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Sample records for alkaloid biosynthetic gene

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

    Science.gov (United States)

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

    2016-01-18

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

  2. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

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    Shinji Kishimoto

    2016-08-01

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

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

    Science.gov (United States)

    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.

  4. 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

  5. Origin of saxitoxin biosynthetic genes in cyanobacteria.

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    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

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

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    Yasuyuki Yamada

    2016-09-01

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

  7. A Papaver somniferum 10-gene cluster for synthesis of the anticancer alkaloid noscapine.

    Science.gov (United States)

    Winzer, Thilo; Gazda, Valeria; He, Zhesi; Kaminski, Filip; Kern, Marcelo; Larson, Tony R; Li, Yi; Meade, Fergus; Teodor, Roxana; Vaistij, Fabián E; Walker, Carol; Bowser, Tim A; Graham, Ian A

    2012-06-29

    Noscapine is an antitumor alkaloid from opium poppy that binds tubulin, arrests metaphase, and induces apoptosis in dividing human cells. Elucidation of the biosynthetic pathway will enable improvement in the commercial production of noscapine and related bioactive molecules. Transcriptomic analysis revealed the exclusive expression of 10 genes encoding five distinct enzyme classes in a high noscapine-producing poppy variety, HN1. Analysis of an F(2) mapping population indicated that these genes are tightly linked in HN1, and bacterial artificial chromosome sequencing confirmed that they exist as a complex gene cluster for plant alkaloids. Virus-induced gene silencing resulted in accumulation of pathway intermediates, allowing gene function to be linked to noscapine synthesis and a novel biosynthetic pathway to be proposed.

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

    Science.gov (United States)

    Liscombe, David K; O'Connor, Sarah E

    2011-11-01

    The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). 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 either 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. A VIGS method was developed herein 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.

  9. Digital Gene Expression Analysis Provides Insight into the Transcript Profile of the Genes Involved in Aporphine Alkaloid Biosynthesis in Lotus (Nelumbo nucifera)

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    Yang, Mei; Zhu, Lingping; Li, Ling; Li, Juanjuan; Xu, Liming; Feng, Ji; Liu, Yanling

    2017-01-01

    The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (NMCH), and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This

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

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    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.

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    Patricia Müller-Moulé

    2016-10-01

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

  13. 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

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

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    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.

  15. Origin of Saxitoxin Biosynthetic Genes in Cyanobacteria

    OpenAIRE

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

    2009-01-01

    BACKGROUND: Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX). STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and th...

  16. 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.

  17. 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

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

    Science.gov (United States)

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

    2013-07-19

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    characterization of terpenoid biosynthetic genes, engineered Physcomitrella can be a green biotechnological platform for production of terpenoids. Here, we describe two complementary and simple procedures for stable nuclear transformation of Physcomitrella with terpenoid biosynthetic genes, selection......Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure...... already been widely recognized as a viable alternative for industrial-scale production of biopharmaceuticals. For expression of terpenoid biosynthetic genes, and reconstruction of heterologous pathways, Physcomitrella has unique attributes that makes it a very promising biotechnological host...

  20. 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

  1. Functional Analysis of the Fusarielin Biosynthetic Gene Cluster

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    Aida Droce

    2016-12-01

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

  2. 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...

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

    Science.gov (United States)

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

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

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    Megan Coomer

    2014-01-01

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

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

    Science.gov (United States)

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

  6. Transcription factors in alkaloid biosynthesis.

    Science.gov (United States)

    Yamada, Yasuyuki; Sato, Fumihiko

    2013-01-01

    Higher plants produce a large variety of low-molecular weight secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used pharmaceutically. Whereas alkaloid chemistry has been intensively investigated, alkaloid biosynthesis, including the relevant biosynthetic enzymes, genes and their regulation, and especially transcription factors, is largely unknown, as only a limited number of plant species produce certain types of alkaloids and they are difficult to study. Recently, however, several groups have succeeded in isolating the transcription factors that are involved in the biosynthesis of several types of alkaloids, including bHLH, ERF, and WRKY. Most of them show Jasmonate (JA) responsiveness, which suggests that the JA signaling cascade plays an important role in alkaloid biosynthesis. Here, we summarize the types and functions of transcription factors that have been isolated in alkaloid biosynthesis, and characterize their similarities and differences compared to those in other secondary metabolite pathways, such as phenylpropanoid and terpenoid biosyntheses. The evolution of this biosynthetic pathway and regulatory network, as well as the application of these transcription factors to metabolic engineering, is discussed.

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

    Science.gov (United States)

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

    2010-11-01

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

  8. Occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants.

    Science.gov (United States)

    Ishida, Mariko; Kitao, Naoko; Mizuno, Kouichi; Tanikawa, Natsu; Kato, Misako

    2009-02-01

    Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-(14)C]adenine and [8-(14)C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue.

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

    OpenAIRE

    2002-01-01

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

  10. Transgenic and mutation-based suppression of a berberine bridge enzyme-like (BBL) gene family reduces alkaloid content in field-grown tobacco.

    Science.gov (United States)

    Lewis, Ramsey S; Lopez, Harry O; Bowen, Steve W; Andres, Karen R; Steede, William T; Dewey, Ralph E

    2015-01-01

    Motivation exists to develop tobacco cultivars with reduced nicotine content for the purpose of facilitating compliance with expected tobacco product regulations that could mandate the lowering of nicotine levels per se, or the reduction of carcinogenic alkaloid-derived tobacco specific nitrosamines (TSNAs). A berberine bridge enzyme-like (BBL) gene family was recently characterized for N. tabacum and found to catalyze one of the final steps in pyridine alkaloid synthesis for this species. Because this gene family acts downstream in the nicotine biosynthetic pathway, it may represent an attractive target for genetic strategies with the objective of reducing alkaloid content in field-grown tobacco. In this research, we produced transgenic doubled haploid lines of tobacco cultivar K326 carrying an RNAi construct designed to reduce expression of the BBL gene family. Field-grown transgenic lines carrying functional RNAi constructs exhibited average cured leaf nicotine levels of 0.684%, in comparison to 2.454% for the untransformed control. Since numerous barriers would need to be overcome to commercialize transgenic tobacco cultivars, we subsequently pursued a mutation breeding approach to identify EMS-induced mutations in the three most highly expressed isoforms of the BBL gene family. Field evaluation of individuals possessing different homozygous combinations of truncation mutations in BBLa, BBLb, and BBLc indicated that a range of alkaloid phenotypes could be produced, with the triple homozygous knockout genotype exhibiting greater than a 13-fold reduction in percent total alkaloids. The novel source of genetic variability described here may be useful in future tobacco breeding for varied alkaloid levels.

  11. 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.

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

    Science.gov (United States)

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

    2003-04-01

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

  13. 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

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

    Science.gov (United States)

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

  15. 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

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

    Science.gov (United States)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    2013-07-01

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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

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

    Science.gov (United States)

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

    2001-02-01

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

  4. 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.

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

    Science.gov (United States)

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

    2012-10-01

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

  6. 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.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G...... abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis....

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

    Science.gov (United States)

    ten Have, A; Woltering, E J

    1997-05-01

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

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

    Science.gov (United States)

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

    2011-04-01

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

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

    Science.gov (United States)

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

    2013-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Dionicia Gloria León-Martínez

    2012-06-01

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

  12. 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.

  13. 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.

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

    OpenAIRE

    SPIERING, MARTIN J.; Moon, Christina D.; Wilkinson, Heather H.; Christopher L Schardl

    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 ...

  15. 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.

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

    Science.gov (United States)

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

    2016-05-12

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

  17. 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.

  18. 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

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

    Science.gov (United States)

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

    2011-04-01

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

  20. 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.

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

    Science.gov (United States)

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

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

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

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

  3. 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

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

    Directory of Open Access Journals (Sweden)

    Dutartre Leslie

    2012-05-01

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

  5. 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.

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

    Science.gov (United States)

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

    2014-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Ester Vilaprinyo

    2010-02-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

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

    Science.gov (United States)

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

    2012-11-01

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

  10. 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.

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

    Science.gov (United States)

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

    2014-10-17

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

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

    OpenAIRE

    2015-01-01

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

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

    Science.gov (United States)

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

    2017-12-01

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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

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

    DEFF Research Database (Denmark)

    Weber, Tilmann; Blin, Kai; Duddela, Srikanth

    2015-01-01

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

  20. 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.

  1. 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

  2. Biosynthesis and accumulation of ergoline alkaloids in a mutualistic association between Ipomoea asarifolia (Convolvulaceae) and a clavicipitalean fungus.

    Science.gov (United States)

    Markert, Anne; Steffan, Nicola; Ploss, Kerstin; Hellwig, Sabine; Steiner, Ulrike; Drewke, Christel; Li, Shu-Ming; Boland, Wilhelm; Leistner, Eckhard

    2008-05-01

    Ergoline alkaloids occur in taxonomically unrelated taxa, such as fungi, belonging to the phylum Ascomycetes and higher plants of the family Convolvulaceae. The disjointed occurrence can be explained by the observation that plant-associated epibiotic clavicipitalean fungi capable of synthesizing ergoline alkaloids colonize the adaxial leaf surface of certain Convolvulaceae plant species. The fungi are seed transmitted. Their capacity to synthesize ergoline alkaloids depends on the presence of an intact differentiated host plant (e.g. Ipomoea asarifolia or Turbina corymbosa [Convolvulaceae]). Here, we present independent proof that these fungi are equipped with genetic material responsible for ergoline alkaloid biosynthesis. The gene (dmaW) for the determinant step in ergoline alkaloid biosynthesis was shown to be part of a cluster involved in ergoline alkaloid formation. The dmaW gene was overexpressed in Saccharomyces cerevisiae, the encoded DmaW protein purified to homogeneity, and characterized. Neither the gene nor the biosynthetic capacity, however, was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Both plants, however, contained the ergoline alkaloids almost exclusively, whereas alkaloids are not detectable in the associated epibiotic fungi. This indicates that a transport system may exist translocating the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant very likely is a symbiotum in which ergoline alkaloids play an essential role.

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

    Directory of Open Access Journals (Sweden)

    Liyao Ji

    2016-12-01

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

  4. 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.

  5. 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.

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

    Science.gov (United States)

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

    2015-04-01

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

  7. 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

  8. Staphylococcal enterotoxin A gene-carrying Staphylococcus aureus isolated from foods and its control by crude alkaloid from papaya leaves.

    Science.gov (United States)

    Handayani, Lita; Faridah, Didah Nur; Kusumaningrum, Harsi D

    2014-11-01

    Staphylococcus aureus is a known pathogen causing intoxication by producing enterotoxins in food. Staphylococcal enterotoxin A is one of the enterotoxins commonly implicated in staphylococcal food poisoning. The ability of crude alkaloid extract from papaya leaves to inhibit the growth of S. aureus and staphylococcal enterotoxin A synthesis was investigated. Staphylococcal enterotoxin A gene-carrying S. aureus was isolated from raw milk and ready-to-eat foods. Crude alkaloid was extracted from ground, dried papaya leaves using ultrasonic-assisted extraction, and a MIC of the alkaloid was determined by the broth macrodilution method. Furthermore, S. aureus isolate was exposed to the crude alkaloid extract at one- and twofold MIC, and the expression of sea was subsequently analyzed using a quantitative reverse transcription real-time PCR. Ten isolates of S. aureus were obtained, and nine of those isolates were sea carriers. The yield of crude alkaloid extract was 0.48 to 1.82% per dry weight of papaya leaves. A MIC of crude alkaloid to S. aureus was 0.25 mg/ml. After exposure to the alkaloid at 0.25 and 0.5 mg/ml for 2 h, a significant increase in cycle threshold values of sea was observed. The sea was expressed 29 and 41 times less when S. aureus was exposed to crude alkaloid at one- and twofold MIC, respectively. This study revealed that crude alkaloid of papaya leaves could control staphylococcal enterotoxin A gene-carrying S. aureus by suppressing the expression of sea, in addition to the ability to inhibit the growth of S. aureus. The expression of sea was successfully quantified.

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

    Directory of Open Access Journals (Sweden)

    F. Jerry Reen

    2015-07-01

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

  10. 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.

  11. 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

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

    Science.gov (United States)

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

    2014-06-19

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  15. 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.

  16. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

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

  18. 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.

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Li Jine

    2012-10-01

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

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

    Science.gov (United States)

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

    2008-05-01

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

  2. Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum.

    Science.gov (United States)

    Dewey, Ralph E; Xie, Jiahua

    2013-10-01

    Alkaloids represent an extensive group of nitrogen-containing secondary metabolites that are widely distributed throughout the plant kingdom. The pyridine alkaloids of tobacco (Nicotiana tabacum L.) have been the subject of particularly intensive investigation, driven largely due to the widespread use of tobacco products by society and the role that nicotine (16) (see Fig. 1) plays as the primary compound responsible for making the consumption of these products both pleasurable and addictive. In a typical commercial tobacco plant, nicotine (16) comprises about 90% of the total alkaloid pool, with the alkaloids nornicotine (17) (a demethylated derivative of nicotine), anatabine (15) and anabasine (5) making up most of the remainder. Advances in molecular biology have led to the characterization of the majority of the genes encoding the enzymes directly responsible the biosynthesis of nicotine (16) and nornicotine (17), while notable gaps remain within the anatabine (15) and anabasine (5) biosynthetic pathways. Several of the genes involved in the transcriptional regulation and transport of nicotine (16) have also been elucidated. Investigations of the molecular genetics of tobacco alkaloids have not only provided plant biologists with insights into the mechanisms underlying the synthesis and accumulation of this important class of plant alkaloids, they have also yielded tools and strategies for modifying the tobacco alkaloid composition in a manner that can result in changing the levels of nicotine (16) within the leaf, or reducing the levels of a potent carcinogenic tobacco-specific nitrosamine (TSNA). This review summarizes recent advances in our understanding of the molecular genetics of alkaloid biosynthesis in tobacco, and discusses the potential for applying information accrued from these studies toward efforts designed to help mitigate some of the negative health consequences associated with the use of tobacco products.

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

    Directory of Open Access Journals (Sweden)

    Gavrilović Ljubica

    2012-01-01

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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  5. 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.

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

    Science.gov (United States)

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

    2016-03-01

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

  7. 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.

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    zhen ezhang

    2015-07-01

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

  10. 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

  11. 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

  12. 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.

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

    Science.gov (United States)

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

    2011-10-01

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

  14. 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.

  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. Early changes in gene expression induced by acute UV exposure in leaves of Psychotria brachyceras, a bioactive alkaloid accumulating plant.

