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Sample records for biosynthetic pathway gene

  1. Detection of additional genes of the patulin biosynthetic pathway in Penicillium griseofulvum

    Science.gov (United States)

    Genes in the patulin biosynthetic pathway are likely to be arranged in a cluster as has been found for biosynthetic pathways of other mycotoxins. The mycotoxin patulin, common in apples and apple juice, is most often associated with Penicillium expansum. However, of 15 fungal species capable of sy...

  2. Differential hexosamine biosynthetic pathway gene expression with type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Megan Coomer

    2014-01-01

    Full Text Available The hexosamine biosynthetic pathway (HBP culminates in the attachment of O-linked β-N-acetylglucosamine (O-GlcNAc onto serine/threonine residues of target proteins. The HBP is regulated by several modulators, i.e. O-linked β-N-acetylglucosaminyl transferase (OGT and β-N-acetylglucosaminidase (OGA catalyze the addition and removal of O-GlcNAc moieties, respectively; while flux is controlled by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFPT, transcribed by two genes, GFPT1 and GFPT2. Since increased HBP flux is glucose-responsive and linked to insulin resistance/type 2 diabetes onset, we hypothesized that diabetic individuals exhibit differential expression of HBP regulatory genes. Volunteers (n = 60; n = 20 Mixed Ancestry, n = 40 Caucasian were recruited from Stellenbosch and Paarl (Western Cape, South Africa and classified as control, pre- or diabetic according to fasting plasma glucose and HbA1c levels, respectively. RNA was purified from leukocytes isolated from collected blood samples and OGT, OGA, GFPT1 and GFPT2 expressions determined by quantitative real-time PCR. The data reveal lower OGA expression in diabetic individuals (P < 0.01, while pre- and diabetic subjects displayed attenuated OGT expression vs. controls (P < 0.01 and P < 0.001, respectively. Moreover, GFPT2 expression decreased in pre- and diabetic Caucasians vs. controls (P < 0.05 and P < 0.01, respectively. We also found ethnic differences, i.e. Mixed Ancestry individuals exhibited a 2.4-fold increase in GFPT2 expression vs. Caucasians, despite diagnosis (P < 0.01. Gene expression of HBP regulators differs between diabetic and non-diabetic individuals, together with distinct ethnic-specific gene profiles. Thus differential HBP gene regulation may offer diagnostic utility and provide candidate susceptibility genes for different ethnic groupings.

  3. Divergent evolutionary pattern of starch biosynthetic pathway genes in grasses and dicots.

    Science.gov (United States)

    Li, Chun; Li, Qi-Gang; Dunwell, Jim M; Zhang, Yuan-Ming

    2012-10-01

    Starch is the most widespread and abundant storage carbohydrate in crops and its production is critical to both crop yield and quality. In regard to the starch content in the seeds of crop plants, there is a distinct difference between grasses (Poaceae) and dicots. However, few studies have described the evolutionary pattern of genes in the starch biosynthetic pathway in these two groups of plants. In this study, therefore, an attempt was made to compare evolutionary rate, gene duplication, and selective pattern of the key genes involved in this pathway between the two groups, using five grasses and five dicots as materials. The results showed 1) distinct differences in patterns of gene duplication and loss between grasses and dicots; duplication in grasses mainly occurred before the divergence of grasses, whereas duplication mostly occurred in individual species within the dicots; there is less gene loss in grasses than in dicots, 2) a considerably higher evolutionary rate in grasses than in dicots in most gene families analyzed, and 3) evidence of a different selective pattern between grasses and dicots; positive selection may have occurred asymmetrically in grasses in some gene families, for example, ADP-glucose pyrophosphorylase small subunit. Therefore, we deduced that gene duplication contributes to, and a higher evolutionary rate is associated with, the higher starch content in grasses. In addition, two novel aspects of the evolution of the starch biosynthetic pathway were observed.

  4. 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

  5. Expression of carotenoid biosynthetic pathway genes and changes in carotenoids during ripening in tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Namitha, Kanakapura Krishnamurthy; Archana, Surya Narayana; Negi, Pradeep Singh

    2011-04-01

    To study the expression pattern of carotenoid biosynthetic pathway genes, changes in their expression at different stages of maturity in tomato fruit (cv. Arka Ahuti) were investigated. The genes regulating carotenoid production were quantified by a dot blot method using a DIG (dioxigenin) labelling and detection kit. The results revealed that there was an increase in the levels of upstream genes of the carotenoid biosynthetic pathway such as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), 4-hydroxy-3-methyl-but-2-enyl diphosphate reductase (Lyt B), phytoene synthase (PSY), phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) by 2-4 fold at the breaker stage as compared to leaf. The lycopene and β-carotene content was analyzed by HPLC at different stages of maturity. The lycopene (15.33 ± 0.24 mg per 100 g) and β-carotene (10.37 ± 0.46 mg per 100 g) content were found to be highest at 5 days post-breaker and 10 days post-breaker stage, respectively. The lycopene accumulation pattern also coincided with the color values at different stages of maturity. These studies may provide insight into devising gene-based strategies for enhancing carotenoid accumulation in tomato fruits.

  6. Phylogenomics of the benzoxazinoid biosynthetic pathway of Poaceae: gene duplications and origin of the Bx cluster

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    Dutartre Leslie

    2012-05-01

    Full Text Available Abstract Background The benzoxazinoids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA and 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA, are key defense compounds present in major agricultural crops such as maize and wheat. Their biosynthesis involves nine enzymes thought to form a linear pathway leading to the storage of DI(MBOA as glucoside conjugates. Seven of the genes (Bx1-Bx6 and Bx8 form a cluster at the tip of the short arm of maize chromosome 4 that includes four P450 genes (Bx2-5 belonging to the same CYP71C subfamily. The origin of this cluster is unknown. Results We show that the pathway appeared following several duplications of the TSA gene (α-subunit of tryptophan synthase and of a Bx2-like ancestral CYP71C gene and the recruitment of Bx8 before the radiation of Poaceae. The origins of Bx6 and Bx7 remain unclear. We demonstrate that the Bx2-like CYP71C ancestor was not committed to the benzoxazinoid pathway and that after duplications the Bx2-Bx5 genes were under positive selection on a few sites and underwent functional divergence, leading to the current specific biochemical properties of the enzymes. The absence of synteny between available Poaceae genomes involving the Bx gene regions is in contrast with the conserved synteny in the TSA gene region. Conclusions These results demonstrate that rearrangements following duplications of an IGL/TSA gene and of a CYP71C gene probably resulted in the clustering of the new copies (Bx1 and Bx2 at the tip of a chromosome in an ancestor of grasses. Clustering favored cosegregation and tip chromosomal location favored gene rearrangements that allowed the further recruitment of genes to the pathway. These events, a founding event and elongation events, may have been the key to the subsequent evolution of the benzoxazinoid biosynthetic cluster.

  7. Reconstructing fungal natural product biosynthetic pathways.

    Science.gov (United States)

    Lazarus, C M; Williams, K; Bailey, A M

    2014-10-01

    Large scale fungal genome sequencing has revealed a multitude of potential natural product biosynthetic pathways that remain uncharted. Here we describe some of the methods that have been used to explore them via heterologous gene expression. We focus on filamentous fungal hosts and discuss the technological challenges and successes behind the reconstruction of fungal natural product pathways. Optimised, efficient heterologous expression of reconstructed biosynthetic pathways promises progress in the discovery of novel compounds that could be utilised by the pharmaceutical and agrochemical industries.

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

    OpenAIRE

    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...

  9. Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells.

    Science.gov (United States)

    Page, Andrew F; Minocha, Rakesh; Minocha, Subhash C

    2012-01-01

    Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the

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

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    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

  11. 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.

  12. 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.

  13. Identification and characterization of genes involved in the jasmonate biosynthetic and signaling pathways in mulberry (Morus notabilis)

    Institute of Scientific and Technical Information of China (English)

    Qing Wang; Bi Ma; Xiwu Qi; Qing Guo; Xuwei Wang; Qiwei Zeng; Ningjia He

    2014-01-01

    Jasmonate (JA) is an important phytohormone regulating growth, development, and environmental response in plants, particularly defense response against herbivorous insects. Recently, completion of the draft genome of the mulberry (Morus notabilis) in conjunction with genome sequencing of silkworm (Bombyx mori) provides an opportuni-ty to study this unique plant-herbivore interaction. Here, we identified genes involved in JA biosynthetic and signaling pathways in the genome of mulberry for the first time, with the majority of samples showing a tissue-biased expression pattern. The analysis of the representative genes 12-oxophy-todienoic acid reductase (OPRs) and jasmonate ZIM-domain (JAZs) was performed and the results indicated that the mulberry genome contains a relatively smal number of JA biosynthetic and signaling pathway genes. A gene encoding an important repressor, MnNINJA, was identified as an alternative splicing variant lacking an ethylene-responsive element binding factor-associated amphiphilic repression motif. Having this fundamental information wil facilitate future functional study of JA-related genes pertaining to mulberry-silkworm interactions.

  14. 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

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

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    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.

  16. 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.

  17. 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.

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

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    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.

  19. Carotenoid profiling, in silico analysis and transcript profiling of miRNAs targeting carotenoid biosynthetic pathway genes in different developmental tissues of tomato.

    Science.gov (United States)

    Koul, Archana; Yogindran, Sneha; Sharma, Deepak; Kaul, Sanjana; Rajam, Manchikatla Venkat; Dhar, Manoj K

    2016-11-01

    Carotenoid biosynthetic pathway is one of the highly significant and very well elucidated secondary metabolic pathways in plants. microRNAs are the potential regulators, widely known for playing a pivotal role in the regulation of various biological as well as metabolic processes. miRNAs may assist in the metabolic engineering of the secondary metabolites for the production of elite genotypes with increased biomass and content of various metabolites. miRNA mediated regulation of carotenoid biosynthetic genes has not been elucidated so far. To illustrate the potential regulatory role of miRNAs in carotenoid biosynthesis, transcript profiling of the known miRNAs and their possible target carotenoid genes was undertaken at eight different developmental stages of tomato, using stem-loop PCR approach combined with quantitative RT-PCR. The inter-relationship amongst carotenoid content, biosynthetic genes and miRNAs was studied in depth. Comparative expression profiles of miRNA and target genes showed variable expression in different tissues studied. The expression level of miRNAs and their target carotenoid genes displayed similar pattern in the vegetative tissues as compared to the reproductive ones, viz. fruit (different stages), indicating the possibility of regulation of carotenoid biosynthesis at various stages of fruit development. This was later confirmed by the HPLC analysis of the carotenoids. The present study has further enhanced the understanding of regulation of carotenoid biosynthetic pathway in plants. The identified miRNAs can be employed to manipulate the biosynthesis of different carotenoids, through metabolic engineering for the production of lycopene rich tomatoes.

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

    NARCIS (Netherlands)

    Liu, Q.; Manzano, D.; Tanic, N.; Pesic, M.; Bankovic, J.; Pateraki, I.; Ricard, L.; Ferrer, A.; Vos, de R.C.H.; Krol, van der A.R.; Bouwmeester, H.J.

    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 that a

  1. 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...... that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.......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...

  2. Characterization of an echinocandin B-producing strain blocked for sterigmatocystin biosynthesis reveals a translocation in the stcW gene of the aflatoxin biosynthetic pathway.

    Science.gov (United States)

    Hodges, R L; Kelkar, H S; Xuei, X; Skatrud, P L; Keller, N P; Adams, T H; Kaiser, R E; Vinci, V A; McGilvray, D

    2000-12-01

    Echinocandin B (ECB), a lipopolypeptide used as a starting material for chemical manufacture of the anti-Candida agent LY303366, is produced by fermentation using a strain of Aspergillus nidulans. In addition to ECB, the wild-type strain also produces a significant level of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Characterization of a mutant designated A42355-OC-1 (OC-1), which is blocked in ST biosynthesis, was the result of a chromosomal translocation. The chromosomal regions containing the breakpoints of the translocation were isolated and DNA sequencing and PCR analysis of the chromosomal breakpoints demonstrated the translocation occurred within the stcW gene of the ST biosynthetic pathway, resulting in disruption of the open reading frame for this gene. Biochemical feeding studies indicate the involvement of this gene product in the conversion of averufin to 1-hydroxy versicolorone. This work demonstrates an effective synergy between classical strain improvement methods and molecular genetics.

  3. 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.

  4. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. Expression of. Arabidopsis tryptophan biosynthetic pathway genes: effect of the 5’ coding region of phosphoribosylanthranilate isomerase gene

    Institute of Scientific and Technical Information of China (English)

    何奕昆; 刘新仿; 李家洋

    1999-01-01

    There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5’ region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants by Agrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5’ coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5’ coding region of PAI1 or PAI3 was 60—100-fold higher than that without the corresponding 5’ region. However, the effect of 5’ coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference). The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by t

  7. The inhibitory effect of Bacillus megaterium on aflatoxin and cyclopiazonic acid biosynthetic pathway gene expression in Aspergillus flavus.

    Science.gov (United States)

    Kong, Qing; Chi, Chen; Yu, Jiujiang; Shan, Shihua; Li, Qiyu; Li, Qianting; Guan, Bin; Nierman, William C; Bennett, Joan W

    2014-06-01

    Aspergillus flavus is one of the major moulds that colonize peanut in the field and during storage. The impact to human and animal health, and to the economy in agriculture and commerce, is significant since this mold produces the most potent known natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect in inhibiting aflatoxin formation in A. flavus through down-regulating aflatoxin pathway gene expression as demonstrated by gene chip analysis. Aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98 %) inhibited by co-cultivation with B. megaterium. Growth was also reduced. Using expression studies, we identified the fungal genes down-regulated by co-cultivation with B. megaterium across the entire fungal genome and specifically within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX). Modulating the expression of these genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was significantly down-regulated during co-cultivation. We present a model showing a hypothesis of the regulatory mechanism of aflatoxin production suppression by AflS and AflR through B. megaterium co-cultivation.

  8. 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.

  9. 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

  10. 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...

  11. Pyrimidine biosynthetic pathway of Baccillus subtilis.

    Science.gov (United States)

    Potvin, B W; Kelleher, R J; Gooder, H

    1975-08-01

    Biochemical and genetic data were obtained from a series of 51 Pyr- strains of Bacillus subtilis. The observed enzymatic deficiencies allowed the mutants to be placed into 12 clases, some of which represent defects in more than one of the six known pyrimidine biosynthetic enzymes. Mapping analysis by transformation has shown that all the Pyr- mutations are located in a single small area of the B. subtilis genome. A correlation of the biochemical defects and the genetic data has been made. Those mutations conferring similar enzymatic deficiencies were found to be contiguous on the B. subtilis map. Regulatory aspects of the pyrimidine pathway have also been investigated and are compared to previously reported results from other organisms. Evidence is presented which bears upon the possible physical association of the first three enzymes and the association of at least some of the enzymes of this pathway with particulate elements of the cell. A model for the organization of the enzymes is presented with dihydroorotate dehydrogenase as the central enzyme in a proposed aggregate.

  12. 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.

  13. Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens

    DEFF Research Database (Denmark)

    Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin

    2014-01-01

    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......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...... 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...

  14. 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.

  15. A biosynthetic pathway for hexanoic acid production in Kluyveromyces marxianus.

    Science.gov (United States)

    Cheon, Yuna; Kim, Jun-Seob; Park, Jun-Bum; Heo, Paul; Lim, Jae Hyung; Jung, Gyoo Yeol; Seo, Jin-Ho; Park, Jin Hwan; Koo, Hyun Min; Cho, Kwang Myung; Park, Jin-Byung; Ha, Suk-Jin; Kweon, Dae-Hyuk

    2014-07-20

    Hexanoic acid can be used for diverse industrial applications and is a precursor for fine chemistry. Although some natural microorganisms have been screened and evolved to produce hexanoic acid, the construction of an engineered biosynthetic pathway for producing hexanoic acid in yeast has not been reported. Here we constructed hexanoic acid pathways in Kluyveromyces marxianus by integrating 5 combinations of seven genes (AtoB, BktB, Crt, Hbd, MCT1, Ter, and TES1), by which random chromosomal sites of the strain are overwritten by the new genes from bacteria and yeast. One recombinant strain, H4A, which contained AtoB, BktB, Crt, Hbd, and Ter, produced 154mg/L of hexanoic acid from galactose as the sole substrate. However, the hexanoic acid produced by the H4A strain was re-assimilated during the fermentation due to the reverse activity of AtoB, which condenses two acetyl-CoAs into a single acetoacetyl-CoA. This product instability could be overcome by the replacement of AtoB with a malonyl CoA-acyl carrier protein transacylase (MCT1) from Saccharomyces cerevisiae. Our results suggest that Mct1 provides a slow but stable acetyl-CoA chain elongation pathway, whereas the AtoB-mediated route is fast but unstable. In conclusion, hexanoic acid was produced for the first time in yeast by the construction of chain elongation pathways comprising 5-7 genes in K. marxianus.

  16. Molecular cloning and characterization of three genes encoding dihydroflavonol-4-reductase from Ginkgo biloba in anthocyanin biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Cheng Hua

    Full Text Available Dihydroflavonol-4-reductase (DFR, EC1.1.1.219 catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins, and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-03-21

    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.

  19. Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage.

    Directory of Open Access Journals (Sweden)

    Rashel V Grindberg

    Full Text Available Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

  20. 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.

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

    Science.gov (United States)

    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.

  2. 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...... of the IPNS by glutathione. The model predicted flux through the pathway corresponds well with the measured rate of penicillin biosynthesis. From the kinetic model the elasticity coefficients and the flux control coefficients are calculated throughout a fed-batch cultivation, and it is found...

  3. Functional Analysis of the Fusarielin Biosynthetic Gene Cluster

    Directory of Open Access Journals (Sweden)

    Aida Droce

    2016-12-01

    Full Text Available Fusarielins are polyketides with a decalin core produced by various species of Aspergillus and Fusarium. Although the responsible gene cluster has been identified, the biosynthetic pathway remains to be elucidated. In the present study, members of the gene cluster were deleted individually in a Fusarium graminearum strain overexpressing the local transcription factor. The results suggest that a trans-acting enoyl reductase (FSL5 assists the polyketide synthase FSL1 in biosynthesis of a polyketide product, which is released by hydrolysis by a trans-acting thioesterase (FSL2. Deletion of the epimerase (FSL3 resulted in accumulation of an unstable compound, which could be the released product. A novel compound, named prefusarielin, accumulated in the deletion mutant of the cytochrome P450 monooxygenase FSL4. Unlike the known fusarielins from Fusarium, this compound does not contain oxygenized decalin rings, suggesting that FSL4 is responsible for the oxygenation.

  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. 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.

  6. New biosynthetic pathway for pink pigments from uncultured oceanic viruses.

    Science.gov (United States)

    Ledermann, Benjamin; Béjà, Oded; Frankenberg-Dinkel, Nicole

    2016-12-01

    The pink open-chain tetrapyrrole pigment phycoerythrobilin (PEB) is employed by marine cyanobacteria, red algae and cryptophytes as a light-harvesting chromophore in phycobiliproteins. Genes encoding biosynthesis proteins for PEB have also been discovered in cyanophages, viruses that infect cyanobacteria, and mimic host pigment biosynthesis with the exception of PebS which combines the enzymatic activities of two host enzymes. In this study, we have identified novel members of the PEB biosynthetic enzyme families, heme oxygenases and ferredoxin-dependent bilin reductases. Encoding genes were found in metagenomic datasets and could be traced back to bacteriophage but not cyanophage origin. While the heme oxygenase exhibited standard activity, a new bilin reductase with highest homology to the teal pigment producing enzyme PcyA revealed PEB biosynthetic activity. Although PcyX possesses PebS-like activity both enzymes share only 9% sequence identity and likely catalyze the reaction via two independent mechanisms. Our data point towards the presence of phycobilin biosynthetic genes in phages that probably infect alphaproteobacteria and, therefore, further support a role of phycobilins outside oxygenic phototrophs.

  7. 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.

  8. 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.

  9. 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.

  10. Enhancement of cordyceps polysaccharide production via biosynthetic pathway analysis in Hirsutella sinensis.

    Science.gov (United States)

    Lin, Shan; Liu, Zhi-Qiang; Baker, Peter James; Yi, Ming; Wu, Hui; Xu, Feng; Teng, Yi; Zheng, Yu-Guo

    2016-11-01

    The addition of various sulfates for enhanced cordyceps polysaccharide (CP) production in submerged cultivation of H. sinensis was investigated, and manganese sulfate was found the most effective. 2mM of manganese sulfate on 0day (d) was investigated as the optimal adding condition, and the CP production reached optimum with 5.33%, increasing by 93.3% compared with the control. Furthermore, the consumption of three main precursors of CP was studied over cultivation under two conditions. Intracellular mannose content decreased by 43.1% throughout 6days cultivation, which corresponded to CP accumulation rate sharply increased from 0 d to 6 d, and mannose was considered as the most preferred precursor for generating CP. Subsequently, mannose biosynthetic pathway was constructed and verified for the first time in H. sinensis, which constituted the important part of CP biosynthesis, and transcriptional levels of the biosynthetic genes were studied. Transcriptional level of gene cpsA was significantly up-regulated 5.35-fold and it was a key gene involved both in mannose and CP biosynthesis. This study demonstrated that manganese sulfate addition is an efficient and simple way to improve CP production. Transcriptional analysis based on biosynthetic pathway was helpful to find key genes and better understand CP biosynthesis.

  11. The c4h, tat, hppr and hppd genes prompted engineering of rosmarinic acid biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.

    Directory of Open Access Journals (Sweden)

    Ying Xiao

    Full Text Available Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1 overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h, tyrosine aminotransferase (tat, and 4-hydroxyphenylpyruvate reductase (hppr, (2 overexpression of both tat and hppr, and (3 suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd. Co-expression of tat/hppr produced the most abundant RA (906 mg/liter and LAB (992 mg/liter, which were 4.3 and 3.2-fold more than in their wild-type (wt counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors.

  12. Nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum.

    Science.gov (United States)

    Nijland, Jeroen G; Ebbendorf, Bjorg; Woszczynska, Marta; Boer, Rémon; Bovenberg, Roel A L; Driessen, Arnold J M

    2010-11-01

    Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the β-lactam biosynthetic pathway. We have analyzed the gene cluster dose effect on penicillin production using the high-yielding P. chrysogenum strain DS17690 that was cured from its native clusters. The amount of penicillin V produced increased with the penicillin biosynthetic gene cluster number but was saturated at high copy numbers. Likewise, transcript levels of the biosynthetic genes pcbAB [δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase], pcbC (isopenicillin N synthase), and penDE (acyltransferase) correlated with the cluster copy number. Remarkably, the protein level of acyltransferase, which localizes to peroxisomes, was saturated already at low cluster copy numbers. At higher copy numbers, intracellular levels of isopenicillin N increased, suggesting that the acyltransferase reaction presents a limiting step at a high gene dose. Since the number and appearance of the peroxisomes did not change significantly with the gene cluster copy number, we conclude that the acyltransferase activity is limiting for penicillin biosynthesis at high biosynthetic gene cluster copy numbers. These results suggest that at a high penicillin production level, productivity is limited by the peroxisomal acyltransferase import activity and/or the availability of coenzyme A (CoA)-activated side chains.

  13. Substrate specificity of the sialic acid biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    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.

  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. Metabolic engineering of biosynthetic pathway for production of renewable biofuels.

    Science.gov (United States)

    Singh, Vijai; Mani, Indra; Chaudhary, Dharmendra Kumar; Dhar, Pawan Kumar

    2014-02-01

    Metabolic engineering is an important area of research that involves editing genetic networks to overproduce a certain substance by the cells. Using a combination of genetic, metabolic, and modeling methods, useful substances have been synthesized in the past at industrial scale and in a cost-effective manner. Currently, metabolic engineering is being used to produce sufficient, economical, and eco-friendly biofuels. In the recent past, a number of efforts have been made towards engineering biosynthetic pathways for large scale and efficient production of biofuels from biomass. Given the adoption of metabolic engineering approaches by the biofuel industry, this paper reviews various approaches towards the production and enhancement of renewable biofuels such as ethanol, butanol, isopropanol, hydrogen, and biodiesel. We have also identified specific areas where more work needs to be done in the future.

  16. Expanding the product profile of a microbial alkane biosynthetic pathway.

    Science.gov (United States)

    Harger, Matthew; Zheng, Lei; Moon, Austin; Ager, Casey; An, Ju Hye; Choe, Chris; Lai, Yi-Ling; Mo, Benjamin; Zong, David; Smith, Matthew D; Egbert, Robert G; Mills, Jeremy H; Baker, David; Pultz, Ingrid Swanson; Siegel, Justin B

    2013-01-18

    Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis , an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli . When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.

  17. Role of the hexosamine biosynthetic pathway in diabetic nephropathy.

    Science.gov (United States)

    Schleicher, E D; Weigert, C

    2000-09-01

    The hexosamine biosynthetic pathway has been hypothesized to be involved in the development of insulin resistance and diabetic vascular complications. In particular, it was demonstrated that hyperglycemia-induced production of transforming growth factor-beta (TGF-beta1), a prosclerotic cytokine causally involved in the development of diabetic nephropathy. Several lines of evidence indicate that TGF-beta1 induction is mediated by the hexosamine pathway. In cultured mesangial cells, high glucose levels induce TGF-beta1 production. This effect is eliminated by inhibition of glutamine: fructose-6-phosphate-amidotransferase (GFAT), the rate-limiting enzyme of this pathway. Furthermore, stable overexpression of GFAT increased levels of TGF-beta1 protein, mRNA, and promoter activity. Inasmuch as stimulation or inhibition of GFAT increased or decreased high glucose-stimulated activity of protein kinase C (PKC), respectively, the observed effects appear to be transduced by PKC. In similar experiments, involvement of the hexosamine pathway in hyperglycemia-induced production of cytokines (TGF-alpha and basic fibroblast growth factor [bFGF]) was demonstrated in vascular smooth muscle cells. These studies also revealed a rapid increase in GFAT activity by treatment with agents that elevated levels of cyclic adenosine 3',5' monophosphate (cAMP), thus indicating that GFAT activity is tightly regulated by cAMP-dependent phosphorylation. Using immunohistochemistry and in situ hybridization, high expression of GFAT was found in human adipocytes, skeletal muscle, vascular smooth muscle cells, and renal tubular epithelial cells. whereas glomerular cells remained essentially unstained. However, significant staining occurred in glomerular cells of patients with diabetic nephropathy. Current data indicate that the flux through the hexosamine pathway, regulated by GFAT, may be causally involved in the development of diabetic vascular disease, particularly diabetic nephropathy.

  18. The biosynthetic gene cluster for the beta-lactam carbapenem thienamycin in Streptomyces cattleya.

    Science.gov (United States)

    Núñez, Luz Elena; Méndez, Carmen; Braña, Alfredo F; Blanco, Gloria; Salas, José A

    2003-04-01

    beta-lactam ring formation in carbapenem and clavam biosynthesis proceeds through an alternative mechanism to the biosynthetic pathway of classic beta-lactam antibiotics. This involves the participation of a beta-lactam synthetase. Using available information from beta-lactam synthetases, we generated a probe for the isolation of the thienamycin cluster from Streptomyces cattleya. Genes homologous to carbapenem and clavulanic acid biosynthetic genes have been identified. They would participate in early steps of thienamycin biosynthesis leading to the formation of the beta-lactam ring. Other genes necessary for the biosynthesis of thienamycin have also been identified in the cluster (methyltransferases, cysteinyl transferases, oxidoreductases, hydroxylase, etc.) together with two regulatory genes, genes involved in exportation and/or resistance, and a quorum sensing system. Involvement of the cluster in thienamycin biosynthesis was demonstrated by insertional inactivation of several genes generating thienamycin nonproducing mutants.

  19. 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.

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

    Science.gov (United States)

    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 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.

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

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere

    andfeed. Secondary metabolites therefore both have a positive and deleterious impact on the human health.The increase in available genome sequences of fungi has revealed that there is a large number of putativesecondary metabolite biosynthetic gene clusters to be discovered and potentially exploited...... 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...... and product discovery. As proof ofconcept the biosynthetic gene cluster for production of the polyketide geodin was identified andtransferred from A. terreus to A. nidulans. The cluster was integrated in a well characterized locus in A.nidulans. Reconstitution of the cluster resulted in the production...

  2. 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.

  3. YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance

    Directory of Open Access Journals (Sweden)

    Patricia Müller-Moulé

    2016-10-01

    Full Text Available Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance.

  4. YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance

    Science.gov (United States)

    Müller-Moulé, Patricia; Nozue, Kazunari; Pytlak, Melissa L.; Palmer, Christine M.; Covington, Michael F.; Wallace, Andreah D.; Harmer, Stacey L.

    2016-01-01

    Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR) light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance. PMID:27761349

  5. Trichodiene Production in a Trichoderma harzianum erg1-Silenced Strain Provides Evidence of the Importance of the Sterol Biosynthetic Pathway in Inducing Plant Defense-Related Gene Expression.

    Science.gov (United States)

    Malmierca, M G; McCormick, S P; Cardoza, R E; Monte, E; Alexander, N J; Gutiérrez, S

    2015-11-01

    Trichoderma species are often used as biocontrol agents against plant-pathogenic fungi. A complex molecular interaction occurs among the biocontrol agent, the antagonistic fungus, and the plant. Terpenes and sterols produced by the biocontrol fungus have been found to affect gene expression in both the antagonistic fungus and the plant. The terpene trichodiene (TD) elicits the expression of genes related to tomato defense and to Botrytis virulence. We show here that TD itself is able to induce the expression of Botrytis genes involved in the synthesis of botrydial (BOT) and also induces terpene gene expression in Trichoderma spp. The terpene ergosterol, in addition to its role as a structural component of the fungal cell membranes, acts as an elicitor of defense response in plants. In the present work, using a transformant of T. harzianum, which is silenced in the erg1 gene and accumulates high levels of squalene, we show that this ergosterol precursor also acts as an important elicitor molecule of tomato defense-related genes and induces Botrytis genes involved in BOT biosynthesis, in both cases, in a concentration-dependent manner. Our data emphasize the importance of a balance of squalene and ergosterol in fungal interactions as well as in the biocontrol activity of Trichoderma spp.

  6. Neurosteroid biosynthetic pathways changes in prefrontal cortex in Alzheimer's disease.

    Science.gov (United States)

    Luchetti, Sabina; Bossers, Koen; Van de Bilt, Saskia; Agrapart, Vincent; Morales, Rafael Ramirez; Frajese, Giovanni Vanni; Swaab, Dick F

    2011-11-01

    Expression of the genes for enzymes involved in neurosteroid biosynthesis was studied in human prefrontal cortex (PFC) in the course of Alzheimer's disease (AD) (n=49). Quantitative RT-PCR (qPCR) revealed that mRNA levels of diazepam binding inhibitor (DBI), which is involved in the first step of steroidogenesis and in GABAergic transmission, were increased, as were mRNA levels for several neurosteroid biosynthetic enzymes. Aromatase, 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) and aldo-keto reductase 1C2 (AKR1C2), were all increased in the late stages of AD. Several GABA-A subunits were significantly reduced in AD. Increased expression of aromatase in the PFC was confirmed by immunohistochemistry and was found to be localized predominantly in astrocytes. These data suggest a role for estrogens and allopregnanolone produced by astrocytes in the PFC in AD, possibly as part of a rescue program. The reduced gene expression of some synaptic and extra-synaptic GABA-A subunits may indicate a deficit of modulation of GABA-A receptors by neuroactive steroids, which may contribute to the neuropsychiatric characteristics of this disease.

  7. 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.

  8. Trichodiene production in a Trichoderma harzianum erg1-silenced strain provides evidence of the importance of the sterol biosynthetic pathway in inducing plant defense-related gene expression

    Science.gov (United States)

    Trichoderma species are often used as biocontrol agents against plant-pathogenic fungi. A complex molecular interaction occurs among the biocontrol agent, the antagonistic fungus, and the plant. Terpenes and sterols produced by the biocontrol fungus have been found to affect gene expression in both ...

  9. 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.

  10. 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.

  11. A Unique TryptophanC-Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

    Science.gov (United States)

    Parajuli, Anirudra; Kwak, Daniel H.; Dalponte, Luca; Leikoski, Niina; Galica, Tomas; Umeobika, Ugochukwu; Trembleau, Laurent; Bent, Andrew; Sivonen, Kaarina; Wahlsten, Matti; Wang, Hao; Rizzi, Ermanno; De Bellis, Gianluca; Naismith, James; Jaspars, Marcel; Liu, Xinyu; Houssen, Wael; Fewer, David Peter

    2016-01-01

    Cyanobactins are are rapidly growing family of linear and cyclic peptides produced by cyanobacteria. Kawaguchipeptins A and B, two macrocyclic undecapeptides reported earlier from Microcystis aeruginosa NIES-88, are shown to be products of the cyanobactin biosynthetic pathway. The 9 kb kawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mb draft genome of Microcystis aeruginosa NIES-88. We verified that this gene cluster is responsible for the production of the kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C-3 of Trp residues in both linear and cyclic peptides in vitro. Our findings serve to further enhance the structural diversity of cyanobactins to include tryptophan-prenylated cyclic peptides. PMID:26846478

  12. Origin of Saxitoxin Biosynthetic Genes in Cyanobacteria

    OpenAIRE

    Ahmed Moustafa; Jeannette E Loram; Hackett, Jeremiah D.; Anderson, Donald M.; F Gerald Plumley; Debashish Bhattacharya

    2009-01-01

    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 th...

  13. Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains

    Science.gov (United States)

    In order to determine the genetic basis for loss of fumonisin B¬2 (FB2) biosynthesis in FB2 non-producing A. niger strains, we developed multiplex PCR primer sets to amplify fragments of eight fumonisin biosynthetic pathway (fum) genes. Fragments of all eight fum genes were amplified in FB2-produci...

  14. 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

  15. Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis

    Science.gov (United States)

    Bennallack, Philip R.; Bewley, Kathryn D.; Burlingame, Mark A.; Robison, Richard A.; Miller, Susan M.

    2016-01-01

    ABSTRACT Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically dissect this pathway, we have reconstituted it in Bacillus subtilis. Successful MP1 production required promoter engineering and the reassembly of essential biosynthetic genes in a modular plasmid. The resulting system allows for rapid pathway manipulation, including protein tagging and gene deletion. We find that 8 processing proteins are sufficient for the production of MP1 and that the tailoring enzyme TclS catalyzes a C-terminal reduction step that distinguishes MP1 from its sister compound micrococcin P2. IMPORTANCE The emergence of antibiotic resistance is one of the most urgent human health concerns of our day. A crucial component in an integrated strategy for countering antibiotic resistance is the ability to engineer pathways for the biosynthesis of natural and derivatized antimicrobial compounds. In this study, the model organism B. subtilis was employed to reconstitute and genetically modularize a 9-gene system for the biosynthesis of micrococcin, the founding member of a growing family of thiopeptide antibiotics. PMID:27381911

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

    Science.gov (United States)

    Stahlhut, Steen G; Siedler, Solvej; Malla, Sailesh; Harrison, Scott J; Maury, Jérôme; Neves, Ana Rute; Forster, Jochen

    2015-09-01

    Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti-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 pathway in Escherichia coli. We propose a novel biosynthetic pathway from the amino acid, tyrosine, utilizing nine heterologous enzymes. The pathway proceeds via the synthesis of two flavanones never produced in microorganisms before--garbanzol and resokaempferol. We show for the first time a functional biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols.

  17. 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.

  18. 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...... screening methods based on the genome sequences of potential producing organisms. The basic principle of such genome mining approaches is to identify genes, which are involved in the biosynthesis of such molecules, and to predict the products of the identified pathways. Thus, bioinformatics methods...... 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....

  19. 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.

  20. Reassembled Biosynthetic Pathway for a Large-scale Synthesis of CMP-Neu5Ac

    Directory of Open Access Journals (Sweden)

    Min Xiao

    2003-11-01

    Full Text Available Abstract: CMP-Neu5Ac is an important sugar nucleotide for biosynthesis of sialic acid and its conjugates. In this paper, a large-scale production system of CMP-Neu5Ac by a single strain is reported. The co-expression of Neu5Ac aldolase (EC4.1.3.3 and CMP-Neu5Ac synthetase (EC 2.7.7.43 was achieved by constructing individual genes into one plasmid and having a single culture that has both NeuAc aldolase and CMP-Neu5Ac synthetase activities. Overall this system only employed N-acetylmannosamine, excess of pyruvate and CTP to produce CMP-Neu5Ac. This work has demonstrated that a large-scale synthesis of sialic acid-derived oligosaccharides could be achieved economically and efficiently through a single, biosynthetic pathway engineered microorganism.

  1. 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.

  2. Engineered Streptomyces avermitilis host for heterologous expression of biosynthetic gene cluster for secondary metabolites.

    Science.gov (United States)

    Komatsu, Mamoru; Komatsu, Kyoko; Koiwai, Hanae; Yamada, Yuuki; Kozone, Ikuko; Izumikawa, Miho; Hashimoto, Junko; Takagi, Motoki; Omura, Satoshi; Shin-ya, Kazuo; Cane, David E; Ikeda, Haruo

    2013-07-19

    An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.

  3. Identification and developmental expression profiling of putative alkaloid biosynthetic genes in Corydalis yanhusuo bulbs.

    Science.gov (United States)

    Liao, Dengqun; Wang, Pengfei; Jia, Chan; Sun, Peng; Qi, Jianjun; Zhou, Lili; Li, Xian'en

    2016-01-18

    Alkaloids in bulbs of Corydalis (C.) yanhusuo are the major pharmacologically active compounds in treatment of blood vessel diseases, tumors and various pains. However, due to the absence of gene sequences in C. yanhusuo, the genes involved in alkaloid biosynthesis and their expression during bulb development remain unknown. We therefore established the first transcriptome database of C. yanhusuo via Illumina mRNA-Sequencing of a RNA composite sample collected at Bulb initiation (Day 0), early enlargement (Day 10) and maturation (Day 30). 25,013,630 clean 90 bp paired-end reads were de novo assembled into 47,081 unigenes with an average length of 489 bp, among which 30,868 unigenes (65.56%) were annotated in four protein databases. Of 526 putative unigenes involved in biosynthesis o f various alkaloids, 187 were identified as the candidate genes involved in the biosynthesis of benzylisoquinoline alkaloids (BIAs), the only alkaloid type reported in C. yanhusuo untill now. BIAs biosynthetic genes were highly upregulated in the overall pathway during bulb development. Identification of alkaloid biosynthetic genes in C. yanhusuo provide insights on pathways and molecular regulation of alkaloid biosynthesis, to initiate metabolic engineering in order to improve the yield of interesting alkaloids and to identify potentially new alkaloids predicted from the transcriptomic information.

  4. 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.

  5. 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

    Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti......-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...... biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols....

  6. Characterization of the CDP-D-mannitol biosynthetic pathway in Streptococcus pneumoniae 35A.

    Science.gov (United States)

    Wang, Quan; Xu, Yanli; Perepelov, Andrei V; Knirel, Yuriy A; Reeves, Peter R; Shashkov, Alexander S; Ding, Peng; Guo, Xi; Feng, Lu

    2012-12-01

    Streptococcus pneumoniae is a major human pathogen associated with diseases worldwide. The capsular polysaccharides (CPSs) are considered a major virulence factor and are targets for a vaccine. d-Mannitol was found to be present in the CPS of several S. pneumoniae serotypes. Two genes, mnp1 and mnp2, which are located in the CPS gene cluster, were proposed to be responsible for the synthesis of NDP-d-mannitol (the nucleotide activated form of d-mannitol). However, the pathway has never been identified by experimental methods and we aimed to characterize it in the present study. To achieve this, the two genes, mnp1 and mnp2, were cloned and the gene products were overexpressed, purified, and analyzed in vitro for their respective enzymatic activities. Products of reactions catalyzed by Mnp1 and Mnp2 were detected by capillary electrophoresis and validated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. We show that Mnp1 is responsible for the transfer of CMP from CTP to d-fructose-6-phosphate (Fru-6-P) to form CDP-d-fructose, whereas Mnp2 catalyzed the conversion of CDP-d-fructose to CDP-d-mannitol. Therefore, Mnp1 (renamed as mnpA) was identified as Fru-6-P cytidylyltransferase-encoding gene, and mnp2 (renamed as mnpB) as a CDP-d-fructose reductase-encoding gene. The kinetics of Mnp1 for the substrate (Fru-6-P and CTP) and of Mnp2 for the substrate (CDP-d-fructose) and the cofactor NADH or NADPH fitted the Michaelis-Menten model. The effects of temperature, pH and cations on the two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-d-mannitol has been identified biochemically.

  7. 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.

  8. 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.

  9. The biosynthetic pathway for a thousand-year-old natural food colorant and citrinin in Penicillium marneffei.

    Science.gov (United States)

    Woo, Patrick C Y; Lam, Ching-Wan; Tam, Emily W T; Lee, Kim-Chung; Yung, Karrie K Y; Leung, Chris K F; Sze, Kong-Hung; Lau, Susanna K P; Yuen, Kwok-Yung

    2014-10-22

    Monascorubrin and its derivatives are polyketides used as natural colorants for a wide range of food for more than one thousand years. Since the biosynthetic pathway for this ancient chemical compound is unknown and genome sequence unavailable for any Monascus species, monascorubrin production has relied on extraction from fungal cultures of Monascus species. In vitro synthesis and genetic manipulation are not possible. Here we report the polyketide gene cluster and pathway for monascorubrin biosynthesis in Penicillium marneffei, a diffusible red pigment-producing, thermal dimorphic fungus, taking advantage of available genome sequence and faster growth rate than Monascus species. We also documented that the red pigment of P. marneffei is a mixture of more than 16 chemical compounds, which are amino acid conjugates of monascorubrin and rubropunctatin, and showed that this polyketide gene cluster and pathway are also responsible for biosynthesis of ankaflavin and citrinin, a mycotoxin with nephrotoxic activity in mammals. The present study on elucidation of the biosynthetic pathway of monascorubrin is a proof-of-the-concept study that serves as a cornerstone for future studies on monascorubrin biosynthesis pathway dissection in Monascus species.

  10. Establishment of pomegranate (Punica granatum) hairy root cultures for genetic interrogation of the hydrolyzable tannin biosynthetic pathway.

    Science.gov (United States)

    Ono, Nadia N; Bandaranayake, Pradeepa C G; Tian, Li

    2012-09-01

    In contrast to the numerous reports on the human therapeutic applications of hydrolyzable tannins (HTs), genes involved in their biosynthesis have not been identified at the molecular level from any plant species. Although we have previously identified candidate HT biosynthetic genes in pomegranate using transcriptomic and bioinformatic analyses, characterization of in planta enzyme function remains a critical step in biochemical pathway elucidation. We here report the establishment of a pomegranate (Punica granatum) hairy root culture system that produces HTs. Agrobacterium rhizogenes strains transformed with a binary vector harboring a yellow fluorescent protein (YFP) gene were used for hairy root induction, allowing visual, non-destructive, detection of transgene incorporation. It also demonstrated that the pomegranate hairy root culture system is suitable for expressing heterologous genes (YFP in this case). Expression of 26 putative UDP-glycosyltransferase (UGT) genes, obtained from a pomegranate fruit peel (a tissue highly abundant in HTs) RNA-Seq library, were verified in wild type and hairy roots. In addition, two candidate UGTs for HT biosynthesis were identified based on HPLC and differential gene expression analyses of various pomegranate tissues. Together with in vitro enzyme activity assays, the hairy root culture system holds great promise for revealing the undivulged HT biosynthetic pathway using pomegranate as a model system.

  11. Alteration of the coenzyme A biosynthetic pathway in neurodegeneration with brain iron accumulation syndromes.

    Science.gov (United States)

    Venco, Paola; Dusi, Sabrina; Valletta, Lorella; Tiranti, Valeria

    2014-08-01

    NBIA (neurodegeneration with brain iron accumulation) comprises a heterogeneous group of neurodegenerative diseases having as a common denominator, iron overload in specific brain areas, mainly basal ganglia and globus pallidus. In the past decade a bunch of disease genes have been identified, but NBIA pathomechanisms are still not completely clear. PKAN (pantothenate kinase-associated neurodegeneration), an autosomal recessive disorder with progressive impairment of movement, vision and cognition, is the most common form of NBIA. It is caused by mutations in the PANK2 (pantothenate kinase 2) gene, coding for a mitochondrial enzyme that phosphorylates vitamin B5 in the first reaction of the CoA (coenzyme A) biosynthetic pathway. A distinct form of NBIA, denominated CoPAN (CoA synthase protein-associated neurodegeneration), is caused by mutations in the CoASY (CoA synthase) gene coding for a bifunctional mitochondrial enzyme, which catalyses the final steps of CoA biosynthesis. These two inborn errors of CoA metabolism further support the concept that dysfunctions in CoA synthesis may play a crucial role in the pathogenesis of NBIA.

  12. 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.

  13. Transcription Factor Families Regulate the Anthocyanin Biosynthetic Pathway in Capsicum

    Science.gov (United States)

    Anthocyanin structural gene transcription requires the expression of at least one member of each of three transcription factor families - MYC, MYB and WD40. These transcription factors form a complex that binds to structural gene promoters, thereby modulating gene expression. Capsicum annuum display...

  14. 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

  15. 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

    2016-06-01

    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.

