

    Dyall, S C; Mandhair, H K; Fincham, R E A; Kerr, D M; Roche, M; Molina-Holgado, F

    2016-08-01

    Emerging evidence suggests a complex interplay between the endocannabinoid system, omega-3 fatty acids and the immune system in the promotion of brain self-repair. However, it is unknown if all omega-3 fatty acids elicit similar effects on adult neurogenesis and if such effects are mediated or regulated by interactions with the endocannabinoid system. This study investigated the effects of DHA and EPA on neural stem cell (NSC) fate and the role of the endocannabinoid signalling pathways in these effects. EPA, but not DHA, significantly increased proliferation of NSCs compared to controls, an effect associated with enhanced levels of the endocannabinoid 2-arachidonylglycerol (2-AG) and p-p38 MAPK, effects attenuated by pre-treatment with CB1 (AM251) or CB2 (AM630) receptor antagonists. Furthermore, in NSCs derived from IL-1β deficient mice, EPA significantly decreased proliferation and p-p38 MAPK levels compared to controls, suggesting a key role for IL-1β signalling in the effects observed. Although DHA similarly increased 2-AG levels in wild-type NSCs, there was no concomitant increase in proliferation or p-p38 MAPK activity. In addition, in NSCs from IL-1β deficient mice, DHA significantly increased proliferation without effects on p-P38 MAPK, suggesting effects of DHA are mediated via alternative signalling pathways. These results provide crucial new insights into the divergent effects of EPA and DHA in regulating NSC proliferation and the pathways involved, and highlight the therapeutic potential of their interplay with endocannabinoid signalling in brain repair. PMID:27044662

  9. Variation in oxygen isotope fractionation during cellulose synthesis: intramolecular and biosynthetic effects.

    Science.gov (United States)

    Sternberg, Leonel; Pinzon, Maria Camila; Anderson, William T; Jahren, A Hope

    2006-10-01

    The oxygen isotopic composition of plant cellulose is commonly used for the interpretations of climate, ecophysiology and dendrochronology in both modern and palaeoenvironments. Further applications of this analytical tool depends on our in-depth knowledge of the isotopic fractionations associated with the biochemical pathways leading to cellulose. Here, we test two important assumptions regarding isotopic effects resulting from the location of oxygen in the carbohydrate moiety and the biosynthetic pathway towards cellulose synthesis. We show that the oxygen isotopic fractionation of the oxygen attached to carbon 2 of the glucose moieties differs from the average fractionation of the oxygens attached to carbons 3-6 from cellulose by at least 9%, for cellulose synthesized within seedlings of two different species (Triticum aestivum L. and Ricinus communis L.). The fractionation for a given oxygen in cellulose synthesized by the Triticum seedlings, which have starch as their primary carbon source, is different than the corresponding fractionation in Ricinus seedlings, within which lipids are the primary carbon source. This observation shows that the biosynthetic pathway towards cellulose affects oxygen isotope partitioning, a fact heretofore undemonstrated. Our findings may explain the species-dependent variability in the overall oxygen isotope fractionation during cellulose synthesis, and may provide much-needed insight for palaeoclimate reconstruction using fossil cellulose.

  10. Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis.

    Science.gov (United States)

    Li, Mai; Zhang, Xiujun; Agrawal, Arpita; San, Ka-Yiu

    2012-07-01

    Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115

  11. Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source.

    OpenAIRE

    Dailey, F E; Cronan, J E

    1986-01-01

    Escherichia coli K-12 has two acetohydroxy acid synthase (AHAS) isozymes (AHAS I and AHAS III). Both of these isozymes catalyze the synthesis of alpha-aceto-alpha-hydroxybutyrate and alpha-acetolactate, which are key intermediates of the isoleucine-valine biosynthetic pathway. Strains lacking either isozyme but not both activities have been previously shown to grow well in minimal media in the absence of isoleucine and valine on any of several commonly used carbon sources (e.g., glucose or su...

  12. Investigations into the biosynthesis of salinosporamide A : new insights on PKS extender units and the origin of a nonproteinogenic amino acid

    OpenAIRE

    Liu, Yuan

    2010-01-01

    Salinosporamide A, a highly bioactive [beta]-lactone from the marine bacterium Salinispora tropica, originates from three biosynthetic building blocks, namely acetate, chloroethylmalonyl-CoA, and the nonproteinogenic amino acid cyclohexenylalanine. The unexpected and unprecedented pathway to chloroethylmalonyl-CoA was illuminated by a multidisciplinary approach involving genetics, organic synthesis, and protein biochemistry, where S-adenosyl-L- methionine (SAM) is converted to chloroethylmalo...

  13. A sequential bioorthogonal dual strategy: ManNAl and SiaNAl as distinct tools to unravel sialic acid metabolic pathways.