    Science.gov (United States)

    do Nascimento, Naíla Cannes; Menguer, Paloma Koprovski; Sperotto, Raul Antonio; de Almeida, Márcia Rodrigues; Fett-Neto, Arthur Germano

    2013-05-01

    UV-B radiation can damage biomolecules, such as DNA, RNA, and proteins, halting essential cellular processes; this damage is partly due to ROS generation. Plant secondary metabolites may protect against UV-B. Psychotria brachyceras Müll. Arg. (Rubiaceae), a subtropical shrub, produces brachycerine, a monoterpene indole alkaloid mainly accumulated in leaf tissues, which displays antioxidant and antimutagenic activities. Exposure of P. brachyceras cuttings to UV-B radiation significantly increases leaf brachycerine concentration. It has been suggested that this alkaloid might contribute to protection against UV-B damage both through its quenching activity on ROS and as UV shield. To identify differentially expressed genes of P. brachyceras in response to UV-B and investigate a possible influence of this stimulus on putative brachycerine-related genes, suppressive subtractive hybridization was applied. Complementary DNA from UV-B-treated leaves for 24 h was used as tester, and cDNA from untreated leaves, as driver. After BLASTX alignments, 134 sequences matched plant genes. Using quantitative RT-PCR, selected genes potentially related to brachycerine showed significant increases in transcription after UV-B exposure: tryptophan decarboxylase, ACC oxidase, UDP-glucose glucosyltransferase, lipase, and serine/threonine kinase. Results suggest a possible involvement of brachycerine in acute UV-B responses and show that alkaloid accumulation seems at least partly regulated at transcriptional level.

  17. 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

    2015-06-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Royah Vaezi

    2013-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Na Zhang

    2016-12-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

  3. 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.

  4. 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.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Jin-Yuan Ho

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

  7. 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.

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

    Science.gov (United States)

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

    2017-01-01

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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

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

    Science.gov (United States)

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

    2010-04-01

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

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

    Science.gov (United States)

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

    2016-11-01

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

  16. 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.

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

    Science.gov (United States)

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

    2017-01-01

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

  18. 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.

  19. 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.

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

    Science.gov (United States)

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

    2008-10-15

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

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

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    Immacolata Coraggio

    2013-06-01

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

  2. 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.

  3. 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.

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

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    Jungsuwadee Paiboon

    2011-02-01

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

  5. 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.

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

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    Sha Xie

    2015-12-01

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

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

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    Castellarin Simone D

    2007-08-01

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

  8. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2011-03-18

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

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

    Science.gov (United States)

    Pulsawat, Nattika; Kitani, Shigeru; Nihira, Takuya

    2007-05-15

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

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

    Science.gov (United States)

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

    2011-04-29

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

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

    Directory of Open Access Journals (Sweden)

    Yong-Zan Wei

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

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

    Directory of Open Access Journals (Sweden)

    Luo Hongmei

    2011-12-01

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

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

    Science.gov (United States)

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

    2011-06-08

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

  16. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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.

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

    Science.gov (United States)

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

    2010-07-01

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

  1. 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

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

    Science.gov (United States)

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

    2008-08-01

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

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

    DEFF Research Database (Denmark)

    Ley, Ana; Frandsen, Rasmus John Normand

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

  4. Vinca alkaloids.

    Science.gov (United States)

    Moudi, Maryam; Go, Rusea; Yien, Christina Yong Seok; Nazre, Mohd

    2013-11-01

    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.

  5. 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.

  6. Racemic alkaloids from the fungus Ganoderma cochlear.

    Science.gov (United States)

    Wang, Xin-Long; Dou, Man; Luo, Qi; Cheng, Li-Zhi; Yan, Yong-Ming; Li, Rong-Tao; Cheng, Yong-Xian

    2017-01-01

    Seven pairs of new alkaloid enantiomers, ganocochlearines C-I (1, 3-8), and three pairs of known alkaloids were isolated from the fruiting bodies of Ganoderma cochlear. The chemical structures of new compounds were elucidated on the basis of 1D and 2D NMR data. The absolute configurations of compounds 1, 3-10 were assigned by ECD calculations. Biological activities of these isolates against renal fibrosis were accessed in rat normal or diseased renal interstitial fibroblast cells. Importantly, the plausible biosynthetic pathway for this class of alkaloids was originally proposed.

  7. 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.

  8. 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.

  9. The Securinega alkaloids.

    Science.gov (United States)

    Chirkin, Eqor; Atkatlian, William; Porée, François-Hugues

    2015-01-01

    Securinega alkaloids represent a family of plant secondary metabolites known for 50 years. Securinine (1), the most abundant and studied alkaloid of this series was isolated by Russian researchers in 1956. In the following years, French and Japanese scientists reported other Securinega compounds and extensive work was done to elucidate their intriguing structures. The homogeneity of this family relies mainly on its tetracyclic chemical backbone, which features a butenolide moiety (cycle D) and an azabicyclo[3.2.1]octane ring system (rings B and C). Interestingly, after a period of latency of 20 years, the Securinega topic reemerged as a prolific source of new natural structures and to date more than 50 compounds have been identified and characterized. The oligomeric subgroup gathering dimeric, trimeric, and tetrameric units is of particular interest. The unprecedented structure of the Securinega alkaloids was the subject of extensive synthetic efforts culminating in several efficient and elegant total syntheses. The botanical distribution of these alkaloids seems limited to the Securinega, Flueggea, Margaritaria, and Breynia genera (Phyllanthaceae). However, only a limited number of plant species have been considered for their alkaloid contents, and additional phytochemical as well as genetic studies are needed. Concerning the biosynthesis, experiments carried out with radiolabelled aminoacids allowed to identify lysine and tyrosine as the precursors of the piperidine ring A and the CD rings of securinine (1), respectively. Besides, plausible biosynthetic pathways were proposed for virosaine A (38) and B (39), flueggine A (46), and also the different oligomers flueggenine A-D (48-51), fluevirosinine A (56), and flueggedine (20). The case of nirurine (45) and secu'amamine (37) remains elusive and additional studies seem necessary to understand their mode of production. The scope of biological of activities of the Securinega alkaloids was mainly centered on the CNS

  10. 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.

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Institute of Scientific and Technical Information of China (English)

    何奕昆; 刘新仿; 李家洋

    1999-01-01

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

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

    Science.gov (United States)

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

    2014-02-01

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

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

    Science.gov (United States)

    Morimoto, Kinuyo; Satake, Honoo

    2013-01-01

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

  15. 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.

  16. 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

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

    OpenAIRE

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2000-12-01

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

  19. Disturbance of gene expression in primary human hepatocytes by hepatotoxic pyrrolizidine alkaloids: A whole genome transcriptome analysis.

    Science.gov (United States)

    Luckert, Claudia; Hessel, Stefanie; Lenze, Dido; Lampen, Alfonso

    2015-10-01

    1,2-unsaturated pyrrolizidine alkaloids (PA) are plant metabolites predominantly occurring in the plant families Asteraceae and Boraginaceae. Acute and chronic PA poisoning causes severe hepatotoxicity. So far, the molecular mechanisms of PA toxicity are not well understood. To analyze its mode of action, primary human hepatocytes were exposed to a non-cytotoxic dose of 100 μM of four structurally different PA: echimidine, heliotrine, senecionine, senkirkine. Changes in mRNA expression were analyzed by a whole genome microarray. Employing cut-off values with a |fold change| of 2 and a q-value of 0.01, data analysis revealed numerous changes in gene expression. In total, 4556, 1806, 3406 and 8623 genes were regulated by echimidine, heliotrine, senecione and senkirkine, respectively. 1304 genes were identified as commonly regulated. PA affected pathways related to cell cycle regulation, cell death and cancer development. The transcription factors TP53, MYC, NFκB and NUPR1 were predicted to be activated upon PA treatment. Furthermore, gene expression data showed a considerable interference with lipid metabolism and bile acid flow. The associated transcription factors FXR, LXR, SREBF1/2, and PPARα/γ/δ were predicted to be inhibited. In conclusion, though structurally different, all four PA significantly regulated a great number of genes in common. This proposes similar molecular mechanisms, although the extent seems to differ between the analyzed PA as reflected by the potential hepatotoxicity and individual PA structure.

  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. The Double-Bond Configuration of Corynanthean Alkaloids and Its Impact on Monoterpenoid Indole Alkaloid Biosynthesis.

    Science.gov (United States)

    Eckermann, Ruben; Gaich, Tanja

    2016-04-11

    Experimental evidence is provided for the coherence of the double-bond geometry and the occurrence of "secondary cyclizations" in the biosynthesis of monoterpenoid indole alkaloids. Biosynthetically, akuammiline, C-mavacurine, and Strychnos alkaloids are proposed to be derived from the corynanthean alkaloid geissoschizine, a key intermediate in the biosynthetic pathway of these monoterpenoid indole alkaloids. This process occurs by so-called "secondary cyclizations" from geissoschizine or its derivatives. Although corynanthean alkaloids like geissoschizine incorporate E or Z double bonds located at C19-C20, the alkaloids downstream in the biosynthesis exclusively exhibit the E double bond. This study shows that secondary cyclizations preferentially occur with the E isomer of geissoschizine or its derivatives. This is attributed to the flexibility of the quinolizidine system of the corynanthean alkaloids, which can adopt a cis or trans conformation. For the secondary cyclization to take place, the cis-quinolizidine conformation is required. Experimental evidence supports the hypothesis that the E double bond of geissoschizine induces the cis conformation, whereas the Z double bond induces the trans conformation, which prohibits secondary cyclization of the Z compounds.

  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. Monoterpene Indole Alkaloids from the Fruit of Tabernaemontana litoralis and Differential Alkaloid Composition in Various Fruit Components.

    Science.gov (United States)

    Qu, Yang; Simonescu, Razvan; De Luca, Vincenzo

    2016-12-23

    Two new monoterpene indole alkaloids, isoakuammiline (1) and 18-hydroxypseudovincadifformine (2), and five known alkaloids, coronaridine (3), heyneanine (4), 3,19-oxidocoronaridine (5), tabersonine, and strictosidine, were identified from the fruit of Tabernaemontana litoralis. The structures of the alkaloids were determined using NMR and MS data analyses. While 18-hydroxypseudovincadifformine (2) showed a new hydroxylation pattern, isoakuammiline (1) revealed a novel skeleton for monoterpene indole alkaloids. In spite of the isolation of stemmadenine from the fruit tissues in other Tabernaemontana species, this vital biosynthetic precursor of iboga, aspidosperma, and pseudoaspidosperma skeletons was not found in T. litoralis.

  4. Pyrrolizidine alkaloids.

    Science.gov (United States)

    Robertson, Jeremy; Stevens, Kiri

    2014-12-01

    This review covers pyrrolizidine alkaloids isolated from natural sources. Topics include: aspects of structure, isolation, and biological/pharmacological studies; total syntheses of necic acids, necine bases and closely-related non-natural analogues.

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

    Institute of Scientific and Technical Information of China (English)

    王辉; 李文丽; 王富

    2015-01-01

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

  6. Engineering biosynthesis of the anticancer alkaloid noscapine in yeast.

    Science.gov (United States)

    Li, Yanran; Smolke, Christina D

    2016-07-05

    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.

  7. 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

  8. 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.

  9. 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.

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

    Science.gov (United States)

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

    2014-06-01

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

  11. Antiprotozoal and antioxidant alkaloids from Alternanthera littoralis.

    Science.gov (United States)

    Koolen, Hector H F; Pral, Elizabeth M F; Alfieri, Silvia C; Marinho, Jane V N; Serain, Alessandra F; Hernández-Tasco, Alvaro J; Andreazza, Nathalia L; Salvador, Marcos J

    2017-02-01

    Five alkaloids, in addition to hydroxytyrosol and uridine, were isolated from aerial parts of Alternanthera littoralis P. Beauv. Among the isolated compounds, alternamide A was an unusual tricyclic alkaloid with a bridged benzoazepine core. All isolated alkaloids have a catechol moiety, indicating a possible common biosynthetic route. Their structures were established by 1D and 2D NMR spectroscopy in combination with extensive tandem MS experiments by collisional induced dissociation (CID). The antiprotozoal activity of the isolated compounds was assayed against trypomastigote forms of Trypanosoma cruzi and amastigotes of Leishmania amazonensis. Alternamine A was the most active compound, reducing markedly the viability of both parasites. Antioxidant capacities evaluated by ORACFL assay showed that the isolated alkaloids (mainly alternamide B) contributed to the high activity recorded for the ethanolic crude extract; possibly, the catechol moiety present in all structures plays a central role in this result.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

    Patil, Prabhu B; Sonti, Ramesh V

    2004-01-01

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

  14. 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

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

    Science.gov (United States)

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

    2015-10-13

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

  16. 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.

  17. 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.

  18. Transcript and metabolite profiling in cell cultures of 18 plant species that produce benzylisoquinoline alkaloids.

    Science.gov (United States)

    Farrow, Scott C; Hagel, Jillian M; Facchini, Peter J

    2012-05-01

    Benzylisoquinoline alkaloids (BIAs) are a large and diverse group of ~2500 specialized metabolites found predominantly in plants of the order Ranunculales. Research focused on BIA metabolism in a restricted number of plant species has identified many enzymes and cognate genes involved in the biosynthesis of compounds such as morphine, sanguinarine and berberine. However, the formation of most BIAs remains uncharacterized at the molecular biochemical level. Herein a compendium of sequence- and metabolite-profiling resources from 18 species of BIA-accumulating cell cultures was established, representing four related plant families. Our integrated approach consisted of the construction of EST libraries each containing approximately 3500 unigenes per species for a total of 58,787 unigenes. The EST libraries were manually triaged using known BIA-biosynthetic genes as queries to identify putative homologs with similar or potentially different functions. Sequence resources were analyzed in the context of the targeted metabolite profiles obtained for each cell culture using electrospray-ionization and collision-induced dissociation mass spectrometry. Fragmentation analysis was used for the identification or structural characterization coupled with the relative quantification of 72 BIAs, which establishes a key resource for future work on alkaloid biosynthesis. The metabolite profile obtained for each species provides a rational basis for the prediction of enzyme function in BIA metabolism. The metabolic frameworks assembled through the integration of transcript and metabolite profiles allow a comparison of BIA metabolism across several plant species and families. Taken together, these data represent an important tool for the discovery of BIA biosynthetic genes.

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

    Science.gov (United States)

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

    2012-05-16

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

  20. Reconstructing fungal natural product biosynthetic pathways.

    Science.gov (United States)

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

    2014-10-01

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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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...

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

    Science.gov (United States)

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

    2014-06-01

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

  8. 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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  10. 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.

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

    Science.gov (United States)

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

    2009-03-10

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

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

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    Tetsuya Yasutake

    2016-03-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Science.gov (United States)

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

    2015-04-01

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

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

    Science.gov (United States)

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

    2015-12-19

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

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

    Science.gov (United States)

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

    2013-03-27

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

  18. 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.

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

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    Zhichao eXu

    2016-02-01

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

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

    Science.gov (United States)

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

    2015-11-11

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

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

    Science.gov (United States)

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

    2016-08-01

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

  2. 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.

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

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    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.

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

    Science.gov (United States)

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

    1998-09-01

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

  5. 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.

  6. 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.

