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Sample records for alkaloid biosynthetic gene

  1. CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes.

    Science.gov (United States)

    Li, Chun Yao; Leopold, Alex L; Sander, Guy W; Shanks, Jacqueline V; Zhao, Le; Gibson, Susan I

    2015-01-01

    Terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Toward this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression of CrBPF1 causes increases in the transcript levels for 11 out of 13 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a "fine-tune" regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression. PMID:26483828

  2. CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes

    Directory of Open Access Journals (Sweden)

    Chun Yao eLi

    2015-10-01

    Full Text Available Terpenoid indole alkaloid (TIA biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Towards this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression of CrBPF1 causes increases in the transcript levels for 11 out of 13 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a fine-tune regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression.

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

    Science.gov (United States)

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

    2016-01-01

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

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

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

    1999-01-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

  7. Alkaloids from Pandanus amaryllifolius: Isolation and Their Plausible Biosynthetic Formation.

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    Tsai, Yu-Chi; Yu, Meng-Lun; El-Shazly, Mohamed; Beerhues, Ludger; Cheng, Yuan-Bin; Chen, Lei-Chin; Hwang, Tsong-Long; Chen, Hui-Fen; Chung, Yu-Ming; Hou, Ming-Feng; Wu, Yang-Chang; Chang, Fang-Rong

    2015-10-23

    Pandanus amaryllifolius Roxb. (Pandanaceae) is used as a flavor and in folk medicine in Southeast Asia. The ethanolic crude extract of the aerial parts of P. amaryllifolius exhibited antioxidant, antibiofilm, and anti-inflammatory activities in previous studies. In the current investigation, the purification of the ethanolic extract yielded nine new compounds, including N-acetylnorpandamarilactonines A (1) and B (2); pandalizines A (3) and B (4); pandanmenyamine (5); pandamarilactones 2 (6) and 3 (7), and 5(E)-pandamarilactonine-32 (8); and pandalactonine (9). The isolated alkaloids, with either a γ-alkylidene-α,β-unsaturated-γ-lactone or γ-alkylidene-α,β-unsaturated-γ-lactam system, can be classified into five skeletons including norpandamarilactonine, indolizinone, pandanamine, pandamarilactone, and pandamarilactonine. A plausible biosynthetic route toward 1-5, 7, and 9 is proposed.

  8. Atropurpuran – Missing Biosynthetic Link Leading to the Hetidine and Arcutine C20-Diterpenoid Alkaloids or an Oxidative Degradation Product?

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    Weber, Manuel; Owens, Kyle; Sarpong, Richmond

    2015-01-01

    A possible biosynthetic link between atropurpuran, the hetidine diterpenoid alkaloids and the alkaloid arcutine and congeners is proposed. The feasibility of aspects of this biosynthesis, especially key 1,2-rearrangements, have been examined computationally. PMID:26028789

  9. Minimum Information about a Biosynthetic Gene cluster

    NARCIS (Netherlands)

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

    2015-01-01

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

  10. Anthocyanin biosynthetic genes in Brassica rapa

    OpenAIRE

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

    2014-01-01

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

  11. A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

    Science.gov (United States)

    Liscombe, David K.; O’Connor, Sarah E.

    2011-01-01

    The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by Madagascar periwinkle (Catharanthus roseus) plants. Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. We have developed a VIGS method to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro. PMID:21802100

  12. Characterization of the Promoter Region of Biosynthetic Enzyme Genes Involved in Berberine Biosynthesis in Coptis japonica

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    Yamada, Yasuyuki; Yoshimoto, Tadashi; Yoshida, Sayumi T.; Sato, Fumihiko

    2016-01-01

    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 TF, CjWRKY1, and a basic helix-loop-helix TF, 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 and by a chromatin immunoprecipitation 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. PMID:27642289

  13. Characterization of the Promoter Region of Biosynthetic Enzyme Genes Involved in Berberine Biosynthesis in Coptis japonica.

    Science.gov (United States)

    Yamada, Yasuyuki; Yoshimoto, Tadashi; Yoshida, Sayumi T; Sato, Fumihiko

    2016-01-01

    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 TF, CjWRKY1, and a basic helix-loop-helix TF, 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 and by a chromatin immunoprecipitation 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. PMID:27642289

  14. 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

  15. Biochemical genomics for gene discovery in benzylisoquinoline alkaloid biosynthesis in opium poppy and related species.

    Science.gov (United States)

    Dang, Thu Thuy T; Onoyovwi, Akpevwe; Farrow, Scott C; Facchini, Peter J

    2012-01-01

    Benzylisoquinoline alkaloids (BIAs) are a large, diverse group of ∼2500 specialized plant metabolites. Many BIAs display potent pharmacological activities, including the narcotic analgesics codeine and morphine, the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine, the antimicrobial agents sanguinarine and berberine, and the muscle relaxant (+)-tubocurarine. Opium poppy remains the sole commercial source for codeine, morphine, and a variety of semisynthetic drugs, including oxycodone and buprenorphine, derived primarily from the biosynthetic pathway intermediate thebaine. Recent advances in transcriptomics, proteomics, and metabolomics have created unprecedented opportunities for isolating and characterizing novel BIA biosynthetic genes. Here, we describe the application of next-generation sequencing and cDNA microarrays for selecting gene candidates based on comparative transcriptome analysis. We outline the basic mass spectrometric techniques to perform deep proteome and targeted metabolite analyses on BIA-producing plant tissues and provide methodologies for functionally characterizing biosynthetic gene candidates through in vitro enzyme assays and transient gene silencing in planta. PMID:22999177

  16. Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA

    OpenAIRE

    Wenli Li; Kui Hong; Weiming Zhu; Jingtao Zhang; Qiu Cui; Yuanyuan Du; Tong Li

    2013-01-01

    The indolocarbazole (ICZ) alkaloids have attracted much attention due to their unique structures and potential therapeutic applications. A series of ICZs were recently isolated and identified from a marine-derived actinomycete strain, Streptomyces sanyensis FMA. To elucidate the biosynthetic machinery associated with ICZs production in S. sanyensis FMA, PCR using degenerate primers was carried out to clone the FAD-dependent monooxygenase gene fragment for ICZ ring formation, which was used as...

  17. Preliminary Studies on Dynamic Change of Three Alkaloids and Expression of The Associated Biosynthetic Genes In Catharathus Roseus Leaves%长春花叶片三种生物碱含量及合成基因表达的初步研究

    Institute of Scientific and Technical Information of China (English)

    田跃胜; 陆平; 王名雪; 唐克轩; 赵静雅

    2011-01-01

    In order to clarify the dynamic change of three alkaloids (TIAs including vindoline, catharanthine and vinblastine) and the expression of the associated biosynthetic genes in the Catharathus Roseus leaves (young,medium and old) at different growing stages, high performance liquid chromatography was applied to determine the contents of TIAs at the leaf period, pre-floweringperiod, bud stage, flowering period, fully-mature period 1 and fully-mature period 2 in five pairs of leaves from Catharathus Roseus.In addition, the expression of the associated biosynthetic genes was analyzed by molecular biological technique (real time PCR, FQ-PCT).The results showed that highest contents of vindoline and catharanthine were presented in young leaves while the content of vinblastine was highest in old leaves.The best time for collecting the leaves of Catharathus Roseus was between the stage of alabastrum and flowering since the maximum contents of TIAs could be extracted in this period.The data from the FQ-PCR determination indicated that the expression of TIAs contents in C.Roseus leaves was more affected by the six genes inclucding GGPP,G10H ,SLS,STR,D4H and DAT while two genes (ASA, TDC) showed less influence on the production TIAs.%为探明长春花叶片中生物碱的动态变化和生物碱合成途径中的关键酶基因的表达量,利用高效液相色谱技术(HPLC)测定了5对真叶期、开花前期、出现花蕾期、出现开花期、成熟期1和成熟期2等6个生长阶段的长春花幼嫩、中等成熟、成熟三种叶片的生物碱(文多灵、长春质碱、长春碱)的含量.幼嫩叶片中文多灵、长春质碱和成熟叶片中长春碱的含量最高.长春花叶片的最佳采集期是花蕾期到开花期.利用荧光定量PCR技术检测了长春花叶片中生物碱合成途径中10个相关基因的相对表达量,GGPP,G10H,SLS,STR,D4H及DAT这6个基因对生物碱含量贡献较大,ASA及TDC基因对生物碱的增加的作用不明显.

  18. Effect of polyamine biosynthetic inhibitors on alkaloids and organogenesis in tobacco callus cultures.

    Science.gov (United States)

    Tiburcio, A F; Kaur-Sawhney, R; Galston, A W

    1987-01-01

    We studied the effects of inhibitors of ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and spermidine synthase (Spd synthase) on organogenesis and the titers of polyamines (PA) and alkaloids in tobacco calli. DL-alpha-diffluromethylarginine (DFMA) and D-arginine (D-Arg), both inhibitors of ADC activity, were more effective than DL-alpha-difluromethylorinithine (DFMO), an inhibitor of ODC, in reducing titers of PA and the putrescine (Put)-derived alkaloids (nornicotine and nicotine). Dicyclohexylammonium sulfate (DCHA), an inhibitor of Spd synthase, was also more efficient than DFMO in reducing PA and alkaloid levels. Root organogenesis is inversely related to the titers of Put and alkaloids. Thus, DFMA and D-Arg, which strongly inhibit Put and alkaloid biosynthesis, markedly promote root organogenesis, while control callus with high Put and alkaloid content showed poor root organization. These results suggest that morphological differentiation is not required for activation of secondary metabolic pathways and support the view that ADC has a major role in the generation of Put going to the pyrrolidine ring of tobacco alkaloids.

  19. Variation in the Trichothecene Mycotoxin Biosynthetic Gene Cluster in Fusarium

    Science.gov (United States)

    Trichothecene mycotoxins are produced by some plant pathogenic species of the fungus Fusarium and can contribute to its virulence on some plants. In Fusarium graminearum and F. sporotrichioides trichothecene biosynthetic enzymes are encoded at three loci: the single-gene TRI101 locus; the two-gene ...

  20. 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.

  1. Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis.

    Science.gov (United States)

    Sievert, Christian; Beuerle, Till; Hollmann, Julien; Ober, Dietrich

    2015-09-01

    Progress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis. PMID:26057225

  2. 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

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

    Science.gov (United States)

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

    2015-07-01

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

  4. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    OpenAIRE

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

    2015-01-01

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

  7. 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

  8. 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

  9. Variability in mycotoxin biosynthetic genes in Fusarium and its effect on mycotoxin contamination of crops

    Science.gov (United States)

    The Fusarium metabolites fumonisins and trichothecenes are among the mycotoxins of greatest concern to food and feed safety worldwide. As is the case for other fungal secondary metabolite biosynthetic genes, mycotoxin biosynthetic genes are often located adjacent to one another in gene clusters. Thu...

  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. Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA

    Directory of Open Access Journals (Sweden)

    Wenli Li

    2013-02-01

    Full Text Available The indolocarbazole (ICZ alkaloids have attracted much attention due to their unique structures and potential therapeutic applications. A series of ICZs were recently isolated and identified from a marine-derived actinomycete strain, Streptomyces sanyensis FMA. To elucidate the biosynthetic machinery associated with ICZs production in S. sanyensis FMA, PCR using degenerate primers was carried out to clone the FAD-dependent monooxygenase gene fragment for ICZ ring formation, which was used as a probe to isolate the 34.6-kb DNA region containing the spc gene cluster. Sequence analysis revealed genes for ICZ ring formation (spcO, D, P, C, sugar unit formation (spcA, B, E, K, J, I, glycosylation (spcN, G, methylation (spcMA, MB, as well as regulation (spcR. Their involvement in ICZ biosynthesis was confirmed by gene inactivation and heterologous expression in Streptomyces coelicolor M1152. This work represents the first cloning and characterization of an ICZ gene cluster isolated from a marine-derived actinomycete strain and would be helpful for thoroughly understanding the biosynthetic mechanism of ICZ glycosides.

  12. Coordinated regulation of biosynthetic and regulatory genes coincides with anthocyanin accumulation in developing eggplant fruit

    Science.gov (United States)

    Violet to black pigmentation of eggplant (Solanum melongena) fruit is attributed to anthocyanin accumulation. Model systems support the interaction of biosynthetic and regulatory genes for anthocyanin biosynthesis. Anthocyanin structural gene transcription requires the expression of at least one m...

  13. Nonlinear Biosynthetic Gene Cluster Dose Effect on Penicillin Production by Penicillium chrysogenum

    NARCIS (Netherlands)

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

    2010-01-01

    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 beta-lactam biosynthetic p

  14. Variability in mycotoxin biosynthetic genes and gene clusters in Fusarium and its implications for mycotoxin contamination of crops

    Science.gov (United States)

    The Fusarium metabolites fumonisins and trichothecenes are among the mycotoxins of greatest concern to food and feed safety worldwide. As with other fungal secondary metabolites, mycotoxin biosynthetic genes are often located adjacent to one another in gene clusters. Thus, fumonisin biosynthetic gen...

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

    OpenAIRE

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

    2015-01-01

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

  16. Unlocking the Diversity of Alkaloids in Catharanthus roseus: Nuclear Localization Suggests Metabolic Channeling in Secondary Metabolism

    OpenAIRE

    Stavrinides, Anna; Tatsis, Evangelos C.; Foureau, Emilien; Caputi, Lorenzo; Kellner, Franziska; Courdavault, Vincent; O’Connor, Sarah E.

    2015-01-01

    Summary The extraordinary chemical diversity of the plant-derived monoterpene indole alkaloids, which include vinblastine, quinine, and strychnine, originates from a single biosynthetic intermediate, strictosidine aglycone. Here we report for the first time the cloning of a biosynthetic gene and characterization of the corresponding enzyme that acts at this crucial branchpoint. This enzyme, an alcohol dehydrogenase homolog, converts strictosidine aglycone to the heteroyohimbine-type alkaloid ...

  17. Bacterial Alkaloids Prevent Amoebal Predation.

    Science.gov (United States)

    Klapper, Martin; Götze, Sebastian; Barnett, Robert; Willing, Karsten; Stallforth, Pierre

    2016-07-25

    Bacterial defense mechanisms have evolved to protect bacteria against predation by nematodes, predatory bacteria, or amoebae. We identified novel bacterial alkaloids (pyreudiones A-D) that protect the producer, Pseudomonas fluorescens HKI0770, against amoebal predation. Isolation, structure elucidation, total synthesis, and a proposed biosynthetic pathway for these structures are presented. The generation of P. fluorescens gene-deletion mutants unable to produce pyreudiones rendered the bacterium edible to a variety of soil-dwelling amoebae. PMID:27294402

  18. Benzylisoquinoline alkaloid biosynthesis in opium poppy.

    Science.gov (United States)

    Beaudoin, Guillaume A W; Facchini, Peter J

    2014-07-01

    Opium poppy (Papaver somniferum) is one of the world's oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy. PMID:24671624

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

    OpenAIRE

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

    2002-01-01

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

  20. 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.

  1. 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; Liang, Zhao-Xun

    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

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

    OpenAIRE

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

    2011-01-01

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

  3. 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

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

    OpenAIRE

    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 are currently being devised to prioritize these gene clusters in terms of their potential for yielding biochemical novelty. High-potential gene clusters from any biological source can then be acti...

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

    Science.gov (United States)

    Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin; Simonsen, Henrik Toft; Hamberger, Björn

    2014-01-01

    Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has already been widely recognized as a viable alternative for industrial-scale production of biopharmaceuticals. For expression of terpenoid biosynthetic genes, and reconstruction of heterologous pathways, Physcomitrella has unique attributes that makes it a very promising biotechnological host. These features include a high native tolerance to terpenoids, a simple endogenous terpenoid profile, convenient genome editing using homologous recombination, and cultivation techniques that allow up-scaling from single cells in microtiter plates to industrial photo-bioreactors. Beyond its use for functional characterization of terpenoid biosynthetic genes, engineered Physcomitrella can be a green biotechnological platform for production of terpenoids. Here, we describe two complementary and simple procedures for stable nuclear transformation of Physcomitrella with terpenoid biosynthetic genes, selection and cultivation of transgenic lines, and metabolite analysis of terpenoids produced in transgenic moss lines. We also provide tools for metabolic engineering through genome editing using homologous recombination. PMID:24777804

  6. 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

  7. Cloning and characterization of the goadsporin biosynthetic gene cluster from Streptomyces sp. TP-A0584.

    Science.gov (United States)

    Onaka, Hiroyasu; Nakaho, Mizuho; Hayashi, Keiko; Igarashi, Yasuhiro; Furumai, Tamotsu

    2005-12-01

    The biosynthetic gene cluster of goadsporin, a polypeptide antibiotic containing thiazole and oxazole rings, was cloned from Streptomyces sp. TP-A0584. The cluster contains a structural gene, godA, and nine god (goadsporin) genes involved in post-translational modification, immunity and transcriptional regulation. Although the gene organization is similar to typical bacteriocin biosynthetic gene clusters, each goadsporin biosynthetic gene shows low homology to these genes. Goadsporin biosynthesis is initiated by the translation of godA, and the subsequent cyclization, dehydration and acetylation are probably catalysed by godD, godE, godF, godG and godH gene products. godI shows high similarity to the 54 kDa subunit of the signal recognition particle and plays an important role in goadsporin immunity. Furthermore, four goadsporin analogues were produced by site-directed mutagenesis of godA, suggesting that this biosynthesis machinery is used for the heterocyclization of peptides. PMID:16339937

  8. Currencies of Mutualisms: Sources of Alkaloid Genes in Vertically Transmitted Epichloae

    Directory of Open Access Journals (Sweden)

    Christopher L. Schardl

    2013-06-01

    Full Text Available The epichloae (Epichloë and Neotyphodium species, a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae. Most epichloae are vertically transmitted in seeds (endophytes, and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens, which are active in vertebrate systems, and lolines and peramine, which are more specific against invertebrates. Several Epichloë species have been described which are sexual and capable of horizontal transmission, and most are vertically transmissible also. Asexual epichloae are mainly or exclusively vertically transmitted, and many are interspecific hybrids with genomic contributions from two or three ancestral Epichloë species. Here we employ genome-scale analyses to investigate the origins of biosynthesis gene clusters for ergot alkaloids (EAS, indole-diterpenes (IDT, and lolines (LOL in 12 hybrid species. In each hybrid, the alkaloid-gene and housekeeping-gene relationships were congruent. Interestingly, hybrids frequently had alkaloid clusters that were rare in their sexual ancestors. Also, in those hybrids that had multiple EAS, IDT or LOL clusters, one cluster lacked some genes, usually for late pathway steps. Possible implications of these findings for the alkaloid profiles and endophyte ecology are discussed.

  9. Currencies of mutualisms: sources of alkaloid genes in vertically transmitted epichloae.

    Science.gov (United States)

    Schardl, Christopher L; Young, Carolyn A; Pan, Juan; Florea, Simona; Takach, Johanna E; Panaccione, Daniel G; Farman, Mark L; Webb, Jennifer S; Jaromczyk, Jolanta; Charlton, Nikki D; Nagabhyru, Padmaja; Chen, Li; Shi, Chong; Leuchtmann, Adrian

    2013-06-01

    The epichloae (Epichloë and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens), which are active in vertebrate systems, and lolines and peramine, which are more specific against invertebrates. Several Epichloë species have been described which are sexual and capable of horizontal transmission, and most are vertically transmissible also. Asexual epichloae are mainly or exclusively vertically transmitted, and many are interspecific hybrids with genomic contributions from two or three ancestral Epichloë species. Here we employ genome-scale analyses to investigate the origins of biosynthesis gene clusters for ergot alkaloids (EAS), indole-diterpenes (IDT), and lolines (LOL) in 12 hybrid species. In each hybrid, the alkaloid-gene and housekeeping-gene relationships were congruent. Interestingly, hybrids frequently had alkaloid clusters that were rare in their sexual ancestors. Also, in those hybrids that had multiple EAS, IDT or LOL clusters, one cluster lacked some genes, usually for late pathway steps. Possible implications of these findings for the alkaloid profiles and endophyte ecology are discussed. PMID:23744053

  10. 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

  11. 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

  12. 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.

  13. A genomics based discovery of secondary metabolite biosynthetic gene clusters in Aspergillus ustus.

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    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.

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

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    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.

  15. 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.

  16. Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.

    OpenAIRE

    Barghouthi, S; Payne, S M; Arceneaux, J E; Byers, B R

    1991-01-01

    Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete n...

  17. Gene Clusters for Insecticidal Loline Alkaloids in the Grass-Endophytic Fungus Neotyphodium uncinatum

    OpenAIRE

    Spiering, Martin J.; Moon, Christina D; Wilkinson, Heather H.; Schardl, Christopher L

    2005-01-01

    Loline alkaloids are produced by mutualistic fungi symbiotic with grasses, and they protect the host plants from insects. Here we identify in the fungal symbiont, Neotyphodium uncinatum, two homologous gene clusters (LOL-1 and LOL-2) associated with loline-alkaloid production. Nine genes were identified in a 25-kb region of LOL-1 and designated (in order) lolF-1, lolC-1, lolD-1, lolO-1, lolA-1, lolU-1, lolP-1, lolT-1, and lolE-1. LOL-2 contained the homologs lolC-2 through lolE-2 in the same ...

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

    Science.gov (United States)

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

    2015-05-01

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

  19. 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.

  20. Evolutionary Conservation of Xylan Biosynthetic Genes in Selaginella moellendorffii and Physcomitrella patens.

    Science.gov (United States)

    Haghighat, Marziyeh; Teng, Quincy; Zhong, Ruiqin; Ye, Zheng-Hua

    2016-08-01

    Xylan is a major cross-linking hemicellulose in secondary walls of vascular tissues, and the recruitment of xylan as a secondary wall component was suggested to be a pivotal event for the evolution of vascular tissues. To decipher the evolution of xylan structure and xylan biosynthetic genes, we analyzed xylan substitution patterns and characterized genes mediating methylation of glucuronic acid (GlcA) side chains in xylan of the model seedless vascular plant, Selaginella moellendorffii, and investigated GT43 genes from S. moellendorffii and the model non-vascular plant, Physcomitrella patens, for their roles in xylan biosynthesis. Using nuclear magentic resonance spectroscopy, we have demonstrated that S. moellendorffii xylan consists of β-1,4-linked xylosyl residues subsituted solely with methylated GlcA residues and that xylans from both S. moellendorffii and P. patens are acetylated at O-2 and O-3. To investigate genes responsible for GlcA methylation of xylan, we identified two DUF579 genes in the S. moellendorffii genome and showed that one of them, SmGXM, encodes a glucuronoxylan methyltransferase capable of adding the methyl group onto the GlcA side chain of xylooligomers. Furthermore, we revealed that the two GT43 genes in S. moellendorffii, SmGT43A and SmGT43B, are functional orthologs of the Arabidopsis xylan backbone biosynthetic genes IRX9 and IRX14, respectively, indicating the evolutionary conservation of the involvement of two functionally non-redundant groups of GT43 genes in xylan backbone biosynthesis between seedless and seed vascular plants. Among the five GT43 genes in P. patens, PpGT43A was found to be a functional ortholog of Arabidopsis IRX9, suggesting that the recruitment of GT43 genes in xylan backbone biosynthesis occurred when non-vascular plants appeared on land. PMID:27345025

  1. Structural Diversification of Lyngbyatoxin A by Host-Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster.

    Science.gov (United States)

    Zhang, Lihan; Hoshino, Shotaro; Awakawa, Takayoshi; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-08-01

    Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk.

  2. 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.

  3. 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.

  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]CGT as ATAF1 consensus binding sequences. Co-expression analysis across publicly available microarray experiments identified 25 genes co-expressed with ATAF1. The promoter regions of ATAF1 co-expressors were significantly enriched for ATAF1 binding sites, and TTGCGTA was identified in the promoter of the key...... abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis....

  5. Unlocking the diversity of alkaloids in Catharanthus roseus: nuclear localization suggests metabolic channeling in secondary metabolism.

    Science.gov (United States)

    Stavrinides, Anna; Tatsis, Evangelos C; Foureau, Emilien; Caputi, Lorenzo; Kellner, Franziska; Courdavault, Vincent; O'Connor, Sarah E

    2015-03-19

    The extraordinary chemical diversity of the plant-derived monoterpene indole alkaloids, which include vinblastine, quinine, and strychnine, originates from a single biosynthetic intermediate, strictosidine aglycone. Here we report for the first time the cloning of a biosynthetic gene and characterization of the corresponding enzyme that acts at this crucial branchpoint. This enzyme, an alcohol dehydrogenase homolog, converts strictosidine aglycone to the heteroyohimbine-type alkaloid tetrahydroalstonine. We also demonstrate how this enzyme, which uses a highly reactive substrate, may interact with the upstream enzyme of the pathway. PMID:25772467

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

    Institute of Scientific and Technical Information of China (English)

    邢世海; 王荃; 潘琪芳; 赵静雅; 唐克轩

    2012-01-01

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

  7. 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.

  8. 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

  9. Expression of Terpenoid Biosynthetic Genes and Accumulation of Chemical Constituents in Valeriana fauriei

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    Yun Ji Park

    2016-05-01

    Full Text Available Valeriana fauriei (V. fauriei, which emits a characteristic and unpleasant odor, is important in traditional medicine. In this study, the expression of terpenoid biosynthetic genes was investigated in different organs that were also screened for volatile compounds including valerenic acid and its derivatives. Specific expression patterns from different parts of V. fauriei were observed using quantitative real-time PCR (qRT-PCR. The highest transcript levels of biosynthetic genes involved in mevalonic acid (MVA and methylerythritol phosphate (MEP production were found in the stem. Although the amounts of volatile compounds were varied by organ, most of the volatile terpenoids were accumulated in the root. Gas chromatography mass spectrometry (GC-MS analysis identified 128 volatile compounds, which represented 65.33% to 95.66% of total volatiles. Certain compounds were only found in specific organs. For example, isovalerenic acid and valerenic acid and its derivatives were restricted to the root. Organs with high transcript levels did not necessarily have high levels of the corresponding chemical constituents. According to these results, we hypothesize that translocation may occur between different organs in V. fauriei.

  10. Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells

    Science.gov (United States)

    Rischer, Heiko; Orešič, Matej; Seppänen-Laakso, Tuulikki; Katajamaa, Mikko; Lammertyn, Freya; Ardiles-Diaz, Wilson; Van Montagu, Marc C. E.; Inzé, Dirk; Oksman-Caldentey, Kirsi-Marja; Goossens, Alain

    2006-01-01

    Rational engineering of complicated metabolic networks involved in the production of biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Whereas comprehensive genome-wide functional genomics approaches can be successfully applied to analyze a select number of model plants, these holistic approaches are not yet available for the study of nonmodel plants that include most, if not all, medicinal plants. We report here a comprehensive profiling analysis of the Madagascar periwinkle (Catharanthus roseus), a source of the anticancer drugs vinblastine and vincristine. Genome-wide transcript profiling by cDNA-amplified fragment-length polymorphism combined with metabolic profiling of elicited C. roseus cell cultures yielded a collection of known and previously undescribed transcript tags and metabolites associated with terpenoid indole alkaloids. Previously undescribed gene-to-gene and gene-to-metabolite networks were drawn up by searching for correlations between the expression profiles of 417 gene tags and the accumulation profiles of 178 metabolite peaks. These networks revealed that the different branches of terpenoid indole alkaloid biosynthesis and various other metabolic pathways are subject to differing hormonal regulation. These networks also served to identify a select number of genes and metabolites likely to be involved in the biosynthesis of terpenoid indole alkaloids. This study provides the basis for a better understanding of periwinkle secondary metabolism and increases the practical potential of metabolic engineering of this important medicinal plant. PMID:16565214

  11. 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.

  12. Evolution of the Structure and Chromosomal Distribution of Histidine Biosynthetic Genes

    Science.gov (United States)

    Fani, Renato; Mori, Elena; Tamburini, Elena; Lazcano, Antonio

    1998-10-01

    A database of more than 100 histidine biosynthetic genes from different organisms belonging to the three primary domains has been analyzed, including those found in the now completely sequenced genomes of Haemophilus influenzae, Mycoplasma genitalium, Synechocystis sp., Methanococcus jannaschii, and Saccharomyces cerevisiae. The ubiquity of his genes suggests that it is a highly conserved pathway that was probably already present in the last common ancestor of all extant life. The chromosomal distribution of the his genes shows that the enterobacterial histidine operon structure is not the only possible organization, and that there is a diversity of gene arrays for the his pathway. Analysis of the available sequences shows that gene fusions (like those involved in the origin of the Escherichia coli and Salmonella typhimurium hisIE and hisB gene structures) are not universal. In contrast, the elongation event that led to the extant hisA gene from two homologous ancestral modules, as well as the subsequent paralogous duplication that originated hisF, appear to be irreversible and are conserved in all known organisms. The available evidence supports the hypothesis that histidine biosynthesis was assembled by a gene recruitment process.

  13. 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.

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

    Science.gov (United States)

    Imanian, Behzad; Keeling, Patrick J

    2014-02-01

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

  15. Stress and developmental responses of terpenoid biosynthetic genes in Cistus creticus subsp. creticus.

    Science.gov (United States)

    Pateraki, Irene; Kanellis, Angelos K

    2010-06-01

    Plants, and specially species adapted in non-friendly environments, produce secondary metabolites that help them to cope with biotic or abiotic stresses. These metabolites could be of great pharmaceutical interest because several of those show cytotoxic, antibacterial or antioxidant activities. Leaves' trichomes of Cistus creticus ssp. creticus, a Mediterranean xerophytic shrub, excrete a resin rich in several labdane-type diterpenes with verified in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. Bearing in mind the properties and possible future exploitation of these natural products, it seemed interesting to study their biosynthesis and its regulation, initially at the molecular level. For this purpose, genes encoding enzymes participating in the early steps of the terpenoids biosynthetic pathways were isolated and their gene expression patterns were investigated in different organs and in response to various stresses and defence signals. The genes studied were the CcHMGR from the mevalonate pathway, CcDXS and CcDXR from the methylerythritol 4-phosphate pathway and the two geranylgeranyl diphosphate synthases (CcGGDPS1 and 2) previously characterized from this species. The present work indicates that the leaf trichomes are very active biosynthetically as far as it concerns terpenoids biosynthesis, and the terpenoid production from this tissue seems to be transcriptionally regulated. Moreover, the CcHMGR and CcDXS genes (the rate-limiting steps of the isoprenoids' pathways) showed an increase during mechanical wounding and application of defence signals (like meJA and SA), which is possible to reflect an increased need of the plant tissues for the corresponding metabolites. PMID:20364257

  16. 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

  17. Terpenoid Indole Alkaloids Biosynthesis and Metabolic Engineering in Catharanthus roseus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Catharanthus roseus L. (Madagascar periwinkle) biosynthesizes a diverse array of secondary metabolites including anticancer dimeric alkaloids (vinblastine and vincristine) and antihypertensive alkaloids (ajmalicine and serpentine). The multi-step terpenoid indole alkaloids (TIAs) biosynthetic pathway in C. roseus is complex and is under strict molecular regulation. Many enzymes and genes involved in the TIAs biosynthesis have been studied in recent decades. Moreover,some regulatory proteins were found recently to control the production of TIAs in C. roseus. Based on mastering the rough scheme of the pathway and cloning the related genes, metabolic engineering of TIAs biosynthesis has been studied in C.roseus aiming at increasing the desired secondary metabolites in the past few years. The present article summarizes recent advances in isolation and characterization of TIAs biosynthesis genes and transcriptional regulators involved in the second metabolic control in C. roseus. Metabolic engineering applications in TIAs pathway via overexpression of these genes and regulators in C. roseus are also discussed.

  18. 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. PMID:27457995

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

    International Nuclear Information System (INIS)

    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 themechanisms 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

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

    OpenAIRE

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

    2009-01-01

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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

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

    Science.gov (United States)

    García-Estrada, Carlos; Martín, Juan-Francisco

    2016-10-01

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

  11. 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.

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    Foley William J

    2009-09-01

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

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

    OpenAIRE

    Wim Van den Ende; An Michiels; Joke De Roover; Andrea Van Laere

    2002-01-01

    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...

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

    OpenAIRE

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

    2015-01-01

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

  16. 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.

  17. 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

  18. Molecular Characterization of Carotenoid Biosynthetic Genes and Carotenoid Accumulation in Lycium chinense

    Directory of Open Access Journals (Sweden)

    Shicheng Zhao

    2014-07-01

    Full Text Available Lycium chinense is a shrub that has health benefits and is used as a source of medicines in Asia. In this study, a full-length cDNA clone encoding β-ring carotene hydroxylase (LcCHXB and partial-length cDNA clones encoding phytoene synthase (LcPSY, phytoene desaturase (LcPDS, ξ-carotene desaturase (LcZDS, lycopene β-cyclase (LcLCYB, lycopene ε-cyclase (LcLCYE, ε-ring carotene hydroxylase (LcCHXE, zeaxanthin epoxidase (LcZEP, carotenoid cleavage dioxygenase (LcCCD1, and 9-cis epoxycarotenoid dioxygenase (LcNCED were identified in L. chinense. The transcripts were constitutively expressed at high levels in leaves, flowers and red fruits, where the carotenoids are mostly distributed. In contrast, most of the carotenoid biosynthetic genes were weakly expressed in the roots and stems, which contained only small amounts of carotenoids. The level of LcLCYE transcripts was very high in leaves and correlated with the abundance of lutein in this plant tissue. During maturation, the levels of lutein and zeaxanthin in L. chinense fruits dramatically increased, concomitant with a rise in the level of β-cryptoxanthin. LcPSY, LcPDS, LcZDS, LcLCYB, and LcCHXE were highly expressed in red fruits, leading to their substantially higher total carotenoid content compared to that in green fruits. Total carotenoid content was high in both the leaves and red fruits of L. chinense. Our findings on the biosynthesis of carotenoids in L. chinense provide insights into the molecular mechanisms involved in carotenoid biosynthesis and may facilitate the optimization of carotenoid production in L. chinense.

  19. 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.

  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. 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...... 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...

  2. A Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in Catharanthus roseus*

    Science.gov (United States)

    Giddings, Lesley-Ann; Liscombe, David K.; Hamilton, John P.; Childs, Kevin L.; DellaPenna, Dean; Buell, C. Robin; O'Connor, Sarah E.

    2011-01-01

    Plant cytochrome P450s are involved in the production of over a hundred thousand metabolites such as alkaloids, terpenoids, and phenylpropanoids. Although cytochrome P450 genes constitute one of the largest superfamilies in plants, many of the catalytic functions of the enzymes they encode remain unknown. Here, we report the identification and functional characterization of a cytochrome P450 gene in a new subfamily of CYP71, CYP71BJ1, involved in alkaloid biosynthesis. Co-expression analysis of putative cytochrome P450 genes in the Catharanthus roseus transcriptome identified candidate genes with expression profiles similar to known terpene indole alkaloid biosynthetic genes. Screening of these candidate genes by functional expression in Saccharomyces cerevisiae yielded a unique P450-dependent enzyme that stereoselectively hydroxylates the alkaloids tabersonine and lochnericine at the 19-position of the aspidosperma-type alkaloid scaffold. Tabersonine, which can be converted to either vindoline or 19-O-acetylhörhammericine, represents a branch point in alkaloid biosynthesis. The discovery of CYP71BJ1, which forms part of the pathway leading to 19-O-acetylhörhammericine, will help illuminate how this branch point is controlled in C. roseus. PMID:21454651

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

    Directory of Open Access Journals (Sweden)

    Rafael da Silva Messias

    2014-01-01

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

  4. Sarpagine and Related Alkaloids.

    Science.gov (United States)

    Namjoshi, Ojas A; Cook, James M

    2016-01-01

    The sarpagine-related macroline and ajmaline alkaloids share a common biosynthetic origin, and bear important structural similarities, as expected. These indole alkaloids are widely dispersed in 25 plant genera, principally in the family Apocynaceae. Very diverse and interesting biological properties have been reported for this group of natural products. Isolation of new sarpagine-related alkaloids and the asymmetric synthesis of these structurally complex molecules are of paramount importance to the synthetic and medicinal chemists. A total of 115 newly isolated sarpagine-related macroline and ajmaline alkaloids, along with their physicochemical properties have been included in this chapter. A general and efficient strategy for the synthesis of these monomeric alkaloids, as well as bisindoles, has been presented, which involves application of the asymmetric Pictet-Spengler reaction (>98% ee) as a key step because of the ease of scale up of the tetracyclic template. Also included in this chapter are the syntheses of the sarpagine-related alkaloids, published since 2000. PMID:26827883

  5. Cloning, sequencing, and functional analysis of the biosynthetic gene cluster of macrolactam antibiotic vicenistatin in Streptomyces halstedii.

    Science.gov (United States)

    Ogasawara, Yasushi; Katayama, Kinya; Minami, Atsushi; Otsuka, Miyuki; Eguchi, Tadashi; Kakinuma, Katsumi

    2004-01-01

    Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning approximately 64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well. PMID:15112997

  6. A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

    OpenAIRE

    Liscombe, David K.; O’Connor, Sarah E.

    2011-01-01

    The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by Madagascar periwinkle (Catharanthus roseus) plants. Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to improve alkaloid content or provide alternative sources of the ...

  7. 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.