    Science.gov (United States)

    Gilormini, P A; Lion, C; Vicogne, D; Levade, T; Potelle, S; Mariller, C; Guérardel, Y; Biot, C; Foulquier, F

    2016-02-01

    Recent methodological developments in metabolic oligosaccharide engineering (MOE) pave the way for tremendous advances in glycobiology. Herein, we propose a Sequential Bioorthogonal Dual Strategy (SBDS) combining the use of two unprotected alkyne-tagged monosaccharide reporters (ManNAl and SiaNAl) with the bioligation of fluorescent probes by copper-catalysed azide-alkyne cycloaddition (CuAAC). With SBDS, we are able to shed light on trafficking and cellular uptake mechanisms of sialic acid. Using their corresponding analogues, we visualized that SiaNAl enters via endocytosis, whereas its biosynthetic intermediate ManNAl uptake is mediated by a yet unknown but specific plasma membrane transporter. Sialin, a lysosomal protein, is shown to be crucial for the export of exogenous sialic acid from lysosomes to the cytosol. Metabolic labeling with alkyne-tagged derivatives of N-acetylneuraminic acid (Neu5Ac) or N-acetylmannosamine (ManNAc) could thus be used to follow endocytosis in physiological vs. pathological conditions. PMID:26727964

  14. Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes.

    Science.gov (United States)

    Boudjelal, M; Wang, Z; Voorhees, J J; Fisher, G J

    2000-04-15

    Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of

  15. Exploring complex pheromone biosynthetic processes in the bumblebee male labial gland by RNA sequencing.

    Science.gov (United States)

    Buček, A; Brabcová, J; Vogel, H; Prchalová, D; Kindl, J; Valterová, I; Pichová, I

    2016-06-01

    Male marking pheromones (MPs) are used by the majority of bumblebee species (Hymenoptera: Apidae), including a commercially important greenhouse pollinator, the buff-tailed bumblebee (Bombus terrestris), to attract conspecific females. MP biosynthetic processes in the cephalic part of the bumblebee male labial gland (LG) are of extraordinary complexity, involving enzymes of fatty acid and isoprenoid biosynthesis, which jointly produce more than 50 compounds. We employed a differential transcriptomic approach to identify candidate genes involved in MP biosynthesis by sequencing Bombus terrestris LG and fat body (FB) transcriptomes. We identified 12 454 abundantly expressed gene products (reads per kilobase of exon model per million mapped reads value > 1) that had significant hits in the GenBank nonredundant database. Of these, 876 were upregulated in the LG (> 4-fold difference). We identified more than 140 candidate genes potentially involved in MP biosynthesis, including esterases, fatty acid reductases, lipases, enzymes involved in limited fatty acid chain shortening, neuropeptide receptors and enzymes involved in biosynthesis of triacylglycerols, isoprenoids and fatty acids. For selected candidates, we confirmed their abundant expression in LG using quantitative real-time reverse transcription-PCR (qRT-PCR). Our study shows that the Bombus terrestris LG transcriptome reflects both fatty acid and isoprenoid MP biosynthetic processes and identifies rational gene targets for future studies to disentangle the molecular basis of MP biosynthesis. Additionally, LG and FB transcriptomes enrich the available transcriptomic resources for Bombus terrestris. PMID:26945888

  16. Deoxygenation of benzoic acid on metal oxides. I. The selective pathway to benzaldehyde

    NARCIS (Netherlands)

    Lange, de M.W.; Ommen, van J.G.; Lefferts, L.

    2001-01-01

    The mechanism of the selective deoxygenation of benzoic acid to benzaldehyde was studied on ZnO and ZrO2. The results show conclusively that the reaction proceeds as a reverse type of Mars and van Krevelen mechanism consisting of two steps: hydrogen activates the oxide by reduction resulting in the

  17. The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway

    Directory of Open Access Journals (Sweden)

    Bourgaud Frédéric

    2009-03-01

    Full Text Available Abstract Background The leaves of globe artichoke and cultivated cardoon (Cynara cardunculus L. have significant pharmaceutical properties, which mainly result from their high content of polyphenolic compounds such as monocaffeoylquinic and dicaffeoylquinic acid (DCQ, and a range of flavonoid compounds. Results Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase (HQT encoding genes have been isolated from both globe artichoke and cultivated cardoon (GenBank accessions DQ915589 and DQ915590, respectively using CODEHOP and PCR-RACE. A phylogenetic analysis revealed that their sequences belong to one of the major acyltransferase groups (anthranilate N-hydroxycinnamoyl/benzoyltransferase. The heterologous expression of globe artichoke HQT in E. coli showed that this enzyme can catalyze the esterification of quinic acid with caffeoyl-CoA or p-coumaroyl-CoA to generate, respectively, chlorogenic acid (CGA and p-coumaroyl quinate. Real time PCR experiments demonstrated an increase in the expression level of HQT in UV-C treated leaves, and established a correlation between the synthesis of phenolic acids and protection against damage due to abiotic stress. The HQT gene, together with a gene encoding hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT previously isolated from globe artichoke, have been incorporated within the developing globe artichoke linkage maps. Conclusion A novel acyltransferase involved in the biosynthesis of CGA in globe artichoke has been isolated, characterized and mapped. This is a good basis for our effort to understand the genetic basis of phenylpropanoid (PP biosynthesis in C. cardunculus.

  18. Profiling the Changes in Signaling Pathways in Ascorbic Acid/beta-Glycerophosphate-Induced Osteoblastic Differentiation

    NARCIS (Netherlands)

    Chaves Neto, Antonio Hernandes; Queiroz, Karla Cristiana; Milani, Renato; Paredes-Gamero, Edgar Julian; Justo, Giselle Zenker; Peppelenbosch, Maikel P.; Ferreira, Carmen Verissima

    2011-01-01

    Despite numerous reports on the ability of ascorbic acid and beta-glycerophosphate (AA/beta-GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential s

  19. Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway.