  16. Mutational analysis of the thienamycin biosynthetic gene cluster from Streptomyces cattleya.

    Science.gov (United States)

    Rodríguez, Miriam; Núñez, Luz Elena; Braña, Alfredo F; Méndez, Carmen; Salas, José A; Blanco, Gloria

    2011-04-01

    The generation of non-thienamycin-producing mutants with mutations in the thnL, thnN, thnO, and thnI genes within the thn gene cluster from Streptomyces cattleya and their involvement in thienamycin biosynthesis and regulation were previously reported. Four additional mutations were independently generated in the thnP, thnG, thnR, and thnT genes by insertional inactivation. Only the first two genes were found to play a role in thienamycin biosynthesis, since these mutations negatively or positively affect antibiotic production. A mutation of thnP results in the absence of thienamycin production, whereas a 2- to 3-fold increase in thienamycin production was observed for the thnG mutant. On the other hand, mutations in thnR and thnT showed that although these genes were previously reported to participate in this pathway, they seem to be nonessential for thienamycin biosynthesis, as thienamycin production was not affected in these mutants. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of all available mutants revealed some putative intermediates in the thienamycin biosynthetic pathway. A compound with a mass corresponding to carbapenam-3-carboxylic acid was detected in some of the mutants, suggesting that the assembly of the bicyclic nucleus of thienamycin might proceed in a way analogous to that of the simplest natural carbapenem, 1-carbapen-2-em-3-carboxylic acid biosynthesis. The accumulation of a compound with a mass corresponding to 2,3-dihydrothienamycin in the thnG mutant suggests that it might be the last intermediate in the biosynthetic pathway. These data, together with the establishment of cross-feeding relationships by the cosynthesis analysis of the non-thienamycin-producing mutants, lead to a proposal for some enzymatic steps during thienamycin assembly.

  17. 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.

  18. 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.

  19. 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.

  20. Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters.

    Science.gov (United States)

    Du, Deyao; Zhu, Yu; Wei, Junhong; Tian, Yuqing; Niu, Guoqing; Tan, Huarong

    2013-07-01

    Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.

  1. Molecular cloning and expression of phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthetic pathway from Acanthamoeba castellanii.

    Science.gov (United States)

    Deng, Yihong; Wu, Duo; Tachibana, Hiroshi; Cheng, Xunjia

    2015-04-01

    Free-living amoebae of the genus Acanthamoeba are widespread protozoans that can cause serious infectious diseases. This study characterised phosphoglycerate dehydrogenase (PGDH) and phosphoserine aminotransferase (PSAT) in the phosphorylated serine biosynthetic pathway of Acanthamoeba castellanii. The PGDH gene encodes a protein of 442 amino acids with a calculated molecular weight of 47.7 kDa and an isoelectric point (pI) of 7.64. Meanwhile, the PSAT gene encodes a protein of 394 amino acids with a calculated molecular weight of 43.8 kDa and a pI of 5.80. Confocal microscopy suggests that PGDH is mainly diffused in the cytoplasm, whereas PSAT is located in the inner part of the cell membrane. The messenger RNA (mRNA) expression levels of PGDH and PSAT vary depending on growth state under consecutive culture conditions. No significant changes in the mRNA expression levels of both PGDH and PSAT occur after the incubation of L-serine with Acanthamoeba. This result indicates that exogenous serine exerts no influence on the expression of these genes and that the so-called feedback inhibition of both PGDH and PSAT in Acanthamoeba differs from that in bacteria or other organisms. We propose that the enzymes in the phosphorylated serine biosynthetic pathway function in amoeba growth and proliferation.

  2. Genetic engineering, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy elucidate the bikaverin biosynthetic pathway in Fusarium fujikuroi.

    Science.gov (United States)

    Arndt, Birgit; Studt, Lena; Wiemann, Philipp; Osmanov, Helena; Kleigrewe, Karin; Köhler, Jens; Krug, Isabel; Tudzynski, Bettina; Humpf, Hans-Ulrich

    2015-11-01

    Secondary metabolites of filamentous fungi can be highly bioactive, ranging from antibiotic to cancerogenic properties. In this study we were able to identify a new, yet unknown metabolite produced by Fusarium fujikuroi, an ascomycetous rice pathogen. With the help of genomic engineering and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS) followed by isolation and detailed structure elucidation, the new substance could be designated as an unknown bikaverin precursor, missing two methyl- and one hydroxy group, hence named oxo-pre-bikaverin. Though the bikaverin gene cluster has been extensively studied in the past, elucidation of the biosynthetic pathway remained elusive due to a negative feedback loop that regulates the genes within the cluster. To decipher the bikaverin biosynthetic pathway and to overcome these negative regulation circuits, the structural cluster genes BIK2 and BIK3 were overexpressed independently in the ΔΔBIK2/BIK3+OE::BIK1 mutant background by using strong constitutive promoters. Using the software tool MZmine 2, the metabolite profile of the generated mutants obtained by HPLC-HRMS was compared, revealing further intermediates.

  3. Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis.

    Science.gov (United States)

    Winterberg, Britta; Uhlmann, Stefanie; Linne, Uwe; Lessing, Franziska; Marahiel, Mohamed A; Eichhorn, Heiko; Kahmann, Regine; Schirawski, Jan

    2010-03-01

    Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron-chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non-ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC-MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl-coenzyme A (CoA)-hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)-CoA synthase Hcs1 in U. maydis that HMG-CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.

  4. Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

    Science.gov (United States)

    Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

    2014-03-13

    The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

  5. Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter.

    Science.gov (United States)

    Kim, Eun Jin; Angell, Scott; Janes, Jeff; Watanabe, Coran M H

    2008-06-01

    Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.

  6. Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Visser, Hans; van Ooyen, Albert J J; Verdoes, Jan C

    2003-12-01

    This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.

  7. Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative.

    Science.gov (United States)

    Bleeker, Petra M; Mirabella, Rossana; Diergaarde, Paul J; VanDoorn, Arjen; Tissier, Alain; Kant, Merijn R; Prins, Marcel; de Vos, Martin; Haring, Michel A; Schuurink, Robert C

    2012-12-04

    Tomato breeding has been tremendously efficient in increasing fruit quality and quantity but did not focus on improving herbivore resistance. The biosynthetic pathway for the production of 7-epizingiberene in a wild tomato was introduced into a cultivated greenhouse variety with the aim to obtain herbivore resistance. 7-Epizingiberene is a specific sesquiterpene with toxic and repellent properties that is produced and stored in glandular trichomes. We identified 7-epizingiberene synthase (ShZIS) that belongs to a new class of sesquiterpene synthases, exclusively using Z-Z-farnesyl-diphosphate (zFPP) in plastids, probably arisen through neo-functionalization of a common ancestor. Expression of the ShZIS and zFPP synthases in the glandular trichomes of cultivated tomato resulted in the production of 7-epizingiberene. These tomatoes gained resistance to several herbivores that are pests of tomato. Hence, introduction of this sesquiterpene biosynthetic pathway into cultivated tomatoes resulted in improved herbivore resistance.

  8. antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters

    DEFF Research Database (Denmark)

    Weber, Tilmann; Blin, Kai; Duddela, Srikanth

    2015-01-01

    Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we...... introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration...... of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products...

  9. 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.

  10. Comparison of carotenoid accumulation and biosynthetic gene expression between Valencia and Rohde Red Valencia sweet oranges

    Science.gov (United States)

    Carotenoid accumulation and biosynthetic gene expression levels during fruit maturation were compared between ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV). The two cultivars exhibited different carotenoid profiles and regulatory mechanisms in flavedo and...

  11. Analysis of carotenoid biosynthetic gene expression during marigold petal development.

    Science.gov (United States)

    Moehs, C P; Tian, L; Osteryoung, K W; Dellapenna, D

    2001-02-01

    Marigold (Tagetes erecta L.) flower petals synthesize and accumulate carotenoids at levels greater than 20 times that in leaves and provide an excellent model system to investigate the molecular biology and biochemistry of carotenoid biosynthesis in plants. In addition, marigold cultivars exist with flower colors ranging from white to dark orange due to >100-fold differences in carotenoid levels, and presumably similar changes in carbon flux through the pathway. To examine the expression of carotenoid genes in marigold petals, we have cloned the majority of the genes in this pathway and used these to assess their steady-state mRNA levels in four marigold cultivars with extreme differences in carotenoid content. We have also cloned genes encoding early steps in the biosynthesis of isopentenyl pyrophosphate (IPP), the precursor of all isoprenoids, including carotenoids, as well as two genes required for plastid division. Differences among the marigold varieties in the expression of these genes suggest that differences in mRNA transcription or stability underlie the vast differences in carotenoid synthesis and accumulation in the different marigold varieties.

  12. Blockage of the pyrimidine biosynthetic pathway affects riboflavin production in Ashbya gossypii.

    Science.gov (United States)

    Silva, Rui; Aguiar, Tatiana Q; Domingues, Lucília

    2015-01-10

    The Ashbya gossypii riboflavin biosynthetic pathway and its connection with the purine pathway have been well studied. However, the outcome of genetic alterations in the pyrimidine pathway on riboflavin production by A. gossypii had not yet been assessed. Here, we report that the blockage of the de novo pyrimidine biosynthetic pathway in the recently generated A. gossypii Agura3 uridine/uracil auxotrophic strain led to improved riboflavin production on standard agar-solidified complex medium. When extra uridine/uracil was supplied, the production of riboflavin by this auxotroph was repressed. High concentrations of uracil hampered this (and the parent) strain growth, whereas excess uridine favored the A. gossypii Agura3 growth. Considering that the riboflavin and the pyrimidine pathways share the same precursors and that riboflavin overproduction may be triggered by nutritional stress, we suggest that overproduction of riboflavin by the A. gossypii Agura3 may occur as an outcome of a nutritional stress response and/or of an increased availability in precursors for riboflavin biosynthesis, due to their reduced consumption by the pyrimidine pathway.

  13. 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

  14. Microbial modulation of bacoside A biosynthetic pathway and systemic defense mechanism in Bacopa monnieri under Meloidogyne incognita stress

    Science.gov (United States)

    Gupta, Rupali; Singh, Akanksha; Srivastava, Madhumita; Singh, Vivek; Gupta, M. M.; Pandey, Rakesh

    2017-01-01

    Plant-associated beneficial microbes have been explored to fulfill the imperative function for plant health. However, their impact on the host secondary metabolite production and nematode disease management remains elusive. Our present work has shown that chitinolytic microbes viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 singly as well as in combination modulated the biosynthetic pathway of bacoside A and systemic defense mechanism against Meloidogyne incognita in Bacopa monnieri. Interestingly, expression of bacoside biosynthetic pathway genes (3-Hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate diphosphate decarboxylase, and squalene synthase) were upregulated in plants treated with the microbial combination in the presence as well as in absence of M. incognita stress. These microbes not only augmented bacoside A production (1.5 fold) but also strengthened host resistance via enhancement in chlorophyll a, defense enzymes and phenolic compounds like gallic acid, syringic acid, ferulic acid and cinnamic acid. Furthermore, elevated lignification and callose deposition in the microbial combination treated plants corroborate well with the above findings. Overall, the results provide novel insights into the underlying mechanisms of priming by beneficial microbes and underscore their capacity to trigger bacoside A production in B. monnieri under biotic stress. PMID:28157221

  15. Global regulation of nucleotide biosynthetic genes by c-Myc.

    Directory of Open Access Journals (Sweden)

    Yen-Chun Liu

    Full Text Available BACKGROUND: The c-Myc transcription factor is a master regulator and integrates cell proliferation, cell growth and metabolism through activating thousands of target genes. Our identification of direct c-Myc target genes by chromatin immunoprecipitation (ChIP coupled with pair-end ditag sequencing analysis (ChIP-PET revealed that nucleotide metabolic genes are enriched among c-Myc targets, but the role of Myc in regulating nucleotide metabolic genes has not been comprehensively delineated. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that the majority of genes in human purine and pyrimidine biosynthesis pathway were induced and directly bound by c-Myc in the P493-6 human Burkitt's lymphoma model cell line. The majority of these genes were also responsive to the ligand-activated Myc-estrogen receptor fusion protein, Myc-ER, in a Myc null rat fibroblast cell line, HO.15 MYC-ER. Furthermore, these targets are also responsive to Myc activation in transgenic mouse livers in vivo. To determine the functional significance of c-Myc regulation of nucleotide metabolism, we sought to determine the effect of loss of function of direct Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2 on c-Myc-induced cell growth and proliferation. In this regard, we used a specific IMPDH inhibitor mycophenolic acid (MPA and found that MPA dramatically inhibits c-Myc-induced P493-6 cell proliferation through S-phase arrest and apoptosis. CONCLUSIONS/SIGNIFICANCE: Taken together, these results demonstrate the direct induction of nucleotide metabolic genes by c-Myc in multiple systems. Our finding of an S-phase arrest in cells with diminished IMPDH activity suggests that nucleotide pool balance is essential for c-Myc's orchestration of DNA replication, such that uncoupling of these two processes create DNA replication stress and apoptosis.

  16. antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters.

    Science.gov (United States)

    Weber, Tilmann; Blin, Kai; Duddela, Srikanth; Krug, Daniel; Kim, Hyun Uk; Bruccoleri, Robert; Lee, Sang Yup; Fischbach, Michael A; Müller, Rolf; Wohlleben, Wolfgang; Breitling, Rainer; Takano, Eriko; Medema, Marnix H

    2015-07-01

    Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

  17. Identification and expression analysis of chitin synthase and related enzymes in the chitin biosynthetic pathway genes of Cnaphalocrocis medinalis%稻纵卷叶螟几丁质合成酶及合成通路相关酶基因的鉴定及表达分析

    Institute of Scientific and Technical Information of China (English)

    余海中; 黄克慧; 汪婉玲; 刘明辉; 杨鑫; 张彦; 徐家萍

    2015-01-01

    [Objectives] The rice leaf folder, Cnaphalocrocis medinalis (Guenee), is one of four rice pest insects that cause serious crop damage. In recent years, chitin synthesis and metabolism has become a focus of pest control research. Cloning and spatio-temporal expression of two chitin synthases, and other two key enzymes in the chitin biosynthetic pathway encoding genes in C. medinalis, were conducted to reveal the function of these genes. [Methods] Based on transcriptome data, we used the PCR and RACE techniques to clone the full length cDNA sequences of 4 key enzymes in the chitin biosynthetic pathway. Prediction of the structure, sequence alignment and phylogenetic analysis of the products of these 4 genes were performed using different bioinformatics software. The relative expression levels of the 4 genes in different developmental stages and larval tissues of C. medinalis were determined with quantitative Real-time PCR. [Results] Two full-length cDNA sequences encoding chitin synthase, and two full-length cDNA sequences encoding other two key enzymes related to the chitin biosynthetic pathway, were obtained. These were; Chitin Synthase A (CHSA), Chitin Synthase B (CHSB), Phosphoacetylglucosamine Mutase (PGM) and UDP-N-acetylglucosamine pyrophosphorylase (UAP) (hereafter CmCHSA, CmCHSB, CmPGM and CmUAP, respectively). Sequence analysis shows that the full length of the CmCHSA gene is 4 868 bp, which encodes a polypeptide of 1 564 amino acids, the full length of the CmCHSB gene is 4 651 bp, which encodes a polypeptide of 1 525 amino acids, the full length of CmPGM gene is 1 934 bp, which encodes a polypeptide of 548 amino acids, and the full length of CmUAP gene is 1 837 bp, which encodes a polypeptide of 487 amino acids. The results of RT-qPCR indicate that CmUAP and CmPGM had higher expression in hemolymph, whereas CmCHSA was more highly expressed in the head and integument than the midgut and CmCHSB was more highly expressed in the midgut than in other tissues

  18. Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli.

    Science.gov (United States)

    Ma, Tian; Zhou, Yuanjie; Li, Xiaowei; Zhu, Fayin; Cheng, Yongbo; Liu, Yi; Deng, Zixin; Liu, Tiangang

    2016-02-01

    As a highly valued keto-carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α-Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole-genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio-Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high-efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4-fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future.

  19. Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy.

    Science.gov (United States)

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey

    2006-08-16

    Axonal degeneration occurs in many neurodegenerative diseases and after traumatic injury and is a self-destructive program independent from programmed cell death. Previous studies demonstrated that overexpression of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) or exogenous application of nicotinamide adenine dinucleotide (NAD) can protect axons of cultured dorsal root ganglion (DRG) neurons from degeneration caused by mechanical or neurotoxic injury. In mammalian cells, NAD can be synthesized from multiple precursors, including tryptophan, nicotinic acid, nicotinamide, and nicotinamide riboside (NmR), via multiple enzymatic steps. To determine whether other components of these NAD biosynthetic pathways are capable of delaying axonal degeneration, we overexpressed each of the enzymes involved in each pathway and/or exogenously administered their respective substrates in DRG cultures and assessed their capacity to protect axons after axotomy. Among the enzymes tested, Nmnat1 had the strongest protective effects, whereas nicotinamide phosphoribosyl transferase and nicotinic acid phosphoribosyl transferase showed moderate protective activity in the presence of their substrates. Strong axonal protection was also provided by Nmnat3, which is predominantly located in mitochondria, and an Nmnat1 mutant localized to the cytoplasm, indicating that the subcellular location of NAD production is not crucial for protective activity. In addition, we showed that exogenous application of the NAD precursors that are the substrates of these enzymes, including nicotinic acid mononucleotide, nicotinamide mononucleotide, and NmR, can also delay axonal degeneration. These results indicate that stimulation of NAD biosynthetic pathways via a variety of interventions may be useful in preventing or delaying axonal degeneration.

  20. Reconstruction of the biosynthetic pathway for the core fungal polyketide scaffold rubrofusarin in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Naesby, Michael; Mortensen, Uffe Hasbro

    2013-01-01

    production in easily fermentable and genetically engineerable organisms, such as Saccharomyces cerevisiae and Escherichia coli are desirable. Rubrofusarin is an orange polyketide pigment that is a common intermediate in many different fungal biosynthetic pathways. RESULTS: In this study, we established......BACKGROUND: Fungal polyketides include commercially important pharmaceuticals and food additives, e.g. the cholesterol-lowering statins and the red and orange monascus pigments. Presently, production relies on isolation of the compounds from the natural producers, and systems for heterologous....... CONCLUSIONS: The reconstructed pathway for rubrofusarin in S. cerevisiae allows the production of a core scaffold molecule with a branch-point role in several fungal polyketide pathways, thus paving the way for production of further natural pigments and bioactive molecules. Furthermore, the reconstruction...

  1. Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Speiser, D.M.; Ortiz, D.F.; Kreppel, L.; Scheel, G.; McDonald, G.; Ow, D.W. (Dept. of Agriculture, Albany, CA (United States) Univ. of California, Berkeley (United States))

    1992-12-01

    Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems. 41 refs., 8 figs., 2 tabs.

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

    Science.gov (United States)

    Verdoes, Jan C; Sandmann, Gerhard; Visser, Hans; Diaz, Maria; van Mossel, Minca; van Ooyen, Albert J J

    2003-07-01

    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 single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.

  3. 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

    that catalyze the terminal hydroxylation steps in the conversion of cholesterol to the molting hormone 20-hydroxyecdysone. These P450s are conserved in other insects and each is thought to function throughout development as the sole mediator of a particular biosynthetic step since, where analyzed, each...... larval stages within the prothoracic gland cells of the ring gland. RNAi mediated reduction in the expression of this heterochromatin localized gene leads to arrest at the first instar stage which can be rescued by feeding the larva 20E, E or ketodiol but not 7dC. In addition, spok expression...

  4. Generation of the natamycin analogs by gene engineering of natamycin biosynthetic genes in Streptomyces chattanoogensis L10.

    Science.gov (United States)

    Liu, Shui-Ping; Yuan, Peng-Hui; Wang, Yue-Yue; Liu, Xiao-Fang; Zhou, Zhen-Xing; Bu, Qing-ting; Yu, Pin; Jiang, Hui; Li, Yong-Quan

    2015-04-01

    The polyene antibiotic natamycin is widely used as an antifungal agent in both human therapy and the food industry. Here we obtained four natamycin analogs with high titers, including two new compounds, by engineering of six post-polyketide synthase (PKS) tailoring enzyme encoding genes in a natamycin industrial producing strain, Streptomyces chattanoogensis L10. Precise analysis of S. chattanoogensis L10 culture identified natamycin and two natamycin analogs, 4,5-deepoxy-natamycin and 4,5-deepoxy-natamycinolide. The scnD deletion mutant of S. chattanoogensis L10 did not produce natamycin but increased the titer of 4,5-deepoxy-natamycin. Inactivation of each of scnK, scnC, and scnJ in S. chattanoogensis L10 abolished natamycin production and accumulated 4,5-deepoxy-natamycinolide. Deletion of scnG in S. chattanoogensis L10 resulted in production of two new compounds, 4,5-deepoxy-12-decarboxyl-12-methyl-natamycin and its dehydration product without natamycin production. Inactivation of the ScnG-associated ferredoxin ScnF resulted in impaired production of natamycin. Bioassay of these natamycin analogs showed that three natamycin analogs remained antifungal activities. We found that homologous glycosyltransferases genes including amphDI and nysDI can partly complement the ΔscnK mutant. Our results here also support that ScnG, ScnK, and ScnD catalyze carboxylation, glycosylation, and epoxidation in turn in the natamycin biosynthetic pathway. Thus this paper provided a method to generate natamycin analogs and shed light on the natamycin biosynthetic pathway.

  5. Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors.

    Science.gov (United States)

    Suzuki, Hiroyoshi; Yokokura, Junpei; Ito, Tsukasa; Arai, Ryoma; Yokoyama, Chiaki; Toshima, Hiroaki; Nagata, Shinji; Asami, Tadao; Suzuki, Yoshihito

    2014-10-01

    Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future.

  6. Accumulation of Kaempferitrin and Expression of Phenyl-Propanoid Biosynthetic Genes in Kenaf (Hibiscus cannabinus

    Directory of Open Access Journals (Sweden)

    Shicheng Zhao

    2014-10-01

    Full Text Available Kenaf (Hibiscus cannabinus is cultivated worldwide for its fiber; however, the medicinal properties of this plant are currently attracting increasing attention. In this study, we investigated the expression levels of genes involved in the biosynthesis of kaempferitrin, a compound with many biological functions, in different kenaf organs. We found that phenylalanine ammonia lyase (HcPAL was more highly expressed in stems than in other organs. Expression levels of cinnamate 4-hydroxylase (HcC4H and 4-coumarate-CoA ligase (Hc4CL were highest in mature leaves, followed by stems and young leaves, and lowest in roots and mature flowers. The expression of chalcone synthase (HcCHS, chalcone isomerase (HcCHI, and flavone 3-hydroxylase (HcF3H was highest in young flowers, whereas that of flavone synthase (HcFLS was highest in leaves. An analysis of kaempferitrin accumulation in the different organs of kenaf revealed that the accumulation of this compound was considerably higher (>10-fold in leaves than in other organs. On the basis of a comparison of kaempferitrin contents with the expression levels of different genes in different organs, we speculate that HcFLS plays an important regulatory role in the kaempferitrin biosynthetic pathway in kenaf.

  7. Revisiting sesquiterpene biosynthetic pathways leading to santalene and its analogues: a comprehensive mechanistic study.

    Science.gov (United States)

    Jindal, Garima; Sunoj, Raghavan B

    2012-10-21

    Santalene and bergamotene are the major olefinic sesquiterpenes responsible for the fragrance of sandalwood oil. Herein we report the details of density functional theory investigations on the biosynthetic pathway of this important class of terpenes. The mechanistic study has been found to be effective toward gaining significant new insight into different possibilities for the formation of the key intermediates involved in santalene and bergamotene biosynthesis. The stereoelectronic features of the transition states and intermediates for (i) ring closure of the initial bisabolyl cation, and (ii) skeletal rearrangements in the ensuing bicyclic carbocationic intermediates leading to (-)-epi-β-santalene, (-)-β-santalene, (-)-α-santalene, (+)-epi-β-santalene, exo-β-bergamotene, endo-β-bergamotene, exo-α-bergamotene, and endo-α-bergamotene are presented. Interesting structural features pertaining to certain new carbocationic intermediates (such as b) resulting from the ring closure of bisabolyl cation are discussed. Extensive conformational sampling of all key intermediates along the biosynthetic pathway offered new insight into the role of the isoprenyl side chain conformation in the formation of santalene and its analogues. Although the major bicyclic products in Santalum album appear to arise from the right or left handed helical form of farnesyl pyrophosphate (FPP), different alternatives for their formation are found to be energetically feasible. The interconversion of the exo and endo isomers of bisabolyl cation and a likely epimerization, both with interesting mechanistic implications, are presented. The exo to endo conversion is identified to be energetically more favorable than another pathway emanating from the left handed helical FPP. The role of pyrophosphate (OPP(-)) in the penultimate deprotonation step leading to olefinic sesquiterpenes is also examined.

  8. Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A

    Science.gov (United States)

    Chakrabortti, Alolika; Li, Jinming

    2016-01-01

    Ochratoxin A (OTA) is a common mycotoxin that contaminates food and agricultural products. Sequencing of the complete genome of Aspergillus westerdijkiae, a major producer of OTA, reveals more than 50 biosynthetic gene clusters, including a putative OTA biosynthetic gene cluster that encodes a dozen of enzymes, transporters, and regulatory proteins. PMID:27635003

  9. Sequencing, physical organization and kinetic expression of the patulin biosynthetic gene cluster from Penicillium expansum.

    Science.gov (United States)

    Tannous, Joanna; El Khoury, Rhoda; Snini, Selma P; Lippi, Yannick; El Khoury, André; Atoui, Ali; Lteif, Roger; Oswald, Isabelle P; Puel, Olivier

    2014-10-17

    Patulin is a polyketide-derived mycotoxin produced by numerous filamentous fungi. Among them, Penicillium expansum is by far the most problematic species. This fungus is a destructive phytopathogen capable of growing on fruit, provoking the blue mold decay of apples and producing significant amounts of patulin. The biosynthetic pathway of this mycotoxin is chemically well-characterized, but its genetic bases remain largely unknown with only few characterized genes in less economic relevant species. The present study consisted of the identification and positional organization of the patulin gene cluster in P. expansum strain NRRL 35695. Several amplification reactions were performed with degenerative primers that were designed based on sequences from the orthologous genes available in other species. An improved genome Walking approach was used in order to sequence the remaining adjacent genes of the cluster. RACE-PCR was also carried out from mRNAs to determine the start and stop codons of the coding sequences. The patulin gene cluster in P. expansum consists of 15 genes in the following order: patH, patG, patF, patE, patD, patC, patB, patA, patM, patN, patO, patL, patI, patJ, and patK. These genes share 60-70% of identity with orthologous genes grouped differently, within a putative patulin cluster described in a non-producing strain of Aspergillus clavatus. The kinetics of patulin cluster genes expression was studied under patulin-permissive conditions (natural apple-based medium) and patulin-restrictive conditions (Eagle's minimal essential medium), and demonstrated a significant association between gene expression and patulin production. In conclusion, the sequence of the patulin cluster in P. expansum constitutes a key step for a better understanding of the mechanisms leading to patulin production in this fungus. It will allow the role of each gene to be elucidated, and help to define strategies to reduce patulin production in apple-based products.

  10. Engineering the central biosynthetic and secondary metabolic pathways of Pseudomonas aeruginosa strain PA1201 to improve phenazine-1-carboxylic acid production.

    Science.gov (United States)

    Jin, Kaiming; Zhou, Lian; Jiang, Haixia; Sun, Shuang; Fang, Yunling; Liu, Jianhua; Zhang, Xuehong; He, Ya-Wen

    2015-11-01

    The secondary metabolite phenazine-1-carboxylic acid (PCA) is an important component of the newly registered biopesticide Shenqinmycin. We used a combined method involving gene, promoter, and protein engineering to modify the central biosynthetic and secondary metabolic pathways in the PCA-producing Pseudomonas aeruginosa strain PA1201. The PCA yield of the resulting strain PA-IV was increased 54.6-fold via the following strategies: (1) blocking PCA conversion and enhancing PCA efflux pumping; (2) increasing metabolic flux towards the PCA biosynthetic pathway through the over-production of two DAHP synthases and blocking the synthesis of 21 secondary metabolites; (3) increasing the PCA precursor supply through the engineering of five chorismate-utilizing enzymes; (4) engineering the promoters of two PCA biosynthetic gene clusters. Strain PA-IV produced 9882 mg/L PCA in fed-batch fermentation, which is twice as much as that produced by the current industrial strain. Strain PA-IV was also genetically stable and comparable to Escherichia coli in cytotoxicity.

  11. Characterization of the fumonisin B2 biosynthetic gene cluster in Aspergillus niger and A. awamori.

    Science.gov (United States)

    Aspergillus niger and A. awamori strains isolated from grapes cultivated in Mediterranean basin were examined for fumonisin B2 (FB2) production and presence/absence of sequences within the fumonisin biosynthetic gene (fum) cluster. Presence of 13 regions in the fum cluster was evaluated by PCR assay...

  12. Design-based re-engineering of biosynthetic gene clusters : plug-and-play in practice

    NARCIS (Netherlands)

    Frasch, Hans-Jörg; Medema, Marnix H.; Takano, Eriko; Breitling, Rainer; Gago, Federico; Parayil, Ajikumar

    2013-01-01

    Synthetic biology is revolutionizing the way in which the biosphere is explored for natural products. Through computational genome mining, thousands of biosynthetic gene clusters are being identified in microbial genomes, which constitute a rich source of potential novel pharmaceuticals. New methods

  13. Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243.

    Science.gov (United States)

    Onaka, Hiroyasu; Taniguchi, Shin-ichi; Igarashi, Yasuhiro; Furumai, Tamotsu

    2003-01-01

    The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.

  14. Elongating internodes of Zea mays (maize): Early steps in the GA biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Y.; Phinney, B.O. (Univ. of California, Los Angeles (USA)); Gaskin, P.; MacMillan, J. (Univ. of Bristol (England))

    1989-04-01

    The early steps in the gibberellin (GA) biosynthetic pathway have yet to be defined for tissues that show a growth response to GAs. To this end we have synthesized the ({sup 13}C,{sup 3}H)-ent-kaurenoids, ent-kaurenol, ent-kaurenal ent-kaukenoic acid. We also have double-labeled ent-kaurene and double-labeled GA{sub 12}-aldehyde. We feed 1 - 10{mu}g of each substrate, individually, to 1.0g diced internodes in the appropriate buffer plus cofactors. We have observed up to 80% metabolism. We have identified (full scan GC-MS) 7{beta}-hydroxy-ent-kaurenoic acid as the major metabolite from double-labeled ent-kaurenoic acid feeds, thus defining the step ent-kaurenoic acid to 7{beta}-hydroxy-ent-kaurenoic acid.

  15. Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity.

    Science.gov (United States)

    Chang, Aram; Singh, Shanteri; Helmich, Kate E; Goff, Randal D; Bingman, Craig A; Thorson, Jon S; Phillips, George N

    2011-10-25

    Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.

  16. Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Aram; Singh, Shanteri; Helmich, Kate E.; Goff, Randal D.; Bingman, Craig A.; Thorson, Jon S.; Phillips, Jr., George N. (UW)

    2012-03-15

    Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.

  17. Sex pheromone biosynthetic pathways are conserved between moths and the butterfly Bicyclus anynana.

    Science.gov (United States)

    Liénard, Marjorie A; Wang, Hong-Lei; Lassance, Jean-Marc; Löfstedt, Christer

    2014-05-27

    Although phylogenetically nested within the moths, butterflies have diverged extensively in a number of life history traits. Whereas moths rely greatly on chemical signals, visual advertisement is the hallmark of mate finding in butterflies. In the context of courtship, however, male chemical signals are widespread in both groups although they likely have multiple evolutionary origins. Here, we report that in males of the butterfly Bicyclus anynana, courtship scents are produced de novo via biosynthetic pathways shared with females of many moth species. We show that two of the pheromone components that play a major role in mate choice, namely the (Z)-9-tetradecenol and hexadecanal, are produced through the activity of a fatty acyl Δ11-desaturase and two specialized alcohol-forming fatty acyl reductases. Our study provides the first evidence of conservation and sharing of ancestral genetic modules for the production of FA-derived pheromones over a long evolutionary timeframe thereby reconciling mate communication in moths and butterflies.

  18. Linking fungal secondary metabolites and pathways to their genes in Aspergillus

    DEFF Research Database (Denmark)

    Petersen, Lene Maj

    organisms for genetic studies and human opportunistic pathogens. The aim of this PhD study has been divided into two major topics: 1) Discovery and characterization of novel SMs from filamentous fungi 2) Linking of fungal SMs to genes and elucidation of biosynthetic pathways The first part of this study......, analytical and natural products chemistry is critical for advances in both the linking of fungal SMs to genes and unraveling the biosynthetic pathways, as well as for the discovery of novel SMs hidden in a treasury of biosynthetic potential of filamentous fungi....

  19. 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

  20. Characterization of the promoter region of biosynthetic enzyme genes involved in berberine biosynthesis in Coptis japonica

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    Yasuyuki Yamada

    2016-09-01

    Full Text Available The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs, a plant-specific WRKY-type transcription factor, CjWRKY1, and a basic helix-loop-helix (bHLH transcription factor, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4’OMT and CYP719A1 were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay (EMSA and by a chromatin immunoprecipitation (ChIP assay. In addition, CjbHLH1 also activated transcription from truncated 4’OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed.

  1. Genetic localization and in vivo characterization of a Monascus azaphilone pigment biosynthetic gene cluster.

    Science.gov (United States)

    Balakrishnan, Bijinu; Karki, Suman; Chiu, Shih-Hau; Kim, Hyun-Ju; Suh, Jae-Won; Nam, Bora; Yoon, Yeo-Min; Chen, Chien-Chi; Kwon, Hyung-Jin

    2013-07-01

    Monascus spp. produce several well-known polyketides such as monacolin K, citrinin, and azaphilone pigments. In this study, the azaphilone pigment biosynthetic gene cluster was identified through T-DNA random mutagenesis in Monascus purpureus. The albino mutant W13 bears a T-DNA insertion upstream of a transcriptional regulator gene (mppR1). The transcription of mppR1 and the nearby polyketide synthase gene (MpPKS5) was significantly repressed in the W13 mutant. Targeted inactivation of MpPKS5 also gave rise to an albino mutant, confirming that mppR1 and MpPKS5 belong to an azaphilone pigment biosynthetic gene cluster. This M. purpureus sequence was used to identify the whole biosynthetic gene cluster in the Monascus pilosus genome. MpPKS5 contains SAT/KS/AT/PT/ACP/MT/R domains, and this domain organization is preserved in other azaphilone polyketide synthases. This biosynthetic gene cluster also encodes fatty acid synthase (FAS), which is predicted to assist the synthesis of 3-oxooactanoyl-CoA and 3-oxodecanoyl-CoA. These 3-oxoacyl compounds are proposed to be incorporated into the azaphilone backbone to complete the pigment biosynthesis. A monooxygenase gene (an azaH and tropB homolog) that is located far downstream of the FAS gene is proposed to be involved in pyrone ring formation. A homology search on other fungal genome sequences suggests that this azaphilone pigment gene cluster also exists in the Penicillium marneffei and Talaromyces stipitatus genomes.

  2. Genome mining of the sordarin biosynthetic gene cluster from Sordaria araneosa Cain ATCC 36386: characterization of cycloaraneosene synthase and GDP-6-deoxyaltrose transferase.

    Science.gov (United States)

    Kudo, Fumitaka; Matsuura, Yasunori; Hayashi, Takaaki; Fukushima, Masayuki; Eguchi, Tadashi

    2016-07-01

    Sordarin is a glycoside antibiotic with a unique tetracyclic diterpene aglycone structure called sordaricin. To understand its intriguing biosynthetic pathway that may include a Diels-Alder-type [4+2]cycloaddition, genome mining of the gene cluster from the draft genome sequence of the producer strain, Sordaria araneosa Cain ATCC 36386, was carried out. A contiguous 67 kb gene cluster consisting of 20 open reading frames encoding a putative diterpene cyclase, a glycosyltransferase, a type I polyketide synthase, and six cytochrome P450 monooxygenases were identified. In vitro enzymatic analysis of the putative diterpene cyclase SdnA showed that it catalyzes the transformation of geranylgeranyl diphosphate to cycloaraneosene, a known biosynthetic intermediate of sordarin. Furthermore, a putative glycosyltransferase SdnJ was found to catalyze the glycosylation of sordaricin in the presence of GDP-6-deoxy-d-altrose to give 4'-O-demethylsordarin. These results suggest that the identified sdn gene cluster is responsible for the biosynthesis of sordarin. Based on the isolated potential biosynthetic intermediates and bioinformatics analysis, a plausible biosynthetic pathway for sordarin is proposed.

  3. 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.

  4. Genomics of iron acquisition in the plant pathogen Erwinia amylovora: insights in the biosynthetic pathway of the siderophore desferrioxamine E.

    Science.gov (United States)

    Smits, Theo H M; Duffy, Brion

    2011-10-01

    Genomics has clarified the biosynthetic pathway for desferrioxamine E critical for iron acquisition in the enterobacterial fire blight pathogen Erwinia amylovora. Evidence for each of the individual steps and the role of desferrioxamine E biosynthesis in pathogen virulence and cell protection from host defenses is presented. Using comparative genomics, it can be concluded that desferrioxamine biosynthesis is ancestral within the genera Erwinia and Pantoea.

  5. Novel Key Metabolites Reveal Further Branching of the Roquefortine/Meleagrin Biosynthetic Pathway

    NARCIS (Netherlands)

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

    2013-01-01

    Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding produ

  6. Engineering salidroside biosynthetic pathway in hairy root cultures of Rhodiola crenulata based on metabolic characterization of tyrosine decarboxylase.

    Directory of Open Access Journals (Sweden)

    Xiaozhong Lan

    Full Text Available Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21-6.84, 1.50-2.19 and 1.27-3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. Calmodulin-mediated suppression of 2-ketoisovalerate reductase in Beauveria bassiana beauvericin biosynthetic pathway.

    Science.gov (United States)

    Kim, Jiyoung; Yoon, Deok-Hyo; Oh, Junsang; Hyun, Min-Woo; Han, Jae-Gu; Sung, Gi-Ho

    2016-11-01

    Ketoisovalerate reductase (KIVR, E.C. 1.2.7.7) mediates the specific reduction of 2-ketoisovalerate (2-Kiv) to d-hydroxyisovalerate (d-Hiv), a precursor for beauvericin biosynthesis. Beauvericin, a famous mycotoxin produced by many fungi, is a cyclooligomer depsipeptide, which has insecticidal, antimicrobial, antiviral, and cytotoxic activities. In this report, we demonstrated that Beauveria bassiana 2-ketoisovalerate reductase (BbKIVR) acts as a typical KIVR enzyme in the entomopathogenic fungus B. bassiana. In addition, we found that BbKIVR interacts with calmodulin (CaM) in vitro and in vivo. The functional role of CaM-binding to BbKIVR was to negatively regulate the BbKIVR activity in B. bassiana. Environmental stimuli such as light and salt stress suppressed BbKIVR activity in B. bassiana. Interestingly, this negative effect of BbKIVR activity by light and salt stress was recovered by CaM inhibitors, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbKIVR plays an important role in the beauvericin biosynthetic pathway mediated by environmental stimuli such as light and salt stress via the CaM signaling pathway.

  12. Engineering the leucine biosynthetic pathway for isoamyl alcohol overproduction in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yuan, Jifeng; Mishra, Pranjul; Ching, Chi Bun

    2017-01-01

    Isoamyl alcohol can be used not only as a biofuel, but also as a precursor for various chemicals. Saccharomyces cerevisiae inherently produces a small amount of isoamyl alcohol via the leucine degradation pathway, but the yield is very low. In the current study, several strategies were devised to overproduce isoamyl alcohol in budding yeast. The engineered yeast cells with the cytosolic isoamyl alcohol biosynthetic pathway produced significantly higher amounts of isobutanol over isoamyl alcohol, suggesting that the majority of the metabolic flux was diverted to the isobutanol biosynthesis due to the broad substrate specificity of Ehrlich pathway enzymes. To channel the key intermediate 2-ketosiovalerate (KIV) towards α-IPM biosynthesis, we introduced an artificial protein scaffold to pull dihydroxyacid dehydratase and α-IPM synthase into the close proximity, and the resulting strain yielded more than twofold improvement of isoamyl alcohol. The best isoamyl alcohol producer yielded 522.76 ± 38.88 mg/L isoamyl alcohol, together with 540.30 ± 48.26 mg/L isobutanol and 82.56 ± 8.22 mg/L 2-methyl-1-butanol. To our best knowledge, our work represents the first study to bypass the native compartmentalized α-IPM biosynthesis pathway for the isoamyl alcohol overproduction in budding yeast. More importantly, artificial protein scaffold based on the feature of quaternary structure of enzymes would be useful in improving the catalytic efficiency and the product specificity of other enzymatic reactions.

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

    Science.gov (United States)

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

    2015-10-01

    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 against planktonic cells and biofilms was determined by redox dye-based viability staining, and the antibiofilm efficacy of the compounds was confirmed by viable counting. A novel antistaphylococcal polyketide, alnumycin D, was identified. Unexpectedly, the C-ribosylated pathway shunt product alnumycin D was more active against planktonic and biofilm cells than the pathway end product alnumycin A, where a ribose unit has been converted into a dioxane moiety. The evaluation of the antibiofilm potential of other alnumycins revealed that the presence of the ribose moiety in pyranose form is essential for high activity against preformed biofilms. Furthermore, the antibiofilm potential of other closely related PNQ polyketides was examined. Based on their previously reported activity against planktonic S. aureus cells, granaticin B, kalafungin, and medermycin were also selected for testing, and among them, granaticin B was found to be the most potent against preformed biofilms. The most active antibiofilm PNQs, alnumycin D and granaticin B, share several structural features that may be important for their antibiofilm activity. They are uncharged, glycosylated, and also contain a similar oxygenation pattern of the lateral naphthoquinone ring. These findings highlight the potential of antibiotic biosynthetic pathways as a source of effective antibiofilm compounds.

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

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    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

  15. Bioinformatic analysis of an unusual gene-enzyme relationship in the arginine biosynthetic pathway among marine gamma proteobacteria: implications concerning the formation of N-acetylated intermediates in prokaryotes

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    Labedan Bernard

    2006-01-01

    Full Text Available Abstract Background The N-acetylation of L-glutamate is regarded as a universal metabolic strategy to commit glutamate towards arginine biosynthesis. Until recently, this reaction was thought to be catalyzed by either of two enzymes: (i the classical N-acetylglutamate synthase (NAGS, gene argA first characterized in Escherichia coli and Pseudomonas aeruginosa several decades ago and also present in vertebrates, or (ii the bifunctional version of ornithine acetyltransferase (OAT, gene argJ present in Bacteria, Archaea and many Eukaryotes. This paper focuses on a new and surprising aspect of glutamate acetylation. We recently showed that in Moritella abyssi and M. profunda, two marine gamma proteobacteria, the gene for the last enzyme in arginine biosynthesis (argH is fused to a short sequence that corresponds to the C-terminal, N-acetyltransferase-encoding domain of NAGS and is able to complement an argA mutant of E. coli. Very recently, other authors identified in Mycobacterium tuberculosis an independent gene corresponding to this short C-terminal domain and coding for a new type of NAGS. We have investigated the two prokaryotic Domains for patterns of gene-enzyme relationships in the first committed step of arginine biosynthesis. Results The argH-A fusion, designated argH(A, and discovered in Moritella was found to be present in (and confined to marine gamma proteobacteria of the Alteromonas- and Vibrio-like group. Most of them have a classical NAGS with the exception of Idiomarina loihiensis and Pseudoalteromonas haloplanktis which nevertheless can grow in the absence of arginine and therefore appear to rely on the arg(A sequence for arginine biosynthesis. Screening prokaryotic genomes for virtual argH-X 'fusions' where X stands for a homologue of arg(A, we retrieved a large number of Bacteria and several Archaea, all of them devoid of a classical NAGS. In the case of Thermus thermophilus and Deinococcus radiodurans, the arg(A-like sequence

  16. Ancient horizontal gene transfer from bacteria enhances biosynthetic capabilities of fungi.

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    Imke Schmitt

    Full Text Available BACKGROUND: Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms. Gene duplication and subfunctionalization, as well as horizontal gene transfer are proposed mechanisms in the evolution of biosynthetic gene clusters. To explain the amount of homology in some polyketide synthases in unrelated organisms such as bacteria and fungi, interkingdom horizontal gene transfer has been evoked as the most likely evolutionary scenario. However, the origin of the genes and the direction of the transfer remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We used comparative phylogenetics to infer the ancestor of a group of polyketide synthase genes involved in antibiotic and mycotoxin production. We aligned keto synthase domain sequences of all available fungal 6-methylsalicylic acid (6-MSA-type PKSs and their closest bacterial relatives. To assess the role of symbiotic fungi in the evolution of this gene we generated 24 6-MSA synthase sequence tags from lichen-forming fungi. Our results support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication. CONCLUSIONS/SIGNIFICANCE: Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi. The large number of 6-MSA-type PKS sequences found in lichen-forming fungi leads us hypothesize that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase.

  17. Knockout of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNA-damaging agents due to change of free radicals scavenging ability.

    Science.gov (United States)

    Zhang, Lei; Yang, Qiao; Luo, Xuesong; Fang, Chengxiang; Zhang, Qiuju; Tang, Yali

    2007-10-01

    Deinococcus radiodurans R1, a red-pigmented strain of the extremely radioresistant genus Deinococcus, contains a major carotenoid namely deinoxanthin. The high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV) has been widely reported. However, the possible antioxidant role of carotenoids in this strain has not been completely elucidated. In this study, we constructed two colorless mutants by knockout of crtB and crtI genes, respectively. Comparative analysis of the two colorless mutants and the wild type showed that the two colorless mutants were more sensitive to ionizing radiation, UV, and hydrogen peroxide, but not to mitomycin-C (MMC). With electron spin resonance (ESR) and spin trapping techniques, we observed that hydroxyl radical signals occurred in the suspensions of UV irradiated Deinococcus radiodurans cells and the intensity of signals was influenced by carotenoids levels. We further showed that the carotenoid extract from the wild type could obviously scavenge superoxide anions generated by the irradiated riboflavin/EDTA system. These results suggest that carotenoids in D. radiodurans R1 function as free radical scavengers to protect this organism against the deleterious effects of oxidative DNA-damaging agents.

  18. 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.

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

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    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.