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

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

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    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

  10. Quinolizidine Alkaloid Biosynthesis in Lupins and Prospects for Grain Quality Improvement.

    Science.gov (United States)

    Frick, Karen M; Kamphuis, Lars G; Siddique, Kadambot H M; Singh, Karam B; Foley, Rhonda C

    2017-01-01

    Quinolizidine alkaloids (QAs) are toxic secondary metabolites found within the genus Lupinus, some species of which are commercially important grain legume crops including Lupinus angustifolius (narrow-leafed lupin, NLL), L. luteus (yellow lupin), L. albus (white lupin), and L. mutabilis (pearl lupin), with NLL grain being the most largely produced of the four species in Australia and worldwide. While QAs offer the plants protection against insect pests, the accumulation of QAs in lupin grain complicates its use for food purposes as QA levels must remain below the industry threshold (0.02%), which is often exceeded. It is not well understood what factors cause grain QA levels to exceed this threshold. Much of the early work on QA biosynthesis began in the 1970-1980s, with many QA chemical structures well-characterized and lupin cell cultures and enzyme assays employed to identify some biosynthetic enzymes and pathway intermediates. More recently, two genes associated with these enzymes have been characterized, however, the QA biosynthetic pathway remains only partially elucidated. Here, we review the research accomplished thus far concerning QAs in lupin and consider some possibilities for further elucidation and manipulation of the QA pathway in lupin crops, drawing on examples from model alkaloid species. One breeding strategy for lupin is to produce plants with high QAs in vegetative tissues while low in the grain in order to confer insect resistance to plants while keeping grain QA levels within industry regulations. With the knowledge achieved on alkaloid biosynthesis in other plant species in recent years, and the recent development of genomic and transcriptomic resources for NLL, there is considerable scope to facilitate advances in our knowledge of QAs, leading to the production of improved lupin crops.

  11. Quinolizidine Alkaloid Biosynthesis in Lupins and Prospects for Grain Quality Improvement

    Science.gov (United States)

    Frick, Karen M.; Kamphuis, Lars G.; Siddique, Kadambot H. M.; Singh, Karam B.; Foley, Rhonda C.

    2017-01-01

    Quinolizidine alkaloids (QAs) are toxic secondary metabolites found within the genus Lupinus, some species of which are commercially important grain legume crops including Lupinus angustifolius (narrow-leafed lupin, NLL), L. luteus (yellow lupin), L. albus (white lupin), and L. mutabilis (pearl lupin), with NLL grain being the most largely produced of the four species in Australia and worldwide. While QAs offer the plants protection against insect pests, the accumulation of QAs in lupin grain complicates its use for food purposes as QA levels must remain below the industry threshold (0.02%), which is often exceeded. It is not well understood what factors cause grain QA levels to exceed this threshold. Much of the early work on QA biosynthesis began in the 1970–1980s, with many QA chemical structures well-characterized and lupin cell cultures and enzyme assays employed to identify some biosynthetic enzymes and pathway intermediates. More recently, two genes associated with these enzymes have been characterized, however, the QA biosynthetic pathway remains only partially elucidated. Here, we review the research accomplished thus far concerning QAs in lupin and consider some possibilities for further elucidation and manipulation of the QA pathway in lupin crops, drawing on examples from model alkaloid species. One breeding strategy for lupin is to produce plants with high QAs in vegetative tissues while low in the grain in order to confer insect resistance to plants while keeping grain QA levels within industry regulations. With the knowledge achieved on alkaloid biosynthesis in other plant species in recent years, and the recent development of genomic and transcriptomic resources for NLL, there is considerable scope to facilitate advances in our knowledge of QAs, leading to the production of improved lupin crops. PMID:28197163

  12. Biosynthetic inorganic chemistry.

    Science.gov (United States)

    Lu, Yi

    2006-08-25

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

  13. 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.

  14. Chromone and Flavonoid Alkaloids: Occurrence and Bioactivity

    Directory of Open Access Journals (Sweden)

    Robin J. Marles

    2011-12-01

    Full Text Available The chromone and flavonoid alkaloids represent an unusual group of structurally diverse secondary metabolites, derived from the convergence of multiple biosynthetic pathways that are widely distributed through the plant and animal kingdoms. Many of them have been discovered through bioassay-guided chemical investigations of traditional medicines, suggesting potential therapeutic significance. Their unique structures and varied pharmacological activities may provide important new leads for the discovery of drugs with novel mechanisms of action. Potential therapeutic indications are as diverse as cancer and viral infections, inflammation and immunomodulation, neurological and psychiatric conditions, and diabetes.

  15. A new view on the codonocarpine type alkaloids of Capparis decidua.

    Science.gov (United States)

    Forster, Yvonne; Ghaffar, Abdul; Bienz, Stefan

    2016-08-01

    Several spermidine alkaloids are described in literature as constituents of the root bark of Capparis decidua. Since some of the proposed structures, however, are in conflict with the expected biosynthetic paths, an extract of the root bark of the plant was re-investigated. Four major spermidine alkaloids of the codonocarpine type were identified and their structures elucidated: of the four compounds, isocodonocarpine was described previously for C. decidua and cadabicine was proposed as a possible constituent as well. Codonocarpine was found for the first time in an extract of C. decidua but was previously isolated from a closely related plant. Capparidisinine, finally, is an alkaloid with a structure that has never been described before. The structures of the four alkaloids are substantiated by NMR and MS data, and the four compounds are in logical agreement with biosynthetic considerations: they would arise from α,ω-bis-adducts of spermidine with coumaric and/or ferulic acids, followed by phenol oxidation.

  16. 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...

  17. 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.

  18. Exploiting plant alkaloids.

    Science.gov (United States)

    Schläger, Sabrina; Dräger, Birgit

    2016-02-01

    Alkaloid-containing plants have been used for medicine since ancient times. Modern pharmaceuticals still rely on alkaloid extraction from plants, some of which grow slowly, are difficult to cultivate and produce low alkaloid yields. Microbial cells as alternative alkaloid production systems are emerging. Before industrial application of genetically engineered bacteria and yeasts, several steps have to be taken. Original alkaloid-forming enzymes have to be elucidated from plants. Their activity in the heterologous host cells, however, may be low. The exchange of individual plant enzymes for alternative catalysts with better performance and optimal fermentation parameters appear promising. The overall aim is enhancement and stabilization of alkaloid yields from microbes in order to replace the tedious extraction of low alkaloid concentrations from intact plants.

  19. 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

  20. 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.

  1. 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.

  2. 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...

  3. Genotoxicity of pyrrolizidine alkaloids.

    Science.gov (United States)

    Chen, Tao; Mei, Nan; Fu, Peter P

    2010-04-01

    Pyrrolizidine alkaloids (PAs) are common constituents of many plant species around the world. PA-containing plants are probably the most common poisonous plants affecting livestock and wildlife. They can inflict harm to humans through contaminated food sources, herbal medicines and dietary supplements. Half of the identified PAs are genotoxic and many of them are tumorigenic. The mutagenicity of PAs has been extensively studied in different biological systems. Upon metabolic activation, PAs produce DNA adducts, DNA cross-linking, DNA breaks, sister chromatid exchange, micronuclei, chromosomal aberrations, gene mutations and chromosome mutations in vivo and in vitro. PAs induced mutations in the cII gene of rat liver and in the p53 and K-ras genes of mouse liver tumors. It has been suggested that all PAs produce a set of (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine-derived DNA adducts and similar types of gene mutations. The signature types of mutations are G : C --> T : A transversion and tandem base substitutions. Overall, PAs are mutagenic in vivo and in vitro and their mutagenicity appears to be responsible for the carcinogenesis of PAs.

  4. 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...

  5. Assessing the limitations to terpenoid indole alkaloid biosynthesis in Catharanthus roseus hairy root cultures through gene expression profiling and precursor feeding.

    Science.gov (United States)

    Goklany, Sheba; Loring, Ralph H; Glick, James; Lee-Parsons, Carolyn W T

    2009-01-01

    The production of pharmaceutically important terpenoid indole alkaloids (TIAs) from Catharanthus roseus is partly regulated at the transcriptional level. In this study, limitations in TIA biosynthesis from C. roseus hairy root cultures were assessed through gene expression profiling and precursor feeding. The transcript levels of key TIA pathway genes (G10h, Tdc, Str, and Sgd) and metabolite levels associated with the TIA pathway (tryptamine, loganin, secologanin, strictosidine, ajmalicine, serpentine, and tabersonine) were monitored using quantitative RT-PCR and HPLC, respectively. In cultures elicited with methyl jasmonate (250 microM MeJA on day 21), G10h, Tdc, Str, and Sgd expression increased by 9.1, 3.1, 6.7, and 8.3-fold, respectively, after 24 h. Up-regulation of gene expression was followed by a 160, 440, and 420% increase in strictosidine, ajmalicine, and tabersonine levels, respectively, after 5 days. Precursors loganin, tryptamine, or their combination were fed to noninduced and MeJA-induced cultures to complement the above studies. TIA production was not significantly enhanced in either noninduced or MeJA-induced cultures with precursor feeding. In noninduced cells, steps downstream of loganin and tryptamine were limiting (SLS, STR, or SGD) because either loganin or tryptamine accumulated in the cells with precursor feeding. These bottlenecks were partly overcome in MeJA-induced cultures as the expression of Str and Sgd genes and TIA production increased. However, secologanin accumulated in MeJA-induced cultures with precursor feeding, suggesting that STR was likely limiting under MeJA-induced conditions.

  6. Unravelling the architecture and dynamics of tropane alkaloid biosynthesis pathways using metabolite correlation networks.

    Science.gov (United States)

    Nguyen, Thi-Kieu-Oanh; Jamali, Arash; Lanoue, Arnaud; Gontier, Eric; Dauwe, Rebecca

    2015-08-01

    The tropane alkaloid spectrum in Solanaceae is highly variable within and between species. Little is known about the topology and the coordination of the biosynthetic pathways leading to the variety of tropine and pseudotropine derived esters in the alkaloid spectrum, or about the metabolic dynamics induced by tropane alkaloid biosynthesis stimulating conditions. A good understanding of the metabolism, including all ramifications, is however necessary for the development of strategies to increase the abundance of pharmacologically interesting compounds such as hyoscyamine and scopolamine. The present study explores the tropane alkaloid metabolic pathways in an untargeted approach involving a correlation-based network analysis. Using GC-MS metabolite profiling, the variation and co-variation among tropane alkaloids and primary metabolites was monitored in 60 Datura innoxia Mill. individuals, of which half were exposed to tropane alkaloid biosynthesis stimulating conditions by co-culture with Agrobacterium rhizogenes. Considerable variation was evident in the relative proportions of the tropane alkaloids. Remodeling of the tropane alkaloid spectrum under co-culture with A. rhizogenes involved a specific and strong increase of hyoscyamine production and revealed that the accumulation of hyoscyamine, 3-tigloyloxy-6,7-epoxytropane, and 3-methylbutyryloxytropane was controlled independently of the majority of tropane alkaloids. Based on correlations between metabolites, we propose a biosynthetic origin of hygrine, the order of esterification of certain di-oxygenated tropanes, and that the rate of acetoxylation contributes to control of hyoscyamine production. Overall, this study shows that the biosynthesis of tropane alkaloids may be far more complex and finely controlled than previously expected.

  7. Alkaloids from Menispermum dauricum.

    Science.gov (United States)

    Yu, Bing-Wu; Chen, Jian-Yong; Wang, Yan-Ping; Cheng, Kin-Fin; Li, Xiao-Yu; Qin, Guo-Wei

    2002-10-01

    The alkaloids, dechloroacutumidine and 1-epidechloroacutumine, together with three known alkaloids, acutumidine, acutumine, and dechloroacutumine, were isolated from the rhizomes of Menispermum dauricum and their structures established by spectral and chemical methods. The cytotoxicity of each compound against the growth of human cell lines was studied, and acutumine selectively inhibited T-cell growth.

  8. [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.

  9. Biosynthetic Relationship between Acutumine and Dechloroacutumine in Menispermum dauricum Root Cultures.

    Science.gov (United States)

    Babiker, H A; Sugimoto, Y; Saisho, T; Inanaga, S; Hashimoto, M; Isogai, A

    1999-01-01

    The biosynthetic relationship between acutumine 1 and dechloroacutumine 2 was studied using (13)C-labeled tyrosine and (3)H-labeled 2 as tracers. (13)C-NMR spectra of (13)C-labeled 1 and 2 showed that the alkaloids, each composed of two molecules of tyrosine, are derived from the same biosynthetic pathway. Feeding Menispermum dauricum (Menispermaceae) roots, cultured in a chloride-enriched medium, with (3)H-labeled 2 demonstrated that 1 is the only alkaloid metabolite of 2. Conversion (5%) of the exogenously applied 2, taken up by the roots, into 1 showed that 2 is the precursor of 1. Incomplete conversion of 2 into 1 suggests accumulation of the exogenously applied 2 in cell organelles and/or compartmentation of the enzymes involved in the biosynthesis of 1.

  10. 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.

  11. 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.

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

    Science.gov (United States)

    Hevir-Kene, Neli; Rižner, Tea Lanišnik

    2015-06-05

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

  13. 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...

  14. 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

  15. 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

  16. Analysis of Ergot Alkaloids

    OpenAIRE

    Colin Crews

    2015-01-01

    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). Ot...

  17. 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.

  18. 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.

  19. 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

  20. Analysis of an inactive cyanobactin biosynthetic gene cluster leads to discovery of new natural products from strains of the genus Microcystis.

    Directory of Open Access Journals (Sweden)

    Niina Leikoski

    Full Text Available Cyanobactins are cyclic peptides assembled through the cleavage and modification of short precursor proteins. An inactive cyanobactin gene cluster has been described from the genome Microcystis aeruginosa NIES843. Here we report the discovery of active counterparts in strains of the genus Microcystis guided by this silent cyanobactin gene cluster. The end products of the gene clusters were structurally diverse cyclic peptides, which we named piricyclamides. Some of the piricyclamides consisted solely of proteinogenic amino acids while others contained disulfide bridges and some were prenylated or geranylated. The piricyclamide gene clusters encoded between 1 and 4 precursor genes. They encoded highly diverse core peptides ranging in length from 7-17 amino acids with just a single conserved amino acid. Heterologous expression of the pir gene cluster from Microcystis aeruginosa PCC7005 in Escherichia coli confirmed that this gene cluster is responsible for the biosynthesis of piricyclamides. Chemical analysis demonstrated that Microcystis strains could produce an array of piricyclamides some of which are geranylated or prenylated. The genetic diversity of piricyclamides in a bloom sample was explored and 19 different piricyclamide precursor genes were found. This study provides evidence for a stunning array of piricyclamides in Microcystis, a worldwide occurring bloom forming cyanobacteria.

  1. 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.

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

    Science.gov (United States)

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

    2011-07-01

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

  3. Belladonna Alkaloid Combinations and Phenobarbital

    Science.gov (United States)

    Belladonna alkaloid combinations and phenobarbital are used to relieve cramping pains in conditions such as irritable bowel syndrome and ... Belladonna alkaloid combinations and phenobarbital come as a regular tablet, a slow-acting tablet, capsule, and liquid to take ...

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

    NARCIS (Netherlands)

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

    2014-01-01

    Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew that a

  5. Complexity generation in fungal peptidyl alkaloid biosynthesis: a two-enzyme pathway to the hexacyclic MDR export pump inhibitor ardeemin.

    Science.gov (United States)

    Haynes, Stuart W; Gao, Xue; Tang, Yi; Walsh, Christopher T

    2013-04-19

    Ardeemins are hexacyclic peptidyl alkaloids isolated from Aspergillus fischeri as agents that block efflux of anticancer drugs by MultiDrug Resistance (MDR) export pumps. To evaluate the biosynthetic logic and enzymatic machinery for ardeemin framework assembly, we sequenced the A. fischeri genome and identified the ardABC gene cluster. Through both genetic deletions and biochemical characterizations of purified ArdA and ArdB we show this ArdAB enzyme pair is sufficient to convert anthranilate (Ant), L-Ala, and L-Trp to ardeemin. ArdA is a 430 kDa trimodular nonribosomal peptide synthase (NRPS) that converts the three building blocks into a fumiquinazoline (FQ) regioisomer termed ardeemin FQ. ArdB is a prenyltransferase that takes tricyclic ardeemin FQ and dimethylallyl diphosphate to the hexacyclic ardeemin scaffold via prenylation at C2 of the Trp-derived indole moiety with intramolecular capture by an amide NH of the fumiquinazoline ring. The two-enzyme ArdAB pathway reveals remarkable efficiency in construction of the hexacyclic peptidyl alkaloid scaffold.