    Science.gov (United States)

    Amonyingcharoen, Sumet; Suriyo, Tawit; Thiantanawat, Apinya; Watcharasit, Piyajit; Satayavivad, Jutamaad

    2015-01-01

    Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1-40 µM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth.

  20. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    Energy Technology Data Exchange (ETDEWEB)

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong

    2011-12-31

    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  1. Isolation and characterisation of 8-hydroxy-3Z,5Z-tetradecadienoic acid, a putative intermediate in Pichia guilliermondii gamma-decalactone biosynthesis from ricinoleic acid.

    Science.gov (United States)

    Iacazio, G; Martini, D; Faure, B; N'Guyen, M H

    2002-03-19

    During a screening procedure for the discovery of a strong gamma-decalactone producer from ricinoleic acid, we observed that the yeast Pichia guilliermondii accumulated transiently 8-hydroxy-3Z,5Z-tetradecadienoic acid 1 during gamma-decalactone biosynthesis in the stationary phase of growth. The structural elucidation of 1 was based on nuclear magnetic resonance, infrared, ultraviolet and gas chromatography-mass spectrometry experiments. The occurrence of 1 is discussed in relation with previously proposed gamma-decalactone biosynthetic pathways. PMID:12007654

  2. NREL Creates New Pathways for Producing Biofuels and Acids from Cyanobacteria (Fact Sheet)

    Energy Technology Data Exchange (ETDEWEB)

    2012-10-01

    Cyanobacteria use photosynthesis to convert carbon dioxide into glycogen, a carbohydrate that is stored in the cells as an energy source. However, researchers at the National Renewable Energy Laboratory (NREL) have discovered that this photosynthesis can be redirected to produce lipids and valuable organic acids. The research could yield a new source of biofuels, because the lipids can potentially be extracted from the bacteria and converted into biodiesel.

  3. Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells

    OpenAIRE

    Wang, Li; Liu, Yuan; Li, Sen; Zai-yun LONG; Wu, Ya-min

    2015-01-01

    Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cor...

  4. Ginkgolic acid suppresses the development of pancreatic cancer by inhibiting pathways driving lipogenesis

    OpenAIRE

    Ma, Jiguang; Duan, Wanxing; Han, Suxia; Lei, Jianjun; Xu, Qinhong; Chen, Xin; Jiang, Zhengdong; Nan, Ligang; Li, Jiahui; Chen, Ke; Han, Liang; Wang, Zheng; Li, Xuqi; Wu, Erxi; HUO, XIONGWEI

    2015-01-01

    Ginkgolic acid (GA) is a botanical drug extracted from the seed coat of Ginkgo biloba L. with a wide range of bioactive properties, including anti-tumor effect. However, whether GA has antitumor effect on pancreatic cancer cells and the underlying mechanisms have yet to be investigated. In this study, we show that GA suppressed the viability of cancer cells but has little toxicity on normal cells, e.g, HUVEC cells. Furthermore, treatment of GA resulted in impaired colony formation, migration,...

  5. Abscisic acid and blue light signaling pathways in chloroplast movements in Arabidopsis mesophyll.

    Science.gov (United States)

    Eckstein, Aleksandra; Krzeszowiec, Weronika; Banaś, Agnieszka Katarzyna; Janowiak, Franciszek; Gabryś, Halina

    2016-01-01

    Abscisic acid (ABA) and phototropins act antagonistically to control stomatal movements. Here, we investigated the role of ABA in phototropin-directed chloroplast movements in mesophyll cells of Arabidopsis thaliana. We analyzed the expression of phototropins at mRNA and protein level under the influence of ABA. PHOT1 mRNA level was decreased by ABA in the dark while it was insensitive to ABA in light. PHOT2 mRNA level was independent of the hormone treatment. The levels of phototropin proteins were down-regulated by ABA, both in darkness and light. No impact of exogenous ABA on amplitudes and kinetics of chloroplast movements was detected. Chloroplast responses in wild type Arabidopsis and three mutants, abi4, abi2 (abscisic acid insensitive4, 2) and aba1 (abscisic acid1), were measured to account for endogenous ABA signaling. The chloroplast responses were slightly reduced in abi2 and aba1 mutants in strong light. To further investigate the effect, abi2 and aba1 mutants were supplemented with exogenous ABA. In the aba1 mutant, the reaction was rescued but in abi2 it was unaffected. Our results show that ABA is not directly involved in phototropin-controlled chloroplast responses in mature leaves of Arabidopsis. However, the disturbance of ABA biosynthesis and signaling in mutants affects some elements of the chloroplast movement mechanism. In line with its role as a stress hormone, ABA appears to enhance plant sensitivity to light and promote the chloroplast avoidance response.