  20. Analysis of Antioxidants Metabolic Pathway and Expression of Anthocyanin Biosynthetic Genes in Blueberry Flower and Fruit%越橘花果抗氧化物代谢通路及花青素合成酶基因表达分析

    Institute of Scientific and Technical Information of China (English)

    张长青; 丁元亮

    2016-01-01

    以越橘花、幼果、成熟果为试材,基于转录组测序开展了抗氧化物代谢通路和花青素生物合成酶基因表达研究。结果表明,抗氧化物代谢通路中,黄酮类、苯丙素类合成通路是活跃的,叶酸类、类胡萝卜素、二苯乙烯类、二芳基庚酸类、姜酚、二萜类化合物的合成通路是部分支路活跃的,而其他抗氧化物通路是不活跃的。花青素合成酶基因中,苯丙氨酸裂解酶、肉桂酸羟化酶、查尔酮合成酶、4-羟查尔酮异构酶、黄烷酮-3-羟化酶、二氢黄酮醇-4-还原酶、花青素合成酶、类黄酮3,5-糖苷转移酶是幼果或成熟果中表达上调的。%Studies on metabolism pathway of antioxidants and expression of anthocyanin biosyn-thetic genes are carried out by using transcriptome sequencing technology to blueberry flower, young fruit and ripe fruit.The results show that,among antioxidant metabolic pathways,the biosynthesises of flavonoids and phenylpropanoid are active,and the biosynthesises of folate,ca-rotenoid,stilbenoid,diarylheptanoid,gingerol,and diterpenoid are partly active,while the other antioxidant pathways are inactive.They also show that,among the enzymes related to anthocy-anin biosynthesis,phenylalanine ammonia-lyase,cinnamate 4-hydroxylase,chalcone synthase, chalcone isomerase,flavanone 3-hydroxylase,dihydroflavanol 4-reductase,anthocyanidin syn-thase,and anthocyanidin/flavonol 3-O-glucosyltransferase are up-regulated in young fruit or ripe fruit,compared with flower.

  1. Review on biosynthetic pathway of secondary metabolites and the related genes in Monascus spp.%红曲菌次生代谢产物生物合成途径及相关基因的研究进展

    Institute of Scientific and Technical Information of China (English)

    李利; 陈莎; 陈福生; 高梦祥

    2013-01-01

    Monascus spp., one of the important microbial resources both for food and medicine in China, can produce kinds of useful secondary metabolites, such as the natural food additive Monascus pigments, the cholesterol-lowering substance monacolin K and so on.Meanwhile, this genus also has the ability to secrete the mycotoxin citrinin, causing a safety risk to Monascus-related products.Therefore, how to promote the production of the useful metabolites and eliminate the production of citrinin has become a hot topic.Over the past decade, with the development and application of molecular biological approaches in Monascus spp., great efforts have been made to explore the basical knowledge about biosynthesis of secondary metabolites, and many genes involved in biosynthesis of Monascus pigments, monacolin K and citrinin have been identified and characterized.The latest achievements were summarized in this paper, in the purpose of providing potential approaches to manipulate and improve industrial Monascus strains efficiently.%红曲菌(Monascus spp.)是我国重要的药食两用微生物资源之一,能够产生天然食品添加剂红曲色素、降血酯活性物质Monacolin K等有益次生代谢产物,但也能分泌真菌毒素桔霉素(Citrinin),红曲菌及其相关产品的安全性由此受到质疑.因此,如何促进红曲菌有益代谢产物的产生,减少或抑制桔霉素的产生成为广大科研工作者研究的重点方向.近年来,红曲菌的分子生物学研究有了较快的发展,红曲菌次生代谢产物生物合成及其调控的研究是热点.本文重点介绍红曲色素、Monacolin K和桔霉素生物合成途径及相关基因的研究进展,以期为有效调控红曲菌次生代谢产物的产生、提高红曲产品的安全性提供参考和借鉴.

  2. Evolutionary rate patterns of the Gibberellin pathway genes

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    Zhang Fu-min

    2009-08-01

    Full Text Available Abstract Background Analysis of molecular evolutionary patterns of different genes within metabolic pathways allows us to determine whether these genes are subject to equivalent evolutionary forces and how natural selection shapes the evolution of proteins in an interacting system. Although previous studies found that upstream genes in the pathway evolved more slowly than downstream genes, the correlation between evolutionary rate and position of the genes in metabolic pathways as well as its implications in molecular evolution are still less understood. Results We sequenced and characterized 7 core structural genes of the gibberellin biosynthetic pathway from 8 representative species of the rice tribe (Oryzeae to address alternative hypotheses regarding evolutionary rates and patterns of metabolic pathway genes. We have detected significant rate heterogeneity among 7 GA pathway genes for both synonymous and nonsynonymous sites. Such rate variation is mostly likely attributed to differences of selection intensity rather than differential mutation pressures on the genes. Unlike previous argument that downstream genes in metabolic pathways would evolve more slowly than upstream genes, the downstream genes in the GA pathway did not exhibited the elevated substitution rate and instead, the genes that encode either the enzyme at the branch point (GA20ox or enzymes catalyzing multiple steps (KO, KAO and GA3ox in the pathway had the lowest evolutionary rates due to strong purifying selection. Our branch and codon models failed to detect signature of positive selection for any lineage and codon of the GA pathway genes. Conclusion This study suggests that significant heterogeneity of evolutionary rate of the GA pathway genes is mainly ascribed to differential constraint relaxation rather than the positive selection and supports the pathway flux theory that predicts that natural selection primarily targets enzymes that have the greatest control on fluxes.

  3. Novel key metabolites reveal further branching of the roquefortine/meleagrin biosynthetic pathway.

    Science.gov (United States)

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

    2013-12-27

    Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding products. Next to the structural characterization of roquefortine F and neoxaline, which are for the first time reported for P. chrysogenum, we identified the novel metabolite roquefortine L, including its degradation products, harboring remarkable chemical structures. Their biosynthesis is discussed, questioning the exclusive role of glandicoline A as key intermediate in the pathway. The results reveal that further enzymes of this pathway are rather unspecific and catalyze more than one reaction, leading to excessive branching in the pathway with meleagrin and neoxaline as end products of two branches.

  4. 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...... 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....

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

    Science.gov (United States)

    Dick, Cynthia A; Buenrostro, Jason; Butler, Timothy; Carlson, Matthew L; Kliebenstein, Daniel J; Whittall, Justen B

    2011-04-07

    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.

  6. 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.

  7. Hypoxia decreases the expression of the two enzymes responsible for producing linear and cyclic tetrapyrroles in the heme biosynthetic pathway.

    Science.gov (United States)

    Vargas, Patrick D; Furuyama, Kazumichi; Sassa, Shigeru; Shibahara, Shigeki

    2008-12-01

    Heme is synthesized in all cell types in aerobic organisms. Hydroxymethylbilane synthase (HMBS) and uroporphyrinogen III synthase (UROS) catalyze two consecutive reactions in the heme biosynthetic pathway, generating the first linear and the first cyclic tetrapyrroles, respectively. Each of the HMBS and UROS genes contains the two separate promoters that generate ubiquitous and erythroid-specific mRNAs. Despite the functional significance of HMBS and UROS, regulation of their gene expression remains to be investigated. Here, we showed that hypoxia (1% O(2)) decreased the expression of ubiquitous mRNAs for HMBS and UROS by three- and twofold, respectively, in human hepatic cells (HepG2 and Hep3B), whereas the expression of ubiquitous and erythroid HMBS and UROS mRNAs remained unchanged in erythroid cells (YN-1 and K562). Unexpectedly, hypoxia did not decrease the half-life of HMBS mRNA (8.4 h under normoxia versus 9.1 h under hypoxia) or UROS mRNA (9.0 versus 10.4 h) in hepatic cells. It is therefore unlikely that a change in mRNA stability is responsible for the hypoxia-mediated decrease in the expression levels of these mRNAs. Furthermore, expression levels of HMBS and UROS mRNAs were decreased under normoxia by treatment with deferoxamine or cobalt chloride in hepatic cells, while hypoxia-inducible factor 1alpha was accumulated. Thus, the decrease in the expression of ubiquitous HMBS and UROS mRNAs is associated with accumulation of hypoxia-inducible factor 1alpha protein. In conclusion, the expression of HMBS and UROS mRNAs may be coordinately regulated, which represents a newly identified mechanism that is important for heme homeostasis.

  8. Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence.

    Science.gov (United States)

    ten Have, A; Woltering, E J

    1997-05-01

    Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC 4.4.1.14) genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity. Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.

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

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    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

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

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    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.

  11. The efflux pump MlcE from the Penicillium solitum compactin biosynthetic gene cluster increases Saccharomyces cerevisiae resistance to natural statins

    DEFF Research Database (Denmark)

    Ley, Ana; Frandsen, Rasmus John Normand

    The use of statins as cholesterol-lowering drugs is based on their ability to inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the key enzyme in the mevalonate pathway, which is responsible for the production of ergosterol in fungi and cholesterol in human. Industrial scale...... integrated a putative efflux pump-encoding gene mlcE from the P. solitum compactin biosynthetic gene cluster into S. cerevisiae genome. The resulting strain was tested for susceptibility to statins by growing the strain on media containing statins. The constructed strain showed an increased resistance...

  12. Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

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    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

  13. Expression of phenazine biosynthetic genes during the arbuscular mycorrhizal symbiosis of Glomus intraradices

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    Dionicia Gloria León-Martínez

    2012-06-01

    Full Text Available To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010. Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.

  14. Treadmill exercise does not change gene expression of adrenal catecholamine biosynthetic enzymes in chronically stressed rats

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    LJUBICA GAVRILOVIC

    2013-09-01

    Full Text Available ABSTRACT Chronic isolation of adult animals represents a form of psychological stress that produces sympatho-adrenomedullar activation. Exercise training acts as an important modulator of sympatho-adrenomedullary system. This study aimed to investigate physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes (tyrosine hydroxylase, dopamine-ß-hydroxylase and phenylethanolamine N-methyltransferase and cyclic adenosine monophosphate response element-binding (CREB in the adrenal medulla, concentrations of catecholamines and corticosterone (CORT in the plasma and the weight of adrenal glands of chronically psychosocially stressed adult rats exposed daily to 20 min treadmill running for 12 weeks. Also, we examined how additional acute immobilization stress changes the mentioned parameters. Treadmill running did not result in modulation of gene expression of catecholamine synthesizing enzymes and it decreased the level of CREB mRNA in the adrenal medulla of chronically psychosocially stressed adult rats. The potentially negative physiological adaptations after treadmill running were recorded as increased concentrations of catecholamines and decreased morning CORT concentration in the plasma, as well as the adrenal gland hypertrophy of chronically psychosocially stressed rats. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. Treadmill exercise does not change the activity of sympatho-adrenomedullary system of chronically psychosocially stressed rats.

  15. 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.

  16. 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.

  17. Identification of a new diterpene biosynthetic gene cluster that produces O-methylkolavelool in Herpetosiphon aurantiacus.

    Science.gov (United States)

    Nakano, Chiaki; Oshima, Misaki; Kurashima, Nodoka; Hoshino, Tsutomu

    2015-03-23

    Diterpenoids are usually found in plants and fungi, but are rare in bacteria. We have previously reported new diterpenes, named tuberculosinol and isotuberculosinol, which are generated from the Mycobacterium tuberculosis gene products Rv3377c and Rv3378c. No homologous gene was found at that time, but we recently found highly homologous proteins in the Herpetosiphon aurantiacus ATCC 23779 genome. Haur_2145 was a class II diterpene cyclase responsible for the conversion of geranylgeranyl diphosphate into kolavenyl diphosphate. Haur_2146, homologous to Rv3378c, synthesized (+)-kolavelool through the nucleophilic addition of a water molecule to the incipient cation formed after the diphosphate moiety was released. Haur_2147 afforded (+)-O-methylkolavelool from (+)-kolavelool, so this enzyme was an O-methyltransferase. This new diterpene was indeed detected in H. aurantiacus cells. This is the first report of the identification of a (+)-O-methylkolavelool biosynthetic gene cluster.

  18. 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.

  19. Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use.

    Science.gov (United States)

    Yoshida, Kiyohito; Hashimoto, Mikako; Hori, Ryuji; Adachi, Takumi; Okuyama, Hidetoshi; Orikasa, Yoshitake; Nagamine, Tadashi; Shimizu, Satoru; Ueno, Akio; Morita, Naoki

    2016-05-12

    The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

  20. 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.

  1. Overexpressions of Lambda Phage Lysis Genes and Biosynthetic Genes of Poly-β-hydroxybutyrate in Recombinant E.coli

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    A plasmid (pTU9) containing the lambda (λ) phage lysis genes S(-)RRz and the biosynthetic genes phbCAB of poly-β-hydroxybutyrate (PHB) was constructed and transformed into E.coli JM109. Cultured in Luria-Bertani (LB) medium with 20 g/L glucose, E.coli JM109 (pTU9) could accumulate PHB in cells up to 40% (g PHB per g dry cells). A chelating agent EDTA was applied to induce a complete cell lysis and PHB granules were released. This method has a potential application in PHB separation.

  2. Effects of Cerium on Accumulation of Anthocyanins and Expression of Anthocyanin Biosynthetic Genes in Potato Cell Tissue Cultures

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The effects of Ce (Ⅳ) on callus growth, anthocyanin content, and expression of anthocyanin biosynthetic genes in callus suspension cultures of Solanum tuberosum cv. Chieftain were studied by the measurement of fresh weight, spectrophotometric assays, and semiquantitative RT-PCR. The results indicate that 0.1 mmol·L-1 Ce (Ⅳ) can promote callus growth, increase the accumulation of anthocyanins, and enhance the expression of five anthocyanin biosynthetic genes (CHS, F3H, F3′5′H, DFR, and 3GT) most efficiently. At high concentrations of 1 mmol·L-1, Ce (Ⅳ) partially inhibits callus growth and at 2 mmol·L-1 eventually lends to cell death. The results show that Ce(Ⅳ) can induce the expression of anthocyanin biosynthetic genes to produce and accumulate anthocyanins and increase the yield of anthocyanins.

  3. Cloning of artemisinin biosynthetic cDNAs and novel ESTs and quantification of low temperature-induced gene overexpression

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To isolate and verify novel genes from qinghao (Artemisia annua) based on the development-specific and environment-induced transcriptomics, leaves have been harvested from the flowering A. annua plants and exposed to low temperature for isolation of total RNAs and cloning of full-length cDNAs and cDNA fragments, or expressed sequence tags (ESTs). After being sequenced and browsed for homol- ogy, these sequences have been submitted to GenBank. Among the accessed 75 sequences, 4 full-length cDNAs are highly homologous to the known A. annua genes, but 71 ESTs are absent in the sequence records of A. annua genes, in which 34 sequences are homologous to other plant genes, including 24 identified protein-coding sequences and 10 unidentified protein-coding sequences, while other 37 sequences are not present in the sequence records of any plant genes, representing the first cloned plant genes. In order to investigate the responsive patterns of A. annua genes to extreme envi- ronmental stresses, especially low temperature, the expression levels of 3 critical qinhaosu (artemisi- nin) biosynthetic genes, ADS, CYP71AV1 and CPR, have been measured in pre- and post-chilling A. annua seedlings cultured in vitro by semi-quantitative PCR (SQ-PCR). Consequently, ADS and CYP71AV1 genes are strongly induced by chilling, but CPR gene is not significantly affected by such treatment. Furthermore, induction of these genes by chilling can be potently suppressed by Ca2+ channel inhibitor LaCl3 or Ca2+ chelator EGTA, suggesting a putative involvement of Ca2+-CaM signal transduction pathway in chilling-induced overexpression of ADS and CYP71AV1 genes. The real-time fluorescent quantitative PCR (RFQ-PCR) assay of A. annua seedlings exposed to chilling has shown that the expression level of CaM gene is up-regulated for more than 2.5 folds, thereby confirming our above inference on the relevance of Ca2+-CaM-mediated signal transduction to chilling-induced gene overexpression. Finally, 7 newly

  4. Cloning of artemisinin biosynthetic cDNAs and novel ESTs and quantification of low temperature-induced gene overexpression

    Institute of Scientific and Technical Information of China (English)

    ZENG QingPing; ZHAO Chang; YIN LuLu; YANG RuiYi; ZENG XiaoMei; HUANG Ying; FENG LiLing; YANG XueQin

    2008-01-01

    To isolate and verify novel genes from qinghao (Artemisia annua) based on the development-specific and environment-induced transcriptomics, leaves have been harvested from the flowering A. annua plants and exposed to low temperature for isolation of total RNAs and cloning of full-length cDNAs and cDNA fragments, or expressed sequence tags (ESTs). After being sequenced and browsed for homology, these sequences have been submitted to GenBank. Among the accessed 75 sequences, 4 full-length cDNAs are highly homologous to the known A. annua genes, but 71 ESTs are absent in the sequence records of A. annua genes, in which 34 sequences are homologous to other plant genes,including 24 identified protein-coding sequences and 10 unidentified protein-coding sequences, while other 37 sequences are not present in the sequence records of any plant genes, representing the first cloned plant genes. In order to investigate the responsive patterns of A. annua genes to extreme environmental stresses, especially low temperature, the expression levels of 3 critical qinhaosu (artemisinin) biosynthetic genes, ADS, CYP71AV1 and CPR, have been measured in pre- and post-chilling A.annua seedlings cultured in vitro by semi-quantitative PCR (SQ-PCR). Consequently, ADS and CYP71AV1 genes are strongly induced by chilling, but CPR gene is not significantly affected by such treatment. Furthermore, induction of these genes by chilling can be potently suppressed by Ca2+channel inhibitor LaCl3 or Ca2+ chelator EGTA, suggesting a putative involvement of Ca2+-CaM signal transduction pathway in chilling-induced overexpression of ADS and CYP71AV1 genes. The real-time fluorescent quantitative PCR (RFQ-PCR) assay of A. annua seedlings exposed to chilling has shown that the expression level of CaM gene is up-regulated for more than 2.5 folds, thereby confirming our above inference on the relevance of Ca2+-CaM-mediated signal transduction to chilling-induced gene overexpression. Finally, 7 newly isolated A

  5. 番茄与西瓜中番茄红素生物合成途径基因的比较分析%Comparison of Lycopene Biosynthetic Genes between Tomato and Watermelon

    Institute of Scientific and Technical Information of China (English)

    王辉; 李文丽; 王富

    2015-01-01

    为了阐明番茄和西瓜中番茄红素生物合成途径的相关基因,借助比较基因组学,在全基因组水平上对两物种间该代谢途径进行了比较分析。在番茄中共鉴定了12个番茄红素生物合成途径基因,在西瓜中发现了14个相应的番茄红素生物合成途径基因,同时将所有基因定位到相应的染色体上。番茄和西瓜中直系同源番茄红素生物合成基因在核酸水平保持在54.8%~75.0%的一致性,而西瓜中相应的旁系同源基因在核酸水平上的一致性为70.5%~74.8%。番茄与西瓜中番茄红素直系同源基因间在基因结构上高度相似。且系统进化分析发现西瓜中番茄红素生物合成关键基因PSY (Cla005425)和LCYE(Cla016840)可能与胡萝卜中的直系同源基因拥有共同的祖先。%In order to elucidate the genes related to the lycopene biosynthetic pathway in tomato and watermelon , we conducted comparative genomic analyses of lycopene biosynthetic pathway in these two crop species at a genome -wide level .We identified 12 lycopene biosynthetic genes in tomato , and found 14 relevant lycopene biosynthetic genes in watermelon .All these genes were suc-cessfully mapped on the related chromosomes .The orthologous lycopene biosynthetic genes between tomato and watermelon shared 54.8%~75.0% nucleotide sequence identity , while the relevant paralogous lycopene biosynthetic genes in watermelon shared 70.5%~74.8%nucleotide sequence identity .The structure of lycopene biosynthetic genes in tomato was highly similar to that of their orthologous genes in watermelon .Moreover, the phylogenetic trees indicated that the lycopene biosynthetic genes PSY (Cla005425) and LCYE (Cla016840) in watermelon maybe shared the same ancestor with their orthologous genes in carrot .

  6. Phenylpropanoids accumulation in eggplant fruit: characterization of biosynthetic genes and regulation by a MYB transcription factor

    Directory of Open Access Journals (Sweden)

    Teresa eDocimo

    2016-01-01

    Full Text Available Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena fruits. Chlorogenic acid (CGA accounts for 70 to 90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena.Higher contents of CGA, Delphinidin 3-rutinoside and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group 6 MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties.In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation.Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9 resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of

  7. 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.

  8. Saponin determination, expression analysis and functional characterization of saponin biosynthetic genes in Chenopodium quinoa leaves.

    Science.gov (United States)

    Fiallos-Jurado, Jennifer; Pollier, Jacob; Moses, Tessa; Arendt, Philipp; Barriga-Medina, Noelia; Morillo, Eduardo; Arahana, Venancio; de Lourdes Torres, Maria; Goossens, Alain; Leon-Reyes, Antonio

    2016-09-01

    Quinoa (Chenopodium quinoa Willd.) is a highly nutritious pseudocereal with an outstanding protein, vitamin, mineral and nutraceutical content. The leaves, flowers and seed coat of quinoa contain triterpenoid saponins, which impart bitterness to the grain and make them unpalatable without postharvest removal of the saponins. In this study, we quantified saponin content in quinoa leaves from Ecuadorian sweet and bitter genotypes and assessed the expression of saponin biosynthetic genes in leaf samples elicited with methyl jasmonate. We found saponin accumulation in leaves after MeJA treatment in both ecotypes tested. As no reference genes were available to perform qPCR in quinoa, we mined publicly available RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana using geNorm, NormFinder and BestKeeper algorithms. The quinoa ortholog of At2g28390 (Monensin Sensitivity 1, MON1) was stably expressed and chosen as a suitable reference gene for qPCR analysis. Candidate saponin biosynthesis genes were screened in the quinoa RNA-Seq data and subsequent functional characterization in yeast led to the identification of CqbAS1, CqCYP716A78 and CqCYP716A79. These genes were found to be induced by MeJA, suggesting this phytohormone might also modulate saponin biosynthesis in quinoa leaves. Knowledge of the saponin biosynthesis and its regulation in quinoa may aid the further development of sweet cultivars that do not require postharvest processing.

  9. 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

  10. 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

  11. Ultraviolet Radiation-Elicited Enhancement of Isoflavonoid Accumulation, Biosynthetic Gene Expression, and Antioxidant Activity in Astragalus membranaceus Hairy Root Cultures.

    Science.gov (United States)

    Jiao, Jiao; Gai, Qing-Yan; Wang, Wei; Luo, Meng; Gu, Cheng-Bo; Fu, Yu-Jie; Ma, Wei

    2015-09-23

    In this work, Astragalus membranaceus hairy root cultures (AMHRCs) were exposed to ultraviolet radiation (UV-A, UV-B, and UV-C) for promoting isoflavonoid accumulation. The optimum enhancement for isoflavonoid production was achieved in 34-day-old AMHRCs elicited by 86.4 kJ/m(2) of UV-B. The resulting isoflavonoid yield was 533.54 ± 13.61 μg/g dry weight (DW), which was 2.29-fold higher relative to control (232.93 ± 3.08 μg/g DW). UV-B up-regulated the transcriptional expressions of all investigated genes involved in isoflavonoid biosynthetic pathway. PAL and C4H were found to be two potential key genes that controlled isoflavonoid biosynthesis. Moreover, a significant increase was noted in antioxidant activity of extracts from UV-B-elicited AMHRCs (IC50 values = 0.85 and 1.08 mg/mL) in comparison with control (1.38 and 1.71 mg/mL). Overall, this study offered a feasible elicitation strategy to enhance isoflavonoid accumulation in AMHRCs and also provided a basis for metabolic engineering of isoflavonoid biosynthesis in the future.

  12. Environmental cues induce changes of steviol glycosides contents and transcription of corresponding biosynthetic genes in Stevia rebaudiana.

    Science.gov (United States)

    Yang, Yongheng; Huang, Suzhen; Han, Yulin; Yuan, Haiyan; Gu, Chunsun; Wang, Zhongwei

    2015-01-01

    Plant growth and secondary metabolism are commonly regulated by external cues such as light, temperature and water availability. In this study, the influences of low and high temperatures, dehydration, photoperiods, and different growing stages on the changes of steviol glycosides (SGs) contents and transcription levels of fifteen genes involved in SGs biosynthesis of Stevia rebaudiana Bertoni were examined using HPLC and RT-PCR. The observations showed that the transcript levels of all the fifteen genes were maximum under 25 °C treatment, and the transcription of SrDXS, SrDXR, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI, SrGGDPS, SrCPPS1, SrUGT85C2 and SrUGT76G1 were restrained both in low temperature (15 °C) and high temperature (35 °C). Most genes in SGs biosynthesis pathway exhibited down-regulation in dehydration. To elucidate the effect of photoperiods, the plants were treated by different simulated photoperiods (8 L/16 D, 1 0L/14 D, 14 L/10 D and 16 L/8 D), but no significant transcription changes were observed. In the study of growing stages, there were evident changes of SGs contents, and the transcript levels of all the fifteen genes were minimal in fast growing period, and exhibited evident increase both in flower-bud appearing stage and flowering stage. The obtained results strongly suggest that the effect of environmental cues on steviol glycosides contents and transcription of corresponding biosynthetic genes in S. rebaudiana is significant. It is worth to study deeply.

  13. 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

  14. Cloning and expression analyses of the anthocyanin biosynthetic genes in mulberry plants.

    Science.gov (United States)

    Qi, Xiwu; Shuai, Qin; Chen, Hu; Fan, Li; Zeng, Qiwei; He, Ningjia

    2014-10-01

    Anthocyanins are natural food colorants produced by plants that play important roles in their growth and development. Mulberry fruits are rich in anthocyanins, which are the most important active components of mulberry and have many potentially beneficial effects on human health. The study of anthocyanin biosynthesis will bring benefits for quality improvement and industrial exploration of mulberry fruits. In the present study, nine putative genes involved in anthocyanin biosynthesis in mulberry plants were identified and cloned. Sequence analysis revealed that the mulberry anthocyanin biosynthetic genes were conserved and had counterparts in other plants. Spatial transcriptional analysis showed detectable expression of eight of these genes in different tissues. The results of expression and UPLC analyses in two mulberry cultivars with differently colored fruit indicated that anthocyanin concentrations correlated with the expression levels of genes associated with anthocyanin biosynthesis including CHS1, CHI, F3H1, F3'H1, and ANS during the fruit ripening process. The present studies provide insight into anthocyanin biosynthesis in mulberry plants and may facilitate genetic engineering for improvement of the anthocyanin content in mulberry fruit.

  15. Minimization of biosynthetic costs in adaptive gene expression responses of yeast to environmental changes.

    Directory of Open Access Journals (Sweden)

    Ester Vilaprinyo

    2010-02-01

    Full Text Available Yeast successfully adapts to an environmental stress by altering physiology and fine-tuning metabolism. This fine-tuning is achieved through regulation of both gene expression and protein activity, and it is shaped by various physiological requirements. Such requirements impose a sustained evolutionary pressure that ultimately selects a specific gene expression profile, generating a suitable adaptive response to each environmental change. Although some of the requirements are stress specific, it is likely that others are common to various situations. We hypothesize that an evolutionary pressure for minimizing biosynthetic costs might have left signatures in the physicochemical properties of proteins whose gene expression is fine-tuned during adaptive responses. To test this hypothesis we analyze existing yeast transcriptomic data for such responses and investigate how several properties of proteins correlate to changes in gene expression. Our results reveal signatures that are consistent with a selective pressure for economy in protein synthesis during adaptive response of yeast to various types of stress. These signatures differentiate two groups of adaptive responses with respect to how cells manage expenditure in protein biosynthesis. In one group, significant trends towards downregulation of large proteins and upregulation of small ones are observed. In the other group we find no such trends. These results are consistent with resource limitation being important in the evolution of the first group of stress responses.

  16. Cloning and expression of anthocyanin biosynthetic genes in red and white pomegranate.

    Science.gov (United States)

    Zhao, Xueqing; Yuan, Zhaohe; Feng, Lijuan; Fang, Yanming

    2015-07-01

    Exterior fruit color is an important trait for the evaluation of pomegranate fruit quality, but the molecular mechanism underlying the variation in color between red- and white-fruited pomegranate is poorly understood. In this study, full-length cDNA clones encoding enzymes involved in anthocyanin biosynthesis-such as chalcone synthase, chalcone isomerase, flavanone 3-hydoxylase, dihydroflavonol 4-reductase, anthocyanidin synthase (ANS), UDP-glucose-flavonoid 3-O-glucosyltransferase, and the R2R3 MYB transcription factor PgMYB-were isolated from fruit peels. In addition, transcript levels of anthocyanin biosynthetic genes were quantitatively measured by real-time PCR in red and white fruits. In both cultivars, two expression peaks for structural genes were detected during fruit development, whereas only one peak was observed-during early development-for PgMYB. While PgMYB is important for flavonoid biosynthesis, other transcription factors appear to also be necessary for the regulation of anthocyanin biosynthesis. No anthocyanins were detected in the white cultivar. Peels of white fruits contained transcripts of all identified genes except for PgANS, suggesting that the lack of PgANS expression may be the main factor responsible for the absence of anthocyanins in white pomegranate. PgANS may be the key gene involved in anthocyanin biosynthesis in pomegranate fruit.

  17. Identification of the Pr1 gene product completes the anthocyanin biosynthesis pathway of maize

    Science.gov (United States)

    In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3'H encoding gene (Zmf3'h1), and showed by segrega...

  18. Labellum transcriptome reveals alkene biosynthetic genes involved in orchid sexual deception and pollination-induced senescence.

    Science.gov (United States)

    Monteiro, Filipa; Sebastiana, Mónica; Figueiredo, Andreia; Sousa, Lisete; Cotrim, Helena C; Pais, Maria Salomé

    2012-11-01

    One of the most remarkable pollination strategy in orchids biology is pollination by sexual deception, in which the modified petal labellum lures pollinators by mimicking the chemical (e.g. sex pheromones), visual (e.g. colour and shape/size) and tactile (e.g. labellum trichomes) cues of the receptive female insect species. The present study aimed to characterize the transcriptional changes occurring after pollination in the labellum of a sexually deceptive orchid (Ophrys fusca Link) in order to identify genes involved on signals responsible for pollinator attraction, the major goal of floral tissues. Novel information on alterations in the orchid petal labellum gene expression occurring after pollination demonstrates a reduction in the expression of alkene biosynthetic genes using O. fusca Link as the species under study. Petal labellum transcriptional analysis revealed downregulation of transcripts involved in both pigment machinery and scent compounds, acting as visual and olfactory cues, respectively, important in sexual mimicry. Regulation of petal labellum senescence was revealed by transcripts related to macromolecules breakdown, protein synthesis and remobilization of nutrients.

  19. 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

  20. Carotenoids of Gemmatimonas aurantiaca (Gemmatimonadetes): identification of a novel carotenoid, deoxyoscillol 2-rhamnoside, and proposed biosynthetic pathway of oscillol 2,2'-dirhamnoside.

    Science.gov (United States)

    Takaichi, Shinichi; Maoka, Takashi; Takasaki, Kazuto; Hanada, Satoshi

    2010-03-01

    Gemmatimonas aurantiaca strain T-27(T) is an orange-coloured, Gram-negative, facultatively aerobic, polyphosphate-accumulating bacterium belonging to a recently proposed phylum, Gemmatimonadetes. We purified its pigments and identified them as carotenoids and their glycoside derivatives using spectral data. The major carotenoid was (2S,2' S)-oscillol 2,2'-di-(alpha-l-rhamnoside), and the minor carotenoids were (2S)-deoxyoscillol 2-( alpha-l-rhamnoside) and didemethylspirilloxanthin. Deoxyoscillol 2-rhamnoside is a novel carotenoid. Oscillol 2,2'-diglycosides have hitherto only been reported in a limited number of cyanobacteria, and this is believed to be the first finding of such carotenoids in another bacterial phylum. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence, we propose a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. We propose the involvement of geranylgeranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI) for lycopene synthesis; and of carotenoid 1,2-hydratase (CruF) and carotenoid 2-O-rhamnosyltransferase (CruG) for oscillol 2,2'-dirhamnoside synthesis. Further, isopentenyl pyrophosphate could be synthesized by a non-mevalonate pathway (DXP pathway).

  1. First Biosynthetic pathway of 1-hepten-3-one in Iporangaia pustulosa (Opiliones)

    Science.gov (United States)

    Rocha, Daniele F. O.; Wouters, Felipe C.; Machado, Glauco; Marsaioli, Anita J.

    2013-11-01

    Arthropods produce a great variety of natural compounds, many of which have unexplored biosynthesis. Among the armored harvestmen (Arachnida: Opiliones) of the suborder Laniatores, the defensive gland exudates contain vinyl ketones and other constituents of supposed polyketide origin. We have studied the biosynthesis of 1-hepten-3-one in the Neotropical harvestman Iporangaia pustulosa by feeding individuals with 13C-labeled precursors, demonstrating its mixed acetate/propionate origin. 13C NMR spectroscopy showed an unusual labeling pattern suggesting different propionate sources for starting and extender units. Our analysis also indicates the presence of methylmalonyl-CoA mutase, converting acetate into propionyl-CoA via succinyl-CoA, together with other C3 unit routes. This is the first biosynthetic study of alkyl vinyl ketones in arthropods. Our results shed light on the origin and diversification of chemical compounds in a major arthropod group.

  2. Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway.

    Science.gov (United States)

    Kopečná, Jana; Cabeza de Vaca, Israel; Adams, Nathan B P; Davison, Paul A; Brindley, Amanda A; Hunter, C Neil; Guallar, Victor; Sobotka, Roman

    2015-11-20

    In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

  3. Polymorphism of starch pathway genes in cassava.

    Science.gov (United States)

    Vasconcelos, L M; Brito, A C; Carmo, C D; Oliveira, E J

    2016-12-02

    The distribution and frequency of single nucleotide polymorphisms (SNPs) can help to understand changes associated with characteristics of interest. We aimed to evaluate nucleotide diversity in six genes involved in starch biosynthesis in cassava using a panel of 96 unrelated accessions. The genes were sequenced, aligned, and used to obtain values for nucleotide diversity (π), segregating sites (θ), Tajima's D test, and neighbor-joining (NJ) clustering. On average, one SNP per 147 and 171 bp was identified in exon and intron regions, respectively. Thirteen heterozygous loci were found. Three of seven SNPs in the exon region resulted in non-synonymous replacement or four synonymous substitutions. However, no associations were noted between SNPs and root dry-matter content. The parameter π ranged from 0.0001 (granule bound starch synthase I) to 0.0033 (α-amylase), averaging 0.0011, while θ ranged from 0.00014 (starch branching enzyme) to 0.00584 (starch synthase I), averaging 0.002353. The θ diversity value was typically double that of the π. Results of the D test did not suggest any evidence of deviance of neutrality in these genes. Among the evaluated accession, 82/96 were clustered using the NJ method but without a clear separation of the root dry-matter content, root pulp coloration, and classification of the cyanogenic compound content. High variation in genes of the starch biosynthetic pathway can be used to identify associations with the functional properties of starch for the use of polymorphisms for selection purposes.

  4. Exploitation of the Streptomyces coelicolor A3(2) genome sequence for discovery of new natural products and biosynthetic pathways.

    Science.gov (United States)

    Challis, Gregory L

    2014-02-01

    Streptomyces, and related genera of Actinobacteria, are renowned for their ability to produce antibiotics and other bioactive natural products with a wide range of applications in medicine and agriculture. Streptomyces coelicolor A3(2) is a model organism that has been used for more than five decades to study the genetic and biochemical basis for the production of bioactive metabolites. In 2002, the complete genome sequence of S. coelicolor was published. This greatly accelerated progress in understanding the biosynthesis of metabolites known or suspected to be produced by S. coelicolor and revealed that streptomycetes have far greater potential to produce bioactive natural products than suggested by classical bioassay-guided isolation studies. In this article, efforts to exploit the S. coelicolor genome sequence for the discovery of novel natural products and biosynthetic pathways are summarized.

  5. Allelic effects on starch structure and properties of six starch biosynthetic genes in a rice recombinant inbred line population

    OpenAIRE

    2015-01-01

    Background The genetic diversity of six starch biosynthetic genes (Wx, SSI, SSIIa, SBEI, SBEIIa and SBEIIb) in indica and japonica rices opens an opportunity to produce a new variety with more favourable grain starch quality. However, there is limited information about the effects of these six gene allele combinations on starch structure and properties. A recombinant inbred line population from a cross between indica and japonica varieties offers opportunities to combine specific alleles of t...

  6. Discovery of putative capsaicin biosynthetic genes by RNA-Seq and digital gene expression analysis of pepper

    Science.gov (United States)

    Zhang, Zi-Xin; Zhao, Shu-Niu; Liu, Gao-Feng; Huang, Zu-Mei; Cao, Zhen-Mu; Cheng, Shan-Han; Lin, Shi-Sen

    2016-01-01

    The Indian pepper ‘Guijiangwang’ (Capsicum frutescens L.), one of the world’s hottest chili peppers, is rich in capsaicinoids. The accumulation of the alkaloid capsaicin and its analogs in the epidermal cells of the placenta contribute to the pungency of Capsicum fruits. To identify putative genes involved in capsaicin biosynthesis, RNA-Seq was used to analyze the pepper’s expression profiles over five developmental stages. Five cDNA libraries were constructed from the total RNA of placental tissue and sequenced using an Illumina HiSeq 2000. More than 19 million clean reads were obtained from each library, and greater than 50% of the reads were assignable to reference genes. Digital gene expression (DGE) profile analysis using Solexa sequencing was performed at five fruit developmental stages and resulted in the identification of 135 genes of known function; their expression patterns were compared to the capsaicin accumulation pattern. Ten genes of known function were identified as most likely to be involved in regulating capsaicin synthesis. Additionally, 20 new candidate genes were identified related to capsaicin synthesis. We use a combination of RNA-Seq and DGE analyses to contribute to the understanding of the biosynthetic regulatory mechanism(s) of secondary metabolites in a nonmodel plant and to identify candidate enzyme-encoding genes. PMID:27756914

  7. Glutamic acid promotes monacolin K production and monacolin K biosynthetic gene cluster expression in Monascus.

    Science.gov (United States)

    Zhang, Chan; Liang, Jian; Yang, Le; Chai, Shiyuan; Zhang, Chenxi; Sun, Baoguo; Wang, Chengtao

    2017-12-01

    This study investigated the effects of glutamic acid on production of monacolin K and expression of the monacolin K biosynthetic gene cluster. When Monascus M1 was grown in glutamic medium instead of in the original medium, monacolin K production increased from 48.4 to 215.4 mg l(-1), monacolin K production increased by 3.5 times. Glutamic acid enhanced monacolin K production by upregulating the expression of mokB-mokI; on day 8, the expression level of mokA tended to decrease by Reverse Transcription-polymerase Chain Reaction. Our findings demonstrated that mokA was not a key gene responsible for the quantity of monacolin K production in the presence of glutamic acid. Observation of Monascus mycelium morphology using Scanning Electron Microscope showed glutamic acid significantly increased the content of Monascus mycelium, altered the permeability of Monascus mycelium, enhanced secretion of monacolin K from the cell, and reduced the monacolin K content in Monascus mycelium, thereby enhancing monacolin K production.

  8. Diurnal Regulation of the Brassinosteroid-Biosynthetic CPD Gene in Arabidopsis1[W

    Science.gov (United States)

    Bancos, Simona; Szatmári, Anna-Mária; Castle, Julie; Kozma-Bognár, László; Shibata, Kyomi; Yokota, Takao; Bishop, Gerard J.; Nagy, Ferenc; Szekeres, Miklós

    2006-01-01

    Plant steroid hormones, brassinosteroids (BRs), are essential for normal photomorphogenesis. However, the mechanism by which light controls physiological functions via BRs is not well understood. Using transgenic plants carrying promoter-luciferase reporter gene fusions, we show that in Arabidopsis (Arabidopsis thaliana) the BR-biosynthetic CPD and CYP85A2 genes are under diurnal regulation. The complex diurnal expression profile of CPD is determined by dual, light-dependent, and circadian control. The severely decreased expression level of CPD in phytochrome-deficient background and the red light-specific induction in wild-type plants suggest that light regulation of CPD is primarily mediated by phytochrome signaling. The diurnal rhythmicity of CPD expression is maintained in brassinosteroid insensitive 1 transgenic seedlings, indicating that its transcriptional control is independent of hormonal feedback regulation. Diurnal changes in the expression of CPD and CYP85A2 are accompanied by changes of the endogenous BR content during the day, leading to brassinolide accumulation at the middle of the light phase. We also show that CPD expression is repressed in extended darkness in a BR feedback-dependent manner. In the dark the level of the bioactive hormone did not increase; therefore, our data strongly suggest that light also influences the sensitivity of plants to BRs. PMID:16531479

  9. Diurnal regulation of the brassinosteroid-biosynthetic CPD gene in Arabidopsis.

    Science.gov (United States)

    Bancos, Simona; Szatmári, Anna-Mária; Castle, Julie; Kozma-Bognár, László; Shibata, Kyomi; Yokota, Takao; Bishop, Gerard J; Nagy, Ferenc; Szekeres, Miklós

    2006-05-01

    Plant steroid hormones, brassinosteroids (BRs), are essential for normal photomorphogenesis. However, the mechanism by which light controls physiological functions via BRs is not well understood. Using transgenic plants carrying promoter-luciferase reporter gene fusions, we show that in Arabidopsis (Arabidopsis thaliana) the BR-biosynthetic CPD and CYP85A2 genes are under diurnal regulation. The complex diurnal expression profile of CPD is determined by dual, light-dependent, and circadian control. The severely decreased expression level of CPD in phytochrome-deficient background and the red light-specific induction in wild-type plants suggest that light regulation of CPD is primarily mediated by phytochrome signaling. The diurnal rhythmicity of CPD expression is maintained in brassinosteroid insensitive 1 transgenic seedlings, indicating that its transcriptional control is independent of hormonal feedback regulation. Diurnal changes in the expression of CPD and CYP85A2 are accompanied by changes of the endogenous BR content during the day, leading to brassinolide accumulation at the middle of the light phase. We also show that CPD expression is repressed in extended darkness in a BR feedback-dependent manner. In the dark the level of the bioactive hormone did not increase; therefore, our data strongly suggest that light also influences the sensitivity of plants to BRs.

  10. 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.

  11. Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways.

    Science.gov (United States)

    Michener, Joshua K; Thodey, Kate; Liang, Joe C; Smolke, Christina D

    2012-05-01

    Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.

  12. Diversity of Culturable Thermophilic Actinobacteria in Hot Springs in Tengchong, China and Studies of their Biosynthetic Gene Profiles.

    Science.gov (United States)

    Liu, Lan; Salam, Nimaichand; Jiao, Jian-Yu; Jiang, Hong-Chen; Zhou, En-Min; Yin, Yi-Rui; Ming, Hong; Li, Wen-Jun

    2016-07-01

    The class Actinobacteria has been a goldmine for the discovery of antibiotics and has attracted interest from both academics and industries. However, an absence of novel approaches during the last few decades has limited the discovery of new microbial natural products useful for industries. Scientists are now focusing on the ecological aspects of diverse environments including unexplored or underexplored habitats and extreme environments in the search for new metabolites. This paper reports on the diversity of culturable actinobacteria associated with hot springs located in Tengchong County, Yunnan Province, southwestern China. A total of 58 thermophilic actinobacterial strains were isolated from the samples collected from ten hot springs distributed over three geothermal fields (e.g., Hehua, Rehai, and Ruidian). Phylogenetic positions and their biosynthetic profiles were analyzed by sequencing 16S rRNA gene and three biosynthetic gene clusters (KS domain of PKS-I, KSα domain of PKS-II and A domain of NRPS). On the basis of 16S rRNA gene phylogenetic analysis, the 58 strains were affiliated with 12 actinobacterial genera: Actinomadura Micromonospora, Microbispora, Micrococcus, Nocardiopsis, Nonomuraea, Promicromonospora, Pseudonocardia, Streptomyces, Thermoactinospora, Thermocatellispora, and Verrucosispora, of which the two novel genera Thermoactinospora and Thermocatellisopora were recently described from among these strains. Considering the biosynthetic potential of these actinobacterial strains, 22 were positive for PCR amplification of at least one of the three biosynthetic gene clusters (PKS-I, PKS-II, and NRPS). These actinobacteria were further subjected to antimicrobial assay against five opportunistic human pathogens (Acinetobacter baumannii, Escherichia coli, Micrococcus luteus, Staphylococcus aureus and Streptococcus faecalis). All of the 22 strains that were positive for PCR amplification of at least one of the biosynthetic gene domains exhibited

  13. Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses.

    Science.gov (United States)

    Kampa, Annette; Gagunashvili, Andrey N; Gulder, Tobias A M; Morinaka, Brandon I; Daolio, Cristina; Godejohann, Markus; Miao, Vivian P W; Piel, Jörn; Andrésson, Ólafur S

    2013-08-13

    Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 μg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites.

  14. 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

  15. Engineering a novel biosynthetic pathway in Escherichia coli for production of renewable ethylene glycol.

    Science.gov (United States)

    Pereira, Brian; Zhang, Haoran; De Mey, Marjan; Lim, Chin Giaw; Li, Zheng-Jun; Stephanopoulos, Gregory

    2016-02-01

    Ethylene glycol (EG) is an important commodity chemical with broad industrial applications. It is presently produced from petroleum or natural gas feedstocks in processes requiring consumption of significant quantities of non-renewable resources. Here, we report a novel pathway for biosynthesis of EG from the renewable sugar glucose in metabolically engineered Escherichia coli. Serine-to-EG conversion was first achieved through a pathway comprising serine decarboxylase, ethanolamine oxidase, and glycolaldehyde reductase. Serine provision in E. coli was then enhanced by overexpression of the serine-biosynthesis pathway. The integration of these two parts into the complete EG-biosynthesis pathway in E. coli allowed for production of 4.1 g/L EG at a cumulative yield of 0.14 g-EG/g-glucose, establishing a foundation for a promising biotechnology.