  6. TaWRKY70 transcription factor in wheat QTL-2DL regulates downstream metabolite biosynthetic genes to resist Fusarium graminearum infection spread within spike

    Science.gov (United States)

    Kage, Udaykumar; Yogendra, Kalenahalli N.; Kushalappa, Ajjamada C.

    2017-01-01

    A semi-comprehensive metabolomics was used to identify the candidate metabolites and genes to decipher mechanisms of resistance in wheat near-isogenic lines (NILs) containing QTL-2DL against Fusarium graminearum (Fg). Metabolites, with high fold-change in abundance, belonging to hydroxycinnamic acid amides (HCAAs): such as coumaroylagmatine, coumaroylputrescine and Fatty acids: phosphatidic acids (PAs) were identified as resistance related induced (RRI) metabolites in rachis of resistant NIL (NIL-R), inoculated with Fg. A WRKY like transcription factor (TF) was identified within the QTL-2DL region, along with three resistance genes that biosynthesized RRI metabolites. Sequencing and in-silico analysis of WRKY confirmed it to be wheat TaWRKY70. Quantitative real time-PCR studies showed a higher expression of TaWRKY70 in NIL-R as compared to NIL-S after Fg inoculation. Further, the functional validation of TaWRKY70 based on virus induced gene silencing (VIGS) in NIL-R, not only confirmed an increased fungal biomass but also decreased expressions of downstream resistance genes: TaACT, TaDGK and TaGLI1, along with decreased abundances of RRI metabolites biosynthesized by them. Among more than 200 FHB resistance QTL identified in wheat, this is the first QTL from which a TF was identified, and its downstream target genes as well as the FHB resistance functions were deciphered. PMID:28198421

  7. Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca.

    Science.gov (United States)

    Docimo, Teresa; Schmidt, Gregor W; Luck, Katrin; Delaney, Sven K; D'Auria, John C

    2013-01-01

    Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca ( Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.

  8. Alkaloids from Hippeastrum papilio

    Directory of Open Access Journals (Sweden)

    Jaume Bastida

    2011-08-01

    Full Text Available Galanthamine, an acetylcholinesterase inhibitor marketed as a hydrobromide salt (Razadyne®, Reminyl® for the treatment of Alzheimer’s disease (AD, is obtained from Amaryllidaceae plants, especially those belonging to the genera Leucojum, Narcissus, Lycoris and Ungernia. The growing demand for galanthamine has prompted searches for new sources of this compound, as well as other bioactive alkaloids for the treatment of AD. In this paper we report the isolation of the new alkaloid 11β-hydroxygalanthamine, an epimer of the previously isolated alkaloid habranthine, which was identified using NMR techniques. It has been shown that 11β-hydroxygalanthamine has an important in vitro acetylcholinesterase inhibitory activity. Additionally, Hippeastrum papilio yielded substantial quantities of galanthamine.

  9. 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.

  10. 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...

  11. 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.

  12. 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 ...

  13. Biosynthesis of the defensive alkaloid cicindeloine in Stenus solutus beetles

    Science.gov (United States)

    Schierling, Andreas; Dettner, Konrad; Schmidt, Jürgen; Seifert, Karlheinz

    2012-08-01

    To protect themselves from predation and microorganismic infestation, rove beetles of the genus Stenus produce and store bioactive alkaloids like stenusine, 3-(2-methyl-1-butenyl)pyridine, and cicindeloine in their pygidial glands. The biosynthesis of stenusine and 3-(2-methyl-1-butenyl)pyridine was previously investigated in Stenus bimaculatus and Stenus similis, respectively. Both molecules follow the same biosynthetic pathway, where the N-heterocyclic ring is derived from l-lysine and the side chain from l-isoleucine. The different alkaloids are finally obtained by slight modifications of shared precursor molecules. The piperideine alkaloid cicindeloine occurs as a main compound additionally to ( E)-3-(2-methyl-1-butenyl)pyridine and traces of stenusine in the pygidial gland secretion of Stenus cicindeloides and Stenus solutus. Feeding of S. solutus beetles with [D,15N]-labeled amino acids followed by GC/MS analysis techniques showed that cicindeloine is synthesized via the identical pathway and precursor molecules as the other two defensive alkaloids.

  14. 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

  15. 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.

  16. 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.

  17. Genetic and Phenotypic Analyses of a Papaver somniferum T-DNA Insertional Mutant with Altered Alkaloid Composition

    Directory of Open Access Journals (Sweden)

    Kayo Yoshimatsu

    2012-02-01

    Full Text Available The in vitro shoot culture of a T-DNA insertional mutant of Papaver somniferum L. established by the infection of Agrobacterium rhizogenes MAFF03-01724 accumulated thebaine instead of morphine as a major opium alkaloid. To develop a non-narcotic opium poppy and to gain insight into its genetic background, we have transplanted this mutant to soil, and analyzed its alkaloid content along with the manner of inheritance of T-DNA insertion loci among its selfed progenies. In the transplanted T0 primary mutant, the opium (latex was found to be rich in thebaine (16.3% of dried opium by HPLC analysis. The analyses on T-DNA insertion loci by inverse PCR, adaptor-ligation PCR, and quantitative real-time PCR revealed that as many as 18 copies of T-DNAs were integrated into a poppy genome in a highly complicated manner. The number of copies of T-DNAs was decreased to seven in the selected T3 progenies, in which the average thebaine content was 2.4-fold that of the wild type plant. This may indicate that the high thebaine phenotype was increasingly stabilized as the number of T-DNA copies was decreased. In addition, by reverse transcription PCR analysis on selected morphine biosynthetic genes, the expression of codeine 6-O-demethylase was clearly shown to be diminished in the T0 in vitro shoot culture, which can be considered as one of the key factors of altered alkaloid composition.

  18. Emerging trends in research on spatial and temporal organization of terpenoid indole alkaloid pathway in Catharanthus roseus: a literature update.

    Science.gov (United States)

    Verma, Priyanka; Mathur, Ajay Kumar; Srivastava, Alka; Mathur, Archana

    2012-04-01

    Catharanthus roseus (The Madagaskar Periwinkle) plant is commercially valued for harbouring more than 130 bioactive terpenoid indole alkaloids (TIAs). Amongst these, two of the leaf-derived bisindole alkaloids-vinblastine and vincristine-are widely used in several anticancer chemotherapies. The great pharmacological values, low in planta occurrence, unavailability of synthetic substitutes and exorbitant market cost of these alkaloids have prompted scientists to understand the basic architecture and regulation of biosynthesis of these TIAs in C. roseus plant and its cultured tissues. The knowledge gathered over a period of 30 years suggests that the TIA biosynthesis is highly regulated by developmental and environmental factors and operates through a complex multi-step enzymatic network. Extensive spatial and temporal cross talking also occurs at inter- and intracellular levels in different plant organs during TIA biogenesis. A close association of indole, methylerythritol phosphate and secoiridoid monoterpenoid pathways and involvement of at least four cell types (epidermis, internal phloem-associated parenchyma, laticifers and idioblasts) and five intracellular compartments (chloroplast, vacuole, nucleus, endoplasmic reticulum and cytosol) have been implicated with this biosynthetic mechanism. Accordingly, the research in this area is primarily advancing today to address and resolve six major issues namely: precise localization and expression of pathway enzymes using modern in situ RNA hybridization tools, mechanisms of intra- and intercellular trafficking of pathway intermediates, cloning and functional validation of genes coding for known or hitherto unknown pathway enzymes, mechanism of global regulation of the pathway by transcription factors, control of relative diversion of metabolite flux at crucial branch points and finally, strategising the metabolic engineering approaches to improve the productivity of the desired TIAs in plant or corresponding cultured

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

    DEFF Research Database (Denmark)

    Liu, Qing; Manzano, David; Tanić, Nikola

    2014-01-01

    Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew...... that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.......4μgg-1 parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding...

  20. 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.

  1. Cytisine-like alkaloids from Ormosia hosiei Hemsl. & E.H. Wilson.

    Science.gov (United States)

    Pouny, Isabelle; Batut, Muriel; Vendier, Laure; David, Bruno; Yi, Sirong; Sautel, François; Arimondo, Paola B; Massiot, Georges

    2014-11-01

    Four alkaloids named hosieines A-D were isolated from the root and stem of Ormosia hosiei. Their flat structures were established by mass spectrometry and by a combination of NMR experiments. These molecules probably share a common biosynthetic origin with the lupin alkaloids but they differ in the formation of the last ring, being here part of a rare 2-azabicyclo[3.2.1]octane system. Their absolute configuration was determined by X-ray crystallography using CuKα radiation. As has been described for cytisine, they display a remarkable affinity towards neuronal nicotinic acetylcholine α4β2 receptor.

  2. Towards a Biosynthetic UAV

    Science.gov (United States)

    Block, Eli; Byemerwa, Jovita; Dispenza, Ross; Doughty, Benjamin; Gillyard, KaNesha; Godbole, Poorwa; Gonzales-Wright, Jeanette; Hull, Ian; Kannappan, Jotthe; Levine, Alexander; Nelakanti, Raman; Ruffner, Lydia; Shumate, Alaina; Sorayya, Aryo; Ugwu, Kyla

    2014-01-01

    We are currently working on a series of projects towards the construction of a fully biological unmanned aerial vehicle (UAV) for use in scientific and humanitarian missions. The prospect of a biologically-produced UAV presents numerous advantages over the current manufacturing paradigm. First, a foundational architecture built by cells allows for construction or repair in locations where it would be difficult to bring traditional tools of production. Second, a major limitation of current research with UAVs is the size and high power consumption of analytical instruments, which require bulky electrical components and large fuselages to support their weight. By moving these functions into cells with biosensing capabilities - for example, a series of cells engineered to report GFP, green fluorescent protein, when conditions exceed a certain threshold concentration of a compound of interest, enabling their detection post-flight - these problems of scale can be avoided. To this end, we are working to engineer cells to synthesize cellulose acetate as a novel bioplastic, characterize biological methods of waterproofing the material, and program this material's systemic biodegradation. In addition, we aim to use an "amberless" system to prevent horizontal gene transfer from live cells on the material to microorganisms in the flight environment.

  3. 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.

  4. Effective use of heterologous hosts for characterization of biosynthetic enzymes allows production of natural products and promotes new natural product discovery.

    Science.gov (United States)

    Watanabe, Kenji

    2014-01-01

    In the past few years, there has been impressive progress in elucidating the mechanism of biosynthesis of various natural products accomplished through the use of genetic, molecular biological and biochemical techniques. Here, we present a comprehensive overview of the current results from our studies on fungal natural product biosynthetic enzymes, including nonribosomal peptide synthetase and polyketide synthase-nonribosomal peptide synthetase hybrid synthetase, as well as auxiliary enzymes, such as methyltransferases and oxygenases. Specifically, biosynthesis of the following compounds is described in detail: (i) Sch210972, potentially involving a Diels-Alder reaction that may be catalyzed by CghA, a functionally unknown protein identified by targeted gene disruption in the wild type fungus; (ii) chaetoglobosin A, formed via multi-step oxidations catalyzed by three redox enzymes, one flavin-containing monooxygenase and two cytochrome P450 oxygenases as characterized by in vivo biotransformation of relevant intermediates in our engineered Saccharomyces cerevisiae; (iii) (-)-ditryptophenaline, formed by a cytochrome P450, revealing the dimerization mechanism for the biosynthesis of diketopiperazine alkaloids; (iv) pseurotins, whose variations in the C- and O-methylations and the degree of oxidation are introduced combinatorially by multiple redox enzymes; and (v) spirotryprostatins, whose spiro-carbon moiety is formed by a flavin-containing monooxygenase or a cytochrome P450 as determined by heterologous de novo production of the biosynthetic intermediates and final products in Aspergillus niger. We close our discussion by summarizing some of the key techniques that have facilitated the discovery of new natural products, production of their analogs and identification of biosynthetic mechanisms in our study.

  5. 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.

  6. 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.

  7. 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.

  8. The Neurospora crassa mutant NcΔEgt-1 identifies an ergothioneine biosynthetic gene and demonstrates that ergothioneine enhances conidial survival and protects against peroxide toxicity during conidial germination.

    Science.gov (United States)

    Bello, Marco H; Barrera-Perez, Viviana; Morin, Dexter; Epstein, Lynn

    2012-02-01

    Ergothioneine (EGT) is a histidine derivative with sulfur on the imidazole ring and a trimethylated amine; it is postulated to have an antioxidant function. Although EGT apparently is only produced by fungi and some prokaryotes, it is acquired by animals and plants from the environment, and is concentrated in animal tissues in cells with an EGT transporter. Monobromobimane derivatives of EGT allowed conclusive identification of EGT by LC/MS and the quantification of EGT in Colletotrichum graminicola and Neurospora crassa conidia and mycelia. EGT concentrations were significantly (α=0.05) higher in conidia than in mycelia, with approximately 17X and 5X more in C. graminicola and N. crassa, respectively. The first EGT biosynthetic gene in a fungus was identified by quantifying EGT in N. crassa wild type and knockouts in putative homologs of actinomycete EGT biosynthetic genes. NcΔEgt-1, a strain with a knockout in gene NCU04343, does not produce EGT, in contrast to the wild type. To determine the effects of EGT in vivo, we compared NcΔEgt-1 to the wild type. NcΔEgt-1 is not pleiotropically affected in rate of hyphal elongation in Vogel's medium either with or without ammonium nitrate and in the rate of germination of macroconidia on Vogel's medium. The superoxide-producer menadione had indistinguishable effects on conidial germination between the two strains. Cupric sulfate also had indistinguishable effects on conidial germination and on hyphal growth between the two strains. In contrast, germination of NcΔEgt-1 conidia was significantly more sensitive to tert-butyl hydroperoxide than the wild type; germination of 50% (GI(50)) of the NcΔEgt-1 conidia was prevented at 2.7 mM tert-butyl hydroperoxide whereas the GI(50) for the wild type was 4.7 mM tert-butyl hydroperoxide, or at a 1.7X greater concentration. In the presence of tert-butyl hydroperoxide and the fluorescent reactive oxygen species indicator 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein

  9. 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....

  10. 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

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

    Directory of Open Access Journals (Sweden)

    Ying Xiao

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

  12. 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

  13. Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

    Science.gov (United States)

    Zhang, Huimin; Zheng, Beiwen; Gao, Rongsui; Feng, Youjun

    2015-09-01

    The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid

  14. Structural determination of Streptococcus pneumoniae repeat units in serotype 41A and 41F capsular polysaccharides to probe gene functions in the corresponding capsular biosynthetic loci.