  6. Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

    Science.gov (United States)

    Goswami, Moloy T; Chen, Guoan; Chakravarthi, Balabhadrapatruni V S K; Pathi, Satya S; Anand, Sharath K; Carskadon, Shannon L; Giordano, Thomas J; Chinnaiyan, Arul M; Thomas, Dafydd G; Palanisamy, Nallasivam; Beer, David G; Varambally, Sooryanarayana

    2015-09-15

    Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention. PMID:26140362

  7. Stress-induced biosynthesis of dicaffeoylquinic acids in globe artichoke.

    Science.gov (United States)

    Moglia, Andrea; Lanteri, Sergio; Comino, Cinzia; Acquadro, Alberto; de Vos, Ric; Beekwilder, Jules

    2008-09-24

    Leaf extracts from globe artichoke ( Cynara cardunculus L. var. scolymus) have been widely used in medicine as hepatoprotectant and choleretic agents. Globe artichoke leaves represent a natural source of phenolic acids with dicaffeoylquinic acids, such as cynarin (1,3-dicaffeoylquinic acid), along with its biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the most abundant molecules. This paper reports the development of an experimental system to induce caffeoylquinic acids. This system may serve to study the regulation of the biosynthesis of (poly)phenolic compounds in globe artichoke and the genetic basis of this metabolic regulation. By means of HPLC-PDA and accurate mass LC-QTOF MS and MS/MS analyses, the major phenolic compounds in globe artichoke leaves were identified: four isomers of dicaffeoylquinic acid, three isomers of caffeoylquinic acid, and the flavone luteolin 7-glucoside. Next, plant material was identified in which the concentration of phenolic compounds was comparable in the absence of particular treatments, with the aim to use this material to test the effect of stress application on the regulation of biosynthesis of caffeoylquinic acids. Using this material, the effect of UV-C, methyl jasmonate, and salicylic acid treatments on (poly)phenolic compounds was tested in different globe artichoke genotypes. UV-C exposure consistently increased the levels of dicaffeoylquinic acids in all genotypes, whereas the effect on compounds from the same biosynthetic pathway, for example, chlorogenic acid and luteolin-7-glucoside, was much less pronounced and was not statistically significant. No effect of methyl jasmonate or salicylic acid was found. Time-response experiments indicated that the level of dicaffeoylquinic acids reached a maximum at 24 h after UV radiation. On the basis of these results a role of dicaffeoylquinic acids in UV protection in globe artichoke is hypothesized. PMID:18710252

  8. Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

    Institute of Scientific and Technical Information of China (English)

    Agus Suryawan; Teresa ADavis

    2014-01-01

    Background:The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the current study, we identified mechanisms involved in protein degradation regulation. In experiment 1, 6-and 26-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic, 2) euinsulinemic-euglycemic-hyperaminoacidemic, and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps for 2 h. In experiment 2, 5-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic-euleucinemic, 2) euinsulinemic-euglycemic-hypoaminoacidemic-hyperleucinemic, and 3) euinsulinemic-euglycemic-euaminoacidemic-hyperleucinemic clamps for 24 h. We determined in muscle indices of ubiquitin-proteasome, i.e., atrogin-1 (MAFbx) and muscle RING-finger protein-1 (MuRF1) and autophagy-lysosome systems, i.e., unc51-like kinase 1 (UKL1), microtubule-associated protein light chain 3 (LC3), and lysosomal-associated membrane protein 2 (Lamp-2). For comparison, we measured ribosomal protein S6 (rpS6) and eukaryotic initiation factor 4E (eIF4E) activation, components of translation initiation. Results:Abundance of atrogin-1, but not MuRF1, was greater in 26-than 6-d-old pigs and was not affected by insulin, amino acids, or leucine. Abundance of ULK1 and LC3 was higher in younger pigs and not affected by treatment. The LC3-II/LC3-I ratio was reduced and ULK1 phosphorylation increased by insulin, amino acids, and leucine. These responses were more profound in younger pigs. Abundance of Lamp-2 was not affected by treatment or development. Abundance of eIF4E, but not rpS6, was higher in 6-than 26-d-old-pigs but unaffected by treatment. Phosphorylation of eIF4E was not affected by treatment, however, insulin, amino acids, and leucine stimulated rpS6 phosphorylation, and the

  9. Expression of Terpenoid Biosynthetic Genes and Accumulation of Chemical Constituents in Valeriana fauriei

    Directory of Open Access Journals (Sweden)

    Yun Ji Park

    2016-05-01

    Full Text Available Valeriana fauriei (V. fauriei, which emits a characteristic and unpleasant odor, is important in traditional medicine. In this study, the expression of terpenoid biosynthetic genes was investigated in different organs that were also screened for volatile compounds including valerenic acid and its derivatives. Specific expression patterns from different parts of V. fauriei were observed using quantitative real-time PCR (qRT-PCR. The highest transcript levels of biosynthetic genes involved in mevalonic acid (MVA and methylerythritol phosphate (MEP production were found in the stem. Although the amounts of volatile compounds were varied by organ, most of the volatile terpenoids were accumulated in the root. Gas chromatography mass spectrometry (GC-MS analysis identified 128 volatile compounds, which represented 65.33% to 95.66% of total volatiles. Certain compounds were only found in specific organs. For example, isovalerenic acid and valerenic acid and its derivatives were restricted to the root. Organs with high transcript levels did not necessarily have high levels of the corresponding chemical constituents. According to these results, we hypothesize that translocation may occur between different organs in V. fauriei.