  16. 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

  17. 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.

  18. Time Dependency of Chemodiversity and Biosynthetic Pathways: An LC-MS Metabolomic Study of Marine-Sourced Penicillium

    Directory of Open Access Journals (Sweden)

    Catherine Roullier

    2016-05-01

    Full Text Available This work aimed at studying metabolome variations of marine fungal strains along their growth to highlight the importance of the parameter “time” for new natural products discovery. An untargeted time-scale metabolomic study has been performed on two different marine-derived Penicillium strains. They were cultivated for 18 days and their crude extracts were analyzed by HPLC-DAD-HRMS (High Performance Liquid Chromatography-Diode Array Detector-High Resolution Mass Spectrometry each day. With the example of griseofulvin biosynthesis, a pathway shared by both strains, this work provides a new approach to study biosynthetic pathway regulations, which could be applied to other metabolites and more particularly new ones. Moreover, the results of this study emphasize the interest of such an approach for the discovery of new chemical entities. In particular, at every harvesting time, previously undetected features were observed in the LC-MS (Liquid Chromatography-Mass Spectrometry data. Therefore, harvesting times for metabolite extraction should be performed at different time points to access the hidden metabolome.

  19. Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn.

    Science.gov (United States)

    Wei, Yong-Zan; Hu, Fu-Chu; Hu, Gui-Bing; Li, Xiao-Jing; Huang, Xu-Ming; Wang, Hui-Cong

    2011-04-29

    Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU), bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m(-2) among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red ('Kuixingqingpitian', 'Xingqiumili', 'Yamulong'and 'Yongxing No. 2'), unevenly red ('Feizixiao' and 'Sanyuehong') and fully red ('Meiguili', 'Baila', Baitangying' 'Guiwei', 'Nuomici' and 'Guinuo'). The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT was found significantly correlated with the

  20. Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn.

    Directory of Open Access Journals (Sweden)

    Yong-Zan Wei

    Full Text Available Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU, bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS, chalcone isomerase (CHI, flavanone 3-hydroxylase (F3H, dihydroflavonol 4-reductase (DFR, anthocyanidin synthase (ANS and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m(-2 among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red ('Kuixingqingpitian', 'Xingqiumili', 'Yamulong'and 'Yongxing No. 2', unevenly red ('Feizixiao' and 'Sanyuehong' and fully red ('Meiguili', 'Baila', Baitangying' 'Guiwei', 'Nuomici' and 'Guinuo'. The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT was found significantly correlated

  1. Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers

    Directory of Open Access Journals (Sweden)

    Luo Hongmei

    2011-12-01

    Full Text Available Abstract Background Panax notoginseng (Burk F.H. Chen is important medicinal plant of the Araliacease family. Triterpene saponins are the bioactive constituents in P. notoginseng. However, available genomic information regarding this plant is limited. Moreover, details of triterpene saponin biosynthesis in the Panax species are largely unknown. Results Using the 454 pyrosequencing technology, a one-quarter GS FLX titanium run resulted in 188,185 reads with an average length of 410 bases for P. notoginseng root. These reads were processed and assembled by 454 GS De Novo Assembler software into 30,852 unique sequences. A total of 70.2% of unique sequences were annotated by Basic Local Alignment Search Tool (BLAST similarity searches against public sequence databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG assignment discovered 41 unique sequences representing 11 genes involved in triterpene saponin backbone biosynthesis in the 454-EST dataset. In particular, the transcript encoding dammarenediol synthase (DS, which is the first committed enzyme in the biosynthetic pathway of major triterpene saponins, is highly expressed in the root of four-year-old P. notoginseng. It is worth emphasizing that the candidate cytochrome P450 (Pn02132 and Pn00158 and UDP-glycosyltransferase (Pn00082 gene most likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis were discovered from 174 cytochrome P450s and 242 glycosyltransferases by phylogenetic analysis, respectively. Putative transcription factors were detected in 906 unique sequences, including Myb, homeobox, WRKY, basic helix-loop-helix (bHLH, and other family proteins. Additionally, a total of 2,772 simple sequence repeat (SSR were identified from 2,361 unique sequences, of which, di-nucleotide motifs were the most abundant motif. Conclusion This study is the first to present a large-scale EST dataset for P. notoginseng root acquired by next

  2. The Sound of Silence: Activating Silent Biosynthetic Gene Clusters in Marine Microorganisms

    Directory of Open Access Journals (Sweden)

    F. Jerry Reen

    2015-07-01

    Full Text Available Unlocking the rich harvest of marine microbial ecosystems has the potential to both safeguard the existence of our species for the future, while also presenting significant lifestyle benefits for commercial gain. However, while significant advances have been made in the field of marine biodiscovery, leading to the introduction of new classes of therapeutics for clinical medicine, cosmetics and industrial products, much of what this natural ecosystem has to offer is locked in, and essentially hidden from our screening methods. Releasing this silent potential represents a significant technological challenge, the key to which is a comprehensive understanding of what controls these systems. Heterologous expression systems have been successful in awakening a number of these cryptic marine biosynthetic gene clusters (BGCs. However, this approach is limited by the typically large size of the encoding sequences. More recently, focus has shifted to the regulatory proteins associated with each BGC, many of which are signal responsive raising the possibility of exogenous activation. Abundant among these are the LysR-type family of transcriptional regulators, which are known to control production of microbial aromatic systems. Although the environmental signals that activate these regulatory systems remain unknown, it offers the exciting possibility of evoking mimic molecules and synthetic expression systems to drive production of potentially novel natural products in microorganisms. Success in this field has the potential to provide a quantum leap forward in medical and industrial bio-product development. To achieve these new endpoints, it is clear that the integrated efforts of bioinformaticians and natural product chemists will be required as we strive to uncover new and potentially unique structures from silent or cryptic marine gene clusters.

  3. 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

  4. 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

  5. Chapter 3: Omics Advances of Biosynthetic Pathways of Isoprenoid Production in Microalgae

    Energy Technology Data Exchange (ETDEWEB)

    Paniagua-Michel, J.; Subramanian, Venkataramanan

    2017-01-01

    In this chapter, the current status of microalgal isoprenoids and the role of omics technologies, or otherwise specified, in bioproducts optimization and applications are reviewed. Emphasis is focused in the metabolic pathways of microalgae involved in the production of commercially important products, namely, hydrocarbons and biofuels, nutraceuticals, and pharmaceuticals.

  6. miRNA Nomenclature : A View Incorporating Genetic Origins, Biosynthetic Pathways, and Sequence Variants

    NARCIS (Netherlands)

    Desvignes, T.; Batzel, P.; Berezikov, E.; Eilbeck, K.; Eppig, J. T.; McAndrews, M. S.; Singer, A.; Postlethwait, J. H.

    2015-01-01

    High-throughput sequencing of miRNAs has revealed the diversity and variability of mature and functional short noncoding RNAs, including their genomic origins, biogenesis pathways, sequence variability, and newly identified products such as miRNA-offset RNAs (moRs). Here we review known cases of alt

  7. SCS3 and YFT2 link transcription of phospholipid biosynthetic genes to ER stress and the UPR.

    Directory of Open Access Journals (Sweden)

    Robyn D Moir

    2012-08-01

    Full Text Available The ability to store nutrients in lipid droplets (LDs is an ancient function that provides the primary source of metabolic energy during periods of nutrient insufficiency and between meals. The Fat storage-Inducing Transmembrane (FIT proteins are conserved ER-resident proteins that facilitate fat storage by partitioning energy-rich triglycerides into LDs. FIT2, the ancient ortholog of the FIT gene family first identified in mammals has two homologs in Saccharomyces cerevisiae (SCS3 and YFT2 and other fungi of the Saccharomycotina lineage. Despite the coevolution of these genes for more than 170 million years and their divergence from higher eukaryotes, SCS3, YFT2, and the human FIT2 gene retain some common functions: expression of the yeast genes in a human embryonic kidney cell line promotes LD formation, and expression of human FIT2 in yeast rescues the inositol auxotrophy and chemical and genetic phenotypes of strains lacking SCS3. To better understand the function of SCS3 and YFT2, we investigated the chemical sensitivities of strains deleted for either or both genes and identified synthetic genetic interactions against the viable yeast gene-deletion collection. We show that SCS3 and YFT2 have shared and unique functions that connect major biosynthetic processes critical for cell growth. These include lipid metabolism, vesicular trafficking, transcription of phospholipid biosynthetic genes, and protein synthesis. The genetic data indicate that optimal strain fitness requires a balance between phospholipid synthesis and protein synthesis and that deletion of SCS3 and YFT2 impacts a regulatory mechanism that coordinates these processes. Part of this mechanism involves a role for SCS3 in communicating changes in the ER (e.g. due to low inositol to Opi1-regulated transcription of phospholipid biosynthetic genes. We conclude that SCS3 and YFT2 are required for normal ER membrane biosynthesis in response to perturbations in lipid metabolism and ER

  8. Signal Transduction Pathways that Regulate CAB Gene Expression

    Energy Technology Data Exchange (ETDEWEB)

    Chory, Joanne

    2006-01-16

    The process of chloroplast differentiation, involves the coordinate regulation of many nuclear and chloroplast genes. The cues for the initiation of this developmental program are both extrinsic (e.g., light) and intrinsic (cell-type and plastid signals). During this project period, we utilized a molecular genetic approach to select for Arabidopsis mutants that did not respond properly to environmental light conditions, as well as mutants that were unable to perceive plastid damage. These latter mutants, called gun mutants, define two retrograde signaling pathways that regulate nuclear gene expression in response to chloroplasts. A major finding was to identify a signal from chloroplasts that regulates nuclear gene transcription. This signal is the build-up of Mg-Protoporphyrin IX, a key intermediate of the chlorophyll biosynthetic pathway. The signaling pathways downstream of this signal are currently being studied. Completion of this project has provided an increased understanding of the input signals and retrograde signaling pathways that control nuclear gene expression in response to the functional state of chloroplasts. These studies should ultimately influence our abilities to manipulate plant growth and development, and will aid in the understanding of the developmental control of photosynthesis.

  9. Signal Transduction Pathways that Regulate CAB Gene Expression

    Energy Technology Data Exchange (ETDEWEB)

    Chory, Joanne

    2004-12-31

    The process of chloroplast differentiation, involves the coordinate regulation of many nuclear and chloroplast genes. The cues for the initiation of this developmental program are both extrinsic (e.g., light) and intrinsic (cell-type and plastid signals). During this project period, we utilized a molecular genetic approach to select for Arabidopsis mutants that did not respond properly to environmental light conditions, as well as mutants that were unable to perceive plastid damage. These latter mutants, called gun mutants, define two retrograde signaling pathways that regulate nuclear gene expression in response to chloroplasts. A major finding was to identify a signal from chloroplasts that regulates nuclear gene transcription. This signal is the build-up of Mg-Protoporphyrin IX, a key intermediate of the chlorophyll biosynthetic pathway. The signaling pathways downstream of this signal are currently being studied. Completion of this project has provided an increased understanding of the input signals and retrograde signaling pathways that control nuclear gene expression in response to the functional state of chloroplasts. These studies should ultimately influence our abilities to manipulate plant growth and development, and will aid in the understanding of the developmental control of photosynthesis.

  10. 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....

  11. Natural product proteomining, a quantitative proteomics platform, allows rapid discovery of biosynthetic gene clusters for different classes of natural products.

    Science.gov (United States)

    Gubbens, Jacob; Zhu, Hua; Girard, Geneviève; Song, Lijiang; Florea, Bogdan I; Aston, Philip; Ichinose, Koji; Filippov, Dmitri V; Choi, Young H; Overkleeft, Herman S; Challis, Gregory L; van Wezel, Gilles P

    2014-06-19

    Information on gene clusters for natural product biosynthesis is accumulating rapidly because of the current boom of available genome sequencing data. However, linking a natural product to a specific gene cluster remains challenging. Here, we present a widely applicable strategy for the identification of gene clusters for specific natural products, which we name natural product proteomining. The method is based on using fluctuating growth conditions that ensure differential biosynthesis of the bioactivity of interest. Subsequent combination of metabolomics and quantitative proteomics establishes correlations between abundance of natural products and concomitant changes in the protein pool, which allows identification of the relevant biosynthetic gene cluster. We used this approach to elucidate gene clusters for different natural products in Bacillus and Streptomyces, including a novel juglomycin-type antibiotic. Natural product proteomining does not require prior knowledge of the gene cluster or secondary metabolite and therefore represents a general strategy for identification of all types of gene clusters.

  12. 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.

  13. 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.

  14. Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin.

    Science.gov (United States)

    Katsumoto, Yukihisa; Fukuchi-Mizutani, Masako; Fukui, Yuko; Brugliera, Filippa; Holton, Timothy A; Karan, Mirko; Nakamura, Noriko; Yonekura-Sakakibara, Keiko; Togami, Junichi; Pigeaire, Alix; Tao, Guo-Qing; Nehra, Narender S; Lu, Chin-Yi; Dyson, Barry K; Tsuda, Shinzo; Ashikari, Toshihiko; Kusumi, Takaaki; Mason, John G; Tanaka, Yoshikazu

    2007-11-01

    Flower color is mainly determined by anthocyanins. Rosa hybrida lacks violet to blue flower varieties due to the absence of delphinidin-based anthocyanins, usually the major constituents of violet and blue flowers, because roses do not possess flavonoid 3',5'-hydoxylase (F3'5'H), a key enzyme for delphinidin biosynthesis. Other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. We analyzed the flavonoid composition of hundreds of rose cultivars and measured the pH of their petal juice in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color change toward blue. Expression of the viola F3'5'H gene in some of the selected cultivars resulted in the accumulation of a high percentage of delphinidin (up to 95%) and a novel bluish flower color. For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, we down-regulated the endogenous dihydroflavonol 4-reductase (DFR) gene and overexpressed the Irisxhollandica DFR gene in addition to the viola F3'5'H gene in a rose cultivar. The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.

  15. 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

  16. IMG-ABC: An Atlas of Biosynthetic Gene Clusters to Fuel the Discovery of Novel Secondary Metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Chen, I-Min; Chu, Ken; Ratner, Anna; Palaniappan, Krishna; Huang, Jinghua; Reddy, T. B.K.; Cimermancic, Peter; Fischbach, Michael; Ivanova, Natalia; Markowitz, Victor; Kyrpides, Nikos; Pati, Amrita

    2014-10-28

    In the discovery of secondary metabolites (SMs), large-scale analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of relevant computational resources. We present IMG-ABC (https://img.jgi.doe.gov/abc/) -- An Atlas of Biosynthetic gene Clusters within the Integrated Microbial Genomes (IMG) system1. IMG-ABC is a rich repository of both validated and predicted biosynthetic clusters (BCs) in cultured isolates, single-cells and metagenomes linked with the SM chemicals they produce and enhanced with focused analysis tools within IMG. The underlying scalable framework enables traversal of phylogenetic dark matter and chemical structure space -- serving as a doorway to a new era in the discovery of novel molecules.

  17. Biosynthetic pathway of aliphatic formates via a Baeyer–Villiger oxidation in mechanism present in astigmatid mites

    Science.gov (United States)

    Shimizu, Nobuhiro; Sakata, Daisuke; Schmelz, Eric A.; Mori, Naoki; Kuwahara, Yasumasa

    2017-01-01

    Astigmatid mites depend on bioactive glandular secretions, pheromones, and defensive agents to mediate intra- and interspecies interactions. Aliphatic formates, such as (Z,Z)-8,11-heptadecadienyl formate (8,11-F17) and (Z)-8-heptadecenyl formate (8-F17), are rarely encountered natural products that are abundant in Sancassania sp. Sasagawa (Acari: Acaridae) mite secretions. Linoleic acid and oleic acid are predicted as key intermediates in the synthesis of the closely related aliphatic formates. To gain insight in this biosynthetic pathway, acarid mite feeding experiments were conducted using 13C-labeled precursors to precisely track incorporation. Analyses using 13C NMR spectroscopy demonstrated that the 13C-labeling pattern of the precursors was detectable on formates in exocrine secretions and likewise on fatty acids in total lipid pools. Curiously, the results demonstrated that the formates were biosynthesized without the dehomologation of corresponding fatty acids. Careful examination of the mass spectra from labeling experiments revealed that the carbonyl carbon of the formates is originally derived from the C-1 position of the fatty acids. Consistent with a Baeyer–Villiger oxidation reaction, labeling studies support the insertion of an oxygen atom between the carbonyl group and carbon chain. Empirical data support the existence of a Baeyer–Villiger monooxygenase responsible for the catalyzation of the Baeyer–Villiger oxidation. The predicted existence of a Baeyer–Villiger monooxygenase capable of converting aliphatic aldehydes to formates represents an exciting opportunity to expand the enzymatic toolbox available for controlled biochemical synthesis. PMID:28223501

  18. 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

    The influence the of dissolved oxygen concentration on penicillin biosynthesis was studied in steady-state continuous cultures of a high-yielding strain of Penicillium chrysogenum operated at a dilution rate of 0.05 h-l. The dissolved oxygen concentration was varied between 0.019 and 0.344 mM (co...... and cysteine decreased at low dissolved oxygen concentrations. On the basis of the intracellular pool measurements, metabolic control analysis is performed, and the flux control coefficients for the first two enzymes in the penicillin biosynthetic pathway, i.e., delta......The influence the of dissolved oxygen concentration on penicillin biosynthesis was studied in steady-state continuous cultures of a high-yielding strain of Penicillium chrysogenum operated at a dilution rate of 0.05 h-l. The dissolved oxygen concentration was varied between 0.019 and 0.344 m......M (corresponding to 7% and 131% air saturation at 1 bar) solely through manipulations of the inlet gas composition. At dissolved oxygen concentrations above 0.06-0.08 mM, a constant specific penicillin productivity of around 22 (mu mol/g of DW)/h is maintained. At lower oxygen concentrations, the specific...

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

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    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.

  20. Clustered array of ochratoxin A biosynthetic genes in Aspergillus steynii and their expression patterns in permissive conditions.

    Science.gov (United States)

    Gil-Serna, Jessica; Vázquez, Covadonga; González-Jaén, María Teresa; Patiño, Belén

    2015-12-01

    Aspergillus steynii is probably the most relevant species of section Circumdati producing ochratoxin A (OTA). This mycotoxin contaminates a wide number of commodities and it is highly toxic for humans and animals. Little is known on the biosynthetic genes and their regulation in Aspergillus species. In this work, we identified and analysed three contiguous genes in A. steynii using 5'-RACE and genome walking approaches which predicted a cytochrome P450 monooxygenase (p450ste), a non-ribosomal peptide synthetase (nrpsste) and a polyketide synthase (pksste). These three genes were contiguous within a 20742 bp long genomic DNA fragment. Their corresponding cDNA were sequenced and their expression was analysed in three A. steynii strains using real time RT-PCR specific assays in permissive conditions in in vitro cultures. OTA was also analysed in these cultures. Comparative analyses of predicted genomic, cDNA and amino acid sequences were performed with sequences of similar gene functions. All the results obtained in these analyses were consistent and point out the involvement of these three genes in OTA biosynthesis by A. steynii and showed a co-ordinated expression pattern. This is the first time that a clustered organization OTA biosynthetic genes has been reported in Aspergillus genus. The results also suggested that this situation might be common in Aspergillus OTA-producing species and distinct to the one described for Penicillium species.

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

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    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

  2. Staphylococcus aureus mevalonate kinase: isolation and characterization of an enzyme of the isoprenoid biosynthetic pathway.

    Science.gov (United States)

    Voynova, Natalya E; Rios, Sandra E; Miziorko, Henry M

    2004-01-01

    It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K(m) values for R,S-mevalonate and ATP were 41 and 339 micro M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K(i) values were 46 and 45 micro M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K(i) difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.

  3. Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI.

    Science.gov (United States)

    Wang, Cheng; Zeng, Jian; Li, Yin; Hu, Wei; Chen, Ling; Miao, Yingjie; Deng, Pengyi; Yuan, Cuihong; Ma, Cheng; Chen, Xi; Zang, Mingli; Wang, Qiong; Li, Kexiu; Chang, Junli; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

    2014-06-01

    Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid levels and trace amounts of provitamin A content. In order to enrich the carotenoid content in wheat grains, the bacterial phytoene synthase gene (CrtB) and carotene desaturase gene (CrtI) were transformed into the common wheat cultivar Bobwhite. Expression of CrtB or CrtI alone slightly increased the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grain phenotype, accompanied by a total carotenoid content increase of approximately 8-fold achieving 4.76 μg g(-1) of seed dry weight, a β-carotene increase of 65-fold to 3.21 μg g(-1) of seed dry weight, and a provitamin A content (sum of α-carotene, β-carotene, and β-cryptoxanthin) increase of 76-fold to 3.82 μg g(-1) of seed dry weight. The high provitamin A content in the transgenic wheat was stably inherited over four generations. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat was also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in the wheat carotenoid biosynthetic pathway. These transgenic wheat lines are not only valuable for breeding wheat varieties with nutritional benefits for human health but also for understanding the mechanism regulating carotenoid biosynthesis in wheat endosperm.

  4. Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.

    Science.gov (United States)

    Kai, Guoyin; Xu, Hui; Zhou, Congcong; Liao, Pan; Xiao, Jianbo; Luo, Xiuqin; You, Lijia; Zhang, Lin

    2011-05-01

    Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control (Ppushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push-pull strategy in S. miltiorrhiza.

  5. Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus corniculatus leaves.

    Science.gov (United States)

    Paolocci, Francesco; Bovone, Tessa; Tosti, Nicola; Arcioni, Sergio; Damiani, Francesco

    2005-04-01

    The effects of increasing light and of a heterologous bHLH transcription factor on the accumulation of condensed tannins (CT) were investigated in leaves of Lotus corniculatus, a model legume species which accumulates these secondary metabolites in leaves as well as reproductive tissues. Light and expression of the transgene increased the level of CT in a synergistic way. To monitor how the changes in accumulation of condensed tannins were achieved, the level of expression of four key genes in the flavonoid pathway was estimated by real-time RT-PCR analysis. Early genes of the pathway (PAL and CHS) were affected less in their expression and so appeared to be less involved in influencing the final level of CT than later genes in the pathway (DFR and ANS). Steady-state levels of DFR and ANS transcripts showed a strong positive correlation with CT and these genes might be considered the first rate-limiting steps in CT biosynthesis in Lotus leaves. However, additional factors mediated by light are limiting CT accumulation once these genes are up-regulated by the transgene. Therefore, the increment of the steady-state mRNA level for DFR and ANS might not be sufficient to up-regulate condensed tannins in leaves. The real-time RT-PCR approach adopted showed that members within the CHS and DFR gene families are differentially regulated by the exogenous bHLH gene and light. This finding is discussed in relation to the approaches for controlling CT biosynthesis and for studying the expression profile of multi-gene families.

  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. Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

    Science.gov (United States)

    Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

    2015-11-20

    The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol.

  8. 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

  9. Genetic control of lithium sensitivity and regulation of inositol biosynthetic genes.

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    Jason King

    Full Text Available Lithium (Li(+ is a common treatment for bipolar mood disorder, a major psychiatric illness with a lifetime prevalence of more than 1%. Risk of bipolar disorder is heavily influenced by genetic predisposition, but is a complex genetic trait and, to date, genetic studies have provided little insight into its molecular origins. An alternative approach is to investigate the genetics of Li(+ sensitivity. Using the social amoeba Dictyostelium, we previously identified prolyl oligopeptidase (PO as a modulator of Li(+ sensitivity. In a link to the clinic, PO enzyme activity is altered in bipolar disorder patients. Further studies demonstrated that PO is a negative regulator of inositol(1,4,5trisphosphate (IP(3 synthesis, a Li(+ sensitive intracellular signal. However, it was unclear how PO could influence either Li(+ sensitivity or risk of bipolar disorder. Here we show that in both Dictyostelium and cultured human cells PO acts via Multiple Inositol Polyphosphate Phosphatase (Mipp1 to control gene expression. This reveals a novel, gene regulatory network that modulates inositol metabolism and Li(+ sensitivity. Among its targets is the inositol monophosphatase gene IMPA2, which has also been associated with risk of bipolar disorder in some family studies, and our observations offer a cellular signalling pathway in which PO activity and IMPA2 gene expression converge.

  10. NF-Y activates genes of metabolic pathways altered in cancer cells.

    Science.gov (United States)

    Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta

    2016-01-12

    The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.

  11. 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

  12. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    Science.gov (United States)

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  13. Cloning, reassembling and integration of the entire nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved nikkomycin production

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    Yang Haihua

    2010-01-01

    Full Text Available Abstract Background Nikkomycins are a group of peptidyl nucleoside antibiotics produced by Streptomyces ansochromogenes. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by S. ansochromogenes. Generation of a high-producing strain is crucial to scale up nikkomycins production for further clinical trials. Results To increase the yields of nikkomycins, an additional copy of nikkomycin biosynthetic gene cluster (35 kb was introduced into nikkomycin producing strain, S. ansochromogenes 7100. The gene cluster was first reassembled into an integrative plasmid by Red/ET technology combining with classic cloning methods and then the resulting plasmid(pNIKwas introduced into S. ansochromogenes by conjugal transfer. Introduction of pNIK led to enhanced production of nikkomycins (880 mg L-1, 4 -fold nikkomycin X and 210 mg L-1, 1.8-fold nikkomycin Z in the resulting exconjugants comparing with the parent strain (220 mg L-1 nikkomycin X and 120 mg L-1 nikkomycin Z. The exconjugants are genetically stable in the absence of antibiotic resistance selection pressure. Conclusion A high nikkomycins producing strain (1100 mg L-1 nikkomycins was obtained by introduction of an extra nikkomycin biosynthetic gene cluster into the genome of S. ansochromogenes. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites.

  14. Novel polyoxins generated by heterologously expressing polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes

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    Li Jine

    2012-10-01

    Full Text Available Abstract Background Polyoxins are potent inhibitors of chitin synthetases in fungi and insects. The gene cluster responsible for biosynthesis of polyoxins has been cloned and sequenced from Streptomyces cacaoi and tens of polyoxin analogs have been identified already. Results The polyoxin biosynthetic gene cluster from Streptomyces cacaoi was heterologously expressed in the sanN inactivated mutant of Streptomyces ansochromogenes as a nikkomycin producer. Besides hybrid antibiotics (polynik A and polyoxin N and some known polyoxins, two novel polyoxin analogs were accumulated. One of them is polyoxin P that has 5-aminohexuronic acid with N-glycosidically bound thymine as the nucleoside moiety and dehydroxyl-carbamoylpolyoxic acid as the peptidyl moiety. The other analog is polyoxin O that contains 5-aminohexuronic acid bound thymine as the nucleoside moiety, but recruits polyoximic acid as the sole peptidyl moiety. Bioassay against phytopathogenic fungi showed that polyoxin P displayed comparatively strong inhibitory activity, whereas the inhibitory activity of polyoxin O was weak under the same testing conditions. Conclusion Two novel polyoxin analogs (polyoxin P and O were generated by the heterologous expression of polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes. Polyoxin P showed potent antifungal activity,while the activity of polyoxin O was weak. The strategy presented here may be available for other antibiotics producers.

  15. Lovastatin biosynthetic genes of Aspergillus terreus are expressed differentially in solid-state and in liquid submerged fermentation.

    Science.gov (United States)

    Barrios-González, J; Baños, J G; Covarrubias, A A; Garay-Arroyo, A

    2008-05-01

    Molecular studies were performed to establish the causes of the superior lovastatin productivity of a novel solid-state fermentation (SSF) process, in relation with liquid submerged fermentation (SmF; 20 mg/g vs. 0.65 mg/ml). In SSF, biosynthetic genes lovE and lovF transcripts accumulated to high levels from day 1 to day 7. In this period, lovE transcript showed 4.6-fold higher accumulation levels (transcription) than the highest level detected in SmF (day 5). lovF transcript showed two-fold higher expression than the highest point in SmF. In SmF, the expression was only detected clearly on day 5 and, showing a 50% decrease, on day 7. These results show that the higher lovastatin production in SSF is related to a more intense transcription of these biosynthetic genes. A strong expression of gldB gene in lovastatin SSF indicated that Aspergillus terreus senses osmotic stress during the course of SSF, but not in SmF. However, when a liquid medium of identical concentration was used in SmF, lovastatin production decreased in SSF.

  16. Early Phenylpropanoid Biosynthetic Steps in Cannabis sativa: Link between Genes and Metabolites

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    Immacolata Coraggio

    2013-06-01

    Full Text Available Phenylalanine ammonia-lyase (PAL, Cinnamic acid 4-hydroxylase (C4H and 4-Coumarate: CoA ligase (4CL catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids and roots (mainly lignin was discussed in relation to gene expression and enzymatic activities data.

  17. Chronic physical stress changes gene expression of catecholamine biosynthetic enzymes in the adrenal medulla of adult rats

    Directory of Open Access Journals (Sweden)

    Gavrilović Ljubica

    2012-01-01

    Full Text Available In this study we examined how chronic forced running (CFR affects the expression of catecholamine biosynthetic enzymes and cAMP response element-binding (CREB in the adrenal medulla and the weight of adrenal glands of rats. Also, we examined how CFR and additional acute immobilization stress affect the expression of catecholamine biosynthetic enzymes in the adrenal medulla and the concentration of catecholamines and corticosterone (CORT in the blood plasma. In this experiment we used as a model forced exercise in rats (treadmill running. We used the most advanced method for determining the level of gene expression, Real-time PCR with TaqMan probes, as well as Western blot analysis (ECL. We found that CFR decreases tyrosine hydroxylase (TH, and dopamine-β-hydroxylase (DBH mRNA and protein levels in the adrenal medulla. The decreased TH and DBH mRNA levels coincide with the reduced expression of CREB in the adrenal medulla and with the reduced plasma CORT level. Additionally, CFR reduces the level of phenylethanolamine N-methyltransferase (PNMT mRNA, but elevates its protein level in the adrenal medulla and increases the concentration of adrenaline (A in the plasma. Reduced level of PNMT mRNA in the adrenal medulla coincides with reduced plasma CORT level. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. The increased synthesis of PNMT enzyme in the adrenal medulla may result in an increased biosynthesis of A under chronic stress conditions. Additionally, increased level of catecholamines in the plasma after chronic physical stress is the allostatic load that may induce numerous diseases and pathological conditions.

  18. Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer Streptomyces collinus Tü 365

    DEFF Research Database (Denmark)

    Iftime, Dumitrita; Kulik, Andreas; Härtner, Thomas;

    2016-01-01

    Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein...... metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC–MS analyses, most of the uncharacterized...... biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary...

  19. Genome of Diaporthe sp. provides insights into the potential inter-phylum transfer of a fungal sesquiterpenoid biosynthetic pathway.

    Science.gov (United States)

    de Sena Filho, Jose Guedes; Quin, Maureen B; Spakowicz, Daniel J; Shaw, Jeffrey J; Kucera, Kaury; Dunican, Brian; Strobel, Scott A; Schmidt-Dannert, Claudia

    2016-08-01

    Fungi have highly active secondary metabolic pathways which enable them to produce a wealth of sesquiterpenoids that are bioactive. One example is Δ6-protoilludene, the precursor to the cytotoxic illudins, which are pharmaceutically relevant as anticancer therapeutics. To date, this valuable sesquiterpene has only been identified in members of the fungal division Basidiomycota. To explore the untapped potential of fungi belonging to the division Ascomycota in producing Δ6-protoilludene, we isolated a fungal endophyte Diaporthe sp. BR109 and show that it produces a diversity of terpenoids including Δ6-protoilludene. Using a genome sequencing and mining approach 17 putative novel sesquiterpene synthases were identified in Diaporthe sp. BR109. A phylogenetic approach was used to predict which gene encodes Δ6-protoilludene synthase, which was then confirmed experimentally. These analyses reveal that the sesquiterpene synthase and its putative sesquiterpene scaffold modifying cytochrome P450(s) may have been acquired by inter-phylum horizontal gene transfer from Basidiomycota to Ascomycota. Bioinformatic analyses indicate that inter-phylum transfer of these minimal sequiterpenoid secondary metabolic pathways may have occurred in other fungi. This work provides insights into the evolution of fungal sesquiterpenoid secondary metabolic pathways in the production of pharmaceutically relevant bioactive natural products.

  20. Fructan Biosynthetic and Breakdown Enzymes in Dicots Evolved From Different Invertases. Expression of Fructan Genes Throughout Chicory Development

    Directory of Open Access Journals (Sweden)

    Wim Van den Ende

    2002-01-01

    Full Text Available Fructans are fructose-based oligo- and polymers that serve as reserve carbohydrates in many plant species. The biochemistry of fructan biosynthesis in dicots has been resolved, and the respective cDNAs have been cloned. Recent progress has now succeeded in elucidating the biochemistry and molecular biology of fructan biodegradation in chicory, an economically important species used for commercial inulin extraction. Unlike fructan biosynthetic genes that originated from vacuolar-type invertase, fructan exohydrolases (FEHs seem to have evolved from a cell-wall invertase ancestor gene that later obtained a low iso-electric point and a vacuolar targeting signal. Expression analysis reveals that fructan enzymes are controlled mainly at the transcriptional level. Using chicory as a model system, northern analysis was consistent with enzymatic activity measurements and observed carbohydrate changes throughout its development.

  1. Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer Streptomyces collinus Tü 365.

    Science.gov (United States)

    Iftime, Dumitrita; Kulik, Andreas; Härtner, Thomas; Rohrer, Sabrina; Niedermeyer, Timo Horst Johannes; Stegmann, Evi; Weber, Tilmann; Wohlleben, Wolfgang

    2016-03-01

    Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC-MS analyses, most of the uncharacterized gene clusters are not expressed under standard laboratory conditions. With this study we aimed to characterize the genome information of S. collinus Tü 365 to make use of gene clusters, which previously have not been described for this strain. We were able to connect the gene clusters of a lanthipeptide, a carotenoid, five terpenoid compounds, an ectoine, a siderophore and a spore pigment-associated gene cluster to their respective biosynthesis products.

  2. 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

    2009-03-04

    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.

  3. Effect of phenolic compounds and osmotic stress on the expression of penicillin biosynthetic genes from Penicillium chrysogenum var. halophenolicum strain

    Directory of Open Access Journals (Sweden)

    Sumaya Ferreira Guedes

    2012-01-01

    Full Text Available Phenol and phenolic compounds are aromatic pollutants that inhibit biological treatment of wastewaters. Penicillium chrysogenum var. halophenolicum is a halotolerant fungus that previously showed the ability to degrade phenol and resorcinol in high salinity conditions. The presence of the penicillin biosynthetic cluster in P. chrysogenum var. halophenolicum was recently described. In this article, we examined the expression of pcbAB, pcbC and penDE, genes responsible for δ-(L-α-aminoadipyl-L-cysteinyl-D-valine synthetase, isopenicillin N synthase and isopenicillin N acyltransferase activities, respectively, in P. chrysogenum var. halophenolicum. A quantitative PCR (qPCR approach was used to determine how these genes were expressed in media with 2% and 5.9% NaCl supplemented with phenol, catechol, hydroquinone and resorcinol as the sole carbon source. The effect of salt on the capability of P. chrysogenum var. halophenolicum to degrade aromatic compounds was measured using HPLC. qPCR analysis of RNA extracted from P. chrysogenum var. halophenolicum indicated that the expression levels of pcbAB, pcbC and penDE decreased in high saline concentrations compared to the levels expressed in media with glucose. High concentrations of salt significantly repress the expression of pcbAB and penDE. The pcbC gene was expressed differentially in catechol containing medium. There was no evident relationship between the expression levels of penicillin biosynthetic genes and yields of penicillin. Meanwhile, the presence of phenol and phenolic compounds seems to positively influence the antibiotic production; high concentrations of salt stimulated penicillin production. These results support the hypothesis that phenol, phenolic compounds and high concentrations of salt could act like a stress factor for P. chrysogenum var. halophenolicum resulting in higher yields of β-lactam antibiotic production.

  4. Transcriptional control of anthocyanin biosynthetic genes in extreme phenotypes for berry pigmentation of naturally occurring grapevines

    Directory of Open Access Journals (Sweden)

    Castellarin Simone D

    2007-08-01

    Full Text Available Abstract Background Fruit coloration of red-skinned grapevines is mainly due to anthocyanin pigments. We analysed a panel of nine cultivars that included extreme phenotypes for berry colour, ranging from green (absence of anthocyanins to red, purple, violet and blue. Expression of six genes of the anthocyanin pathway coding for flavanone-hydroxylase (F3H, flavonoid 3'-hydroxylase (F3'H, flavonoid 3',5'-hydroxylase (F3'5'H, UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT, glutathione-S-transferase (GST, O-methyltransferase (OMT and four transcription factors (MybA, MybB, MybC, MybD was analysed by quantitative RT-PCR at four developmental stages from before the onset of ripening until full maturity and compared to anthocyanin metabolites. Results Total anthocyanin content at full maturity correlated well with the cumulative expression of F3H, UFGT and GST throughout ripening. Transcripts of the last two genes were absent in the green-skinned cultivar 'Sauvignonasse', also known as 'Tocai friulano', and were at least 10-fold less abundant in pale red cultivars, such as 'Pinot gris' and 'Gewürztraminer', compared to fully coloured cultivars. Predominance of tri-hydroxylated anthocyanins (delphinidin, petunidin and malvidin in cultivars bearing dark berries with violet and blue hue was associated with higher ratios of F3'5'H/F3'H transcription, compared to red-skinned cultivars. Higher levels of OMT transcripts were observed in berries of cultivars that accumulated methoxylated forms of anthocyanins more abundantly than non-methoxylated forms. Conclusion Colour variation of the grape berry conforms to a peculiar pattern of genotype-specific expression of the whole set of anthocyanin genes in a direct transcript-metabolite-phenotype relationship. Cumulative mRNA levels of the structural genes and their relative abundance throughout ripening explained per se the final phenotype for anthocyanin content, anthocyanin composition, colour intensity

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

    Energy Technology Data Exchange (ETDEWEB)

    Tange, K. (Hiroshima Univ. (Japan). Faculty of Science)

    1981-09-01

    Radioisotopically labeled L-valine, DL-alanine, sodium acetate, and DL-mevalonic acid were incorporated into linalool by the intact plant of Cinnamomum camphora Sieb. var. linalooliferum Fujita and into geraniol and citronellol by that of Pelargonium roseum Bourbon. The uptake of leucine and valine resulted in the preferential location of the radioactivity on the 3,3-dimethylallyl pyrophosphate-derived moiety of these acyclic monoterpenoids, whereas the uptake of alanine resulted in the preferential location on the isopentenyl pyrophosphate-derived moiety, much as in the cases of mevalonic acid and sodium acetate. A biosynthetic pathway leading to the monoterpenoids from the amino acids is discussed.

  6. 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.

  7. Violet/blue chrysanthemums--metabolic engineering of the anthocyanin biosynthetic pathway results in novel petal colors.

    Science.gov (United States)

    Brugliera, Filippa; Tao, Guo-Qing; Tems, Ursula; Kalc, Gianna; Mouradova, Ekaterina; Price, Kym; Stevenson, Kim; Nakamura, Noriko; Stacey, Iolanda; Katsumoto, Yukihisa; Tanaka, Yoshikazu; Mason, John G

    2013-10-01

    Chrysanthemums (Chrysanthemum×morifolium Ramat.) are an important cut-flower and potted plant crop in the horticultural industry world wide. Chrysanthemums express the flavonoid 3'-hydroxylase (F3'H) gene and thus accumulate anthocyanins derived from cyanidin in their inflorescences which appear pink/red. Delphinidin-based anthocyanins are lacking due to the deficiency of a flavonoid 3', 5'-hydroxylase (F3'5'H), and so violet/blue chrysanthemum flower colors are not found. In this study, together with optimization of transgene expression and selection of the host cultivars and gene source, F3'5'H genes have been successfully utilized to produce transgenic bluish chrysanthemums that accumulate delphinidin-based anthocyanins. HPLC analysis and feeding experiments with a delphinidin precursor identified 16 cultivars of chrysanthemums out of 75 that were predicted to turn bluish upon delphinidin accumulation. A selection of eight cultivars were successfully transformed with F3'5'H genes under the control of different promoters. A pansy F3'5'H gene under the control of a chalcone synthase promoter fragment from rose resulted in the effective diversion of the anthocyanin pathway to produce delphinidin in transgenic chrysanthemum flower petals. The resultant petal color was bluish, with 40% of total anthocyanidins attributed to delphinidin. Increased delphinidin levels (up to 80%) were further achieved by hairpin RNA interference-mediated silencing of the endogenous F3'H gene. The resulting petal colors were novel bluish hues, not possible by hybridization breeding. This is the first report of the production of anthocyanins derived from delphinidin in chrysanthemum petals leading to novel flower color.

  8. Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data

    Directory of Open Access Journals (Sweden)

    Regiane Kawasaki

    2016-01-01

    Full Text Available Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA and elementary modes (EM, were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments.

  9. VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in Chili pepper leaves

    Directory of Open Access Journals (Sweden)

    zhen ezhang

    2015-07-01

    Full Text Available The purple coloration of pepper leaves arises from the accumulation of anthocyanin. Three regulatory and 12 structural genes have been characterized for their involvement in the anthocyanin biosynthesis. Examination of the abundance of these genes in leaves showed that the majority of them differed between anthocyanin pigmented line Z1 and non-pigmented line A3. Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation. Moreover, the expression of multiple genes was altered in the silenced leaves. The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern. Most structural genes including CHS, CHI, F3H, F3’5’H, DFR, ANS, UFGT, ANP and GST were repressed in CaMYB-silenced foliage with the exception of PAL, C4H and 4CL. These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes. Besides CaMYB silenced leaves rendered more sporulation of Phytophthora capsici Leonian indicating that CaMYB might be involved in the defense response to pathogens.

  10. Autism: Many Genes, Common Pathways?

    OpenAIRE

    Geschwind, Daniel H.

    2008-01-01

    Autism is a heterogeneous neurodevelopmental syndrome with a complex genetic etiology. It is still not clear whether autism comprises a vast collection of different disorders akin to intellectual disability or a few disorders sharing common aberrant pathways. Unifying principles among cases of autism are likely to be at the level of brain circuitry in addition to molecular pathways.

  11. Autism: many genes, common pathways?

    Science.gov (United States)

    Geschwind, Daniel H

    2008-10-31

    Autism is a heterogeneous neurodevelopmental syndrome with a complex genetic etiology. It is still not clear whether autism comprises a vast collection of different disorders akin to intellectual disability or a few disorders sharing common aberrant pathways. Unifying principles among cases of autism are likely to be at the level of brain circuitry in addition to molecular pathways.

  12. Widespread occurrence and lateral transfer of the cyanobactin biosynthesis gene cluster in cyanobacteria.

    Science.gov (United States)

    Leikoski, Niina; Fewer, David P; Sivonen, Kaarina

    2009-02-01

    Cyanobactins are small cyclic peptides produced by cyanobacteria. Here we demonstrate the widespread but sporadic occurrence of the cyanobactin biosynthetic pathway. We detected a cyanobactin biosynthetic gene in 48 of the 132 strains included in this study. Our results suggest that cyanobactin biosynthetic genes have a complex evolutionary history in cyanobacteria punctuated by a series of ancient horizontal gene transfer events.

  13. Widespread Occurrence and Lateral Transfer of the Cyanobactin Biosynthesis Gene Cluster in Cyanobacteria ▿ †

    OpenAIRE

    Leikoski, Niina; Fewer, David P.; Sivonen, Kaarina

    2008-01-01

    Cyanobactins are small cyclic peptides produced by cyanobacteria. Here we demonstrate the widespread but sporadic occurrence of the cyanobactin biosynthetic pathway. We detected a cyanobactin biosynthetic gene in 48 of the 132 strains included in this study. Our results suggest that cyanobactin biosynthetic genes have a complex evolutionary history in cyanobacteria punctuated by a series of ancient horizontal gene transfer events.

  14. Exploring the Transcriptome Landscape of Pomegranate Fruit Peel for Natural Product Biosynthetic Gene and SSR Marker Discovery(F).

    Science.gov (United States)

    Ono, Nadia Nicole; Britton, Monica Therese; Fass, Joseph Nathaniel; Nicolet, Charles Meyer; Lin, Dawei; Tian, Li

    2011-10-01

    Pomegranate fruit peel is rich in bioactive plant natural products, such as hydrolyzable tannins and anthocyanins. Despite their documented roles in human nutrition and fruit quality, genes involved in natural product biosynthesis have not been cloned from pomegranate and very little sequence information is available on pomegranate in the public domain. Shotgun transcriptome sequencing of pomegranate fruit peel cDNA was performed using RNA-Seq on the Illumina Genome Analyzer platform. Over 100 million raw sequence reads were obtained and assembled into 9,839 transcriptome assemblies (TAs) (>200 bp). Candidate genes for hydrolyzable tannin, anthocyanin, flavonoid, terpenoid and fatty acid biosynthesis and/or regulation were identified. Three lipid transfer proteins were obtained that may contribute to the previously reported IgE reactivity of pomegranate fruit extracts. In addition, 115 SSR markers were identified from the pomegranate fruit peel transcriptome and primers were designed for 77 SSR markers. The pomegranate fruit peel transcriptome set provides a valuable platform for natural product biosynthetic gene and SSR marker discovery in pomegranate. This work also demonstrates that next-generation transcriptome sequencing is an economical and effective approach for investigating natural product biosynthesis, identifying genes controlling important agronomic traits, and discovering molecular markers in non-model specialty crop species.