    Science.gov (United States)

    Petersen, Bent O; Skovsted, Ian C; Paulsen, Berit Smestad; Redondo, Antonio R; Meier, Sebastian

    2014-12-05

    We report the repeating unit structures of the native capsular polysaccharides of Streptococcus pneumoniae serotypes 41A and 41F. Structural determinations yielded six carbohydrate units in the doubly branched repeating unit to give the following structure for serotype 41A: The structure determinations were motivated (1) by an ambition to help close the remaining gaps in S. pneumoniae capsular polysaccharide structures, and (2) by the attempt to derive functional annotations of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides from the determined structures. An activity present in 41F but not 41A is identified as an acetyltransferase acting on the rhamnopyranosyl sidechain E. The genes encoding the formation of the six glycosidic bonds in serogroup 41 were determined from the capsular polysaccharide structures of serotype 41A, 41F, and genetically related serotypes, in conjunction with corresponding genomic information and computational homology searches. In combination with complementary information, NMR spectroscopy considerably simplifies the functional annotation of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides.

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

    Directory of Open Access Journals (Sweden)

    Rashel V Grindberg

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

  16. Quinolizidine alkaloids from Lupinus lanatus

    Science.gov (United States)

    Neto, Alexandre T.; Oliveira, Carolina Q.; Ilha, Vinicius; Pedroso, Marcelo; Burrow, Robert A.; Dalcol, Ionara I.; Morel, Ademir F.

    2011-10-01

    In this study, one new quinolizidine alkaloid, lanatine A ( 1), together with three other known alkaloids, 13-α- trans-cinnamoyloxylupanine ( 2), 13-α-hydroxylupanine ( 3), and (-)-multiflorine ( 4) were isolated from the aerial parts of Lupinus lanatus (Fabaceae). The structures of alkaloids 1- 4 were elucidated by spectroscopic data analysis. The stereochemistry of 1 was determined by single crystal X-ray analysis. Bayesian statistical analysis of the Bijvoet differences suggests the absolute stereochemistry of 1. In addition, the antimicrobial potential of alkaloids 1- 4 is also reported.

  17. Analysis of Indole Alkaloids from Rhazya stricta Hairy Roots by Ultra-Performance Liquid Chromatography-Mass Spectrometry.

    Science.gov (United States)

    Akhgari, Amir; Laakso, Into; Seppänen-Laakso, Tuulikki; Yrjönen, Teijo; Vuorela, Heikki; Oksman-Caldentey, Kirsi-Marja; Rischer, Heiko

    2015-12-17

    Rhazya stricta Decne. (Apocynaceae) contains a large number of terpenoid indole alkaloids (TIAs). This study focused on the composition of alkaloids obtained from transformed hairy root cultures of R. stricta employing ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). In the UPLC-MS analyses, a total of 20 TIAs were identified from crude extracts. Eburenine and vincanine were the main alkaloids followed by polar glucoalkaloids, strictosidine lactam and strictosidine. Secodine-type alkaloids, tetrahydrosecodinol, tetrahydro- and dihydrosecodine were detected too. The occurrence of tetrahydrosecodinol was confirmed for the first time for R. stricta. Furthermore, two isomers of yohimbine, serpentine and vallesiachotamine were identified. The study shows that a characteristic pattern of biosynthetically related TIAs can be monitored in Rhazya hairy root crude extract by this chromatographic method.

  18. Analysis of Indole Alkaloids from Rhazya stricta Hairy Roots by Ultra-Performance Liquid Chromatography-Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Amir Akhgari

    2015-12-01

    Full Text Available Rhazya stricta Decne. (Apocynaceae contains a large number of terpenoid indole alkaloids (TIAs. This study focused on the composition of alkaloids obtained from transformed hairy root cultures of R. stricta employing ultra-performance liquid chromatography-mass spectrometry (UPLC-MS. In the UPLC-MS analyses, a total of 20 TIAs were identified from crude extracts. Eburenine and vincanine were the main alkaloids followed by polar glucoalkaloids, strictosidine lactam and strictosidine. Secodine-type alkaloids, tetrahydrosecodinol, tetrahydro- and dihydrosecodine were detected too. The occurrence of tetrahydrosecodinol was confirmed for the first time for R. stricta. Furthermore, two isomers of yohimbine, serpentine and vallesiachotamine were identified. The study shows that a characteristic pattern of biosynthetically related TIAs can be monitored in Rhazya hairy root crude extract by this chromatographic method.

  19. 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.

  20. Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica

    Science.gov (United States)

    Shitan, Nobukazu; Bazin, Ingrid; Dan, Kazuyuki; Obata, Kazuaki; Kigawa, Koji; Ueda, Kazumitsu; Sato, Fumihiko; Forestier, Cyrille; Yazaki, Kazufumi

    2003-01-01

    Alkaloids comprise one of the largest groups of plant secondary metabolites. Berberine, a benzylisoquinoline alkaloid, is preferentially accumulated in the rhizome of Coptis japonica, a ranunculaceous plant, whereas gene expression for berberine biosynthetic enzymes has been observed specifically in root tissues, which suggests that berberine synthesized in the root is transported to the rhizome, where there is high accumulation. We recently isolated a cDNA encoding a multidrug-resistance protein (MDR)-type ATP-binding cassette (ABC) transporter (Cjmdr1) from berberine-producing cultured C. japonica cells, which is highly expressed in the rhizome. Functional analysis of Cjmdr1 by using a Xenopus oocyte expression system showed that CjMDR1 transported berberine in an inward direction, resulting in a higher accumulation of berberine in Cjmdr1-injected oocytes than in the control. Typical inhibitors of ABC proteins, such as vanadate, nifedipine, and glibenclamide, as well as ATP depletion, clearly inhibited this CjMDR1-dependent berberine uptake, suggesting that CjMDR1 functioned as an ABC transporter. Conventional membrane separation methods showed that CjMDR1 was localized in the plasma membrane of C. japonica cells. In situ hybridization indicated that Cjmdr1 mRNA was expressed preferentially in xylem tissues of the rhizome. These findings strongly suggest that CjMDR1 is involved in the translocation of berberine from the root to the rhizome. PMID:12524452

  1. 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.

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

    Science.gov (United States)

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

    2016-12-01

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

  3. 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.

  4. Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca [v1; ref status: indexed, http://f1000r.es/y1

    Directory of Open Access Journals (Sweden)

    Teresa Docimo

    2013-02-01

    Full Text Available Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves, and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca (Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3. The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper. From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced, while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.

  5. Assessing the tobacco-rattle-virus-based vectors system as an efficient gene silencing technique in Datura stramonium (Solanaceae).

    Science.gov (United States)

    Eftekhariyan Ghamsari, Mohammad Reza; Karimi, Farah; Mousavi Gargari, Seyed Latif; Hosseini Tafreshi, Seyed Ali; Salami, Seyed Alireza

    2014-12-01

    Datura stramonium is a well-known medicinal plant, which is important for its alkaloids. There are intrinsic limitations for the natural production of alkaloids in plants; metabolic engineering methods can be effectively used to conquer these limitations. In order for this the genes involved in corresponding pathways need to be studied. Virus-Induced Gene Silencing is known as a functional genomics technique to knock-down expression of endogenous genes. In this study, we silenced phytoene desaturase as a marker gene in D. stramonium in a heterologous and homologous manner by tobacco-rattle-virus-based VIGS vectors. Recombinant TRV vector containing pds gene from D. stramonium (pTRV2-Dspds) was constructed and injected into seedlings. The plants injected with pTRV2-Dspds showed photobleaching 2 weeks after infiltration. Spectrophotometric analysis demonstrated that the amount of chlorophylls and carotenoids in leaves of the bleached plants decreased considerably compared to that of the control plants. Semi-Quantitative RT-PCR results also confirmed that the expression of pds gene in the silenced plants was significantly reduced in comparison with the control plants. The results showed that the viral vector was able to influence the levels of total alkaloid content in D. stramonium. Our results illustrated that TRV-based VIGS vectors are able to induce effective and reliable functional gene silencing in D. stramonium as an alternative tool for studying the genes of interest in this plant, such as the targeted genes in tropane alkaloid biosynthetic pathway. The present work is the first report of establishing VIGS as an efficient method for transient silencing of any gene of interest in D. stramonium.

  6. 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.

  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. 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.

  9. 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.

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

    Science.gov (United States)

    Wang, Linzhu; Beuerle, Till; Timbilla, James; Ober, Dietrich

    2012-01-01

    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.

  11. Loganin and secologanin derived tryptamine-iridoid alkaloids from Palicourea crocea and Palicourea padifolia (Rubiaceae).

    Science.gov (United States)

    Berger, Andreas; Kostyan, Maria Katharina; Klose, Simon Immo; Gastegger, Michael; Lorbeer, Eberhard; Brecker, Lothar; Schinnerl, Johann

    2015-08-01

    During comparative analysis on Palicourea species from Costa Rica, two unusual loganin derived tryptamine-iridoid alkaloids were isolated from an accession of Palicourea crocea. Besides the already known brachycerine (2), palicroceaine (1) features a novel hexacyclic backbone. A second provenance, however, yielded strictosidinic acid (3), belonging to the more common secologanin derived tryptamine-iridoid alkaloids, such as those found in Palicourea padifolia. From this species, strictosidine (4), lyaloside (5) and its derivative (E)-O-(6')-(4″-hydroxy-3″,5″-dimethoxy)-cinnamoyl lyaloside (6) could be isolated. A herbarium specimen-based screening was performed, indicating some degree of regional differentiation in alkaloid content and biosynthetic pathways within the widespread and variable Pal. crocea. It further shows its differentiation from the related strictosidine containing Palicourea croceoides. The occurrence of loganin derived tryptamine-iridoid alkaloids in Pal. crocea, Psychotria brachyceras and Psychotria brachypoda, all putatively unrelated members of the Palicourea s.l. clade, is a noteworthy exception within the genus, otherwise largely characterized by secologanin-derived tryptamine-iridoid alkaloids.

  12. Pyrrolizidine alkaloids from Heliotropium indicum

    Energy Technology Data Exchange (ETDEWEB)

    Souza, Joao Sammy N.; Machado, Luciana L.; Pessoa, Otilia D.L.; Lemos, Telma L.G. [Ceara Univ., Fortaleza, CE (Brazil). Dept. de Quimica Organica e Inorganica]. E-mail: tlemos@dqoi.ufc.br; Braz-Filho, Raimundo [Universidade Estadual do Norte Fluminense (UENF), Campos dos Goytacases, RJ (Brazil). Setor de Quimica de Produtos Naturais; Overk, Cassia R.; Ping Yao; Cordell, Geoffrey A. [University of Illinois at Chicago, IL (United States). College of Pharmacy. Dept. of Medicinal Chemistry and Pharmacognosy

    2005-11-15

    ndicine (1), a new pyrrolizidine alkaloid with unusual structural features, together with the known lycopsamine (2), were isolated from the roots of Heliotropium indicum (Boraginaceae). The structures were established by a combination of 1D and 2D NMR methods (COSY, HMQC, HMBC, and NOESY) and HREIMS. This is the first report of a lactone pyrrolizidine alkaloid in the genus Heliotropium. Compounds 1 and 2 were assayed for antioxidant activity and showed moderate activity. (author)

  13. 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].

  14. 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.

  15. 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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  17. Tropine forming tropinone reductase gene from Withania somnifera (Ashwagandha: biochemical characteristics of the recombinant enzyme and novel physiological overtones of tissue-wide gene expression patterns.

    Directory of Open Access Journals (Sweden)

    Amit Kumar Kushwaha

    Full Text Available Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ~60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation. Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[(14C]-sucrose to orphan shoot (twigs and [(14C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid and on mechanical injury. The enzyme's catalytic and structural properties as well as gene

  18. 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.

  19. Erythroidine alkaloids: a novel class of phytoestrogens.

    Science.gov (United States)

    Djiogue, Sefirin; Halabalaki, Maria; Njamen, Dieudonné; Kretzschmar, Georg; Lambrinidis, George; Hoepping, Josephine; Raffaelli, Francesca M; Mikros, Emmanuel; Skaltsounis, Alexios-Leandros; Vollmer, Günter

    2014-07-01

    Erythrina poeppigiana is a medicinal plant which is widely used in Asia, Latin America, and Africa in traditional remedies for gynecological complications and maladies. In continuation of studies for the discovery of novel phytoestrogens, four erythroidine alkaloids, namely α-erythroidine, β-erythroidine, and their oxo-derivatives 8-oxo-α-erythroidine and 8-oxo-β-erythroidine, were isolated and structurally characterized from the methanolic extract of the stem bark of E. poeppigiana. Due to the high amounts of erythroidines in the extract and considering the widespread utilization of Erythrina preparations in traditional medicine, the exploration of their estrogenic properties was performed. The estrogenicity of the isolated erythroidines was assayed in various estrogen receptor-(ER)-dependent test systems, including receptor binding affinity, cell culture based ER-dependent reporter gene assays, and gene expression studies in cultured cells using reverse transcription polymerase chain reaction techniques. α-Erythroidine and β-erythroidine showed binding affinity values for ERα of 0.015 ± 0.010% and 0.005 ± 0.010%, respectively, whereas only β-erythroidine bound to ERβ (0.006 ± 0.010%). In reporter gene assays, both erythroidines exhibited a significant dose-dependent estrogenic stimulation of ER-dependent reporter gene activity in osteosarcoma cells detectable already at 10 nM. Results were confirmed in the MVLN cells, a bioluminescent variant of MCF-7 breast cancer cells. Further, α-erythroidine and β-erythroidine both induced the enhanced expression of the specific ERα-dependent genes trefoil factor-1 and serum/glucocorticoid regulated kinase 3 in MCF-7 cells, confirming estrogenicity. Additionally, using molecular docking simulations, a potential mode of binding on ERα, is proposed, supporting the experimental evidences. This is the first time that an estrogenic profile is reported for erythroidine alkaloids, potentially a new class of

  20. The Chemistry of the Akuammiline Alkaloids.

    Science.gov (United States)

    Adams, Gregory L; Smith, Amos B

    2016-01-01

    An update on the literature covering the akuammiline family of alkaloids is presented. This chapter begins with a summary of new akuammiline alkaloids reported since 2000 and is followed by an overview of new reported bioactivities of akuammiline alkaloids since 2000. The remainder of the chapter comprises a comprehensive review of the synthetic chemistry that has been reported in the last 50 years concerning akuammiline alkaloids and their structural motifs.

  1. 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

  2. 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.

  3. 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.

  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. 27 CFR 21.99 - Brucine alkaloid.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Brucine alkaloid. 21.99... Brucine alkaloid. (a) Identification test. Add a few drops of concentrated nitric acid to about 10 mg of brucine alkaloid. A vivid red color is produced. Dilute the red solution with a few drops of water and...

  7. Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma.