  8. Plant-like biosynthesis of isoquinoline alkaloids in Aspergillus fumigatus.

    Science.gov (United States)

    Baccile, Joshua A; Spraker, Joseph E; Le, Henry H; Brandenburger, Eileen; Gomez, Christian; Bok, Jin Woo; Macheleidt, Juliane; Brakhage, Axel A; Hoffmeister, Dirk; Keller, Nancy P; Schroeder, Frank C

    2016-06-01

    Natural product discovery efforts have focused primarily on microbial biosynthetic gene clusters (BGCs) containing large multimodular polyketide synthases and nonribosomal peptide synthetases; however, sequencing of fungal genomes has revealed a vast number of BGCs containing smaller NRPS-like genes of unknown biosynthetic function. Using comparative metabolomics, we show that a BGC in the human pathogen Aspergillus fumigatus named fsq, which contains an NRPS-like gene lacking a condensation domain, produces several new isoquinoline alkaloids known as the fumisoquins. These compounds derive from carbon-carbon bond formation between two amino acid-derived moieties followed by a sequence that is directly analogous to isoquinoline alkaloid biosynthesis in plants. Fumisoquin biosynthesis requires the N-methyltransferase FsqC and the FAD-dependent oxidase FsqB, which represent functional analogs of coclaurine N-methyltransferase and berberine bridge enzyme in plants. Our results show that BGCs containing incomplete NRPS modules may reveal new biosynthetic paradigms and suggest that plant-like isoquinoline biosynthesis occurs in diverse fungi. PMID:27065235

  9. Coregulated expression of loline alkaloid-biosynthesis genes in Neotyphodium uncinatum cultures.

    Science.gov (United States)

    Zhang, Dong-Xiu; Stromberg, Arnold J; Spiering, Martin J; Schardl, Christopher L

    2009-08-01

    Epichloë endophytes (holomorphic Epichloë spp. and anamorphic Neotyphodium spp.) are systemic, often heritable symbionts of cool-season grasses (subfamily Pooideae). Many epichloae provide protection to their hosts by producing anti-insect compounds. Among these are the loline alkaloids (LA), which are toxic and deterrent to a broad range of herbivorous insects but not to mammalian herbivores. LOL, a gene cluster containing nine genes, is associated with LA biosynthesis. We investigated coordinate regulation between LOL-gene expression and LA production in minimal medium (MM) cultures of Neotyphodium uncinatum. Expression of all LOL genes significantly fit temporal quadratic patterns during LA production. LOL-gene expression started before LA were detectable, and increased while LA accumulated. The highest gene expression level was reached at close to the time of most rapid LA accumulation, and gene expression declined to a very low level as amounts of LA plateaued. Temporal expression profiles of the nine LOL genes were tightly correlated with each other, but not as tightly correlated with proC and metE (genes for biosynthesis of precursor amino acids). Furthermore, the start days and peak days of expression significantly correlated with the order of the LOL-cluster genes in the genome. Hierarchical cluster analysis indicated three pairs of genes-lolA and lolC, lolO and lolD, and lolT and lolE-expression of which was especially tightly correlated. Of these, lolA and lolC tended to be expressed early, and lolT and lolE tended to be expressed late, in keeping with the putative roles of the respective gene products in the LA-biosynthesis pathway. Several common transcriptional binding sites were discovered in the LOL upstream regions. However, low expression of P(lolC2)uidA and P(lolA2)uidA in N. uncinatum transformants suggested induced expression of LOL genes might be subject to position effect at the LOL locus. PMID:19366635

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Phylogenomic Analysis of Natural Products Biosynthetic Gene Clusters Allows Discovery of Arseno-Organic Metabolites in Model Streptomycetes.

    Science.gov (United States)

    Cruz-Morales, Pablo; Kopp, Johannes Florian; Martínez-Guerrero, Christian; Yáñez-Guerra, Luis Alfonso; Selem-Mojica, Nelly; Ramos-Aboites, Hilda; Feldmann, Jörg; Barona-Gómez, Francisco

    2016-01-01

    Natural products from microbes have provided humans with beneficial antibiotics for millennia. However, a decline in the pace of antibiotic discovery exerts pressure on human health as antibiotic resistance spreads, a challenge that may better faced by unveiling chemical diversity produced by microbes. Current microbial genome mining approaches have revitalized research into antibiotics, but the empirical nature of these methods limits the chemical space that is explored.Here, we address the problem of finding novel pathways by incorporating evolutionary principles into genome mining. We recapitulated the evolutionary history of twenty-three enzyme families previously uninvestigated in the context of natural product biosynthesis in Actinobacteria, the most proficient producers of natural products. Our genome evolutionary analyses where based on the assumption that expanded-repurposed enzyme families-from central metabolism, occur frequently and thus have the potential to catalyze new conversions in the context of natural products biosynthesis. Our analyses led to the discovery of biosynthetic gene clusters coding for hidden chemical diversity, as validated by comparing our predictions with those from state-of-the-art genome mining tools; as well as experimentally demonstrating the existence of a biosynthetic pathway for arseno-organic metabolites in Streptomyces coelicolor and Streptomyces lividans, Using a gene knockout and metabolite profile combined strategy.As our approach does not rely solely on sequence similarity searches of previously identified biosynthetic enzymes, these results establish the basis for the development of an evolutionary-driven genome mining tool termed EvoMining that complements current platforms. We anticipate that by doing so real 'chemical dark matter' will be unveiled. PMID:27289100

  12. Phylogenomic Analysis of Natural Products Biosynthetic Gene Clusters Allows Discovery of Arseno-Organic Metabolites in Model Streptomycetes

    Science.gov (United States)

    Cruz-Morales, Pablo; Kopp, Johannes Florian; Martínez-Guerrero, Christian; Yáñez-Guerra, Luis Alfonso; Selem-Mojica, Nelly; Ramos-Aboites, Hilda; Feldmann, Jörg; Barona-Gómez, Francisco

    2016-01-01

    Natural products from microbes have provided humans with beneficial antibiotics for millennia. However, a decline in the pace of antibiotic discovery exerts pressure on human health as antibiotic resistance spreads, a challenge that may better faced by unveiling chemical diversity produced by microbes. Current microbial genome mining approaches have revitalized research into antibiotics, but the empirical nature of these methods limits the chemical space that is explored. Here, we address the problem of finding novel pathways by incorporating evolutionary principles into genome mining. We recapitulated the evolutionary history of twenty-three enzyme families previously uninvestigated in the context of natural product biosynthesis in Actinobacteria, the most proficient producers of natural products. Our genome evolutionary analyses where based on the assumption that expanded—repurposed enzyme families—from central metabolism, occur frequently and thus have the potential to catalyze new conversions in the context of natural products biosynthesis. Our analyses led to the discovery of biosynthetic gene clusters coding for hidden chemical diversity, as validated by comparing our predictions with those from state-of-the-art genome mining tools; as well as experimentally demonstrating the existence of a biosynthetic pathway for arseno-organic metabolites in Streptomyces coelicolor and Streptomyces lividans, Using a gene knockout and metabolite profile combined strategy. As our approach does not rely solely on sequence similarity searches of previously identified biosynthetic enzymes, these results establish the basis for the development of an evolutionary-driven genome mining tool termed EvoMining that complements current platforms. We anticipate that by doing so real ‘chemical dark matter’ will be unveiled. PMID:27289100

  13. Phylogenomic Analysis of Natural Products Biosynthetic Gene Clusters Allows Discovery of Arseno-Organic Metabolites in Model Streptomycetes.

    Science.gov (United States)

    Cruz-Morales, Pablo; Kopp, Johannes Florian; Martínez-Guerrero, Christian; Yáñez-Guerra, Luis Alfonso; Selem-Mojica, Nelly; Ramos-Aboites, Hilda; Feldmann, Jörg; Barona-Gómez, Francisco

    2016-01-01

    Natural products from microbes have provided humans with beneficial antibiotics for millennia. However, a decline in the pace of antibiotic discovery exerts pressure on human health as antibiotic resistance spreads, a challenge that may better faced by unveiling chemical diversity produced by microbes. Current microbial genome mining approaches have revitalized research into antibiotics, but the empirical nature of these methods limits the chemical space that is explored.Here, we address the problem of finding novel pathways by incorporating evolutionary principles into genome mining. We recapitulated the evolutionary history of twenty-three enzyme families previously uninvestigated in the context of natural product biosynthesis in Actinobacteria, the most proficient producers of natural products. Our genome evolutionary analyses where based on the assumption that expanded-repurposed enzyme families-from central metabolism, occur frequently and thus have the potential to catalyze new conversions in the context of natural products biosynthesis. Our analyses led to the discovery of biosynthetic gene clusters coding for hidden chemical diversity, as validated by comparing our predictions with those from state-of-the-art genome mining tools; as well as experimentally demonstrating the existence of a biosynthetic pathway for arseno-organic metabolites in Streptomyces coelicolor and Streptomyces lividans, Using a gene knockout and metabolite profile combined strategy.As our approach does not rely solely on sequence similarity searches of previously identified biosynthetic enzymes, these results establish the basis for the development of an evolutionary-driven genome mining tool termed EvoMining that complements current platforms. We anticipate that by doing so real 'chemical dark matter' will be unveiled.

  14. Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

    Institute of Scientific and Technical Information of China (English)

    Lei ZHANG; Guo-Yin KAI; Bei-Bei LU; Han-Ming ZHANG; Ke-Xuan TANG; Ji-Hong JIANG; Wan-Sheng CHEN

    2005-01-01

    Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.

  15. Regulation of the tryptophan biosynthetic genes in Bacillus halodurans: common elements but different strategies than those used by Bacillus subtilis.

    Science.gov (United States)

    Szigeti, Reka; Milescu, Mirela; Gollnick, Paul

    2004-02-01

    In Bacillus subtilis, an RNA binding protein called TRAP regulates both transcription and translation of the tryptophan biosynthetic genes. Bacillus halodurans is an alkaliphilic Bacillus species that grows at high pHs. Previous studies of this bacterium have focused on mechanisms of adaptation for growth in alkaline environments. We have characterized the regulation of the tryptophan biosynthetic genes in B. halodurans and compared it to that in B. subtilis. B. halodurans encodes a TRAP protein with 71% sequence identity to the B. subtilis protein. Expression of anthranilate synthetase, the first enzyme in the pathway to tryptophan, is regulated significantly less in B. halodurans than in B. subtilis. Examination of the control of the B. halodurans trpEDCFBA operon both in vivo and in vitro shows that only transcription is regulated, whereas in B. subtilis both transcription of the operon and translation of trpE are controlled. The attenuation mechanism that controls transcription in B. halodurans is similar to that in B. subtilis, but there are some differences in the predicted RNA secondary structures in the B. halodurans trp leader region, including the presence of a potential anti-antiterminator structure. Translation of trpG, which is within the folate operon in both bacilli, is regulated similarly in the two species. PMID:14729709

  16. 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.

  17. 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.

  18. Cloning and Expression Analysis of a Brassinosteroid Biosynthetic Enzyme Gene, GhDWF1, from Cotton (Gossypium hirsuturm L.)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Brassinosteroids (BRs) are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. To determine the effects of BRs on the development of cotton fibers, through screening cotton fiber EST database and contigging the candidate ESTs, a key gene (GhDWF1) involved in the upstream biosynthetic pathway of BRs was cloned from developing fibers of upland cotton (Gossypium hirsutum L.) cv. Xuzhou 142. The full length of the cloned cDNA is 1 849 bp, including a 37 bp 5'-untranslated region, an ORF of 1692 bp, and a 120 bp 3'-untranslated region.The cDNA encodes a polypeptide of 563 amino acid residues with a predicted molecular mass of 65 kD. The deduced amino acid sequence has high homology with the BR biosynthetic enzyme, DWARF1/DIMINUTO, from rice, maize, pea,tomato, and Arabidopsis. Furthermore, the typical conserved structures, such as the transmembrane domain, the FAD-dependent oxidase domain, and the FAD-binding site, are present in the GhDWF1 protein. The Southern blot indicated that the GhDWF1 gene is a single copy in upland cotton genome. RT-PCR analysis revealed that the highest level of GhDWF1 expression was detected in 0 DPA (day post anthesis) ovule (with fibers) while the lowest level was observed in cotyledon. The GhDWF1 gene presents high expression levels in root, young stem, and fiber, especially, at the fiber developmental stage of secondary cell wall accumulation. Moreover, the expression level was higher in ovules (with fibers) of wildtype (Xuzhou 142) than in ovules of fuzzless-lintless mutant at the same developmental stages (0 and 4 DPA). The results suggest that the GhDWF1 gene plays a crucial role in fiber development.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  20. Insights into the evolution of macrolactam biosynthesis through cloning and comparative analysis of the biosynthetic gene cluster for a novel macrocyclic lactam, ML-449.

    Science.gov (United States)

    Jørgensen, Hanne; Degnes, Kristin F; Dikiy, Alexander; Fjaervik, Espen; Klinkenberg, Geir; Zotchev, Sergey B

    2010-01-01

    A new compound, designated ML-449, structurally similar to the known 20-membered macrolactam BE-14106, was isolated from a marine sediment-derived Streptomyces sp. Cloning and sequencing of the 83-kb ML-449 biosynthetic gene cluster revealed its high level of similarity to the BE-14106 gene cluster. Comparison of the respective biosynthetic pathways indicated that the difference in the compounds' structures stems from the incorporation of one extra acetate unit during the synthesis of the acyl side chain. A phylogenetic analysis of the beta-ketosynthase (KS) domains from polyketide synthases involved in the biosynthesis of macrolactams pointed to a common ancestry for the two clusters. Furthermore, the analysis demonstrated the formation of a macrolactam-specific subclade for the majority of the KS domains from several macrolactam-biosynthetic gene clusters, indicating a closer relationship between macrolactam clusters than with the macrolactone clusters included in the analysis. Some KS domains from the ML-449, BE-14106, and salinilactam gene clusters did, however, show a closer relationship with KS domains from the polyene macrolide clusters, suggesting potential acquisition rather than duplication of certain PKS genes. Comparison of the ML-449, BE-14106, vicenistatin, and salinilactam biosynthetic gene clusters indicated an evolutionary relationship between them and provided new insights into the processes governing the evolution of small-ring macrolactam biosynthesis. PMID:19854930

  1. Insights into the Evolution of Macrolactam Biosynthesis through Cloning and Comparative Analysis of the Biosynthetic Gene Cluster for a Novel Macrocyclic Lactam, ML-449 ▿ †

    Science.gov (United States)

    Jørgensen, Hanne; Degnes, Kristin F.; Dikiy, Alexander; Fjærvik, Espen; Klinkenberg, Geir; Zotchev, Sergey B.

    2010-01-01

    A new compound, designated ML-449, structurally similar to the known 20-membered macrolactam BE-14106, was isolated from a marine sediment-derived Streptomyces sp. Cloning and sequencing of the 83-kb ML-449 biosynthetic gene cluster revealed its high level of similarity to the BE-14106 gene cluster. Comparison of the respective biosynthetic pathways indicated that the difference in the compounds' structures stems from the incorporation of one extra acetate unit during the synthesis of the acyl side chain. A phylogenetic analysis of the β-ketosynthase (KS) domains from polyketide synthases involved in the biosynthesis of macrolactams pointed to a common ancestry for the two clusters. Furthermore, the analysis demonstrated the formation of a macrolactam-specific subclade for the majority of the KS domains from several macrolactam-biosynthetic gene clusters, indicating a closer relationship between macrolactam clusters than with the macrolactone clusters included in the analysis. Some KS domains from the ML-449, BE-14106, and salinilactam gene clusters did, however, show a closer relationship with KS domains from the polyene macrolide clusters, suggesting potential acquisition rather than duplication of certain PKS genes. Comparison of the ML-449, BE-14106, vicenistatin, and salinilactam biosynthetic gene clusters indicated an evolutionary relationship between them and provided new insights into the processes governing the evolution of small-ring macrolactam biosynthesis. PMID:19854930

  2. Currencies of Mutualisms: Sources of Alkaloid Genes in Vertically Transmitted Epichloae

    OpenAIRE

    Schardl, Christopher L; Young, Carolyn A; Juan Pan; Simona Florea; Takach, Johanna E.; Panaccione, Daniel G.; Farman, Mark L.; Webb, Jennifer S.; Jolanta Jaromczyk; Charlton, Nikki D.; Padmaja Nagabhyru; Li Chen; Chong Shi; Adrian Leuchtmann

    2013-01-01

    The epichloae (Epichloë and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens), which are active in vertebrate systems, and lolines and peramine, which are...

  3. Biosynthetic Functional Gene Analysis of Bis-Indole Metabolites from 25D7, a Clone Derived from a Deep-Sea Sediment Metagenomic Library

    Science.gov (United States)

    Yan, Xia; Tang, Xi-Xiang; Qin, Dan; Yi, Zhi-Wei; Fang, Mei-Juan; Wu, Zhen; Qiu, Ying-Kun

    2016-01-01

    This work investigated the metabolites and their biosynthetic functional hydroxylase genes of the deep-sea sediment metagenomic clone 25D7. 5-Bromoindole was added to the 25D7 clone derived Escherichia coli fermentation broth. The new-generated metabolites and their biosynthetic byproducts were located through LC-MS, in which the isotope peaks of brominated products emerged. Two new brominated bis-indole metabolites, 5-bromometagenediindole B (1), and 5-bromometagenediindole C (2) were separated under the guidance of LC-MS. Their structures were elucidated on the basis of 1D and 2D NMR spectra (COSY, HSQC, and HMBC). The biosynthetic functional genes of the two new compounds were revealed through LC-MS and transposon mutagenesis analysis. 5-Bromometagenediindole B (1) also demonstrated moderately cytotoxic activity against MCF7, B16, CNE2, Bel7402, and HT1080 tumor cell lines in vitro. PMID:27258289

  4. VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in chili pepper leaves.

    Science.gov (United States)

    Zhang, Zhen; Li, Da-Wei; Jin, Jing-Hao; Yin, Yan-Xu; Zhang, Huai-Xia; Chai, Wei-Guo; Gong, Zhen-Hui

    2015-01-01

    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. PMID:26217354

  5. New lessons for combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene cluster of Stigmatella aurantiaca DW4/3-1.

    Science.gov (United States)

    Silakowski, B; Schairer, H U; Ehret, H; Kunze, B; Weinig, S; Nordsiek, G; Brandt, P; Blöcker, H; Höfle, G; Beyer, S; Müller, R

    1999-12-24

    The biosynthetic mta gene cluster responsible for myxothiazol formation from the fruiting body forming myxobacterium Stigmatella aurantiaca DW4/3-1 was sequenced and analyzed. Myxothiazol, an inhibitor of the electron transport via the bc(1)-complex of the respiratory chain, is biosynthesized by a unique combination of several polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS), which are activated by the 4'-phosphopantetheinyl transferase MtaA. Genomic replacement of a fragment of mtaB and insertion of a kanamycin resistance gene into mtaA both impaired myxothiazol synthesis. Genes mtaC and mtaD encode the enzymes for bis-thiazol(ine) formation and chain extension on one pure NRPS (MtaC) and on a unique combination of PKS and NRPS (MtaD). The genes mtaE and mtaF encode PKSs including peptide fragments with homology to methyltransferases. These methyltransferase modules are assumed to be necessary for the formation of the proposed methoxy- and beta-methoxy-acrylate intermediates of myxothiazol biosynthesis. The last gene of the cluster, mtaG, again resembles a NRPS and provides insight into the mechanism of the formation of the terminal amide of myxothiazol. The carbon backbone of an amino acid added to the myxothiazol-acid is assumed to be removed via an unprecedented module with homology to monooxygenases within MtaG. PMID:10601310

  6. 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.

  7. 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

  8. 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

  9. Biosynthesis and regulation of terpenoid indole alkaloids in Catharanthus roseus.

    Science.gov (United States)

    Zhu, Jianhua; Wang, Mingxuan; Wen, Wei; Yu, Rongmin

    2015-01-01

    Catharanthus roseus produces a wide range of terpenoid indole alkaloids (TIA). Many of them, such as vinblastine and vincristine, have significant bioactivity. They are valuable chemotherapy drugs used in combination with other drugs to treat lymphoma and leukemia. The TIA biosynthetic pathway has been investigated for many years, for scientific interest and for their potential in manufacturing applications, to fulfill the market demand. In this review, the progress and perspective of C. roseus TIA biosynthesis and its regulating enzymes are described. In addition, the culture condition, hormones, signaling molecules, precursor feeding on the accumulation of TIA, and gene expression are also evaluated and discussed. PMID:26009689

  10. Biosynthesis and regulation of terpenoid indole alkaloids in Catharanthus roseus

    Directory of Open Access Journals (Sweden)

    Jianhua Zhu

    2015-01-01

    Full Text Available Catharanthus roseus produces a wide range of terpenoid indole alkaloids (TIA. Many of them, such as vinblastine and vincristine, have significant bioactivity. They are valuable chemotherapy drugs used in combination with other drugs to treat lymphoma and leukemia. The TIA biosynthetic pathway has been investigated for many years, for scientific interest and for their potential in manufacturing applications, to fulfill the market demand. In this review, the progress and perspective of C. roseus TIA biosynthesis and its regulating enzymes are described. In addition, the culture condition, hormones, signaling molecules, precursor feeding on the accumulation of TIA, and gene expression are also evaluated and discussed.

  11. Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    XU MaoJun; DONG JuFang

    2007-01-01

    Bax, a mammalian pro-apoptotic member of the Bcl-2 family, triggers hypersensitive reactions when expressed in plants. To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells, we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter. The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied. Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0- and 5.5-fold of the control cells. Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str, two key genes in terpenoid indole alkaloid biosynthetic pathway of Catharanthus roseus cells, and stimulates the accumulation of defense-related protein PR1 in the cells, showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway. Thus, our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

  12. Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bax,a mammalian pro-apoptotic member of the Bcl-2 family,triggers hypersensitive reactions when expressed in plants.To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells,we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter.The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied.Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0-and 5.5-fold of the control cells.Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str,two key genes in terpenoid indole alkaloid bio-synthetic pathway of Catharanthus roseus cells,and stimulates the accumulation of defense-related protein PR1 in the cells,showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway.Thus,our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Samiha Sioud

    2007-01-01

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

  15. 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.

  16. Comparative analysis of transcription factor gene families from Papaver somniferum: identification of regulatory factors involved in benzylisoquinoline alkaloid biosynthesis.

    Science.gov (United States)

    Agarwal, Parul; Pathak, Sumya; Lakhwani, Deepika; Gupta, Parul; Asif, Mehar Hasan; Trivedi, Prabodh Kumar

    2016-05-01

    Opium poppy (Papaver somniferum L.), known for biosynthesis of several therapeutically important benzylisoquinoline alkaloids (BIAs), has emerged as the premier organism to study plant alkaloid metabolism. The most prominent molecules produced in opium poppy include narcotic analgesic morphine, the cough suppressant codeine, the muscle relaxant papaverine and the anti-microbial agent sanguinarine and berberine. Despite several health benefits, biosynthesis of some of these molecules is very low due to tight temporal and spatial regulation of the genes committed to their biosynthesis. Transcription factors, one of the prime regulators of secondary plant product biosynthesis, might be involved in controlled biosynthesis of BIAs in P. somniferum. In this study, identification of members of different transcription factor gene families using transcriptome datasets of 10 cultivars of P. somniferum with distinct chemoprofile has been carried out. Analysis suggests that most represented transcription factor gene family in all the poppy cultivars is WRKY. Comparative transcriptome analysis revealed differential expression pattern of the members of a set of transcription factor gene families among 10 cultivars. Through analysis, two members of WRKY and one member of C3H gene family were identified as potential candidates which might regulate thebaine and papaverine biosynthesis, respectively, in poppy. PMID:26108744

  17. Genotoxic effect of alkaloids

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    J. A. P. Henriques

    1991-01-01

    Full Text Available Because of the increase use of alkaloids in general medical practice in recent years, it is of interest to determine genotoxic, mutagenic and recombinogenic response to different groups of alkaloids in prokaryotic and eucaryotic organisms. Reserpine, boldine and chelerythrine did not show genotoxicity response in the SOS-Chromotest whereas skimmianine showed genotixicity in the presence of a metabolic activation mixture. Voacristine isolated fromthe leaves of Ervatamia coronaria shows in vivo cytostatic and mutagenic effects in Saccharomyces cerevisiae hapioids cells. The Rauwolfia alkaloid (reserpine was not able to induce reverse mutation and recombinational mitotic events (crossing-over and gene conversion in yeast diploid strain XS2316.

  18. Organization and characterization of a biosynthetic gene cluster for bafilomycin from Streptomyces griseus DSM 2608

    OpenAIRE

    Hwang, Jae Yoon; Kim, Hyo Sun; Kim, Soo Hee; Oh, Hye Ryeung; Nam, Doo Hyun

    2013-01-01

    Streptomyces griseus DSM 2608 produces bafilomycin, an antifungal plecomacrolide antibiotic. We cloned and sequenced an 87.4-kb region, including a polyketide synthase (PKS) region, methoxymalonate genes, flavensomycinate genes, and other putative regulatory genes. The 58.5kb of PKS region consisting 12 PKS modules arranged in five different PKS genes, was assumed to be responsible for the biosynthesis of plecomacrolide backbone including 16-membered macrocyclic lactone. All the modules showe...

  19. Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters

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    Qin Zhongjun

    2011-10-01

    Full Text Available Abstract Background Streptomyces species are a major source of antibiotics. They usually grow slowly at their optimal temperature and fermentation of industrial strains in a large scale often takes a long time, consuming more energy and materials than some other bacterial industrial strains (e.g., E. coli and Bacillus. Most thermophilic Streptomyces species grow fast, but no gene cloning systems have been developed in such strains. Results We report here the isolation of 41 fast-growing (about twice the rate of S. coelicolor, moderately thermophilic (growing at both 30°C and 50°C Streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, pTSC1, from one of them. pTSC1-derived pCWH1 could replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons function in thermophilic Streptomyces species. By examining ten well-sporulating strains, we found two promising cloning hosts, 2C and 4F. A gene cloning system was established by using the two strains. The actinorhodin and anthramycin biosynthetic gene clusters from mesophilic S. coelicolor A3(2 and thermophilic S. refuineus were heterologously expressed in one of the hosts. Conclusions We have developed a gene cloning and expression system in a fast-growing and moderately thermophilic Streptomyces species. Although just a few plasmids and one antibiotic biosynthetic gene cluster from mesophilic Streptomyces were successfully expressed in thermophilic Streptomyces species, we expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed.

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

    Science.gov (United States)

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

    2013-12-01

    In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative "front-end" desaturases (Δ6 and Δ4) from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of putative open reading frames (ORFs) in yeast revealed that the encoded enzyme activities efficiently convert their respective substrates: 54.1% conversion of α-linolenic acid for Δ6-desaturase, 15.1% conversion of 22:5n-3 for Δ4-desaturase and 38.1% conversion of γ-linolenic acid for Δ6-elongase. The Δ6-desaturase from Ostreococcus RCC809 displays a very strong substrate preference resulting in the predominant synthesis of stearidonic acid (C18:4Δ6,9,12,15). These data confirm the functional characterization of omega-3 long chain polyunsaturated fatty acid biosynthetic genes from these two species which have until now not been investigated for such activities. The identification of these new genes will also serve to expand the repertoire of activities available for metabolically engineering the omega-3 trait in heterologous hosts as well as providing better insights into the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in marine microalgae. PMID:24351909

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

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    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.

  2. 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.

  3. Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

    Science.gov (United States)

    Carqueijeiro, Inês; Guimarães, Ana Luísa; Bettencourt, Sara; Martínez-Cortés, Teresa; Guedes, Joana G; Gardner, Rui; Lopes, Telma; Andrade, Cláudia; Bispo, Cláudia; Martins, Nuno Pimpão; Andrade, Paula; Valentão, Patrícia; Valente, Inês M; Rodrigues, José A; Duarte, Patrícia; Sottomayor, Mariana

    2016-08-01

    Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus. PMID:27356972

  4. 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. PMID:27072286

  5. 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.

  6. Transcriptome profiling of khat (Catha edulis and Ephedra sinica reveals gene candidates potentially involved in amphetamine-type alkaloid biosynthesis.

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    Ryan A Groves

    Full Text Available Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis, and include the widely used decongestants and appetite suppressants (1S,2S-pseudoephedrine and (1R,2S-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications.

  7. Binary stress induces an increase in indole alkaloid biosynthesis in Catharanthus roseus.

    Science.gov (United States)

    Zhu, Wei; Yang, Bingxian; Komatsu, Setsuko; Lu, Xiaoping; Li, Ximin; Tian, Jingkui

    2015-01-01

    Catharanthus roseus is an important medicinal plant, which produces a variety of indole alkaloids of significant pharmaceutical relevance. In the present study, we aimed to investigate the potential stress-induced increase of indole alkaloid biosynthesis in C. roseus using proteomic technique. The contents of the detectable alkaloids ajmalicine, vindoline, catharanthine, and strictosidine in C. roseus were significantly increased under binary stress. Proteomic analysis revealed that the abundance of proteins related to tricarboxylic acid cycle and cell wall was largely increased; while, that of proteins related to tetrapyrrole synthesis and photosynthesis was decreased. Of note, 10-hydroxygeraniol oxidoreductase, which is involved in the biosynthesis of indole alkaloid was two-fold more abundant in treated group compared to the control. In addition, mRNA expression levels of genes involved in the indole alkaloid biosynthetic pathway indicated an up-regulation in their transcription in C. roseus under UV-B irradiation. These results suggest that binary stress might negatively affect the process of photosynthesis in C. roseus. In addition, the induction of alkaloid biosynthesis appears to be responsive to binary stress. PMID:26284098

  8. Binary Stress Induces an Increase in Indole Alkaloid Biosynthesis in Catharanthus roseus

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    Wei eZhu

    2015-07-01

    Full Text Available Catharanthus roseus is an important medicinal plant, which produces a variety of indole alkaloids of significant pharmaceutical relevance. In the present study, we aimed to investigate the potential stress-induced increase of indole alkaloid biosynthesis in C. roseus using proteomic technique. The contents of the detectable alkaloids ajmalicine, vindoline, catharanthine, and strictosidine in C. roseus were significantly increased under binary stress. Proteomic analysis revealed that the abundance of proteins related to tricarboxylic acid cycle and cell wall was largely increased; while, that of proteins related to tetrapyrrole synthesis and photosynthesis was decreased. Of note, 10-hydroxygeraniol oxidoreductase, which is involved in the biosynthesis of indole alkaloid was two-fold more abundant in treated group compared to that in control. In addition, mRNA expression levels of genes involved in the indole alkaloid biosynthetic pathway indicated an up-regulation in their transcription in C. roseus under UV-B irradiation. These results suggest that binary stress might negatively affect the process of photosynthesis in C. roseus. In addition, the induction of alkaloid biosynthesis appears to be responsive to binary stress.

  9. 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. PMID:25500454

  10. 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.

  11. Towards a Molecular Understanding of the Biosynthesis of Amaryllidaceae Alkaloids in Support of Their Expanding Medical Use

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    Adam M. Takos

    2013-05-01

    Full Text Available The alkaloids characteristically produced by the subfamily Amaryllidoideae of the Amaryllidaceae, bulbous plant species that include well know genera such as Narcissus (daffodils and Galanthus (snowdrops, are a source of new pharmaceutical compounds. Presently, only the Amaryllidaceae alkaloid galanthamine, an acetylcholinesterase inhibitor used to treat symptoms of Alzheimer’s disease, is produced commercially as a drug from cultivated plants. However, several Amaryllidaceae alkaloids have shown great promise as anti-cancer drugs, but their further clinical development is restricted by their limited commercial availability. Amaryllidaceae species have a long history of cultivation and breeding as ornamental bulbs, and phytochemical research has focussed on the diversity in alkaloid content and composition. In contrast to the available pharmacological and phytochemical data, ecological, physiological and molecular aspects of the Amaryllidaceae and their alkaloids are much less explored and the identity of the alkaloid biosynthetic genes is presently unknown. An improved molecular understanding of Amaryllidaceae alkaloid biosynthesis would greatly benefit the rational design of breeding programs to produce cultivars optimised for the production of pharmaceutical compounds and enable biotechnology based approaches.

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

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    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.

  13. Overexpression of the brassinosteroid biosynthetic gene DWF4 in Brassica napus simultaneously increases seed yield and stress tolerance.

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    Sahni, Sangita; Prasad, Bishun D; Liu, Qing; Grbic, Vojislava; Sharpe, Andrew; Singh, Surinder P; Krishna, Priti

    2016-01-01

    As a resource allocation strategy, plant growth and defense responses are generally mutually antagonistic. Brassinosteroid (BR) regulates many aspects of plant development and stress responses, however, genetic evidence of its integrated effects on plant growth and stress tolerance is lacking. We overexpressed the Arabidopsis BR biosynthetic gene AtDWF4 in the oilseed plant Brassica napus and scored growth and stress response phenotypes. The transgenic B. napus plants, in comparison to wild type, displayed increased seed yield leading to increased overall oil content per plant, higher root biomass and root length, significantly better tolerance to dehydration and heat stress, and enhanced resistance to necrotrophic fungal pathogens Leptosphaeria maculans and Sclerotinia sclerotiorum. Transcriptome analysis supported the integrated effects of BR on growth and stress responses; in addition to BR responses associated with growth, a predominant plant defense signature, likely mediated by BES1/BZR1, was evident in the transgenic plants. These results establish that BR can interactively and simultaneously enhance abiotic and biotic stress tolerance and plant productivity. The ability to confer pleiotropic beneficial effects that are associated with different agronomic traits suggests that BR-related genes may be important targets for simultaneously increasing plant productivity and performance under stress conditions. PMID:27324083

  14. 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.

  15. The genome of tolypocladium inflatum: evolution, organization, and expression of the cyclosporin biosynthetic gene cluster.

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    Kathryn E Bushley

    2013-06-01

    Full Text Available The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921, the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS that encodes for cyclosporin synthetase (simA and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc., and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further

  16. 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.

  17. Discovery of diversity in xylan biosynthetic genes by transcriptional profiling of a heteroxylan containing mucilaginous tissue

    Directory of Open Access Journals (Sweden)

    Jacob Kruger Jensen

    2013-06-01

    Full Text Available The exact biochemical steps of xylan backbone synthesis remain elusive. In Arabidopsis, three non-redundant genes from two glycosyltransferase (GT families, IRX9 and IRX14 from GT43 and IRX10 from GT47, are candidates for forming the xylan backbone. In other plants, evidence exists that different tissues express these three genes at widely different levels, which suggests that diversity in the makeup of the xylan synthase complex exists. Recently we have profiled the transcripts present in the developing mucilaginous tissue of psyllium (Plantago ovata Forsk. This tissue was found to have high expression levels of an IRX10 homolog, but very low levels of the two GT43 family members. This contrasts with recent wheat endosperm tissue profiling that found a relatively high abundance of the GT43 family members. We have performed an in-depth analysis of all GTs genes expressed in four developmental stages of the psyllium mucilagenous layer and in a single stage of the psyllium stem using RNA-Seq. This analysis revealed several IRX10 homologs, an expansion in GT61 (homologs of At3g18170/At3g18180, and several GTs from other GT families that are highly abundant and specifically expressed in the mucilaginous tissue. Our current hypothesis is that the four IRX10 genes present in the mucilagenous tissues have evolved to function without the GT43 genes. These four genes represent some of the most divergent IRX10 genes identified to date. Conversely, those present in the psyllium stem are very similar to those in other eudicots. This suggests these genes are under selective pressure, likely due to the synthesis of the various xylan structures present in mucilage that has a different biochemical role than that present in secondary walls. The numerous GT61 family members also show a wide sequence diversity and may be responsible for the larger number of side chain structures present in the psyllium mucilage.

  18. Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis

    OpenAIRE

    Ikeda, Haruo; Nonomiya, Tomoko; Usami, Masayo; Ohta, Toshio; Ōmura, Satoshi

    1999-01-01

    Analysis of the gene cluster from Streptomyces avermitilis that governs the biosynthesis of the polyketide anthelmintic avermectin revealed that it contains four large ORFs encoding giant multifunctional polypeptides of the avermectin polyketide synthase (AVES 1, AVES 2, AVES 3, and AVES 4). These clustered polyketide synthase genes responsible for avermectin biosynthesis together encode 12 homologous sets of enzyme activities (modules), each catalyzing a specific round of polyketide chain el...