  15. Exploring the Transcriptome Landscape of Pomegranate Fruit Peel for Natural Product Biosynthetic Gene and SSR Marker Discovery

    Institute of Scientific and Technical Information of China (English)

    Nadia Nicole Ono; Monica Therese Britton; Joseph Nathaniel Fass; Charles Meyer Nicolet; Dawei Lin; Li Tian

    2011-01-01

    Pomegranate fruit peel is rich in bioactive plant natural products,such as hydrolyzable tannins and anthocyanins.Despite their documented roles in human nutrition and fruit quality,genes involved in natural product biosynthesis have not been cloned from pomegranate and very little sequence information is available on pomegranate in the public domain.Shotgun transcriptome sequencing of pomegranate fruit peel cDNA was performed using RNA-Seq on the Illumina Genome Analyzer platform.Over 100 million raw sequence reads were obtained and assembled into 9,839 transcriptome assemblies (TAs) (>200 bp).Candidate genes for hydrolyzable tannin,anthocyanin,flavonoid,terpenoid and fatty acid biosynthesis and/or regulation were identified.Three lipid transfer proteins were obtained that may contribute to the previously reported IgE reactivity of pomegranate fruit extracts.In addition,115 SSR markers were identified from the pomegranate fruit peel transcriptome and primers were designed for 77 SSR markers.The pomegranate fruit peel transcriptome set provides a valuable platform for natural product biosynthetic gene and SSR marker discovery in pomegranate.This work also demonstrates that next-generation transcriptome sequencing is an economical and effective approach for investigating natural product biosynthesis,identifying genes controlling important agronomic traits,and discovering molecular markers in non-model specialty crop species.

  16. Tannerella forsythia strains display different cell-surface nonulosonic acids: biosynthetic pathway characterization and first insight into biological implications.

    Science.gov (United States)

    Friedrich, Valentin; Janesch, Bettina; Windwarder, Markus; Maresch, Daniel; Braun, Matthias L; Megson, Zoë A; Vinogradov, Evgeny; Goneau, Marie-France; Sharma, Ashu; Altmann, Friedrich; Messner, Paul; Schoenhofen, Ian C; Schäffer, Christina

    2016-12-16

    Tannerella forsythia is an anaerobic, Gram-negative periodontal pathogen. A unique O-linked oligosaccharide decorates the bacterium's cell surface proteins and was shown to modulate the host immune response. In our study, we investigated the biosynthesis of the nonulosonic acid (NulO) present at the terminal position of this glycan. A bioinformatic analysis of T. forsythia genomes revealed a gene locus for the synthesis of pseudaminic acid (Pse) in the type strain ATCC 43037 while strains FDC 92A2 and UB4 possess a locus for the synthesis of legionaminic acid (Leg) instead. In contrast to the NulO in ATCC 43037, which has been previously identified as a Pse derivative (5-N-acetimidoyl-7-N-glyceroyl-3,5,7,9-tetradeoxy-l-glycero-l-manno-NulO), glycan analysis of strain UB4 performed in this study indicated a 350-Da, possibly N-glycolyl Leg (3,5,7,9-tetradeoxy-d-glycero-d-galacto-NulO) derivative with unknown C5,7 N-acyl moieties. We have expressed, purified and characterized enzymes of both NulO pathways to confirm these genes' functions. Using capillary electrophoresis (CE), CE-mass spectrometry and NMR spectroscopy, our studies revealed that Pse biosynthesis in ATCC 43037 essentially follows the UDP-sugar route described in Helicobacter pylori, while the pathway in strain FDC 92A2 corresponds to Leg biosynthesis in Campylobacter jejuni involving GDP-sugar intermediates. To demonstrate that the NulO biosynthesis enzymes are functional in vivo, we created knockout mutants resulting in glycans lacking the respective NulO. Compared to the wild-type strains, the mutants exhibited significantly reduced biofilm formation on mucin-coated surfaces, suggestive of their involvement in host-pathogen interactions or host survival. This study contributes to understanding possible biological roles of bacterial NulOs.

  17. Genistein: A Novel Anthocyanin Synthesis Promoter that Directly Regulates Biosynthetic Genes in Red Cabbage in a Light-Dependent Way

    Science.gov (United States)

    Zhang, Na; Qi, Yan; Zhang, Hai-Jun; Wang, Xiaoyun; Li, Hongfei; Shi, Yantong; Guo, Yang-Dong

    2016-01-01

    Genistein (GNT), an isoflavone, is used in the clinical treatment of various health disorders. GNT is found in primary food source plants and some medical plants. However, studies on the functions of GNT in plants are rarely reported. In this study, we demonstrated that GNT plays an important role in promoting anthocyanin accumulation in red cabbage. GNT solutions (10, 20, 30, 40, and 50 mg/L) as foliar fertilizers were applied to red cabbage. Consequently, anthocyanin accumulation in red cabbage increased in a light-dependent manner. GNT solution at 30 mg/L exhibited the optimal effect on anthocyanin accumulation, which was twice that of the control. Quantitative real-time PCR analysis indicated that GNT application upregulated the expression of all structural genes, contributing to anthocyanin biosynthesis under light conditions. Under dark conditions, GNT exerted no significant promotive effect on anthocyanin accumulation; only early biosynthetic genes of anthocyanin biosynthesis responded to GNT. The promotive effect of GNT on anthocyanin biosynthesis is directly attributable to the regulation of structural gene expression. Transcription factors exhibited no response to GNT. The levels of anthocyanin in red cabbage positively correlated with the enzyme activities of antioxidant systems. This finding correlation suggested that the promotive effect of GNT on anthocyanin levels was correlated with improved antioxidant activity in the red cabbage. PMID:27990149

  18. 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.

  19. Genistein: a novel anthocyanin synthesis promoter that directly regulates biosynthetic genes in red cabbage in a light-dependent way

    Directory of Open Access Journals (Sweden)

    Na Zhang

    2016-12-01

    Full Text Available Genistein (GNT, an isoflavone, is used in the clinical treatment of various health disorders. GNT is found in primary food source plants and some medical plants. However, studies on the functions of GNT in plants are rarely reported. In this study, we demonstrated that GNT plays an important role in promoting anthocyanin accumulation in red cabbage. GNT solutions (10, 20, 30, 40, and 50 mg/L as foliar fertilizers were applied to red cabbage. Consequently, anthocyanin accumulation in red cabbage increased in a light-dependent manner. GNT solution at 30 mg/L exhibited the optimal effect on anthocyanin accumulation, which was twice that of the control. Quantitative real-time PCR analysis indicated that GNT application upregulated the expression of all structural genes, contributing to anthocyanin biosynthesis under light conditions. Under dark conditions, GNT exerted no significant promotive effect on anthocyanin accumulation; only early biosynthetic genes of anthocyanin biosynthesis responded to GNT. The promotive effect of GNT on anthocyanin biosynthesis is directly attributable to the regulation of structural gene expression. Transcription factors exhibited no response to GNT. The levels of anthocyanin in red cabbage positively correlated with the enzyme activities of antioxidant systems. This finding correlation suggested that the promotive effect of GNT on anthocyanin levels was correlated with improved antioxidant activity in the red cabbage.

  20. An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis

    Directory of Open Access Journals (Sweden)

    Song Cai

    2011-07-01

    Full Text Available Abstract Background Siraitia grosvenorii (Luohanguo is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in S. grosvenorii, especially the late steps of the pathway. Results In this study, a cDNA library generated from of equal amount of RNA taken from S. grosvenorii fruit at 50 days after flowering (DAF and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9% unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450 and ninety UDP-glucosyltransferase (UDPG unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven CYP450s and five UDPGs were selected as the candidates most likely to be involved in mogrosides biosynthesis. Conclusion A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying

  1. Functions of some capsular polysaccharide biosynthetic genes in Klebsiella pneumoniae NTUH K-2044.

    Directory of Open Access Journals (Sweden)

    Jin-Yuan Ho

    Full Text Available The growing number of Klebsiella pneumoniae infections, commonly acquired in hospitals, has drawn great concern. It has been shown that the K1 and K2 capsular serotypes are the most detrimental strains, particularly to those with diabetes. The K1 cps (capsular polysaccharide locus in the NTUH-2044 strain of the pyogenic liver abscess (PLA K. pneumoniae has been identified recently, but little is known about the functions of the genes therein. Here we report characterization of a group of cps genes and their roles in the pathogenesis of K1 K. pneumoniae. By sequential gene deletion, the cps gene cluster was first re-delimited between genes galF and ugd, which serve as up- and down-stream ends, respectively. Eight gene products were characterized in vitro and in vivo to be involved in the syntheses of UDP-glucose, UDP-glucuronic acid and GDP-fucose building units. Twelve genes were identified as virulence factors based on the observation that their deletion mutants became avirulent or lost K1 antigenicity. Furthermore, deletion of kp3706, kp3709 or kp3712 (ΔwcaI, ΔwcaG or Δatf, respectively, which are all involved in fucose biosynthesis, led to a broad range of transcriptional suppression for 52 upstream genes. The genes suppressed include those coding for unknown regulatory membrane proteins and six multidrug efflux system proteins, as well as proteins required for the K1 CPS biosynthesis. In support of the suppression of multidrug efflux genes, we showed that these three mutants became more sensitive to antibiotics. Taken together, the results suggest that kp3706, kp3709 or kp3712 genes are strongly related to the pathogenesis of K. pneumoniae K1.

  2. Developmental and Genotypic Variation in Leaf Wax Content and Composition, and in Expression of Wax Biosynthetic Genes in Brassica oleracea var. capitata

    Science.gov (United States)

    Laila, Rawnak; Robin, Arif Hasan Khan; Yang, Kiwoung; Park, Jong-In; Suh, Mi Chung; Kim, Juyoung; Nou, Ill-Sup

    2017-01-01

    Cuticular waxes act as a protective barrier against environmental stresses. In the present study, we investigated developmental and genotypic variation in wax formation of cabbage lines, with a view to understand the related morphology, genetics and biochemistry. Our studies revealed that the relative expression levels of wax biosynthetic genes in the first-formed leaf of the highest-wax line remained constantly higher but were decreased in other genotypes with leaf aging. Similarly, the expression of most of the tested genes exhibited decrease from the inner leaves to the outer leaves of 5-month-old cabbage heads in the low-wax lines in contrast to the highest-wax line. In 10-week-old plants, expression of wax biosynthetic genes followed a quadratic function and was generally increased in the early developing leaves but substantially decreased at the older leaves. The waxy compounds in all cabbage lines were predominately C29-alkane, -secondary alcohol, and -ketone. Its deposition was increased with leaf age in 5-month-old plants. The high-wax lines had dense, prominent and larger crystals on the leaf surface compared to low-wax lines under scanning electron microscopy. Principal component analysis revealed that the higher expression of LTP2 genes in the lowest-wax line and the higher expression of CER3 gene in the highest-wax line were probably associated with the comparatively lower and higher wax content in those two lines, respectively. This study furthers our understanding of the relationships between the expression of wax biosynthetic genes and the wax deposition in cabbage lines. Highlight: In cabbage, expression of wax-biosynthetic genes was generally decreased in older and senescing leaves, while wax deposition was increased with leaf aging, and C29-hydrocarbon was predominant in the wax crystals. PMID:28119701

  3. Inhibitory Effect of Cinnamaldehyde, Citral, and Eugenol on Aflatoxin Biosynthetic Gene Expression and Aflatoxin B1 Biosynthesis in Aspergillus flavus.

    Science.gov (United States)

    Liang, Dandan; Xing, Fuguo; Selvaraj, Jonathan Nimal; Liu, Xiao; Wang, Limin; Hua, Huijuan; Zhou, Lu; Zhao, Yueju; Wang, Yan; Liu, Yang

    2015-12-01

    In order to reveal the inhibitory effects of cinnamaldehyde, citral, and eugenol on aflatoxin biosynthesis, the expression levels of 5 key aflatoxin biosynthetic genes were evaluated by real-time PCR. Aspergillus flavus growth and AFB1 production were completely inhibited by 0.80 mmol/L of cinnamaldehyde and 2.80 mmol/L of citral. However, at lower concentration, cinnamaldehyde (0.40 mmol/L), eugenol (0.80 mmol/L), and citral (0.56 mmol/L) significantly reduced AFB1 production with inhibition rate of 68.9%, 95.4%, and 41.8%, respectively, while no effect on fungal growth. Real-time PCR showed that the expressions of aflR, aflT, aflD, aflM, and aflP were down-regulated by cinnamaldehyde (0.40 mmol/L), eugenol (0.80 mmol/L), and citral (0.56 mmol/L). In the presence of cinnamaldehyde, AflM was highly down-regulated (average of 5963 folds), followed by aflP, aflR, aflD, and aflT with the average folds of 55, 18, 6.5, and 5.8, respectively. With 0.80 mmol/L of eugenol, aflP was highly down-regulated (average of 2061-folds), followed by aflM, aflR, aflD, and aflT with average of 138-, 15-, 5.2-, and 4.8-folds reduction, respectively. With 0.56 mmol/L of citral, aflT was completely inhibited, followed by aflM, aflP, aflR, and aflD with average of 257-, 29-, 3.5-, and 2.5-folds reduction, respectively. These results suggest that the reduction in AFB1 production by cinnamaldehyde, eugenol, and citral at low concentration may be due to the down-regulations of the transcription level of aflatoxin biosynthetic genes. Cinnamaldehyde and eugenol may be employed successfully as a good candidate in controlling of toxigenic fungi and subsequently contamination with aflatoxins in practice.

  4. Indole-Diterpene Biosynthetic Capability of Epichloë Endophytes as Predicted by ltm Gene Analysis▿

    Science.gov (United States)

    Young, Carolyn A.; Tapper, Brian A.; May, Kimberley; Moon, Christina D.; Schardl, Christopher L.; Scott, Barry

    2009-01-01

    Bioprotective alkaloids produced by Epichloë and closely related asexual Neotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. The LTM locus of Neotyphodium lolii (Lp19) and Epichloë festuce (Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10 ltm genes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10 ltm genes were present in only three epichloë endophytes. A predominance of the asexual Neotyphodium spp. examined contained 8 of the 10 ltm genes, with only one N. lolii containing the entire LTM locus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present in ltm clusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement. PMID:19181837

  5. Exploration of geosmin synthase from Streptomyces peucetius ATCC 27952 by deletion of doxorubicin biosynthetic gene cluster.

    Science.gov (United States)

    Singh, Bijay; Oh, Tae-Jin; Sohng, Jae Kyung

    2009-10-01

    Thorough investigation of Streptomyces peucetius ATCC 27952 genome revealed a sesquiterpene synthase, named spterp13, which encodes a putative protein of 732 amino acids with significant similarity to S. avermitilis MA-4680 (SAV2163, GeoA) and S. coelicolor A3(2) (SCO6073). The proteins encoded by SAV2163 and SCO6073 produce geosmin in the respective strains. However, the spterp13 gene seemed to be silent in S. peucetius. Deletion of the doxorubicin gene cluster from S. peucetius resulted in increased cell growth rate along with detectable production of geosmin. When we over expressed the spterp13 gene in S. peucetius DM07 under the control of an ermE* promoter, 2.4 +/- 0.4-fold enhanced production of geosmin was observed.

  6. 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.

  7. DMPD: Signalling pathways mediating type I interferon gene expression. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17904888 Signalling pathways mediating type I interferon gene expression. Edwards M...csml) Show Signalling pathways mediating type I interferon gene expression. PubmedID 17904888 Title Signalling pathways media

  8. Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

    Directory of Open Access Journals (Sweden)

    L. Gavrilovic

    2009-12-01

    Full Text Available Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH, dopamine-β-hydroxylase (DBH and phenylethanolamine N-methyltransferase (PNMT and protein levels in the right and left heart auricles of naive control and long-term (12 weeks socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70% compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62% and left (about 81% auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%, DBH (about 37% and PNMT (about 60% only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

  9. Diversity and distribution of a key sulpholipid biosynthetic gene in marine microbial assemblages

    NARCIS (Netherlands)

    Villanueva, L.; Hopmans, E.C.; Bale, N.; Schouten, S.; Sinninghe Damsté, J.S.

    2014-01-01

    Sulphoquinovosyldiacylglycerols (SQDG) are polar sulphur-containing membrane lipids, whose presence has been related to a microbial strategy to adapt to phosphate deprivation. In this study, we have targeted the sqdB gene coding the uridine 5-diphosphate-sulphoquinovose (UDP-SQ) synthase involved in

  10. Genes of primary sulfate assimilation are part of the glucosinolate biosynthetic network in Arabidopsis thaliana.

    Science.gov (United States)

    Yatusevich, Ruslan; Mugford, Sarah G; Matthewman, Colette; Gigolashvili, Tamara; Frerigmann, Henning; Delaney, Sean; Koprivova, Anna; Flügge, Ulf-Ingo; Kopriva, Stanislav

    2010-04-01

    Glucosinolates are plant secondary metabolites involved in responses to biotic stress. The final step of their synthesis is the transfer of a sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) onto a desulfo precursor. Thus, glucosinolate synthesis is linked to sulfate assimilation. The sulfate donor for this reaction is synthesized from sulfate in two steps catalyzed by ATP sulfurylase (ATPS) and adenosine 5'-phosphosulfate kinase (APK). Here we demonstrate that R2R3-MYB transcription factors, which are known to regulate both aliphatic and indolic glucosinolate biosynthesis in Arabidopsis thaliana, also control genes of primary sulfate metabolism. Using trans-activation assays we found that two isoforms of APK, APK1, and APK2, are regulated by both classes of glucosinolate MYB transcription factors; whereas two ATPS genes, ATPS1 and ATPS3, are differentially regulated by these two groups of MYB factors. In addition, we show that the adenosine 5'-phosphosulfate reductases APR1, APR2, and APR3, which participate in primary sulfate reduction, are also activated by the MYB factors. These observations were confirmed by analysis of transgenic lines with modulated expression levels of the glucosinolate MYB factors. The changes in transcript levels also affected enzyme activities, the thiol content and the sulfate reduction rate in some of the transgenic plants. Altogether the data revealed that the MYB transcription factors regulate genes of primary sulfate metabolism and that the genes involved in the synthesis of activated sulfate are part of the glucosinolate biosynthesis network.

  11. IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes

    Science.gov (United States)

    Hadjithomas, Michalis; Chen, I-Min A.; Chu, Ken; Huang, Jinghua; Ratner, Anna; Palaniappan, Krishna; Andersen, Evan; Markowitz, Victor; Kyrpides, Nikos C.; Ivanova, Natalia N.

    2017-01-01

    Secondary metabolites produced by microbes have diverse biological functions, which makes them a great potential source of biotechnologically relevant compounds with antimicrobial, anti-cancer and other activities. The proteins needed to synthesize these natural products are often encoded by clusters of co-located genes called biosynthetic gene clusters (BCs). In order to advance the exploration of microbial secondary metabolism, we developed the largest publically available database of experimentally verified and predicted BCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc/). Here, we describe an update of IMG-ABC, which includes ClusterScout, a tool for targeted identification of custom biosynthetic gene clusters across 40 000 isolate microbial genomes, and a new search capability to query more than 700 000 BCs from isolate genomes for clusters with similar Pfam composition. Additional features enable fast exploration and analysis of BCs through two new interactive visualization features, a BC function heatmap and a BC similarity network graph. These new tools and features add to the value of IMG-ABC's vast body of BC data, facilitating their in-depth analysis and accelerating secondary metabolite discovery. PMID:27903896

  12. Pathways: Strategies for Susceptibility Genes in SLE

    Science.gov (United States)

    Kelley, James M.; Edberg, Jeffrey C.; Kimberly, Robert P.

    2010-01-01

    Systemic lupus erythematosus (SLE) is a complex autoimmune disorder marked by an inappropriate immune response to nuclear antigens. Recent whole genome association and more focused studies have revealed numerous genes implicated in this disease process, including ITGAM, Fc gamma receptors, complement components, C-reactive protein, and others. One common feature of these molecules is their involvement in the immune opsonins pathway and phagocytic clearing of nuclear antigens and apoptotic debris which provide excessive exposure of lupus-related antigens to immune cells. Analysis of gene-gene interactions in the opsonin pathway and its relationship to SLE may provide a systems-based approach to identify additional candidate genes associated with disease able to account for a larger part of lupus susceptibility. PMID:20144911

  13. Engineering of avermectin biosynthetic genes to improve production of ivermectin in Streptomyces avermitilis.

    Science.gov (United States)

    Li, Meng; Chen, Zhi; Lin, Xiuping; Zhang, Xuan; Song, Yuan; Wen, Ying; Li, Jilun

    2008-10-15

    Two new recombinants of avermectin polyketide synthases were constructed by domain and module swapping in Streptomyces avermitilis 73-12. However, only the strain, S. avermitilis OI-31, formed by domain substitution could produce ivermectin. Analysis of the ivermectin synthesized gene cluster showed that decreased amount of aveC transcripts was one of the factors causing low yield of ivermectin. Overexpression of aveC could improve ivermectin yield.

  14. Characterization of the Biosynthetic Gene Cluster for Benzoxazole Antibiotics A33853 Reveals Unusual Assembly Logic.

    Science.gov (United States)

    Lv, Meinan; Zhao, Junfeng; Deng, Zixin; Yu, Yi

    2015-10-22

    A33853, which shows excellent bioactivity against Leishmania, is a benzoxazole-family compound formed from two moieties of 3-hydroxyanthranilic acid and one 3-hydroxypicolinic acid. In this study, we have identified the gene cluster responsible for the biosynthesis of A33853 in Streptomyces sp. NRRL12068 through genome mining and heterologous expression. Bioinformatics analysis and functional characterization of the orfs contained in the gene cluster revealed that the biosynthesis of A33853 is directed by a group of unusual enzymes. In particular, BomK, annotated as a ketosynthase, was found to catalyze the amide bond formation between 3-hydroxypicolinic and 3-hydroxyanthranilic acid during the assembly of A33853. BomJ, a putative ATP-dependent coenzyme A ligase, and BomN, a putative amidohydrolase, were further proposed to be involved in the benzoxazole formation in A33853 according to gene deletion experiments. Finally, we have successfully utilized mutasynthesis to generate two analogs of A33853, which were reported previously to possess excellent anti-leishmanial activity.

  15. 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.

  16. Effects of white, blue, and red light-emitting diodes on carotenoid biosynthetic gene expression levels and carotenoid accumulation in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.).

    Science.gov (United States)

    Tuan, Pham Anh; Thwe, Aye Aye; Kim, Yeon Bok; Kim, Jae Kwang; Kim, Sun-Ju; Lee, Sanghyun; Chung, Sun-Ok; Park, Sang Un

    2013-12-18

    In this study, the optimum wavelengths of light required for carotenoid biosynthesis were determined by investigating the expression levels of carotenoid biosynthetic genes and carotenoid accumulation in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.) exposed to white, blue, and red light-emitting diodes (LEDs). Most carotenoid biosynthetic genes showed higher expression in sprouts irradiated with white light at 8 days after sowing than in those irradiated with blue and red lights. The dominant carotenoids in tartary buckwheat sprouts were lutein and β-carotene. The richest accumulation of total carotenoids was observed in sprouts grown under white light (1282.63 μg g(-1) dry weight), which was relatively higher than that in sprouts grown under blue and red lights (940.86 and 985.54 μg g(-1), respectively). This study might establish an effective strategy for maximizing the production of carotenoids and other important secondary metabolites in tartary buckwheat sprouts by using LED technology.

  17. Tissue- Specific Expression Analysis of Anthocyanin Biosynthetic Genes in White- and Red-Fleshed Grape Cultivars

    Directory of Open Access Journals (Sweden)

    Sha Xie

    2015-12-01

    Full Text Available Yan73, a teinturier (dyer grape variety in China, is one of the few Vitis vinifera cultivars with red-coloured berry flesh. To examine the tissue-specific expression of genes associated with berry colour in Yan73, we analysed the differential accumulation of anthocyanins in the skin and flesh tissues of two red-skinned grape varieties with either red (Yan73 or white flesh (Muscat Hamburg based on HPLC-MS analysis, as well as the differential expression of 18 anthocyanin biosynthesis genes in both varieties by quantitative RT-PCR. The results revealed that the transcripts of GST, OMT, AM3, CHS3, UFGT, MYBA1, F3′5′H, F3H1 and LDOX were barely detectable in the white flesh of Muscat Hamburg. In particular, GST, OMT, AM3, CHS3 and F3H1 showed approximately 50-fold downregulation in the white flesh of Muscat Hamburg compared to the red flesh of Yan73. A correlation analysis between the accumulation of different types of anthocyanins and gene expression indicated that the cumulative expression of GST, F3′5′H, LDOX and MYBA1 was more closely associated with the acylated anthocyanins and the 3′5′-OH anthocyanins, while OMT and AM3 were more closely associated with the total anthocyanins and methoxylated anthocyanins. Therefore, the transcripts of OMT, AM3, GST, F3′5′H, LDOX and MYBA1 explained most of the variation in the amount and composition of anthocyanins in skin and flesh of Yan73. The data suggest that the specific localization of anthocyanins in the flesh tissue of Yan73 is most likely due to the tissue-specific expression of OMT, AM3, GST, F3′5′H, LDOX and MYBA1 in the flesh.

  18. Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens.

    Science.gov (United States)

    Morea, A; Mathee, K; Franklin, M J; Giacomini, A; O'Regan, M; Ohman, D E

    2001-10-31

    The organization of the alginate gene cluster in Pseudomonas fluorescens was characterized. A bank of genomic DNA from P. fluorescens was mobilized to a strain of Pseudomonas aeruginosa with a transposon insertion (algJ::Tn501) in the alginate biosynthetic operon that rendered it non-mucoid. Phenotypic complementation in this heterologous host was observed, and a complementing clone containing 32 kb of P. fluorescens DNA was obtained. Southern hybridization studies showed that genes involved in alginate biosynthesis (e.g. algD, algG, and algA) were approximately in the same order and position as in P. aeruginosa. When the clone was mobilized to a P. aeruginosa algG mutant that produced alginate as polymannuronate due to its C5-epimerase defect, complementation was observed and the alginate from the recombinant strain contained L-guluronate as determined by proton nuclear magnetic resonance spectroscopy. A sequence analysis of the P. fluorescens DNA containing algG revealed sequences similar to P. aeruginosa algG that were also flanked by algE- and algX-like sequences. The predicted AlgG amino acid sequence of P. fluorescens was 67% identical (80% similar) to P. aeruginosa AlgG and 60% identical (76% similar) to Azotobacter vinelandii AlgG. As in P. aeruginosa, AlgG from P. fluorescens appeared to have a signal sequence that would localize it to the periplasm where AlgG presumably acts as a C5-epimerase at the polymer level. Non-polar algG knockout mutants of P. fluorescens were defective in alginate production, suggesting a potential role for this protein in polymer formation.

  19. Effects of methyl jasmonate and salicylic acid on tanshinone production and biosynthetic gene expression in transgenic Salvia miltiorrhiza hairy roots.

    Science.gov (United States)

    Hao, Xiaolong; Shi, Min; Cui, Lijie; Xu, Chao; Zhang, Yanjie; Kai, Guoyin

    2015-01-01

    Tanshinone is a group of active diterpenes, which are widely used in the treatment of cardiovascular disease. In this study, methyl jasmonate (MJ) and salicylic acid (SA) were used to investigate their effects on tanshinone accumulation and biosynthetic gene expression in the hairy roots of geranylgeranyl diphosphate synthase (SmGGPPS) overexpression line (G50) in Salvia miltiorrhiza. High-performance liquid chromatography analysis showed that total tanshinone content in G50 was obviously increased by 3.10-fold (11.33 mg/g) with MJ at 36 H and 1.63 times (5.95 mg/g) after SA treatment for 36 H in comparison with their mimic treatment control. Furthermore, quantitative reverse-transcription PCR analysis showed that the expression of isopentenyl-diphosphate delta-isomerase (SmIPPI), SmGGPPS, copalyl diphosphate synthase (SmCPS), and kaurene synthase-like (SmKSL) increased significantly with MJ treatment. However, the expression of SmIPPI reached the highest level at 144 H, whereas those of SmGGPPS, SmCPS, and SmKSL only increased slightly with SA treatment. The two elicitor treatments suggested that tanshinone accumulation positively correlated to the expression of key genes such as SmGGPPS, SmCPS, and SmKSL. Meanwhile, the study also indicated that it was a feasible strategy to combine elicitor treatment with transgenic technology for the enhancement of tanshinone, which paved the way for further metabolic engineering of tanshinone biosynthesis.

  20. Cloning and characterization of the biosynthetic gene cluster of the bacterial RNA polymerase inhibitor tirandamycin from marine-derived Streptomyces sp. SCSIO1666.

    Science.gov (United States)

    Mo, Xuhua; Wang, Zhongwen; Wang, Bo; Ma, Junying; Huang, Hongbo; Tian, Xinpeng; Zhang, Si; Zhang, Changsheng; Ju, Jianhua

    2011-03-18

    Tirandamycins are bacterial RNA polymerase inhibitors holding great potential for antibacterial agent design. To elucidate the biosynthetic machinery and generate new derivatives, the tirandamycin biosynthetic gene cluster was cloned and sequenced from marine-derived Streptomyces sp. SCSIO1666. The biosynthetic gene cluster of tirandamycin spans a DNA region of ∼56kb and consists of 15 open reading frames (ORFs) which encode three type I polyketide synthases (TrdAI, AII, AIII), one non-ribosomal peptide synthetase (TrdD), one phosphopantetheinyl transferase (TrdM), one Type II thioesterase (TrdB), one FAD-dependent oxidoreductase (TrdL), one cytochrome P450 monooxygenase (TrdI), three proteins related to resistance and regulations (TrdHJK), and four proteins with unknown function (TrdCEFG). To investigate the roles of the genes played in the biosynthetic machinery, seven genes (trdAI and trdBDFHIK) were inactivated via in frame replacement with an apramycin gene cassette using λ-RED recombination technology. The ΔtrdAI and ΔtrdD mutants targeting the ketosynthase and adenylation domain of TrdAI and TrdD, respectively, abolished the production of tirandamycins, confirming their involvement in the tirandamycin biosynthesis. TrdH showed high homology to LuxR family transcriptional regulatory proteins, disruption of which abolished the production of tirandamycins, indicating that TrdH is a positive regulator for tirandamycin biosynthesis. On the other hand, TrdK showed high homology to TetR-family transcriptional regulatory proteins, disruption of which significantly increased the yields of tirandamycins almost one-fold, implicating that TrdK is a negative regulator for tirandamycin biosynthesis. Disruption of the gene trdI resulted in the accumulation of the intermediate tirandamycin C (3) and a trace amount of new product tirandamycin C2 (5). A model of tirandamycin biosynthesis was proposed based on bioinformatics analyses, gene inactivation experiments and

  1. Characterization of biosynthetic gene cluster for the production of virginiamycin M, a streptogramin type A antibiotic, in Streptomyces virginiae.

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    Pulsawat, Nattika; Kitani, Shigeru; Nihira, Takuya

    2007-05-15

    Virginiamycin M (VM) of Streptomyces virginiae is a hybrid polyketide-peptide antibiotic with peptide antibiotic virginiamycin S (VS) as its synergistic counterpart. VM and VS belong to the Streptogramin family, which is characterized by strong synergistic antibacterial activity, and their water-soluble derivatives are a new therapeutic option for combating vancomycin-resistant Gram-positive bacteria. Here, the VM biosynthetic gene cluster was isolated from S. virginiae in the 62-kb region located in the vicinity of the regulatory island for virginiamycin production. Sequence analysis revealed that the region consists of 19 complete open reading frames (ORFs) and one C-terminally truncated ORF, encoding hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS), typical PKS, enzymes synthesizing precursors for VM, transporters for resistance, regulatory proteins, and auxiliary enzymes. The involvement of the cloned gene cluster in VM biosynthesis was confirmed by gene disruption of virA encoding a hybrid PKS-NRPS megasynthetase, which resulted in complete loss of VM production without any effect on VS production. To assemble the VM core structure, VirA, VirF, VirG, and VirH consisting, as a whole, of 24 domains in 8 PKS modules and 7 domains in 2 NRPS modules were predicted to act as an acyltransferase (AT)-less hybrid PKS-NRPS, whereas VirB, VirC, VirD, and VirE are likely to be essential for the incorporation of the methyl group into the VM framework by a HMG-CoA synthase-based reaction. Among several uncommon features of gene organization in the VM gene cluster, the lack of AT domain in every PKS module and the presence of a discrete AT encoded by virI are notable. AT-overexpression by an additional copy of virI driven by ermEp() resulted in 1.5-fold increase of VM production, suggesting that the amount of VirI is partly limiting VM biosynthesis.

  2. Anthocyanin accumulation and expression of anthocyanin biosynthetic genes in radish (Raphanus sativus).

    Science.gov (United States)

    Park, Nam Il; Xu, Hui; Li, Xiaohua; Jang, In Hyuk; Park, Suhyoung; Ahn, Gil Hwan; Lim, Yong Pyo; Kim, Sun Ju; Park, Sang Un

    2011-06-08

    Radish [Raphanus sativus (Rs)] is an important dietary vegetable in Asian countries, especially China, Japan, and Korea. To elucidate the molecular mechanisms of anthocyanin accumulation in radish, the gene expression of enzymes directly involved in anthocyanin biosynthesis was analyzed. These genes include phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), and anthocyanidin synthase (ANS). RsDFR and RsANS were found to accumulate in the flesh or skin of two radish cultivars (Man Tang Hong and Hong Feng No.1). Radish skin contained higher CHS, CHI, and F3H transcript levels than radish flesh in all three cultivars. In the red radish, 16 anthocyanins were separated and identified by high-performance liquid chromatography (HPLC) and elctrospray ionization-tandem mass spectrometry (ESI-MS/MS). Some of them were acylated with coumaroyl, malonoyl, feruoyl, and caffeoyl moieties. Furthermore (-)-epicatechin and ferulic acid were also identified in the three cultivars.

  3. Water-deficit inducible expression of a cytokinin biosynthetic gene IPT improves drought tolerance in cotton.

    Directory of Open Access Journals (Sweden)

    Sundaram Kuppu

    Full Text Available Water-deficit stress is a major environmental factor that limits agricultural productivity worldwide. Recent episodes of extreme drought have severely affected cotton production in the Southwestern USA. There is a pressing need to develop cotton varieties with improved tolerance to water-deficit stress for sustainable production in water-limited regions. One approach to engineer drought tolerance is by delaying drought-induced senescence via up-regulation of cytokinin biosynthesis. The isopentenyltransferase gene (IPT that encodes a rate limiting enzyme in cytokinin biosynthesis, under the control of a water-deficit responsive and maturation specific promoter P(SARK was introduced into cotton and the performance of the P(SARK::IPT transgenic cotton plants was analyzed in the greenhouse and growth chamber conditions. The data indicate that P(SARK::IPT-transgenic cotton plants displayed delayed senescence under water deficit conditions in the greenhouse. These plants produced more root and shoot biomass, dropped fewer flowers, maintained higher chlorophyll content, and higher photosynthetic rates under reduced irrigation conditions in comparison to wild-type and segregated non-transgenic lines. Furthermore, P(SARK::IPT-transgenic cotton plants grown in growth chamber condition also displayed greater drought tolerance. These results indicate that water-deficit induced expression of an isopentenyltransferase gene in cotton could significantly improve drought tolerance.

  4. Gene Discovery for Synthetic Biology: Exploring the Novel Natural Product Biosynthetic Capacity of Eukaryotic Microalgae.

    Science.gov (United States)

    O'Neill, E C; Saalbach, G; Field, R A

    2016-01-01

    Eukaryotic microalgae are an incredibly diverse group of organisms whose sole unifying feature is their ability to photosynthesize. They are known for producing a range of potent toxins, which can build up during harmful algal blooms causing damage to ecosystems and fisheries. Genome sequencing is lagging behind in these organisms because of their genetic complexity, but transcriptome sequencing is beginning to make up for this deficit. As more sequence data becomes available, it is apparent that eukaryotic microalgae possess a range of complex natural product biosynthesis capabilities. Some of the genes concerned are responsible for the biosynthesis of known toxins, but there are many more for which we do not know the products. Bioinformatic and analytical techniques have been developed for natural product discovery in bacteria and these approaches can be used to extract information about the products synthesized by algae. Recent analyses suggest that eukaryotic microalgae produce many complex natural products that remain to be discovered.

  5. Transcriptional Regulation of Cytosolic Sulfotransferase 1C2 by Intermediates of the Cholesterol Biosynthetic Pathway in Primary Cultured Rat Hepatocytes.

    Science.gov (United States)

    Rondini, Elizabeth A; Pant, Asmita; Kocarek, Thomas A

    2015-12-01

    Cytosolic sulfotransferase 1C2 (SULT1C2) is expressed in the kidney, stomach, and liver of rats; however, the mechanisms regulating expression of this enzyme are not known. We evaluated transcriptional regulation of SULT1C2 by mevalonate (MVA)-derived intermediates in primary cultured rat hepatocytes using several cholesterol synthesis inhibitors. Blocking production of mevalonate with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin (30 μM), reduced SULT1C2 mRNA content by ∼40% whereas the squalene synthase inhibitor squalestatin (SQ1, 0.1 μM), which causes accumulation of nonsterol isoprenoids, increased mRNA content by 4-fold. Treatment with MVA (10 mM) strongly induced SULT1C2 mRNA by 12-fold, and this effect was blocked by inhibiting squalene epoxidase but not by more distal cholesterol inhibitors, indicating the effects of MVA are mediated by postsqualene metabolites. Using rapid amplification of cDNA ends (RACE), we characterized the 5' end of SULT1C2 mRNA and used this information to generate constructs for promoter analysis. SQ1 and MVA increased reporter activity by ∼1.6- and 3-fold, respectively, from a construct beginning 49 base pairs (bp) upstream from the longest 5'-RACE product (-3140:-49). Sequence deletions from this construct revealed a hepatocyte nuclear factor 1 (HNF1) element (-2558), and mutation of this element reduced basal (75%) and MVA-induced (30%) reporter activity and attenuated promoter activation following overexpression of HNF1α or 1β. However, the effects of SQ1 were localized to a more proximal promoter region (-281:-49). Collectively, our findings demonstrate that cholesterol biosynthetic intermediates influence SULT1C2 expression in rat primary hepatocytes. Further, HNF1 appears to play an important role in mediating basal and MVA-induced SULT1C2 transcription.

  6. Variation in siderophore biosynthetic gene distribution and production across environmental and faecal populations of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Laura J Searle

    Full Text Available Iron is essential for Escherichia coli growth and survival in the host and the external environment, but its availability is generally low due to the poor solubility of its ferric form in aqueous environments and the presence of iron-withholding proteins in the host. Most E. coli can increase access to iron by excreting siderophores such as enterobactin, which have a very strong affinity for Fe3+. A smaller proportion of isolates can generate up to 3 additional siderophores linked with pathogenesis; aerobactin, salmochelin, and yersiniabactin. However, non-pathogenic E. coli are also able to synthesise these virulence-associated siderophores. This raises questions about their role in the ecology of E. coli, beyond virulence, and whether specific siderophores might be linked with persistence in the external environment. Under the assumption that selection favours phenotypes that confer a fitness advantage, we compared siderophore production and gene distribution in E. coli isolated either from agricultural plants or the faeces of healthy mammals. This population-level comparison has revealed that under iron limiting growth conditions plant-associated isolates produced lower amounts of siderophores than faecal isolates. Additionally, multiplex PCR showed that environmental isolates were less likely to contain loci associated with aerobactin and yersiniabactin synthesis. Although aerobactin was linked with strong siderophore excretion, a significant difference in production was still observed between plant and faecal isolates when the analysis was restricted to strains only able to synthesise enterobactin. This finding suggests that the regulatory response to iron limitation may be an important trait associated with adaptation to the non-host environment. Our findings are consistent with the hypothesis that the ability to produce multiple siderophores facilitates E. coli gut colonisation and plays an important role in E. coli commensalism.

  7. Impact of bacterial biocontrol agents on aflatoxin biosynthetic genes, aflD and aflR expression, and phenotypic aflatoxin B₁ production by Aspergillus flavus under different environmental and nutritional regimes.

    Science.gov (United States)

    Al-Saad, Labeed A; Al-Badran, Adnan I; Al-Jumayli, Sami A; Magan, Naresh; Rodríguez, Alicia

    2016-01-18

    The objectives of this study were to examine the efficacy of four bacterial antagonists against Aspergillus flavus using 50:50 ratio of bacterial cells/conidia for the control of aflatoxin B1 (AFB1) production on two different nutritional matrices, nutrient and maize-based media at different water availabilities (0.98, 0.94 water activity (aw) on nutrient medium; 0.995, 0.98 aw on maize meal agar medium) at 35°C. The indicators of efficacy used were the relative expression of one structural and regulatory gene in the biosynthetic pathway (aflD and aflR respectively) and the production of AFB1. These studies showed that some of the bacterial species could significantly inhibit the relative expression of the aflD and aflR genes at both 0.98 and 0.94 aw on nutrient agar. On maize-based media some of the bacterial antagonists reduced the activity of both genes at 0.94 aw and some at 0.995 aw. However, the results for AFB1 production were not consistent with the effects on gene expression. Some bacterial species stimulated AFB1 production on both nutrient and maize-based media regardless of aw. However, some bacterial treatments did inhibit AFB1 production significantly when compared to the control. Overall, this study suggests that temporal studies are required on the biosynthetic genes under different environmental and nutritional conditions to evaluate the potential of antagonists to control AFB1.

  8. Sulphate as a xylem-borne chemical signal precedes the expression of ABA biosynthetic genes in maize roots.

    Science.gov (United States)

    Ernst, Laura; Goodger, Jason Q D; Alvarez, Sophie; Marsh, Ellen L; Berla, Bert; Lockhart, Eric; Jung, Jiyul; Li, Pinghua; Bohnert, Hans J; Schachtman, Daniel P

    2010-07-01

    Recent reports suggest that early sensing of soil water stress by plant roots and the concomitant reduction in stomatal conductance may not be mediated by root-sourced abscisic acid (ABA), but that other xylem-borne chemicals may be the primary stress signal(s). To gain more insight into the role of root-sourced ABA, the timing and location of the expression of genes for key enzymes involved in ABA biosynthesis in Zea mays roots was measured and a comprehensive analysis of root xylem sap constituents from the early to the later stages of water stress was conducted. Xylem sap and roots were sampled from plants at an early stage of water stress when only a reduction in leaf conductance was measured, as well as at later stages when leaf xylem pressure potential decreased. It was found that the majority of ABA biosynthetic genes examined were only significantly expressed in the elongation region of roots at a later stage of water stress. Apart from ABA, sulphate was the only xylem-borne chemical that consistently showed significantly higher concentrations from the early to the later stages of stress. Moreover, there was an interactive effect of ABA and sulphate in decreasing maize transpiration rate and Vicia faba stomatal aperture, as compared to ABA alone. The expression of a sulphate transporter gene was also analysed and it was found that it had increased in the elongation region of roots from the early to the later stages of water stress. Our results support the suggestion that in the early stage of water stress, increased levels of ABA in xylem sap may not be due to root biosynthesis, ABA glucose ester catabolism or pH-mediated redistribution, but may be due to shoot biosynthesis and translocation to the roots. The analysis of xylem sap mineral content and bioassays indicate that the anti-transpirant effect of the ABA reaching the stomata at the early stages of water stress may be enhanced by the increased concentrations of sulphate in the xylem which is also

  9. CYP99A3: Functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice

    Science.gov (United States)

    Wang, Qiang; Hillwig, Matthew L.; Peters, Reuben J.

    2013-01-01

    SUMMARY Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochromes P450 mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNAi double knock-down of this pair of closely related CYP reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which ultimately was achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that, while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-γ-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiological relevance for the observed activity of CYP99A3. In addition, we found that CYP99A3 also oxidized syn-stemod-13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis. PMID:21175892

  10. 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.

  11. Identification of transcriptional activators for thienamycin and cephamycin C biosynthetic genes within the thienamycin gene cluster from Streptomyces cattleya.

    Science.gov (United States)

    Rodríguez, Miriam; Núñez, Luz Elena; Braña, Alfredo F; Méndez, Carmen; Salas, José A; Blanco, Gloria

    2008-08-01

    Two regulatory genes, thnI and thnU, were identified in the thienamycin (thn) gene cluster from Streptomyces cattleya. ThnI resembles LysR-type transcriptional activators and ThnU belongs to the SARP family of transcriptional activators. Their functional role was established after independent inactivation by gene replacement together with transcriptional analysis involving reverse transcription polymerase chain reaction (RT-PCR). Deletion of thnI abolished thienamycin production showing its involvement in thienamycin biosynthesis. Gene expression analysis applied to the thn gene cluster demonstrated that ThnI is a transcriptional activator essential for thienamycin biosynthesis that regulates the expression of nine genes involved in thienamycin assembly and export (thnH, thnJ, thnK, thnL, thnM, thnN, thnO, thnP and thnQ). Unexpectedly, the thnU disrupted mutant was not affected in thienamycin production but turned out to be essential for cephamycin C biosynthesis. Transcript analysis applied to early and late structural genes for cephamycin C biosynthesis (pcbAB and cmcI), revealed that ThnU is the transcriptional activator of these cephamycin C genes although they are not physically linked to the thn cluster. In addition, it was shown that deletion of thnI has an upregulatory effect on pcbAB and cmcI transcription consistent with a significant increase in cephamycin C biosynthesis in this mutant.

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

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    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

  13. NF-Y activates genes of metabolic pathways altered in cancer cells

    Science.gov (United States)

    Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A.; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta

    2016-01-01

    The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells. PMID:26646448

  14. 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.

  15. 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.

  16. Studies on the evolution of complex natural products biosynthetic pathways on basis of Taxol biosynthesis in plants and endophytic fungi

    OpenAIRE

    Heinig, Uwe Herbert

    2012-01-01

    Focus of the present PhD thesis was the examination of the interesting and only poorly understood phenomenon of detection of identical natural products in distantly related organisms with regard to metabolite pathway evolution. Due to the impact of Taxol as one of the most important anti-cancer drugs today intensive research efforts were made with respect to improvement of production systems in order to overcome the supply problems. During the search for new production systems in the early 19...