  10. Competitive fragmentation pathways of acetic acid dimer explored by synchrotron VUV photoionization mass spectrometry and electronic structure calculations

    International Nuclear Information System (INIS)

    In present study, photoionization and dissociation of acetic acid dimers have been studied with the synchrotron vacuum ultraviolet photoionization mass spectrometry and theoretical calculations. Besides the intense signal corresponding to protonated cluster ions (CH3COOH)n·H+, the feature related to the fragment ions (CH3COOH)H+·COO (105 amu) via β-carbon-carbon bond cleavage is observed. By scanning photoionization efficiency spectra, appearance energies of the fragments (CH3COOH)·H+ and (CH3COOH)H+·COO are obtained. With the aid of theoretical calculations, seven fragmentation channels of acetic acid dimer cations were discussed, where five cation isomers of acetic acid dimer are involved. While four of them are found to generate the protonated species, only one of them can dissociate into a C–C bond cleavage product (CH3COOH)H+·COO. After surmounting the methyl hydrogen-transfer barrier 10.84 ± 0.05 eV, the opening of dissociative channel to produce ions (CH3COOH)+ becomes the most competitive path. When photon energy increases to 12.4 eV, we also found dimer cations can be fragmented and generate new cations (CH3COOH)·CH3CO+. Kinetics, thermodynamics, and entropy factors for these competitive dissociation pathways are discussed. The present report provides a clear picture of the photoionization and dissociation processes of the acetic acid dimer in the range of the photon energy 9–15 eV.

  11. Arsenite tolerance in rice (Oryza sativa L.) involves coordinated role of metabolic pathways of thiols and amino acids.

    Science.gov (United States)

    Tripathi, Preeti; Tripathi, Rudra Deo; Singh, Rana Pratap; Dwivedi, Sanjay; Chakrabarty, Debasis; Trivedi, Prabodh K; Adhikari, Bijan

    2013-02-01

    Thiolic ligands and several amino acids (AAs) are known to build up in plants against heavy metal stress. In the present study, alteration of various AAs in rice and its synchronized role with thiolic ligand was explored for arsenic (As) tolerance and detoxification. To understand the mechanism of As tolerance and stress response, rice seedlings of one tolerant (Triguna) and one sensitive (IET-4786) cultivar were exposed to arsenite (0-25 μM) for 7 days for various biochemical analyses using spectrophotometer, HPLC and ICPMS. Tolerant and sensitive cultivars respond differentially in terms of thiol metabolism, essential amino acids (EEAs) and nonessential amino acids (NEEAs) vis-á-vis As accumulation. Thiol biosynthesis-related enzymes were positively correlated to As accumulation in Triguna. Conversely, these enzymes, cysteine content and GSH/GSSG ratio declined significantly in IET-4786 upon As exposure. The level of identified phytochelatin (PC) species (PC(2), PC(3) and PC(4)) and phytochelatin synthase activity were also more pronounced in Triguna than IET-4786. Nearly all EAAs were negatively affected by As-induced oxidative stress (except phenylalanine in Triguna), but more significantly in IET-4786 than Triguna. However, most of the stress-responsive NEAAs like glutamic acid, histidine, alanine, glycine, tyrosine, cysteine and proline were enhanced more prominently in Triguna than IET-4786 upon As exposure. The study suggests that IET-4786 appears sensitive to As due to reduction of AAs and thiol metabolic pathway. However, a coordinated response of thiolic ligands and stress-responsive AAs seems to play role for As tolerance in Triguna to achieve the effective complexation of As by PCs.

  12. Biochemical and structural characterization of Klebsiella pneumoniae oxamate amidohydrolase in the uric acid degradation pathway

    Energy Technology Data Exchange (ETDEWEB)

    Hicks, Katherine A.; Ealick, Steven E.

    2016-05-25

    HpxW from the ubiquitous pathogenKlebsiella pneumoniaeis involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.

  13. Gallic acid induces necroptosis via TNF-α signaling pathway in activated hepatic stellate cells.

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    Ya Ju Chang

    Full Text Available Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA, a natural phenolic acid widely found in gallnuts, tea leaves and various fruits, possesses several bioactivities against inflammation, oxidation, and carcinogenicity. The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property. However, the cytotoxicity of GA autoxidation causing cell death has also been reported. Herein, we postulated that GA might target activated hepatic stellate cells (aHSCs, the cell type responsible for hepatofibrosis, to mitigate the process of fibrosis. The molecular cytotoxic mechanisms that GA exerted on aHSCs were then analyzed. The results indicated that GA elicited aHSC programmed cell death through TNF-α-mediated necroptosis. GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH. Elevated oxidative stress triggered the production of TNF-α facilitating the undergoing of necroptosis through the up-regulation of key necroptotic regulatory proteins TRADD and receptor-interacting protein 3 (RIP3, and the inactivation of caspase-8. Calmodulin and calpain-1 activation were engaged, which promoted subsequent lysosomal membrane permeabilization (LMP. The TNF-α antagonist (SPD-304 and the RIP1 inhibitor (necrostatin-1, Nec-1 confirmed GA-induced TNFR1-mediated necroptosis. The inhibition of RIP1 by Nec-1 diverted the cell death from necroptosis to apoptosis, as the activation of caspase 3 and the increase of cytochrome c. Collectively, this is the first report indicating that GA induces TNF signaling-triggered necroptosis in aHSCs, which may offer an alternative strategy for the amelioration of liver fibrosis.