  19. Pictet-Spengler reaction-based biosynthetic machinery in fungi.

    Science.gov (United States)

    Yan, Wei; Ge, Hui Ming; Wang, Gang; Jiang, Nan; Mei, Ya Ning; Jiang, Rong; Li, Sui Jun; Chen, Chao Jun; Jiao, Rui Hua; Xu, Qiang; Ng, Seik Weng; Tan, Ren Xiang

    2014-12-23

    The Pictet-Spengler (PS) reaction constructs plant alkaloids such as morphine and camptothecin, but it has not yet been noticed in the fungal kingdom. Here, a silent fungal Pictet-Spenglerase (FPS) gene of Chaetomium globosum 1C51 residing in Epinephelus drummondhayi guts is described and ascertained to be activable by 1-methyl-L-tryptophan (1-MT). The activated FPS expression enables the PS reaction between 1-MT and flavipin (fungal aldehyde) to form "unnatural" natural products with unprecedented skeletons, of which chaetoglines B and F are potently antibacterial with the latter inhibiting acetylcholinesterase. A gene-implied enzyme inhibition (GIEI) strategy has been introduced to address the key steps for PS product diversifications. In aggregation, the work designs and validates an innovative approach that can activate the PS reaction-based fungal biosynthetic machinery to produce unpredictable compounds of unusual and novel structure valuable for new biology and biomedicine. PMID:25425666

  20. Pictet–Spengler reaction-based biosynthetic machinery in fungi

    Science.gov (United States)

    Yan, Wei; Ge, Hui Ming; Wang, Gang; Jiang, Nan; Mei, Ya Ning; Jiang, Rong; Li, Sui Jun; Chen, Chao Jun; Jiao, Rui Hua; Xu, Qiang; Ng, Seik Weng; Tan, Ren Xiang

    2014-01-01

    The Pictet–Spengler (PS) reaction constructs plant alkaloids such as morphine and camptothecin, but it has not yet been noticed in the fungal kingdom. Here, a silent fungal Pictet–Spenglerase (FPS) gene of Chaetomium globosum 1C51 residing in Epinephelus drummondhayi guts is described and ascertained to be activable by 1-methyl-l-tryptophan (1-MT). The activated FPS expression enables the PS reaction between 1-MT and flavipin (fungal aldehyde) to form “unnatural” natural products with unprecedented skeletons, of which chaetoglines B and F are potently antibacterial with the latter inhibiting acetylcholinesterase. A gene-implied enzyme inhibition (GIEI) strategy has been introduced to address the key steps for PS product diversifications. In aggregation, the work designs and validates an innovative approach that can activate the PS reaction-based fungal biosynthetic machinery to produce unpredictable compounds of unusual and novel structure valuable for new biology and biomedicine. PMID:25425666

  1. Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi

    Directory of Open Access Journals (Sweden)

    Ralph A Cacho

    2015-01-01

    Full Text Available Genomics has revolutionized the research on fungal secondary metabolite biosynthesis. To elucidate the molecular and enzymatic mechanisms underlying the biosynthesis of a specific secondary metabolite compound, the important first step is often to find the genes that responsible for its synthesis. The accessibility to fungal genome sequences allows the bypass of the cumbersome traditional library construction and screening approach. The advance in next-generation sequencing (NGS technologies have further improved the speed and reduced the cost of microbial genome sequencing in the past few years, which has accelerated the research in this field. Here, we will present an example work flow for identifying the gene cluster encoding the biosynthesis of secondary metabolites of interest using an NGS approach. We will also review the different strategies that can be employed to pinpoint the targeted gene clusters rapidly by giving several examples stemming from our work.

  2. 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.

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

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    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.

  4. 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.

  5. 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

  6. 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.

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Jungsuwadee Paiboon

    2011-02-01

    Full Text Available Abstract Background Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. Results A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC and Adenosine Triphosphate Binding Cassette (ABC superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175. Conclusions We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and

  9. 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.

  10. Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi

    OpenAIRE

    Cacho, Ralph A.; Yi eTang; Yit-Heng eChooi

    2015-01-01

    Genomics has revolutionized the research on fungal secondary metabolite biosynthesis. To elucidate the molecular and enzymatic mechanisms underlying the biosynthesis of a specific secondary metabolite compound, the important first step is often to find the genes that responsible for its synthesis. The accessibility to fungal genome sequences allows the bypass of the cumbersome traditional library construction and screening approach. The advance in next-generation sequencing (NGS) technologies...

  11. Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi

    OpenAIRE

    Cacho, Ralph A.; Tang, Yi; Chooi, Yit-Heng

    2015-01-01

    Genomics has revolutionized the research on fungal secondary metabolite (SM) biosynthesis. To elucidate the molecular and enzymatic mechanisms underlying the biosynthesis of a specific SM compound, the important first step is often to find the genes that responsible for its synthesis. The accessibility to fungal genome sequences allows the bypass of the cumbersome traditional library construction and screening approach. The advance in next-generation sequencing (NGS) technologies have further...

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

    OpenAIRE

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

    2001-01-01

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

  13. 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.

  14. Biosynthetic Investigations of Lactonamycin and Lactonamycin Z: Cloning of the Biosynthetic Gene Clusters and Discovery of an Unusual Starter Unit▿ †

    OpenAIRE

    ZHANG, XIUJUN; Lawrence B. Alemany; Fiedler, Hans-Peter; Goodfellow, Michael; Parry, Ronald J.

    2007-01-01

    The antibiotics lactonamycin and lactonamycin Z provide attractive leads for antibacterial drug development. Both antibiotics contain a novel aglycone core called lactonamycinone. To gain insight into lactonamycinone biosynthesis, cloning and precursor incorporation experiments were undertaken. The lactonamycin gene cluster was initially cloned from Streptomyces rishiriensis. Sequencing of ca. 61 kb of S. rishiriensis DNA revealed the presence of 57 open reading frames. These included genes c...

  15. 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.

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

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    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

  17. Cyclic Lipopeptide Biosynthetic Genes and Products, and Inhibitory Activity of Plant-Associated Bacillus against Phytopathogenic Bacteria.

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    Isabel Mora

    Full Text Available The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP biosynthetic genes ituC (iturin, bmyB (bacillomycin, fenD (fengycin and srfAA (surfactin, and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP genes were bmyB, srfAA and fenD (34-50% of isolates. Most isolates (98.4% produced surfactin isoforms, 90.6% iturins and 79.7% fengycins. The antibacterial activity was very frequent and generally intense among the collection of strains because 75% of the isolates were active against at least 6 of the 8 bacterial plant pathogens tested. Hierarchical and correspondence analysis confirmed the presence of two clearly differentiated groups. One group consisted of Bacillus strains that showed a strong antibacterial activity, presented several cLPs genes and produced several isoforms of cLPs simultaneously, mainly composed of B. subtilis and B. amyloliquefaciens, although the last one was exclusive to this group. Another group was characterized by strains with very low or none antibacterial activity, that showed one or none of the cLP genes and produced a few or none of the corresponding cLPs, and was the most heterogenous group including B. subtilis, B. licheniformis, B. megaterium, B. pumilus, B. cereus and B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillus against plant pathogenic bacteria is related to the presence of cLP genes and to the production of the corresponding cLPs, and it is mainly associated to the species B. subtilis and B. amyloliquefaciens. Our findings would help to increase the yield and efficiency of screening methods to obtain candidate strains to biocontrol agents with a mechanism of action relaying on the

  18. Cyclic Lipopeptide Biosynthetic Genes and Products, and Inhibitory Activity of Plant-Associated Bacillus against Phytopathogenic Bacteria.

    Science.gov (United States)

    Mora, Isabel; Cabrefiga, Jordi; Montesinos, Emilio

    2015-01-01

    The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP) biosynthetic genes ituC (iturin), bmyB (bacillomycin), fenD (fengycin) and srfAA (surfactin), and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP) genes were bmyB, srfAA and fenD (34-50% of isolates). Most isolates (98.4%) produced surfactin isoforms, 90.6% iturins and 79.7% fengycins. The antibacterial activity was very frequent and generally intense among the collection of strains because 75% of the isolates were active against at least 6 of the 8 bacterial plant pathogens tested. Hierarchical and correspondence analysis confirmed the presence of two clearly differentiated groups. One group consisted of Bacillus strains that showed a strong antibacterial activity, presented several cLPs genes and produced several isoforms of cLPs simultaneously, mainly composed of B. subtilis and B. amyloliquefaciens, although the last one was exclusive to this group. Another group was characterized by strains with very low or none antibacterial activity, that showed one or none of the cLP genes and produced a few or none of the corresponding cLPs, and was the most heterogenous group including B. subtilis, B. licheniformis, B. megaterium, B. pumilus, B. cereus and B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillus against plant pathogenic bacteria is related to the presence of cLP genes and to the production of the corresponding cLPs, and it is mainly associated to the species B. subtilis and B. amyloliquefaciens. Our findings would help to increase the yield and efficiency of screening methods to obtain candidate strains to biocontrol agents with a mechanism of action relaying on the production of

  19. 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.

  20. Neurotoxic Alkaloids: Saxitoxin and Its Analogs

    Directory of Open Access Journals (Sweden)

    Troco K. Mihali

    2010-07-01

    Full Text Available Saxitoxin (STX and its 57 analogs are a broad group of natural neurotoxic alkaloids, commonly known as the paralytic shellfish toxins (PSTs. PSTs are the causative agents of paralytic shellfish poisoning (PSP and are mostly associated with marine dinoflagellates (eukaryotes and freshwater cyanobacteria (prokaryotes, which form extensive blooms around the world. PST producing dinoflagellates belong to the genera Alexandrium, Gymnodinium and Pyrodinium whilst production has been identified in several cyanobacterial genera including Anabaena, Cylindrospermopsis, Aphanizomenon Planktothrix and Lyngbya. STX and its analogs can be structurally classified into several classes such as non-sulfated, mono-sulfated, di-sulfated, decarbamoylated and the recently discovered hydrophobic analogs—each with varying levels of toxicity. Biotransformation of the PSTs into other PST analogs has been identified within marine invertebrates, humans and bacteria. An improved understanding of PST transformation into less toxic analogs and degradation, both chemically or enzymatically, will be important for the development of methods for the detoxification of contaminated water supplies and of shellfish destined for consumption. Some PSTs also have demonstrated pharmaceutical potential as a long-term anesthetic in the treatment of anal fissures and for chronic tension-type headache. The recent elucidation of the saxitoxin biosynthetic gene cluster in cyanobacteria and the identification of new PST analogs will present opportunities to further explore the pharmaceutical potential of these intriguing alkaloids.

  1. T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis.

    Science.gov (United States)

    Brill, Jeanette; Hoffmann, Tamara; Putzer, Harald; Bremer, Erhard

    2011-04-01

    Bacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ-ProA-ProH route is responsible for the production of proline as an osmoprotectant, and the ProB-ProA-ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB-treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine. PMID:21233158

  2. 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.

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

    Science.gov (United States)

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

    2001-03-13

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

  4. 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

  5. Characterization of indigoidine biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity.

    Science.gov (United States)

    Reverchon, Sylvie; Rouanet, Carine; Expert, Dominique; Nasser, William

    2002-02-01

    In the plant-pathogenic bacterium Erwinia chrysanthemi production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins. One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine. Since production of this pigment is cryptic in the wild-type strain, E. chrysanthemi ind mutants deficient in indigoidine synthesis were isolated by screening a library of Tn5-B21 insertions in a pecS mutant. These ind mutations were localized close to the regulatory pecS-pecM locus, immediately downstream of pecM. Sequence analysis of this DNA region revealed three open reading frames, indA, indB, and indC, involved in indigoidine biosynthesis. No specific function could be assigned to IndA. In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS). The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS. These data suggest that glutamine is the precursor of indigoidine. We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated. Expression of ind genes is strongly derepressed in the pecS background, indicating that PecS is the main regulator of this secondary metabolite synthesis. DNA band shift assays support a model whereby the PecS protein represses indA and indC expression by binding to indA and indC promoter regions. The regulatory link, via pecS, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in E. chrysanthemi pathogenicity. Mutants impaired in indigoidine production were unable to cause systemic invasion of potted Saintpaulia ionantha

  6. 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.

  7. Vinca Alkaloids

    Directory of Open Access Journals (Sweden)

    Maryam Moudi

    2013-01-01

    Full Text Available Vinca alkaloids are a subset of drugs obtained from the Madagascar periwinkle plant. They are naturally extracted from the pink periwinkle plant, Catharanthus roseus G. Don and have a hypoglycemic as well as cytotoxic effects. They have been used to treat diabetes, high blood pressure and have been used as disinfectants. The vinca alkaloids are also important for being cancer fighters. There are four major vinca alkaloids in clinical use: Vinblastine (VBL, vinorelbine (VRL, vincristine (VCR and vindesine (VDS. VCR, VBL and VRL have been approved for use in the United States. Vinflunine is also a new synthetic vinca alkaloid, which has been approved in Europe for the treatment of second-line transitional cell carcinoma of the urothelium is being developed for other malignancies. Vinca alkaloids are the second-most-used class of cancer drugs and will stay among the original cancer therapies. Different researches and studies for new vinca alkaloid applications will be carried out in this regard.

  8. Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis.

    Science.gov (United States)

    Pandey, Shiv S; Singh, Sucheta; Babu, C S Vivek; Shanker, Karuna; Srivastava, N K; Shukla, Ashutosh K; Kalra, Alok

    2016-01-01

    Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229-403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant. PMID:27220774

  9. 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.

  10. 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.

  11. Engineering biosynthesis of the anticancer alkaloid noscapine in yeast

    Science.gov (United States)

    Li, Yanran; Smolke, Christina D.

    2016-01-01

    Noscapine is a potential anticancer drug isolated from the opium poppy Papaver somniferum, and genes encoding enzymes responsible for the synthesis of noscapine have been recently discovered to be clustered on the genome of P. somniferum. Here, we reconstitute the noscapine gene cluster in Saccharomyces cerevisiae to achieve the microbial production of noscapine and related pathway intermediates, complementing and extending previous in planta and in vitro investigations. Our work provides structural validation of the secoberberine intermediates and the description of the narcotoline-4′-O-methyltransferase, suggesting this activity is catalysed by a unique heterodimer. We also reconstitute a 14-step biosynthetic pathway of noscapine from the simple alkaloid norlaudanosoline by engineering a yeast strain expressing 16 heterologous plant enzymes, achieving reconstitution of a complex plant pathway in a microbial host. Other engineered yeasts produce previously inaccessible pathway intermediates and a novel derivative, thereby advancing protoberberine and noscapine related drug discovery. PMID:27378283

  12. Products of the iron-molybdenum cofactor-specific biosynthetic genes, nifE and nifN, are structurally homologous to the products of the nitrogenase molybdenum-iron protein genes, nifD and nifK.

    OpenAIRE

    Brigle, K E; Weiss, M C; Newton, W E; Dean, D R

    1987-01-01

    The genes from Azotobacter vinelandii, which are homologous to the iron-molybdenum cofactor biosynthetic genes, nifE and nifN, from Klebsiella pneumoniae, have been cloned and sequenced. These genes comprise a single transcription unit and are located immediately downstream from the nitrogenase structural gene cluster (nifHDK). DNA sequence analysis has revealed that the products of the nifE and nifN genes contain considerable homology when compared with the nifD (MoFe protein alpha subunit) ...

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

    Science.gov (United States)

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

    2015-07-01

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

  14. 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. 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. PMID:23832493

  16. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

    Science.gov (United States)

    Rizvi, Noreen F; Weaver, Jessica D; Cram, Erin J; Lee-Parsons, Carolyn W T

    2016-01-01

    The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis. PMID:27467510

  17. 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

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

    International Nuclear Information System (INIS)

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

  19. Structures and comparative characterization of biosynthetic gene clusters for cyanosporasides, enediyne-derived natural products from marine actinomycetes

    OpenAIRE

    Lane, Amy L.; Nam, Sang Jip; Fukuda, Takashi; Yamanaka, Kazuya; Kauffman, Christopher A.; Jensen, Paul R; Fenical, William; Moore, Bradley S.

    2013-01-01

    Cyanosporasides are marine bacterial natural products containing a chlorinated cyclopenta[a]indene core of suspected enediyne polyketide biosynthetic origin. Herein, we report the isolation and characterization of novel cyanosporasides C–F (3–6) from the marine actinomycetes “Salinispora pacifica” CNS-143 and Streptomyces sp. CNT-179, highlighted by the unprecedented C-2' N-acetylcysteamine functionalized hexose group of 6. Cloning, sequencing, and mutagenesis of homologous ~50 kb cyanosporas...

  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. Hemlock alkaloids from Socrates to poison aloes.

    Science.gov (United States)

    Reynolds, Tom

    2005-06-01

    Hemlock (Conium maculatum L. Umbelliferae) has long been known as a poisonous plant. Toxicity is due to a group of piperidine alkaloids of which the representative members are coniine and gamma-coniceine. The latter is the more toxic and is the first formed biosynthetically. Its levels in relation to coniine vary widely according to environmental conditions and to provenance of the plants. Surprisingly, these piperidine alkaloids have turned up in quite unrelated species in the monocotyledons as well as the dicotyledons. Aloes, for instance, important medicinal plants, are not regarded as poisonous although some species are very bitter. Nevertheless a small number of mostly local species contain the alkaloids, especially gamma-coniceine and there have been records of human poisoning. The compounds are recognized by their characteristic mousy smell. Both acute and chronic symptoms have been described. The compounds are neurotoxins and death results from respiratory failure, recalling the effects of curare. Chronic non-lethal ingestion by pregnant livestock leads to foetal malformation. Both acute and chronic toxicity are seen with stock in damp meadows and have been recorded as problems especially in North America. The alkaloids derive biosynthetically from acetate units via the polyketide pathway in contrast to other piperidine alkaloids which derive from lysine.

  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. 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

  4. A vacuolar class III peroxidase and the metabolism of anticancer indole alkaloids in Catharanthus roseus

    Science.gov (United States)

    Duarte, Patrícia; Figueiredo, Raquel; Ros Barceló, Alfonso

    2008-01-01

    Plants possess a unique metabolic diversity commonly designated as secondary metabolism, of which the anticancer alkaloids from Catharanthus roseus are among the most studied. Recently, in a classical function-to-protein-to-gene approach, we have characterized the main class III peroxidase (Prx) expressed in C. roseus leaves, CrPrx1, implicated in a key biosynthetic step of the anticancer alkaloids. We have shown the vacuolar sorting determination of CrPrx1 using GFP fusions and we have obtained further evidence supporting the role of this enzyme in alkaloid biosynthesis, indicating the potential of CrPrx1 as a molecular tool for the manipulation of alkaloid metabolism. Here, we discuss how plant cells may regulate Prx reactions. In fact, Prxs form a large multigenic family whose members accept a broad range of substrates and, in their two subcellular localizations, the cell wall and the vacuole, Prxs co-locate with a large variety of secondary metabolites which can be accepted as substrates. How then, are Prx reactions regulated? Localization data obtained in our lab suggest that arabinogalactan proteins (AGPs) and Prxs may be associated in membrane microdomains, evocative of lipid rafts. Whether plasma membrane and/or tonoplast microcompartmentation involve AGPs and Prxs and whether this enables metabolic channeling determining Prx substrate selection are challenging questions ahead. PMID:19704535

  5. Identification and characterization of a new erythromycin biosynthetic gene cluster in Actinopolyspora erythraea YIM90600, a novel erythronolide-producing halophilic actinomycete isolated from salt field.

    Directory of Open Access Journals (Sweden)

    Dandan Chen

    Full Text Available Erythromycins (Ers are clinically potent macrolide antibiotics in treating pathogenic bacterial infections. Microorganisms capable of producing Ers, represented by Saccharopolyspora erythraea, are mainly soil-dwelling actinomycetes. So far, Actinopolyspora erythraea YIM90600, a halophilic actinomycete isolated from Baicheng salt field, is the only known Er-producing extremophile. In this study, we have reported the draft genome sequence of Ac. erythraea YIM90600, genome mining of which has revealed a new Er biosynthetic gene cluster encoding several novel Er metabolites. This Er gene cluster shares high identity and similarity with the one of Sa. erythraea NRRL2338, except for two absent genes, eryBI and eryG. By correlating genotype and chemotype, the biosynthetic pathways of 3'-demethyl-erythromycin C, erythronolide H (EH and erythronolide I have been proposed. The formation of EH is supposed to be sequentially biosynthesized via C-6/C-18 epoxidation and C-14 hydroxylation from 6-deoxyerythronolide B. Although an in vitro enzymatic activity assay has provided limited evidence for the involvement of the cytochrome P450 oxidase EryFAc (derived from Ac. erythraea YIM90600 in the catalysis of a two-step oxidation, resulting in an epoxy moiety, the attempt to construct an EH-producing Sa. erythraea mutant via gene complementation was not successful. Characterization of EryKAc (derived from Ac. erythraea YIM90600 in vitro has confirmed its unique role as a C-12 hydroxylase, rather than a C-14 hydroxylase of the erythronolide. Genomic characterization of the halophile Ac. erythraea YIM90600 will assist us to explore the great potential of extremophiles, and promote the understanding of EH formation, which will shed new insights into the biosynthesis of Er metabolites.

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

    Directory of Open Access Journals (Sweden)

    Sonti Ramesh V

    2004-10-01

    Full Text Available Abstract 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

  7. Biosynthesis of Antinutritional Alkaloids in Solanaceous Crops Is Mediated by Clustered Genes

    NARCIS (Netherlands)

    Itkin, M.; Heinig, U.; Tzfadia, O.; Bhide, A.J.; Shinde, B.; Cardenas, P.D.; Bocobza, S.E.; Unger, T.; Malitsky, S.; Finkers, H.J.; Tikunov, Y.M.; Bovy, A.G.; Chikate, Y.; Singh, P.; Rogachev, I.; Beekwilder, J.; Giri, A.P.; Aharoni, A.

    2013-01-01

    Steroidal glycoalkaloids (SGAs) such as a-solanine found in solanaceous food plants—as, for example, potato—are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthes

  8. Comparison of 454-ESTs from Huperzia serrata and Phlegmariurus carinatus reveals putative genes involved in lycopodium alkaloid biosynthesis and developmental regulation

    Directory of Open Access Journals (Sweden)

    Steinmetz André

    2010-09-01

    Full Text Available Abstract Background Plants of the Huperziaceae family, which comprise the two genera Huperzia and Phlegmariurus, produce various types of lycopodium alkaloids that are used to treat a number of human ailments, such as contusions, swellings and strains. Huperzine A, which belongs to the lycodine type of lycopodium alkaloids, has been used as an anti-Alzheimer's disease drug candidate. Despite their medical importance, little genomic or transcriptomic data are available for the members of this family. We used massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform to generate a substantial EST dataset for Huperzia serrata (H. serrata and Phlegmariurus carinatus (P. carinatus as representative members of the Huperzia and Phlegmariurus genera, respectively. H. serrata and P. carinatus are important plants for research on the biosynthesis of lycopodium alkaloids. We focused on gene discovery in the areas of bioactive compound biosynthesis and transcriptional regulation as well as genetic marker detection in these species. Results For H. serrata, 36,763 unique putative transcripts were generated from 140,930 reads totaling over 57,028,559 base pairs; for P. carinatus, 31,812 unique putative transcripts were generated from 79,920 reads totaling over 30,498,684 base pairs. Using BLASTX searches of public databases, 16,274 (44.3% unique putative transcripts from H. serrata and 14,070 (44.2% from P. carinatus were assigned to at least one protein. Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG orthology annotations revealed that the functions of the unique putative transcripts from these two species cover a similarly broad set of molecular functions, biological processes and biochemical pathways. In particular, a total of 20 H. serrata candidate cytochrome P450 genes, which are more abundant in leaves than in roots and might be involved in lycopodium alkaloid biosynthesis, were found based on the comparison of H

  9. 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

  10. Effects of Adding Vindoline and MeJA on Production of Vincristine and Vinblastine, and Transcription of their Biosynthetic Genes in the Cultured CMCs of Catharanthus roseus.

    Science.gov (United States)

    Zhang, Wenjin; Yang, Jiazeng; Zi, Jiachen; Zhu, Jianhua; Song, Liyan; Yu, Rongmin

    2015-12-01

    Vincristine and vinblastine were found by Liquid Chromatography-Mass Spectrometry (LC-MS) in Catharanthus roseuscambial meristem cells (CMCs) jointly treated with 0.25 mM vindoline and methyl jasmonate (MeJA), suggesting that C. roseus CMCs contain a complete set of the enzymes which are in response to convert vindoline into vincristine and vinblastine. Based on the facts that the transcript levels of vindoline-biosynthetic genes (STR, SGD and D4H) were up-regulated instead of being down-regulated by adding itself to the culture, and that the transcriptional factor ORCA3 was up-regulated simultaneously, we further confirmed that the transcription of STR, SGD, D4H was manipulated by ORCA3. PMID:26882673

  11. Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells

    Science.gov (United States)

    Augustin, Megan M.; Ruzicka, Dan R.; Shukla, Ashutosh K.; Augustin, Jörg M.; Starks, Courtney M.; O’Neil-Johnson, Mark; McKain, Michael R.; Evans, Bradley S.; Barrett, Matt D.; Smithson, Ann; Wong, Gane Ka-Shu; Deyholos, Michael K.; Edger, Patrick P.; Pires, J. Chris; Leebens-Mack, James H.; Mann, David A.; Kutchan, Toni M.

    2015-01-01

    Summary Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol. PMID:25939370

  12. Cloning and sequencing of the kedarcidin biosynthetic gene cluster from Streptoalloteichus sp. ATCC 53650 revealing new insights into biosynthesis of the enediyne family of antitumor antibiotics†

    Science.gov (United States)

    Lohman, Jeremy R.; Huang, Sheng-Xiong; Horsman, Geoffrey P.; Dilfer, Paul E.; Huang, Tingting; Chen, Yihua; Wendt-Pienkowski, Evelyn; Shen, Ben

    2013-01-01

    Enediyne natural product biosynthesis is characterized by a convergence of multiple pathways, generating unique peripheral moieties that are appended onto the distinctive enediyne core. Kedarcidin (KED) possesses two unique peripheral moieties, a (R)-2-aza-3-chloro-β-tyrosine and an iso-propoxy-bearing 2-naphthonate moiety, as well as two deoxysugars. The appendage pattern of these peripheral moieties to the enediyne core in KED differs from the other enediynes studied to date with respect to stereochemical configuration. To investigate the biosynthesis of these moieties and expand our understanding of enediyne core formation, the biosynthetic gene cluster for KED was cloned from Streptoalloteichus sp. ATCC 53650 and sequenced. Bioinformatics analysis of the ked cluster revealed the presence of the conserved genes encoding for enediyne core biosynthesis, type I and type II polyketide synthase loci likely responsible for 2-aza-L-tyrosine and 3,6,8-trihydroxy-2-naphthonate formation, and enzymes known for deoxysugar biosynthesis. Genes homologous to those responsible for the biosynthesis, activation, and coupling of the L-tyrosine-derived moieties from C-1027 and maduropeptin and of the naphthonate moiety from neocarzinostatin are present in the ked cluster, supporting 2-aza-L-tyrosine and 3,6,8-trihydroxy-2-naphthoic acid as precursors, respectively, for the (R)-2-aza-3-chloro-β-tyrosine and the 2-naphthonate moieties in KED biosynthesis. PMID:23360970

  13. Cloning and sequencing of the kedarcidin biosynthetic gene cluster from Streptoalloteichus sp. ATCC 53650 revealing new insights into biosynthesis of the enediyne family of antitumor antibiotics.

    Science.gov (United States)

    Lohman, Jeremy R; Huang, Sheng-Xiong; Horsman, Geoffrey P; Dilfer, Paul E; Huang, Tingting; Chen, Yihua; Wendt-Pienkowski, Evelyn; Shen, Ben

    2013-03-01

    Enediyne natural product biosynthesis is characterized by a convergence of multiple pathways, generating unique peripheral moieties that are appended onto the distinctive enediyne core. Kedarcidin (KED) possesses two unique peripheral moieties, a (R)-2-aza-3-chloro-β-tyrosine and an iso-propoxy-bearing 2-naphthonate moiety, as well as two deoxysugars. The appendage pattern of these peripheral moieties to the enediyne core in KED differs from the other enediynes studied to date with respect to stereochemical configuration. To investigate the biosynthesis of these moieties and expand our understanding of enediyne core formation, the biosynthetic gene cluster for KED was cloned from Streptoalloteichus sp. ATCC 53650 and sequenced. Bioinformatics analysis of the ked cluster revealed the presence of the conserved genes encoding for enediyne core biosynthesis, type I and type II polyketide synthase loci likely responsible for 2-aza-l-tyrosine and 3,6,8-trihydroxy-2-naphthonate formation, and enzymes known for deoxysugar biosynthesis. Genes homologous to those responsible for the biosynthesis, activation, and coupling of the l-tyrosine-derived moieties from C-1027 and maduropeptin and of the naphthonate moiety from neocarzinostatin are present in the ked cluster, supporting 2-aza-l-tyrosine and 3,6,8-trihydroxy-2-naphthoic acid as precursors, respectively, for the (R)-2-aza-3-chloro-β-tyrosine and the 2-naphthonate moieties in KED biosynthesis. PMID:23360970

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Woo Tae Park

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

  17. Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis.

    Science.gov (United States)

    Salim, Vonny; Yu, Fang; Altarejos, Joaquín; De Luca, Vincenzo

    2013-12-01

    Iridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g(-1) fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus. PMID:24103035

  18. Alkaloids from Delphinium pentagynum.

    Science.gov (United States)

    Díaz, Jesús G; Ruiz, Juan García; Herz, Werner

    2004-07-01

    Aerial parts of a collection of Delphinium pentagynum Lam. from Niebla, Southern Spain, furnished one diterpene alkaloid, 2-dehydrodeacetylheterophylloidine, two norditerpene alkaloids, 14-demethyl-14-isobutyrylanhweidelphinine and 14-demethyl-14-acetylanhweidelphinine, the known alkaloids 14-deacetylnudicauline, methyllycaconitine, 14-deacetyl-14-isobutyrylnudicauline, 14-acetylbrowniine, browniine, delcosine, lycoctonine, 18-methoxygadesine, neoline, karakoline and the aporphine alkaloid magnoflorine. Structures of the alkaloids were established by MS, 1D and 2-D NMR techniques.

  19. 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

    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...

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Identification and expression analysis of glucosinolate biosynthetic genes and estimation of glucosinolate contents in edible organs of Brassica oleracea subspecies.

    Science.gov (United States)

    Yi, Go-Eun; Robin, Arif Hasan Khan; Yang, Kiwoung; Park, Jong-In; Kang, Jong-Goo; Yang, Tae-Jin; Nou, Ill-Sup

    2015-01-01

    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. PMID:26205053

  2. 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...

  3. Endocytotic uptake, processing, and retroendocytosis of human biosynthetic proinsulin by rat fibroblasts transfected with the human insulin receptor gene.

    OpenAIRE

    Levy, J R; Ullrich, A; Olefsky, J M

    1988-01-01

    The cellular itinerary and processing of insulin and proinsulin were studied to elucidate possible mechanisms for the observed in vivo differences in the biologic half-lives of these two hormones. A rat fibroblast cell line transfected with a normal human insulin receptor gene was used. Due to gene amplification, the cells express large numbers of receptors and are ideal for studying a ligand, such as proinsulin, that has a low affinity for the insulin receptor. Competitive binding at 4 degre...

  4. 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.

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

    Science.gov (United States)

    Robin, Arif Hasan Khan; Yi, Go-Eun; Laila, Rawnak; Yang, Kiwoung; Park, Jong-In; Kim, Hye Ran; Nou, Ill-Sup

    2016-01-01

    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 glucosinolates detected

  7. Phytochemical genomics of the Madagascar periwinkle: Unravelling the last twists of the alkaloid engine.

    Science.gov (United States)

    Dugé de Bernonville, Thomas; Clastre, Marc; Besseau, Sébastien; Oudin, Audrey; Burlat, Vincent; Glévarec, Gaëlle; Lanoue, Arnaud; Papon, Nicolas; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Courdavault, Vincent

    2015-05-01

    The Madagascar periwinkle produces a large palette of Monoterpenoid Indole Alkaloids (MIAs), a class of complex alkaloids including some of the most valuable plant natural products with precious therapeutical values. Evolutionary pressure on one of the hotspots of biodiversity has obviously turned this endemic Malagasy plant into an innovative alkaloid engine. Catharanthus is a unique taxon producing vinblastine and vincristine, heterodimeric MIAs with complex stereochemistry, and also manufactures more than 100 different MIAs, some shared with the Apocynaceae, Loganiaceae and Rubiaceae members. For over 60 years, the quest for these powerful anticancer drugs has inspired biologists, chemists, and pharmacists to unravel the chemistry, biochemistry, therapeutic activity, cell and molecular biology of Catharanthus roseus. Recently, the "omics" technologies have fuelled rapid progress in deciphering the last secret of strictosidine biosynthesis, the central precursor opening biosynthetic routes to several thousand MIA compounds. Dedicated C. roseus transcriptome, proteome and metabolome databases, comprising organ-, tissue- and cell-specific libraries, and other phytogenomic resources, were developed for instance by PhytoMetaSyn, Medicinal Plant Genomic Resources and SmartCell consortium. Tissue specific library screening, orthology comparison in species with or without MIA-biochemical engines, clustering of gene expression profiles together with various functional validation strategies, largely contributed to enrich the toolbox for plant synthetic biology and metabolic engineering of MIA biosynthesis. PMID:25146650

  8. Deep Sequencing of the Scutellaria baicalensis Georgi Transcriptome Reveals Flavonoid Biosynthetic Profiling and Organ-Specific Gene Expression.

    Directory of Open Access Journals (Sweden)

    Jinxin Liu

    Full Text Available Scutellaria baicalensis Georgi has long been used in traditional medicine to treat various such widely varying diseases and has been listed in the Chinese Pharmacopeia, the Japanese Pharmacopeia, the Korean Pharmacopoeia and the European Pharmacopoeia. Flavonoids, especially wogonin, wogonoside, baicalin, and baicalein, are its main functional ingredients with various pharmacological activities. Although pharmaological studies for these flavonoid components have been well conducted, the molecular mechanism of their biosynthesis remains unclear in S. baicalensis. In this study, Illumina/Solexa deep sequencing generated more than 91 million paired-end reads and 49,507 unigenes from S. baicalensis roots, stems, leaves and flowers. More than 70% unigenes were annotated in at least one of the five public databases and 13,627 unigenes were assigned to 3,810 KEGG genes involved in 579 different pathways. 54 unigenes that encode 12 key enzymes involved in the pathway of flavonoid biosynthesis were discovered. One baicalinase and three baicalein 7-O-glucuronosyltransferases genes potentially involved in the transformation between baicalin/wogonoside and baicalein/wogonin were identified. Four candidate 6-hydroxylase genes for the formation of baicalin/baicalein and one candidate 8-O-methyltransferase gene for the biosynthesis of wogonoside/wogonin were also recognized. Our results further support the conclusion that, in S. baicalensis, 3,5,7-trihydroxyflavone was the precursor of the four above compounds. Then, the differential expression models and simple sequence repeats associated with these genes were carefully analyzed. All of these results not only enrich the gene resource but also benefit research into the molecular genetics and functional genomics in S. baicalensis.

  9. Biosynthetic Gene Cluster of Cetoniacytone A, an Unusual Aminocyclitol from the Endosymbiotic Bacterium Actinomyces sp. Lu 9419

    OpenAIRE

    Wu, Xiumei; Flatt, Patricia M.; Xu, Hui; Mahmud, Taifo

    2009-01-01

    A gene cluster responsible for the biosynthesis of the antitumor agent cetoniacytone A was identified in Actinomyces sp. strain Lu 9419, an endosymbiotic bacteria isolated from the intestines of the rose chafer beetle (Cetonia aurata). The nucleotide sequence analysis of the 46 kb DNA region revealed the presence of 31 complete ORFs, including genes predicted to encode a 2-epi-5-epi-valiolone synthase (CetA), a glyoxalase/bleomycin resistance protein (CetB), an acyltransferase (CetD), an FAD-...

  10. Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

    Directory of Open Access Journals (Sweden)

    Schriemer David C

    2010-11-01

    Full Text Available Abstract Background Papaver somniferum (opium poppy is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. Results A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. Conclusions The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a

  11. Identification of Quorum-Sensing Signal Molecules and a Biosynthetic Gene in Alicycliphilus sp. Isolated from Activated Sludge.

    Science.gov (United States)

    Morohoshi, Tomohiro; Okutsu, Noriya; Xie, Xiaonan; Ikeda, Tsukasa

    2016-01-01

    Activated sludge is a complicated mixture of various microorganisms that is used to treat sewage and industrial wastewater. Many bacteria produce N-acylhomoserine lactone (AHL) as a quorum-sensing signal molecule to regulate the expression of the exoenzymes used for wastewater treatment. Here, we isolated an AHL-producing bacteria from an activated sludge sample collected from an electronic component factory, which we named Alicycliphilus sp. B1. Clone library analysis revealed that Alicycliphilus was a subdominant genus in this sample. When we screened the activated sludge sample for AHL-producing strains, 12 of 14 the AHL-producing isolates were assigned to the genus Alicycliphilus. A putative AHL-synthase gene, ALISP_0667, was cloned from the genome of B1 and transformed into Escherichia coli DH5α. The AHLs were extracted from the culture supernatants of the B1 strain and E. coli DH5α cells harboring the ALISP_0667 gene and were identified by liquid chromatography-mass spectrometry as N-(3-hydroxydecanoyl)-l-homoserine lactone and N-(3-hydroxydodecanoyl)-l-homoserine lactone. The results of comparative genomic analysis suggested that the quorum-sensing genes in the B1 strain might have been acquired by horizontal gene transfer within activated sludge. PMID:27490553

  12. 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.

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

    OpenAIRE

    Royah Vaezi; Napier, Johnathan A.; Olga Sayanova

    2013-01-01

    In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative “front-end” desaturases (Δ6 and Δ4) from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of...

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

    OpenAIRE

    Go-Eun Yi; Arif Hasan Khan Robin; Kiwoung Yang; Jong-In Park; Jong-Goo Kang; Tae-Jin Yang; Ill-Sup Nou

    2015-01-01

    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 t...

  15. Differential Expression of Anthocyanin Biosynthetic Genes in Relation to Anthocyanin Accumulation in the Pericarp of Litchi Chinensis Sonn

    OpenAIRE

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

    2011-01-01

    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...

  16. Organization, Evolution, and Expression Analysis of the Biosynthetic Gene Cluster for Scytonemin, a Cyanobacterial UV-Absorbing Pigment▿ †

    OpenAIRE

    Sorrels, Carla M.; Proteau, Philip J.; Gerwick, William H.