  17. Identification of key pathways and genes influencing prognosis in bladder urothelial carcinoma

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    Ning X

    2017-03-01

    Full Text Available Xin Ning, Yaoliang Deng Department of Urology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Province, People’s Republic of China Background: Genomic profiling can be used to identify the predictive effect of genomic subsets for determining prognosis in bladder urothelial carcinoma (BUC after radical cystectomy. This study aimed to investigate potential gene and pathway markers associated with prognosis in BUC.Methods: A microarray dataset of BUC was obtained from The Cancer Genome Atlas database. Differentially expressed genes (DEGs were identified by DESeq of the R platform. Kaplan–Meier analysis was applied for prognostic markers. Key pathways and genes were identified using bioinformatics tools, such as gene set enrichment analysis, gene ontology, the Kyoto Encyclopedia of Genes and Genomes, gene multiple association network integration algorithm (GeneMANIA, Search Tool for the Retrieval of Interacting Genes/Proteins, and Molecular Complex Detection.Results: A comparative gene set enrichment analysis of tumor and adjacent normal tissues suggested BUC tumorigenesis resulted mainly from enrichment of cell cycle and DNA damage and repair-related biological processes and pathways, including TP53 and mitotic recombination. Two hundred and fifty-six genes were identified as potential prognosis-related DEGs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the potential prognosis-related DEGs were enriched in angiogenesis, including the cyclic adenosine monophosphate biosynthetic process, cyclic guanosine monophosphate-protein kinase G, mitogen-activated protein kinase, Rap1, and phosphoinositide-3-kinase-AKT signaling pathway. Nine hub genes, TAGLN, ACTA2, MYH11, CALD1, MYLK, GEM, PRELP, TPM2, and OGN, were identified from the intersection of protein–protein interaction and GeneMANIA networks. Module analysis of protein–protein interaction and GeneMANIA networks mainly showed

  18. Identification and Expression Analysis of Glucosinolate Biosynthetic Genes and Estimation of Glucosinolate Contents in Edible Organs of Brassica oleracea Subspecies

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    Go-Eun Yi

    2015-07-01

    Full Text Available Glucosinolates are anti-carcinogenic, anti-oxidative biochemical compounds that defend plants from insect and microbial attack. Glucosinolates are abundant in all cruciferous crops, including all vegetable and oilseed Brassica species. Here, we studied the expression of glucosinolate biosynthesis genes and determined glucosinolate contents in the edible organs of a total of 12 genotypes of Brassica oleracea: three genotypes each from cabbage, kale, kohlrabi and cauliflower subspecies. Among the 81 genes analyzed by RT-PCR, 19 are transcription factor-related, two different sets of 25 genes are involved in aliphatic and indolic biosynthesis pathways and the rest are breakdown-related. The expression of glucosinolate-related genes in the stems of kohlrabi was remarkably different compared to leaves of cabbage and kale and florets of cauliflower as only eight genes out of 81 were expressed in the stem tissues of kohlrabi. In the stem tissue of kohlrabi, only one aliphatic transcription factor-related gene, Bol036286 (MYB28 and one indolic transcription factor-related gene, Bol030761 (MYB51, were expressed. The results indicated the expression of all genes is not essential for glucosinolate biosynthesis. Using HPLC analysis, a total of 16 different types of glucosinolates were identified in four subspecies, nine of them were aliphatic, four of them were indolic and one was aromatic. Cauliflower florets measured the highest number of 14 glucosinolates. Among the aliphatic glucosinolates, only gluconapin was found in the florets of cauliflower. Glucoiberverin and glucobrassicanapin contents were the highest in the stems of kohlrabi. The indolic methoxyglucobrassicin and aromatic gluconasturtiin accounted for the highest content in the florets of cauliflower. A further detailed investigation and analyses is required to discern the precise roles of each of the genes for aliphatic and indolic glucosinolate biosynthesis in the edible organs.

  19. Application of an Efficient Gene Targeting System Linking Secondary Metabolites to their Biosynthetic Genes in Aspergillus terreus

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Chun-Jun; Knox, Benjamin P.; Sanchez, James F.; Chiang, Yi-Ming; Bruno, Kenneth S.; Wang, Clay C.

    2013-07-19

    Nonribosomal peptides (NRPs) are natural products biosynthesized by NRP synthetases. A kusA-, pyrG- mutant strain of Aspergillusterreus NIH 2624 was developed that greatly facilitated the gene targeting efficiency in this organism. Application of this tool allowed us to link four major types of NRP related secondary metabolites to their responsible genes in A. terreus. In addition, an NRP related melanin synthetase was also identified in this species.

  20. Tissue-specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding.

    Science.gov (United States)

    Drew, Janice E; Farquharson, Andrew J; Horgan, Graham W; Williams, Lynda M

    2016-11-01

    The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D.

  1. Identification of anrF gene, a homology of admM of andrimid biosynthetic gene cluster related to the antagonistic activity of Enterobacter cloacae B8

    Institute of Scientific and Technical Information of China (English)

    Xu-Ping Yu; Jun-Li Zhu; Xue-Ping Yao; Shi-Cheng He; Hai-Ning Huang; Wei-Liang Chen; Yong-Hao Hu; De-Bao Li

    2005-01-01

    AIM: To identify the gene (s) related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism.METHODS: Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain(s) of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame (ORF) finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www. ncbi.nlm.nih.gov. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrFgene to the mutant B8F strain was used.RESULTS: A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrFgene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglornerans andrimid biosynthetic gene cluster (AY192157). The Tn5was inserted at 2 420 bp of the gene corresponding to the COG3319 (the thioesterase domain of type Ⅰ polyketide synthase) coding region on B8F. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrFgene to the mutant B8F.CONCLUSION: The anrFgene obtained is related to the antagonistic activity of B8, and the antagonistic substances produced by B8 are andrimid and/or its analogs.

  2. Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway.

    Science.gov (United States)

    Yan, Jinyong; Yan, Yunjun; Madzak, Catherine; Han, Bingnan

    2017-02-01

    Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.

  3. The Biosynthesis of Capuramycin-type Antibiotics: IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5'-CARBOXAMIDE.

    Science.gov (United States)

    Cai, Wenlong; Goswami, Anwesha; Yang, Zhaoyong; Liu, Xiaodong; Green, Keith D; Barnard-Britson, Sandra; Baba, Satoshi; Funabashi, Masanori; Nonaka, Koichi; Sunkara, Manjula; Morris, Andrew J; Spork, Anatol P; Ducho, Christian; Garneau-Tsodikova, Sylvie; Thorson, Jon S; Van Lanen, Steven G

    2015-05-29

    A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.

  4. 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.

  5. Method development and analysis of free HS and HS in proteoglycans from pre- and postmenopausal women: evidence for biosynthetic pathway changes in sulfotransferase and sulfatase enzymes.

    Science.gov (United States)

    Wei, Wei; Miller, Rebecca L; Leary, Julie A

    2013-06-18

    Heparan sulfate (HS) is one of the most complex and informative biopolymers found on the cell surface or in the extracellular matrix as either free HS fragments or constituents of HS proteoglycans (HSPGs). Analysis of free HS and HSPG sugar chains in human serum at the disaccharide level has great potential for early disease diagnosis and prognosis; however, the low concentration of HS in human serum, together with the complexity of the serum matrix, limits the information on HS. In this study, we present and validate the development of a new sensitive method for in-depth compositional analysis of free HS and HSPG sugar chains. This protocol involved several steps including weak anion exchange chromatography, ultrafiltration, and solid-phase extraction for enhanced detection prior to LC-MS/MS analysis. Using this protocol, a total of 51 serum samples from 26 premenopausal and 25 postmenopausal women were analyzed. Statistically significant differences in heparin/HS disaccharide profiles were observed. The proportion of N-acetylation and N-sulfation in both free HS and HSPG sugar chains were significantly different between pre- and postmenopausal women, indicating changes in N-deacetylase/N-sulfotransferases (NDSTs), the enzymes involved in the initial step of the biosynthetic pathway. Differences in the proportion of 6-O-sulfation suggest that 6-O-sulfotransferase and/or 6-O-sulfatase enzymes may also be implicated.

  6. Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes.

    Science.gov (United States)

    Mandal, Shantanu; Upadhyay, Shivangi; Singh, Ved Pal; Kapoor, Rupam

    2015-04-01

    Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs)--stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-D-xylulose 5-phospate synthase (DXS), 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants.

  7. Genome-scale transcriptome analysis in response to nitric oxide in birch cells: implications of the triterpene biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Fansuo Zeng

    Full Text Available Evidence supporting nitric oxide (NO as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in Betula platyphylla cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10-5 sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374 were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis.

  8. Structural characterization of the polar lipids of Clostridium novyi NT. Further evidence for a novel anaerobic biosynthetic pathway to plasmalogens.

    Science.gov (United States)

    Guan, Ziqiang; Johnston, Norah C; Aygun-Sunar, Semra; Daldal, Fevzi; Raetz, Christian R H; Goldfine, Howard

    2011-03-01

    A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1'-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1'-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism.

  9. 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.

  10. Hydroxycinnamic acids and UV-B depletion: Profiling and biosynthetic gene expression in flesh and peel of wild-type and hp-1.

    Science.gov (United States)

    Calvenzani, Valentina; Castagna, Antonella; Ranieri, Annamaria; Tonelli, Chiara; Petroni, Katia

    2015-06-01

    Hydroxycinnamic acids (HCAs) are phenolic compounds widely found in most plant families. Aim of the present work was to investigate their accumulation and biosynthetic gene expression in presence or absence of UV-B radiation in tomato fruits of wild-type and hp-1, a mutant characterized by exaggerated photoresponsiveness and increased fruit pigmentation. Gene expression and HCAs content were higher in hp-1 than in wild type peel and UV-B depletion determined a decrease in HCAs accumulation in wild-type and an increase in hp-1 fruits, generally in accordance with biosynthetic gene expression. In flesh, despite a similar transcript level of most genes between the two genotypes, HCAs content was generally higher in wild type than in hp-1, although remaining at a lower level with respect to wild type peel. Under UV-B depletion, a general reduction of HCAs content was observed in wild-type flesh, whereas an increase in the content of p-coumaric acid and caffeic acid was observed in hp-1 flesh.

  11. 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.

  12. An Integrated Metabolomic and Genomic Mining Workflow to Uncover the Biosynthetic Potential of Bacteria

    DEFF Research Database (Denmark)

    Månsson, Maria; Vynne, Nikolaj Grønnegaard; Klitgaard, Andreas

    2016-01-01

    considerable diversity: only 2% of the chemical features and 7% of the biosynthetic genes were common to all strains, while 30% of all features and 24% of the genes were unique to single strains. The list of chemical features was reduced to 50 discriminating features using a genetic algorithm and support...... vector machines. Features were dereplicated by tandem mass spectrometry (MS/MS) networking to identify molecular families of the same biosynthetic origin, and the associated pathways were probed using comparative genomics. Most of the discriminating features were related to antibacterial compounds...

  13. Genome mining of the Streptomyces avermitilis genome and development of genome-minimized hosts for heterologous expression of biosynthetic gene clusters.

    Science.gov (United States)

    Ikeda, Haruo; Kazuo, Shin-ya; Omura, Satoshi

    2014-02-01

    To date, several actinomycete genomes have been completed and annotated. Among them, Streptomyces microorganisms are of major pharmaceutical interest because they are a rich source of numerous secondary metabolites. S. avermitilis is an industrial microorganism used for the production of an anthelmintic agent, avermectin, which is a commercially important antiparasitic agent in human and veterinary medicine, and agricultural pesticides. Genome analysis of S. avermitilis provides significant information for not only industrial applications but also understanding the features of this genus. On genome mining of S. avermitilis, the microorganism has been found to harbor at least 38 secondary metabolic gene clusters and 46 insertion sequence (IS)-like sequences on the genome, which have not been searched so far. A significant use of the genome data of Streptomyces microorganisms is the construction of a versatile host for heterologous expression of exogenous biosynthetic gene clusters by genetic engineering. Since S. avermitilis is used as an industrial microorganism, the microorganism is already optimized for the efficient supply of primary metabolic precursors and biochemical energy to support multistep biosynthesis. The feasibility of large-deletion mutants of S. avermitilis has been confirmed by heterologous expression of more than 20 exogenous biosynthetic gene clusters.

  14. Seasonal alteration in amounts of lignans and their glucosides and gene expression of the relevant biosynthetic enzymes in the Forsythia suspense leaf.

    Science.gov (United States)

    Morimoto, Kinuyo; Satake, Honoo

    2013-01-01

    Lignans of Forsythia spp. are essential components of various Chinese medicines and health diets. However, the seasonal alteration in lignan amounts and the gene expression profile of lignan-biosynthetic enzymes has yet to be investigated. In this study, we have assessed seasonal alteration in amounts of major lignans, such as pinoresinol, matairesinol, and arctigenin, and examined the gene expression profile of pinoresinol/lariciresinol reductase (PLR), pinoresinol-glucosylating enzyme (UGT71A18), and secoisolariciresinol dehydrogenase (SIRD) in the leaf of Forsythia suspense from April to November. All of the lignans in the leaf continuously increased from April to June, reached the maximal level in June, and then decreased. Ninety percent of pinoresinol and matairesinol was converted into glucosides, while approximately 50% of arctigenin was aglycone. PLR was stably expressed from April to August, whereas the PLR expression was not detected from September to November. In contrast, the UGT71A18 expression was found from August to November, but not from April to July. The SIRD expression was prominent from April to May, not detected in June to July, and then increased again from September to November. These expression profiles of the lignan-synthetic enzymes are largely compatible with the alteration in lignan contents. Furthermore, such seasonal lignan profiles are in good agreement with the fact that the Forsythia leaves for Chinese medicinal tea are harvested in June. This is the first report on seasonal alteration in lignans and the relevant biosynthetic enzyme genes in the leaf of Forsythia species.

  15. Vitis vinifera secondary metabolism as affected by sulfate depletion: diagnosis through phenylpropanoid pathway genes and metabolites.

    Science.gov (United States)

    Tavares, Sílvia; Vesentini, Damiano; Fernandes, João Carlos; Ferreira, Ricardo B; Laureano, Olga; Ricardo-Da-Silva, Jorge M; Amâncio, Sara

    2013-05-01

    Grapevine (Vitis vinifera L.) is rich in phenylpropanoid compounds, namely flavonoids and stilbenes which, present in most tissues, are described as antioxidants and known to accumulate in response to biotic and abiotic stress. Grapevine is then a choice model for studying the interplay between the phenylpropanoid pathway and nutrient deficiency. Here we report the response to sulfur deficiency (-S) of flavonoids and stilbenes biosynthetic pathways in chlorophyll tissues (plantlets) and cell culture. Anthocyanins and trans-resveratrol accumulated in plantlets and trans-resveratrol glucoside in cell cultures in response to sulfur deficiency, while a significant decrease in chlorophyll was observed in -S plantlets. The up-regulation of chalcone synthase gene and the downstream flavonoid biosynthesis genes dihydroflavonol reductase and anthocyanidin synthase matched the accumulation of anthocyanins in -S V. vinifera plantlets. The mRNA level of stilbene synthase gene(s) was correlated tightly with the increase in trans-resveratrol and trans-resveratrol glucoside levels, respectively in -S plantlets and cell cultures. As a whole, the present study unveil that V. vinifera under sulfur deficiency allocates resources to the phenylpropanoid pathway, probably consecutive to inhibition of protein synthesis, which can be advantageous to resist against oxidative stress symptoms evoked by -S conditions.

  16. Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli

    OpenAIRE

    Ma, Tian; Zhou, Yuanjie; Li, Xiaowei; Zhu, Fayin; Cheng, Yongbo; Liu, Yi; Deng, Zixin; Liu, Tiangang

    2015-01-01

    Abstract As a highly valued keto‐carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α‐Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin n...

  17. 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.

  18. Characterisation of betalain biosynthesis in Parakeelya flowers identifies the key biosynthetic gene DOD as belonging to an expanded LigB gene family that is conserved in betalain-producing species

    Directory of Open Access Journals (Sweden)

    Hsiao-Hang eChung

    2015-07-01

    Full Text Available Plant betalain pigments are intriguing because they are restricted to the Caryophyllales and are mutually exclusive with the more common anthocyanins. However, betalain biosynthesis is poorly understood compared to that of anthocyanins. In this study, betalain production and betalain-related genes were characterized in Parakeelya mirabilis (Montiaceae. RT-PCR and transcriptomics identified three sequences related to the key biosynthetic enzyme Dopa 4,5-dioxgenase (DOD. In addition to a LigB gene similar to that of non-Caryophyllales species (Class I genes, two other P. mirabilis LigB genes were found (DOD and DOD-like, termed Class II. PmDOD and PmDOD-like had 70% amino acid identity. Only PmDOD was implicated in betalain synthesis based on transient assays of enzyme activity and correlation of transcript abundance to spatio-temporal betalain accumulation. The role of PmDOD-like remains unknown. The striking pigment patterning of the flowers was due to distinct zones of red betacyanin and yellow betaxanthin production. The major betacyanin was the unglycosylated betanidin rather than the commonly found glycosides, an occurrence for which there are a few previous reports. The white petal zones lacked pigment but had DOD activity suggesting alternate regulation of the pathway in this tissue. DOD and DOD-like sequences were also identified in other betalain-producing species but not in examples of anthocyanin-producing Caryophyllales or non-Caryophyllales species. A Class I LigB sequence from the anthocyanin-producing Caryophyllaceae species Dianthus superbus and two DOD-like sequences from the Amaranthaceae species Beta vulgaris and Ptilotus spp. did not show DOD activity in the transient assay. The additional sequences suggests that DOD is part of a larger LigB gene family in betalain-producing Caryophyllales taxa, and the tandem genomic arrangement of two of the three B. vulgaris LigB genes suggests the involvement of duplication in the gene

  19. Separate enrichment analysis of pathways for up- and downregulated genes.

    Science.gov (United States)

    Hong, Guini; Zhang, Wenjing; Li, Hongdong; Shen, Xiaopei; Guo, Zheng

    2014-03-06

    Two strategies are often adopted for enrichment analysis of pathways: the analysis of all differentially expressed (DE) genes together or the analysis of up- and downregulated genes separately. However, few studies have examined the rationales of these enrichment analysis strategies. Using both microarray and RNA-seq data, we show that gene pairs with functional links in pathways tended to have positively correlated expression levels, which could result in an imbalance between the up- and downregulated genes in particular pathways. We then show that the imbalance could greatly reduce the statistical power for finding disease-associated pathways through the analysis of all-DE genes. Further, using gene expression profiles from five types of tumours, we illustrate that the separate analysis of up- and downregulated genes could identify more pathways that are really pertinent to phenotypic difference. In conclusion, analysing up- and downregulated genes separately is more powerful than analysing all of the DE genes together.

  20. Pyocyanine Biosynthetic Genes in Clinical and Environmental Isolates of Pseudomonas aeruginosa and Detection of Pyocyanine’s Antimicrobial Effects with or without Colloidal Silver Nanoparticles

    Directory of Open Access Journals (Sweden)

    Afrooz Rashnonejad

    2012-01-01

    Full Text Available Objective: Pyocyanine plays an important role in the pathogenesis of Pseudomonas aeruginosa, (P. aeruginosa and is known to have inhibitory and bactericidal effects. This study has aimed to detect the phenazine biosynthetic operon (phz ABCDEFG and two phenazine modifying genes (phzM and phzS by polymerase chain reaction (PCR and detection of its possible protein bands by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE. The antimicrobial effects of pyocyanine alone and mixed with colloidal silver nanoparticles were studied.Materials and Methods: In this descriptive study, clinical and environmental species of P. aeruginosa were isolated by thioglycollate medium culture and cetrimide agar, respectively. The existence of a phenazine biosynthetic operon and two phenazine modifying genes as well as their protein products were confirmed by PCR and SDS-PAGE, respectively. Pyocyanine was extracted with chloroform and its antimicrobial effects against bacteria such as; Escherichia coli (E. coli, P. aeruginosaand Staphylococcus aureus (S. aureus bacteria and yeast Candida albicans (C. albicans were tested using well, spot and disk diffusion methods.Results: In this study, 3 out of 48 clinical strains were unable to produce pyocyanine on cetrimide and Mueller Hinton (MH agar. Two strains did not have phenazine modifying gene bands. Another strain did not have the possible protein band of the phzM gene. Pyocyanine had antimicrobial effects against the microbial strains, which increased in the presence of silver nanoparticles.Conclusion: According to the results of the present study, some P. aeruginosa strains are unable to produce pyocyanine due to the absence of the phzM or phzS genes. Therefore, these genes have an important role in pyocyanine production in P. aeruginosa. Pyocyanine shows synergistic antimicrobial effects in the presence of silver nanoparticles against microbial strains.

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

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    Muhammad Tariq Saeed

    2016-09-01

    Full Text Available 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.

  2. Variation in Fumonisin and Ochratoxin Production Associated with Differences in Biosynthetic Gene Content in Aspergillus niger and A. welwitschiae Isolates from Multiple Crop and Geographic Origins

    Science.gov (United States)

    Susca, Antonia; Proctor, Robert H.; Morelli, Massimiliano; Haidukowski, Miriam; Gallo, Antonia; Logrieco, Antonio F.; Moretti, Antonio

    2016-01-01

    The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that (i) isolates of both species differed in ability to produce the mycotoxins; (ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum) cluster; (iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and (iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas, a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin. PMID:27667988

  3. Global identification of the full-length transcripts and alternative splicing related to phenolic acid biosynthetic genes in Salvia miltiorrhiza

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    Zhichao eXu

    2016-02-01

    Full Text Available Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and 4 alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that 6 candidate cytochrome P450s and 5 candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza.

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

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    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

  5. Reducing AsA leads to leaf lesion and defence response in knock-down of the AsA biosynthetic enzyme GDP-D-mannose pyrophosphorylase gene in tomato plant.

    Science.gov (United States)

    Zhang, Chanjuan; Ouyang, Bo; Yang, Changxian; Zhang, Xiaohui; Liu, Hui; Zhang, Yuyang; Zhang, Junhong; Li, Hanxia; Ye, Zhibiao

    2013-01-01

    As a vital antioxidant, L-ascorbic acid (AsA) affects diverse biological processes in higher plants. Lack of AsA in cell impairs plant development. In the present study, we manipulated a gene of GDP-mannose pyrophosphorylase which catalyzes the conversion of D-mannose-1-P to GDP-D-mannose in AsA biosynthetic pathway and found out the phenotype alteration of tomato. In the tomato genome, there are four members of GMP gene family and they constitutively expressed in various tissues in distinct expression patterns. As expected, over-expression of SlGMP3 increased total AsA contents and enhanced the tolerance to oxidative stress in tomato. On the contrary, knock-down of SlGMP3 significantly decreased AsA contents below the threshold level and altered the phenotype of tomato plants with lesions and further senescence. Further analysis indicated the causes for this symptom could result from failing to instantly deplete the reactive oxygen species (ROS) as decline of free radical scavenging activity. More ROS accumulated in the leaves and then triggered expressions of defence-related genes and mimic symptom occurred on the leaves similar to hypersensitive responses against pathogens. Consequently, the photosynthesis of leaves was dramatically fallen. These results suggested the vital roles of AsA as an antioxidant in leaf function and defence response of tomato.

  6. Reducing AsA leads to leaf lesion and defence response in knock-down of the AsA biosynthetic enzyme GDP-D-mannose pyrophosphorylase gene in tomato plant.

    Directory of Open Access Journals (Sweden)

    Chanjuan Zhang

    Full Text Available As a vital antioxidant, L-ascorbic acid (AsA affects diverse biological processes in higher plants. Lack of AsA in cell impairs plant development. In the present study, we manipulated a gene of GDP-mannose pyrophosphorylase which catalyzes the conversion of D-mannose-1-P to GDP-D-mannose in AsA biosynthetic pathway and found out the phenotype alteration of tomato. In the tomato genome, there are four members of GMP gene family and they constitutively expressed in various tissues in distinct expression patterns. As expected, over-expression of SlGMP3 increased total AsA contents and enhanced the tolerance to oxidative stress in tomato. On the contrary, knock-down of SlGMP3 significantly decreased AsA contents below the threshold level and altered the phenotype of tomato plants with lesions and further senescence. Further analysis indicated the causes for this symptom could result from failing to instantly deplete the reactive oxygen species (ROS as decline of free radical scavenging activity. More ROS accumulated in the leaves and then triggered expressions of defence-related genes and mimic symptom occurred on the leaves similar to hypersensitive responses against pathogens. Consequently, the photosynthesis of leaves was dramatically fallen. These results suggested the vital roles of AsA as an antioxidant in leaf function and defence response of tomato.

  7. Differentially Expressed Genes and Signature Pathways of Human Prostate Cancer.

    Directory of Open Access Journals (Sweden)

    Jennifer S Myers

    Full Text Available Genomic technologies including microarrays and next-generation sequencing have enabled the generation of molecular signatures of prostate cancer. Lists of differentially expressed genes between malignant and non-malignant states are thought to be fertile sources of putative prostate cancer biomarkers. However such lists of differentially expressed genes can be highly variable for multiple reasons. As such, looking at differential expression in the context of gene sets and pathways has been more robust. Using next-generation genome sequencing data from The Cancer Genome Atlas, differential gene expression between age- and stage- matched human prostate tumors and non-malignant samples was assessed and used to craft a pathway signature of prostate cancer. Up- and down-regulated genes were assigned to pathways composed of curated groups of related genes from multiple databases. The significance of these pathways was then evaluated according to the number of differentially expressed genes found in the pathway and their position within the pathway using Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis. The "transforming growth factor-beta signaling" and "Ran regulation of mitotic spindle formation" pathways were strongly associated with prostate cancer. Several other significant pathways confirm reported findings from microarray data that suggest actin cytoskeleton regulation, cell cycle, mitogen-activated protein kinase signaling, and calcium signaling are also altered in prostate cancer. Thus we have demonstrated feasibility of pathway analysis and identified an underexplored area (Ran for investigation in prostate cancer pathogenesis.

  8. Variation suggestive of horizontal gene transfer at a lipopolysaccharide (lps) biosynthetic locus in Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen of rice

    Science.gov (United States)

    Patil, Prabhu B; Sonti, Ramesh V

    2004-01-01

    Background In animal pathogenic bacteria, horizontal gene transfer events (HGT) have been frequently observed in genomic regions that encode functions involved in biosynthesis of the outer membrane located lipopolysaccharide (LPS). As a result, different strains of the same pathogen can have substantially different lps biosynthetic gene clusters. Since LPS is highly antigenic, the variation at lps loci is attributed to be of advantage in evading the host immune system. Although LPS has been suggested as a potentiator of plant defense responses, interstrain variation at lps biosynthetic gene clusters has not been reported for any plant pathogenic bacterium. Results We report here the complete sequence of a 12.2 kb virulence locus of Xanthomonas oryzae pv. oryzae (Xoo) encoding six genes whose products are homologous to functions involved in LPS biosynthesis and transport. All six open reading frames (ORFs) have atypical G+C content and altered codon usage, which are the hallmarks of genomic islands that are acquired by horizontal gene transfer. The lps locus is flanked by highly conserved genes, metB and etfA, respectively encoding cystathionine gamma lyase and electron transport flavoprotein. Interestingly, two different sets of lps genes are present at this locus in the plant pathogens, Xanthomonas campestris pv. campestris (Xcc) and Xanthomonas axonopodis pv. citri (Xac). The genomic island is present in a number of Xoo strains from India and other Asian countries but is not present in two strains, one from India (BXO8) and another from Nepal (Nepal624) as well as the closely related rice pathogen, Xanthomonas oryzae pv. oryzicola (Xoor). TAIL-PCR analysis indicates that sequences related to Xac are present at the lps locus in both BXO8 and Nepal624. The Xoor strain has a hybrid lps gene cluster, with sequences at the metB and etfA ends, being most closely related to sequences from Xac and the tomato pathogen, Pseudomonas syringae pv. tomato respectively

  9. Enhanced C30 carotenoid production in Bacillus subtilis by systematic overexpression of MEP pathway genes

    NARCIS (Netherlands)

    Xue, Dan; Abdallah, Ingy I.; de Haan, Ilse E.M.; Sibbald, Mark J.J.B.; Quax, Wim J.

    2015-01-01

    Creating novel biosynthetic pathways and modulating the synthesis of important compounds are one of the hallmarks of synthetic biology. Understanding the key parameters controlling the flux of chemicals throughout a metabolic pathway is one of the challenges ahead. Isoprenoids are the most functiona

  10. De Novo Assembly and Genome Analyses of the Marine-Derived Scopulariopsis brevicaulis Strain LF580 Unravels Life-Style Traits and Anticancerous Scopularide Biosynthetic Gene Cluster

    Science.gov (United States)

    Kumar, Abhishek; Henrissat, Bernard; Arvas, Mikko; Syed, Muhammad Fahad; Thieme, Nils; Benz, J. Philipp; Sørensen, Jens Laurids; Record, Eric; Pöggeler, Stefanie; Kempken, Frank

    2015-01-01

    The marine-derived Scopulariopsis brevicaulis strain LF580 produces scopularides A and B, which have anticancerous properties. We carried out genome sequencing using three next-generation DNA sequencing methods. De novo hybrid assembly yielded 621 scaffolds with a total size of 32.2 Mb and 16298 putative gene models. We identified a large non-ribosomal peptide synthetase gene (nrps1) and supporting pks2 gene in the same biosynthetic gene cluster. This cluster and the genes within the cluster are functionally active as confirmed by RNA-Seq. Characterization of carbohydrate-active enzymes and major facilitator superfamily (MFS)-type transporters lead to postulate S. brevicaulis originated from a soil fungus, which came into contact with the marine sponge Tethya aurantium. This marine sponge seems to provide shelter to this fungus and micro-environment suitable for its survival in the ocean. This study also builds the platform for further investigations of the role of life-style and secondary metabolites from S. brevicaulis. PMID:26505484

  11. Direct cloning and heterologous expression of the salinomycin biosynthetic gene cluster from Streptomyces albus DSM41398 in Streptomyces coelicolor A3(2).

    Science.gov (United States)

    Yin, Jia; Hoffmann, Michael; Bian, Xiaoying; Tu, Qiang; Yan, Fu; Xia, Liqiu; Ding, Xuezhi; Stewart, A Francis; Müller, Rolf; Fu, Jun; Zhang, Youming

    2015-10-13

    Linear plus linear homologous recombination-mediated recombineering (LLHR) is ideal for obtaining natural product biosynthetic gene clusters from pre-digested bacterial genomic DNA in one or two steps of recombineering. The natural product salinomycin has a potent and selective activity against cancer stem cells and is therefore a potential anti-cancer drug. Herein, we separately isolated three fragments of the salinomycin gene cluster (salO-orf18) from Streptomyces albus (S. albus) DSM41398 using LLHR and assembled them into intact gene cluster (106 kb) by Red/ET and expressed it in the heterologous host Streptomyces coelicolor (S. coelicolor) A3(2). We are the first to report a large genomic region from a Gram-positive strain has been cloned using LLHR. The successful reconstitution and heterologous expression of the salinomycin gene cluster offer an attractive system for studying the function of the individual genes and identifying novel and potential analogues of complex natural products in the recipient strain.

  12. De Novo Assembly and Genome Analyses of the Marine-Derived Scopulariopsis brevicaulis Strain LF580 Unravels Life-Style Traits and Anticancerous Scopularide Biosynthetic Gene Cluster.

    Science.gov (United States)

    Kumar, Abhishek; Henrissat, Bernard; Arvas, Mikko; Syed, Muhammad Fahad; Thieme, Nils; Benz, J Philipp; Sørensen, Jens Laurids; Record, Eric; Pöggeler, Stefanie; Kempken, Frank

    2015-01-01

    The marine-derived Scopulariopsis brevicaulis strain LF580 produces scopularides A and B, which have anticancerous properties. We carried out genome sequencing using three next-generation DNA sequencing methods. De novo hybrid assembly yielded 621 scaffolds with a total size of 32.2 Mb and 16298 putative gene models. We identified a large non-ribosomal peptide synthetase gene (nrps1) and supporting pks2 gene in the same biosynthetic gene cluster. This cluster and the genes within the cluster are functionally active as confirmed by RNA-Seq. Characterization of carbohydrate-active enzymes and major facilitator superfamily (MFS)-type transporters lead to postulate S. brevicaulis originated from a soil fungus, which came into contact with the marine sponge Tethya aurantium. This marine sponge seems to provide shelter to this fungus and micro-environment suitable for its survival in the ocean. This study also builds the platform for further investigations of the role of life-style and secondary metabolites from S. brevicaulis.

  13. The endometrial cancer cell lines Ishikawa and HEC-1A, and the control cell line HIEEC, differ in expression of estrogen biosynthetic and metabolic genes, and in androstenedione and estrone-sulfate metabolism.

    Science.gov (United States)

    Hevir-Kene, Neli; Rižner, Tea Lanišnik

    2015-06-05

    Estrogens have important roles in the pathogenesis of endometrial cancer. They can have carcinogenic effects through stimulation of cell proliferation or formation of DNA-damaging species. To characterize model cell lines of endometrial cancer, we determined the expression profiles of the estrogen receptors (ERs) ESR1, ESR2 and GPER, and 23 estrogen biosynthetic and metabolic genes, and investigated estrogen biosynthesis in the control HIEEC cell line and the Ishikawa and HEC-1A EC cell lines. HIEEC and Ishikawa expressed all ERs to different extents, while HEC-1A cells lacked expression of ESR1. Considering the estrogen biosynthetic and metabolic enzymes, these cells showed statistically significant different gene expression profiles for SULT2B1, HSD3B2, CYP19A1, AKR1C3, HSD17B1, HSD17B7, HSD17B12, CYP1B1, CYP3A5, COMT, SULT1A1, GSTP1 and NQO2. In these cells, E2 was formed from E1S and E1, while androstenedione was not converted to estrogens. HIEEC and Ishikawa had similar profiles of androstenedione and E1 metabolism, but hydrolysis of E1S to E1 was weaker in Ishikawa cells. HEC-1A cells were less efficient for activation of E1 into the potent E2, but metabolized androstenedione to other androgenic metabolites better than HIEEC and Ishikawa cells. This study reveals that HIEEC, Ishikawa, and HEC-1A cells can all form estrogens only via the sulfatase pathway. HIEEC, Ishikawa, and HEC-1A cells expressed all the major genes in the production of hydroxyestrogens and estrogen quinones, and in their conjugation. Significantly higher CYP1B1 mRNA levels in Ishikawa cells compared to HEC-1A cells, together with lack of UGT2B7 expression, indicate that Ishikawa cells can accumulate more toxic estrogen-3,4-quinones than HEC-1A cells, as also for HIEEC cells. This study provides further characterization of HIEEC, Ishikawa, and HEC-1A cells, and shows that they differ greatly in expression of the genes investigated and in their capacity for E2 formation, and thus they

  14. The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence.

    Science.gov (United States)

    Hissen, Anna H T; Wan, Adrian N C; Warwas, Mark L; Pinto, Linda J; Moore, Margo M

    2005-09-01

    Aspergillus fumigatus is the leading cause of invasive mold infection and is a serious problem in immunocompromised populations worldwide. We have previously shown that survival of A. fumigatus in serum may be related to secretion of siderophores. In this study, we identified and characterized the sidA gene of A. fumigatus, which encodes l-ornithine N(5)-oxygenase, the first committed step in hydroxamate siderophore biosynthesis. A. fumigatus sidA codes for a protein of 501 amino acids with significant homology to other fungal l-ornithine N(5)-oxygenases. A stable DeltasidA strain was created by deletion of A. fumigatus sidA. This strain was unable to synthesize the siderophores N',N",N'''-triacetylfusarinine C (TAF) and ferricrocin. Growth of the DeltasidA strain was the same as that of the wild type in rich media; however, the DeltasidA strain was unable to grow in low-iron defined media or media containing 10% human serum unless supplemented with TAF or ferricrocin. No significant differences in ferric reduction activities were observed between the parental strain and the DeltasidA strain, indicating that blocking siderophore secretion did not result in upregulation of this pathway. Unlike the parental strain, the DeltasidA strain was unable to remove iron from human transferrin. A rescued strain (DeltasidA + sidA) was constructed; it produced siderophores and had the same growth as the wild type on iron-limited media. Unlike the wild-type and rescued strains, the DeltasidA strain was avirulent in a mouse model of invasive aspergillosis, indicating that sidA is necessary for A. fumigatus virulence.

  15. Carotenoid profiling and biosynthetic gene expression in flesh and peel of wild-type and hp-1 tomato fruit under UV-B depletion.

    Science.gov (United States)

    Lazzeri, Valerio; Calvenzani, Valentina; Petroni, Katia; Tonelli, Chiara; Castagna, Antonella; Ranieri, Annamaria

    2012-05-16

    Although light is recognized as one of the main factors influencing fruit carotenogenesis, the specific role of UV-B radiation has been poorly investigated. The present work is addressed to assess the molecular events underlying carotenoid accumulation in presence or absence of ultraviolet-B (UV-B) light in tomato fruits of wild-type and high pigment-1 (hp-1), a mutant characterized by exaggerated photoresponsiveness and increased fruit pigmentation. Gene expression analyses indicated that in wild-type fruits UV-B radiation mainly negatively affects the carotenoid biosynthetic genes encoding enzymes downstream of lycopene both in flesh and peel, suggesting that the down-regulation of genes CrtL-b and CrtL-e and the subsequent accumulation of lycopene during tomato ripening are determined at least in part by UV-B light. In contrast to wild-type, UV-B depletion did not greatly affect carotenoid accumulation in hp-1 and generally determined minor differences in gene expression between control and UV-B-depleted conditions.

  16. Conservation of Male Sterility 2 function during spore and pollen wall development supports an evolutionarily early recruitment of a core component in the sporopollenin biosynthetic pathway.

    Science.gov (United States)

    Wallace, Simon; Chater, Caspar C; Kamisugi, Yasuko; Cuming, Andrew C; Wellman, Charles H; Beerling, David J; Fleming, Andrew J

    2015-01-01

    The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.

  17. 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.

  18. Reprogramming Hansenula polymorpha for penicillin production : expression of the Penicillium chrysogenum pcl gene

    NARCIS (Netherlands)

    Gidijala, Loknath; van der Klei, Ida J.; Veenhuis, Marten; Kiel, Jan A. K. W.

    2007-01-01

    We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host-vector system based on

  19. Ligand-dependent regulation of the activity of the orphan nuclear receptor, small heterodimer partner (SHP), in the repression of bile acid biosynthetic CYP7A1 and CYP8B1 genes.

    Science.gov (United States)

    Miao, Ji; Choi, Sung-E; Seok, Sun Mi; Yang, Linda; Zuercher, William J; Xu, Yong; Willson, Timothy M; Xu, H Eric; Kemper, Jongsook Kim

    2011-07-01

    Small heterodimer partner (SHP) plays important roles in diverse biological processes by directly interacting with transcription factors and inhibiting their activities. SHP has been designated an orphan nuclear receptor, but whether its activity can be modulated by ligands has been a long-standing question. Recently, retinoid-related molecules, including 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3Cl-AHPC), were shown to bind to SHP and enhance apoptosis. We have examined whether 3Cl-AHPC acts as an agonist and increases SHP activity in the repression of bile acid biosynthetic CYP7A1 and CYP8B1 genes and delineated the underlying mechanisms. Contrary to this expectation, micromolar concentrations of 3Cl-AHPC increased CYP7A1 expression but indirectly via p38 kinase signaling. Nanomolar concentrations, however, repressed CYP7A1 expression and decreased bile acid levels in HepG2 cells, and little repression was observed when SHP was down-regulated by small hairpin RNA. Mechanistic studies revealed that 3Cl-AHPC bound to SHP, increased the interaction of SHP with liver receptor homologue (LRH)-1, a hepatic activator for CYP7A1 and CYP8B1 genes, and with repressive cofactors, Brahma, mammalian Sin3a, and histone deacetylase-1, and, subsequently, increased the occupancy of SHP and these cofactors at the promoters. Mutation of Leu-100, predicted to contact 3Cl-AHPC within the SHP ligand binding pocket by molecular modeling, severely impaired the increased interaction with LRH-1, and repression of LRH-1 activity mediated by 3Cl-AHPC. 3Cl-AHPC repressed SHP metabolic target genes in a gene-specific manner in human primary hepatocytes and HepG2 cells. These data suggest that SHP may act as a ligand-regulated receptor in metabolic pathways. Modulation of SHP activity by synthetic ligands may be a useful therapeutic strategy.

  20. Inference of gene pathways using mixture Bayesian networks

    Directory of Open Access Journals (Sweden)

    Ko Younhee

    2009-05-01

    Full Text Available Abstract Background Inference of gene networks typically relies on measurements across a wide range of conditions or treatments. Although one network structure is predicted, the relationship between genes could vary across conditions. A comprehensive approach to infer general and condition-dependent gene networks was evaluated. This approach integrated Bayesian network and Gaussian mixture models to describe continuous microarray gene expression measurements, and three gene networks were predicted. Results The first reconstructions of a circadian rhythm pathway in honey bees and an adherens junction pathway in mouse embryos were obtained. In addition, general and condition-specific gene relationships, some unexpected, were detected in these two pathways and in a yeast cell-cycle pathway. The mixture Bayesian network approach identified all (honey bee circadian rhythm and mouse adherens junction pathways or the vast majority (yeast cell-cycle pathway of the gene relationships reported in empirical studies. Findings across the three pathways and data sets indicate that the mixture Bayesian network approach is well-suited to infer gene pathways based on microarray data. Furthermore, the interpretation of model estimates provided a broader understanding of the relationships between genes. The mixture models offered a comprehensive description of the relationships among genes in complex biological processes or across a wide range of conditions. The mixture parameter estimates and corresponding odds that the gene network inferred for a sample pertained to each mixture component allowed the uncovering of both general and condition-dependent gene relationships and patterns of expression. Conclusion This study demonstrated the two main benefits of learning gene pathways using mixture Bayesian networks. First, the identification of the optimal number of mixture components supported by the data offered a robust approach to infer gene relationships and

  1. Identification and Characterization of Multiple Intermediate Alleles of the Key Genes Regulating Brassinosteroid Biosynthesis Pathways

    Science.gov (United States)

    Du, Junbo; Zhao, Baolin; Sun, Xin; Sun, Mengyuan; Zhang, Dongzhi; Zhang, Shasha; Yang, Wenyu

    2017-01-01

    Most of the early identified brassinosteroid signaling and biosynthetic mutants are null mutants, exhibiting extremely dwarfed phenotypes and male sterility. These null mutants are usually unable to be directly transformed via a routinely used Agrobacterium-mediated gene transformation system and therefore are less useful for genetic characterization of the brassinosteroid (BR)-related pathways. Identification of intermediate signaling mutants such as bri1–5 and bri1–9 has contributed drastically to the elucidation of BR signaling pathway using both genetic and biochemical approaches. However, intermediate mutants of key genes regulating BR biosynthesis have seldom been reported. Here we report identification of several intermediate BR biosynthesis mutants mainly resulted from leaky transcriptions due to the insertions of T-DNAs in the introns. These mutants are semi-dwarfed and fertile and capable to be transformed. These intermediate mutants could be useful tools for future discovery and analyses of novel components regulating BR biosynthesis and catabolism via genetic modifier screen. PMID:28138331

  2. Text mining in cancer gene and pathway prioritization.

    Science.gov (United States)

    Luo, Yuan; Riedlinger, Gregory; Szolovits, Peter

    2014-01-01

    Prioritization of cancer implicated genes has received growing attention as an effective way to reduce wet lab cost by computational analysis that ranks candidate genes according to the likelihood that experimental verifications will succeed. A multitude of gene prioritization tools have been developed, each integrating different data sources covering gene sequences, differential expressions, function annotations, gene regulations, protein domains, protein interactions, and pathways. This review places existing gene prioritization tools against the backdrop of an integrative Omic hierarchy view toward cancer and focuses on the analysis of their text mining components. We explain the relatively slow progress of text mining in gene prioritization, identify several challenges to current text mining methods, and highlight a few directions where more effective text mining algorithms may improve the overall prioritization task and where prioritizing the pathways may be more desirable than prioritizing only genes.

  3. Sterol Composition and Biosynthetic Genes of Vitrella brassicaformis, a Recently Discovered Chromerid: Comparison to Chromera velia and Phylogenetic Relationship with Apicomplexan Parasites.

    Science.gov (United States)

    Khadka, Manoj; Salem, Mohamed; Leblond, Jeffrey D

    2015-01-01

    Vitrella brassicaformis is the second discovered species in the Chromerida, and first in the family Vitrellaceae. Chromera velia, the first discovered species, forms an independent photosynthetic lineage with V. brassicaformis, and both are closely related to peridinin-containing dinoflagellates and nonphotosynthetic apicomplexans; both also show phylogenetic closeness with red algal plastids. We have utilized gas chromatography/mass spectrometry to identify two free sterols, 24-ethylcholest-5-en-3β-ol, and a minor unknown sterol which appeared to be a C(28:4) compound. We have also used RNA Seq analysis to identify seven genes found in the nonmevalonate/methylerythritol pathway (MEP) for sterol biosynthesis. Subsequent genome analysis of V. brassicaformis showed the presence of two mevalonate (MVA) pathway genes, though the genes were not observed in the transcriptome analysis. Transcripts from four genes (dxr, ispf, ispd, and idi) were selected and translated into proteins to study the phylogenetic relationship of sterol biosynthesis in V. brassicaformis and C. velia to other groups of algae and apicomplexans. On the basis of our genomic and transcriptomic analyses, we hypothesize that the MEP pathway was the primary pathway that apicomplexans used for sterol biosynthesis before they lost their sterol biosynthesis ability, although contribution of the MVA pathway cannot be discounted.

  4. Polymorphisms in monolignol biosynthetic genes are associated with biomass yield and agronomic traits in European maize (Zea mays L.)

    DEFF Research Database (Denmark)

    Chen, Yongsheng; Zein, Imad; Brenner, Everton A;

    2010-01-01

    Background Reduced lignin content leads to higher cell wall digestibility and, therefore, better forage quality and increased conversion of lignocellulosic biomass into ethanol. However, reduced lignin content might lead to weaker stalks, lodging, and reduced biomass yield. Genes encoding enzymes...