    Science.gov (United States)

    Simkin, Andrew J; Miettinen, Karel; Claudel, Patricia; Burlat, Vincent; Guirimand, Grégory; Courdavault, Vincent; Papon, Nicolas; Meyer, Sophie; Godet, Stéphanie; St-Pierre, Benoit; Giglioli-Guivarc'h, Nathalie; Fischer, Marc J C; Memelink, Johan; Clastre, Marc

    2013-01-01

    Madagascar periwinkle (Catharanthus roseus [L.] G. Don, Apocynaceae) produces monoterpene indole alkaloids (MIAs), secondary metabolites of high interest due to their therapeutic value. A key step in the biosynthesis is the generation of geraniol from geranyl diphosphate (GPP) in the monoterpenoid branch of the MIA pathway. Here we report on the cloning and functional characterization of C. roseus geraniol synthase (CrGES). The full-length CrGES was over-expressed in Escherichia coli and the purified recombinant protein catalyzed the conversion of GPP into geraniol with a K(m) value of 58.5 μM for GPP. In vivo CrGES activity was evaluated by heterologous expression in a Saccharomyces cerevisiae strain mutated in the farnesyl diphosphate synthase gene. Analysis of culture extracts by gas chromatography-mass spectrometry confirmed the excretion of geraniol into the growth medium. Transient transformation of C. roseus cells with a Yellow Fluorescent Protein-fusion construct revealed that CrGES is localized in plastid stroma and stromules. In aerial plant organs, RNA in situ hybridization showed specific labeling of CrGES transcripts in the internal phloem associated parenchyma as observed for other characterized genes involved in the early steps of MIA biosynthesis. Finally, when cultures of Catharanthus cells were treated with the alkaloid-inducing hormone methyl jasmonate, an increase in CrGES transcript levels was observed. This observation coupled with the tissue-specific expression and the subcellular compartmentalization support the idea that CrGES initiates the monoterpenoid branch of the MIA biosynthetic pathway.

  8. 1-Methylpyrrolidine-2-acetic Acid is not a Precursor of Tropane Alkaloids.

    Science.gov (United States)

    Huang, M N; Abraham, T W; Kim, S H; Leete, E

    1996-02-01

    1-Methylpyrrolidine-2-acetic acid and related compounds were studied as precursors in the biosynthesis of the tropane alkaloids in Erythroxylum coca and Datura innoxia. (R,S)-[1',2-(13)C2,2-(14)C,(15)N]-1-methylpyrrolidine-2- acid, (R,S)-[1',2'-(13)C2,1'-(14)C]-1-methylpyrrolidine-2-acetic acid, (R,S) [1',2'-(13)C2,1-(14)C]-1-methylpyrrolidine-2-acetate, and (R,S)-+2'-(14)C] methylpyrrolidine-2-acetic acid N-acetylcysteamine thioester were synthesized an intact plants by leaf-planting or hydroponic-feeding. Specific incorporation of compounds into ( - )-hyoscyamine, ( - )-scopolamine, ( - )-cocaine and the biosynthetically related cuscohygrine were very low. These results indicate that 1-methylpyrrolidine acid is not an efficient precursor of tropane alkaloids.

  9. Unprecedented Utilization of Pelargonidin and Indole for the Biosynthesis of Plant Indole Alkaloids.

    Science.gov (United States)

    Warskulat, Anne-Christin; Tatsis, Evangelos C; Dudek, Bettina; Kai, Marco; Lorenz, Sybille; Schneider, Bernd

    2016-02-15

    Nudicaulins are a group of indole alkaloid glycosides responsible for the color of yellow petals of Papaver nudicaule (Iceland poppy). The unique aglycone scaffold of these alkaloids attracted our interest as one of the most unusual flavonoid-indole hybrid structures that occur in nature. Stable isotope labeling experiments with sliced petals identified free indole, but not tryptamine or l-tryptophan, as one of the two key biosynthetic precursors of the nudicaulin aglycone. Pelargonidin was identified as the second key precursor, contributing the polyphenolic unit to the nudicaulin molecule. This finding was inferred from the temporary accumulation of pelargonidin glycosides in the petals during flower bud development and a drop at the point in time when nudicaulin levels start to increase. The precursor-directed incorporation of cyanidin into a new 3'-hydroxynudicaulin strongly supports the hypothesis that anthocyanins are involved in the biosynthesis of nudicaulins.

  10. Detection of a new piperideine alkaloid in the pygidial glands of some Stenus beetles.

    Science.gov (United States)

    Wittmann, Isabel; Schierling, Andreas; Dettner, Konrad; Göhl, Matthias; Schmidt, Jürgen; Seifert, Karlheinz

    2015-09-01

    Rove beetles of the genus Stenus produce and store bioactive alkaloids like stenusine (3), 3-(2-methylbut-1-enyl)pyridine (4), and cicindeloine (5) in their pygidial glands to protect themselves from predation and microorganismic infestation. The biosynthesis of stenusine (3), 3-(2-methylbut-1-enyl)pyridine (4), and cicindeloine (5) was previously investigated in Stenus bimaculatus, Stenus similis, and Stenus solutus, respectively. The piperideine alkaloid cicindeloine (5) occurs also as a major compound in the pygidial gland secretion of Stenus cicindeloides. The three metabolites follow the same biosynthetic pathway, where the N-heterocyclic ring is derived from L-lysine and the side chain from L-isoleucine. The different alkaloids are finally obtained by few modifications of shared precursor molecules, such as 2,3,4,5-tetrahydro-5-(2-methylbutylidene)pyridine (1). This piperideine alkaloid was synthesized and detected by GC/MS and GC at a chiral phase in the pygidial glands of Stenus similis, Stenus tarsalis, and Stenus cicindeloides.

  11. 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.

  12. Alkaloids with antioxidant activities from Aconitum handelianum.

    Science.gov (United States)

    Yin, Tian-Peng; Cai, Le; Xing, Yun; Yu, Jing; Li, Xue-Jiao; Mei, Rui-Feng; Ding, Zhong-Tao

    2016-06-01

    A new C20-diterpenoid alkaloid handelidine (1) and twenty-seven known alkaloids (2-28) were isolated from the roots of Aconitum handelianum. Their structures were established on the basis of extensive spectroscopic analyses. The study indicated that denudatine-type C20-diterpenoid alkaloids with vicinal-triol system and benzyltetrahydroisoquinoline alkaloids exhibited significant antioxidant activities measured by three antioxidant test systems. The aconitine-type C19-diterpenoid alkaloids could serve as potential secondary antioxidants for their strong binding effects to metal ions.

  13. 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.

  14. Structural and quantitative analysis of Equisetum alkaloids.

    Science.gov (United States)

    Cramer, Luise; Ernst, Ludger; Lubienski, Marcus; Papke, Uli; Schiebel, Hans-Martin; Jerz, Gerold; Beuerle, Till

    2015-08-01

    Equisetum palustre L. is known for its toxicity for livestock. Several studies in the past addressed the isolation and identification of the responsible alkaloids. So far, palustrine (1) and N(5)-formylpalustrine (2) are known alkaloids of E. palustre. A HPLC-ESI-MS/MS method in combination with simple sample work-up was developed to identify and quantitate Equisetum alkaloids. Besides the two known alkaloids six related alkaloids were detected in different Equisetum samples. The structure of the alkaloid palustridiene (3) was derived by comprehensive 1D and 2D NMR experiments. N(5)-Acetylpalustrine (4) was also thoroughly characterized by NMR for the first time. The structure of N(5)-formylpalustridiene (5) is proposed based on mass spectrometry results. Twenty-two E. palustre samples were screened by a HPLC-ESI-MS/MS method after development of a simple sample work-up and in most cases the set of all eight alkaloids were detected in all parts of the plant. A high variability of the alkaloid content and distribution was found depending on plant organ, plant origin and season ranging from 88 to 597mg/kg dried weight. However, palustrine (1) and the alkaloid palustridiene (3) always represented the main alkaloids. For the first time, a comprehensive identification, quantitation and distribution of Equisetum alkaloids was achieved.

  15. 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

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

    Science.gov (United States)

    Schardl, Christopher L; Young, Carolyn A; Hesse, Uljana; Amyotte, Stefan G; Andreeva, Kalina; Calie, Patrick J; Fleetwood, Damien J; Haws, David C; Moore, Neil; Oeser, Birgitt; Panaccione, Daniel G; Schweri, Kathryn K; Voisey, Christine R; Farman, Mark L; Jaromczyk, Jerzy W; Roe, Bruce A; O'Sullivan, Donal M; Scott, Barry; Tudzynski, Paul; An, Zhiqiang; Arnaoudova, Elissaveta G; Bullock, Charles T; Charlton, Nikki D; Chen, Li; Cox, Murray; Dinkins, Randy D; Florea, Simona; Glenn, Anthony E; Gordon, Anna; Güldener, Ulrich; Harris, Daniel R; Hollin, Walter; Jaromczyk, Jolanta; Johnson, Richard D; Khan, Anar K; Leistner, Eckhard; Leuchtmann, Adrian; Li, Chunjie; Liu, JinGe; Liu, Jinze; Liu, Miao; Mace, Wade; Machado, Caroline; Nagabhyru, Padmaja; Pan, Juan; Schmid, Jan; Sugawara, Koya; Steiner, Ulrike; Takach, Johanna E; Tanaka, Eiji; Webb, Jennifer S; Wilson, Ella V; Wiseman, Jennifer L; Yoshida, Ruriko; Zeng, Zheng

    2013-01-01

    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 of the

  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.

  18. 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)

  19. Historical view on ergot alkaloids.

    Science.gov (United States)

    Hofmann, A

    1978-01-01

    A short survey of the history of ergot, of the original and, for a long time, only source of ergot alkaloids, is given. Once a dreaded poison, ergot has changed its role over the centuries to become a rich treasure house of valuable pharmaceuticals. In the Middle Ages it was the cause of epidemics of ergotism, which cost tens of thousands of people their lives. Ergot was first mentioned by the German physician Lonitzer in 1582 as a remedy used by midwives for quickening childbirth. The isolation of pharmacologically useful alkaloids started in 1906 with the discovery of ergotoxine and its adrenolytic activity by Barger, Carr and Dale. In 1918, Stoll isolated ergotamine, the first chemically pure ergot alkaloid, which found widespread therapeutic use in obstetrics and internal medicine. In 1935 the specific oxytocic principle of ergot, ergonovine, was discovered simultaneously in four separate laboratories. Since then, worldwide investigations on ergot alkaloids resulted in the elucidation of their structures and total syntheses and preparation of valuable therapeutics such as Methergine, Hydergine, Dihydergot, and others.

  20. 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.

  1. 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.

  2. [Effects of alkaloids from Coptidis Rhizoma on mouse peritoneal macrophages in vitro].

    Science.gov (United States)

    Zhou, Xia; Peng, Yao-zong; Huang, Tao; Li, Ling; Mou, Shao-xia; Kou, Shu-ming; Li, Xue-gang

    2015-12-01

    This work was mainly studied the effects of the four alkaloids from Coptidis Rhizoma on the mouse peritoneal macrophages in vitro and preliminarily discussed the regulating mechanisms. The effect of alkaloids from Coptidis Rhizoma on the vitality of macrophages was measured by the MTT assay. The effect of alkaloids on the phagocytosis of macrophages was determined by neutral red trial and respiratory burst activity was tested by NBT. The expressions of respiratory-burst-associated genes influenced by alkaloids were detected by qRT-PCR. The conformation change of membrane protein in macrophages by the impact of alkaloids was studied by fluorospectro-photometer. Results showed that the four alkaloids from Coptidis Rhizoma could increase the phagocytosis of macrophages in different level and berberine had the best effect. Berberine, coptisine and palmatine had up-regulation effects on respiratory burst activity of mouse peritoneal macrophages stimulated by PMA and regulatory activity on the mRNA expression of PKC, p40phox or p47phox, whereas the epiberberine had no significant influence on respiratory burst. Moreover, alkaloids from Coptidis Rhizoma could change the conformation of membrane protein and the berberine showed the strongest activity. The results suggested that the four alkaloids from Coptidis Rhizoma might activate macrophages through changing the conformation of membrane protein of macrophages and then enhanced the phagocytosis and respiratory burst activity of macrophages. Furthermore, the regulatory mechanism of alkaloids on the respiratory burst activity of macrophages may be also related to the expression level of PKC, p40phox and p47phox.

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

    Institute of Scientific and Technical Information of China (English)

    余海中; 黄克慧; 汪婉玲; 刘明辉; 杨鑫; 张彦; 徐家萍

    2015-01-01

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

  4. Structure, Biosynthesis, and Occurrence of Bacterial Pyrrolizidine Alkaloids.

    Science.gov (United States)

    Schimming, Olivia; Challinor, Victoria L; Tobias, Nicholas J; Adihou, Hélène; Grün, Peter; Pöschel, Laura; Richter, Christian; Schwalbe, Harald; Bode, Helge B

    2015-10-19

    Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non-ribosomal peptide synthetase. The occurrence of these biosynthesis gene clusters in Gram-negative and Gram-positive bacteria indicates an important biological function in bacteria.

  5. 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.

  6. Comparative analysis of Papaver somniferum genotypes having contrasting latex and alkaloid profiles.

    Science.gov (United States)

    Chaturvedi, Nidarshana; Singh, Mridula; Shukla, Ashutosh K; Shasany, Ajit K; Shanker, Karuna; Lal, Raj K; Khanuja, Suman P S

    2014-07-01

    Papaver somniferum produces therapeutically useful benzylisoquinoline alkaloids (BIAs) like papaverine, thebaine, codeine, and morphine that accumulate in its capsular latex. Morphine is a potent analgesic but is also abused as a narcotic, which has increased the demand for non-narcotic thebaine that can be converted into various analgesics. To curtail the narcotic menace, many distinct genotypes of the plant have been developed that are deficient in morphine and/or latex. Sujata is one such latex-less low alkaloid-producing variety developed from the alkaloid-rich gum harvest variety Sampada. Its utility for gene prospecting and studying differential gene regulation responsible for its low alkaloid, nutritive seed oil, and latex-less phenotype has been exploited in this study. BIA profiling of Sujata and Sampada capsules at the early and late stages indicated that except for thebaine, Sujata had a depressed alkaloid phenotype as compared to Sampada. Comparative transcript-based analysis of the two genotypes was carried out in the early stage capsule (higher thebaine) using subtractive hybridization and microarray. Interrogation of a P. somniferum array yielded many differentially expressing transcripts. Their homology-based annotation classified them into categories--latex related, oil/lipid related, alkaloid related, cell wall related, and others. These leads will be useful to characterize the highly sought after Sujata phenotype.

  7. 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.

  8. Angustilobine and andranginine type indole alkaloids and an uleine-secovallesamine bisindole alkaloid from Alstonia angustiloba.

    Science.gov (United States)

    Ku, Wai-Foong; Tan, Shin-Jowl; Low, Yun-Yee; Komiyama, Kanki; Kam, Toh-Seok

    2011-12-01

    A total of 20 alkaloids were isolated from the leaf and stem-bark extracts of Alstonia angustiloba, of which two are hitherto unknown. One is an alkaloid of the angustilobine type (angustilobine C), while the other is a bisindole alkaloid angustiphylline, derived from the union of uleine and secovallesamine moieties. The structures of these alkaloids were established using NMR and MS analysis. Angustilobine C showed moderate cytotoxicity towards KB cells.

  9. 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)

  10. 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.

  11. Pyrrolizidine alkaloids of senecio sp from Peru

    OpenAIRE

    2011-01-01

    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. lycopers...

  12. Four new fluorenone alkaloids and one new dihydroazafluoranthene alkaloid from Caulophyllum robustum Maxim.

    Science.gov (United States)

    Wang, Xiao-Ling; Liu, Bing-Rui; Chen, Chien-Kuang; Wang, Jun-Ru; Lee, Shoei-Sheng

    2011-09-01

    Four new fluorenone alkaloids, caulophylline A-D (1-4), and one new dihydroazafluoranthene alkaloid, caulophylline E (5) were isolated from the roots of Caulophyllum robustum Maxim. Their structures were elucidated by spectroscopic analysis. Among the isolated alkaloids, Caulophylline E showed good scavenging effects against DPPH radical with IC(50) of 39 μM.