    2009-01-01

    Cyanobacteria are photosynthetic prokaryotes capable of protecting themselves from UV radiation through the biosynthesis of UV-absorbing secondary metabolites, such as the mycosporines and scytonemin. Scytonemin, a novel indolic-phenolic pigment, is found sequestered in the sheath, where it provides protection to the subtending cells during exposure to UV radiation. The biosynthesis of scytonemin is encoded by a previously identified gene cluster that is present in six cyanobacterial species ...

  17. 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.

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

    Science.gov (United States)

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

    2015-09-01

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

  19. Total synthesis, biosynthesis and biological profiles of clavine alkaloids.

    Science.gov (United States)

    McCabe, Stephanie R; Wipf, Peter

    2016-07-01

    This review highlights noteworthy synthetic and biological aspects of the clavine subfamily of ergot alkaloids. Recent biosynthetic insights have laid the groundwork for a better understanding of the diverse biological pathways leading to these indole derivatives. Ergot alkaloids were among the first fungal-derived natural products identified, inspiring pharmaceutical applications in CNS disorders, migraine, infective diseases, and cancer. Pergolide, for example, is a semi-synthetic clavine alkaloid that has been used to treat Parkinson's disease. Synthetic activities have been particularly valuable to facilitate access to rare members of the Clavine family and empower medicinal chemistry research. Improved molecular target identification tools and a better understanding of signaling pathways can now be deployed to further extend the biological and medical utility of Clavine alkaloids. PMID:27215547

  20. 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. PMID:25734328

  1. 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.

  2. Biosynthesis of ambiguine indole alkaloids in cyanobacterium Fischerella ambigua.

    Science.gov (United States)

    Hillwig, Matthew L; Zhu, Qin; Liu, Xinyu

    2014-02-21

    Ambiguines belong to a family of hapalindole-type indole alkaloid natural products, with many of the members possessing up to eight consecutive carbon stereocenters in a fused pentacyclic 6-6-6-5-7 ring scaffold. Here, we report the identification of a 42 kbp ambiguine (amb) biosynthetic gene cluster that harbors 32 protein-coding genes in its native producer Fischerella ambigua UTEX1903. Association of the amb cluster with ambiguine biosynthesis was confirmed by both bioinformatic analysis and in vitro characterizations of enzymes responsible for 3-((Z)-2'-isocyanoethenyl) indole and geranyl pyrophosphate biosynthesis and a C-2 indole dimethylallyltransferase that regiospecifically tailors hapalindole G to ambiguine A. The presence of five nonheme iron-dependent oxygenase coding genes (including four Rieske-type oxygenases) within the amb cluster suggests late-stage C-H activations are likely responsible for the structural diversities of ambiguines by regio- and stereospecific chlorination, hydroxylation, epoxidation, and sp(2)-sp(3) C-C bond formation. PMID:24180436

  3. Cytotoxicity, genotoxicity and mechanism of action (via gene expression analysis) of the indole alkaloid aspidospermine (antiparasitic) extracted from Aspidosperma polyneuron in HepG2 cells.

    Science.gov (United States)

    Coatti, Giuliana Castello; Marcarini, Juliana Cristina; Sartori, Daniele; Fidelis, Queli Cristina; Ferreira, Dalva Trevisan; Mantovani, Mário Sérgio

    2016-08-01

    Aspidospermine is an indole alkaloid with biological properties associated with combating parasites included in the genera Plasmodium, Leishmania and Trypanossoma. The present study evaluated the cytotoxicity (resazurin test), genotoxicity (comet assay) and mechanism of action (gene expression analysis via qRT-PCR) of this alkaloid in human HepG2 cells. The results demonstrated that treatment with aspidospermine was both cytotoxic (starting at 75 μM) and genotoxic (starting at 50 μM). There was no significant modulation of the expression of the following genes: GSTP1 and GPX1 (xenobiotic metabolism); CAT (oxidative stress); TP53 and CCNA2 (cell cycle); HSPA5, ERN1, EIF2AK3 and TRAF2 (endoplasmic reticulum stress); CASP8, CASP9, CASP3, CASP7, BCL-2, BCL-XL BAX and BAX (apoptosis); and PCBP4, ERCC4, OGG1, RAD21 and MLH1 (DNA repair). At a concentration of 50 μM (non-cytotoxic, but genotoxic), there was a significant increase in the expression of CYP1A1 (xenobiotic metabolism) and APC (cell cycle), and at a concentration of 100 μM, a significant increase in the expression of CYP1A1 (xenobiotic metabolism), GADD153 (endoplasmic reticulum stress) and SOD (oxidative stress) was detected, with repression of the expression of GR (xenobiotic metabolism and oxidative stress). The results of treatment with aspidospermine at a 100 μM concentration (the dose indicated in the literature to achieve 89 % reduction of the growth of L. amazonensis) suggest that increased oxidative stress and an unfolded protein response (UPR) occurred in HepG2 cells. For the therapeutic use of aspidospermine (antiparasitic), chemical alteration of the molecule to achieve a lower cytotoxicity/genotoxicity in host cells is recommended. PMID:25894792

  4. 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.

  5. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis

    Science.gov (United States)

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKYY115E phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  6. Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world.

    Science.gov (United States)

    Hagel, Jillian M; Facchini, Peter J

    2013-05-01

    Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant specialized metabolites with a long history of investigation. Although the ecophysiological functions of most BIAs are unknown, the medicinal properties of many compounds have been exploited for centuries. These include the narcotic analgesics codeine and morphine, the antimicrobial agents sanguinarine and berberine, and the antitussive and anticancer drug noscapine. BIA biosynthesis involves a restricted number of enzyme types that catalyze landmark coupling reactions and subsequent functional group modifications. A pathogenesis-related (PR)10/Bet v1 'Pictet-Spenglerase', several O-methyl-, N-methyl- and O-acetyltransferases, cytochromes P450, FAD-dependent oxidases, non-heme dioxygenases and NADPH-dependent reductases have been implicated in the multistep pathways leading to structurally diverse alkaloids. A small number of plant species, including opium poppy (Papaver somniferum) and other members of the Ranunculales, have emerged as model systems to study BIA metabolism. The expansion of resources to include a wider range of plant species is creating an opportunity to investigate previously uncharacterized BIA pathways. Contemporary knowledge of BIA metabolism reflects over a century of research coupled with the development of key innovations such as radioactive tracing, enzyme isolation and molecular cloning, and functional genomics approaches such as virus-induced gene silencing. Recently, the emergence of transcriptomics, proteomics and metabolomics has expedited the discovery of new BIA biosynthetic genes. The growing repository of BIA biosynthetic genes is providing the parts required to apply emerging synthetic biology platforms to the development of production systems in microbes as an alternative to plants as a commecial source of valuable BIAs.

  7. 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.

  8. 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.

  9. Taichunamides: Prenylated Indole Alkaloids from Aspergillus taichungensis (IBT 19404)

    DEFF Research Database (Denmark)

    Kagiyama, Ippei; Kato, Hikaru; Nehira, Tatsuo;

    2016-01-01

    Seven new prenylated indole alkaloids, taichunamides A–G, were isolated from the fungus Aspergillus taichungensis (IBT 19404). Taichunamides A and B contained an azetidine and 4‐pyridone units, respectively, and are likely biosynthesized from notoamide S via (+)‐6‐epi‐stephacidin A. Taichunamides C...... and D contain endoperoxide and methylsulfonyl units, respectively. This fungus produced indole alkaloids containing an anti‐bicyclo[2.2.2]diazaoctane core, whereas A. protuberus and A. amoenus produced congeners with a syn‐bicyclo[2.2.2]diazaoctane core. Plausible biosynthetic pathways to access...

  10. HPLC-ESI-MS/MS of Imidazole Alkaloids in Pilocarpus microphyllus

    OpenAIRE

    Sawaya, Alexandra; Abreu, Ilka Nacif; Andreazza, Nathalia Luiza; Marcos N Eberlin; Mazzafera, Paulo

    2008-01-01

    Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae), known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MSn was developed for their qualitative and quantitative analysis. This meth...

  11. Alkaloids in Marine Algae

    OpenAIRE

    Ekrem Sezik; Aline Percot; Kasım Cemal Güven

    2010-01-01

    This paper presents the alkaloids found in green, brown and red marine algae. Algal chemistry has interested many researchers in order to develop new drugs, as algae include compounds with functional groups which are characteristic from this particular source. Among these compounds, alkaloids present special interest because of their pharmacological activities. Alkaloid chemistry has been widely studied in terrestrial plants, but the number of studies in algae is insignificant. In this review...

  12. Reviewing colchicaceae alkaloids – perspectives of evolution on medicinal chemistry

    DEFF Research Database (Denmark)

    Larsson, Sonny; Rønsted, Nina

    2014-01-01

    . In this review an approach of taking phylogenetic classification into account in evaluating colchicine and related phenethylisoquinoline alkaloids from the family Colchicaceae will be applied. Following on the trends of utilizing evolutionary reasoning in inferring mechanisms in eg. drug resistance in cancer...... and infections, this will exemplify how thinking about evolution can influence selection of plant material in drug lead discovery, and how knowledge about phylogenetic relationships may be used to evaluate predicted biosynthetic pathways...

  13. Antimalarial diterpene alkaloids from the seeds of Caesalpinia minax.

    Science.gov (United States)

    Ma, Guoxu; Sun, Zhaocui; Sun, Zhonghao; Yuan, Jingquan; Wei, Hua; Yang, Junshan; Wu, Haifeng; Xu, Xudong

    2014-06-01

    Two new diterpene alkaloids, caesalminines A (1) and B (2), possessing a tetracyclic cassane-type furanoditerpenoid skeleton with γ-lactam ring, were isolated from the seeds of Caesalpinia minax. Their structures were determined by different spectroscopic methods and ECD calculation. The plausible biosynthetic pathway of caesalminines A and B was proposed. The anti-malarial activity of compounds 1 and 2 is presented with IC50 values of 0.42 and 0.79 μM, respectively.

  14. Biosynthesis and regulation of terpenoid indole alkaloids in Catharanthus roseus

    OpenAIRE

    Jianhua Zhu; Mingxuan Wang; Wei Wen; Rongmin Yu

    2015-01-01

    Catharanthus roseus produces a wide range of terpenoid indole alkaloids (TIA). Many of them, such as vinblastine and vincristine, have significant bioactivity. They are valuable chemotherapy drugs used in combination with other drugs to treat lymphoma and leukemia. The TIA biosynthetic pathway has been investigated for many years, for scientific interest and for their potential in manufacturing applications, to fulfill the market demand. In this review, the progress and perspective of C. rose...

  15. Reviewing Colchicaceae Alkaloids – Perspectives of Evolution on Medicinal Chemistry

    OpenAIRE

    Larsson, Sonny; Rønsted, Nina

    2014-01-01

    The subject of chemosystematics has provided insight to both botanical classification and drug development. However, degrees of subjectivity in botanical classifications and limited understanding of the evolution of chemical characters and their biosynthetic pathways has often hampered such studies. In this review an approach of taking phylogenetic classification into account in evaluating colchicine and related phenethylisoquinoline alkaloids from the family Colchicaceae will be applied. Fol...

  16. 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.

  17. A look inside an alkaloid multisite plant: the Catharanthus logistics.

    Science.gov (United States)

    Courdavault, Vincent; Papon, Nicolas; Clastre, Marc; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Burlat, Vincent

    2014-06-01

    Environmental pressures forced plants to diversify specialized metabolisms to accumulate noxious molecules such as alkaloids constituting one of the largest classes of defense metabolites. Catharanthus roseus produces monoterpene indole alkaloids via a highly elaborated biosynthetic pathway whose characterization greatly progressed with the recent expansion of transcriptomic resources. The complex architecture of this pathway, sequentially distributed in at least four cell types and further compartmentalized into several organelles, involves partially identified inter-cellular and intra-cellular translocation events acting as potential key-regulators of metabolic fluxes. The description of this spatial organization and the inherent secretion and sequestration of metabolites not only provide new insight into alkaloid cell biology and its involvement in plant defense processes but also present new biotechnological challenges for synthetic biology. PMID:24727073

  18. Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: overproduction of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene.

    OpenAIRE

    Sá-Correia, I.; Darzins, A; Wang, S K; Berry, A.; Chakrabarty, A M

    1987-01-01

    The specific activities of phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP), and GDP-mannose dehydrogenase (GMD) were compared in a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa and in two spontaneous nonmucoid revertants. In both revertants some or all of the alginate biosynthetic enzymes we examined appeared to be repressed, indicating that the loss of the mucoid phenotype may be a result of decreased formation of sugar-nucleotide prec...

  19. [Alkaloids of Annonaceae. XXIX. Alkaloids of Annona muricata].

    Science.gov (United States)

    Leboeuf, M; Legueut, C; Cavé, A; Desconclois, J F; Forgacs, P; Jacquemin, H

    1981-05-01

    From leaves, root - and stem - barks of Annona muricata L., seven isoquinoline alkaloids have been isolated: reticuline (main alkaloid), coclaurine, coreximine, atherosperminine, stepharine. Anomurine and anomuricine, two minor alkaloids, are new tetrahydrobenzylisoquinolines, with 5, 6, 7 substituted ring A. The phytochemical significance of these alkaloids is discussed.

  20. HPLC-ESI-MS/MS of Imidazole Alkaloids in Pilocarpus microphyllus

    Directory of Open Access Journals (Sweden)

    Paulo Mazzafera

    2008-07-01

    Full Text Available Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae, known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MSn was developed for their qualitative and quantitative analysis. This method permits the chromatographic separation of the imidazole alkaloids found in extracts of jaborandi, as well as the MS/MS analysis of the individual compounds. Thus two samples: leaves of P. microphyllus and a paste that is left over after the industrial extraction of pilocarpine; were compared. The paste was found to contain significant amounts of pilocarpine and other imidazole alkaloids, but had a slightly different alkaloid profile than the leaf extract. The method is suitable for the routine analysis of samples containing these alkaloids, as well as for the separation and identification of known and novel alkaloids from this family, and may be applied to further studies of the biosynthetic pathway of pilocarpine in P. microphyllus.

  1. HPLC-ESI-MS/MS of imidazole alkaloids in Pilocarpus microphyllus.

    Science.gov (United States)

    Sawaya, Alexandra C H F; Abreu, Ilka Nacif; Andreazza, Nathalia Luiza; Eberlin, Marcos N; Mazzafera, Paulo

    2008-01-01

    Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae), known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MS(n) was developed for their qualitative and quantitative analysis. This method permits the chromatographic separation of the imidazole alkaloids found in extracts of jaborandi, as well as the MS/MS analysis of the individual compounds. Thus two samples: leaves of P. microphyllus and a paste that is left over after the industrial extraction of pilocarpine; were compared. The paste was found to contain significant amounts of pilocarpine and other imidazole alkaloids, but had a slightly different alkaloid profile than the leaf extract. The method is suitable for the routine analysis of samples containing these alkaloids, as well as for the separation and identification of known and novel alkaloids from this family, and may be applied to further studies of the biosynthetic pathway of pilocarpine in P. microphyllus. PMID:18719522

  2. Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus1

    Science.gov (United States)

    Whitmer, Serap; Canel, Camilo; Hallard, Didier; Gonçalves, Cecilia; Verpoorte, Robert

    1998-01-01

    We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures. PMID:9490777

  3. Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors.

    Science.gov (United States)

    Carqueijeiro, I; Masini, E; Foureau, E; Sepúlveda, L J; Marais, E; Lanoue, A; Besseau, S; Papon, N; Clastre, M; Dugé de Bernonville, T; Glévarec, G; Atehortùa, L; Oudin, A; Courdavault, V

    2015-11-01

    Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses. PMID:26284695

  4. 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

  5. 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. PMID:26513252

  6. Cloning and reconstitution of rhamose and forosamine biosynthetic gene of Saccharopolyspora spinosa NRRL18395%刺糖多孢菌鼠李糖和福乐糖胺合成基因的克隆和组装

    Institute of Scientific and Technical Information of China (English)

    郭航; 白亭丽; 陶美凤

    2012-01-01

    Rhamnose and forosamine are two essential deoxysugars components of spinosad. Four genes involved in the biosynthesis of rhamnose and forosamine, I. E. Gtt (encoding NDP-glucose syn-thase) ,gdh (encoding NDP-glucose dehydratase), epi (encoding epimerase) and kre (encoding 4 -ke-toreductase),are not located together with the spinosad biosynthetic gene cluster, but distributed in 3 scattered loci in the chromosome of Saccharopolyspora spinosa NRRL18395. To get an integral spinosad biosynthetic pathway, the four genes were cloned by constructing a genomic library and PCR amplifying from S. Spinosa NRRL18395. The 4 genes were successively assembled into an integrative vector to construct an expression vector for biosynthesis of rhamnose and forosamine in heterologous hosts.%鼠李糖和福乐糖胺是多杀菌素生物合成必需的2个脱氧糖结构单元,负责其合成的4种酶的基因(gtt、epi、gdh、kre)与多杀菌素生物合成基因簇并不在一起,而是分散分布在染色体的3个位点上.为克隆到完整的多杀菌素生物合成基因簇,采用构建基因组文库、PCR扩增等手段从刺糖多孢菌NRRL18395染色体分别克隆gtt、epi、gdh和kre基因,并将其克隆组装在同一整合型载体上,以便于构建用于表达多杀菌素糖单元合成基因的表达载体.

  7. Novel Euglenoid Derived Alkaloid

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Disclosed herein is a purified toxin isolated from Euglena sanguinea. More specifically the toxin, termed euglenophycin, is an alkaloid having herbicidal and...

  8. Occurrence of halogenated alkaloids.

    Science.gov (United States)

    Gribble, Gordon W

    2012-01-01

    Once considered to be isolation artifacts or chemical "mistakes" of nature, the number of naturally occurring organohalogen compounds has grown from a dozen in 1954 to >5000 today. Of these, at least 25% are halogenated alkaloids. This is not surprising since nitrogen-containing pyrroles, indoles, carbolines, tryptamines, tyrosines, and tyramines are excellent platforms for biohalogenation, particularly in the marine environment where both chloride and bromide are plentiful for biooxidation and subsequent incorporation into these electron-rich substrates. This review presents the occurrence of all halogenated alkaloids, with the exception of marine bromotyrosines where coverage begins where it left off in volume 61 of The Alkaloids. Whereas the biological activity of these extraordinary compounds is briefly cited for some examples, a future volume of The Alkaloids will present full coverage of this topic and will also include selected syntheses of halogenated alkaloids. Natural organohalogens of all types, especially marine and terrestrial halogenated alkaloids, comprise a rapidly expanding class of natural products, in many cases expressing powerful biological activity. This enormous proliferation has several origins: (1) a revitalization of natural product research in a search for new drugs, (2) improved compound characterization methods (multidimensional NMR, high-resolution mass spectrometry), (3) specific enzyme-based and other biological assays, (4) sophisticated collection methods (SCUBA and remote submersibles for deep ocean marine collections), (5) new separation and purification techniques (HPLC and countercurrent separation), (6) a greater appreciation of traditional folk medicine and ethobotany, and (7) marine bacteria and fungi as novel sources of natural products. Halogenated alkaloids are truly omnipresent in the environment. Indeed, one compound, Q1 (234), is ubiquitous in the marine food web and is found in the Inuit from their diet of whale

  9. Marine Indole Alkaloids

    Directory of Open Access Journals (Sweden)

    Natalie Netz

    2015-08-01

    Full Text Available Marine indole alkaloids comprise a large and steadily growing group of secondary metabolites. Their diverse biological activities make many compounds of this class attractive starting points for pharmaceutical development. Several marine-derived indoles were found to possess cytotoxic, antineoplastic, antibacterial and antimicrobial activities, in addition to the action on human enzymes and receptors. The newly isolated indole alkaloids of marine origin since the last comprehensive review in 2003 are reported, and biological aspects will be discussed.

  10. Biosynthetic engineering of nonribosomal peptide synthetases.

    Science.gov (United States)

    Kries, Hajo

    2016-09-01

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

  11. Activation of the cryptic secondary metabolite biosynthetic gene clusters (CSMGs) in Streptomyces%链霉菌隐性次级代谢产物生物合成基因簇的激活

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    链霉菌基因组中存在许多隐性次级代谢产物生物合成基因簇(cryptic secondary metabolite biosynthetic gene cluster,CSMG),在实验室条件下,它们多数处于沉默状态或者很低的表达水平.但在一定条件下这些CSMG可以被激活合成次级代谢产物,这为寻找新的活性天然产物提供新来源.本文总结了目前激活链霉菌CSMG所使用的方法,包括改变发酵条件、核糖体工程、共培养、异源表达、CSR(cluster-situated regulator)基因的遗传改造等.

  12. Analysis of Ergot Alkaloids

    Directory of Open Access Journals (Sweden)

    Colin Crews

    2015-06-01

    Full Text Available The principles and application of established and newer methods for the quantitative and semi-quantitative determination of ergot alkaloids in food, feed, plant materials and animal tissues are reviewed. The techniques of sampling, extraction, clean-up, detection, quantification and validation are described. The major procedures for ergot alkaloid analysis comprise liquid chromatography with tandem mass spectrometry (LC-MS/MS and liquid chromatography with fluorescence detection (LC-FLD. Other methods based on immunoassays are under development and variations of these and minor techniques are available for specific purposes.

  13. 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.

  14. 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.

  15. 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

  16. Biogenetically inspired synthesis and skeletal diversification of indole alkaloids.

    Science.gov (United States)

    Mizoguchi, Haruki; Oikawa, Hideaki; Oguri, Hiroki

    2014-01-01

    To access architecturally complex natural products, chemists usually devise a customized synthetic strategy for constructing a single target skeleton. In contrast, biosynthetic assembly lines often employ divergent intramolecular cyclizations of a polyunsaturated common intermediate to produce diverse arrays of scaffolds. With the aim of integrating such biogenetic strategies, we show the development of an artificial divergent assembly line generating unprecedented numbers of scaffold variations of terpenoid indole alkaloids. This approach not only allows practical access to multipotent intermediates, but also enables systematic diversification of skeletal, stereochemical and functional group properties without structural simplification of naturally occurring alkaloids. Three distinct modes of [4+2] cyclizations and two types of redox-mediated annulations provided divergent access to five skeletally distinct scaffolds involving iboga-, aspidosperma-, andranginine- and ngouniensine-type skeletons and a non-natural variant within six to nine steps from tryptamine. The efficiency of our approach was demonstrated by successful total syntheses of (±)-vincadifformine, (±)-andranginine and (-)-catharanthine.

  17. Isolation, biosynthesis and biological activity of alkaloids of Tylophora asthmatica, a versatile medicinal plant

    International Nuclear Information System (INIS)

    Tylophorine and related new alkaloids have been isolated from Tylophora asthmatics, Pergularia pallida and Ficus hispida plants. Biosynthesis of this group of alkaloids has been carried out using various labelled precursors for the first time and from the systematic degradation of the isolated radiolabelled tylophorine, it has been concluded that these alkaloids arise from one molecule each of tyrosine, phenylalanine and ornithine. The interactions of Tylophora alkaloids particularly tylophorinidine with biomolecules such as lysozyme and bovine serum albumin have also been studied and binding characteristics determined. It was found that Tylophora alkaloid extract possesses antianaphylactic activity as observed in passive peritoneal anaphylaxis in rats. The drug also possessed mild antihistaminic and anticholinergic activities. Studies of the extract on the bronchial smooth muscle both in vivo and in vitro did not reveal bronchiodilator potential of the drug. In addition, the distribution and metabolism of the drug was studied in vivo using 14C radiolabelled alkaloids prepared by biosynthetic method. This study further revealed its usefulness since the drug is absorbed by vital organs and also it is not metabolised into fragments which could cause some other damage. Tylophora alkaloids have also been found to be anti-mutagenic. 10 tables, 5 figures, 24 refs. (author)

  18. Cloning, sequencing, and characterization of the lipopolysaccharide biosynthetic enzyme heptosyltransferase I gene (waaC) from Campylobacter jejuni and Campylobacter coli.

    Science.gov (United States)

    Klena, J D; Gray, S A; Konkel, M E

    1998-11-19

    Campylobacter jejuni and Campylobacter coli are common causes of gastrointestinal disease and a proportion of C. jejuni infections have been shown to be associated with the Guillain-Barré syndrome. The waaC gene from Campylobacter coli, involved in lipopolysaccharide core biosynthesis, was cloned by complementation of a heptose-deficient strain of Salmonella typhimurium, as judged by novobiocin sensitivity, lipopolysaccharide (LPS)-specific phage sensitivity, and polyacrylamide-resolved lipopolysaccharide profiles. The C. jejuni waaC gene was subsequently cloned using the waaC gene isolated from C. coli as a probe. The C. jejuni and C. coli waaC genes are capable of encoding proteins of 342 amino acids with calculated molecular masses of 39381Da and 39317Da, respectively. Sequence and in-vitro analyses suggested that the C. coli waaC gene may be transcribed from its own promoter. Translation of the C. coli waaC gene in a cell-free system yielded a protein with a Mr of 39000. The waaC gene was detected in every C. jejuni and C. coli isolate tested as judged by dot-blot hybridization analysis. Southern hybridization analysis indicated that both Campylobacter species contain a single copy of the waaC gene. Unlike Escherichia coli and S. typhimurium isolates, the waaC gene in C. jejuni and C. coli isolates does not appear to be linked to the waaF (rfaF) gene.

  19. Simple Indolizidine and Quinolizidine Alkaloids.

    Science.gov (United States)

    Michael, Joseph P

    2016-01-01

    This review of simple indolizidine and quinolizidine alkaloids (i.e., those in which the parent bicyclic systems are in general not embedded in polycyclic arrays) is an update of the previous coverage in Volume 55 of this series (2001). The present survey covers the literature from mid-1999 to the end of 2013; and in addition to aspects of the isolation, characterization, and biological activity of the alkaloids, much emphasis is placed on their total synthesis. A brief introduction to the topic is followed by an overview of relevant alkaloids from fungal and microbial sources, among them slaframine, cyclizidine, Steptomyces metabolites, and the pantocins. The important iminosugar alkaloids lentiginosine, steviamine, swainsonine, castanospermine, and related hydroxyindolizidines are dealt with in the subsequent section. The fourth and fifth sections cover metabolites from terrestrial plants. Pertinent plant alkaloids bearing alkyl, functionalized alkyl or alkenyl substituents include dendroprimine, anibamine, simple alkaloids belonging to the genera Prosopis, Elaeocarpus, Lycopodium, and Poranthera, and bicyclic alkaloids of the lupin family. Plant alkaloids bearing aryl or heteroaryl substituents include ipalbidine and analogs, secophenanthroindolizidine and secophenanthroquinolizidine alkaloids (among them septicine, julandine, and analogs), ficuseptine, lasubines, and other simple quinolizidines of the Lythraceae, the simple furyl-substituted Nuphar alkaloids, and a mixed quinolizidine-quinazoline alkaloid. The penultimate section of the review deals with the sizable group of simple indolizidine and quinolizidine alkaloids isolated from, or detected in, ants, mites, and terrestrial amphibians, and includes an overview of the "dietary hypothesis" for the origin of the amphibian metabolites. The final section surveys relevant alkaloids from marine sources, and includes clathryimines and analogs, stellettamides, the clavepictines and pictamine, and bis

  20. 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.

  1. 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.

  2. 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

  3. "Self" and "non-self" in the control of phytoalexin biosynthesis: plant phospholipases A2 with alkaloid-specific molecular fingerprints.

    Science.gov (United States)

    Heinze, Michael; Brandt, Wolfgang; Marillonnet, Sylvestre; Roos, Werner

    2015-02-01

    The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the "self" plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate "self-made" from "foreign" alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins. PMID:25670767

  4. 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.

  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. PMID:26168138

  6. Cloning and Characterization of Genes Encoded in 187 dTDP-D-mycaminose Biosynthetic Pathway from a Midecamycin-producing Strain,Streptomyces mycarofaciens

    Institute of Scientific and Technical Information of China (English)

    Lina CONG; Wolfgang PIEPERSBERG

    2007-01-01

    Two subclusters from Streptomyces mycarofaciens,a midecamycin producer,were cloned and partially sequenced.One region was located at the 5'end of the mid polyketide synthase (PKS) genes and contained the genes midA,midB and midC.The other region was at the 3'end of the PKS genes and contained midK,midI and midH.Analysis of the nucleotide sequence revealed that these genes encode dTDP-glucose synthase (midA),dTDP-glucose dehydratase(midB),aminotransferase (midC),methyltransferase (midK),glycosyltransferase(midI)and an assistant gene(midH).All of these genes are involved in the biosynthesis of dTDP-D-mycaminose,the first deoxysugar of midecamycin,and in transferring the mycaminose to the midecamycin aglycone in S.mycarofaciens.Similar to gene pairs des VIII/des VII in S.venezuelae and tylMIII/tylMII in S.fradiae,the product of midH probably functions as an auxiliary protein required by the MidI protein for efficient glycosyltransfer in midecamycin biosynthesis.

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

    OpenAIRE

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

    2016-01-01

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

  8. Functional expression of the FeMo-cofactor-specific biosynthetic genes nifEN as a NifE-N fusion protein synthesizing unit in Azotobacter vinelandii.

    Science.gov (United States)

    Suh, Man Hee; Pulakat, Lakshmi; Gavini, Nara

    2002-11-29

    The nifEN encodes an E2N2 tetrameric metalloprotein complex that serves as scaffold for assembly of the FeMo cofactor of nitrogenase. In most diazotrophs, the NifE and NifN are translated as separate polypeptides and then assembled into tetrameric E2N2 complex. However, in Anabaena variabilis which has two nif clusters that encode two different NifEN complexes, the NifEN2 is encoded by a single nifE-N like gene, which has high homology to the NifE at amino-terminus and to the NifN at the carboxy-terminus. These observations implied that a metalloprotein like NifEN can accommodate large variations in their amino acid composition and also in the way they are synthesized (as two separate proteins or as a single protein) and yet remain functional. In Azotobacter vinelandii NifE and NifN are synthesized separately. To test whether NifEN could retain its functionality when encoded by a single gene, we generated a translational fusion of the nifE and nifN genes of A. vinelandii that could encode a large NifE-N fusion protein. When expressed in the nifEN-minus strain of A. vinelandii, the nifE-N gene fusion could complement the NifEN function. Western blot analysis by using polyclonal NifEN antibodies revealed that the complementing nifEN product is a large NifE-N fusion protein unit. The fact that the gene fusion of nifE-N specifies a functional NifE-N fusion protein reflects that these metalloproteins can accommodate a wide range of flexibility in their gene organization, structure, and assembly. PMID:12437975

  9. Regulation of Gene Expression of Catecholamine Biosynthetic Enzymes in Dopamine-β-Hydroxylase- and CRH-Knockout Mice Exposed to Stress

    OpenAIRE

    Richard, Kvetnansky; Olga, Krizanova; Andrej, Tillinger; Sabban Esther, L.; Thomas Steven, A; Lucia, Kubovcakova

    2008-01-01

    Norepinephrine-deficient mice harbor a disruption of the gene for dopamine-β-hydroxylase (DBH-KO). Corticotropin-releasing hormone knockout mice (CRH-KO) have markedly reduced HPA activity. The aim of the present work was to study how deficiency of DBH and CRH would affect tyrosine hydroxylase (TH), DBH, and phenylethanolamine N-methyltransferase (PNMT) gene expression and protein levels in the adrenal medulla (AM) and stellate ganglia (SG) of control and stressed mice. Both in AM and SG, sin...

  10. Patterns of indole alkaloids synthesis in response to heat shock, 5-azacytidine and Na-butyrate treatment of cultured catharanthus roseus mesophyll protoplasts

    International Nuclear Information System (INIS)

    Alkaloids of C. roseus are in high demand for therapeutic and other reasons. Cultured Catharanthus cells can produce limited quantities of these alkaloids. The authors have found that cultured mesophyll protoplasts in the presence of 14C-Tryptamine are capable of synthesizing alkaloids. The pattern of alkaloids synthesis changes when protoplasts are subjected to a heat shock at 370C. The heat shocked protoplasts incorporated 33% more 14C-Tryptamine and produced 3 new types of alkaloids. Treatment of protoplasts with 5-azacytidine, a DNA hypomethylating agent and Na-butyrate which induces hyperacetylation of histones produced qualitative and quantitative changes in the alkaloid pattern. Four new alkaloids following the above treatments were detected by TLC and HPLC of the extracts. It is suggested that the alkaloid pattern of the cultured protoplasts can be altered by treatment with compounds known as regulators of gene expression. Work is in progress to isolate and identify these new alkaloids

  11. Patterns of indole alkaloids synthesis in response to heat shock, 5-azacytidine and Na-butyrate treatment of cultured catharanthus roseus mesophyll protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Saleem, M.; Cutler, A.J.

    1986-04-01

    Alkaloids of C. roseus are in high demand for therapeutic and other reasons. Cultured Catharanthus cells can produce limited quantities of these alkaloids. The authors have found that cultured mesophyll protoplasts in the presence of /sup 14/C-Tryptamine are capable of synthesizing alkaloids. The pattern of alkaloids synthesis changes when protoplasts are subjected to a heat shock at 37/sup 0/C. The heat shocked protoplasts incorporated 33% more /sup 14/C-Tryptamine and produced 3 new types of alkaloids. Treatment of protoplasts with 5-azacytidine, a DNA hypomethylating agent and Na-butyrate which induces hyperacetylation of histones produced qualitative and quantitative changes in the alkaloid pattern. Four new alkaloids following the above treatments were detected by TLC and HPLC of the extracts. It is suggested that the alkaloid pattern of the cultured protoplasts can be altered by treatment with compounds known as regulators of gene expression. Work is in progress to isolate and identify these new alkaloids.

  12. The major volatile compound 2-phenylethanol from the biocontrol yeast, Pichia anomala, inhibits growth and expression of aflatoxin biosynthetic genes of Aspergillus flavus.

    Science.gov (United States)

    Hua, Sui Sheng T; Beck, John J; Sarreal, Siov Bouy L; Gee, Wai

    2014-05-01

    Aspergillus flavus is a ubiquitous saprophyte that is able to produce the most potent natural carcinogenic compound known as aflatoxin B1 (AFB1). This toxin frequently contaminates crops including corn, cotton, peanuts, and tree nuts causing substantial economic loss worldwide. Consequently, more than 100 countries have strict regulations limiting AFB1 in foodstuffs and feedstuffs. Plants and microbes are able to produce volatile compounds that act as a defense mechanism against other organisms. Pichia anomala strain WRL-076 is a biocontrol yeast currently being tested to reduce AF contamination of tree nuts in California. We used the SPME-GC/MS analysis and identified the major volatile compound produced by this strain to be 2-phenylethanol (2-PE). It inhibited spore germination and AF production of A. flavus. Inhibition of AF formation by 2-PE was correlated with significant down regulation of clustering AF biosynthesis genes as evidenced by several to greater than 10,000-fold decrease in gene expression. In a time-course analysis we found that 2-PE also altered the expression patterns of chromatin modifying genes, MYST1, MYST2, MYST3, gcn5, hdaA and rpdA. The biocontrol capacity of P. anomala can be attributed to the production of 2-PE, which affects spore germination, growth, toxin production, and gene expression in A. flavus. PMID:24504634

  13. 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...

  14. 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.

  15. 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 ...

  16. 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.

  17. 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

  18. 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.

  19. Erwinia carotovora ssp. carotovora Infection Induced "Defense Lignin" Accumulation and Lignin Biosynthetic Gene Expression in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Erwinia carotovora subsp. carotovora (Ecc) infects and causes soft rot disease in hundreds of crop species including vegetables, flowers and fruits. Lignin biosynthesis has been implicated in defensive reactions to injury and pathogen Infection in plants. In this work, variations of lignin content and gene expression in the molecular interaction between Chinese cabbage and Ecc were investigated. H2O2 accumulation and peroxidase activity were detected by 3, 3'-Dimethoxybenzidine staining at mocked and Ecc-inoculated sites of Chinese cabbage leafstalks. Klason lignin content in inoculated plants increased by about 7.84%, 40.37%, and 43.13% more than that of the mocked site at 12, 24 and 72 h after inoculation, respectively. Gas chromatography detected more p-coumaryl (H) and less coniferyl (G) and sinapyl (S)monolignins in leafstalks of Chinese cabbage. All three monomers increased in Ecc-infected leafstalks, and the Ecc-induced "defense lignin" were composed of more G and H monolignins, and less S monolignin. After searching the expressed sequence tags (EST) data of Chinese cabbage, 12 genes putatively encoding enzymes involved in lignin biosynthesis were selected to study their expression. All of these genes could be Induced by mock inoculation and Ecc infection, while the gene expression lasted for several more hours in the infected samples than in mocked and untreated plants. Our results indicated that "defense lignin" was different from the developmental lignin in composition; G and S monolignins were significantly induced in plants in response to the soft rot Ecc; thus, lignin biosynthesis was differentially regulated and played a role in plant response to the soft rot Ecc.

  20. 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.

  1. 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.

  2. 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.

  3. 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....