  5. Expression Profiling of Glucosinolate Biosynthetic Genes in Brassica oleracea L. var. capitata Inbred Lines Reveals Their Association with Glucosinolate Content

    Directory of Open Access Journals (Sweden)

    Arif Hasan Khan Robin

    2016-06-01

    Full Text Available Glucosinolates are the biochemical compounds that provide defense to plants against pathogens and herbivores. In this study, the relative expression level of 48 glucosinolate biosynthesis genes was explored in four morphologically-different cabbage inbred lines by qPCR analysis. The content of aliphatic and indolic glucosinolate molecules present in those cabbage lines was also estimated by HPLC analysis. The possible association between glucosinolate accumulation and related gene expression level was explored by principal component analysis (PCA. The genotype-dependent variation in the relative expression level of different aliphatic and indolic glucosinolate biosynthesis genes is the novel result of this study. A total of eight different types of glucosinolates, including five aliphatic and three indolic glucosinolates, was detected in four cabbage lines. Three inbred lines BN3383, BN4059 and BN4072 had no glucoraphanin, sinigrin and gluconapin detected, but the inbred line BN3273 had these three aliphatic glucosinolate compounds. PCA revealed that a higher expression level of ST5b genes and lower expression of GSL-OH was associated with the accumulation of these three aliphatic glucosinolate compounds. PCA further revealed that comparatively higher accumulation of neoglucobrassicin in the inbred line, BN4072, was associated with a high level of expression of MYB34 (Bol017062 and CYP81F1 genes. The Dof1 and IQD1 genes probably trans-activated the genes related to biosynthesis of glucoerucin and methoxyglucobrassicin for their comparatively higher accumulation in the BN4059 and BN4072 lines compared to the other two lines, BN3273 and BN3383. A comparatively higher progoitrin level in BN3273 was probably associated with the higher expression level of the GSL-OH gene. The cabbage inbred line BN3383 accounted for the significantly higher relative expression level for the 12 genes out of 48, but this line had comparatively lower total

  6. Two genes, rif15 and rif16, of the rifamycin biosynthetic gene cluster in Amycolatopsis mediterranei likely encode a transketolase and a P450 monooxygenase,respectively, both essential for the conversion of rifamycin SV into B

    Institute of Scientific and Technical Information of China (English)

    Hua Yuan; Wei Zhao; Yi Zhong; Jin Wang; Zhongiun Qin; Xiaoming Ding; Guo-Ping Zhao

    2011-01-01

    Amycolatopsis mediterranei produces an important antibiotic rifamycin,the biosynthesis of which involves many unusual modifications.Previous work suggested a putative P450 enzyme encoded by rif16 within the rifamycin biosynthetic gene cluster (rif) was required for the conversion of the intermediate rifamycin SV into the end product rifamycin B.In this study,we genetically proved that a putative transketolase encoded by rif15 is another essential enzyme for this conversion.Expression of merely rif15 and rif16 in a rif cluster null mutant ofA.mediterranei U32 was able to convert rifamycin SV into B.However,this Rifl5- and Rifl6-mediated conversion was only detected in intact cells of A.meidterranei,but not in Streptomyce coelicolor or Mycobacterium smegmatis,suggesting that yet-characterized gene(s) in A.mediterranei other than those encoded by the rif cluster should be involved in this process.

  7. Selection for phase variation of LOS biosynthetic genes frequently occurs in progression of non-typeable Haemophilus influenzae infection from the nasopharynx to the middle ear of human patients.

    Directory of Open Access Journals (Sweden)

    Kate L Fox

    Full Text Available Surface structures in Haemophilus influenzae are subject to rapid ON/OFF switching of expression, a process termed phase variation. We analyse tetranucleotide repeats controlling phase variation in lipo-oligosaccharide (LOS genes of H. influenzae in paired isolates from both the nasopharynx and middle ears of paediatric patients with chronic or recurrent otitis media. A change in expression of at least one of the seven phase variable LOS biosynthesis genes was seen in 12 of the 21 strain pairs. Several strains showed switching of expression in multiple LOS genes, consistent with a key role for phase variable LOS biosynthetic genes in human infection.

  8. CLUSEAN: a computer-based framework for the automated analysis of bacterial secondary metabolite biosynthetic gene clusters.

    Science.gov (United States)

    Weber, T; Rausch, C; Lopez, P; Hoof, I; Gaykova, V; Huson, D H; Wohlleben, W

    2009-03-10

    Bacterial secondary metabolites are an important source of antimicrobial and cytostatic drugs. These molecules are often synthesized in a stepwise fashion by multimodular megaenzymes that are encoded in clusters of genes encoding enzymes for precursor supply and modification. In this work,we present an open source software pipeline, CLUSEAN (CLUster SEquence ANalyzer) that helps to annotate and analyze such gene clusters. CLUSEAN integrates standard analysis tools, like BLAST and HMMer, with specific tools for the identification of the functional domains and motifs in nonribosomal peptide synthetases (NRPS)/type I polyketide synthases (PKS) and the prediction of specificities of NRPS.

  9. The influence of salt (NaCl) on ochratoxin A biosynthetic genes, growth and ochratoxin A production by three strains of Penicillium nordicum on a dry-cured ham-based medium.

    Science.gov (United States)

    Rodríguez, Alicia; Medina, Ángel; Córdoba, Juan J; Magan, Naresh

    2014-05-16

    Iberian dry-cured ham is colonised by moulds during the ripening process. The environmental conditions occurring during the process including the salt content predisposes the surface to colonisation by Penicillium species, including Penicillium nordicum which can contaminate the curing ham with ochratoxin A (OTA). The objective of this study was to examine the effect of NaCl (10% and 22%=0.94 and 0.87 water activity (aw)) on the activation of two genes involved in the biosynthetic pathway for OTA production, otapksPN and otanpsPN, relative growth and phenotypic OTA production by three strains of P. nordicum (CBS 110.769, FHSCC1 and FHSCC2) on a ham-based medium over a period of 12days at 25°C. Growth of the three strains was faster at 0.87 than 0.94 aw on the ham-based media. However, some intra- and inter-strain differences were observed. Of the three strains, only two (CBS 110.789; FHSCC2) were able to express the two genes involved in the biosynthesis of OTA in the two salt treatments. RT-qPCR showed that the temporal expression of the two genes (otapksPN and otanpsPN) was relatively similar for the wild type strain (FHSCC2) at both 0.94 and 0.87 aw over the 12day period. However, in the type strain (CBS 110.769) expression increased rapidly at 0.94 aw but was significantly lower at 0.87 aw. Expression of these two genes occurred after 3day incubation, while phenotypic OTA production was observed only after 6days in the two toxigenic strains. The other strain did not produce any OTA. The OTA concentrations confirmed the results observed with the molecular tools. This suggests that the RT-qPCR gene expression of these two genes may be a good early indicator of potential contamination of dry-cured ham with OTA during dry-cured ham ripening.

  10. Phase variable O antigen biosynthetic genes control expression of the major protective antigen and bacteriophage receptor in Vibrio cholerae O1.

    Directory of Open Access Journals (Sweden)

    Kimberley D Seed

    2012-09-01

    Full Text Available The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage.

  11. Characterization of the LP28 strain-specific exopolysaccharide biosynthetic gene cluster found in the whole circular genome of Pediococcus pentosaceus

    Directory of Open Access Journals (Sweden)

    Tetsuya Yasutake

    2016-03-01

    As a first step to deduce the probiotic function of the EPS together with the biosynthesis, we determined the whole genome sequence of the LP28 strain, demonstrating that the genome is a circular DNA, which is composed of 1,774,865 bp (1683 ORFs with a GC content of 37.1%. We also found that the LP28 strain harbors a plasmid carrying 6 ORFs composed of 5366 bp with a GC content of 36.5%. By comparing all of the genome sequences among the LP28 strain and four strains of P. pentosaceus reported previously, we found that 53 proteins in the LP28 strain display a similarity of less than 50% with those in the four P. pentosaceus strains. Significantly, 4 of the 53 proteins, which may be enzymes necessary for the EPS production on the LP28 strain, were absent in the other four P. pentosaceus strains and displayed less than 50% similarity with other LAB species. The EPS-biosynthetic gene cluster detected only in the LP28 genome consisted of 12 ORFs containing a priming enzyme, five glycosyltransferases, and a putative polysaccharide pyruvyltransferase.

  12. Phylogenetic Origin and Diversification of RNAi Pathway Genes in Insects.

    Science.gov (United States)

    Dowling, Daniel; Pauli, Thomas; Donath, Alexander; Meusemann, Karen; Podsiadlowski, Lars; Petersen, Malte; Peters, Ralph S; Mayer, Christoph; Liu, Shanlin; Zhou, Xin; Misof, Bernhard; Niehuis, Oliver

    2017-01-06

    RNA interference (RNAi) refers to the set of molecular processes found in eukaryotic organisms in which small RNA molecules mediate the silencing or down-regulation of target genes. In insects, RNAi serves a number of functions, including regulation of endogenous genes, anti-viral defense, and defense against transposable elements. Despite being well studied in model organisms, such as Drosophila, the distribution of core RNAi pathway genes and their evolution in insects is not well understood. Here we present the most comprehensive overview of the distribution and diversity of core RNAi pathway genes across 100 insect species, encompassing all currently recognized insect orders. We inferred the phylogenetic origin of insect-specific RNAi pathway genes and also identified several hitherto unrecorded gene expansions using whole-body transcriptome data from the international 1KITE (1000 Insect Transcriptome Evolution) project as well as other resources such as i5K (5000 Insect Genome Project). Specifically, we traced the origin of the double stranded RNA binding protein R2D2 to the last common ancestor of winged insects (Pterygota), the loss of Sid-1/Tag-130 orthologs in Antliophora (fleas, flies and relatives, and scorpionflies in a broad sense), and confirm previous evidence for the splitting of the Argonaute proteins Aubergine and Piwi in Brachyceran flies (Diptera, Brachycera). Our study offers new reference points for future experimental research on RNAi-related pathway genes in insects.

  13. Distribution, structure and biosynthetic gene families of (1,3;1,4)-β-glucan in Sorghum bicolor.

    Science.gov (United States)

    Ermawar, Riksfardini A; Collins, Helen M; Byrt, Caitlin S; Betts, Natalie S; Henderson, Marilyn; Shirley, Neil J; Schwerdt, Julian; Lahnstein, Jelle; Fincher, Geoffrey B; Burton, Rachel A

    2015-04-01

    In cereals, the presence of soluble polysaccharides including (1,3;1,4)-β-glucan has downstream implications for human health, animal feed and biofuel applications. Sorghum bicolor (L.) Moench is a versatile crop, but there are limited reports regarding the content of such soluble polysaccharides. Here, the amount of (1,3;1,4)-β-glucan present in sorghum tissues was measured using a Megazyme assay. Very low amounts were present in the grain, ranging from 0.16%-0.27% (w/w), while there was a greater quantity in vegetative tissues at 0.12-1.71% (w/w). The fine structure of (1,3;1,4)-β-glucan, as denoted by the ratio of cellotriosyl and cellotetraosyl residues, was assessed by high performance liquid chromatography (HPLC) and ranged from 2.6-3:1 in the grain, while ratios in vegetative tissues were lower at 2.1-2.6:1. The distribution of (1,3;1,4)-β-glucan was examined using a specific antibody and observed with fluorescence and transmission electron microscopy. Micrographs showed a variable distribution of (1,3;1,4)-β-glucan influenced by temporal and spatial factors. The sorghum orthologs of genes implicated in the synthesis of (1,3;1,4)-β-glucan in other cereals, such as the Cellulose synthase-like (Csl) F and H gene families were defined. Transcript profiling of these genes across sorghum tissues was carried out using real-time quantitative polymerase chain reaction, indicating that, as in other cereals, CslF6 transcripts dominated.

  14. Tissue-Specific Expression Analysis of Anthocyanin Biosynthetic Genes in White- and Red-Fleshed Grape Cultivars.

    Science.gov (United States)

    Xie, Sha; Song, Changzheng; Wang, Xingjie; Liu, Meiying; Zhang, Zhenwen; Xi, Zhumei

    2015-12-19

    Yan73, a teinturier (dyer) grape variety in China, is one of the few Vitis vinifera cultivars with red-coloured berry flesh. To examine the tissue-specific expression of genes associated with berry colour in Yan73, we analysed the differential accumulation of anthocyanins in the skin and flesh tissues of two red-skinned grape varieties with either red (Yan73) or white flesh (Muscat Hamburg) based on HPLC-MS analysis, as well as the differential expression of 18 anthocyanin biosynthesis genes in both varieties by quantitative RT-PCR. The results revealed that the transcripts of GST, OMT, AM3, CHS3, UFGT, MYBA1, F3'5'H, F3H1 and LDOX were barely detectable in the white flesh of Muscat Hamburg. In particular, GST, OMT, AM3, CHS3 and F3H1 showed approximately 50-fold downregulation in the white flesh of Muscat Hamburg compared to the red flesh of Yan73. A correlation analysis between the accumulation of different types of anthocyanins and gene expression indicated that the cumulative expression of GST, F3'5'H, LDOX and MYBA1 was more closely associated with the acylated anthocyanins and the 3'5'-OH anthocyanins, while OMT and AM3 were more closely associated with the total anthocyanins and methoxylated anthocyanins. Therefore, the transcripts of OMT, AM3, GST, F3'5'H, LDOX and MYBA1 explained most of the variation in the amount and composition of anthocyanins in skin and flesh of Yan73. The data suggest that the specific localization of anthocyanins in the flesh tissue of Yan73 is most likely due to the tissue-specific expression of OMT, AM3, GST, F3'5'H, LDOX and MYBA1 in the flesh.

  15. Modification of carotenoid levels by abscission agents and expression of carotenoid biosynthetic genes in 'valencia' sweet orange.

    Science.gov (United States)

    Alferez, Fernando; Pozo, Luis V; Rouseff, Russell R; Burns, Jacqueline K

    2013-03-27

    The effect of 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) and ethephon on peel color, flavedo carotenoid gene expression, and carotenoid accumulation was investigated in mature 'Valencia' orange ( Citrus sinensis L. Osbeck) fruit flavedo at three maturation stages. Abscission agent application altered peel color. CMNP was more effective than ethephon in promoting green-to-red (a) and blue-to-yellow (b) color at the middle and late maturation stages and total carotenoid changes at all maturation stages. Altered flow of carotenoid precursors during maturation due to abscission agents was suggested by changes in phytoene desaturase (Pds) and ζ-carotene desaturase (Zds) gene expression. However, each abscission agent affected downstream expression differentially. Ethephon application increased β-carotene hydroxilase (β-Chx) transcript accumulation 12-fold as maturation advanced from the early to middle and late stages. CMNP markedly increased β- and ε-lycopene cyclase (Lcy) transcript accumulation 45- and 15-fold, respectively, at midmaturation. Patterns of carotenoid accumulation in flavedo were supported in part by gene expression changes. CMNP caused greater accumulation of total flavedo carotenoids at all maturation stages when compared with ethephon or controls. In general, CMNP treatment increased total red carotenoids more than ethephon or the control but decreased total yellow carotenoids at each maturation stage. In control fruit flavedo, total red carotenoids increased and yellow carotenoids decreased as maturation progressed. Trends in total red carotenoids during maturation were consistent with measured a values. Changes in carotenoid accumulation and expression patterns in flavedo suggest that regulation of carotenoid accumulation is under transcriptional, translational, and post-translational control.

  16. The neomycin biosynthetic gene cluster of Streptomyces fradiae NCIMB 8233: genetic and biochemical evidence for the roles of two glycosyltransferases and a deacetylase.

    Science.gov (United States)

    Fan, Qingzhi; Huang, Fanglu; Leadlay, Peter F; Spencer, Jonathan B

    2008-09-21

    An efficient protocol has been developed for the genetic manipulation of Streptomyces fradiae NCIMB 8233, which produces the 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic neomycin. This has allowed the in vivo analysis of the respective roles of the glycosyltransferases Neo8 and Neo15, and of the deacetylase Neo16 in neomycin biosynthesis. Specific deletion of each of the neo8, neo15 and neo16 genes confirmed that they are all essential for neomycin biosynthesis. The pattern of metabolites produced by feeding putative pathway intermediates to these mutants provided unambiguous support for a scheme in which Neo8 and Neo15, whose three-dimensional structures are predicted to be highly similar, have distinct roles: Neo8 catalyses transfer of N-acetylglucosamine to 2-DOS early in the pathway, while Neo15 catalyses transfer of the same aminosugar to ribostamycin later in the pathway. The in vitro substrate specificity of Neo15, purified from recombinant Escherichia coli, was fully consistent with these findings. The in vitro activity of Neo16, the only deacetylase so far recognised in the neo gene cluster, showed that it is capable of acting in tandem with both Neo8 and Neo15 as previously proposed. However, the deacetylation of N-acetylglucosaminylribostamycin was still observed in a strain deleted of the neo16 gene and fed with suitable pathway precursors, providing evidence for the existence of a second enzyme in S. fradiae with this activity.

  17. Molecular Cloning, Expression Pattern and Genotypic Effects on Glucoraphanin Biosynthetic Related Genes in Chinese Kale (Brassica oleracea var. alboglabra Bailey).

    Science.gov (United States)

    Yin, Ling; Chen, Changming; Chen, Guoju; Cao, Bihao; Lei, Jianjun

    2015-11-11

    Glucoraphanin is a plant secondary metabolite that is involved in plant defense and imparts health-promoting properties to cruciferous vegetables. In this study, three genes involved in glucoraphanin metabolism, branched-chain aminotransferase 4 (BCAT4), methylthioalkylmalate synthase 1 (MAM1) and dihomomethionine N-hydroxylase (CYP79F1), were cloned from Chinese kale (Brassica oleracea var. alboglabra Bailey). Sequence homology and phylogenetic analysis identified these genes and confirmed the evolutionary status of Chinese kale. The transcript levels of BCAT4, MAM1 and CYP79F1 were higher in cotyledon, leaf and stem compared with flower and silique. BCAT4, MAM1 and CYP79F1 were expressed throughout leaf development with lower transcript levels during the younger stages. Glucoraphanin content varied extensively among different varieties, which ranged from 0.25 to 2.73 µmol·g(-1) DW (dry weight). Expression levels of BCAT4 and MAM1 were high at vegetative-reproductive transition phase, while CYP79F1 was expressed high at reproductive phase. BCAT4, MAM1 and CYP79F1 were expressed significantly high in genotypes with high glucoraphanin content. All the results provided a better understanding of the roles of BCAT4, MAM1 and CYP79F1 in the glucoraphanin biosynthesis of Chinese kale.

  18. Enhancement of poly-3-hydroxybutyrate production in Synechocystis sp. PCC 6803 by overexpression of its native biosynthetic genes.

    Science.gov (United States)

    Khetkorn, Wanthanee; Incharoensakdi, Aran; Lindblad, Peter; Jantaro, Saowarath

    2016-08-01

    Synechocystis sp. PCC 6803 strains overexpressing pha genes were constructed and characterized for poly-3-hydroxybutyrate (PHB) production. These pha overexpressing strains showed slightly reduced growth rates. Under N-deprived condition, the strains overexpressing (OE) phaAB, phaEC and phaABEC showed significantly higher PHB contents than the wild type. The maximum PHB content, a 2.6-fold increase producing 26% PHB (dcw), was observed in OE phaAB cells grown for 9days in N-deprived medium. Under this condition, these OE phaAB cells increased PHB production to 35% PHB (dcw) upon addition of 0.4% (w/v) acetate. Higher PHB granules in OE phaAB cells were clearly visualized by both Nile red staining and TEM imaging. All OE strains under N-deficient condition had increased glgX transcript levels. Overall results demonstrate an enhanced PHB production in Synechocystis cells overexpressing pha genes, particularly phaA and phaB, when grown in N-deprived medium containing 0.4% (w/v) acetate.

  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. Congenital erythropoietic porphyria: prolonged high-level expression and correction of the heme biosynthetic defect by retroviral-mediated gene transfer into porphyric and erythroid cells.

    Science.gov (United States)

    Kauppinen, R; Glass, I A; Aizencang, G; Astrin, K H; Atweh, G F; Desnick, R J

    1998-09-01

    Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder resulting from the deficient activity of the heme biosynthetic enzyme uroporphyrinogen III synthase (UROS). Severely affected patients are transfusion dependent and have mutilating cutaneous manifestations. Successful bone marrow transplantation has proven curative, providing the rationale for stem cell gene therapy. Toward this goal, two retroviral MFG vectors containing the UROS cDNA were constructed, one with the wild-type sequence (MFG-UROS-wt) and a second with an optimized Kozak consensus sequence (MFG-UROS-K). Following transduction of CEP fibroblasts, the MFG-UROS-wt and MFG-UROS-K vectors increased the endogenous activity without selection to levels that were 18- and 5-fold greater, respectively, than the mean activity in normal fibroblasts. Notably, the MFG-UROS-wt vector expressed UROS activity in CEP fibroblasts at these high levels for over 6 months without cell toxicity. Addition of either delta-aminolevulinic acid (ALA) or ferric chloride did not affect expression of the transduced UROS gene nor did the increased concentrations of uroporphyrin isomers or porphyrin intermediates affect cell viability. Similarly, transduction of CEP lymphoblasts with the MFG-UROS-wt vector without G418 selection increased the endogenous UROS activity by 7-fold or almost 2-fold greater than that in normal lymphoblasts. Transduction of K562 erythroleukemia cells by cocultivation with the MFG-UROS-wt producer cells increased their high endogenous UROS activity by 1.6-fold without selection. Clonally isolated K562 cells expressed UROS for over 4 months at mean levels 4.7-fold greater than the endogenous activity without cell toxicity. Thus, the prolonged, high-level expression of UROS in transduced CEP fibroblasts and lymphoblasts, as well as in transduced K562 erythroid cells, demonstrated that the enzymatic defect in CEP cells could be corrected by retroviral-mediated gene therapy without

  1. Metabolomic analysis and differential expression of anthocyanin biosynthetic genes in white- and red-flowered buckwheat cultivars (Fagopyrum esculentum).

    Science.gov (United States)

    Kim, Yeon Bok; Park, Soo-Yun; Thwe, Aye Aye; Seo, Jeong Min; Suzuki, Tastsuro; Kim, Sun-Ju; Kim, Jae Kwang; Park, Sang Un

    2013-11-01

    Red-flowered buckwheat ( Fagopyrum esculentum ) is used in the production of tea, juice, and alcohols after the detoxification of fagopyrin. In order to investigate the metabolomics and regulatory of anthocyanin production in red-flowered (Gan-Chao) and white-flowered (Tanno) buckwheat cultivars, quantitative real-time RT-PCR (qRT-PCR), gas chromatography time-of-flight mass spectrometry (GC-TOFMS), and high performance liquid chromatography (HPLC) were conducted. The transcriptions of FePAL, FeC4H, Fe4CL1, FeF3H, FeANS, and FeDFR increased gradually from flowering stage 1 and reached their highest peaks at flowering stage 3 in Gan-Chao flower. In total 44 metabolites, 18 amino acids, 15 organic acids, 7 sugars, 3 sugar alcohols, and 1 amine were detected in Gan-Chao flowers. Two anthocyanins, cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside, were identified in Gan-Chao cultivar. The first component of the partial least-squares to latent structures-discriminate analysis (PLS-DA) indicated that high amounts of phenolic, shikimic, and pyruvic acids were present in Gan-Chao. We suggest that transcriptions of genes involved in anthocyanin biosynthesis, anthocyanin contents, and metabolites have correlation in the red-flowered buckwheat Gan-Chao flowers. Our results may be helpful to understand anthocyanin biosynthesis in red-flowered buckwheat.

  2. Metabolic flux between unsaturated and saturated fatty acids is controlled by the FabA:FabB ratio in the fully reconstituted fatty acid biosynthetic pathway of Escherichia coli.

    Science.gov (United States)

    Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

    2013-11-19

    The entire fatty acid biosynthetic pathway of Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from 14 purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H to the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multienzyme system. At steady state, a maximal turnover rate of 0.5 s(-1) was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. Via changes in these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximal turnover rate of the pathway. Our reconstituted system provides a powerful tool for understanding and engineering rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.

  3. Design and synthesis of pathway genes for polyketide biosynthesis.

    Science.gov (United States)

    Peirú, Salvador; Gramajo, Hugo; Menzella, Hugo G

    2009-01-01

    In this chapter we describe novel methods for the design and assembly of synthetic pathways for the synthesis of polyketides and tailoring sugars. First, a generic design for type I polyketide synthase genes is presented that allows their facile assembly for the expression of chimeric enzymes in an engineered Escherichia coli host. The sequences of the synthetic genes are based on naturally occurring polyketide synthase genes but they are redesigned by custom-made software to optimize codon usage to maximize expression in E. coli and to provide a standard set of restriction sites to allow combinatorial assembly into unnatural enzymes. The methodology has been validated by building a large number of bimodular mini-PKSs that make easily assayed triketide products. Learning from the successful bimodules, a conceptual advance was made by assembling genes encoding functional trimodular enzymes, capable of making tetraketide products. Second, methods for the rapid assembly and exchange of sugar pathway genes into functional operons are described. The approach was validated by the assembly of the 15 genes for the synthesis of mycarose and desosamine in two operons, which yielded erythromycin C when coexpressed with the corresponding PKS genes. These methods are important enabling steps toward the goals of making designer drugs by polyketide synthase and sugar pathway engineering and, in the shorter term, producing by fermentation advanced intermediates for the synthesis of compounds that otherwise require large numbers of chemical steps.

  4. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

    Science.gov (United States)

    Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Gingras, Marie-Claude; Muthuswamy, Lakshmi B; Johns, Amber L; Miller, David K; Wilson, Peter J; Patch, Ann-Marie; Wu, Jianmin; Chang, David K; Cowley, Mark J; Gardiner, Brooke B; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J; Gill, Anthony J; Pinho, Andreia V; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R Scott; Humphris, Jeremy L; Kaplan, Warren; Jones, Marc D; Colvin, Emily K; Nagrial, Adnan M; Humphrey, Emily S; Chou, Angela; Chin, Venessa T; Chantrill, Lorraine A; Mawson, Amanda; Samra, Jaswinder S; Kench, James G; Lovell, Jessica A; Daly, Roger J; Merrett, Neil D; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M; Fisher, William E; Brunicardi, F Charles; Hodges, Sally E; Reid, Jeffrey G; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R; Dinh, Huyen; Buhay, Christian J; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E; Yung, Christina K; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A; Petersen, Gloria M; Gallinger, Steven; Hruban, Ralph H; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Schulick, Richard D; Wolfgang, Christopher L; Morgan, Richard A; Lawlor, Rita T; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A; Mann, Karen M; Jenkins, Nancy A; Perez-Mancera, Pedro A; Adams, David J; Largaespada, David A; Wessels, Lodewyk F A; Rust, Alistair G; Stein, Lincoln D; Tuveson, David A; Copeland, Neal G; Musgrove, Elizabeth A; Scarpa, Aldo; Eshleman, James R; Hudson, Thomas J; Sutherland, Robert L; Wheeler, David A; Pearson, John V; McPherson, John D; Gibbs, Richard A; Grimmond, Sean M

    2012-11-15

    Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

  5. Components of complex lipid biosynthetic pathways in developing castor (Ricinus communis) seeds identified by MudPIT analysis of enriched endoplasmic reticulum.

    Science.gov (United States)

    Brown, Adrian P; Kroon, Johan T M; Topping, Jennifer F; Robson, Joanne L; Simon, William J; Slabas, Antoni R

    2011-08-05

    Ricinoleic acid is a feedstock for nylon-11 (N11) synthesis which is currently obtained from castor (Ricinus communis) oil. Production of this fatty acid in a temperate oilseed crop is of great commercial interest, but the highest reported level in transgenic plant oils is 30%, below the 90% observed in castor and insufficient for commercial exploitation. To identify castor oil-biosynthetic enzymes and inform strategies to improve ricinoleic acid yields, we performed MudPIT analysis on endoplasmic reticulum (ER) purified from developing castor bean endosperm. Candidate enzymes for all steps of triacylglycerol synthesis were identified among 72 proteins in the data set related to complex-lipid metabolism. Previous reported proteomic data from oilseeds had not included any membrane-bound enzyme that might incorporate ricinoleic acid into oil. Analysis of enriched ER enabled determination of which protein isoforms for these enzymes were in developing castor seed. To complement this data, quantitative RT-PCR experiments with castor seed and leaf RNA were performed for orthologues of Arabidopsis oil-synthetic enzymes, determining which were highly expressed in the seed. These data provide important information for further manipulation of ricinoleic acid content in oilseeds and peptide data for future quantification strategies.

  6. Exploring two plant hosts for expression of diterpenoid pathway genes

    DEFF Research Database (Denmark)

    Bach, Søren Spanner

    by humanity in biopharmaceuticals or as industrial bioproducts. Yields and purity of diterpenoids purified from natural sources or made by chemical synthesis are generally insufficient for large-volume or high-end applications, thus alternative sources are needed. Synthetic biology, where heterologous pathways...... have been reconstructed in host production organisms is an attractive lternative, which holds the promise to enable a scalable, costeffective and table supply of natural products. Knowledge about the genes and mechanisms nvolved in the original pathway is a prerequisite for such heterologous production...... is compatible with native codon usage, and through the conserved mechanisms of protein targeting and posttranslational odifications, has the capacity to produce functional enzymes. To further explore plant based expression and characterization of diterpenoid pathway genes, two different plant expression hosts...

  7. Integrative analysis of RUNX1 downstream pathways and target genes

    Directory of Open Access Journals (Sweden)

    Liu Marjorie

    2008-07-01

    Full Text Available Abstract Background The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML. The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1 cell lines with RUNX1 mutations from FPD-AML patients, 2 over-expression of RUNX1 and CBFβ, and 3 Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease

  8. Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis.

    Science.gov (United States)

    Wang, Yaya; Deng, Zixin; Qu, Xudong

    2014-01-01

    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 ( K m 4153 μM, k cat 0.073 min (-1)) and SAM ( K m 416 μM, k cat 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.

  9. Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

    Institute of Scientific and Technical Information of China (English)

    Yan LIU; Hong WANG; He-Chun YE; Guo-Feng LI

    2005-01-01

    Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently.With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis path way are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosnthesis are discussed.

  10. Molecular pathways: targeting ETS gene fusions in cancer.

    Science.gov (United States)

    Feng, Felix Y; Brenner, J Chad; Hussain, Maha; Chinnaiyan, Arul M

    2014-09-01

    Rearrangements, or gene fusions, involving the ETS family of transcription factors are common driving events in both prostate cancer and Ewing sarcoma. These rearrangements result in pathogenic expression of the ETS genes and trigger activation of transcriptional programs enriched for invasion and other oncogenic features. Although ETS gene fusions represent intriguing therapeutic targets, transcription factors, such as those comprising the ETS family, have been notoriously difficult to target. Recently, preclinical studies have demonstrated an association between ETS gene fusions and components of the DNA damage response pathway, such as PARP1, the catalytic subunit of DNA protein kinase (DNAPK), and histone deactylase 1 (HDAC1), and have suggested that ETS fusions may confer sensitivity to inhibitors of these DNA repair proteins. In this review, we discuss the role of ETS fusions in cancer, the preclinical rationale for targeting ETS fusions with inhibitors of PARP1, DNAPK, and HDAC1, as well as ongoing clinical trials targeting ETS gene fusions.

  11. Evolutionary Origins of the Eukaryotic Shikimate Pathway: Gene Fusions, Horizontal Gene Transfer, and Endosymbiotic Replacements†

    OpenAIRE

    2006-01-01

    Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These ...

  12. 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...

  13. Biosynthetic inorganic chemistry.

    Science.gov (United States)

    Lu, Yi

    2006-08-25

    Inorganic chemistry and biology can benefit greatly from each other. Although synthetic and physical inorganic chemistry have been greatly successful in clarifying the role of metal ions in biological systems, the time may now be right to utilize biological systems to advance coordination chemistry. One such example is the use of small, stable, easy-to-make, and well-characterized proteins as ligands to synthesize novel inorganic compounds. This biosynthetic inorganic chemistry is possible thanks to a number of developments in biology. This review summarizes the progress in the synthesis of close models of complex metalloproteins, followed by a description of recent advances in using the approach for making novel compounds that are unprecedented in either inorganic chemistry or biology. The focus is mainly on synthetic "tricks" learned from biology, as well as novel structures and insights obtained. The advantages and disadvantages of this biosynthetic approach are discussed.

  14. Standardized Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica.

    Science.gov (United States)

    Schwartz, Cory; Shabbir-Hussain, Murtaza; Frogue, Keith; Blenner, Mark; Wheeldon, Ian

    2016-12-22

    The yeast Yarrowia lipolytica is a promising microbial host due to its native capacity to produce lipid-based chemicals. Engineering stable production strains requires genomic integration of modified genes, avoiding episomal expression that requires specialized media to maintain selective pressures. Here, we develop a CRISPR-Cas9-based tool for targeted, markerless gene integration into the Y. lipolytica genome. A set of genomic loci was screened to identify sites that were accepting of gene integrations without impacting cell growth. Five sites were found to meet these criteria. Expression levels from a GFP expression cassette were consistent when inserted into AXP, XPR2, A08, and D17, with reduced expression from MFE1. The standardized tool is comprised of five pairs of plasmids (one homologous donor plasmid and a CRISPR-Cas9 expression plasmid), with each pair targeting gene integration into one of the characterized sites. To demonstrate the utility of the tool we rapidly engineered a semisynthetic lycopene biosynthesis pathway by integrating four different genes at different loci. The capability to integrate multiple genes without the need for marker recovery and into sites with known expression levels will enable more rapid and reliable pathway engineering in Y. lipolytica.

  15. Hepatic gene expression profiling reveals key pathways involved in leptin-mediated weight loss in ob/ob mice.

    Directory of Open Access Journals (Sweden)

    Ashok Sharma

    Full Text Available BACKGROUND: Leptin, a cytokine-like protein, plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure. Leptin circulates in blood and acts on the brain, which sends downstream signals to regulate body weight. Leptin therapy has been successful in treating leptin deficient obese patients. However, high levels of leptin have been observed in more common forms of obesity indicating a state of leptin resistance which limits the application of leptin in the treatment of obesity. If the central effect of leptin could be by-passed and genes which respond to leptin treatment could be regulated directly, new therapeutic targets for the treatment of obesity may be possible. The purpose of this study was to identify genes and subsequent pathways correlated with leptin-mediated weight loss. METHODOLOGY/PRINCIPAL FINDINGS: WE UTILIZED MICROARRAY TECHNOLOGY TO COMPARE HEPATIC GENE EXPRESSION CHANGES AFTER TWO TYPES OF LEPTIN ADMINISTRATION: one involving a direct stimulatory effect when administered peripherally (subcutaneous: SQ and another that is indirect, involving a hypothalamic relay that suppresses food intake when leptin is administered centrally (intracerebroventricular: ICV. We identified 214 genes that correlate with leptin mediated weight loss. Several biological processes such as mitochondrial metabolic pathways, lipid metabolic and catabolic processes, lipid biosynthetic processes, carboxylic acid metabolic processes, iron ion binding and glutathione S-transferases were downregulated after leptin administration. In contrast, genes involved in the immune system inflammatory response and lysosomal activity were found to be upregulated. Among the cellular compartments mitochondrion (32 genes, endoplasmic reticulum (22 genes and vacuole (8 genes were significantly over represented. CONCLUSIONS/SIGNIFICANCE: In this study we have identified key molecular pathways and downstream

  16. Vitamin D metabolic pathway genes and pancreatic cancer risk.

    Directory of Open Access Journals (Sweden)

    Hannah Arem

    Full Text Available Evidence on the association between vitamin D status and pancreatic cancer risk is inconsistent. This inconsistency may be partially attributable to variation in vitamin D regulating genes. We selected 11 vitamin D-related genes (GC, DHCR7, CYP2R1, VDR, CYP27B1, CYP24A1, CYP27A1, RXRA, CRP2, CASR and CUBN totaling 213 single nucleotide polymorphisms (SNPs, and examined associations with pancreatic adenocarcinoma. Our study included 3,583 pancreatic cancer cases and 7,053 controls from the genome-wide association studies of pancreatic cancer PanScans-I-III. We used the Adaptive Joint Test and the Adaptive Rank Truncated Product statistic for pathway and gene analyses, and unconditional logistic regression for SNP analyses, adjusting for age, sex, study and population stratification. We examined effect modification by circulating vitamin D concentration (≤50, >50 nmol/L for the most significant SNPs using a subset of cohort cases (n = 713 and controls (n = 878. The vitamin D metabolic pathway was not associated with pancreatic cancer risk (p = 0.830. Of the individual genes, none were associated with pancreatic cancer risk at a significance level of p<0.05. SNPs near the VDR (rs2239186, LRP2 (rs4668123, CYP24A1 (rs2762932, GC (rs2282679, and CUBN (rs1810205 genes were the top SNPs associated with pancreatic cancer (p-values 0.008-0.037, but none were statistically significant after adjusting for multiple comparisons. Associations between these SNPs and pancreatic cancer were not modified by circulating concentrations of vitamin D. These findings do not support an association between vitamin D-related genes and pancreatic cancer risk. Future research should explore other pathways through which vitamin D status might be associated with pancreatic cancer risk.

  17. Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of the semidwarf ga4 and ga5 mutants

    Energy Technology Data Exchange (ETDEWEB)

    Talon, M. Zeevaart, J.A.D. (Michigan State Univ., East Lansing (USA)); Koornneef, M. (Agricultural Univ., (Netherlands))

    1990-10-01

    Twenty gibberellins (GAs) have been identified in extracts from shoots of the Landsberg erecta line of Arabidopsis thaliana by full-scan gas chromatography-mass spectrometry and Kovats retention indices. Eight of them are members of the early-13-hydroxylation pathway (GA{sub 53}, GA{sub 44}, GA{sub 19}, GA{sub 17}, GA{sub 20}, GA{sub 1}, GA{sub 29}, and GA{sub 8}), six are members of the early-3-hydroxylation pathway (GA{sub 37}, GA{sub 27}, GA{sub 36}, GA{sub 13}, GA{sub 4}, and GA{sub 34}), and the remaining six are members of the non-3,13-hydroxylation pathway (GA{sub 12}, GA{sub 15}, GA{sub 24}, GA{sub 25}, GA{sub 9}, and GFA{sub 51}). Seven of these GAs were quantified in the Landsberg erecta line of Arabidopsis and in the semidwarf ga4 and ga5 mutants by gas chromatography-selected ion monitoring (SIM) using internal standards. The relative levels of the remaining 13 GAs were compared by the use of ion intensities only. The growth-response data, as well as the accumulation of GA{sub 9} in the ga4 mutant, indicate that GA{sub 9} is not active in Arabidopsis, but it must be 3{beta}-hydroxytlated to GA{sub 4} to become bioactive. It is concluded that the reduced levels of the 3{beta}-hydroxy-GAs, GA{sub 1} and GA{sub 4}, are the cause of the semidwarf growth habit of both mutants.

  18. ATP citrate lyase activity is post-translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways.

    Science.gov (United States)

    Xing, Shufan; van Deenen, Nicole; Magliano, Pasqualina; Frahm, Lea; Forestier, Edith; Nawrath, Christiane; Schaller, Hubert; Gronover, Christian S; Prüfer, Dirk; Poirier, Yves

    2014-07-01

    Cytosolic acetyl-CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over-expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl-CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl-CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl-CoA to endogenous pathways. Increasing the ACL activity via the over-expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady-state mRNA or protein level, indicating a post-translational regulation of ACL activity in response to sink strength. Over-expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over-expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four- and two-fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over-expression of ACL also changed the amount of the cutin monomer octadecadien-1,18-dioic acid, revealing an unsuspected link between cytosolic acetyl-CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl-CoA, including wax and polyisoprenoids.

  19. Transcription of meiotic-like-pathway genes in Giardia intestinalis

    OpenAIRE

    2008-01-01

    The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasite's genome cast doubt on exclusively ...

  20. Overexpression of the Trichoderma brevicompactum tri5 Gene: Effect on the Expression of the Trichodermin Biosynthetic Genes and on Tomato Seedlings

    Directory of Open Access Journals (Sweden)

    Josefina Aleu

    2011-09-01

    Full Text Available Trichoderma brevicompactum IBT 40841 produces trichodermin, a trichothecene-type toxin that shares most of the steps of its biosynthesis with harzianum A, another trichothecene produced by several Trichoderma species. The first specific step in the trichothecene biosynthesis is carried out by a terpene cylcase, trichodiene synthase, that catalyzes the conversion of farnesyl pyrophosphate to trichodiene and that is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin production, but also in an increase in tyrosol and hydroxytyrosol production, two antioxidant compounds that may play a regulatory role in trichothecene biosynthesis, and also in a higher expression of three trichothecene genes, tri4, tri6 and tri10, and of the erg1 gene, which participates in the synthesis of triterpenes. The effect of tri5 overexpression on tomato seedling disease response was also studied.

  1. Gene Expression Profiling of Biological Pathway Alterations by Radiation Exposure

    Directory of Open Access Journals (Sweden)

    Kuei-Fang Lee

    2014-01-01

    Full Text Available Though damage caused by radiation has been the focus of rigorous research, the mechanisms through which radiation exerts harmful effects on cells are complex and not well-understood. In particular, the influence of low dose radiation exposure on the regulation of genes and pathways remains unclear. In an attempt to investigate the molecular alterations induced by varying doses of radiation, a genome-wide expression analysis was conducted. Peripheral blood mononuclear cells were collected from five participants and each sample was subjected to 0.5 Gy, 1 Gy, 2.5 Gy, and 5 Gy of cobalt 60 radiation, followed by array-based expression profiling. Gene set enrichment analysis indicated that the immune system and cancer development pathways appeared to be the major affected targets by radiation exposure. Therefore, 1 Gy radioactive exposure seemed to be a critical threshold dosage. In fact, after 1 Gy radiation exposure, expression levels of several genes including FADD, TNFRSF10B, TNFRSF8, TNFRSF10A, TNFSF10, TNFSF8, CASP1, and CASP4 that are associated with carcinogenesis and metabolic disorders showed significant alterations. Our results suggest that exposure to low-dose radiation may elicit changes in metabolic and immune pathways, potentially increasing the risk of immune dysfunctions and metabolic disorders.

  2. Transcription of meiotic-like-pathway genes in Giardia intestinalis

    Directory of Open Access Journals (Sweden)

    Sandra P Melo

    2008-06-01

    Full Text Available The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasite's genome cast doubt on exclusively asexual nature of Giardia. In this work, semiquantitative RT-PCR analysis was used to assess the transcription pattern of three meiosis-like-specific genes involved in homologues recombination: dmc1, hop1 and spo11. The mRNAs were amplified during the parasite's differentiation processes, encystation and excystation, and expression was found at each stage of its life cycle. A semiquantitative assessment also suggests that expression of some of the genes is regulated during encystation process.

  3. Transcription of meiotic-like-pathway genes in Giardia intestinalis.

    Science.gov (United States)

    Melo, Sandra P; Gómez, Vanessa; Castellanos, Isabel C; Alvarado, Magda E; Hernández, Paula C; Gallego, Amanda; Wasserman, Moisés

    2008-06-01

    The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasite's genome cast doubt on exclusively asexual nature of Giardia. In this work, semiquantitative RT-PCR analysis was used to assess the transcription pattern of three meiosis-like-specific genes involved in homologues recombination: dmc1, hop1 and spo11. The mRNAs were amplified during the parasite's differentiation processes, encystation and excystation, and expression was found at each stage of its life cycle. A semiquantitative assessment also suggests that expression of some of the genes is regulated during encystation process.

  4. Characterization of a silent sesquiterpenoid biosynthetic pathway in Streptomyces avermitilis controlling epi-isozizaene albaflavenone biosynthesis and isolation of a new oxidized epi-isozizaene metabolite.

    Science.gov (United States)

    Takamatsu, Satoshi; Lin, Xin; Nara, Ayako; Komatsu, Mamoru; Cane, David E; Ikeda, Haruo

    2011-03-01

    The genome-sequenced, Gram-positive bacterium Streptomyces avermitilis harbours an orthologue (SAV_3032) of the previously identified epi-isozizaene synthase (SCO5222) in Streptomyces coelicolor A3(2). The sav3032 is translationally coupled with the downstream sav3031 gene encoding the cytochrome P450 CYP170A2 analogous to SCO5223 (CYP170A1) of S. coelicolor A3(2), which exhibits a similar translation coupling. Streptomyces avermitilis did not produce epi-isozizaene or any of its oxidized derivatives, albaflavenols and albaflavenone, under in any culture conditions examined. Nonetheless, recombinant SAV_3032 protein expressed in Escherichia coli catalysed the Mg²+-dependent cyclization of farnesyl diphosphate to epi-isozizaene. To effect the production of epi-isozizaene in S. avermitilis, the sav3032 gene was cloned and placed under control of a copy of the native S. avermitilis promoter rpsJp (sav4925). The derived expression construct was introduced by transformation into a large-deletion mutant of S. avermitilis SUKA16 and the resulting transformants accumulated epi-isozizaene. The previously characterized oxidized epi-isozizaene metabolites (4R)- and (4S)-albaflavenols and albaflavenone, as well as a previously undescribed doubly oxidized epi-isozizaene derivative were isolated from cultures of S. avermitilis SUKA16 transformants in which sav3032 was coexpressed with the P450-encoding sav3031. This new metabolite was identified as 4β,5β-epoxy-2-epi-zizaan-6β-ol which is most likely formed by oxidation of (4S)-albaflavenol.