  14. Chlorogenic Acid Improves Late Diabetes through Adiponectin Receptor Signaling Pathways in db/db Mice

    OpenAIRE

    Shasha Jin; Cuiqing Chang; Lantao Zhang; Yang Liu; Xianren Huang; Zhimin Chen

    2015-01-01

    The aim of this study was to examine the effects of chlorogenic acid (CGA) on glucose and lipid metabolism in late diabetic db/db mice, as well as on adiponectin receptors and their signaling molecules, to provide evidence for CGA in the prevention of type 2 diabetes. We randomly divided 16 female db/db mice into db/db-CGA and db/db-control (CON) groups equally; db/m mice were used as control mice. The mice in both the db/db-CGA and db/m-CGA groups were administered 80 mg/kg/d CGA by lavage f...

  15. Alleviation of Drought Stress by Hydrogen Sulfide Is Partially Related to the Abscisic Acid Signaling Pathway in Wheat

    Science.gov (United States)

    Wang, Chenyang; Qin, Haixia; Han, Qiaoxia; Hou, Junfeng; Lu, Hongfang; Xie, Yingxin; Guo, Tiancai

    2016-01-01

    Little information is available describing the effects of exogenous H2S on the ABA pathway in the acquisition of drought tolerance in wheat. In this study, we investigated the physiological parameters, the transcription levels of several genes involved in the abscisic acid (ABA) metabolism pathway, and the ABA and H2S contents in wheat leaves and roots under drought stress in response to exogenous NaHS treatment. The results showed that pretreatment with NaHS significantly increased plant height and the leaf relative water content of seedlings under drought stress. Compared with drought stress treatment alone, H2S application increased antioxidant enzyme activities and reduced MDA and H2O2 contents in both leaves and roots. NaHS pretreatment increased the expression levels of ABA biosynthesis and ABA reactivation genes in leaves; whereas the expression levels of ABA biosynthesis and ABA catabolism genes were up-regulated in roots. These results indicated that ABA participates in drought tolerance induced by exogenous H2S, and that the responses in leaves and roots are different. The transcription levels of genes encoding ABA receptors were up-regulated in response to NaHS pretreatment under drought conditions in both leaves and roots. Correspondingly, the H2S contents in leaves and roots were increased by NaHS pretreatment, while the ABA contents of leaves and roots decreased. This implied that there is complex crosstalk between these two signal molecules, and that the alleviation of drought stress by H2S, at least in part, involves the ABA signaling pathway. PMID:27649534

  16. Alleviation of Drought Stress by Hydrogen Sulfide Is Partially Related to the Abscisic Acid Signaling Pathway in Wheat.

    Science.gov (United States)

    Ma, Dongyun; Ding, Huina; Wang, Chenyang; Qin, Haixia; Han, Qiaoxia; Hou, Junfeng; Lu, Hongfang; Xie, Yingxin; Guo, Tiancai

    2016-01-01

    Little information is available describing the effects of exogenous H2S on the ABA pathway in the acquisition of drought tolerance in wheat. In this study, we investigated the physiological parameters, the transcription levels of several genes involved in the abscisic acid (ABA) metabolism pathway, and the ABA and H2S contents in wheat leaves and roots under drought stress in response to exogenous NaHS treatment. The results showed that pretreatment with NaHS significantly increased plant height and the leaf relative water content of seedlings under drought stress. Compared with drought stress treatment alone, H2S application increased antioxidant enzyme activities and reduced MDA and H2O2 contents in both leaves and roots. NaHS pretreatment increased the expression levels of ABA biosynthesis and ABA reactivation genes in leaves; whereas the expression levels of ABA biosynthesis and ABA catabolism genes were up-regulated in roots. These results indicated that ABA participates in drought tolerance induced by exogenous H2S, and that the responses in leaves and roots are different. The transcription levels of genes encoding ABA receptors were up-regulated in response to NaHS pretreatment under drought conditions in both leaves and roots. Correspondingly, the H2S contents in leaves and roots were increased by NaHS pretreatment, while the ABA contents of leaves and roots decreased. This implied that there is complex crosstalk between these two signal molecules, and that the alleviation of drought stress by H2S, at least in part, involves the ABA signaling pathway. PMID:27649534

  17. Ursolic acid simultaneously targets multiple signaling pathways to suppress proliferation and induce apoptosis in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Jingshu Wang

    Full Text Available Ursolic acid (UA, a natural pentacyclic triterpenoid carboxylic acid distributed in medical herbs, exerts antitumor effects and is emerging as a promising compound for cancer prevention and therapy, but its excise mechanisms of action in colon cancer cells remains largely unknown. Here, we identified the molecular mechanisms by which UA inhibited cell proliferation and induced apoptosis in human colon cancer SW480 and LoVo cells. Treatment with UA led to significant inhibitions in cell viability and clone formation and changes in cell morphology and spreading. UA also suppressed colon cancer cell migration by inhibiting MMP9 and upregulating CDH1 expression. Further studies showed that UA inhibited the phosphorylation of Akt and ERK proteins. Pretreatment with an Akt or ERK-specific inhibitor considerably abrogated the proliferation inhibition by UA. UA also significantly inhibited colon cancer cell COX-2 expression and PGE2 production. Pretreatment with a COX-2 inhibitor (celecoxib abrogated the UA-induced cell proliferation. Moreover, we found that UA effectively promoted NF-κB and p300 translocation from cell nuclei to cytoplasm, and attenuated the p300-mediated acetylation of NF-κB and CREB2. Pretreatment with a p300 inhibitor (roscovitine abrogated the UA-induced cell proliferation, which is reversed by p300 overexpression. Furthermore, UA treatment induced colon cancer cell apoptosis, increased the cleavage of PARP, caspase-3 and 9, and trigged the release of cytochrome c from mitochondrial inter-membrane space into cytosol. These results indicate that UA inhibits cell proliferation and induces apoptosis in colon cancer cells through simultaneous modulation of the multiple signaling pathways such as MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase pathways.