  4. Alkaloids from Galanthus nivalis.

    Science.gov (United States)

    Berkov, Strahil; Codina, Carles; Viladomat, Francesc; Bastida, Jaume

    2007-07-01

    Phytochemical studies on Galanthus nivalis of Bulgarian origin resulted in the isolation of five compounds: 11-O-(3'-hydroxybutanoyl)hamayne, 3,11-O-(3',3''-dihydroxybutanoyl)hamayne, 3-O-(2''-butenoyl)-11-O-(3'-hydroxybutanoyl)hamayne, 3,11,3''-O-(3',3'',3'''-trihydroxybutanoyl)hamayne, and 2-O-(3'-acetoxybutanoyl)lycorine, together with five known alkaloids: ungeremine, lycorine, tazettine, hamayne, and ismine. Their structures were determined by (1)H and (13)C NMR spectroscopy and two-dimensional (1)H-(1)H and (1)H-(13)C chemical shift correlation experiments.

  5. Regulation of purine biosynthetic genes expression in Salmonella typhimurium Ⅳ O~c mutation site of purG and its function analysis

    Institute of Scientific and Technical Information of China (English)

    刘奔; 黄谊; 王敖全

    1997-01-01

    Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded by purG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu-tamine and ATP for the de novo purine nucleotide biosynthesis. purG gene is negatively regulated by a repressor-oper-ator system. The O+ purG and OC purG were cloned respectively in vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O + ) and pLBG-2 (OC) were carried out. The hybrid plasmids pLB1933 (O+ ) and pLB1927 (OC) containing 5 control region of purG were constructed and the DNA sequences were determined respectively. DNA se-quences data showed that Oc mutation of purG occurred at the 3rd position of 16 bp PUR box in the 5’ control region ( G→A). Gel retardation experiment indicated that the repressor bound well with O+ PUR box, but not with Oc PUR box. The result strongly supported the idea that PUR box is the binding region of represser protein and the 3rd posi-tion base G of PUR bo

  6. Cactus alkaloids. XXXVI. Mescaline and related compounds from Trichocereus peruvianus.

    Science.gov (United States)

    Pardanani, J H; McLaughlin, J L; Kondrat, R W; Cooks, R G

    1977-01-01

    Agurell has previously detected (tlc, glc-ms) tyramine, 3-methoxytyramine, and two unknown alkaloids in the Peruvian cactus, Trichocereus peruvianus Br. and R. The presence of mescaline in other similar Trichocereus species prompted us to reinvestigate this species, which is commercially available in the United States. The nonphenolic alkaloid extracts yielded an abundance of crystalline mescaline hydrochloride (0.82% yield) and a trace of 3,4-dimethoxyphenethylamine (tlc-ms). Crystalline tyramine hydrochloride, 3-methoxytyramine hydrochloride, and 3,5 dimethoxy-4-hydroxphenethylamine hydrochloride were isolated from the phenolic alkaloid extracts; the last compound has not been previously crystallized from nature, although it is the immediate biosynthetic precursor of mescaline. Crystalline 2-chloromescaline hydrochloride was isolated drom the nonphenolic extracts; but, as determined by mass-analyzed ion kinetic energy spectrometry, this new compound is an extraction artifact. Both 2-chloromescaline and 2.6-dichloromescaline hydrochlorides were prepared synthetically from mescaline. This cactus species has a mescaline content equal or superior to peyote and should be legally controlled as an item of drug abuse.

  7. Integration of Transcriptome and Proteome Reveals the Alkaloids Biosynthesis in Macleaya cordata and Macleaya microcarpa

    Institute of Scientific and Technical Information of China (English)

    Yisong Liu; Wei Liu; Xiubing Liu; Peng Huang; Pengcheng Zhu; Pi Cheng; Jing Zeng

    2012-01-01

    The Macleaya spp.,including Macleaya cordata and Macleaya microcarpa,are traditional anti-virus,inflammation eliminating,and insecticide herb medicines for their isoquinoline alkaloids.The studies of their alkaloids biosyntheses are urgent for better application.To further characterize their alkaloids biosyntheses,we elaborately designed the transcriptome,proteome and metabolism profiling for 10 samples of both species to explore their alkaloids biosyntheses.From the transcriptome data,we obtained 69367 and 78255 unigenes for M.cordata and M.microcarpa,which two thirds of them were similar to sequences in public databases.By metabolism profiling,we observed reverse patterns in different organs of two species for alkaloids sanguinarine,chelerythrine,protopine,and allocryptopine.Thus,the expression of enzymes in alkaloid biosynthesis pathways and the differential gene expression for multiple interesting comparisons were analyzed.We identified more than 1000 proteins and hundreds of differentially expressed proteins from iTRAQ proteome data.Furthermore,the ultrastructure of laticifers by SEM proved the alkaloids accumulation in the mature roots.This study suggests strongly that root maybe the organ for major alkaloids biosynthesis.Except for biosynthesis,the alkaloids storage and transport were also important for their accumulation.This work provided the first genome scale analysis for Macleaya spp.and shed light on researches for non-model plants by integrating different high-throughput technologies.

  8. Independent recruitment of a flavin-dependent monooxygenase for safe accumulation of sequestered pyrrolizidine alkaloids in grasshoppers and moths.

    Directory of Open Access Journals (Sweden)

    Linzhu Wang

    Full Text Available Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.

  9. Biosynthesis of tylophora alkaloids

    International Nuclear Information System (INIS)

    Using labelled precursors, biosynthesis of the tylophora alkaloids, tylophorine, tylophorinidine and tylophorinide has been investigated in Tylophora asthmatica plants. The radioactive precursors, phenylalanine-2-14C, benzoic acid-1-14C, benzoic acid-ring 14C, acetate-2-14C, ornithine-5-14C, acetate-2-14C, ornithine-5-14C and cinnamic acid-2-14C were administered to the plants individually by wick technique. Tylophorine was isolated in each case and assayed for its radioactivity to find out the incorporation of the label into it. The results indicate that: (1) phenylalanine via cinnamic acid is an important precursor in the biosynthesis of tylophorine (2) orinithine participates in tylophorine biosynthesis via pyrroline and (3) tylophorinidine may be a direct precursor of tylophorine. (M.G.B.)

  10. Dimeric Matrine-Type Alkaloids from the Roots of Sophora flavescens and Their Anti-Hepatitis B Virus Activities.

    Science.gov (United States)

    Zhang, Yu-Bo; Zhan, Li-Qin; Li, Guo-Qiang; Wang, Feng; Wang, Ying; Li, Yao-Lan; Ye, Wen-Cai; Wang, Guo-Cai

    2016-08-01

    Six unusual matrine-type alkaloid dimers, flavesines A-F (1-6, respectively), together with three proposed biosynthetic intermediates (7-9) were isolated from the roots of Sophora flavescens. Compounds 1-5 were the first natural matrine-type alkaloid dimers, and compound 6 represented an unprecedented dimerization pattern constructed by matrine and (-)-cytisine. Their structures were elucidated by NMR, MS, single-crystal X-ray diffraction, and a chemical method. The hypothetical biogenetic pathways of 1-6 were also proposed. Compounds 1-9 exhibited inhibitory activities against hepatitis B virus.

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

    Science.gov (United States)

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

    2010-06-29

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

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

    Science.gov (United States)

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

    2013-12-01

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

  14. Mining and engineering natural-product biosynthetic pathways.

    Science.gov (United States)

    Wilkinson, Barrie; Micklefield, Jason

    2007-07-01

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

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

    OpenAIRE

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

    2006-01-01

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

  16. Total Synthesis of Securinega Alkaloids

    Institute of Scientific and Technical Information of China (English)

    T. Honda

    2005-01-01

    @@ 1Introduction Naturally occurring Securinega alkaloids (1-4) (Fig. 1)[1], with their wide range of structural and stereochemical features, continue to provide challenging synthetic targets, since these alkaloids exhibit attractive biological activities. Securinine (1), isolated from Securinega suffruticosa [2], was structurally determined to contain an indolizidine skeleton with an azabicyclo[3.2.1 ]octane system together with an α, β-unsaturated γ-lactone ring. This alkaloid has been clinically used in Russia as a CNS stimulating drug[3], and has been shown to act as a stereospecific antagonist at the GABA binding site of the GABAA-receptor complex[4].Viroallosecurinine (2), a diastereoisomeric alkaloid of securinine, was also isolated from the leaves of Securinega virosa[5] as a cytotoxic alkaloid exhibiting a MIC of 0.48 μg/mL for Ps. aeruginosa and Staph.aureus[6]. This alkaloid is recognized to be bactericidal since the yields of MIC/MBC were less than 1[7].

  17. Didemnin Biosynthetic Gene Cluster In Tistrella Mobilis

    KAUST Repository

    Qian, Pei-Yuan

    2014-10-02

    A novel Tistrella mobilis strain having Accession Deposit Number NRRL B-50531 is provided. A method of producing a didemnin precursor, didemnin or didemnin derivative by using the Tistrella mobilis strain, and the therapeutic composition comprising at least one didemnin or didemnin derivative produced from the strain or modified strain thereof are also provided.

  18. A Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in Catharanthus roseus*

    OpenAIRE

    Giddings, Lesley-Ann; Liscombe, David K.; Hamilton, John P; Childs, Kevin L.; DellaPenna, Dean; Buell, C. Robin; O'Connor, Sarah E.

    2011-01-01

    Plant cytochrome P450s are involved in the production of over a hundred thousand metabolites such as alkaloids, terpenoids, and phenylpropanoids. Although cytochrome P450 genes constitute one of the largest superfamilies in plants, many of the catalytic functions of the enzymes they encode remain unknown. Here, we report the identification and functional characterization of a cytochrome P450 gene in a new subfamily of CYP71, CYP71BJ1, involved in alkaloid biosynthesis. Co-expression analysis ...

  19. Jasmonate-dependent alkaloid biosynthesis in Catharanthus Roseus hairy root cultures is correlated with the relative expression of Orca and Zct transcription factors.

    Science.gov (United States)

    Goklany, Sheba; Rizvi, Noreen F; Loring, Ralph H; Cram, Erin J; Lee-Parsons, Carolyn W T

    2013-01-01

    The effects of methyl jasmonate (MJ) dosage on terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus are correlated with the relative levels of specific MJ-responsive transcription factors. In this study, the expression of transcription factors (Orca, Zct, Gbf, Myc2, At-hook, and Wrky1), TIA pathway genes (G10h, Tdc, Str, and Sgd), and TIA metabolites (secologanin, strictosidine, and tabersonine) were investigated in C. roseus hairy root cultures elicited with a range of MJ dosages (0-1,000 µM) during mid-exponential growth. The highest production of TIA metabolites occurs at 250 μM MJ, increasing by 150-370% compared with untreated controls. At this MJ dosage, the expression of the transcriptional activators (Orca) is dramatically increased (29-40 fold) while the levels of the transcriptional repressors (Zct) remain low (2-7 fold). Simultaneously, the expression of genes coding for key enzymes involved in TIA biosynthesis increases by 8-15 fold. In contrast, high MJ dosages (1,000 µM) inhibit the production of TIA metabolites. This dosage is correlated with elevated expression levels of Zct (up to 40-fold) relative to Orca (13-19-fold) and minimal induction of the TIA biosynthetic genes (0-6 fold). The significant changes in the expression of Orca and Zct with MJ dosage do not correspond to changes in the expression of the early-response transcription factors (AT-hook, Myc2, and Wrky1) believed to regulate Orca and Zct. In summary, these observations suggest that the dependence of alkaloid production on MJ dosage in C. roseus may be partly mediated through the relative levels of Orca and Zct family transcription factors. PMID:23970483

  20. Copper catalysed synthesis of indolylquinazolinone alkaloid bouchardatine

    Indian Academy of Sciences (India)

    Mayavan Viji; Rajagopal Nagarajan

    2014-07-01

    We describe the total synthesis of indolylquinazolinone alkaloid bouchardatine and some of the quinazolinone derivatives. The aerobic oxidation induced by copper(I) bromide, followed by Vilsmeier-Haack formylation gives the natural product bouchardatine alkaloid in good yield.

  1. Development of transcriptomic resources for interrogating the biosynthesis of monoterpene indole alkaloids in medicinal plant species.

    Directory of Open Access Journals (Sweden)

    Elsa Góngora-Castillo

    Full Text Available The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs, includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin, hypertension (reserpine, ajmalicine, malaria (quinine, and as analgesics (7-hydroxymitragynine. Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  3. Antimicrobial activity of benzylisoquinoline alkaloids.

    Science.gov (United States)

    Villar, A; Mares, M; Rios, J L; Canton, E; Gobernado, M

    1987-04-01

    The antimicrobial in vitro activity of 14 benzylisoquinoline alkaloids was investigated by agar diffusion and agar dilution methods against several genera of microorganisms that included Streptococcus, Staphylococcus, Bacillus, Lysteria, Escherichia, Salmonella, Klebsiella, Pseudomonas, Enterobacter, Serratia, Shigella, Mycobacterium and Candida. Anolobine was the most active compound against grampositive bacteria with MIC90 between 12 and 50 mg/l; less active were anonaine, lysicamine and liriodenine. All the alkaloids of the noraporphine and oxoaporphine groups, with the exception of isopiline, showed activity against Mycobacterium phlei (MIC 6-25 mg/l). Candida albicans ATCC26555 was inhibited by anonaine, nornantenine and xylopine (MIC 3-12 mg/l). None of the alkaloids tested had a significant activity against gramnegative rods. The action against susceptible microorganisms was bactericidal. PMID:3615557

  4. Alkaloids from Stems of Ervatamia yunnanensis (Ⅱ)

    Institute of Scientific and Technical Information of China (English)

    Shuang LIANG; Xin Gen LUO; Hai Sheng CHEN; Xiao Dong ZHANG; Mao HUANG; Wen Yong LIU

    2006-01-01

    Six indole alkaloids were isolated from the stems of Ervatamia yunnanensis. Among them, 10-hydroxy-19, 20-dihydroisositsirikine (Ⅰ) is new. The other five are known alkaloids,namely: matrine (Ⅱ), 19, 20-dihydroisositsuikine (Ⅲ), 19-s-voacangarine (Ⅳ), 11'-methoxyl- 19s-heyneanine (Ⅴ), conodurine (Ⅵ). The structural elucidation of the alkaloids was based on spectral means.

  5. Alkaloids from stems of Ervatamia yunnanensis

    Institute of Scientific and Technical Information of China (English)

    Xin Gen Luo; Hai Sheng Chen; Shuang Liang; Mao Huang; Wei Dong Xuan; Li Jin

    2007-01-01

    Six indole alkaloids were isolated from the stems of Ervatamia yunnanensis.Among them, yunnanensine (Ⅰ) is new.The other five are known alkaloids, namely: 19,20-E-vallesamine (Ⅱ), 19s-heyneanine (Ⅲ), ibogaine (Ⅳ), ibogamine (Ⅴ), coronaridine (Ⅵ).The structural elucidation of the alkaloids was based on spectral means.

  6. FIVE NEW NORDITERPENOID ALKALOIDS FROM ACONITUM SINOMONTANUM

    Institute of Scientific and Technical Information of China (English)

    FENG-PENG WANG; CHONG-SHENG PENG; XI-XIAN JIAN; DONG-LIN CHEN

    2001-01-01

    From the roots of A conitum sinomontanum, five new norditerpenoid alkaloids, sinomontanitines A (1) and B (2), sinomontanines A (3), B (4) and C (5), were isolated together with the known alkaloids lappaconitine (6) and ranaconitine (7), The structures of the new alkaloids were determined by spectral analysis.

  7. THE ALKALOID CYTISINE IN THE CELL CULTURE

    Directory of Open Access Journals (Sweden)

    Gazaliev A.M.

    2012-08-01

    Full Text Available Alkaloids are vegetative establishments of complex and original structure with nitrous heterocycles in the basis. For a long time they drew researchers’ attention because of their unique and specific physiological effect on alive organisms. Not all the representatives of the globe’s flora contain these unique substances. Alkaloid cytisine is to be found mainly in the plants of the fabaceous family - Fabaceae. For the cytisine production the seeds of Thermopsis lanceolata R.Br (T. lanceolata R.Br and Cytisus laburnum (C. laburnum are used as a raw material. The object of the research is T. lanceolata cell culture. Sterile sprouts are used at the first stage of the experiment. Callus genesis is accompanied with dedifferentiation. It leads to the cellular organization simplification. Based on an important property of a plant cell, such as totipotency, there appears the formation of the “de novo” biosynthetic device. The cultivation algorithm consists of two basic stages: (i the cultivation conditions optimization of callus with a high level of the primary metabolites biosynthesis (Aspartat – lysine; (ii the research of cultivation chemical and physical factors influence on the secondary metabolite (cytisine biosynthesis and accumulation. During the cultivation the Murashige and Skoog classical recipe of nutrient medium will be used. Optimization of the cultivation conditions will concern the phytohormones, macro- and micronutrients content, as the purpose of optimization is the production of the determined high-level competence embriogenical callus. The main problem is genetic heterogeneity of a cellular population and instability of morpho-physiological processes. The correct management of higher plants cells population is possible at the synchronization of a cellular cycle phases. The references analysis has shown that it is almost impossible to synchronize cellular cycles in the culture of plant tissue. The application of chemical

  8. I. Cyclopeptide alkaloids; II. Phycocyanobilipeptides

    Energy Technology Data Exchange (ETDEWEB)

    Lagarias, J.C.

    1979-09-01

    Several examples of the 14-membered, para-bridged ring system of the cyclopeptide alkaloids have been synthesized via an active ester cyclization. The yield of monomeric cyclopeptide varied from 1 to 33% and was affected by the amino acid substitution pattern and amide conformation of the linear peptide precursors. Both the synthetic models and a naturally cyclopeptide alkaloid, ceanothine B, bind monovalent (Li/sup +/) and divalent (Ca/sup + +/, Mg/sup + +/) cations. Seven cyclopeptide alkaloids, phencyclopeptines 1-7, have been found distributed among three forms of the shrub Ceanothus integerrimus. Chemical degradation, mass spectroscopy, and /sup 1/H NMR spectroscopy have established structures for these seven compounds, three of which have been previously reported. The utility of cyclopeptide alkaloid structure and the distribution for chemotaxonomic assignments is discussed. Field desorption mass spectroscopy has identified five phencyclopeptines in the crude alkaloidal extracts of Ceanothus sanguineus. A new paired-ion HPLC system for the separation of these alkaloids is discussed. Amino acid analysis, electron impact mass spectroscopy, and /sup 1/H NMR spectroscopy have established the structures of six phencyclopeptines including two isomeric compounds 5 and 6. The structure of 2 has not been previously reported. The smallest cyanogen bromide fragment derived from the ..beta..-subunit of Synechococcus sp 6301 C-phycocyanin, the blue heptapeptide 2, has been investigated by 360 MHz /sup 1/H NMR spectroscopy. The peptide portion, heptapeptide 3, was synthesized independently and used in comparative spectroscopic analysis.These studies have led to complete assignment of the structure of the peptide-linked phycocyanobilin and elucidation of the nature of the thioether chromophore-peptide linkage.

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

    Institute of Scientific and Technical Information of China (English)

    陈雪岚; 汤立; 焦海涛; 徐峰; 熊勇华

    2013-01-01

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

  10. A putative gene cluster from a Lyngbya wollei bloom that encodes paralytic shellfish toxin biosynthesis.

    Directory of Open Access Journals (Sweden)

    Troco K Mihali

    Full Text Available Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds.

  11. Genotoxicity of the boldine aporphine alkaloid in prokaryotic and eukaryotic organisms.

    Science.gov (United States)

    Moreno, P R; Vargas, V M; Andrade, H H; Henriques, A T; Henriques, J A

    1991-06-01

    The aporphine alkaloid boldine, present in Peumus boldus (boldo-do-Chile) widely used all over the world, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in microorganisms. This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest and Ames tester strains TA100, TA98 and TA102. It was not able to induce point and frameshift mutations in haploid Saccharomyces cerevisiae cells. However, mitotic recombinational events such as crossing-over and gene conversion were weakly induced in diploid yeast cells by this alkaloid. Also, boldine was able to induce weakly cytoplasmic 'petite' mutation in haploid yeast cells. PMID:2046695

  12. The alkaloid profiles of Lupinus sulphureus.

    Science.gov (United States)

    Cook, Daniel; Lee, Stephen T; Gardner, Dale R; Pfister, James A; Welch, Kevin D; Green, Benedict T; Davis, T Zane; Panter, Kip E

    2009-02-25

    Lupines are common plants on the rangelands in the western United States. Lupines contain alkaloids that can be toxic and teratogenic causing congenital birth defects (crooked calf disease). One such lupine, Lupinus sulphureus, occurs in parts of Oregon, Washington, and British Columbia. Specimens of L. sulphureus from field collections and herbaria were evaluated taxonomically and by chemical means. A total of seven distinct alkaloid profiles and the individual alkaloids associated with each profile were identified. Each alkaloid profile was unique in its geographical distribution and its potential risk to livestock. In conclusion, taxonomic classification is not sufficient to determine risk, as chemical characterization of the alkaloids must also be performed.

  13. Pyridinoacridine alkaloids of marine origin: NMR and MS spectral data, synthesis, biosynthesis and biological activity

    Directory of Open Access Journals (Sweden)

    Louis P. Sandjo

    2015-09-01

    Full Text Available This review focuses on pyridoacridine-related metabolites as one biologically interesting group of alkaloids identified from marine sources. They are produced by marine sponges, ascidians and tunicates, and they are structurally comprised of four to eight fused rings including heterocycles. Acridine, acridone, dihydroacridine, and quinolone cores are features regularly found in these alkaloid skeletons. The lack of hydrogen atoms next to quaternary carbon atoms for two or three rings makes the chemical shift assignment a difficult task. In this regard, one of the aims of this review is the compilation of previously reported, pyridoacridine 13C NMR data. Observations have been made on the delocalization of electrons and the presence of some functional groups that lead to changes in the chemical shift of some carbon resonances. The lack of mass spectra information for these alkaloids due to the compactness of their structures is further discussed. Moreover, the biosynthetic pathways of some of these metabolites have been shown since they could inspire biomimetic synthesis. The synthesis routes used to prepare members of these marine alkaloids (as well as their analogues, which are synthesized for biological purposes are also discussed. Pyridoacridines were found to have a large spectrum of bioactivity and this review highlights and compares the pharmacophores that are responsible for the observed bioactivity.

  14. Lycopodium alkaloids from Palhinhaea cernua

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Fu-Wei [Graduate University of Chinese Academy of Sciences, Beijing (China); Luo, Ji-Feng; Wang, Yue-Hu, E-mail: wangyuehu@mail.kib.ac.cn [Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences (China); Sun, Qian-Yun; Yang, Fu-Mei [Key Laboratory of Chemistry for Natural Products, Guizhou Province and Chinese Academy of Sciences (China); Liu, Fang [College of Landscape and Horticulture, Yunnan Agricultural University (China); Long, Chun-Lin, E-mail: long@mail.kib.ac.cn [College of Life and Environmental Sciences, Minzu University of China, Beijing, (China)

    2012-07-01

    Two new Lycopodium alkaloids, acetyllycoposerramine M and palcernine A were isolated from whole plant extracts of Palhinhaea cernua L. together with ten previously identified compounds. The structures of the new compounds were elucidated by spectroscopic methods and single-crystal X-ray diffraction analyses using the Flack parameter. (author)

  15. Quaternary alkaloids of tinospora species.

    Science.gov (United States)

    Bisset, N G; Nwaiwu, J

    1983-08-01

    The occurrence of quaternary alkaloids in TINOSPORA (and PARABAENA) species (Menispermaceae) has been studied. The main components were generally the protoberberine bases berberine and palmatine, with jatrorrhizine an occasional minor constituent, and the aporphine base magnoflorine. Choline was also often present. Only magnoflorine was detected in the PARABAENA material examined. PMID:17404996

  16. Validation of a housekeeping gene for use in bovine vascular gene expression studies

    Science.gov (United States)

    Exposure of ungulate vasculature to ergot alkaloids while grazing endophyte (Neotyphodium coenophialum)-infected tall fescue (Lolium arundinaceum) affects vasoactivity and causes vasoconstriction. Bovine vascular gene expression as affected by exposure to ergot alkaloids in tall fescue is largely un...

  17. Alkaloids: an overview of their antibacterial, antibiotic-enhancing and antivirulence activities.

    Science.gov (United States)

    Cushnie, T P Tim; Cushnie, Benjamart; Lamb, Andrew J

    2014-11-01

    With reports of pandrug-resistant bacteria causing untreatable infections, the need for new antibacterial therapies is more pressing than ever. Alkaloids are a large and structurally diverse group of compounds that have served as scaffolds for important antibacterial drugs such as metronidazole and the quinolones. In this review, we highlight other alkaloids with development potential. Natural, semisynthetic and synthetic alkaloids of all classes are considered, looking first at those with direct antibacterial activity and those with antibiotic-enhancing activity. Potent examples include CJ-13,136, a novel actinomycete-derived quinolone alkaloid with a minimum inhibitory concentration of 0.1 ng/mL against Helicobacter pylori, and squalamine, a polyamine alkaloid from the dogfish shark that renders Gram-negative pathogens 16- to >32-fold more susceptible to ciprofloxacin. Where available, information on toxicity, structure-activity relationships, mechanisms of action and in vivo activity is presented. The effects of alkaloids on virulence gene regulatory systems such as quorum sensing and virulence factors such as sortases, adhesins and secretion systems are also described. The synthetic isoquinoline alkaloid virstatin, for example, inhibits the transcriptional regulator ToxT in Vibrio cholerae, preventing expression of cholera toxin and fimbriae and conferring in vivo protection against intestinal colonisation. The review concludes with implications and limitations of the described research and directions for future research. PMID:25130096

  18. Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

    Directory of Open Access Journals (Sweden)

    Christopher L Schardl

    Full Text Available The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species, which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne, and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species, a morning-glory symbiont (Periglandula ipomoeae, and a bamboo pathogen (Aciculosporium take, and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories

  19. Investigations into the molecular evolution of plant terpene, alkaloid, and urushiol biosynthetic enzymes

    OpenAIRE

    Weisberg, Alexandra Jamie

    2014-01-01

    Plants produce a vast number of low-molecular-weight chemicals (so called secondary or specialized metabolites) that confer a selective advantage to the plant, such as defense against herbivory or protection from changing environmental conditions. Many of these specialized metabolites are used for their medicinal properties, as lead compounds in drug discovery, or to impart our food with different tastes and scents. These chemicals are produced by various pathways of enzyme-mediated reactions...

  20. Alkaloids of in vitro cultures of Rhazya stricta

    OpenAIRE

    Akhgari, Amir

    2015-01-01

    Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in the Middle East and South Asia. It produces a large number of terpenoid indole alkaloids(TIAs), some of which possess important pharmacological properties. This study focused on the establishment of biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA path...

  1. The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus.

    Science.gov (United States)

    Van Moerkercke, Alex; Steensma, Priscille; Schweizer, Fabian; Pollier, Jacob; Gariboldi, Ivo; Payne, Richard; Vanden Bossche, Robin; Miettinen, Karel; Espoz, Javiera; Purnama, Purin Candra; Kellner, Franziska; Seppänen-Laakso, Tuulikki; O'Connor, Sarah E; Rischer, Heiko; Memelink, Johan; Goossens, Alain

    2015-06-30

    Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix-loop-helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures. PMID:26080427

  2. CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids

    Directory of Open Access Journals (Sweden)

    Raina Susheel

    2012-08-01

    Full Text Available Abstract Background Mitogen activated protein kinase (MAPK cascade is an important signaling cascade that operates in stress signal transduction in plants. The biologically active monoterpenoid indole alkaloids (MIA produced in Catharanthus roseus are known to be induced under several abiotic stress conditions such as wounding, UV-B etc. However involvement of any signaling component in the accumulation of MIAs remains poorly investigated so far. Here we report isolation of a novel abiotic stress inducible Catharanthus roseus MAPK, CrMPK3 that may have role in accumulation of MIAs in response to abiotic stress. Results CrMPK3 expressed in bacterial system is an active kinase as it showed auto-phosphorylation and phosphorylation of Myelin Basic Protein. CrMPK3 though localized in cytoplasm, moves to nucleus upon wounding. Wounding, UV treatment and MeJA application on C. roseus leaves resulted in the transcript accumulation of CrMPK3 as well as activation of MAPK in C. roseus leaves. Immuno-precipitation followed by immunoblot analysis revealed that wounding, UV treatment and methyl jasmonate (MeJA activate CrMPK3. Transient over-expression of CrMPK3 in C. roseus leaf tissue showed enhanced expression of key MIA biosynthesis pathway genes and also accumulation of specific MIAs. Conclusion Results from our study suggest a possible involvement of CrMPK3 in abiotic stress signal transduction towards regulation of transcripts of key MIA biosynthetic pathway genes, regulators and accumulation of major MIAs.

  3. CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids

    Science.gov (United States)

    2012-01-01

    Background Mitogen activated protein kinase (MAPK) cascade is an important signaling cascade that operates in stress signal transduction in plants. The biologically active monoterpenoid indole alkaloids (MIA) produced in Catharanthus roseus are known to be induced under several abiotic stress conditions such as wounding, UV-B etc. However involvement of any signaling component in the accumulation of MIAs remains poorly investigated so far. Here we report isolation of a novel abiotic stress inducible Catharanthus roseus MAPK, CrMPK3 that may have role in accumulation of MIAs in response to abiotic stress. Results CrMPK3 expressed in bacterial system is an active kinase as it showed auto-phosphorylation and phosphorylation of Myelin Basic Protein. CrMPK3 though localized in cytoplasm, moves to nucleus upon wounding. Wounding, UV treatment and MeJA application on C. roseus leaves resulted in the transcript accumulation of CrMPK3 as well as activation of MAPK in C. roseus leaves. Immuno-precipitation followed by immunoblot analysis revealed that wounding, UV treatment and methyl jasmonate (MeJA) activate CrMPK3. Transient over-expression of CrMPK3 in C. roseus leaf tissue showed enhanced expression of key MIA biosynthesis pathway genes and also accumulation of specific MIAs. Conclusion Results from our study suggest a possible involvement of CrMPK3 in abiotic stress signal transduction towards regulation of transcripts of key MIA biosynthetic pathway genes, regulators and accumulation of major MIAs. PMID:22871174

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

    Institute of Scientific and Technical Information of China (English)

    郑夏生; 罗秀秀; 徐晖; 詹若挺; 陈蔚文

    2014-01-01

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

  5. Pyrrolizidine alkaloids of senecio sp from Peru

    Directory of Open Access Journals (Sweden)

    Liliana Ruiz Vásquez and Matías Reina Artiles

    2011-01-01

    Full Text Available Six pyrrolizidine alkaloids (PAs (two saturated macrocyclic, three unsaturated macrocyclic and one unsaturated seco-macrocyclic were isolated from native Peruvian Senecio species. The structures of these alkaloids were established by a complete NMR spectroscopic analysis, chemical transformations and comparison of their NMR data with those published for similar alkaloids. Three PAs were then tested for antifungal activity against Fusarium moniliforme, F. (Sheldon, F. oxysporum fs. lycopersici (Scheldt and F. solani (Mart, no significant activity being observed.

  6. Pyrrolizidine alkaloids of Senecio sp from Peru

    International Nuclear Information System (INIS)

    Six pyrrolizidine alkaloids (PAs) (two saturated macrocyclic, three unsaturated macrocyclic and one unsaturated seco-macrocyclic) were isolated from native Peruvian Senecio species. The structures of these alkaloids were established by a complete NMR spectroscopic analysis, chemical transformations and comparison of their NMR data with those published for similar alkaloids. Three PAs were then tested for antifungal activity against Fusarium moniliforme, F. (Sheldon), F. oxysporum fs. lycopersici (Scheldt) and F. solani (Mart), no significant activity being observed. (author)

  7. Pyrrolizidine alkaloids of Senecio sp from Peru

    Energy Technology Data Exchange (ETDEWEB)

    Ruiz Vasquez, Liliana; Reina Artiles, Matias [Instituto de Productos Naturales y Agrobiologia, CSIC, Tenerife (Spain); Gonzalez Coloma, Azucena [Instituto de Ciencias Agrarias (ICA), CSIC, Madrid (Spain); Cabrera Perez, Raimundo [Universidad de La Laguna (ULL), Tenerife (Spain). Unidad de Fitopatologia, Facultad de Biologia; Ruiz Mesia, Lastenia [Universidad Nacional de la Amazonia Peruana (LIPNAA-UNAP), AA.HH. Nuevo San Lorenzo, San Juan, Iquitos (Peru). Lab. de Investigacion en Productos Naturales Antiparasitarios de la Amazonia

    2011-07-01

    Six pyrrolizidine alkaloids (PAs) (two saturated macrocyclic, three unsaturated macrocyclic and one unsaturated seco-macrocyclic) were isolated from native Peruvian Senecio species. The structures of these alkaloids were established by a complete NMR spectroscopic analysis, chemical transformations and comparison of their NMR data with those published for similar alkaloids. Three PAs were then tested for antifungal activity against Fusarium moniliforme, F. (Sheldon), F. oxysporum fs. lycopersici (Scheldt) and F. solani (Mart), no significant activity being observed. (author)

  8. Indole alkaloids and terpenoids from Tabernaemontana markgrafiana

    DEFF Research Database (Denmark)

    Nielsen, H.B.; Hazell, A.; Hazell, R.;

    1994-01-01

    The bark of Tabernaemontana markgrafiana yielded five acetylated pentacyclic triterpenes and 24 monoterpene indole alkaloids. The major triterpene was baurenyl acetate, which constituted ca 6% of the crude petrol extract. An X-ray study of iso-ursenyl acetate was carried out for the first time....... The indole alkaloids were primarily of the iboga-type and constituted ca 3% of the dried bark and 20% of the total extracts. The major alkaloids were coronaridine, (19S)-heyneanine, voacangine and ibogamine. Among the minor components, four new alkaloids were identified: 5,6-dehydro-coronaridine, 3R...

  9. Intraspecific variability in the alkaloid metabolism of Galanthus elwesii.

    Science.gov (United States)

    Berkov, Strahil; Sidjimova, Borjana; Evstatieva, Luba; Popov, Simeon

    2004-03-01

    Alkaloid pattern of individuals from 16 Bulgarian Galanthus elwesii populations was investigated by GC/MS and TLC. Twenty-one Amaryllidaceae alkaloids were detected and 14 of them were identified. Crinane type alkaloids, haemanthamine or crinine, dominated alkaloid metabolism in most of the populations. With exception of one population, where the separate individuals showed variable alkaloid profiles (dominated by crinine or haemanthamine) the individuals of the rest of populations have identical and characteristic alkaloid profiles. Some populations showed remarkable differences in respect to their alkaloid pattern-type of biosynthesis, main alkaloids and number of alkaloids. Populations dominated by galanthamine type alkaloids were found as well. These data demonstrate that like the morphological features, the alkaloid metabolism of G. elwesii is also variable.

  10. Tropane alkaloids in food: poisoning incidents

    NARCIS (Netherlands)

    Adamse, P.; Egmond, van H.P.; Noordam, M.Y.; Mulder, P.P.J.; Nijs, de W.C.M.

    2014-01-01

    A large number of wild and cultured plants produce secondary metabolites that can be toxic to humans and animals. The present study aims to provide insight into the routes of (un)intentional poisonings of humans by tropane alkaloids. Poisonings of humans by tropane alkaloids occur as unintended inge

  11. Alkaloids from the Roots of Saccopetalum prolificum

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A new alkaloid, named prolifine (1), was isolated along with four known alkaloids, liriodenine (2), 6-hydroxyonychine (3), isooncodine (4) and discretamine (5) from the roots of Saccopetalum prolificum. The structure of 1 was elucidated on the basis of spectroscopic and chemical methods.

  12. Alkaloids of some Asian Sedum species

    NARCIS (Netherlands)

    Kim, JH; THart, H; Stevens, JF

    1996-01-01

    The leafy parts of 16 Asian species belonging to the three sections of Sedum were investigated for the presence of alkaloids. Only in seven species of Sedum sect. Sedum were alkaloids found. Sedum bulbiferum, S. japonicum, S. lepidopodium, S. morrisomensis, S. oryzifolium, S. polytrichoides and S. s

  13. 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.

  14. 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.

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

    Science.gov (United States)

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

    2002-01-01

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

  16. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

    KAUST Repository

    Ross, Avena C.

    2013-01-23

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-05-20

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

  18. Homospermidine in transgenic tobacco results in considerably reduced spermidine levels but is not converted to pyrrolizidine alkaloid precursors.

    Science.gov (United States)

    Abdelhady, Mohamed I S; Beuerle, Till; Ober, Dietrich

    2009-09-01

    Homospermidine synthase is the first specific enzyme in the biosynthesis of pyrrolizidine alkaloids. Whereas the substrates putrescine and spermidine are part of the highly dynamic polyamine pool of plants, the product homospermidine is incorporated exclusively into the necine base moiety of pyrrolizidine alkaloids. Recently, the gene encoding homospermidine synthase has been shown to have been recruited several times independently during angiosperm evolution by the duplication of the gene encoding deoxyhypusine synthase. To test whether high levels of homospermidine suffice for conversion, at least in traces, to precursors of pyrrolizidine alkaloids, transgenic tobacco plants were generated expressing homospermidine synthase. Analyses of the polyamine content revealed that, in the transgenic plants, about 80% of spermidine was replaced by homospermidine without any conspicuous modifications of the phenotype. Tracer-feeding experiments and gas chromatographic analyses suggested that these high levels of homospermidine were not sufficient to explain the formation of alkaloid precursors. These results are discussed with respect to current models of pathway evolution. PMID:19543980

  19. Integration of transcriptome, proteome and metabolism data reveals the alkaloids biosynthesis in Macleaya cordata and Macleaya microcarpa.