  13. Genetic diversity, phylogeny and evolution of alkaloid sequestering in Cuban miniaturized frogs of the Eleutherodactylus limbatus group.

    Science.gov (United States)

    Rodríguez, Ariel; Poth, Dennis; Schulz, Stefan; Gehara, Marcelo; Vences, Miguel

    2013-09-01

    The miniaturized frogs of the Eleutherodactylus limbatus group, endemic to Cuba, have recently been shown to contain alkaloids in their skin, representing the fifth independent evolutionary origin of alkaloid sequestering in anurans. Based on a comprehensive sampling of all known species of the group we here assess their range-wide phylogeography using DNA sequences of the mitochondrial cytochrome b and the nuclear Rag-1 gene. We found E. etheridgei, E. cubanus, and E. orientalis to be genetically highly distinct, with uncorrected pair-wise distances >12% in the cytochrome b gene and >5% in the 16S rRNA gene, and without haplotype sharing in the Rag-1 gene. On the contrary, E. iberia, E. jaumei, E. limbatus, and one unnamed lineage from Gran Piedra mountains had lower mitochondrial divergences to each other (alkaloids revealed the presence of alkaloids in all lineages of the group, but alkaloids known to be sequestered by other anurans were not found in the basalmost E. etheridgei. Furthermore, individuals of the derived lineages of the E. iberia-jaumei-limbatus clade had a much larger variety of different alkaloid compounds (3-18) than the other three species (0-4). Traces of alkaloids were found in other species only remotely related to the E. limbatus group, i.e., E. caspari, E. goini, E. ricordii, and E. tetajulia. According to these findings, it can be hypothesized that the capacity to sequester dietary alkaloids into the skin evolved first in an ancestral, litter-dwelling and small-sized Cuban Eleutherodactylus, followed by evolution of miniaturization and diurnality in the early representatives of the E. limbatus group, and eventually by increased brightness of color with probable aposematic function and a highly efficient mechanism of alkaloid sequestering.

  14. Pyrrolizidine alkaloids from Onosma erecta.

    Science.gov (United States)

    Damianakos, Harilaos; Sotiroudis, Georgios; Chinou, Ioanna

    2013-10-25

    The MeOH extract of the aerial parts of Onosma erecta afforded four new pyrrolizidine alkaloids, 7-O-acetylechinatine N-oxide (1), a viridinatine N-oxide stereoisomer (2), 7-epi-echimiplatine N-oxide (3), and onosmerectine N-oxide (4), and two additional new natural products, the acid 2,3-dimethyl-2,3,4-trihydroxypentanoic acid (5) and the acyloin 4-methyl-2-hydroxypentanone (6).

  15. 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

  16. 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...

  17. Alkaloids produced by endophytic fungi: a review.

    Science.gov (United States)

    Zhang, Yanyan; Han, Ting; Ming, Qianliang; Wu, Lingshang; Rahman, Khalid; Qin, Luping

    2012-07-01

    In recent years, a number of alkaloids have been discovered from endophytic fungi in plants, which exhibited excellent biological properties such as antimicrobial, insecticidal, cytotoxic, and anticancer activities. This review mainly deals with the research progress on endophytic fungi for producing bioactive alkaloids such as quinoline and isoquinoline, amines and amides, indole derivatives, pyridines, and quinazolines. The biological activities and action mechanisms of these alkaloids from endophytic fungi are also introduced. Furthermore, the relationships between alkaloid-producing endophytes and their host plants, as well as their potential applications in the future are discussed.

  18. Six new alkaloids from Melodinus henryi.

    Science.gov (United States)

    Ma, Ke; Wang, Jun-Song; Luo, Jun; Kong, Ling-Yi

    2015-01-01

    A total of six new alkaloids, melodinhenines A-F (1-6), were isolated from Melodinus henryi. Melodinhenines A and B are new eburnan-vindolinine-type bisindole alkaloids and melodinhenines C-F are new quinolinic melodinus alkaloids. Their structures were elucidated through extensive spectroscopic methods including 2D NMR and HRESIMS analyses. The absolute configuration of 1 and 2 was determined using ECD exciton chirality method. To the best of our knowledge, this is the first report on the determination of the absolute configuration of eburnan-vindolinine-type bisindole alkaloid using this method.

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

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere

    andfeed. Secondary metabolites therefore both have a positive and deleterious impact on the human health.The increase in available genome sequences of fungi has revealed that there is a large number of putativesecondary metabolite biosynthetic gene clusters to be discovered and potentially exploited...... of secondary metabolites and 2) Developing A. nidulans as a model systemfor protein production with human-like glycan structure.  The first part of this study resulted in the development of a method for the transfer and expression ofintact biosynthetic gene clusters to A. nidulans to facilitate pathway...... and product discovery. As proof ofconcept the biosynthetic gene cluster for production of the polyketide geodin was identified andtransferred from A. terreus to A. nidulans. The cluster was integrated in a well characterized locus in A.nidulans. Reconstitution of the cluster resulted in the production...

  20. 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.

  1. 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.

  2. Evolutionary recruitment of a flavin-dependent monooxygenase for stabilization of sequestered pyrrolizidine alkaloids in arctiids.

    Science.gov (United States)

    Langel, Dorothee; Ober, Dietrich

    2011-09-01

    Pyrrolizidine alkaloids are secondary metabolites that are produced by certain plants as a chemical defense against herbivores. They represent a promising system to study the evolution of pathways in plant secondary metabolism. Recently, a specific gene of this pathway has been shown to have originated by duplication of a gene involved in primary metabolism followed by diversification and optimization for its specific function in the defense machinery of these plants. Furthermore, pyrrolizidine alkaloids are one of the best-studied examples of a plant defense system that has been recruited by several insect lineages for their own chemical defense. In each case, this recruitment requires sophisticated mechanisms of adaptations, e.g., efficient excretion, transport, suppression of toxification, or detoxification. In this review, we briefly summarize detoxification mechanism known for pyrrolizidine alkaloids and focus on pyrrolizidine alkaloid N-oxidation as one of the mechanisms allowing insects to accumulate the sequestered toxins in an inactivated protoxic form. Recent research into the evolution of pyrrolizidine alkaloid N-oxygenases of adapted arctiid moths (Lepidoptera) has shown that this enzyme originated by the duplication of a gene encoding a flavin-dependent monooxygenase of unknown function early in the arctiid lineage. The available data suggest several similarities in the molecular evolution of this adaptation strategy of insects to the mechanisms described previously for the evolution of the respective pathway in plants.

  3. 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...... alkaloid in Laureliopsis supports its close relationship to Atherosperma and its taxonomic segregation from Laurelia....

  4. 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.

  5. Plant alkaloids of the polymethyleneamine series

    Energy Technology Data Exchange (ETDEWEB)

    Rogoza, Ludmila N; Salakhutdinov, Nariman F; Tolstikov, Genrikh A [N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk (Russian Federation)

    2005-04-30

    The published data on the structures and biological activities of the plant alkaloids of the biogenic polymethyleneamine series, viz., putrescine (1,4-diaminobutane), spermidine (1,8-diamino-4 -azaoctane), and spermine (1,12-diamino-4,9-diazadodecane), are considered and systematised. The structures and biological activities of some synthetic analogues of these alkaloids are also presented.

  6. Alkaloids of Nelumbo lutea (Wild.) pers. (Nymphaeaceae)

    Science.gov (United States)

    Zelenski, S G

    1977-11-01

    A phytochemical investigation of an alcoholic extract of the petioles of Nelumbo lutea resulted in the identification of the alkaloids N-methylasimilobine, anonaine, and roemerine. The alkaloids nuciferine, armepavine, N-nornuciferine, and N-norarmepavine, previously previously reported in the whole plant, were also identified.

  7. 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

  8. Cytotoxic oxoisoaporphine alkaloids from Menispermum dauricum.

    Science.gov (United States)

    Yu, B W; Meng, L H; Chen, J Y; Zhou, T X; Cheng, K F; Ding, J; Qin, G W

    2001-07-01

    Four new oxoisoaporphine alkaloids, daurioxoisoporphines A-D (1-4), were isolated from the rhizomes of Menispermum dauricum. The structures of these alkaloids were established by spectroscopic methods. The cytotoxic evaluation of 1 and 2 is reported against four cancer cell lines.

  9. 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

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

    Science.gov (United States)

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

    2009-02-01

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

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

    OpenAIRE

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

    2008-01-01

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

  12. Piperidine alkaloids: human and food animal teratogens.

    Science.gov (United States)

    Green, Benedict T; Lee, Stephen T; Panter, Kip E; Brown, David R

    2012-06-01

    Piperidine alkaloids are acutely toxic to adult livestock species and produce musculoskeletal deformities in neonatal animals. These teratogenic effects include multiple congenital contracture (MCC) deformities and cleft palate in cattle, pigs, sheep, and goats. Poisonous plants containing teratogenic piperidine alkaloids include poison hemlock (Conium maculatum), lupine (Lupinus spp.), and tobacco (Nicotiana tabacum) [including wild tree tobacco (Nicotiana glauca)]. There is abundant epidemiological evidence in humans that link maternal tobacco use with a high incidence of oral clefting in newborns; this association may be partly attributable to the presence of piperidine alkaloids in tobacco products. In this review, we summarize the evidence for piperidine alkaloids that act as teratogens in livestock, piperidine alkaloid structure-activity relationships and their potential implications for human health.

  13. Re-investigation of venom chemistry of Solenopsis fire ants. II. Identification of novel alkaloids in S. invicta.

    Science.gov (United States)

    Chen, Li; Fadamiro, Henry Y

    2009-04-01

    The venom of the red imported fire ant, Solenopsis invicta, is dominated by trans stereoisomers of 2,6-dialkylpiperidines. cis Stereoisomers of alkaloids in the venom of S. invicta were separated from trans stereoisomers by using silica gel short column chromatography and identified by coupled gas chromatography mass spectrometry (GC-MS). Seven pairs of cis and trans sterecoisomers were identified based on relative retention times and mass spectral data. The GC trace of the cis stereoisomers of S. invicta alkaloids was presented for the first time. In addition to the previously described 2,6-dialkylpiperideines, eleven novel 2,6-dialkyl-delta1,2-piperideines and 2,6-dialkyl-delta1,6-piperideines were identified from S. invicta venom. The results are discussed in relation to the evolutionary significance of these piperideines and their possible biosynthetic pathways in Solenopsis fire ants.

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

    Science.gov (United States)

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

    2016-11-01

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

  15. 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.

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

    Science.gov (United States)

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

    2014-07-20

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

  17. 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...

  18. Medicinally important secondary metabolites in recombinant microorganisms or plants: progress in alkaloid biosynthesis.

    Science.gov (United States)

    Schäfer, Holger; Wink, Michael

    2009-12-01

    Plants produce a high diversity of natural products or secondary metabolites which are important for the communication of plants with other organisms. A prominent function is the protection against herbivores and/or microbial pathogens. Some natural products are also involved in defence against abiotic stress, e.g. UV-B exposure. Many of the secondary metabolites have interesting biological properties and quite a number are of medicinal importance. Because the production of the valuable natural products, such as the anticancer drugs paclitaxel, vinblastine or camptothecin in plants is a costly process, biotechnological alternatives to produce these alkaloids more economically become increasingly important. This review provides an overview of the state of art to produce alkaloids in recombinant microorganisms, such as bacteria or yeast. Some progress has been made in metabolic engineering usually employing a single recombinant alkaloid gene. More importantly, for benzylisoquinoline, monoterpene indole and diterpene alkaloids (taxanes) as well as some terpenoids and phenolics the proof of concept for production of complex alkaloids in recombinant Escherichia coli and yeast has already been achieved. In a long-term perspective, it will probably be possible to generate gene cassettes for complete pathways, which could then be used for production of valuable natural products in bioreactors or for metabolic engineering of crop plants. This will improve their resistance against herbivores and/or microbial pathogens.

  19. 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.

  20. Genetic variation in alkaloid accumulation in leaves of Nicotiana.

    Science.gov (United States)

    Sun, Bo; Zhang, Fen; Zhou, Guo-jun; Chu, Guo-hai; Huang, Fang-fang; Wang, Qiao-mei; Jin, Li-feng; Lin, Fu-cheng; Yang, Jun

    2013-12-01

    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 all six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were observed both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R(2)=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. Comparative Study of Alkaloid Pattern of Four Bulgarian Fumaria species.

    Science.gov (United States)

    Doncheva, Tsvetelina; Yordanova, Gabriela; Vutov, Vassil; Kostova, Nadezhda; Philipov, Stefan

    2016-02-01

    The alkaloid pattern of four Fumaria species (Fumaria kralikii, Fumaria rostellata, Fumaria schleicherii, Fumaria thureii) growing in Bulgaria was investigated by GC-MS and twenty isoquinoline alkaloids were determined. Phytochemical investigation of the alkaloid composition on Fumaria thuretii Boiss was made for the first time. The alkaloid profile of the species was compared at two levels, between different species and within two species from different habitats. Two chemotypical groups, based on the types of isoquinoline alkaloids were suggested. To group A belong species F. kralikii, F. rostellata (F. r. 1) and F. thuretii containing more than 50% spirobenzylisoquinoline alkaloids of the crude alkaloid mixtures. To group B belong species F. rostellata (F. r. 2) and F. schleicherii containing more than 40% protopine alkaloids and relatively high percentage phthaldeisoquinoline alkaloids (11-19%). In group A phthaldeisoquinoline alkaloids were not detected.

  2. 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

  3. 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.

  4. eSNaPD: a versatile, web-based bioinformatics platform for surveying and mining natural product biosynthetic diversity from metagenomes.

    Science.gov (United States)

    Reddy, Boojala Vijay B; Milshteyn, Aleksandr; Charlop-Powers, Zachary; Brady, Sean F

    2014-08-14

    Environmental Surveyor of Natural Product Diversity (eSNaPD) is a web-based bioinformatics and data aggregation platform that aids in the discovery of gene clusters encoding both novel natural products and new congeners of medicinally relevant natural products using (meta)genomic sequence data. Using PCR-generated sequence tags, the eSNaPD data-analysis pipeline profiles biosynthetic diversity hidden within (meta)genomes by comparing sequence tags to a reference data set of characterized gene clusters. Sample mapping, molecule discovery, library mapping, and new clade visualization modules facilitate the interrogation of large (meta)genomic sequence data sets for diverse downstream analyses, including, but not limited to, the identification of environments rich in untapped biosynthetic diversity, targeted molecule discovery efforts, and chemical ecology studies. eSNaPD is designed to generate a global atlas of biosynthetic diversity that can facilitate a systematic, sequence-based interrogation of nature's biosynthetic potential.

  5. Bromopyrrole Alkaloids from Okinawan Marine Sponges Agelas spp.

    Science.gov (United States)

    Tanaka, Naonobu; Kusama, Taishi; Kashiwada, Yoshiki; Kobayashi, Jun'ichi

    2016-01-01

    In our continuing study for structurally and biogenetically interesting natural products from marine organisms, Okinawan marine sponges Agelas spp. were investigated, resulting in the isolation of 18 unique alkaloids including five dimeric bromopyrrole alkaloids (1-5), ten monomeric bromopyrrole alkaloids (6-15), and three conjugates of monomeric bromopyrrole alkaloid and hydroxykynurenine (16-18). In this mini-review, the isolation, structure elucidation, and antimicrobial activities of these alkaloids are summarized.