  18. Induction of G2/M arrest by pseudolaric acid B is mediated by activation of the ATM signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Ai-guo MENG; Ling-lingJIANG

    2009-01-01

    Aim: The aim of this study was to investigate the mechanism of pseudolaric acid B (PLAB)-induced cell cycle arrest in human melanoma SK-28 cells. Methods: Cell growth inhibition was detected by MTT assay, the cell cycle was analyzed by flow cytometry, and protein expression was examined by Western blot analysis.Results: PLAB inhibited the growth of human melanoma ceils and induced G2/M arrest in SK-28 cells, accompanied by an up-regulation of Cdc2 phosphorylation and a subsequent down-regulation of Cdc2 expression. Furthermore, PLAB decreased the expression of Cdc25C phosphatase and increased the expression of Wee1 kinase. Meanwhile, a reduction in Cdc2 activity was party due to induction of the expression of p21wsaf1/cip1 in a p53-dependent manner. In addition, PLAB activated the checkpoint kinase, Chk2, and increased the expression of p53, two major targets of ATM kinase. These effects were inhibited by caffeine, an ATM kinase inhibitor. We also found that PLAB significantly enhanced ATM kinase activity. Conclusion: Taken together, these results suggest that PLAB induced G2/M arrest in human melanoma cells via a mechanism involving the activation of ATM, and the effect of PLAB on Cdc2 activity was mediated via interactions with the Chk2-Cdc25C and p53 signalling pathways, two distinct downstream pathways of ATM. PLAB may be a promising chemopreventive agent for treating human melanoma.

  19. UV-C-Induced alleviation of transcriptional gene silencing through plant-plant communication: Key roles of jasmonic acid and salicylic acid pathways.

    Science.gov (United States)

    Xu, Wei; Wang, Ting; Xu, Shaoxin; Li, Fanghua; Deng, Chenguang; Wu, Lijun; Wu, Yuejin; Bian, Po

    2016-08-01

    Plant stress responses at the epigenetic level are expected to allow more permanent changes of gene expression and potentially long-term adaptation. While it has been reported that plants subjected to adverse environments initiate various stress responses in their neighboring plants, little is known regarding epigenetic responses to external stresses mediated by plant-plant communication. In this study, we show that DNA repetitive elements of Arabidopsis thaliana, whose expression is inhibited epigenetically by transcriptional gene silencing (TGS) mechanism, are activated by UV-C irradiation through airborne plant-plant and plant-plant-plant communications, accompanied by DNA demethylation at CHH sites. Moreover, the TGS is alleviated by direct treatments with exogenous methyl jasmonate (MeJA) and methyl salicylate (MeSA). Further, the plant-plant and plant-plant-plant communications are blocked by mutations in the biosynthesis or signaling of jasmonic acid (JA) or salicylic acid (SA), indicating that JA and SA pathways are involved in the interplant communication for epigenetic responses. For the plant-plant-plant communication, stress cues are relayed to the last set of receiver plants by promoting the production of JA and SA signals in relaying plants, which exhibit upregulated expression of genes for JA and SA biosynthesis and enhanced emanation of MeJA and MeSA. PMID:27131397

  20. An Oral Load of [13C3]Glycerol and Blood NMR Analysis Detect Fatty Acid Esterification, Pentose Phosphate Pathway, and Glycerol Metabolism through the Tricarboxylic Acid Cycle in Human Liver.

    Science.gov (United States)

    Jin, Eunsook S; Sherry, A Dean; Malloy, Craig R

    2016-09-01

    Drugs and other interventions for high impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis, or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-(13)C3]glycerol, and blood was drawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution (13)C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-(13)C3]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis, and flux through anaplerotic pathways in mitochondria of human liver.

  1. Partial Decay of Thiamine Signal Transduction Pathway Alters Growth Properties of Candida glabrata

    Science.gov (United States)

    Shaik, Noor F.; Neal, Erin M.; Leone, Sarah G.; Cali, Brian J.; Peel, Michael T.; Grannas, Amanda M.; Wykoff, Dennis D.