    Directory of Open Access Journals (Sweden)

    Jianguo Zeng

    Full Text Available BACKGROUND: The Macleaya spp., including Macleaya cordata and Macleaya microcarpa, are traditional anti-virus, inflammation eliminating, and insecticide herb medicines for their isoquinoline alkaloids. They are also known as the basis of the popular natural animal food addictive in Europe. However, few studies especially at genomics level were conducted on them. Hence, we performed the Macleaya spp. transcriptome and integrated it with iTRAQ proteome analysis in order to identify potential genes involved in alkaloids biosynthesis. METHODOLOGY AND PRINCIPAL FINDINGS: We elaborately designed the transcriptome, proteome and metabolism profiling for 10 samples of both species to explore their alkaloids biosynthesis. From the transcriptome data, we obtained 69367 and 78255 unigenes for M. cordata and M. microcarpa, in which about two thirds of them were similar to sequences in public databases. By metabolism profiling, reverse patterns for alkaloids sanguinarine, chelerythrine, protopine, and allocryptopine were observed in different organs of two species. We characterized the expressions of enzymes in alkaloid biosynthesis pathways. We also identified more than 1000 proteins from iTRAQ proteome data. Our results strongly suggest that the root maybe the organ for major alkaloids biosynthesis of Macleaya spp. Except for biosynthesis, the alkaloids storage and transport were also important for their accumulation. The ultrastructure of laticifers by SEM helps us to prove the alkaloids maybe accumulated in the mature roots. CONCLUSIONS/SIGNIFICANCE: To our knowledge this is the first study to elucidate the genetic makeup of Macleaya spp. This work provides clues to the identification of the potential modulate genes involved in alkaloids biosynthesis in Macleaya spp., and sheds light on researches for non-model medicinal plants by integrating different high-throughput technologies.

  20. Genetic variation in alkaloid accumulation in leaves of Nicotiana

    Institute of Scientific and Technical Information of China (English)

    Bo SUN; Fen ZHANG; Guo-jun ZHOU; Guo-hai CHU; Fang-fang HUANG; Qiao-mei WANG; Li-feng JIN; Fu-cheng LIN; Jun YANG

    2013-01-01

    Alkaloids are plant secondary metabolites that are widely distributed in Nicotiana species and contribute greatly to the quality of tobacco leaves. Some alkaloids, such as nornicotine and myosmine, have adverse effects on human health. To reduce the content of harmful alkaloids in tobacco leaves through conventional breeding, a genetic study of the alkaloid variation among different genotypes is required. In this study, alkaloid profiles in leaves of five Nicotiana tabacum cultivars and Nicotiana tomentosiformis were investigated. Six alkaloids were identified from al six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were ob-served both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R2=0.881), and were also separated from those of other alkaloids by clustering. Thus, the genotype plays a major role in alkaloid accumulation, indicating a high potential for manipulation of alkaloid content through traditional breeding.

  1. 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

  2. Horizontal acquisition of toxic alkaloid synthesis in a clade of plant associated fungi.

    Science.gov (United States)

    Marcet-Houben, Marina; Gabaldón, Toni

    2016-01-01

    Clavicipitaceae is a fungal group that comprises species that closely interact with plants as pathogens, parasites or symbionts. A key factor in these interactions is the ability of these fungi to synthesize toxic alkaloid compounds that contribute to the protection of the plant host against herbivores. Some of these compounds such as ergot alkaloids are toxic to humans and have caused important epidemics throughout history. The gene clusters encoding the proteins responsible for the synthesis of ergot alkaloids and lolines in Clavicipitaceae have been elucidated. Notably, homologs to these gene clusters can be found in distantly related species such as Aspergillus fumigatus and Penicillium expansum, which diverged from Clavicipitaceae more than 400 million years ago. We here use a phylogenetic approach to analyze the evolution of these gene clusters. We found that the gene clusters conferring the ability to synthesize ergot alkaloids and loline emerged first in Eurotiomycetes and were then likely transferred horizontally to Clavicipitaceae. Horizontal gene transfer is known to play a role in shaping the distribution of secondary metabolism clusters across distantly related fungal species. We propose that HGT events have played an important role in the capability of Clavicipitaceae to produce two key secondary metabolites that have enhanced the ability of these species to protect their plant hosts, therefore favoring their interactions. PMID:26688468

  3. Bioactive acridone alkaloids from Swinglea glutinosa.

    Science.gov (United States)

    Weniger, B; Um, B H; Valentin, A; Estrada, A; Lobstein, A; Anton, R; Maillé, M; Sauvain, M

    2001-09-01

    A new prenylated acridone alkaloid, 1,3,5-trihydroxy-2,8-bis(3-methylbut-2-enyl)-10-methyl-9-acridone (1), was isolated from the stembark of Swinglea glutinosa, along with three known acridone alkaloids, 5-hydroxynoracronycine (2), 1,3,5-trihydroxy-4-methoxy-2-(3-methylbut-2-enyl)-10-methyl-9-acridone (3), and 1,3,5-trihydroxy-4-methoxy-10-methylacridone (4). The isolated alkaloids were assessed in vitro against chloroquine-sensitive and -resistant Plasmodium falciparum strains and for cytotoxicity using HeLa cells. PMID:11575960

  4. Norditerpenoid Alkaloids from Aconitum spicatum Stapf

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To search for pharmacologically and structurally interesting substances from traditional Chinese medicines,we investigated the chemical compounds of Aconitum spicatum Stapf. Two new norditerpenoid alkaloids,namely spicatine A (compound 1) and spicatine B (compound 2), as well as 11 known norditerpenoid alkaloids were isolated from the CHCl3 portion of the 90% ethanol extract of the roots of A. spicatum. The structures of the alkaloids were characterized on the basis of their spectral data, One of the isolated compounds showed significant cytotoxic activities (IC50 values < 200 μmol/L) against the HL-60 cell line.

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

    Science.gov (United States)

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

    2014-03-01

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

  6. Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

    Directory of Open Access Journals (Sweden)

    Zuiter Afnan

    2012-08-01

    Full Text Available Abstract Background Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Findings Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. Conclusion To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

  7. 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

    in bacteria and mine the associated chemical diversity. Thirteen strains closely related to Pseudoalteromonas luteoviolacea isolated from all over the Earth were analyzed using an untargeted metabolomics strategy, and metabolomic profiles were correlated with whole-genome sequences of the strains. We found...... 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......, integrative strategy for elucidating the chemical richness of a given set of bacteria and link the chemistry to biosynthetic genes....

  8. Synthesis of the isoquinoline alkaloid, crispine C

    OpenAIRE

    Blair, A.; Stevenson, L; Sutherland, A

    2012-01-01

    The first total synthesis of the isoquinoline alkaloid, crispine C is described in seven steps using a Henry reaction and the Pictet–Gams variant of the Bischler–Napieralski reaction to effect the key transformations.

  9. Anxiolytic Activity of Diterpene Alkaloid Songorine.

    Science.gov (United States)

    Nesterova, Yu V; Povet'eva, T N; Suslov, N I; Shults, E E; Ziuz'kov, G N; Aksinenko, S G; Afanas'eva, O G; Krapivin, A V; Kharina, T G

    2015-09-01

    Antianxiety action of diterpene alkaloid songorine was studied using Vogel conflict test. Songorine in a dose of 0.25 mg/kg demonstrated high anxiolytic activity comparable to that of phenazepam and produced no sedative effect.

  10. Polycyclic alkaloids via transannular Mannich reactions

    DEFF Research Database (Denmark)

    Vital, Paulo; Hosseini, Masood; Shanmugham, M. S.;

    2009-01-01

    The tricyclic compound 13, representing the framework of the cylindricine 4 and lepadiformine 5 alkaloids, was prepared in a single operation via the first example of a transannular Mannich reaction involving a macrocyclic diketoamine 12.......The tricyclic compound 13, representing the framework of the cylindricine 4 and lepadiformine 5 alkaloids, was prepared in a single operation via the first example of a transannular Mannich reaction involving a macrocyclic diketoamine 12....

  11. Alkaloids from Fissistigma latifolium (Dunal) Merr.

    OpenAIRE

    Asmah Alias; Hazrina Hazni; Faridahanim Mohd Jaafar; Khalijah Awang; Nor Hadiani Ismail

    2010-01-01

    A phytochemical study of the bark of Fissistigma latifolium (Annonaceae) yielded a new aporphine alkaloid, (-)-N-methylguattescidine (1), and eight known alkaloids: liriodenine (2), oxoxylopine (3), (-)-asimilobine (4), dimethyltryptamine (5), (-)-remerine (6), (-)-anonaine (7), columbamine (8) and lysicamine (9). The compounds were isolated using various chromatographic methods and structural elucidation was accomplished by means of spectroscopic methods, notably 1D-NMR (1H, 13C, DEPT), 2D-N...

  12. Alkaloids from Fissistigma latifolium (Dunal Merr.

    Directory of Open Access Journals (Sweden)

    Asmah Alias

    2010-06-01

    Full Text Available A phytochemical study of the bark of Fissistigma latifolium (Annonaceae yielded a new aporphine alkaloid, (--N-methylguattescidine (1, and eight known alkaloids: liriodenine (2, oxoxylopine (3, (--asimilobine (4, dimethyltryptamine (5, (--remerine (6, (--anonaine (7, columbamine (8 and lysicamine (9. The compounds were isolated using various chromatographic methods and structural elucidation was accomplished by means of spectroscopic methods, notably 1D-NMR (1H, 13C, DEPT, 2D-NMR (COSY, HMQC, HMBC, UV, IR and MS.

  13. Ether bridge formation in loline alkaloid biosynthesis

    OpenAIRE

    Pan, Juan; Bhardwaj, Minakshi; Faulkner, Jerome R.; Nagabhyru, Padmaja; Charlton, Nikki D.; Higashi, Richard M.; Miller, Anne-Frances; Young, Carolyn A; Grossman, Robert B.; Schardl, Christopher L

    2013-01-01

    Lolines are potent insecticidal agents produced by endophytic fungi of cool-season grasses. These alkaloids are composed of a pyrrolizidine ring system and an uncommon ether bridge linking carbons 2 and 7. Previous results indicated that 1-aminopyrrolizidine was a pathway intermediate. We used RNA interference to knock down expression of lolO, resulting in the accumulation of a novel alkaloid identified as exo-1-acetamidopyrrolizidine based on high-resolution MS and NMR. Genomes of endophytes...

  14. Two new diterpene alkaloids from Delphinium chrysotrichum

    Institute of Scientific and Technical Information of China (English)

    Yang Qing He; Xiao Mei Wei; Yi Li Han; Li Ming Gao

    2007-01-01

    Chemical investigation on the ethanol extract from the whole plants of Delphinium chrysotrichum resulted in the isolation of two new diterpene alkaloids named delphatisine A (1) and delphatisine B (2), respectively. The structures of the new compounds were deduced on the basis of their spectral data (IR, HREIMS, EIMS, 1D, 2D-NMR). This is the first report on the isolation of diterpenoid alkaloids from the D. Chrysotrichum.

  15. Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5

    Directory of Open Access Journals (Sweden)

    Neilan Brett A

    2009-03-01

    Full Text Available Abstract Background Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. Results We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. Conclusion The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved

  16. Visual identification of alkaloids in some medicinal plants: common alkaloid reagents versus bromocresol green

    Directory of Open Access Journals (Sweden)

    Shamsa F, Esfahani HR, Gamooshi RA

    2008-07-01

    Full Text Available "n Normal 0 false false false MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} Background: Alkaloids are a group of nitrogenous compounds with potential effects on the physiological behavior of human and animals. Some of these compounds are considered important drugs in modern medicine, such as atropine and morphine. Plants are considered the most important source of alkaloids. Therefore, investigating the presence of alkaloids in different plants is very important. Usually, alkaloids in plants are identified by methods such as those of Dragendorf, Wagner and Meyer, among others, which require milligrams of alkaloids for identification. In the present study, a fast and sensitive procedure for detecting of alkaloids in plants is presented.   "n"nMethods: Twelve dried plants samples were investigated for the presence alkaloids. After extracting the total alkaloid into methanol using a Soxhlet extractor, a few milligrams of the extract was transferred to a separatory funnel, buffered to pH 4.7, the bromocresol green (BCG solution (10-4 M was added, mixed and extracted with CHCl3 until a yellow color was observed in the CHCl3 layer, indicating the presence of the alkaloid. The crude extracts were also investigated by the standard methods of Dragendorf, Wagner and Meyer for the presence of alkaloids.   "n"nResults: Investigation of the 12 plant samples for the presence of alkaloids by the standard reagents of Dragendorf, Wagner, and Meyer showed that only Camelia sinensis (flowers, Echium amoenum Fisch & Mey (flowers, and Stachys (aerial parts are devoid

  17. Overexpression of ORCA3 and G10H in Catharanthus roseus Plants Regulated Alkaloid Biosynthesis and Metabolism Revealed by NMR-Metabolomics

    OpenAIRE

    Qifang Pan; Quan Wang; Fang Yuan; Shihai Xing; Jingya Zhao; Young Hae Choi; Robert Verpoorte; Yuesheng Tian; Guofeng Wang; Kexuan Tang

    2012-01-01

    In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plan...

  18. Cytochrome P450 as dimerization catalyst in diketopiperazine alkaloid biosynthesis.

    Science.gov (United States)

    Saruwatari, Takayoshi; Yagishita, Fumitoshi; Mino, Takashi; Noguchi, Hiroshi; Hotta, Kinya; Watanabe, Kenji

    2014-03-21

    As dimeric natural products frequently exhibit useful biological activities, identifying and understanding their mechanisms of dimerization is of great interest. One such compound is (−)-ditryptophenaline, isolated from Aspergillus flavus, which inhibits substance P receptor for potential analgesic and anti-inflammatory activity. Through targeted gene knockout in A. flavus and heterologous yeast gene expression, we determined for the first time the gene cluster and pathway for the biosynthesis of a dimeric diketopiperazine alkaloid. We also determined that a single cytochrome P450, DtpC, is responsible not only for pyrroloindole ring formation but also for concurrent dimerization of N-methylphenylalanyltryptophanyl diketopiperazine monomers into a homodimeric product. Furthermore, DtpC exhibits relaxed substrate specificity, allowing the formation of two new dimeric compounds from a non-native monomeric precursor, brevianamide F. A radical-mediated mechanism of dimerization is proposed.

  19. Emergent biosynthetic capacity in simple microbial communities.

    Directory of Open Access Journals (Sweden)

    Hsuan-Chao Chiu

    2014-07-01

    Full Text Available Microbes have an astonishing capacity to transform their environments. Yet, the metabolic capacity of a single species is limited and the vast majority of microorganisms form complex communities and join forces to exhibit capabilities far exceeding those achieved by any single species. Such enhanced metabolic capacities represent a promising route to many medical, environmental, and industrial applications and call for the development of a predictive, systems-level understanding of synergistic microbial capacity. Here we present a comprehensive computational framework, integrating high-quality metabolic models of multiple species, temporal dynamics, and flux variability analysis, to study the metabolic capacity and dynamics of simple two-species microbial ecosystems. We specifically focus on detecting emergent biosynthetic capacity--instances in which a community growing on some medium produces and secretes metabolites that are not secreted by any member species when growing in isolation on that same medium. Using this framework to model a large collection of two-species communities on multiple media, we demonstrate that emergent biosynthetic capacity is highly prevalent. We identify commonly observed emergent metabolites and metabolic reprogramming patterns, characterizing typical mechanisms of emergent capacity. We further find that emergent secretion tends to occur in two waves, the first as soon as the two organisms are introduced, and the second when the medium is depleted and nutrients become limited. Finally, aiming to identify global community determinants of emergent capacity, we find a marked association between the level of emergent biosynthetic capacity and the functional/phylogenetic distance between community members. Specifically, we demonstrate a "Goldilocks" principle, where high levels of emergent capacity are observed when the species comprising the community are functionally neither too close, nor too distant. Taken together

  20. Pseudomonas corrugata crpCDE is part of the cyclic lipopeptide corpeptin biosynthetic gene cluster and is involved in bacterial virulence in tomato and in hypersensitive response in Nicotiana benthamiana

    NARCIS (Netherlands)

    Strano, C.P.; Bella, P.; Licciardello, G.; Fiore, A.; Piero, Lo A.R.; Fogliano, V.; Fogliano, V.; Catara, V.

    2015-01-01

    Pseudomonas corrugata CFBP 5454 produces two kinds of cyclic lipopeptides (CLPs), cormycin A and corpeptins, both of which possess surfactant, antimicrobial and phytotoxic activities. In this study, we identified genes coding for a putative non-ribosomal peptide synthetase and an ABC-type transport

  1. Polymerase chain reaction (PCR)-based methods for detection/identification of mycotoxigenic fungi targeting fumonisin biosynthetic genes: Use of variation in FUM cluster location to distinguish between and quantify

    Science.gov (United States)

    The fungus Fusarium is an agricultural problem because it can cause disease on most crop plants and can contaminate crops with mycotoxins. There is considerable variation in the presence/absence and genomic location of gene clusters responsible for synthesis of mycotoxins and other secondary metabol...

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  3. Fast induction of biosynthetic polysaccharide genes lpxA, lpxE, and rkpI of Rhizobium sp. strain PRF 81 by common bean seed exudates is indicative of a key role in symbiosis.

    Science.gov (United States)

    Oliveira, Luciana Ruano; Rodrigues, Elisete Pains; Marcelino-Guimarães, Francismar Corrêa; Oliveira, André Luiz Martinez; Hungria, Mariangela

    2013-06-01

    Rhizobial surface polysaccharides (SPS) are, together with nodulation (Nod) factors, recognized as key molecules for establishment of rhizobia-legume symbiosis. In Rhizobium tropici, an important nitrogen-fixing symbiont of common bean (Phaseolus vulgaris L.), molecular structures and symbiotic roles of the SPS are poorly understood. In this study, Rhizobium sp. strain PRF 81 genes, belonging to the R. tropici group, were investigated: lpxA and lpxE, involved in biosynthesis and modification of the lipid-A anchor of lipopolysaccharide (LPS), and rkpI, involved in synthesis of a lipid carrier required for production of capsular polysaccharides (KPS). Reverse transcription quantitative PCR (RT-qPCR) analysis revealed, for the first time, that inducers released from common bean seeds strongly stimulated expression of all three SPS genes. When PRF 81 cells were grown for 48 h in the presence of seed exudates, twofold increases (p PRF81 and of the type strain of R. tropici CIAT899(T)clustered with orthologous Rhizobium radiobacter and were more related to R. etli and Rhizobium leguminosarum, while rkpI was closer to the Sinorhizobium sp. group. Upregulation of lpxE, lpxA, and rkpI genes suggests that seed exudates can modulate production of SPS of Rhizobium sp. PRF81, leading to cell wall changes necessary for symbiosis establishment.

  4. ABA-Mediated Inhibition of Germination Is Related to the Inhibition of Genes Encoding Cell-Wall Biosynthetic and Architecture:Modifying Enzymes and Structural Proteins in Medicago truncatula Embryo Axis

    Institute of Scientific and Technical Information of China (English)

    Christine Gimeno-Gilles; Eric Lelièvre; Laure Viau; Mustafa Malik-Ghulam; Claudie Ricoult; Andreas Niebel; Nathalie Leduc; Anis M. Limami

    2009-01-01

    Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid (ABA) but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.

  5. Transcriptome analysis of stem wood of Nothapodytes nimmoniana (Graham) Mabb. identifies genes associated with biosynthesis of camptothecin, an anti-carcinogenic molecule

    Indian Academy of Sciences (India)

    BL Manjunatha; HR Singh; G Ravikanth; Karaba N Nataraja; Ravi Shankar; Sanjay Kumar; R Uma Shaanker

    2016-03-01

    Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of DNA topoisomerase I and has applications in treating ovarian, small lung and refractory ovarian cancers. Stem wood tissue of Nothapodytes nimmoniana (Graham) Mabb. (family Icacinaceae) is one of the richest sources of CPT. Since there is no genomic or transcriptome data available for the species, the present work sequenced and analysed transcriptome of stem wood tissue on an Illumina platform. From a total of 77,55,978 reads, 9,187 transcripts were assembled with an average length of 255 bp. Functional annotation and categorization of these assembled transcripts unraveled the transcriptome architecture and also a total of 13 genes associated with CPT biosynthetic pathway were identified in the stem wood tissue. Four genes of the pathway were cloned to full length by RACE to validate the transcriptome data. Expression analysis of 13 genes associated with CPT biosynthetic pathway in 11 different tissues vis-a-vis CPT content analysis suggested an important role of NnPG10H, NnPSLS and NnPSTR genes in the biosynthesis of CPT. These results indicated that CPT might be synthesized in the leaves and then perhaps exported to stem wood tissue for storage.

  6. 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

    Directory of Open Access Journals (Sweden)

    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

  7. Two New C19-Diterpenoid Alkaloids from Delphinium davidii Franch.

    Institute of Scientific and Technical Information of China (English)

    Xiao Xia LIANG; Dong Lin CHEN; Feng Peng WANG

    2006-01-01

    Two new C19-diterpenoid alkaloids, davidisines A (1) and B (2) along with thirteen known alkaloids were isolated from the whole herb of Delphinium davidii Franch. Their structures were established by spectral methods, especially 2D NMR techniques.

  8. Biosynthesis of Monoterpenoid Indole Alkaloid Ajmaline Catalyzed by Novel Reductases

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ Introduction One of the major root alkaloids of the Indian medicinal plant Rauvolfia serpenlina Benth. Ex Kurz is named ajmaline. The enzymatic biosynthesisof this alkaloid has been studied for a long time by our group[1].

  9. Biological activity of alkaloids from Solanum dulcamara L.

    Science.gov (United States)

    Kumar, Padma; Sharma, Bindu; Bakshi, Nidhi

    2009-01-01

    Alkaloids are well known for their antimicrobial activity. Though all natural alkaloids come from plants, not all plants produce alkaloids. Plants of the Solanaceae family are known for their high alkaloid content. Alkaloids are found in all plant parts like roots, stems, leaves, flowers, fruits and seeds. In the present study, those plant parts of Solanum dulcamara were selected which have been reported to produce a high content of a specific alkaloid: solanine (from unripe fruits), solasodine (from flowers) and beta-solamarine (from roots). These alkaloids were extracted from various parts of S. dulcamara by well-established methods and were screened for their antibacterial activity. Human pathogenic bacteria, viz., Enterobacter aerogenes, Escherichia coli, Staphylococcus aureus, were selected for the study. All three alkaloids inhibited the growth of E. coli and S. aureus. However, no significant activity was observed against E. aerogenes. Minimum inhibitory concentration and minimum bactericidal concentration were also evaluated.

  10. Monoterpenoid Indole Alkaloids from Catharanthus roseus Cultivated in Yunnan.

    Science.gov (United States)

    Wang, Bei; Liu, Lu; Chen, Ying-ying; Li, Qiong; Li, Dan; Liu, Va-ping; Luo, Xiao-dong

    2015-12-01

    A new monoterpenoid indole alkaloid, 15,20-dehydro-3α-(2-oxopropyl) coronaridine (1), along with sixteen analogues (2-17) were isolated from the leaves of Catharanthus roseus cultivated in Yunnan. The new alkaloid was elucidated on the basis of extensive spectroscopic analysis, and the known alkaloids were identified by comparison with the reported spectroscopic data. Among them, alkaloid 16 was isolated from Catharanthus for the first time. PMID:26882670

  11. An efficient synthesis of loline alkaloids

    Science.gov (United States)

    Cakmak, Mesut; Mayer, Peter; Trauner, Dirk

    2011-07-01

    Loline (1) is a small alkaloid that, in spite of its simple-looking structure, has posed surprising challenges to synthetic chemists. It has been known for more than a century and has been the subject of extensive biological investigations, but only two total syntheses have been achieved to date. Here, we report an asymmetric total synthesis of loline that, with less then ten steps, is remarkably short. Our synthesis incorporates a Sharpless epoxidation, a Grubbs olefin metathesis and an unprecedented transannular aminobromination, which converts an eight-membered cyclic carbamate into a bromopyrrolizidine. The synthesis is marked by a high degree of chemo- and stereoselectivity and gives access to several members of the loline alkaloid family. It delivers sufficient material to support a programme aimed at studying the complex interactions between plants, fungi, insects and bacteria brokered by loline alkaloids.

  12. Two New Alkaloids from Narcissus serotinus L.

    Directory of Open Access Journals (Sweden)

    Francesc Viladomat

    2010-10-01

    Full Text Available The Amaryllidaceae family is well known for the presence of an exclusive group of alkaloids with a wide range of biological activities. Narcissus serotinus L. is a plant belonging to this family and its geographical distribution is mainly located along the Mediterranean coast. In the present work, specimens collected near Casablanca (Morocco were used to study the alkaloid content of this species. Starting with 350 g of the whole plant we used standard extraction and purification procedures to obtain fractions and compounds for GC-MS and NMR analysis. As well as five known alkaloids, we isolated two new compounds: 1-O-(3´-acetoxybutanoyllycorine and narseronine. The latter has been previously published, but with an erroneous structure.

  13. Aporphine alkaloids from Ocotea macrophylla (Lauraceae)

    Energy Technology Data Exchange (ETDEWEB)

    Pabon, Ludy Cristina; Cuca, Luis Enrique, E-mail: lcpabonb@unal.edu.c [Universidad Nacional de Bogota (Colombia). Facultad de Ciencias. Dept. de Quimica

    2010-07-01

    Four aporphine alkaloids from the wood of Ocotea macrophylla (Lauraceae) were isolated and characterized as (S)-3-methoxy-nordomesticine (1), (S)-N-ethoxycarbonyl-3-methoxy-nordomesticine (2), (S)-N-formyl-3-methoxy-nordomesticine (3) and (S)-N-methoxycarbonyl-3-methoxy-nordomesticine (4); alkaloids 2-4 are being report for the first time. The structure the isolated compounds were determined based on their spectral data and by comparison of their spectral data with values described in literature. The alkaloid fraction and compound 1 showed antifungal activity against Fusarium oxysporum f. sp. lycopersici and also compound 1 showed antimicrobial activity towards Staphylococcus aureus, Enterococcus faecalis as well. (author)

  14. Aporphine alkaloids from Ocotea macrophylla (Lauraceae

    Directory of Open Access Journals (Sweden)

    Ludy Cristina Pabon

    2010-01-01

    Full Text Available Four aporphine alkaloids from the wood of Ocotea macrophylla (Lauraceae were isolated and characterized as (S-3-methoxy-nordomesticine (1, (S-N-ethoxycarbonyl-3-methoxy-nordomesticine (2, (S-N-formyl-3-methoxy-nordomesticine (3 and (S-N-methoxycarbonyl-3-methoxy-nordomesticine (4; alkaloids 2-4 are being report for the first time. The structure the isolated compounds were determined based on their spectral data and by comparison of their spectral data with values described in literature. The alkaloid fraction and compound 1 showed antifungal activity against Fusarium oxysporum f. sp. lycopersici and also compound 1 showed antimicrobial activity towards Staphylococcus aureus, Enterococcus faecalis as well.

  15. Antibacterial and antifungal activity of liriodenine and related oxoaporphine alkaloids.

    Science.gov (United States)

    Hufford, C D; Sharma, A S; Oguntimein, B O

    1980-10-01

    Liriodenine was evaluated for its antibacterial and antifungal activity against several microorganisms. Other related oxoaporphine alkaloids also were evaluated. Attempts to prepare oxoaporphine alkaloids from N-acetylnoraporphines were unsuccessful, but an unexpected phenanthrene alkaloid was obtained. A novel N-demethylation reaction was noted when oxogaucine methiodide and liriodenine methiodide were treated with alumina. PMID:7420287

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

  17. Absence of steroid biosynthetic defects in heterozygote individuals for classic 11{beta}-hydroxylase deficiency due to a R448H mutation in the CYP11B1 gene

    Energy Technology Data Exchange (ETDEWEB)

    Roesler, A.; Cohen, H. [Hadassah-Hebrew Univ. Medical Center, Jerusalem (Israel)

    1995-12-01

    Steroid 11{beta}-hydroxylase deficiency (failure to convert 11-deoxycortisol to cortisol) is responsible for less than 5% of cases of classic congenital adrenal hyperplasia, but it is relatively frequent in Israel, among Jews of Moroccan origin. Affected individuals have a single base substitution in exon 8 of CYP11B1 gene, codon 448, from CGC (arginine) to CAC (histidine) (R448H), a mutation that abolishes enzyme activity completely. We studied the hormonal response to ACTH stimulation in individuals genotyped to have the R448H mutation in one allele only (heterozygotes), and who were therefore assumed to have 50% of 11{beta}-hydroxylase activity. No demonstrable hormonal abnormalities were found in the 6 adults (3 mothers and 3 fathers) and 2 sons studied, suggesting that a quantitatively reduced 11{beta}-hydroxylase is still enough for normal adrenal biosynthesis. 19 refs., 1 fig., 2 tabs.

  18. Indole Alkaloids of the Stigonematales (Cyanophyta: Chemical Diversity, Biosynthesis and Biological Activity

    Directory of Open Access Journals (Sweden)

    Katherine Walton

    2016-04-01

    Full Text Available The cyanobacteria are well recognized as producers of a wide array of bioactive metabolites including toxins, and potential drug candidates. However, a limited number of taxa are generally considered with respect to both of these aspects. That said, the order Stigonematales, although largely overlooked in this regard, has become increasingly recognized as a source of bioactive metabolites relevant to both human and environmental health. In particular, the hapalindoles and related indole alkaloids (i.e., ambiguines, fischerindoles, welwitindolinones from the order, represent a diverse, and phylogenetically characteristic, class of secondary metabolites with biological activity suggestive of potential as both environmental toxins, and promising drug discovery leads. The present review gives an overview of the chemical diversity of biologically active metabolites from the Stigonematales—and particularly the so-called hapalindole-type alkaloids—including their biosynthetic origins, and their pharmacologically and toxicologically relevant bioactivities. Taken together, the current evidence suggests that these alkaloids, and the associated cyanobacterial taxa from the order, warrant future consideration as both potentially harmful (i.e., “toxic” algae, and as promising leads for drug discovery.

  19. NAA和ABA处理对‘京优’葡萄花色苷生物合成相关基因表达的影响%Effects of NAA and ABA treatments on the expression of anthocyanin biosynthetic genes in 'Jing You' grape berry skin

    Institute of Scientific and Technical Information of China (English)

    周莉; 王军

    2011-01-01

    为人工调控花色苷合成,以‘京优’葡萄为实验材料,应用液相色谱-质谱(HPLC-MS)技术和荧光定量PCR,研究了萘乙酸(NAA)和脱落酸(ABA)处理对葡萄果皮花色苷积累及其生物合成相关基因表达的影响。结果表明:在‘京优’葡萄果皮中,可检测到16种花色苷;ABA处理的花色苷含量高于对照,NAA处理低于对照,并且ABA处理的果皮比对照和NAA多出3种花色苷;葡萄果实发育进入转色期,花色苷合成过程中类黄酮途径的结构基因上调表达,完熟后下调表达;ABA处理的果皮花色苷结构基因相对表达量高于对照,而NAA则低于对%In order to control anthocyanin synthesis of 'Jingyou' grape,we applied high performance liquid chromatography-mass spectrometry(HPLC-MS) and real time reverse transcription-polymerase chain reaction(RT-PCR) for studying the effects of naphthaleneacetic aicd(NAA) and abscisic acid(ABA) treatments on the accumulation of anthocyanins,as well as the expression of anthocyanin biosynthetic correlative genes.The results showed that there were 16 sorts of anthocyanins in 'Jing You' berry skin.The anthocyanin contents of ABA treatment were higher but NAA treatment lower than that of control.And there were three extra anthocyanins in ABA treatment of berry skin.The structural genes in flavonoid pathway of anthocyanin synthesis were up-regulated at veraison,but down-regulated after complete maturity.The expression of anthocyanin structural genes was higher in ABA treatment than control,but lower for NAA treatment than control.The accumulation of anthocyanin and the correlative genes of anthocyanin synthesis were promoted by exogenous ABA.And NAA treatment had the opposite impacts.

  20. Antimicrobial hasubanalactam alkaloid from Stephania glabra.

    Science.gov (United States)

    Semwal, Deepak Kumar; Rawat, Usha

    2009-03-01

    A novel hasubanalactam alkaloid, named glabradine, has been isolated from the tubers of Stephania glabra, together with three known quaternary protoberberine alkaloids, palmatine, dehydrocorydalmine and stepharanine. The structure of glabradine was assigned as 7-O-demethyl-N,O-dimethyloxostephinine, by means of rigorous spectroscopic analysis including 2 D NMR measurements. It was evaluated for antimicrobial activity against Staphylococcus aureus, S. mutans, Microsporum gypseum, M. canis and Trichophyton rubrum and displayed potent antimicrobial activity superior to those of novobiocin and erythromycin used as positive controls. PMID:19148860

  1. Antiprotozoal alkaloids from Psychotria prunifolia (Kunth) Steyerm

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Lucilia; Oliveira, Cecilia M.A. de; Faria, Emiret O.; Ribeiro, Laryssa C.; Carvalho, Brenda G., E-mail: lucilia@quimica.ufg.br [Instituto de Quimica, Universidade Federal de Goias, Campus II, Samambaia, Goiania, GO (Brazil); Silva, Cleuza C. da; Santin, Silvana M.O. [Departamento de Quimica, Universidade Estadual de Maringa, Maringa, PR (Brazil); Schuque, Ivania T.A.; Nakamura, Celso V.; Britta, Elisandra A.; Miranda, Nathielle [Departamento de Farmacia e Farmacologia, Universidade Estadual de Maringa, PR (Brazil); Iglesias, Amadeu H. [Waters Technologies do Brasil LTDA, Barueri, SP (Brazil); Delprete, Piero G. [VHerbier de Guyane, Institut de Recherche pour le Developpement (IRD), UMR AMAP, French Guiana (France)

    2012-07-01

    The continuity of the phyto chemical study of crude extracts of P. prunifolia's roots and branches led to the isolation of five indole-{beta}-carboline alkaloids. Among them, the 10-hydroxy-iso-deppeaninol and N-oxide-10-hydroxy-antirhine derivatives are described here for the first time. The structures were achieved through 1D and 2D NMR, IR and HRMS analyses. The branches and roots crude extracts and the alkaloids 14-oxoprunifoleine and strictosamide showed selective activity against L. amazonensis, with IC{sub 50} values of 16.0 and 40.7 {mu}g per mL, respectively. (author)

  2. Total Synthesis of the Zoanthamine Alkaloid

    Institute of Scientific and Technical Information of China (English)

    M. Miyashita

    2005-01-01

    @@ 1Introduction The zoanthamine alkaloids, a type of heptacyclic marine alkaloid isolated from colonial zoanthids of the genus Zoanthus sp., have attracted much attention from a wide area of science, because of their distinctive biological and pharmacological properties as well as their chemical structures with stereochemical complexity.Namely, norzoanthamine (1)[1] can suppress the loss of bone weight and strength in ovariectomized mice and has been considered a promising candidate for an antiosteoporotic drug[1], whereas zoanthamine (2)[2] has exhibited potent inhibitory activity toward phorbol myristate-induced inflammation in addition to powerful analgesic effects[2]. See Fig. 1.

  3. Identification and characterization of GDP-d-mannose 4,6-dehydratase and GDP-l-fucose snthetase in a GDP-l-fucose biosynthetic gene cluster from Helicobacter pylori.

    Science.gov (United States)

    Wu, B; Zhang, Y; Wang, P G

    2001-07-13

    In this study two open reading frames, namely HP0044 and HP0045 from H. pylori, were cloned and overexpressed in E. coli. The two recombinant proteins were demonstrated to have GDP-d-mannose 4,6-dehydratase (GMD) and GDP-l-fucose synthetase (GFS) activities, respectively. The recombinant GMD was a tetramer and had an optimum pH of 6.5. Exogenous NADP(+) was essential for its activity. The K(m) and K(cat) for GDP-d-mannose were 117.3 microM and 0.27 s(-1), respectively. The recombinant GFS was a homodimer with an optimum pH of 8.0. The K(m) and K(cat) for GDP-4-keto-6-deoxy-d-mannose were 64.08 microM and 0.75 s(-1), respectively. It can use both NADPH and NADH, but less efficient with the latter. Amino acid sequence alignment and phylogenetic analysis showed that H. pylori GFS was highly homologous to the GFS of E. coli O111 and both of them were located on a separate phylogenetic branch from other GFS. The unique clustering and origin of the two genes were also discussed. PMID:11444851

  4. Alkaloid diversity in Galanthus elwesii and Galanthus nivalis.

    Science.gov (United States)

    Berkov, Strahil; Bastida, Jaume; Sidjimova, Borjana; Viladomat, Francesc; Codina, Carles

    2011-01-01

    Seventy alkaloids of galanthamine, lycorine, homolycorine, tazettine, haemanthamine, narciclasine, and tyramine types were detected by GC/MS in 25 Galanthus elwesii and seven Galanthus nivalis populations, collected from different locations in Bulgaria. Intraspecies diversity in the alkaloid profiles regarding the main alkaloid types (chemotypes) was observed. Tyramine-type protoalkaloids (namely, hordenine and its derivatives) were dominant in 19 populations of G. elwesii. In other populations of G. elwesii, the plants accumulated mainly homolycorine-, lycorine-, and galanthamine-type alkaloids. The alkaloid profiles of G. nivalis were dominated by narciclasine-, galanthamine-, lycorine-, haemanthamine-, or tazettine-type compounds. Geographical distribution of chemotypes indicated a relationship between populations, since adjacent populations often displayed similar alkaloid profiles. The results from year-to-year sampling and transplantation experiments imply genetic determination of alkaloid synthesis in the two studied species of Galanthus.