  5. Gene pathways that delay Caenorhabditis elegans reproductive senescence.

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    Meng C Wang

    2014-12-01

    Full Text Available Reproductive senescence is a hallmark of aging. The molecular mechanisms regulating reproductive senescence and its association with the aging of somatic cells remain poorly understood. From a full genome RNA interference (RNAi screen, we identified 32 Caenorhabditis elegans gene inactivations that delay reproductive senescence and extend reproductive lifespan. We found that many of these gene inactivations interact with insulin/IGF-1 and/or TGF-β endocrine signaling pathways to regulate reproductive senescence, except nhx-2 and sgk-1 that modulate sodium reabsorption. Of these 32 gene inactivations, we also found that 19 increase reproductive lifespan through their effects on oocyte activities, 8 of them coordinate oocyte and sperm functions to extend reproductive lifespan, and 5 of them can induce sperm humoral response to promote reproductive longevity. Furthermore, we examined the effects of these reproductive aging regulators on somatic aging. We found that 5 of these gene inactivations prolong organismal lifespan, and 20 of them increase healthy life expectancy of an organism without altering total life span. These studies provide a systemic view on the genetic regulation of reproductive senescence and its intersection with organism longevity. The majority of these newly identified genes are conserved, and may provide new insights into age-associated reproductive senescence during human aging.

  6. Automation of gene assignments to metabolic pathways using high-throughput expression data

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    Yona Golan

    2005-08-01

    Full Text Available Abstract Background Accurate assignment of genes to pathways is essential in order to understand the functional role of genes and to map the existing pathways in a given genome. Existing algorithms predict pathways by extrapolating experimental data in one organism to other organisms for which this data is not available. However, current systems classify all genes that belong to a specific EC family to all the pathways that contain the corresponding enzymatic reaction, and thus introduce ambiguity. Results Here we describe an algorithm for assignment of genes to cellular pathways that addresses this problem by selectively assigning specific genes to pathways. Our algorithm uses the set of experimentally elucidated metabolic pathways from MetaCyc, together with statistical models of enzyme families and expression data to assign genes to enzyme families and pathways by optimizing correlated co-expression, while minimizing conflicts due to shared assignments among pathways. Our algorithm also identifies alternative ("backup" genes and addresses the multi-domain nature of proteins. We apply our model to assign genes to pathways in the Yeast genome and compare the results for genes that were assigned experimentally. Our assignments are consistent with the experimentally verified assignments and reflect characteristic properties of cellular pathways. Conclusion We present an algorithm for automatic assignment of genes to metabolic pathways. The algorithm utilizes expression data and reduces the ambiguity that characterizes assignments that are based only on EC numbers.

  7. 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...

  8. 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

  9. 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.

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    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.

  10. MicroRNA-gene signaling pathways in pancreatic cancer

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    Alexandra Drakaki

    2013-10-01

    Full Text Available Pancreatic cancer is the fourth most frequent cause of cancer-related deaths and is characterized by early metastasis and pronounced resistance to chemotherapy and radiation therapy. Despite extensive esearch efforts, there is not any substantial progress regarding the identification of novel drugs against pancreatic cancer. Although the introduction of the chemotherapeutic agent gemcitabine improved clinical response, the prognosis of these patients remained extremely poor with a 5-year survival rate of 3-5%. Thus, the identification of the novel molecular pathways involved in pancreatic oncogenesis and the development of new and potent therapeutic options are highly desirable. Here, we describe how microRNAs control signaling pathways that are frequently deregulated during pancreatic oncogenesis. In addition, we provide evidence that microRNAs could be potentially used as novel pancreatic cancer therapeutics through reversal of chemotherapy and radiotherapy resistance or regulation of essential molecular pathways. Further studies should integrate the deregulated genes and microRNAs into molecular networks in order to identify the central regulators of pancreatic oncogenesis. Targeting these central regulators could lead to the development of novel targeted therapeutic approaches for pancreatic cancer patients.

  11. Characterization of purine catabolic pathway genes in coelacanths.

    Science.gov (United States)

    Forconi, Mariko; Biscotti, Maria Assunta; Barucca, Marco; Buonocore, Francesco; De Moro, Gianluca; Fausto, Anna Maria; Gerdol, Marco; Pallavicini, Alberto; Scapigliati, Giuseppe; Schartl, Manfred; Olmo, Ettore; Canapa, Adriana

    2014-09-01

    Coelacanths are a critically valuable species to explore the gene changes that took place in the transition from aquatic to terrestrial life. One interesting and biologically relevant feature of the genus Latimeria is ureotelism. However not all urea is excreted from the body; in fact high concentrations are retained in plasma and seem to be involved in osmoregulation. The purine catabolic pathway, which leads to urea production in Latimeria, has progressively lost some steps, reflecting an enzyme loss during diversification of terrestrial species. We report the results of analyses of the liver and testis transcriptomes of the Indonesian coelacanth Latimeria menadoensis and of the genome of Latimeria chalumnae, which has recently been fully sequenced in the framework of the coelacanth genome project. We describe five genes, uricase, 5-hydroxyisourate hydrolase, parahox neighbor B, allantoinase, and allantoicase, each coding for one of the five enzymes involved in urate degradation to urea, and report the identification of a putative second form of 5-hydroxyisourate hydrolase that is characteristic of the genus Latimeria. The present data also highlight the activity of the complete purine pathway in the coelacanth liver and suggest its involvement in the maintenance of high plasma urea concentrations.

  12. Altered Pathway Analyzer: A gene expression dataset analysis tool for identification and prioritization of differentially regulated and network rewired pathways

    Science.gov (United States)

    Kaushik, Abhinav; Ali, Shakir; Gupta, Dinesh

    2017-01-01

    Gene connection rewiring is an essential feature of gene network dynamics. Apart from its normal functional role, it may also lead to dysregulated functional states by disturbing pathway homeostasis. Very few computational tools measure rewiring within gene co-expression and its corresponding regulatory networks in order to identify and prioritize altered pathways which may or may not be differentially regulated. We have developed Altered Pathway Analyzer (APA), a microarray dataset analysis tool for identification and prioritization of altered pathways, including those which are differentially regulated by TFs, by quantifying rewired sub-network topology. Moreover, APA also helps in re-prioritization of APA shortlisted altered pathways enriched with context-specific genes. We performed APA analysis of simulated datasets and p53 status NCI-60 cell line microarray data to demonstrate potential of APA for identification of several case-specific altered pathways. APA analysis reveals several altered pathways not detected by other tools evaluated by us. APA analysis of unrelated prostate cancer datasets identifies sample-specific as well as conserved altered biological processes, mainly associated with lipid metabolism, cellular differentiation and proliferation. APA is designed as a cross platform tool which may be transparently customized to perform pathway analysis in different gene expression datasets. APA is freely available at http://bioinfo.icgeb.res.in/APA. PMID:28084397

  13. pathDIP: an annotated resource for known and predicted human gene-pathway associations and pathway enrichment analysis

    Science.gov (United States)

    Rahmati, Sara; Abovsky, Mark; Pastrello, Chiara; Jurisica, Igor

    2017-01-01

    Molecular pathway data are essential in current computational and systems biology research. While there are many primary and integrated pathway databases, several challenges remain, including low proteome coverage (57%), low overlap across different databases, unavailability of direct information about underlying physical connectivity of pathway members, and high fraction of protein-coding genes without any pathway annotations, i.e. ‘pathway orphans’. In order to address all these challenges, we developed pathDIP, which integrates data from 20 source pathway databases, ‘core pathways’, with physical protein–protein interactions to predict biologically relevant protein–pathway associations, referred to as ‘extended pathways’. Cross-validation determined 71% recovery rate of our predictions. Data integration and predictions increase coverage of pathway annotations for protein-coding genes to 86%, and provide novel annotations for 5732 pathway orphans. PathDIP (http://ophid.utoronto.ca/pathdip) annotates 17 070 protein-coding genes with 4678 pathways, and provides multiple query, analysis and output options. PMID:27899558

  14. ZCT1 and ZCT2 transcription factors repress the activity of a gene promoter from the methyl erythritol phosphate pathway in Madagascar periwinkle cells.

    Science.gov (United States)

    Chebbi, Mouadh; Ginis, Olivia; Courdavault, Vincent; Glévarec, Gaëlle; Lanoue, Arnaud; Clastre, Marc; Papon, Nicolas; Gaillard, Cécile; Atanassova, Rossitza; St-Pierre, Benoit; Giglioli-Guivarc'h, Nathalie; Courtois, Martine; Oudin, Audrey

    2014-10-15

    In Catharanthus roseus, accumulating data highlighted the existence of a coordinated transcriptional regulation of structural genes that takes place within the secoiridoid biosynthetic branch, including the methyl erythritol phosphate (MEP) pathway and the following steps leading to secologanin. To identify transcription factors acting in these pathways, we performed a yeast one-hybrid screening using as bait a promoter region of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene involved in the responsiveness of C. roseus cells to hormonal signals inducing monoterpene indole alkaloid (MIA) production. We identified that ZCT2, one of the three members of the zinc finger Catharanthus protein (ZCT) family, can bind to a HDS promoter region involved in hormonal responsiveness. By trans-activation assays, we demonstrated that ZCT1 and ZCT2 but not ZCT3 repress the HDS promoter activity. Gene expression analyses in C. roseus cells exposed to methyljasmonate revealed a persistence of induction of ZCT2 gene expression suggesting the existence of feed-back regulatory events acting on HDS gene expression in correlation with the MIA production.

  15. A novel 4-hydroxycoumarin biosynthetic pathway.

    Science.gov (United States)

    Liu, Benye; Raeth, Torben; Beuerle, Till; Beerhues, Ludger

    2010-01-01

    Coumarin forms in melilotoside (trans-ortho-coumaric acid glucoside)-containing plant species upon cell damage. In moldy melilotoside-containing plant material, trans-ortho-coumaric acid is converted by fungi to 4-hydroxycoumarin, two molecules of which spontaneously combine with formaldehyde to give dicoumarol. Dicoumarol causes internal bleeding in livestock and is the forerunner of the warfarin group of medicinal anticoagulants. Here, we report 4-hydroxycoumarin formation by biphenyl synthase (BIS). Two new BIS cDNAs were isolated from elicitor-treated Sorbus aucuparia cell cultures. The encoded isoenzymes preferred ortho-hydroxybenzoyl (salicoyl)-CoA as a starter substrate and catalyzed a single decarboxylative condensation with malonyl-CoA to give 4-hydroxycoumarin. When elicitor-treated S. aucuparia cell cultures were fed with the N-acetylcysteamine thioester of salicylic acid, 4-hydroxycoumarin accumulated in the culture medium. Incubation of the BIS isoenzymes with benzoyl-CoA and malonyl-CoA resulted in the formation of 3,5-dihydroxybiphenyl which is the precursor of the phytoalexins of the Maloideae.

  16. Genes of the mitochondrial apoptotic pathway in Mytilus galloprovincialis.

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    Noelia Estévez-Calvar

    Full Text Available Bivalves play vital roles in marine, brackish, freshwater and terrestrial habitats. In recent years, these ecosystems have become affected through anthropogenic activities. The ecological success of marine bivalves is based on the ability to modify their physiological functions in response to environmental changes. One of the most important mechanisms involved in adaptive responses to environmental and biological stresses is apoptosis, which has been scarcely studied in mollusks, although the final consequence of this process, DNA fragmentation, has been frequently used for pollution monitoring. Environmental stressors induce apoptosis in molluscan cells via an intrinsic pathway. Many of the proteins involved in vertebrate apoptosis have been recognized in model invertebrates; however, this process might not be universally conserved. Mytilus galloprovincialis is presented here as a new model to study the linkage between molecular mechanisms that mediate apoptosis and marine bivalve ecological adaptations. Therefore, it is strictly necessary to identify the key elements involved in bivalve apoptosis. In the present study, six mitochondrial apoptotic-related genes were characterized, and their gene expression profiles following UV irradiation were evaluated. This is the first step for the development of potential biomarkers to assess the biological responses of marine organisms to stress. The results confirmed that apoptosis and, more specifically, the expression of the genes involved in this process can be used to assess the biological responses of marine organisms to stress.

  17. Candidate pathways and genes for prostate cancer: a meta-analysis of gene expression data

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    Efstathiou Eleni

    2009-08-01

    Full Text Available Abstract Backgound The genetic mechanisms of prostate tumorigenesis remain poorly understood, but with the advent of gene expression array capabilities, we can now produce a large amount of data that can be used to explore the molecular and genetic mechanisms of prostate tumorigenesis. Methods We conducted a meta-analysis of gene expression data from 18 gene array datasets targeting transition from normal to localized prostate cancer and from localized to metastatic prostate cancer. We functionally annotated the top 500 differentially expressed genes and identified several candidate pathways associated with prostate tumorigeneses. Results We found the top differentially expressed genes to be clustered in pathways involving integrin-based cell adhesion: integrin signaling, the actin cytoskeleton, cell death, and cell motility pathways. We also found integrins themselves to be downregulated in the transition from normal prostate tissue to primary localized prostate cancer. Based on the results of this study, we developed a collagen hypothesis of prostate tumorigenesis. According to this hypothesis, the initiating event in prostate tumorigenesis is the age-related decrease in the expression of collagen genes and other genes encoding integrin ligands. This concomitant depletion of integrin ligands leads to the accumulation of ligandless integrin and activation of integrin-associated cell death. To escape integrin-associated death, cells suppress the expression of integrins, which in turn alters the actin cytoskeleton, elevates cell motility and proliferation, and disorganizes prostate histology, contributing to the histologic progression of prostate cancer and its increased metastasizing potential. Conclusion The results of this study suggest that prostate tumor progression is associated with the suppression of integrin-based cell adhesion. Suppression of integrin expression driven by integrin-mediated cell death leads to increased cell

  18. Global genome analysis of the shikimic acid pathway reveals greater gene loss in host-associated than in free-living bacteria

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    Amin Bijal

    2010-11-01

    Full Text Available Abstract Background A central tenet in biochemistry for over 50 years has held that microorganisms, plants and, more recently, certain apicomplexan parasites synthesize essential aromatic compounds via elaboration of a complete shikimic acid pathway, whereas metazoans lacking this pathway require a dietary source of these compounds. The large number of sequenced bacterial and archaean genomes now available for comparative genomic analyses allows the fundamentals of this contention to be tested in prokaryotes. Using Hidden Markov Model profiles (HMM profiles to identify all known enzymes of the pathway, we report the presence of genes encoding shikimate pathway enzymes in the hypothetical proteomes constructed from the genomes of 488 sequenced prokaryotes. Results Amongst free-living prokaryotes most Bacteria possess, as expected, genes encoding a complete shikimic acid pathway, whereas of the culturable Archaea, only one was found to have a complete complement of recognisable enzymes in its predicted proteome. It may be that in the Archaea, the primary amino-acid sequences of enzymes of the pathway are highly divergent and so are not detected by HMM profiles. Alternatively, structurally unrelated (non-orthologous proteins might be performing the same biochemical functions as those encoding recognized genes of the shikimate pathway. Most surprisingly, 30% of host-associated (mutualistic, commensal and pathogenic bacteria likewise do not possess a complete shikimic acid pathway. Many of these microbes show some degree of genome reduction, suggesting that these host-associated bacteria might sequester essential aromatic compounds from a parasitised host, as a 'shared metabolic adaptation' in mutualistic symbiosis, or obtain them from other consorts having the complete biosynthetic pathway. The HMM results gave 84% agreement when compared against data in the highly curated BioCyc reference database of genomes and metabolic pathways. Conclusions

  19. Tissue Non-Specific Genes and Pathways Associated with Diabetes: An Expression Meta-Analysis.

    Science.gov (United States)

    Mei, Hao; Li, Lianna; Liu, Shijian; Jiang, Fan; Griswold, Michael; Mosley, Thomas

    2017-01-21

    We performed expression studies to identify tissue non-specific genes and pathways of diabetes by meta-analysis. We searched curated datasets of the Gene Expression Omnibus (GEO) database and identified 13 and five expression studies of diabetes and insulin responses at various tissues, respectively. We tested differential gene expression by empirical Bayes-based linear method and investigated gene set expression association by knowledge-based enrichment analysis. Meta-analysis by different methods was applied to identify tissue non-specific genes and gene sets. We also proposed pathway mapping analysis to infer functions of the identified gene sets, and correlation and independent analysis to evaluate expression association profile of genes and gene sets between studies and tissues. Our analysis showed that PGRMC1 and HADH genes were significant over diabetes studies, while IRS1 and MPST genes were significant over insulin response studies, and joint analysis showed that HADH and MPST genes were significant over all combined data sets. The pathway analysis identified six significant gene sets over all studies. The KEGG pathway mapping indicated that the significant gene sets are related to diabetes pathogenesis. The results also presented that 12.8% and 59.0% pairwise studies had significantly correlated expression association for genes and gene sets, respectively; moreover, 12.8% pairwise studies had independent expression association for genes, but no studies were observed significantly different for expression association of gene sets. Our analysis indicated that there are both tissue specific and non-specific genes and pathways associated with diabetes pathogenesis. Compared to the gene expression, pathway association tends to be tissue non-specific, and a common pathway influencing diabetes development is activated through different genes at different tissues.

  20. Transcriptomic analysis in the developing zebrafish embryo after compound exposure: Individual gene expression and pathway regulation

    Energy Technology Data Exchange (ETDEWEB)

    Hermsen, Sanne A.B., E-mail: Sanne.Hermsen@rivm.nl [Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Department of Toxicogenomics, Maastricht University, P.O. Box 616, 6200 MD, Maastricht (Netherlands); Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.178, 3508 TD, Utrecht (Netherlands); Pronk, Tessa E. [Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Department of Toxicogenomics, Maastricht University, P.O. Box 616, 6200 MD, Maastricht (Netherlands); Brandhof, Evert-Jan van den [Centre for Environmental Quality, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Ven, Leo T.M. van der [Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Piersma, Aldert H. [Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.178, 3508 TD, Utrecht (Netherlands)

    2013-10-01

    The zebrafish embryotoxicity test is a promising alternative assay for developmental toxicity. Classically, morphological assessment of the embryos is applied to evaluate the effects of compound exposure. However, by applying differential gene expression analysis the sensitivity and predictability of the test may be increased. For defining gene expression signatures of developmental toxicity, we explored the possibility of using gene expression signatures of compound exposures based on commonly expressed individual genes as well as based on regulated gene pathways. Four developmental toxic compounds were tested in concentration-response design, caffeine, carbamazepine, retinoic acid and valproic acid, and two non-embryotoxic compounds, D-mannitol and saccharin, were included. With transcriptomic analyses we were able to identify commonly expressed genes, which were mostly development related, after exposure to the embryotoxicants. We also identified gene pathways regulated by the embryotoxicants, suggestive of their modes of action. Furthermore, whereas pathways may be regulated by all compounds, individual gene expression within these pathways can differ for each compound. Overall, the present study suggests that the use of individual gene expression signatures as well as pathway regulation may be useful starting points for defining gene biomarkers for predicting embryotoxicity. - Highlights: • The zebrafish embryotoxicity test in combination with transcriptomics was used. • We explored two approaches of defining gene biomarkers for developmental toxicity. • Four compounds in concentration-response design were tested. • We identified commonly expressed individual genes as well as regulated gene pathways. • Both approaches seem suitable starting points for defining gene biomarkers.

  1. Gene-Gene Interactions in the Folate Metabolic Pathway and the Risk of Conotruncal Heart Defects

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    Philip J. Lupo

    2010-01-01

    Full Text Available Conotruncal and related heart defects (CTRD are common, complex malformations. Although there are few established risk factors, there is evidence that genetic variation in the folate metabolic pathway influences CTRD risk. This study was undertaken to assess the association between inherited (i.e., case and maternal gene-gene interactions in this pathway and the risk of CTRD. Case-parent triads (n=727, ascertained from the Children's Hospital of Philadelphia, were genotyped for ten functional variants of nine folate metabolic genes. Analyses of inherited genotypes were consistent with the previously reported association between MTHFR A1298C and CTRD (adjusted P=.02, but provided no evidence that CTRD was associated with inherited gene-gene interactions. Analyses of the maternal genotypes provided evidence of a MTHFR C677T/CBS 844ins68 interaction and CTRD risk (unadjusted P=.02. This association is consistent with the effects of this genotype combination on folate-homocysteine biochemistry but remains to be confirmed in independent study populations.

  2. Uniform designation for genes of the Calvin-Benson-Bassham reductive pentose phosphate pathway of bacteria

    NARCIS (Netherlands)

    Tabita, F. Robert; Gibson, Janet L.; Bowien, Botho; Dijkhuizen, Lubbert; Meijer, Wilhelmus

    1992-01-01

    Structural and regulatory genes encoding enzymes and proteins of the reductive pentose phosphate pathway have been isolated from a number of bacteria recently. In the phototroph Rhodobacter sphaeroides, and in two chemoautotrophic bacteria, Alcaligenes eutrophus and Xanthobacter flavus, these genes

  3. GeneAnalytics Pathway Analysis and Genetic Overlap among Autism Spectrum Disorder, Bipolar Disorder and Schizophrenia

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    Naveen S. Khanzada

    2017-02-01

    Full Text Available Bipolar disorder (BPD and schizophrenia (SCH show similar neuropsychiatric behavioral disturbances, including impaired social interaction and communication, seen in autism spectrum disorder (ASD with multiple overlapping genetic and environmental influences implicated in risk and course of illness. GeneAnalytics software was used for pathway analysis and genetic profiling to characterize common susceptibility genes obtained from published lists for ASD (792 genes, BPD (290 genes and SCH (560 genes. Rank scores were derived from the number and nature of overlapping genes, gene-disease association, tissue specificity and gene functions subdivided into categories (e.g., diseases, tissues or functional pathways. Twenty-three genes were common to all three disorders and mapped to nine biological Superpathways including Circadian entrainment (10 genes, score = 37.0, Amphetamine addiction (five genes, score = 24.2, and Sudden infant death syndrome (six genes, score = 24.1. Brain tissues included the medulla oblongata (11 genes, score = 2.1, thalamus (10 genes, score = 2.0 and hypothalamus (nine genes, score = 2.0 with six common genes (BDNF, DRD2, CHRNA7, HTR2A, SLC6A3, and TPH2. Overlapping genes impacted dopamine and serotonin homeostasis and signal transduction pathways, impacting mood, behavior and physical activity level. Converging effects on pathways governing circadian rhythms support a core etiological relationship between neuropsychiatric illnesses and sleep disruption with hypoxia and central brain stem dysfunction.

  4. Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR.

    Science.gov (United States)

    Pulsawat, Nattika; Kitani, Shigeru; Fukushima, Eriko; Nihira, Takuya

    2009-04-01

    Two regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.

  5. 武夷菌素部分生物合成基因簇的克隆和分析%Cloning and Analysis of Wuyiencin Partial Biosynthetic Gene Cluster of Streptomyces ahygroscopicus var. wuyiensis CK-15

    Institute of Scientific and Technical Information of China (English)

    葛蓓孛; 杨振娟; 檀贝贝; 刘彦彦; 刘艳; 孙蕾; 张克诚

    2014-01-01

    不吸水链霉菌武夷变种Streptomyces ahygroscopicus var. wuyiensis CK-15是从福建省武夷山土样中分离得到的一株链霉菌,其代谢产物武夷菌素对果蔬真菌病害具有良好的防治效果,但是因其产量低的缺点限制了武夷菌素工业化生产和农业生产中的应用。为了实现利用基因工程培育高产新菌株的目标,首先要获得武夷菌素的生物合成基因。根据大环内酯类抗生素聚酮合成酶基因设计引物筛选菌株CK-15的基因组文库,共获得9个阳性克隆。克隆和测序获得3个较长scaffold片段,序列总长度达53.291 kb,其中包含了14个可能阅读框,通过同源比对证实该序列与S. noursei ATCC 11455的制霉素生物合成基因有很高的同源性。本研究为进一步研究武夷菌素生物合成基因的功能,并通过基因工程培育高产新菌株奠定了基础。%A wuyiencin producing strain Streptomyces ahygroscopicus var. wuyiensis CK-15 was isolated and purified from Wuyi mountain soil in Fujian province. Wuyiencin as secondary metabolites has good control effect on fruit and vegetable fungal diseases whereas which is limited on its low production disadvantage in industrial production and agricultural application. In order to achieve the aim of breeding high yield strain by genetic engineering, an attempt to obtain the biosynthetic gene cluster of wuyiencin generated strain was made. In this study, primers were designed according to a sequence of macrolide antibiotic polyketone synthetase gene, which was used for screening CK-15 genomic library. Nine positive clones were identified from the Streptomyces ahygroscopicus var. wuyiensis CK-15 fosmid genomic library. The positive clones were sequenced. There were three large scaffolds with approximately 53.291 kb of gene sequence. This sequence contains 14 possible ORFs and show high homology with nystatin biosynthetic gene of S. noursei ATCC 11455. The research will

  6. Analysis of gene evolution and metabolic pathways using the Candida Gene Order Browser

    LENUS (Irish Health Repository)

    Fitzpatrick, David A

    2010-05-10

    Abstract Background Candida species are the most common cause of opportunistic fungal infection worldwide. Recent sequencing efforts have provided a wealth of Candida genomic data. We have developed the Candida Gene Order Browser (CGOB), an online tool that aids comparative syntenic analyses of Candida species. CGOB incorporates all available Candida clade genome sequences including two Candida albicans isolates (SC5314 and WO-1) and 8 closely related species (Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Lodderomyces elongisporus, Debaryomyces hansenii, Pichia stipitis, Candida guilliermondii and Candida lusitaniae). Saccharomyces cerevisiae is also included as a reference genome. Results CGOB assignments of homology were manually curated based on sequence similarity and synteny. In total CGOB includes 65617 genes arranged into 13625 homology columns. We have also generated improved Candida gene sets by merging\\/removing partial genes in each genome. Interrogation of CGOB revealed that the majority of tandemly duplicated genes are under strong purifying selection in all Candida species. We identified clusters of adjacent genes involved in the same metabolic pathways (such as catabolism of biotin, galactose and N-acetyl glucosamine) and we showed that some clusters are species or lineage-specific. We also identified one example of intron gain in C. albicans. Conclusions Our analysis provides an important resource that is now available for the Candida community. CGOB is available at http:\\/\\/cgob.ucd.ie.

  7. Analysis of gene evolution and metabolic pathways using the Candida Gene Order Browser

    Directory of Open Access Journals (Sweden)

    Byrne Kevin P

    2010-05-01

    Full Text Available Abstract Background Candida species are the most common cause of opportunistic fungal infection worldwide. Recent sequencing efforts have provided a wealth of Candida genomic data. We have developed the Candida Gene Order Browser (CGOB, an online tool that aids comparative syntenic analyses of Candida species. CGOB incorporates all available Candida clade genome sequences including two Candida albicans isolates (SC5314 and WO-1 and 8 closely related species (Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Lodderomyces elongisporus, Debaryomyces hansenii, Pichia stipitis, Candida guilliermondii and Candida lusitaniae. Saccharomyces cerevisiae is also included as a reference genome. Results CGOB assignments of homology were manually curated based on sequence similarity and synteny. In total CGOB includes 65617 genes arranged into 13625 homology columns. We have also generated improved Candida gene sets by merging/removing partial genes in each genome. Interrogation of CGOB revealed that the majority of tandemly duplicated genes are under strong purifying selection in all Candida species. We identified clusters of adjacent genes involved in the same metabolic pathways (such as catabolism of biotin, galactose and N-acetyl glucosamine and we showed that some clusters are species or lineage-specific. We also identified one example of intron gain in C. albicans. Conclusions Our analysis provides an important resource that is now available for the Candida community. CGOB is available at http://cgob.ucd.ie.

  8. Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria.

    Science.gov (United States)

    Dittmann, Elke; Gugger, Muriel; Sivonen, Kaarina; Fewer, David P

    2015-10-01

    Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with intricate chemical structures and potent biological activities. The bulk of these natural products are known from just a handful of genera. Recent efforts have elucidated the mechanisms underpinning the biosynthesis of a diverse array of natural products from cyanobacteria. Many of the biosynthetic mechanisms are unique to cyanobacteria or rarely described from other organisms. Advances in genome sequence technology have precipitated a deluge of genome sequences for cyanobacteria. This makes it possible to link known natural products to biosynthetic gene clusters but also accelerates the discovery of new natural products through genome mining. These studies demonstrate that cyanobacteria encode a huge variety of cryptic gene clusters for the production of natural products, and the known chemical diversity is likely to be just a fraction of the true biosynthetic capabilities of this fascinating and ancient group of organisms.

  9. Identification of sugarcane genes involved in the purine synthesis pathway

    Directory of Open Access Journals (Sweden)

    Mario A. Jancso

    2001-12-01

    Full Text Available Nucleotide synthesis is of central importance to all cells. In most organisms, the purine nucleotides are synthesized de novo from non-nucleotide precursors such as amino acids, ammonia and carbon dioxide. An understanding of the enzymes involved in sugarcane purine synthesis opens the possibility of using these enzymes as targets for chemicals which may be effective in combating phytopathogen. Such an approach has already been applied to several parasites and types of cancer. The strategy described in this paper was applied to identify sugarcane clusters for each step of the de novo purine synthesis pathway. Representative sequences of this pathway were chosen from the National Center for Biotechnology Information (NCBI database and used to search the translated sugarcane expressed sequence tag (SUCEST database using the available basic local alignment search tool (BLAST facility. Retrieved clusters were further tested for the statistical significance of the alignment by an implementation (PRSS3 of the Monte Carlo shuffling algorithm calibrated using known protein sequences of divergent taxa along the phylogenetic tree. The sequences were compared to each other and to the sugarcane clusters selected using BLAST analysis, with the resulting table of p-values indicating the degree of divergence of each enzyme within different taxa and in relation to the sugarcane clusters. The results obtained by this strategy allowed us to identify the sugarcane proteins participating in the purine synthesis pathway.A via de síntese de purino nucleotídeos é considerada uma via de central importância para todas as células. Na maioria dos organismos, os purino nucleotídeos são sintetizados ''de novo'' a partir de precursores não-nucleotídicos como amino ácidos, amônia e dióxido de carbono. O conhecimento das enzimas envolvidas na via de síntese de purinas da cana-de-açúcar vai abrir a possibilidade do uso dessas enzimas como alvos no desenho

  10. Evolution of variation in presence and absence of genes in bacterial pathways

    Directory of Open Access Journals (Sweden)

    Francis Andrew R

    2012-04-01

    Full Text Available Abstract Background Bacterial genomes exhibit a remarkable degree of variation in the presence and absence of genes, which probably extends to the level of individual pathways. This variation may be a consequence of the significant evolutionary role played by horizontal gene transfer, but might also be explained by the loss of genes through mutation. A challenge is to understand why there would be variation in gene presence within pathways if they confer a benefit only when complete. Results Here, we develop a mathematical model to study how variation in pathway content is produced by horizontal transfer, gene loss and partial exposure of a population to a novel environment. Conclusions We discuss the possibility that variation in gene presence acts as cryptic genetic variation on which selection acts when the appropriate environment occurs. We find that a high level of variation in gene presence can be readily explained by decay of the pathway through mutation when there is no longer exposure to the selective environment, or when selection becomes too weak to maintain the genes. In the context of pathway variation the role of horizontal gene transfer is probably the initial introduction of a complete novel pathway rather than in building up the variation in a genome without the pathway.

  11. 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.

  12. Characterization of the juvenile hormone pathway in the viviparous cockroach, Diploptera punctata.

    Directory of Open Access Journals (Sweden)

    Juan Huang

    Full Text Available Juvenile hormones (JHs are key regulators of insect development and reproduction. The JH biosynthetic pathway is known to involve 13 discrete enzymatic steps. In the present study, we have characterized the JH biosynthetic pathway in the cockroach Diploptera punctata. The effect of exogenous JH precursors on JH biosynthesis was also determined. Based on sequence similarity, orthologs for the genes directly involved in the pathway were cloned, and their spatial and temporal transcript profiles were determined. The effect of shutting down the JH pathway in adult female cockroaches was studied by knocking down genes encoding HMG-CoA reductase (HMGR and Juvenile hormone acid methyltransferase (JHAMT. As a result, oocyte development slowed as a consequence of reduction in JH biosynthesis. Oocyte length, fat body transcription of Vg and ovarian vitellin content significantly decreased. In addition, silencing HMGR and JHAMT resulted in a decrease in the transcript levels of other genes in the pathway.

  13. Analysis of an inactive cyanobactin biosynthetic gene cluster leads to discovery of new natural products from strains of the genus Microcystis.

    Directory of Open Access Journals (Sweden)

    Niina Leikoski

    Full Text Available Cyanobactins are cyclic peptides assembled through the cleavage and modification of short precursor proteins. An inactive cyanobactin gene cluster has been described from the genome Microcystis aeruginosa NIES843. Here we report the discovery of active counterparts in strains of the genus Microcystis guided by this silent cyanobactin gene cluster. The end products of the gene clusters were structurally diverse cyclic peptides, which we named piricyclamides. Some of the piricyclamides consisted solely of proteinogenic amino acids while others contained disulfide bridges and some were prenylated or geranylated. The piricyclamide gene clusters encoded between 1 and 4 precursor genes. They encoded highly diverse core peptides ranging in length from 7-17 amino acids with just a single conserved amino acid. Heterologous expression of the pir gene cluster from Microcystis aeruginosa PCC7005 in Escherichia coli confirmed that this gene cluster is responsible for the biosynthesis of piricyclamides. Chemical analysis demonstrated that Microcystis strains could produce an array of piricyclamides some of which are geranylated or prenylated. The genetic diversity of piricyclamides in a bloom sample was explored and 19 different piricyclamide precursor genes were found. This study provides evidence for a stunning array of piricyclamides in Microcystis, a worldwide occurring bloom forming cyanobacteria.

  14. The Pathway From Genes to Gene Therapy in Glaucoma: A Review of Possibilities for Using Genes as Glaucoma Drugs.

    Science.gov (United States)

    Borrás, Teresa

    2017-01-01

    Treatment of diseases with gene therapy is advancing rapidly. The use of gene therapy has expanded from the original concept of re-placing the mutated gene causing the disease to the use of genes to con-trol nonphysiological levels of expression or to modify pathways known to affect the disease. Genes offer numerous advantages over conventional drugs. They have longer duration of action and are more specific. Genes can be delivered to the target site by naked DNA, cells, nonviral, and viral vectors. The enormous progress of the past decade in molecular bi-ology and delivery systems has provided ways for targeting genes to the intended cell/tissue and safe, long-term vectors. The eye is an ideal organ for gene therapy. It is easily accessible and it is an immune-privileged site. Currently, there are clinical trials for diseases affecting practically every tissue of the eye, including those to restore vision in patients with Leber congenital amaurosis. However, the number of eye trials compared with those for systemic diseases is quite low (1.8%). Nevertheless, judg-ing by the vast amount of ongoing preclinical studies, it is expected that such number will increase considerably in the near future. One area of great need for eye gene therapy is glaucoma, where a long-term gene drug would eliminate daily applications and compliance issues. Here, we review the current state of gene therapy for glaucoma and the possibilities for treating the trabecular meshwork to lower intraocular pressure and the retinal ganglion cells to protect them from neurodegeneration.

  15. Reconstruction of phylogenetic relationships from metabolic pathways based on the enzyme hierarchy and the gene ontology.

    Science.gov (United States)

    Clemente, José C; Satou, Kenji; Valiente, Gabriel

    2005-01-01

    There has been much interest in the structural comparison and alignment of metabolic pathways. Several techniques have been conceived to assess the similarity of metabolic pathways of different organisms. In this paper, we show that the combination of a new heuristic algorithm for the comparison of metabolic pathways together with any of three enzyme similarity measures (hierarchical, information content, and gene ontology) can be used to derive a metabolic pathway similarity measure that is suitable for reconstructing phylogenetic relationships from metabolic pathways. Experimental results on the Glycolysis pathway of 73 organisms representing the three domains of life show that our method outperforms previous techniques.

  16. Differential gene expression by fiber-optic beadarray and pathway in adrenocorticotrophin-secreting pituitary adenomas

    Institute of Scientific and Technical Information of China (English)

    JIANG Zhi-quan; GUI Song-bo; ZHANG Ya-zhuo

    2010-01-01

    Background Adrenocorticotrophin (ACTH)-secreting pituitary adenomas account for approximately 7%-14% of all pituitary adenomas, but its pathogenesis is still enigmatic. This study aimed to explore mechanisms underlying the pathogenesis of ACTH-secreting pituitary adenomas.Methods We used fiber-optic beadarray to examine gene expression in three ACTH-secreting adenomas compared with three normal pituitaries. Four differentially expressed genes from the three ACTH-secreting adenomas and three normal pituitaries were chosen randomly for validation by reverse transcriptase-real time quantitative polymerase chain reaction (RT-qPCR). We then analyzed the differentially expressed gene profile with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.Results Fiber-optic beadarray analysis showed that the expression of 28 genes and 8 expressed sequence tags (ESTs)were significantly increased and the expression of 412 genes and 31 ESTs were significantly decreased. Bioinformatic and pathway analysis showed that the genes HIGD1B, EPS8, HPGD, DAPK2, and IGFBP3 and the transforming growth factor (TGF)-β signaling pathway and extracellular matrix (ECM)-receptor interaction pathway may play important roles in tumorigenesis and progression of ACTH-secreting pituitary adenomas.Conclusions Our data suggest that numerous aberrantly expressed genes and several pathways are involved in the pathogenesis of ACTH-secreting pituitary adenomas. Fiber-optic beadarray combined with pathway analysis of differential gene expression appears to be a valid method of investigating tumour pathogenesis.

  17. TaWRKY70 transcription factor in wheat QTL-2DL regulates downstream metabolite biosynthetic genes to resist Fusarium graminearum infection spread within spike

    Science.gov (United States)

    Kage, Udaykumar; Yogendra, Kalenahalli N.; Kushalappa, Ajjamada C.

    2017-01-01

    A semi-comprehensive metabolomics was used to identify the candidate metabolites and genes to decipher mechanisms of resistance in wheat near-isogenic lines (NILs) containing QTL-2DL against Fusarium graminearum (Fg). Metabolites, with high fold-change in abundance, belonging to hydroxycinnamic acid amides (HCAAs): such as coumaroylagmatine, coumaroylputrescine and Fatty acids: phosphatidic acids (PAs) were identified as resistance related induced (RRI) metabolites in rachis of resistant NIL (NIL-R), inoculated with Fg. A WRKY like transcription factor (TF) was identified within the QTL-2DL region, along with three resistance genes that biosynthesized RRI metabolites. Sequencing and in-silico analysis of WRKY confirmed it to be wheat TaWRKY70. Quantitative real time-PCR studies showed a higher expression of TaWRKY70 in NIL-R as compared to NIL-S after Fg inoculation. Further, the functional validation of TaWRKY70 based on virus induced gene silencing (VIGS) in NIL-R, not only confirmed an increased fungal biomass but also decreased expressions of downstream resistance genes: TaACT, TaDGK and TaGLI1, along with decreased abundances of RRI metabolites biosynthesized by them. Among more than 200 FHB resistance QTL identified in wheat, this is the first QTL from which a TF was identified, and its downstream target genes as well as the FHB resistance functions were deciphered. PMID:28198421

  18. Characterization of genes and pathways that respond to heat stress in Holstein calves through transcriptome analysis.

    Science.gov (United States)

    Srikanth, Krishnamoorthy; Kwon, Anam; Lee, Eunjin; Chung, Hoyoung

    2017-01-01

    This study aimed to investigate the genes and pathways that respond to heat stress in Holstein bull calves exposed to severe ranges of temperature and humidity. A total of ten animals from 4 to 6 months of age were subjected to heat stress at 37 °C and 90 % humidity for 12 h. Skin and rectal temperatures were measured before and after heat stress; while no correlation was found between them before heat stress, a moderate correlation was detected after heat stress, confirming rectal temperature to be a better barometer for monitoring heat stress. RNAseq analysis identified 8567 genes to be differentially regulated, out of which 465 genes were significantly upregulated (≥2-fold, P heat stress. Significant terms and pathways enriched in response to heat stress included chaperones, cochaperones, cellular response to heat stress, phosphorylation, kinase activation, immune response, apoptosis, Toll-like receptor signaling pathway, Pi3K/AKT activation, protein processing in endoplasmic reticulum, interferon signaling, pathways in cancer, estrogen signaling pathway, and MAPK signaling pathway. The differentially expressed genes were validated by quantitative real-time PCR analysis, which confirmed the tendency of the expression. The genes and pathways identified in this analysis extend our understanding of transcriptional response to heat stress and their likely functioning in adapting the animal to hyperthermic stress. The identified genes could be used as candidate genes for association studies to select and breed animals with improved heat tolerance.

  19. Identification of gene networks and pathways associated with Guillain-Barre syndrome.

    Directory of Open Access Journals (Sweden)

    Kuo-Hsuan Chang

    Full Text Available BACKGROUND: The underlying change of gene network expression of Guillain-Barré syndrome (GBS remains elusive. We sought to identify GBS-associated gene networks and signaling pathways by analyzing the transcriptional profile of leukocytes in the patients with GBS. METHODS AND FINDINGS: Quantitative global gene expression microarray analysis of peripheral blood leukocytes was performed on 7 patients with GBS and 7 healthy controls. Gene expression profiles were compared between patients and controls after standardization. The set of genes that significantly correlated with GBS was further analyzed by Ingenuity Pathways Analyses. 256 genes and 18 gene networks were significantly associated with GBS (fold change ≥2, P<0.05. FOS, PTGS2, HMGB2 and MMP9 are the top four of 246 significantly up-regulated genes. The most significant disease and altered biological function genes associated with GBS were those involved in inflammatory response, infectious disease, and respiratory disease. Cell death, cellular development and cellular movement were the top significant molecular and cellular functions involved in GBS. Hematological system development and function, immune cell trafficking and organismal survival were the most significant GBS-associated function in physiological development and system category. Several hub genes, such as MMP9, PTGS2 and CREB1 were identified in the associated gene networks. Canonical pathway analysis showed that GnRH, corticotrophin-releasing hormone and ERK/MAPK signaling were the most significant pathways in the up-regulated gene set in GBS. CONCLUSIONS: This study reveals the gene networks and canonical pathways associated with GBS. These data provide not only networks between the genes for understanding the pathogenic properties of GBS but also map significant pathways for the future development of novel therapeutic strategies.

  20. Analysis of vascular endothelial dysfunction genes and related pathways in obesity through systematic bioinformatics.

    Science.gov (United States)

    Zhang, Hui; Wang, Jing; Sun, Ling; Xu, Qiuqin; Hou, Miao; Ding, Yueyue; Huang, Jie; Chen, Ye; Cao, Lei; Zhang, Jianmin; Qian, Weiguo; Lv, Haitao

    2015-01-01

    Obesity has become an increasingly serious health problem and popular research topic. It is associated with many diseases, especially cardiovascular disease (CVD)-related endothelial dysfunction. This study analyzed genes related to endothelial dysfunction and obesity and then summarized their most significant signaling pathways. Genes related to vascular endothelial dysfunction and obesity were extracted from a PubMed database, and analyzed by STRING, DAVID, and Gene-Go Meta-Core software. 142 genes associated with obesity were found to play a role in endothelial dysfunction in PubMed. A significant pathway (Angiotensin system maturation in protein folding and maturation) associated with obesity and endothelial dysfunction was explored. The genes and the pathway explored may play an important role in obesity. Further studies about preventing vascular endothelial dysfunction obesity should be conducted through targeting these loci and pathways.

  1. Association and gene-gene interactions study of reelin signaling pathway related genes with autism in the Han Chinese population.

    Science.gov (United States)

    Shen, Yidong; Xun, Guanglei; Guo, Hui; He, Yiqun; Ou, Jianjun; Dong, Huixi; Xia, Kun; Zhao, Jingping

    2016-04-01

    Autism is a neurodevelopmental disorder with unclear etiology. Reelin had been proposed to participate in the etiology of autism due to its important role in brain development. The goal of this study was to explore the association and gene-gene interactions of reelin signaling pathway related genes (RELN, VLDLR, LRP8, DAB1, FYN, and CDK5) with autism in Han Chinese population. Genotyping data of the six genes were obtained from a recent genome-wide association study performed in 430 autistic children who fulfilled the DSM-IV-TR criteria for autistic disorder, and 1,074 healthy controls. Single marker case-control association analysis and haplotype case-control association analysis were conducted after the data was screened. Multifactor dimensionality reduction (MDR) was applied to further test gene-gene interactions. Neither the single marker nor the haplotype association tests found any significant difference between the autistic group and the control group after permutation test of 1,000 rounds. The 4-locus MDR model (comprising rs6143734, rs1858782, rs634500, and rs1924267 which belong to RELN and DAB1) was determined to be the model with the highest cross-validation consistency (CVC) and testing balanced accuracy. The results indicate that an interaction between RELN and DAB1 may increase the risk of autism in the Han Chinese population. Furthermore, it can also be inferred that the involvement of RELN in the etiology of autism would occur through interaction with DAB1.

  2. Identification of the Biosynthetic Gene Clusters for the Lipopeptides Fusaristatin A and W493 B in Fusarium graminearum and F. pseudograminearum

    DEFF Research Database (Denmark)

    Sørensen, Jens Laurids; Sondergaard, Teis Esben; Covarelli, Lorenzo;

    2014-01-01

    The closely related species Fusarium graminearum and Fusarium pseudograminearum differ in that each contains a gene cluster with a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) that is not present in the other species. To identify their products, we deleted PKS6 and NRPS7...

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  5. Recursive random forest algorithm for constructing multilayered hierarchical gene regulatory networks that govern biological pathways

    Science.gov (United States)

    Zhang, Kui; Busov, Victor; Wei, Hairong

    2017-01-01

    Background Present knowledge indicates a multilayered hierarchical gene regulatory network (ML-hGRN) often operates above a biological pathway. Although the ML-hGRN is very important for understanding how a pathway is regulated, there is almost no computational algorithm for directly constructing ML-hGRNs. Results A backward elimination random forest (BWERF) algorithm was developed for constructing the ML-hGRN operating above a biological pathway. For each pathway gene, the BWERF used a random forest model to calculate the importance values of all transcription factors (TFs) to this pathway gene recursively with a portion (e.g. 1/10) of least important TFs being excluded in each round of modeling, during which, the importance values of all TFs to the pathway gene were updated and ranked until only one TF was remained in the list. The above procedure, termed BWERF. After that, the importance values of a TF