    2016-01-01

    The phosphorylated form of thiamine (Vitamin B1), thiamine pyrophosphate (TPP) is essential for the metabolism of amino acids and carbohydrates in all organisms. Plants and microorganisms, such as yeast, synthesize thiamine de novo whereas animals do not. The thiamine signal transduction (THI) pathway in Saccharomyces cerevisiae is well characterized. The ~10 genes required for thiamine biosynthesis and uptake are transcriptionally upregulated during thiamine starvation by THI2, THI3, and PDC2. Candida glabrata, a human commensal and opportunistic pathogen, is closely related to S. cerevisiae but is missing half of the biosynthetic pathway, which limits its ability to make thiamine. We investigated the changes to the THI pathway in C. glabrata, confirming orthologous functions. We found that C. glabrata is unable to synthesize the pyrimidine subunit of thiamine as well as the thiamine precursor vitamin B6. In addition, THI2 (the gene encoding a transcription factor) is not present in C. glabrata, indicating a difference in the transcriptional regulation of the pathway. Although the pathway is upregulated by thiamine starvation in both species, C. glabrata appears to upregulate genes involved in thiamine uptake to a greater extent than S. cerevisiae. However, the altered regulation of the THI pathway does not alter the concentration of thiamine and its vitamers in the two species as measured by HPLC. Finally, we demonstrate potential consequences to having a partial decay of the THI biosynthetic and regulatory pathway. When the two species are co-cultured, the presence of thiamine allows C. glabrata to rapidly outcompete S. cerevisiae, while absence of thiamine allows S. cerevisiae to outcompete C. glabrata. This simplification of the THI pathway in C. glabrata suggests its environment provides thiamine and/or its precursors to cells, whereas S. cerevisiae is not as reliant on environmental sources of thiamine. PMID:27015653

  2. Partial Decay of Thiamine Signal Transduction Pathway Alters Growth Properties of Candida glabrata.

    Directory of Open Access Journals (Sweden)

    Christine L Iosue

    Full Text Available The phosphorylated form of thiamine (Vitamin B1, thiamine pyrophosphate (TPP is essential for the metabolism of amino acids and carbohydrates in all organisms. Plants and microorganisms, such as yeast, synthesize thiamine de novo whereas animals do not. The thiamine signal transduction (THI pathway in Saccharomyces cerevisiae is well characterized. The ~10 genes required for thiamine biosynthesis and uptake are transcriptionally upregulated during thiamine starvation by THI2, THI3, and PDC2. Candida glabrata, a human commensal and opportunistic pathogen, is closely related to S. cerevisiae but is missing half of the biosynthetic pathway, which limits its ability to make thiamine. We investigated the changes to the THI pathway in C. glabrata, confirming orthologous functions. We found that C. glabrata is unable to synthesize the pyrimidine subunit of thiamine as well as the thiamine precursor vitamin B6. In addition, THI2 (the gene encoding a transcription factor is not present in C. glabrata, indicating a difference in the transcriptional regulation of the pathway. Although the pathway is upregulated by thiamine starvation in both species, C. glabrata appears to upregulate genes involved in thiamine uptake to a greater extent than S. cerevisiae. However, the altered regulation of the THI pathway does not alter the concentration of thiamine and its vitamers in the two species as measured by HPLC. Finally, we demonstrate potential consequences to having a partial decay of the THI biosynthetic and regulatory pathway. When the two species are co-cultured, the presence of thiamine allows C. glabrata to rapidly outcompete S. cerevisiae, while absence of thiamine allows S. cerevisiae to outcompete C. glabrata. This simplification of the THI pathway in C. glabrata suggests its environment provides thiamine and/or its precursors to cells, whereas S. cerevisiae is not as reliant on environmental sources of thiamine.

  3. Partial Decay of Thiamine Signal Transduction Pathway Alters Growth Properties of Candida glabrata.

    Science.gov (United States)

    Iosue, Christine L; Attanasio, Nicholas; Shaik, Noor F; Neal, Erin M; Leone, Sarah G; Cali, Brian J; Peel, Michael T; Grannas, Amanda M; Wykoff, Dennis D

    2016-01-01

    The phosphorylated form of thiamine (Vitamin B1), thiamine pyrophosphate (TPP) is essential for the metabolism of amino acids and carbohydrates in all organisms. Plants and microorganisms, such as yeast, synthesize thiamine de novo whereas animals do not. The thiamine signal transduction (THI) pathway in Saccharomyces cerevisiae is well characterized. The ~10 genes required for thiamine biosynthesis and uptake are transcriptionally upregulated during thiamine starvation by THI2, THI3, and PDC2. Candida glabrata, a human commensal and opportunistic pathogen, is closely related to S. cerevisiae but is missing half of the biosynthetic pathway, which limits its ability to make thiamine. We investigated the changes to the THI pathway in C. glabrata, confirming orthologous functions. We found that C. glabrata is unable to synthesize the pyrimidine subunit of thiamine as well as the thiamine precursor vitamin B6. In addition, THI2 (the gene encoding a transcription factor) is not present in C. glabrata, indicating a difference in the transcriptional regulation of the pathway. Although the pathway is upregulated by thiamine starvation in both species, C. glabrata appears to upregulate genes involved in thiamine uptake to a greater extent than S. cerevisiae. However, the altered regulation of the THI pathway does not alter the concentration of thiamine and its vitamers in the two species as measured by HPLC. Finally, we demonstrate potential consequences to having a partial decay of the THI biosynthetic and regulatory pathway. When the two species are co-cultured, the presence of thiamine allows C. glabrata to rapidly outcompete S. cerevisiae, while absence of thiamine allows S. cerevisiae to outcompete C. glabrata. This simplification of the THI pathway in C. glabrata suggests its environment provides thiamine and/or its precursors to cells, whereas S. cerevisiae is not as reliant on environmental sources of thiamine. PMID:27015653

  4. The Entner–Doudoroff pathway is an overlooked glycolytic route in cyanobac