  5. A New Quinolizidine Alkaloid from Boehmeria siamensis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A new quinolizidine alkaloid, 3-(4-hydroxyphenyl)-4-(3-methoxy-4-hydroxyphenyl)-3, 4-dehydroquinolizidine (1), was isolated from the ethanol extract of the whole plants of Boehmeria siamensis Craib. Its structure was elucidated on the analysis of 1D NMR and 2D NMR spectrum.

  6. Two New Oxoaporphine Alkaloids from Thalictrum elegans

    Institute of Scientific and Technical Information of China (English)

    梁志远; 杨小生; 汪冶; 郝小江

    2005-01-01

    Two new oxoaporphine alkaloids, 1,2,3,10-tetramethoxy-9-(2-hydroxy-4,5-dimethoxybenzyloxy)oxoaporphine (1) and 1,2,3,10-tetramethoxy-9-(4,5-dimethoxy-2-formylphenoxy)oxoaporphine (2), were isolated from Thalictrum elegans. Their structures were elucidated based on spectroscopic analysis including 1D, 2D NMR, IR and MS.

  7. Saponins and alkaloids from Abuta grandifolia

    OpenAIRE

    Sayagh, C.; Long, C; Moretti, Christian; Lavaud, C.

    2012-01-01

    Five new bidesmosidic saponins (1-5), along with six known triterpenoid saponins, were isolated from the methanolic extracts of the leaves and twigs of Abuta grandifolia. The ethyl acetate extracts allowed the isolation of three tropoloisoquinolines and of one isoquinoline; all were known alkaloids. The structures of all compounds were elucidated on the basis of extensive spectroscopic data analysis.

  8. A New Alkaloid from Sinomenium acutum

    Institute of Scientific and Technical Information of China (English)

    Wei Ming CHENG; Feng QIU; Li Jun WU; Xin Sheng YAO

    2005-01-01

    Sinomenium acutum is widely used in East and South Asia for the treatment of many diseases, especially rheumatoid arthritis (RA). The chemical research on Sinomenium acutum led to the isolation of a new alkaloid compound (1). On the basis of chemical evidences and spectral analysis, 1 was identified as N-(1, 7-dimethoxylphenanthren-2-yl)acetamide.

  9. Histrionicotoxin alkaloids finally detected in an ant

    DEFF Research Database (Denmark)

    Jones, Tappey H.; Adams, Rachelle Martha Marie; Spande, Thomas F.;

    2012-01-01

    Workers of the ant Carebarella bicolor collected in Panama were found to have two major poison-frog alkaloids, cis- and trans-fused decahydroquinolines (DHQs) of the 269AB type, four minor 269AB isomers, two minor 269B isomers, and three isomers of DHQ 271D. For the first time in an ant, however......, the DHQs were accompanied by six histrionicotoxins (HTXs), viz., 283A, 285A, 285B, 285C, 287A, and 287D. This co-occurrence of the HTX and DHQ alkaloids is the usual pattern seen in dendrobatid frogs. This finding contrasts with our earlier study, where workers of a Brazilian ant, Solenopsis (Diplorhoptrum......) sp., were found to have a very similar DHQ complex but failed to show HTXs. Several new DHQ alkaloids of MW 271 (named in the frog as 271G) are reported from the above ants that have both m/z 202 and 204 as major fragment ions, unlike the spectrum seen for the poison-frog alkaloid 271D, which has...

  10. Acetylcholinesterase-inhibiting Alkaloids from Zephyranthes concolor

    Directory of Open Access Journals (Sweden)

    Sebastien Arseneau

    2011-11-01

    Full Text Available The bulbs and aerial parts of Zephyranthes concolor (Lindl. Benth. & Hook. f. (Amaryllidaceae, an endemic species to Mexico, were found to contain the alkaloids chlidanthine, galanthamine, galanthamine N-oxide, lycorine, galwesine, and epinorgalanthamine. Since currently only partial and low resolution 1H-NMR data for chlidanthine acetate are available, and none for chlidanthine, its 1D and 2D high resolution 1H- and 13C-NMR spectra were recorded. Unambiguous assignations were achieved with HMBC, and HSQC experiments, and its structure was corroborated by X-ray diffraction. Minimum energy conformation for structures of chlidanthine, and its positional isomer galanthamine, were calculated by molecular modelling. Galanthamine is a well known acetylcholinesterase inhibitor; therefore, the isolated alkaloids were tested for this activity. Chlidanthine and galanthamine N-oxide inhibited electric eel acetylcholinesterase (2.4 and 2.6 × 10−5 M, respectively, indicating they are about five times less potent than galanthamine, while galwesine was inactive at 10−3 M. Inhibitory activity of HIV-1 replication, and cytotoxicity of the isolated alkaloids were evaluated in human MT-4 cells; however, the alkaloids showed poor activity as compared with standard anti-HIV drugs, but most of them were not cytotoxic.

  11. Vindoline Formation in Shoot Cultures of Catharanthus roseus is Synchronously Activated with Morphogenesis Through the Last Biosynthetic Step

    Science.gov (United States)

    Campos-Tamayo, Freddy; Hernández-Domínguez, Elizabeta; Vázquez-Flota, Felipe

    2008-01-01

    Background and Aims The Madagascar periwinkle (Catharanthus roseus) produces the monoterpenoid alkaloid vindoline, which requires a specialized cell organization present only in the aerial tissues. Vindoline content can be affected by photoperiod and this effect seems to be associated with the morphogenetic capacity of branches; this association formed the basis of the study reported here. Methods Vindoline-producing in vitro shoot cultures were exposed either to continuous light or a 16-h photoperiod regime. New plantlet formation and alkaloid biosynthesis were analysed throughout a culture cycle. Key Results In cultures under the photoperiod, the formation of new plantlets occurred in a more synchronized fashion as compared to those under continuous light. The accumulation of vindoline in cultures under the photoperiod occurred in co-ordination with plantlet formation, in constrast to cultures under continuous light, and coincided with a peak of activity of deacetylvindoline acetyl CoA acetyltransferase (DAT), the enzyme that catalyses the last step in vindoline biosynthesis. When new plantlet formation was blocked in cultures under the photoperiod by treatment with phytoregulators, vindoline synthesis was also reduced via an effect on DAT activity. No association between plantlet formation and other biosynthetic enzymes, such as tryptophan decarboxylase (TDC) and deacetoxyvindoline 4-hydroxylase (D4H), was found. Effects of light treatment on vindoline synthesis were not mediated by ORCA-3 proteins (which are involved in the induction of alkaloid synthesis in response to elicitation), suggesting that the presence of a different set of regulatory proteins. Conclusions The data suggest that vindoline biosynthesis is associated with morphogenesis in shoot cultures of C. roseus. PMID:18587132

  12. Alkaloids in the pharmaceutical industry: Structure, isolation and application

    Directory of Open Access Journals (Sweden)

    Nikolić Milan

    2003-01-01

    Full Text Available By the end of the 18th and the beginning of the 19th century a new era began in medicine, pharmaceutics and chemistry that was strongly connected with alkaloids and alkaloid drugs. Even before that it was known that certain drugs administered in limited doses were medicines, and toxic if taken in larger doses (opium, coke leaves, belladonna roots, monkshood tubers crocus or hemlock seeds. However, the identification, isolation and structural characterization of the active ingredients of the alkaloid drugs was only possible in the mid 20th century by the use of modern extraction equipment and instrumental methods (NMR, X-ray diffraction and others.In spite of continuing use over a long time, there is still great interest in investigating new drugs, potential raw materials for the pharmaceutical industry, as well as the more detailed investigation and definition of bio-active components and the indication of their activity range, and the partial synthesis of new alkaloid molecules based on natural alkaloids. The scope of these investigations, especially in the field of semi-synthesis is to make better use of the bio-active ingredients of alkaloid drugs, i.e. to improve the pharmacological effect (stronger and prolonged effect of the medicine, decreased toxicity and side effects, or to extend or change the applications. A combined classification of alkaloids was used, based on the chemical structure and origin, i.e. the source of their isolation to study alkaloid structure. For practical reasons, the following classification of alkaloids was used: ergot alkaloids, poppy alkaloids, tropanic alkaloids purine derivative alkaloids, carbon-cyclic alkaloids, and other alkaloids. The second part of this report presents a table of general procedures for alkaloid isolation from plant drugs (extraction by water non-miscible solvents, extraction by water-miscible solvents and extraction by diluted acid solutions. Also, methods for obtaining chelidonine and

  13. Biosynthetic porphyrins and the origin of photosynthesis

    Science.gov (United States)

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

    1986-01-01

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

  14. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    Energy Technology Data Exchange (ETDEWEB)

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong

    2011-12-31

    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  15. Rhizoma Coptidis alkaloids alleviate hyperlipidemia in B6 mice by modulating gut microbiota and bile acid pathways.

    Science.gov (United States)

    He, Kai; Hu, Yinran; Ma, Hang; Zou, Zongyao; Xiao, Yubo; Yang, Yong; Feng, Min; Li, Xuegang; Ye, Xiaoli

    2016-09-01

    It is hypothesized that Rhizoma Coptidis (RC) alkaloids exert their hypolipidemic effects primarily by targeting the gastrointestinal tract and liver. Thus, this study was conducted to evaluate the antihyperlipidemic mechanisms of RC alkaloids (at a daily dose of 140mg/kg for 35days) in high-fat and high-cholesterol induced hyperlipidemic B6 mice. After treatment, serum lipid parameters were determined, the expression of lipid metabolism related genes and pathways such as the sterol regulatory element binding proteins (SREBPs) and bile acid signaling in mice were also investigated. Meanwhile, Illumina sequencing was used to investigate the differences in gut microbiota of B6 mice. The results indicated that RC alkaloids reduced the body weight gain and serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), total bile acids (TBA) and lipopolysaccharide of B6 mice. Liver fat deposition and epididymal adipose cell size were also deceased in therapy group. RC alkaloids feeding significantly promoted the abundance of Sporobacter termitidis, Alcaligenes faecalis, Akkermansia muciniphila in the gut of mice, whereas, the abundance of Escherichia coli, Desulfovibrio C21_c20, Parabacteroides distasonis was suppressed. The observed antihyperlipidemic effects of RC alkaloids can also be attributed to their action as agonists of FXR and TGR5, activators for SREBP2, LDLR, UCP2 and CYP7A1, inhibitors of HMGCR, TXNIP, TLR4 and JNK. Therefore, this study expands current knowledge on hypolipidemic mechanisms of RC alkaloids and presents new evidence supporting a key role for RC alkaloids as regulators of lipid homeostasis by modulation gut microbiota and hepatic lipid metabolism. PMID:27287254

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

    OpenAIRE

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

    2010-01-01

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

  17. Diversity and abundance of phosphonate biosynthetic genes in nature

    OpenAIRE

    Yu, Xiaomin; Doroghazi, James R.; Janga, Sarath C.; Zhang, Jun Kai; Circello, Benjamin; Griffin, Benjamin M.; Labeda, David P.; Metcalf, William W.

    2013-01-01

    Phosphonic acids are organophosphorus molecules containing direct carbon–phosphorus bonds that are often perceived as biological rarities. The data presented here show that the ability to synthesize diverse phosphonates is both widespread and relatively common among microbes. These findings are consistent with recent evidence suggesting that phosphonates are important intermediates in the global phosphorus cycle. Moreover, they support the idea that these molecules play a significant role in ...

  18. Exploring complex pheromone biosynthetic processes in the bumblebee male labial gland by RNA sequencing.

    Science.gov (United States)

    Buček, A; Brabcová, J; Vogel, H; Prchalová, D; Kindl, J; Valterová, I; Pichová, I

    2016-06-01

    Male marking pheromones (MPs) are used by the majority of bumblebee species (Hymenoptera: Apidae), including a commercially important greenhouse pollinator, the buff-tailed bumblebee (Bombus terrestris), to attract conspecific females. MP biosynthetic processes in the cephalic part of the bumblebee male labial gland (LG) are of extraordinary complexity, involving enzymes of fatty acid and isoprenoid biosynthesis, which jointly produce more than 50 compounds. We employed a differential transcriptomic approach to identify candidate genes involved in MP biosynthesis by sequencing Bombus terrestris LG and fat body (FB) transcriptomes. We identified 12 454 abundantly expressed gene products (reads per kilobase of exon model per million mapped reads value > 1) that had significant hits in the GenBank nonredundant database. Of these, 876 were upregulated in the LG (> 4-fold difference). We identified more than 140 candidate genes potentially involved in MP biosynthesis, including esterases, fatty acid reductases, lipases, enzymes involved in limited fatty acid chain shortening, neuropeptide receptors and enzymes involved in biosynthesis of triacylglycerols, isoprenoids and fatty acids. For selected candidates, we confirmed their abundant expression in LG using quantitative real-time reverse transcription-PCR (qRT-PCR). Our study shows that the Bombus terrestris LG transcriptome reflects both fatty acid and isoprenoid MP biosynthetic processes and identifies rational gene targets for future studies to disentangle the molecular basis of MP biosynthesis. Additionally, LG and FB transcriptomes enrich the available transcriptomic resources for Bombus terrestris. PMID:26945888

  19. Synthesis of the Marine Pyrroloiminoquinone Alkaloids, Discorhabdins

    Directory of Open Access Journals (Sweden)

    Yasufumi Wada

    2010-04-01

    Full Text Available Manynatural products with biologically interesting structures have been isolated from marine animals and plants such as sponges, corals, worms, etc. Some of them are discorhabdin alkaloids. The discorhabdin alkaloids (discorhabdin A-X, isolated from marine sponges, have a unique structure with azacarbocyclic spirocyclohexanone and pyrroloiminoquinone units. Due to their prominent potent antitumor activity, discorhabdins have attracted considerable attention. Many studies have been reported toward the synthesis of discorhabdins. We have accomplished the first total synthesis of discorhabdin A (1, having the strongest activity in vitro among discorhabdins in 2003. In 2009, we have also accomplished the first total synthesis of prianosin B (2, having the 16,17-dehydropyrroloiminoquinone moiety, by a novel dehydrogenation reaction with a catalytic amount of NaN3. These synthetic studies, as well as syntheses of the discorhabdins by various chemists to-date, are reviewed here.

  20. Alkaloids Isolated from Natural Herbs as the Anticancer Agents

    Directory of Open Access Journals (Sweden)

    Jin-Jian Lu

    2012-01-01

    Full Text Available Alkaloids are important chemical compounds that serve as a rich reservoir for drug discovery. Several alkaloids isolated from natural herbs exhibit antiproliferation and antimetastasis effects on various types of cancers both in vitro and in vivo. Alkaloids, such as camptothecin and vinblastine, have already been successfully developed into anticancer drugs. This paper focuses on the naturally derived alkaloids with prospective anticancer properties, such as berberine, evodiamine, matrine, piperine, sanguinarine, and tetrandrine, and summarizes the mechanisms of action of these compounds. Based on the information in the literature that is summarized in this paper, the use of alkaloids as anticancer agents is very promising, but more research and clinical trials are necessary before final recommendations on specific alkaloids can be made.

  1. Toxicosis by Plant Alkaloids in Humans and Animals in Colombia.

    Science.gov (United States)

    Diaz, Gonzalo J

    2015-12-11

    Due to its tropical location, chains of mountains, inter-Andean valleys, Amazon basin area, eastern plains and shores on both the Atlantic and Pacific Oceans, Colombia has many ecosystems and the second largest plant biodiversity in the world. Many plant species, both native and naturalized, are currently recognized as toxic for both animals and humans, and some of them are known to cause their toxic effects due to their alkaloid content. Among these, there are plants containing the hepatotoxic pyrrolizidine alkaloids, neurotoxins such as the indolizidine alkaloid swainsonine and the piperidine alkaloids coniine and γ-coniceine and tropane alkaloids. Unfortunately, the research in toxic plants in Colombia is not nearly proportional to its plant biodiversity and the scientific information available is only very scarce. The present review aims at summarizing the scarce information about plant alkaloid toxicosis in animals and humans in Colombia.

  2. Toxicosis by Plant Alkaloids in Humans and Animals in Colombia

    Directory of Open Access Journals (Sweden)

    Gonzalo J. Diaz

    2015-12-01

    Full Text Available Due to its tropical location, chains of mountains, inter-Andean valleys, Amazon basin area, eastern plains and shores on both the Atlantic and Pacific Oceans, Colombia has many ecosystems and the second largest plant biodiversity in the world. Many plant species, both native and naturalized, are currently recognized as toxic for both animals and humans, and some of them are known to cause their toxic effects due to their alkaloid content. Among these, there are plants containing the hepatotoxic pyrrolizidine alkaloids, neurotoxins such as the indolizidine alkaloid swainsonine and the piperidine alkaloids coniine and γ-coniceine and tropane alkaloids. Unfortunately, the research in toxic plants in Colombia is not nearly proportional to its plant biodiversity and the scientific information available is only very scarce. The present review aims at summarizing the scarce information about plant alkaloid toxicosis in animals and humans in Colombia.

  3. Antimicrobial alkaloids from Zanthoxylum tetraspermum and caudatum.

    Science.gov (United States)

    Nissanka, A P; Karunaratne, V; Bandara, B M; Kumar, V; Nakanishi, T; Nishi, M; Inada, A; Tillekeratne, L M; Wijesundara, D S; Gunatilaka, A A

    2001-04-01

    Two benzophenanthrene alkaloids, 8-acetonyldihydronitidine and 8-acetonyldihydroavicine were isolated from Zanthoxylum tetraspermum stem bark along with liriodenine, sesamin, lichexanthone and (+)-piperitol-gamma,gamma-dimethylallylether. The species endemic to Sri Lanka, Z. caudatum, contained sesamin, savinin, liriodenine, decarine and 8-O-desmethyl-N-nornitidine. 8-Acetonyldihydronitidine and 8-acetonyldihydroavicine showed significant antibacterial activity while the former along with liriodenine was strongly antifungal. Savinin exhibited potent spermicidal activity. Both savinin and sesamin exhibited significant insecticidal activity. PMID:11324918

  4. Synthesis studies on the Melodinus alkaloid meloscine

    OpenAIRE

    Feldman, Ken S.; Antoline, Joshua F.

    2013-01-01

    The pentacyclic Melodinus alkaloid (±)-meloscine was synthesized in 19 chemical steps from 2-bromobenzaldehyde through a route featuring an allenyl azide cyclization cascade to deliver the core azabicyclo[3.3.0]octane substructure. Peripheral functionalization of this core included a Tollens-type aldol condensation to set the quaternary center at C(20) and a diastereoselective ring closing metathesis to forge the tetrahydropyridine ring.

  5. A new alkaloid from Solanum cathayanum

    Institute of Scientific and Technical Information of China (English)

    Fan Cheng; Xiang Li; Jun Zhi Wang

    2008-01-01

    A new alkaloid compound I was obtained from Solanum cathayanum, a folk medicine of Hubei Province, together with a knownalkaloid, orotic acid ethyl ester. On the basis of spectroscopic data and chemical methods, I was identified as 8-hydroxy-3-methoxy-5H-pyrido[2,1-c]pyrazin-5-one. Compound 1 can inhibit the production of NO in peritoneal macrophage of mice induced with LPS.The content of NO was determined by enzyme methods.

  6. Potential antimalarial activity of indole alkaloids

    OpenAIRE

    Frederich, Michel; Tits, Monique; Angenot, Luc

    2008-01-01

    New antimalarial treatments are now urgently required, following the emergence of resistance to the most used drugs. Natural products contribute greatly to the therapeutic arsenal in this area, including artemisinin and quinine (and atovaquone, semi-synthetic). Among the natural products, indole alkaloids represent an interesting class of compounds. Screening carried out to date has revealed several substances active in vitro under the micromolar range and with a good selectivity index. This ...

  7. Pyrrolizidine alkaloids and diterpenes from Villasenoria orcuttii

    Energy Technology Data Exchange (ETDEWEB)

    Arciniegas, Amira; Perez-Castorena, Ana L.; Gonzalez, Karina; Vivar, Alfonso Romo de, E-mail: alperezc@unam.mx [Instituto de Quimica, Universidad Nacional Autonoma de Mexico, Circuito Exterior, Ciudad Universitaria, Coyoacan, DF (Mexico); Reyes-Lezama, Marisol [Centro Conjunto de Investigacion en Quimica Sustentable, Universidad Autonoma del Estado de Mexico-Universidad Nacional Autonoma de Mexico (UAEM-UNAM), Carretera Toluca-Atlacomulco, Estado de Mexico (Mexico); Villasenor, Jose Luis [Instituto de Biologia, Universidad Nacional Autonoma de Mexico, Circuito Exterior, Ciudad Universitaria, Coyoacan, DF, (Mexico)

    2013-07-15

    The chemical study of Villasenoria orcuttii, the only species of the genus Villasenoria, afforded three acyclic diterpenes, two of them described for the first time. Two pyrrolizidine alkaloids, florosenine and floridanine, among other known compounds were also isolated. The absolute configuration of floridanine was determined by X-ray analysis using anomalous dispersion with Cu K{sub {alpha}} radiation, and its {sup 1}H and {sup 13}C nuclear magnetic resonance (NMR) data were corrected. (author)

  8. A new diketopiperazine alkaloid from Aspergillus oryzae.

    Science.gov (United States)

    Shaaban, Mohamed; El-Metwally, Mohammad Magdy; Nasr, Hamdi

    2014-01-01

    Investigation of bioactive secondary metabolites from terrestrial Aspergillus oryzae sp. MMAO1 using M2 medium afforded a new diketopiperazine alkaloid, 7,9-dihydroxy-3-(1H-indol-3-ylmethyl)-8-methoxy-2,3,11,11a-tetrahydro-6H-pyrazino[1,2-b]isoquinoline-1,4-dione (1a), containing the unusual amino acid L-6,8-dihydroxy-7-methoxyphenylalanine. This was co-isolated with ditryptophenaline (2), cyclo-(Tryp,Tyr) (4), cyclo-(Pro,Val), α-cyclopiazonic acid (3), kojic acid and uridine. Re-cultivation of the fungal strain on Dox medium led to the production of bisdethio(bismethylthio)gliotoxin (5), pseurotin A (6) along with linoleic acid, α-cyclopiazonic acid (3) and kojic acid. The chemical structure of the new diketopiperazine alkaloid including the relative configuration was determined by 1D and 2D NMR spectroscopy and HR-ESI-MS spectrometry, and by comparison with the related literature. The new alkaloid (1a) showed no antimicrobial activity or cytotoxicity against brine shrimps.

  9. Two New Alkaloids from Roots of Stemona tuberosa

    Directory of Open Access Journals (Sweden)

    Ligen Lin

    2014-07-01

    Full Text Available The roots of Stemona tuberosa ha ve been used as an antitussive and insecticide remedy for thousands of years. On the chemical investigation of the roots of S . tuberosa , two new tuberostemonine-type alkaloids, epoxy-tuberostemonol (1, and neotuberostemoenone (2, together with a known alkaloid, n eotuberostemonine, were isolated and identified. The structures of new alkaloids were established on the basis of 1D and 2D NMR and other spectroscopic analyses.

  10. Initial Studies on Alkaloids from Lombok Medicinal Plants

    Directory of Open Access Journals (Sweden)

    John B. Bremner

    2001-01-01

    Full Text Available Initial investigation of medicinal plants from Lombok has resulted in the collection of 100 plant species predicted to have antimicrobial, including antimalarial, properties according to local medicinal uses. These plants represent 49 families and 80 genera; 23% of the plants tested positively for alkaloids. Among the plants testing positive, five have been selected for further investigation involving structure elucidation and antimicrobial testing on the extracted alkaloids. Initial work on structural elucidation of some of the alkaloids is reported briefly.

  11. New Perspectives in the Chemistry of Marine Pyridoacridine Alkaloids

    OpenAIRE

    Alois Plodek; Franz Bracher

    2016-01-01

    Secondary metabolites from marine organisms are a rich source of novel leads for drug development. Among these natural products, polycyclic aromatic alkaloids of the pyridoacridine type have attracted the highest attention as lead compounds for the development of novel anti-cancer and anti-infective drugs. Numerous sophisticated total syntheses of pyridoacridine alkaloids have been worked out, and many of them have also been extended to the synthesis of libraries of analogues of the alkaloids...

  12. A new monoterpenoid oxindole alkaloid from Hamelia patens micropropagated plantlets.

    Science.gov (United States)

    Paniagua-Vega, David; Cerda-García-Rojas, Carlos M; Ponce-Noyola, Teresa; Ramos-Valdivia, Ana C

    2012-11-01

    Chemical studies on Hamelia patens (Rubiaceae) micropropagated plantlets allowed production of a new monoterpenoid oxindole alkaloid, named (-)-hameline (7), together with eight known alkaloids, tetrahydroalstonine (1), aricine (2), pteropodine (3), isopteropodine (4), uncarine F (5), speciophylline (6), palmirine (8), and rumberine (9). The structure of the new alkaloid was assigned on the basis of 1D and 2D NMR spectroscopy, mass spectrometry, and molecular modeling.

  13. Binding Parameters of Alkaloids Berberine and Sanguinarine with DNA

    CERN Document Server

    Gumenyuk, V G; Kutovyy, S Yu; Yashchuk, V M; Zaika, L A

    2012-01-01

    We study the interaction of berberine and sanguinarine (plant alkaloids) with DNA in aqueous solutions, by using optical spectroscopy methods (absorption and fluorescence). The dependencies of alkaloid spectral characteristics on the concentration ratio N/c between the DNA base pairs and alkaloid molecules in the solutions are considered, and the manifestations of the alkaloid-DNA binding are revealed. The character of binding is found to depend on N/c. The parameters of the binding of berberine and sanguinarine with DNA are determined, by using the modified Scatchard and McGhee-von Hippel equations

  14. New Perspectives in the Chemistry of Marine Pyridoacridine Alkaloids

    Science.gov (United States)

    Plodek, Alois; Bracher, Franz

    2016-01-01

    Secondary metabolites from marine organisms are a rich source of novel leads for drug development. Among these natural products, polycyclic aromatic alkaloids of the pyridoacridine type have attracted the highest attention as lead compounds for the development of novel anti-cancer and anti-infective drugs. Numerous sophisticated total syntheses of pyridoacridine alkaloids have been worked out, and many of them have also been extended to the synthesis of libraries of analogues of the alkaloids. This review summarizes the progress in the chemistry of pyridoacridine alkaloids that was made in the last one-and-a-half decades. PMID:26821033

  15. Vitamin K2 Biosynthetic Enzyme, UBIAD1 Is Essential for Embryonic Development of Mice

    OpenAIRE

    Kimie Nakagawa; Natsumi Sawada; Yoshihisa Hirota; Yuri Uchino; Yoshitomo Suhara; Tomoka Hasegawa; Norio Amizuka; Tadashi Okamoto; Naoko Tsugawa; Maya Kamao; Nobuaki Funahashi; Toshio Okano

    2014-01-01

    UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhib...

  16. Genomics of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon mobility within gram-negative bacteria.

    Science.gov (United States)

    Costa, Rodrigo; van Aarle, Ingrid M; Mendes, Rodrigo; van Elsas, Jan Dirk

    2009-01-01

    Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway that converts tryptophan to PRN is composed of four genes, prnA through D, whose diversity, genomic context and spread over bacterial genomes are poorly understood. Therefore, we launched an endeavour aimed at retrieving, by in vitro and in silico means, diverse bacteria carrying the prnABCD biosynthetic loci in their genomes. Analysis of polymorphisms of the prnD gene sequences revealed a high level of conservation between Burkholderia, Pseudomonas and Serratia spp. derived sequences. Whole-operon- and prnD-based phylogeny resulted in tree topologies that are incongruent with the taxonomic status of the evaluated strains as predicted by 16S rRNA gene phylogeny. The genomic composition of c. 20 kb DNA fragments containing the PRN operon varied in different strains. Highly conserved and distinct transposase-encoding genes surrounding the PRN biosynthetic operons of Burkholderia pseudomallei strains were found. A prnABCD-deprived genomic region in B. pseudomallei strain K96243 contained the same gene composition as, and shared high homology with, the flanking regions of the PRN operon in B. pseudomallei strains 668, 1106a and 1710b. Our results strongly suggest that the PRN biosynthetic operon is mobile. The extent, frequency and promiscuity of this mobility remain to be understood. PMID:18793314

  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. Studies of interaction between two alkaloids and double helix DNA

    International Nuclear Information System (INIS)

    This article presents the study on the interaction of two alkaloids (matrine and evodiamine) and hs-DNA by absorption, fluorescence, circular dichroism (CD), DNA melting and viscosity experiments. The spectroscopic studies suggested that two alkaloids can bind to DNA through an intercalative mode. The viscosity measurement and thermal denaturation also indicated that two alkaloids can intercalate to DNA. The binding constants (KA) and the number of binding sites (n) were determined. At the same time, some significant thermodynamic parameters of the binding of the alkaloids to DNA were obtained. Competitive binding studies revealed that alkaloids had an effect on ethidium bromide (EB) bound DNA. In addition, it was also proved that the fluorescence quenching was influenced by ionic strength. - Highlights: • Interaction between two alkaloids and DNA is studied by spectral methods. • The binding constant and the binding sites between two alkaloids and DNA are obtained. • There are a classical intercalative mode between alkaloids and DNA. • The binding of matrine with DNA is weaker than that of evodiamine. • It is important for us to understand the alkaloids–DNA interactions at a molecular level

  19. Pyrrolizidine alkaloids from Bulgarian species of the genus Senecio

    Directory of Open Access Journals (Sweden)

    NADEZHDA KOSTOVA

    2006-12-01

    Full Text Available Nine Bulgarian species from the genus Senecio were studied phytochemically and/or by GC-MS analysis. Senecivernine-N-oxide was isolated and identified by spectral data for the first time. Different types of pyrrolizidine alkaloids were tested for cytotoxicity on murine lymphocytes. At a concentration of 100 µg/ml, the alkaloid retroisosenine showed immunosuppressive effect.

  20. Galanthindole: a new indole alkaloid from Galanthus plicatus ssp. byzantinus.

    Science.gov (United States)

    Unver, Nehir; Kaya, G Irem; Werner, Christa; Verpoorte, Robert; Gözler, Belkis

    2003-09-01

    A new indole alkaloid, galanthindole, was isolated from Galanthus plicatus ssp. byzantinus (Amaryllidaceae), a plant native to northwestern Turkey. Incorporating a non-fused indole ring, galanthindole may represent the prototype of a new subgroup of the Amaryllidaceae alkaloids. Two other bases, (+)-11-hydroxyvittatine and hordenine, are also reported from the same plant.

  1. Two New Norditerpenoid Alkaloids from Aconitum spicatum Stapf

    Institute of Scientific and Technical Information of China (English)

    Li Ming GAO; Xiao Mei WEI; Li YANG

    2005-01-01

    Two new norditerpenoid alkaloids, spicatine A (1) and spicatine B (2) were isolated from the root of Aconitum spicatum. The new compounds were deduced on the basis of their spectral data (IR, HREIMS, EIMS, 1D, 2D-NMR). This is the first whole report on the isolation of diterpenoid alkaloids from the A. spicatum Stapf.

  2. Studies of interaction between two alkaloids and double helix DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yantao [Key Laboratory of Preparation and Applications of Environmentally Friendly Materials (Jilin Normal University), Ministry of Education China, Siping 136000 (China); College of Chemistry, Jilin Normal University, Siping 136000 (China); Peng, Tingting [College of Chemistry, Jilin Normal University, Siping 136000 (China); College of Chemistry, Baicheng Normal University, Baicheng, 130032 (China); Zhao, Lei [Siping Institute for Food and Drug Control, Siping 136000 (China); Jiang, Dayu [Key Laboratory of Preparation and Applications of Environmentally Friendly Materials (Jilin Normal University), Ministry of Education China, Siping 136000 (China); College of Chemistry, Jilin Normal University, Siping 136000 (China); Cui, Yuncheng, E-mail: 1979yanzi@163.com [Key Laboratory of Preparation and Applications of Environmentally Friendly Materials (Jilin Normal University), Ministry of Education China, Siping 136000 (China); College of Chemistry, Jilin Normal University, Siping 136000 (China)

    2014-12-15

    This article presents the study on the interaction of two alkaloids (matrine and evodiamine) and hs-DNA by absorption, fluorescence, circular dichroism (CD), DNA melting and viscosity experiments. The spectroscopic studies suggested that two alkaloids can bind to DNA through an intercalative mode. The viscosity measurement and thermal denaturation also indicated that two alkaloids can intercalate to DNA. The binding constants (K{sub A}) and the number of binding sites (n) were determined. At the same time, some significant thermodynamic parameters of the binding of the alkaloids to DNA were obtained. Competitive binding studies revealed that alkaloids had an effect on ethidium bromide (EB) bound DNA. In addition, it was also proved that the fluorescence quenching was influenced by ionic strength. - Highlights: • Interaction between two alkaloids and DNA is studied by spectral methods. • The binding constant and the binding sites between two alkaloids and DNA are obtained. • There are a classical intercalative mode between alkaloids and DNA. • The binding of matrine with DNA is weaker than that of evodiamine. • It is important for us to understand the alkaloids–DNA interactions at a molecular level.

  3. ALKALOIDS OF SOME EUROPEAN AND MACARONESIAN SEDOIDEAE AND SEMPERVIVOIDEAE (CRASSULACEAE)

    NARCIS (Netherlands)

    STEVENS, JF; THART, H; HENDRIKS, H; MALINGRE, TM

    1992-01-01

    Some 22 pyrrolidine and piperdine alkaloids were detected in the leafy parts of Sedum acre, S. aetnense, S. anglicum, S. brissemoreti, S. farinosum, S. fusiforme, S. lancerottense, S. melanantherum, and S. nudum. In addition to the alkaloids known from S. acre, 1-(2-pyrrolidyl)-propan-2-one and 2-mo

  4. Extraction of alkaloids for NMR-based profiling

    DEFF Research Database (Denmark)

    Yilmaz, Ali; Nyberg, Nils; Jaroszewski, Jerzy W.

    2012-01-01

    of the well-known cinchona alkaloids quinine, cinchonine and cinchonidine without any apparent clustering. Signals from analogues were detected but not in substantial amounts. The main variation was related to the absolute amounts of extracted alkaloids, which was attributed to the evolution of the Cinchona...

  5. New monoterpenoid alkaloids from the aerial parts of Uncaria hirsuta.

    Science.gov (United States)

    Jia, Jun-Feng; Zhang, Yuan; Huang, Xiao-Jun; Zhang, Sheng-Yuan; Tian, Hai-Yan; Wang, Lei; Ye, Wen-Cai

    2014-01-01

    To investigate the chemical constituents of medicinal plant Uncaria hirsuta, three new monoterpenoid alkaloids, named hirsutanines A-C (1-3), were isolated. Their structures with absolute configurations were elucidated by means of NMR, X-ray diffraction and CD analysis. Compound 3 was the first dimeric monoterpenoid alkaloid obtained from genus Uncaria. PMID:24684175

  6. Effects of Ergot Alkaloids on Bovine Sperm Motility In Vitro

    Science.gov (United States)

    Ergot alkaloids are synthesized by endophyte-infected (Neotyphodium coenophialum) tall fescue (Lolium arundinaceum (Schreb.) S.J. Darbyshire). Our objective was to determine direct effects of ergot alkaloids (ergotamine, dihydroergotamine and ergonovine) on the motility of bovine spermatozoa in vit...

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

    Directory of Open Access Journals (Sweden)

    Nori Satoh

    2013-02-01

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

  8. Biosynthesis, asymmetric synthesis, and pharmacology, including cellular targets, of the pyrrole-2-aminoimidazole marine alkaloids

    Digital Repository Service at National Institute of Oceanography (India)

    Al-Mourabit, A.; Zancanella, M.A.; Tilvi, S.; Romo, D.

    biosynthetic intermediates e.g. clathrodin (5) or oroidin (6). While biosynthetic details are completely lacking, biomechanistic analysis of isolated P-2-AIs and successful synthetic strategies premised on speculative biosynthetic pathways have provided some... indirect evidence regarding biosynthetic feasibility. From the standpoint that inherent reactivity of biosynthetic intermediates may offer clues about actual biosynthetic pathways leading to various complex P-2-AIs, isolation of an increasing number...

  9. Receptor binding of biosynthetic human insulin on isolated pig hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gammeltoft, S.

    Biosynthetic human insulin (BHI) and pancreatic human insulin were compared with respect to receptor binding in a heterologous assay system: displacement of pork A14-/sup 125/I-monoiodoinsulin from receptors on pig hepatocytes. The concentrations of human insulin giving half-maximal displacement were identical for both preparations, i.e., 0.5 nM. Their relative potency was 1.01 +/- 0.14 (SD, N . 5), suggesting that biosynthetic and pancreatic human insulin exert the same biologic activity.

  10. Receptor binding of biosynthetic human insulin on isolated pig hepatocytes

    International Nuclear Information System (INIS)

    Biosynthetic human insulin (BHI) and pancreatic human insulin were compared with respect to receptor binding in a heterologous assay system: displacement of pork A14-125I-monoiodoinsulin from receptors on pig hepatocytes. The concentrations of human insulin giving half-maximal displacement were identical for both preparations, i.e., 0.5 nM. Their relative potency was 1.01 +/- 0.14 (SD, N . 5), suggesting that biosynthetic and pancreatic human insulin exert the same biologic activity

  11. Quantitative and qualitative transcriptome analysis of four industrial strains of Claviceps purpurea with respect to ergot alkaloid production.

    Science.gov (United States)

    Majeská Čudejková, Mária; Vojta, Petr; Valík, Josef; Galuszka, Petr

    2016-09-25

    The fungus Claviceps purpurea is a biotrophic phytopathogen widely used in the pharmaceutical industry for its ability to produce ergot alkaloids (EAs). The fungus attacks unfertilized ovaries of grasses and forms sclerotia, which represent the only type of tissue where the synthesis of EAs occurs. The biosynthetic pathway of EAs has been extensively studied; however, little is known concerning its regulation. Here, we present the quantitative transcriptome analysis of the sclerotial and mycelial tissues providing a comprehensive view of transcriptional differences between the tissues that produce EAs and those that do not produce EAs and the pathogenic and non-pathogenic lifestyle. The results indicate metabolic changes coupled with sclerotial differentiation, which are likely needed as initiation factors for EA biosynthesis. One of the promising factors seems to be oxidative stress. Here, we focus on the identification of putative transcription factors and regulators involved in sclerotial differentiation, which might be involved in EA biosynthesis. To shed more light on the regulation of EA composition, whole transcriptome analysis of four industrial strains differing in their alkaloid spectra was performed. The results support the hypothesis proposing the composition of the amino acid pool in sclerotia to be an important factor regulating the final structure of the ergopeptines produced by Claviceps purpurea. PMID:26827914

  12. 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.

  13. Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes.

    Directory of Open Access Journals (Sweden)

    Stephen D Bentley

    2006-03-01

    Full Text Available Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement.

  14. 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.

  15. Identification of a novel sesquiterpene biosynthetic machinery involved in astellolide biosynthesis

    Science.gov (United States)

    Shinohara, Yasutomo; Takahashi, Shunji; Osada, Hiroyuki; Koyama, Yasuji

    2016-01-01

    Esterified drimane-type sesquiterpene lactones such as astellolides display various biological activities and are widely produced by plants and fungi. Given their low homology to known sesquiterpene cyclases, the genes responsible for their biosynthesis have not been uncovered yet. Here, we identified the astellolide gene cluster from Aspergillus oryzae and discovered a novel sesquiterpene biosynthetic machinery consisting of AstC, AstI, and AstK. All these enzymes are annotated as haloacid dehalogenase-like hydrolases, whereas AstC also contains a DxDTT motif conserved in class II diterpene cyclases. Based on enzyme reaction analyses, we found that AstC catalysed the protonation-initiated cyclisation of farnesyl pyrophosphate into drimanyl pyrophosphate. This was successively dephosphorylated by AstI and AstK to produce drim-8-ene-11-ol. Moreover, we also identified and characterised a unique non-ribosomal peptide synthetase, AstA, responsible for esterifying aryl acids to drimane-type sesquiterpene lactones. In this study, we highlight a new biosynthetic route for producing sesquiterpene and its esterified derivative. Our findings shed light on the identification of novel sesquiterpenes via genome mining. PMID:27628599

  16. Internalization of /sup 125/I-insulin by IM-9 cultured human lymphocytes: a comparison between A14-monoiodo-pork and biosynthetic human insulin

    Energy Technology Data Exchange (ETDEWEB)

    Carpentier, J.L.; Halban, P.A.; Renold, A.E.; Orci, L.

    Human insulin synthesized from A- and B-chains separately produced in Escherichia coli from cloned genes was characterized by examining its interaction biochemically and morphologically with IM-9 cultured human lymphocytes. Biosynthetic human insulin (BHI) behaves similarly to pork insulin with respect both to its binding properties and to the rate and magnitude at which it is internalized by the cells.

  17. Internalization of 125I-insulin by IM-9 cultured human lymphocytes: a comparison between A14-monoiodo-pork and biosynthetic human insulin

    International Nuclear Information System (INIS)

    Human insulin synthesized from A- and B-chains separately produced in Escherichia coli from cloned genes was characterized by examining its interaction biochemically and morphologically with IM-9 cultured human lymphocytes. Biosynthetic human insulin (BHI) behaves similarly to pork insulin with respect both to its binding properties and to the rate and magnitude at which it is internalized by the cells

  18. CRINUM; AN ENDLESS SOURCE OF BIOACTIVE PRINCIPLES: A REVIEW. PART 1- CRINUM ALKALOIDS: LYCORINE-TYPE ALKALOIDS

    Directory of Open Access Journals (Sweden)

    John Refaat*, Mohamed S. Kamel , Mahmoud A. Ramadan and Ahmed A. Ali

    2012-07-01

    Full Text Available Crinum is an important Amaryllidaceous plant thanks to the valuable biological and therapeutic activities of its chemical constituents, especially alkaloids. Many Crinum species have been commonly used in traditional medicines worldwide. Long ago, they have been subjected to extensive chemical, cytological and pharmacological investigations. Accordingly, this work comprehensively comprises both the alkaloidal and non-alkaloidal principles of Crinums isolated from 1950 and up to now, together with various biological and toxicological studies conducted on both the total extracts and individual compounds. As being a major common class of Crinum alkaloids, the current part of this review work highlights the lycorine-type alkaloids isolated so far from this plant in addition to their distribution in different Crinum species.

  19. Synergy Potential of Indole Alkaloids and Its Derivative against Drug-resistant Escherichia coli.

    Science.gov (United States)

    Dwivedi, Gaurav Raj; Gupta, Shikha; Maurya, Anupam; Tripathi, Shubhandra; Sharma, Ashok; Darokar, Mahendra P; Srivastava, Santosh K

    2015-12-01

    Antibacterial and synergy potential of naturally occurring indole alkaloids (IA): 10-methoxy tetrahydroalstonine (1), isoreserpiline (2), 10 and 11 demethoxyreserpiline (3), reserpiline (4), serpentine (5), ajmaline (6), ajmalicine (7), yohimbine (8), and α-yohimbine (9) was evaluated using microbroth dilution assay. Further, α-yohimbine (9) was chemically transformed into six semisynthetic derivatives (9A-9F), and their antibacterial and synergy potential in combination with nalidixic acid (NAL) against E. coli strains CA8000 and DH5α were also evaluated. The IA 1, 2, 4, 5, 9 and the derivative 9F showed eightfold reduction in the MIC of NAL against the DH5α and four- to eightfold reduction against CA8000. These alkaloids also reduced MIC of another antibiotic, tetracycline up to 8folds, against the MDREC-KG4, a multidrug-resistant clinical isolate of E. coli. Mode of action study of these alkaloids showed efflux pumps inhibitory potential, which was supported by their in silico binding affinity and downregulation of efflux pump genes. These results may be of great help in the development of cost-effective antibacterial combinations for treating patients infected with multidrug-resistant Gram-negative infections.

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

    Directory of Open Access Journals (Sweden)

    Jiahn Chou eGuan

    2014-08-01

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

  1. Dual responsive physical networks from asymmetric biosynthetic triblock copolymers

    NARCIS (Netherlands)

    Pham, T.H.T.

    2013-01-01

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

  2. The preliminary research for biosynthetic engineering by radiation fusion technology

    Energy Technology Data Exchange (ETDEWEB)

    Roh, Chang Hyun; Jung, U Hee; Park, Hae Ran [KAERI, Daejeon (Korea, Republic of)

    2012-01-15

    The purpose of this project is to elucidate the solution to the production of bioactive substance using biotransformation process from core technology of biosynthetic engineering by radiation fusion technology. And, this strategy will provide core technology for development of drugs as new concept and category. Research scopes and contents of project include 1) The development of mutant for biosynthetic engineering by radiation fusion technology 2) The development of host for biosynthetic engineering by radiation fusion technology 3) The preliminary study for biosynthetic engineering of isoflavone by radiation fusion technology. The results are as follows. Isoflavone compounds(daidzein, hydroxylated isoflavone) were analyzed by GC-MS. The study of radiation doses and p-NCA high-throughput screening for mutant development were elucidated. And, it was carried out the study of radiation doses for host development. Furthermore, the study of redox partner and construction of recombinant strain for region-specific hydroxylation(P450, redox partner). In addition, the biological effect of 6,7,4'-trihydroxyisoflavone as an anti-obesity agent was elucidated in this study.

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

    Science.gov (United States)

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

    2012-01-01

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

  4. Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

    Science.gov (United States)

    Goswami, Moloy T; Chen, Guoan; Chakravarthi, Balabhadrapatruni V S K; Pathi, Satya S; Anand, Sharath K; Carskadon, Shannon L; Giordano, Thomas J; Chinnaiyan, Arul M; Thomas, Dafydd G; Palanisamy, Nallasivam; Beer, David G; Varambally, Sooryanarayana

    2015-09-15

    Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention. PMID:26140362

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

    Science.gov (United States)

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

    2016-02-18

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

  6. 特异茶树资源生物碱测定及相关基因表达分析%Determination of Alkaloid and Analysis of Gene Expression in Peculiar Tea Plant

    Institute of Scientific and Technical Information of China (English)

    夏丽飞; 陈林波; 梁名志; 邓威威; 田易萍; 刘本英; 刘军

    2013-01-01

    The component of caffeine and theobromine of five tea plants was determined by high performance liquid chromatography.The results showed that the theobromine content was low,but caffeine content was high in the same tea plant.The theobromine was accumulated in the tea resource of low caffeine content.The expression level of sAMS and TIDH were nearly affected by the change of caffeine content,according to the differences of expression level of S-adenosylmethionine Synthetase gene(sAMS),Inosine 5'-monophosphate Dehydroge-nase (TIDH) and Caffeine Synthase gene (TCSI) analyzed by RT-PCR in different tea species and the date of caffeine content.TCSI could be expressed whatever in the high caffeine content material or in the low one,but its expression level was higher in the high caffeine content material.The reason could be that the Caffeine Synthase of vitality was low in the low caffeine content tea plant,or other regulation mechanisms could lead to the blockage of biosynthesis of caffeine and the accumulation of theobromine.%利用高效液相色谱对5份茶树资源中的咖啡碱、可可碱组分进行测定,发现咖啡碱含量高的茶树资源可可碱含量低,而咖啡碱含量低茶树资源的可可碱被积累.利用RT-PCR分析了S-腺苷甲硫氨酸合成酶基因(sAMS)、次黄嘌呤苷-5-单酸脱氢酶基因(TIDH)、咖啡碱合成酶基因(TCSI)在咖啡碱含量不同的茶树品种间的表达水平差异,并结合咖啡碱含量数据发现,sAMS和TIDH的表达水平对咖啡碱含量变化影响不明显.TCSI在高咖啡碱和低咖啡碱材料中均有表达,且TCSI在高咖啡碱材料中的表达比在低咖啡碱材料中高.推测可能是低咖啡碱茶树资源中咖啡碱合成酶活性低或者存在其他的调控机制,导致咖啡碱的生物合成受阻,使得可可碱被积累.

  7. Indolopyridoquinazoline alkaloids from Esenbeckia grandiflora mart. (Rutaceae); Alkaloides {beta}-indolopiridoquinazolinicos de Esenbeckia grandiflora mart. (Rutaceae)

    Energy Technology Data Exchange (ETDEWEB)

    Januario, Ana Helena; Vieira, Paulo Cezar; Silva, Maria Fatima das Gracas Fernandes da; Fernandes, Joao Batista [Universidade Federal de Sao Carlos (UFSCAR), SP (Brazil). Dept. de Quimica], e-mail: anahjanuario@unifran.br; Silva, Jorge Jose de Brito; Conserva, Lucia Maria [Universidade Federal de Alagoas (UFAL), Maceio, AL (Brazil). Inst. de Quimica e Biotecnologia

    2009-07-01

    The chemical composition of two specimens of Esenbeckia grandiflora, collected in the south and northeast regions of Brazil, was investigated. In this study, three b-indolopyridoquinazoline alkaloids from the leaves (rutaecarpine, 1-hydroxyrutaecarpine) and roots (euxylophoricine D) were isolated for the first time in this genus. In addition, the triterpenes {alpha}-amyrin, {beta}-amyrin, {alpha}-amyrenonol, {beta}-amyrenonol, 3{alpha}-hydroxy-ursan-12-one, and 3{alpha}-hydroxy-12,13-epoxy-oleanane, the coumarins auraptene, umbelliferone, pimpinelin, and xanthotoxin, the furoquinoline alkaloids delbine and kokusaginine, and the phytosteroids sitosterol, stigmasterol, campesterol and 3{beta}-O-{beta}-D-glucopyranosylsitosterol were also isolated from the leaves, twigs, roots and stems of this species. Structures of these compounds were established by spectral analysis. (author)

  8. The Epipolythiodiketopiperazine Gene Cluster in Claviceps purpurea: Dysfunctional Cytochrome P450 Enzyme Prevents Formation of the Previously Unknown Clapurines.

    Directory of Open Access Journals (Sweden)

    Julian Dopstadt

    Full Text Available Claviceps purpurea is an important food contaminant and well known for the production of the toxic ergot alkaloids. Apart from that, little is known about its secondary metabolism and not all toxic substances going along with the food contamination with Claviceps are known yet. We explored the metabolite profile of a gene cluster in C. purpurea with a high homology to gene clusters, which are responsible for the formation of epipolythiodiketopiperazine (ETP toxins in other fungi. By overexpressing the transcription factor, we were able to activate the cluster in the standard C. purpurea strain 20.1. Although all necessary genes for the formation of the characteristic disulfide bridge were expressed in the overexpression mutants, the fungus did not produce any ETPs. Isolation of pathway intermediates showed that the common biosynthetic pathway stops after the first steps. Our results demonstrate that hydroxylation of the diketopiperazine backbone is the critical step during the ETP biosynthesis. Due to a dysfunctional enzyme, the fungus is not able to produce toxic ETPs. Instead, the pathway end-products are new unusual metabolites with a unique nitrogen-sulfur bond. By heterologous expression of the Leptosphaeria maculans cytochrome P450 encoding gene sirC, we were able to identify the end-products of the ETP cluster in C. purpurea. The thioclapurines are so far unknown ETPs, which might contribute to the toxicity of other C. purpurea strains with a potentially intact ETP cluster.

  9. The Epipolythiodiketopiperazine Gene Cluster in Claviceps purpurea: Dysfunctional Cytochrome P450 Enzyme Prevents Formation of the Previously Unknown Clapurines

    Science.gov (United States)

    Tudzynski, Paul; Humpf, Hans-Ulrich

    2016-01-01

    Claviceps purpurea is an important food contaminant and well known for the production of the toxic ergot alkaloids. Apart from that, little is known about its secondary metabolism and not all toxic substances going along with the food contamination with Claviceps are known yet. We explored the metabolite profile of a gene cluster in C. purpurea with a high homology to gene clusters, which are responsible for the formation of epipolythiodiketopiperazine (ETP) toxins in other fungi. By overexpressing the transcription factor, we were able to activate the cluster in the standard C. purpurea strain 20.1. Although all necessary genes for the formation of the characteristic disulfide bridge were expressed in the overexpression mutants, the fungus did not produce any ETPs. Isolation of pathway intermediates showed that the common biosynthetic pathway stops after the first steps. Our results demonstrate that hydroxylation of the diketopiperazine backbone is the critical step during the ETP biosynthesis. Due to a dysfunctional enzyme, the fungus is not able to produce toxic ETPs. Instead, the pathway end-products are new unusual metabolites with a unique nitrogen-sulfur bond. By heterologous expression of the Leptosphaeria maculans cytochrome P450 encoding gene sirC, we were able to identify the end-products of the ETP cluster in C. purpurea. The thioclapurines are so far unknown ETPs, which might contribute to the toxicity of other C. purpurea strains with a potentially intact ETP cluster. PMID:27390873

  10. 2-Oxoglutarate: linking TCA cycle function with amino acid, glucosinolate, flavonoid, alkaloid and gibberellin biosynthesis

    Directory of Open Access Journals (Sweden)

    Wagner L. Araújo

    2014-10-01

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

  11. Mechanisms of growth inhibition of Phytomonas serpens by the alkaloids tomatine and tomatidine

    Directory of Open Access Journals (Sweden)

    Jorge Mansur Medina

    2015-02-01

    Full Text Available Phytomonas serpens are flagellates in the family Trypanosomatidae that parasitise the tomato plant (Solanum lycopersicum L., which results in fruits with low commercial value. The tomato glycoalkaloid tomatine and its aglycone tomatidine inhibit the growth of P. serpens in axenic cultures. Tomatine, like many other saponins, induces permeabilisation of the cell membrane and a loss of cell content, including the cytosolic enzyme pyruvate kinase. In contrast, tomatidine does not cause permeabilisation of membranes, but instead provokes morphological changes, including vacuolisation. Phytomonas treated with tomatidine show an increased accumulation of labelled neutral lipids (BODYPY-palmitic, a notable decrease in the amount of C24-alkylated sterols and an increase in zymosterol content. These results are consistent with the inhibition of 24-sterol methyltransferase (SMT, which is an important enzyme that is responsible for the methylation of sterols at the 24 position. We propose that the main target of tomatidine is the sterols biosynthetic pathway, specifically, inhibition of the 24-SMT. Altogether, the results obtained in the present paper suggest a more general effect of alkaloids in trypanosomatids, which opens potential therapeutic possibilities for the treatment of the diseases caused by these pathogens.

  12. The distribution of inole alkaloids in different organs of Catharanthus roseus G. Don. (Vinca rosea L)

    OpenAIRE

    Hassan Ebrahimzadeh; Azra Ataei -Azimi4 Mohammad-Reza Noori-Dafoi

    1996-01-01

    The study of alkaloid extract of the leaf of Vinca rosea by TLC showed the existence of 13 bands, 7 bands of them were: Serpentine, Vincristine, Vinblastine, Ajmaline, Catharantine, Vindoline and Ajmalicine. The existence of these 7 alkaloids was confirmed in the young leaves, old leaves and the roots of the plant by HPLC. Furthermore, two alkaloids with the short retention times and one alkaloid with the medium retention time in the young leaf, three alkaloids with the short retention time a...

  13. 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

  14. γ-Lactam alkaloids from the flower buds of daylily.

    Science.gov (United States)

    Matsumoto, Takahiro; Nakamura, Seikou; Nakashima, Souichi; Ohta, Tomoe; Yano, Mamiko; Tsujihata, Junichiro; Tsukioka, Junko; Ogawa, Keiko; Fukaya, Masashi; Yoshikawa, Masayuki; Matsuda, Hisashi

    2016-07-01

    Four new alkaloids, hemerocallisamines IV-VII, were isolated from the methanol extract of flower buds of daylily. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. The absolute stereochemistry of the hemerocallisamines IV-VI was elucidated by the application of the modified Mosher's method, HPLC analysis, and optical rotation. In the present study, the isolated alkaloids significantly inhibited the aggregation of Aβ42 in vitro. This is the first report about bioactive alkaloids with a γ-lactam ring from daylily. In addition, isolated nucleosides showed accelerative effects on neurite outgrowth under the non-fasting condition.

  15. Genotoxic and tumorigenic pyrrolizidine alkaloids in Chinese herbal plants

    Institute of Scientific and Technical Information of China (English)

    P.P. Fu; Q. Xia; M.W. Chou; G. Lin

    2005-01-01

    Pyrrolizidine alkaloids are a class of hepatotoxic and tumorigenic compounds detected in Chinese herbal plants,contaminated foods, and dietary supplements. In this review, the sources, toxicity, genotoxicity, tumorigenicity, and the metabolic pathways,particular the activation pathways leading to hepatotoxicity and tumorigenicity, of pyrrolizidine alkaloids are briefly discussed, with a focus on the most recent important findings concerning the genotoxic mechanism by which riddelliine liver tumors. This mechanism involves the formation of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts and may be general to most carcinogenic pyrrolizidine alkaloids.

  16. Prenylindole alkaloids from Raputia praetermissa (Rutaceae) and their chemosystematic significance

    Energy Technology Data Exchange (ETDEWEB)

    Rosas, Lisandra V.; Veiga, Thiago Andre M.; Fernandes, Joao B.; Vieira, Paulo C.; Silva, M. Fatima das G.F. da, E-mail: dmfs@power.ufscar.b [Universidade Federal de Sao Carlos (DQ/UFSCar), SP (Brazil). Dept. de Quimica

    2011-07-01

    The dichloromethane extract from the stems of Raputia praetermissa afforded four new compounds, 4-deoxyraputindole C (1), raputimonoindole A-B (2, 3), and hexadecanyl 2-hydroxy- 4-methoxy-cinnamate (5), besides the alkaloids 5-(4-methoxymethylfuran-2-yl)-1H-indole (raputimonoindole C), furoquinolines maculosidine, robustine, evolitrine and dictamnine. The hexane extract yielded N-methyl-4-methoxyquinoline-2(1H)-one, skimmianine, cycloartenone, sitosterol, stigmasterol and sitostenone. The anthranilate alkaloid content indicates that the genus is strongly related to those included in Cusparieae tribe, but differs from Neoraputia by the absence of prenylindole alkaloids in the late, whose species have previously been placed in Raputia. (author)

  17. Neisseria gonorrhoeae Heme Biosynthetic Mutants Utilize Heme and Hemoglobin as a Heme Source but Fail To Grow within Epithelial Cells

    OpenAIRE

    Turner, Paul C; Thomas, Christopher E.; Elkins, Christopher; Clary, Susan; Sparling, P F

    1998-01-01

    Many bacterial pathogens, including pathogenic neisseriae, can use heme as an iron source for growth. To study heme utilization by Neisseria gonorrhoeae, two heme biosynthetic mutants were constructed, one with a mutation in hemH (the gene encoding ferrochelatase) and one with a mutation in hemA (the gene encoding γ-glutamyl tRNA reductase). The hemH mutant failed to grow without an exogenous supply of heme or hemoglobin, whereas the hemA mutant failed to grow unless heme, hemoglobin, or heme...

  18. Chromosome-End Knockoff Strategy to Reshape Alkaloid Profiles of a Fungal Endophyte

    Science.gov (United States)

    Florea, Simona; Phillips, Timothy D.; Panaccione, Daniel G.; Farman, Mark L.; Schardl, Christopher L.

    2016-01-01

    Molecular genetic techniques to precisely eliminate genes in asexual filamentous fungi require the introduction of a marker gene into the target genome. We developed a novel strategy to eliminate genes or gene clusters located in subterminal regions of chromosomes, and then eliminate the marker gene and vector backbone used in the transformation procedure. Because many toxin gene clusters are subterminal, this method is particularly suited to generating nontoxic fungal strains. We tested this technique on Epichloë coenophiala, a seed-transmissible symbiotic fungus (endophyte) of the important forage grass, tall fescue (Lolium arundinaceum). The endophyte is necessary for maximal productivity and sustainability of this grass but can produce ergot alkaloids such as ergovaline, which are toxic to livestock. The genome sequence of E. coenophiala strain e19 revealed two paralogous ergot alkaloid biosynthesis gene clusters, designated EAS1 and EAS2. EAS1 was apparently subterminal, and the lpsB copy in EAS2 had a frame-shift mutation. We designed a vector with a fungal-active hygromycin phosphotransferase gene (hph), an lpsA1 gene fragment for homologous recombination at the telomere-distal end of EAS1, and a telomere repeat array positioned to drive spontaneous loss of hph and other vector sequences, and to stabilize the new chromosome end. We transformed E. coenophiala with this vector, then selected “knockoff” endophyte strains, confirmed by genome sequencing to lack 162 kb of a chromosome end including most of EAS1, and also to lack vector sequences. These ∆EAS1 knockoff strains produced no detectable ergovaline, whereas complementation with functional lpsB restored ergovaline production. PMID:27334939

  19. The nuclear genome of Rhazya stricta and the evolution of alkaloid diversity in a medically relevant clade of Apocynaceae

    Science.gov (United States)

    Sabir, Jamal S. M.; Jansen, Robert K.; Arasappan, Dhivya; Calderon, Virginie; Noutahi, Emmanuel; Zheng, Chunfang; Park, Seongjun; Sabir, Meshaal J.; Baeshen, Mohammed N.; Hajrah, Nahid H.; Khiyami, Mohammad A.; Baeshen, Nabih A.; Obaid, Abdullah Y.; Al-Malki, Abdulrahman L.; Sankoff, David; El-Mabrouk, Nadia; Ruhlman, Tracey A.

    2016-01-01

    Alkaloid accumulation in plants is activated in response to stress, is limited in distribution and specific alkaloid repertoires are variable across taxa. Rauvolfioideae (Apocynaceae, Gentianales) represents a major center of structural expansion in the monoterpenoid indole alkaloids (MIAs) yielding thousands of unique molecules including highly valuable chemotherapeutics. The paucity of genome-level data for Apocynaceae precludes a deeper understanding of MIA pathway evolution hindering the elucidation of remaining pathway enzymes and the improvement of MIA availability in planta or in vitro. We sequenced the nuclear genome of Rhazya stricta (Apocynaceae, Rauvolfioideae) and present this high quality assembly in comparison with that of coffee (Rubiaceae, Coffea canephora, Gentianales) and others to investigate the evolution of genome-scale features. The annotated Rhazya genome was used to develop the community resource, RhaCyc, a metabolic pathway database. Gene family trees were constructed to identify homologs of MIA pathway genes and to examine their evolutionary history. We found that, unlike Coffea, the Rhazya lineage has experienced many structural rearrangements. Gene tree analyses suggest recent, lineage-specific expansion and diversification among homologs encoding MIA pathway genes in Gentianales and provide candidate sequences with the potential to close gaps in characterized pathways and support prospecting for new MIA production avenues. PMID:27653669

  20. 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. PMID:25108262

  1. Mycotoxin fumonisins: Health impacts and biosynthetic mechanism

    Institute of Scientific and Technical Information of China (English)

    WANG Qiaomei; WANG Jiansheng; YU Fengan; ZHU Xiangcheng; Zaleta-Rivera Kathia; DU Liangcheng

    2006-01-01

    Fumonisins are one of the most important groups of mycotoxins in agriculture and the food industry. They are produced by several widespread fungal pathogens of corn. Fumonisin contamination in maize-derived food and feeds causes several fatal diseases in livestock and poses a significant cancer risk to humans (Group 2B carcinogen). In the recent years, fumonisins have become a hot area in mycotoxin research. This review attempts to highlight the progress in the studies of molecular mechanisms for fumonisin biosynthesis and mode of action. The current understandings in the molecular basis for the cellular effects induced by fumonisins are discussed. Special attentions are paid to introduce the progress in the characterization of individual genes and enzymes involved in the biosynthesis of fumonisins.

  2. 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.

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

    Science.gov (United States)

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

    2016-08-01

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

  4. Structure determination and interception of biosynthetic intermediates for the plantazolicin class of highly discriminating antibiotics.

    Science.gov (United States)

    Molohon, Katie J; Melby, Joel O; Lee, Jaeheon; Evans, Bradley S; Dunbar, Kyle L; Bumpus, Stefanie B; Kelleher, Neil L; Mitchell, Douglas A

    2011-12-16

    The soil-dwelling, plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42 is a prolific producer of complex natural products. Recently, a new FZB42 metabolite, plantazolicin (PZN), has been described as a member of the growing thiazole/oxazole-modified microcin (TOMM) family. TOMMs are biosynthesized from inactive, ribosomal peptides and undergo a series of cyclodehydrations, dehydrogenations, and other modifications to become bioactive natural products. Using high-resolution mass spectrometry, chemoselective modification, genetic interruptions, and other spectroscopic tools, we have determined the molecular structure of PZN. In addition to two conjugated polyazole moieties, the amino-terminus of PZN has been modified to N(α),N(α)-dimethylarginine. PZN exhibited a highly selective antibiotic activity toward Bacillus anthracis, but no other tested human pathogen. By altering oxygenation levels during fermentation, PZN analogues were produced that bear variability in their heterocycle content, which yielded insight into the order of biosynthetic events. Lastly, genome-mining has revealed the existence of four additional PZN-like biosynthetic gene clusters. Given their structural uniqueness and intriguing antimicrobial specificity, the PZN class of antibiotics may hold pharmacological value. PMID:21950656

  5. 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.

  6. Antifouling Alkaloids from Crinum augustum (Amaryllidaceae

    Directory of Open Access Journals (Sweden)

    John Refaat

    2009-01-01

    Full Text Available Fractionation and purification of the ethanolic extract of the bulbs of Crinum augustum Rox. (Amaryllidaceae cultivated in Egypt yielded five alkaloids 6-methoxy-crinamine (1 , crinamine (2 , buphanisine (3 , ungeremine (4 , and hippadine (5 ; two fatty acid derivatives: myristic acid ethyl ester (6 and palmitic acid ethyl ester (7 ; four terpenoidal and steroidal compounds: ursolic acid (8 , β-sitosterol-O- β glucopyranoside (9 and mixture of β--sitosterol (10 and stigmasterol (11 . The structures of all compounds were determined by interpretation of their spectroscopic data; 1D ( 1 H and 13 C, 2D (HSQC, COSY, DQF, NOE and HMBC NMR; MS and UV analyses. The compounds (1 -4 and (6-8 were tested towards biofouling activity using larvae of barnacle Balance amphitrie. Significant activities of 1, 2 and 3 with EC 50 1.8, 1.2 and 0.75 μg/mL respectively, were observed.

  7. Pyrrolidonyl and pyridyl alkaloids in Lymantria dispar.

    Science.gov (United States)

    Deml, Reinhold

    2003-01-01

    The occurrence and metabolism of nicotine and related N-containing compounds in body fluids of the gipsy moth were addressed. Thin layer chromatographic studies clearly showed the simultaneous presence of GABA and 2-pyrrolidone but not of GABamide in the larval haemolymph and osmeterial secretion of Lymantria dispar as well as in the corresponding body fluids of the saturniids, Saturnia pavonia and Attacus atlas. Furthermore, feeding and injection experiments using alkylated precursors and combined gas chromatography/mass spectrometry gave evidence of the transformation of 2-pyrrolidone to nicotine and of nicotinic acid to nicotinamide in caterpillars of L. dispar. Based on these results, on the earlier described variation of the secondary-compound patterns of L. dispar during its development, and on literature data, metabolic pathways for the hitherto detected pyridyl and pyrrolidonyl alkaloids in Lymantriidae (and possibly Saturniidae) are proposed.

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

    OpenAIRE

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

    1994-01-01

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

  9. Investigation of Aconitine-type Alkaloids from Processed Tuber of Aconitum carmiechaeli by HPLC-ESI-MS/MSn

    Institute of Scientific and Technical Information of China (English)

    YUE Hao; PI Zi-feng; ZHAO Yu-feng; SONG Feng-rui; LIU Zhi-qiang; LIU Shu-ying

    2007-01-01

    @@ Introduction Aconitine-type alkaloids isolated from the roots of Aconitum carmiechaeli show a potential toxicity and a broad spectrum of bioactivity[1-4]. On the basis of the C8-substituent of C19-diterpenoid skeleton, aconitinetype alkaloids can be divided into diester-diterpenoid alkaloids( DDAs), monoester-diterpenoid alkaloids(MDAs), and lipo-alkaloids( Fig. 1 ).

  10. A New Binary Carbazole Alkaloid from Murraya koenigii

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A new binary carbazole alkaloid, 8, 8"-biskoenigine (1), along with its monomer,kocnigine, was isolated from the dried leaves of Murraya koenigii collected in Xishuangbanna,Yunnan province. The structure of 1 was established by spectroscopic methods.

  11. Activity of pyrrolizidine alkaloids against biofilm formation and Trichomonas vaginalis

    Science.gov (United States)

    Crotalaria genus belongs to the subfamily Papilionoideae comprising about 600 species spread throughout tropical, neotropical and subtropical regions. In this study, seeds of Crolatalaria pallida were used to the isolation of usaramine, a pyrrolizidine alkaloid. Thus, Pseudomonas aeruginosa and Stap...

  12. New bisbenzylisoquinoline alkaloid from Laureliopsis philippiana

    DEFF Research Database (Denmark)

    Stærk, Dan; Thi, Loi Pham; Rasmussen, Hasse Bonde;

    2009-01-01

    Phytochemical investigation of Laureliopsis philippiana resulted in isolation of a new bisbenzylisoquinoline alkaloid (1) named laureliopsine A. The structure was established by spectroscopic methods, including 2D homo- and heteronuclear NMR experiments. This finding of a bisbenzylisoquinoline al...

  13. A new pyrrole alkaloid from seeds of Castanea sativa.

    Science.gov (United States)

    Hiermann, Alois; Kedwani, Samir; Schramm, Hans Wolfgang; Seger, Christoph

    2002-02-01

    A new pyrrole alkaloid, methyl-(5-formyl-1H-pyrrole-2-yl)-4-hydroxybutyrate (1), was isolated from sweet chestnut seeds and its structure elucidated on the basis of data from NMR spectroscopy and by comparison with synthetic analogues.

  14. NEW NORDITERPENOID ALKALOIDS FROM THE ROOTS OF ACONITUM GENICULATUM

    Institute of Scientific and Technical Information of China (English)

    ZHENG-BANG LI; LIANG XU; XI-XIAN JIAN; FENG-PENG WANG

    2001-01-01

    Four new norditerpenoid alkaloids, geniculatines A (1), B (4), C (7) and D (8), were isolated from the roots of Aconitum geniculatum Fletcher, and their structures were elucidated by spectral methods.

  15. Marine bromopyrrole alkaloids: synthesis and diverse medicinal applications.

    Science.gov (United States)

    Rane, Rajesh; Sahu, Niteshkumar; Shah, Chetan; Karpoormath, Rajshekhar

    2014-01-01

    Marine organisms have been found to be a very rich source of bioactive molecules. Among marine organisms, sponges have been proven to be excellent producers of secondary metabolites. More than 5,300 compounds have been isolated from sponges with around 200 new molecules reported each year. Bromopyrrole alkaloids constitute a family of exclusively marine alkaloids and represent a fascinating example of the large variety of compounds formed by marine sponges which exhibit different biological activities such as antifeedent, anti-biofilm, anticancer, antiinflammatory, antimicrobial, immunomodulatory, analgesic, antiserotonergic, antiangiogenic, antihistaminic, chitinase inhibitor and actimyosin ATPase activator. More than 140 derivatives with different structures and biological activities, have been isolated from more than 20 different sponges. Most of these alkaloids share a key building block, pyrrole-imidazole with oroidin being their underlying structural motif. In this review detailed account of isolation and medicinal application of marine bromopyrrole alkaloids and their synthetic derivatives are discussed. PMID:24359195

  16. Two bromotyrosine alkaloids from the sponge Psammaplysilla purpurea

    Digital Repository Service at National Institute of Oceanography (India)

    Tilvi, S.; Parameswaran, P.S.; Naik, C.G.

    The sponge Psammaplysilla purpurea (Order: Verongidae, Family: Aplysinellidae) is a well known source for several bromotyrosine alkaloids of unique structural features and exhibiting promising biological activities such as cytotoxicity...

  17. 3-Methoxysampangine, a novel antifungal copyrine alkaloid from Cleistopholis patens.

    OpenAIRE

    S. C. Liu; Oguntimein, B; Hufford, C D; Clark, A M

    1990-01-01

    Further examination of the active ethanolic extract of the root bark of Cleistopholis patens by using bioassay-directed fractionation resulted in the isolation of a new alkaloid, 3-methoxysampangine (compound I), together with three known alkaloids, eupolauridine (compound II), liriodenine (compound III), and eupolauridine N-oxide (compound IV). The proposed structure of compound I was based on its physicochemical properties and spectral data. 3-Methoxysampangine exhibited significant antifun...

  18. Initial Studies on Alkaloids from Lombok Medicinal Plants

    OpenAIRE

    John B. Bremner; Surya Hadi

    2001-01-01

    Initial investigation of medicinal plants from Lombok has resulted in the collection of 100 plant species predicted to have antimicrobial, including antimalarial, properties according to local medicinal uses. These plants represent 49 families and 80 genera; 23% of the plants tested positively for alkaloids. Among the plants testing positive, five have been selected for further investigation involving structure elucidation and antimicrobial testing on the extracted alkaloids. Initial work on ...

  19. Pyrrolizidine alkaloid poisoning of sheep in New South Wales.

    Science.gov (United States)

    Seaman, J T

    1987-06-01

    Pyrrolizidine alkaloid poisoning of sheep in New South Wales was reviewed, based on the records of the New South Wales Department of Agriculture's Regional Veterinary Laboratories. The plant species causing significant mortalities were Echium plantagineum and Heliotropium europaeum. The syndrome of hepatogenous chronic copper poisoning was more frequently diagnosed than primary pyrrolizidine alkaloid poisoning, particularly when grazing E. plantagineum. The data indicated that adult crossbred ewes were the most commonly affected class of sheep. PMID:3632498

  20. Alkaloids in the pharmaceutical industry: Structure, isolation and application

    OpenAIRE

    Nikolić Milan; Đorđević Siniša M.

    2003-01-01

    By the end of the 18th and the beginning of the 19th century a new era began in medicine, pharmaceutics and chemistry that was strongly connected with alkaloids and alkaloid drugs. Even before that it was known that certain drugs administered in limited doses were medicines, and toxic if taken in larger doses (opium, coke leaves, belladonna roots, monkshood tubers crocus or hemlock seeds). However, the identification, isolation and structural characterization of the active ingredients of the ...