  6. 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.

  7. 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 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.

  8. 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

  9. 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.

  10. Polycyclic Guanidine Alkaloids from Poecilosclerida Marine Sponges.

    Science.gov (United States)

    Sfecci, Estelle; Lacour, Thierry; Amade, Philippe; Mehiri, Mohamed

    2016-04-09

    Sessile marine sponges provide an abundance of unique and diversified scaffolds. In particular, marine guanidine alkaloids display a very wide range of biological applications. A large number of cyclic guanidine alkaloids, including crambines, crambescins, crambescidins, batzelladines or netamins have been isolated from Poecilosclerida marine sponges. In this review, we will explore the chemodiversity of tri- and pentacyclic guanidine alkaloids. NMR and MS data tools will also be provided, and an overview of the wide range of bioactivities of crambescidins and batzelladines derivatives will be given.

  11. Marine Pyridoacridine Alkaloids: Biosynthesis and Biological Activities.

    Science.gov (United States)

    Ibrahim, Sabrin R M; Mohamed, Gamal A

    2016-01-01

    Pyridoacridines are a class of strictly marine-derived alkaloids that constitute one of the largest chemical families of marine alkaloids. During the last few years, both natural pyridoacridines and their analogues have constituted excellent targets for synthetic works. They have been the subject of intense study due to their significant biological activities; cytotoxic, antibacterial, antifungal, antiviral, insecticidal, anti-HIV, and anti-parasitic activities. In the present review, 95 pyridoacridine alkaloids isolated from marine organisms are discussed in term of their occurrence, biosynthesis, biological activities, and structural assignment.

  12. Isoquinoline and isoindole alkaloids from Menispermum dauricum.

    Science.gov (United States)

    Zhang, Xiaoqi; Ye, Wencai; Zhao, Shouxun; Che, Chun-Tao

    2004-04-01

    Three isoquinoline alkaloids and an isoindole alkaloid, along with eight known compounds, were isolated from the roots of Menispermum dauricum (Menispermacese). The alkaloids were characterized as 7-hydroxy-6-methoxy-1(2H)-isoquinolinone, 6,7-dimethoxy-N-methyl-3,4-dioxo-1(2H)-isoquinolinone, 1-(4-hydroxybenzoyl)-7-hydroxy-6-methoxy-isoquinoline and 6-hydroxy-5-methoxy-N-methylphthalimide, on the basis of spectral evidence including 1D- and 2D-NMR and MS analyses.

  13. 反义托品酮还原酶Ⅱ基因对三分三的遗传转化及生物碱含量测定%Genetic Transformation of Antisense Tropinone Reductase Ⅱ Gene into Anisodus acutangulus and Alkaloid Content Determination

    Institute of Scientific and Technical Information of China (English)

    彭静叶; 彭梅芳

    2011-01-01

    [ Objective ] The research aimed to increase the contents of hyoscyamine in Anisodus acutangulus by using the antisense tropinone reductase Ⅱ gene (ant-TRⅡ) ,to improve the expression level of tropinone reductase I (TRI), which provided the theory basis for the largescale development of tropane alkaloids. [ Method] The recombinant bacteria C58C1-p1304+ -antTRⅡ which carried ant-TR Ⅱ gene was transformed into Anisodus acutangulus by using the leaf disc method. The hygromycin was used to screen the transgenic hairy root. PCR was used to detect if the genome of transgenic hairy root simultaneously contained rolB, rolC and Hygr resistance genes. After PCR positive transgenic hairy root was screened to culture, the alkaloid was extracted, and HPLC was used to measure the alkaloids content. [ Result] rolB, rolC and Hygr resistance genes in the genomes of nine transgenic hairy roots were simultaneously detected. The hyoscyamine content in the nine transgenic hairy roots had the different degrees of increase when compared with the control. [ Conclusion] ant-TR Ⅱ gene had the certain positive regulation effect on the metabolic pathway of tropane alkaloids.%[目的]通过反义托品酮还原酶Ⅱ基因提高托品酮还原酶I(TRI)的表达水平,从而增加莨菪碱在三分三中的含量,为大规模开发生产托品烷生物碱提供理论基础.[方法]将携带反义托品酮还原酶Ⅱ基因的重组工程菌C58C1-p1304-anTRII采用叶盘法对三分三进行遗传转化.潮霉素筛选转基因发根,PCR检测转基因发根基因组中是否同时含有rolB、rolC和Hygr抗性基因.PCR阳性转基因发根扩大培养后,提取生物碱,并采用高效液相色谱法进行生物碱含量测定.[结果]共有9个转基因发根的基因组同时检测到rolB、rolC和Hygr抗性基因,且这9个转基因发根中的莨菪碱含量较对照有不同程度的提高.[结论]反义托品酮还原酶Ⅱ基因对托品烷生物碱代谢途径起到一定程度的正调控作用.

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

    Science.gov (United States)

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

    2011-11-01

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

  15. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Elena Fossati

    Full Text Available Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S-reticuline starting from (R,S-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS, salutaridine reductase (PsSAR and salutaridinol acetyltransferase (PsSAT were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R-reticuline. Yeast cell feeding assays using (R-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes.

  16. 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.

  17. 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.

  18. Steroidal alkaloid toxicity to fish embryos.

    Science.gov (United States)

    Crawford, L; Kocan, R M

    1993-02-01

    Embryos of two species of fish were evaluated for their suitability as model systems for steroidal alkaloid toxicity, the Japanese rice fish, medaka (Oryzius latipes) and the rainbow trout (Oncorhynchus mykiss). Additionally, the equine neurotoxic sesquiterpene lactone repin, was also tested. A PROBIT program was used to evaluate the EC1, EC50 and EC99 as well as the associated confidence limits. The steroidal alkaloids tested were the Solanum potato glycoalkaloids alpha-chaconine, alpha-solanine, the aglyclones solanidine and solasodine and the Veratrum alkaloid, jervine. Embryo mortality, likely due to structural or functional abnormalities in the early development stages of the embryo, were the only response observed in both species. The rainbow trout exhibited a toxic response to chaconine, solasidine, repin and solanine but the medaka embryos were only affected by the compounds, chaconine and solanine. Rainbow trout may indeed serve as a good lower vertebrate model for studying the toxicity of steroidal alkaloids.

  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. 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....

  1. 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.

  2. 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.

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

    Science.gov (United States)

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

    2007-10-01

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

  4. 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

  5. Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae.

    Science.gov (United States)

    Jirschitzka, Jan; Schmidt, Gregor W; Reichelt, Michael; Schneider, Bernd; Gershenzon, Jonathan; D'Auria, John Charles

    2012-06-26

    The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as atropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-tropinone) stereospecifically to 2-carbomethoxy-3β-tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms.

  6. 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.

  7. An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole.

    Science.gov (United States)

    Payne, Richard M E; Xu, Deyang; Foureau, Emilien; Teto Carqueijeiro, Marta Ines Soares; Oudin, Audrey; Bernonville, Thomas Dugé de; Novak, Vlastimil; Burow, Meike; Olsen, Carl-Erik; Jones, D Marc; Tatsis, Evangelos C; Pendle, Ali; Ann Halkier, Barbara; Geu-Flores, Fernando; Courdavault, Vincent; Nour-Eldin, Hussam Hassan; O'Connor, Sarah E

    2017-01-13

    Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

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

    Institute of Scientific and Technical Information of China (English)

    李利; 陈莎; 陈福生; 高梦祥

    2013-01-01

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

  9. 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.

  10. Pyrimidine biosynthetic pathway of Baccillus subtilis.

    Science.gov (United States)

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

    1975-08-01

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

  11. 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.

  12. 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.

  13. 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].

  14. An Acetylenic Alkaloid from the Calcareous Sponge Leucetta sp.

    OpenAIRE

    Nicole J de Voogd; Idam Hermawan; Junichi Tanaka

    2011-01-01

    A new acetylenic alkaloid was isolated from the sponge Leucetta sp. The structure was established by analyzing spectroscopic data. The alkaloid showed cytotoxicity IC50 2.5 mg/mL against NBT-T2 cells.

  15. [Study on optimum extraction conditions of alkaloids from Pinellia ternate].

    Science.gov (United States)

    Zeng, Jianhong; Peng, Zhengsong; Mao, Zicheng; Wei, Shuhong

    2003-05-01

    The optimum extraction conditions of alkaloids from Pinellia ternate (Thunb.) Breit were studied by orthogonal test. The results showed that the highest extraction rate of the alkaloids could be obtained by smashing the material in 60 (sieve number) of fragmentation and socking the material in 2.575 mol/L ammonia water, extracting alkaloids with 18 times as much chlorolform at room temperature for 25 hours. The highest extraction rate of alkaloids was 0.0817%.

  16. Simulation of the type of coralin alkaloid-DNA binding

    Science.gov (United States)

    Kulikov, K. G.; Koshlan, T. V.

    2015-05-01

    Interaction between a synthesized coralin protoberberine alkaloid and the DNA double helix of the calf's thymus in a salt solution is studied by optical absorption spectroscopy and spectropolarimetry. The dependence of the spectral characteristics of the alkaloid on a ratio between the DNA base pair concentration and the alkaloid molecule concentration is considered. The parameters of bonds between the coralin alkaloid and the DNA double helix are determined using modified McGhee-von Hippel equations.

  17. TOXIC PYRROLIZIDINE ALKALOIDS OF ECHIUM AMOENUM FISCH. & MEY.

    OpenAIRE

    2006-01-01

    Toxic pyrrolizidine alkaloids are present in some species of Echium (Boraginaceae). In this study petals of Echium amoenum Fisch. & Mey. (Gol-e-Gavzaban) as a popular herbal medicine in Iran, were investigated for pyrrolizidine alkaloids (PAs). The alkaloids were separated and purified by preparative TLC and characterized by IR, one and two dimensional 1H and 13C-NMR and Mass spectroscopy. Four toxic alkaloids namely: echimidine I, echimidine isomer II, 7-angeloyl retronecine III and 7-ti...

  18. The alkaloids of the madangamine group.

    Science.gov (United States)

    Amat, Mercedes; Pérez, Maria; Ballette, Roberto; Proto, Stefano; Bosch, Joan

    2015-01-01

    This chapter is focused on madangamines, a small group of complex diamine alkaloids isolated from marine sponges of the order Haplosclerida, and covers their isolation, characterization, biogenesis, biological activity, and synthesis. Structurally, madangamines are pentacyclic alkaloids with an unprecedented skeletal type, characterized by a common diazatricyclic core and two peripheral macrocyclic rings. The isolation of these alkaloids from Xestospongia ingens (madangamines A-E) and Pachychalina alcaloidifera (madangamine F) is described in detail. Physical and complete spectroscopic 1H and 13C NMR data are included. The proposed biogenesis of madangamines from ammonia, a functionalized three-carbon unit, and saturated or unsaturated linear long-chain dialdehydes, via partially reduced bis-alkylpyridine macrocycles, is discussed. The synthesis of alkaloids of the madangamine group has been little explored, with only one total synthesis reported so far, that of (+)-madangamine D. This review also describes several model synthetic approaches to the diazatricyclic ABC core of these alkaloids, as well as model studies on the construction of the (Z,Z)-unsaturated 11-membered E macrocycle common to madangamines A-E, the 13- and 14-membered D rings of madangamines C-E, and the all-cis-triunsaturated 15-membered D ring of madangamine A. Some members of this group have shown significant in vitro cytotoxicity against a number of cancer cell lines.

  19. 21 CFR 119.1 - Dietary supplements containing ephedrine alkaloids.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 2 2010-04-01 2010-04-01 false Dietary supplements containing ephedrine alkaloids... UNREASONABLE RISK § 119.1 Dietary supplements containing ephedrine alkaloids. Dietary supplements containing ephedrine alkaloids present an unreasonable risk of illness or injury under conditions of use recommended...

  20. An Unusual Pentacyclic Dinitrogenous Alkaloid from Galanthus gracilis

    OpenAIRE

    ÜNVER, Nehir; KAYA, G. İrem

    2005-01-01

    A minor alkaloid, namely gracilamine, was isolated from Galanthus gracilis. This was the first example of a pentacyclic dinitrogenous alkaloid isolated from a member of Amaryllidaceae. The structure of this alkaloid was elucidated by means of comprehensive spectroscopic methods (1D and 2D NMR, MS, UV, IR).

  1. Characterization of virginiamycin S biosynthetic genes from Streptomyces virginiae.

    Science.gov (United States)

    Namwat, Wises; Kamioka, Yuji; Kinoshita, Hiroshi; Yamada, Yasuhiro; Nihira, Takuya

    2002-03-20

    Streptomyces virginiae produces -butyrolactone autoregulators (virginiae butanolide, VB), which control the biosynthesis of virginiamycin M1 and S. A 6.3-kb region downstream of the virginiamycin S (VS)-resistance operon in S. virginiae was sequenced, and four plausible open reading frames (ORFs) (visA, 1,260 bp; visB, 1,656 bp; visC, 888 bp; visD, 1209 bp) were identified. Homology analysis revealed significant similarities with enzymes involved in the biosynthesis of cyclopeptolide antibiotics: VisA (53% identity, 65% similarity) to -lysine 2-aminotransferase (NikC) of nikkomycin D biosynthesis, VisB (66% identity, 72% similarity) to 3-hydroxypicolinic acid:AMP ligase of pristinamycin I biosynthesis, VisC (48% identity, 59% similarity) to lysine cyclodeaminase of ascomycin biosynthesis, and VisD (43% identity, 56% similarity) to erythromycin C-22 hydroxylase of erythromycin biosynthesis. Northern blotting as well as high-resolution S1 analysis of the ORFs revealed that they were transcribed as two bicistronic transcripts, namely 3.0-kb visB-visA and another 2.7-kb visC-visD transcript, with promoters locating upstream of visB and visC, respectively. Transcription of the two operons was observed only 1 h after the VB production, which was 2 h before the virginiamycin production. Furthermore, prompt induction of the transcription was observed as a result of external VB addition, suggesting that the expression of the two operons was under the control of VB.

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

    Science.gov (United States)

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

    2003-07-01

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

  3. 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.

  4. 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)

  5. 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.

  6. 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.

  7. 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.

  8. 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)

  9. Chemotaxonomy and geographical distribution of tropane alkaloids.

    Science.gov (United States)

    Griffin, W J; Lin, G D

    2000-03-01

    This review illustrates the distribution of tropane alkaloids within the families Solanaceae, Erythroxylaceae, Proteaceae, Euphorbiaceae, Rhizophoraceae, Convolvulaceae and Cruciferae. Whereas tropane alkaloids are characteristic of the genera Datura, Brugmansia (tree datura) and Duboisia of the Solanaceae, the distribution is more widespread with novel tropane derivatives in families not traditionally associated with these bases. The chemical nature of more recently discovered water-soluble calystegines and the di- and trimeric forms from the Convolvulaceae (e.g. schizanthines from Schizanthus spp.), truxillines from Bolivian coca leaves and moonines of Erythroxylum moonii are highlighted. Where possible and appropriate, links between the phytochemistry and taxonomy are discussed.

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

    Institute of Scientific and Technical Information of China (English)

    张长青; 丁元亮

    2016-01-01

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

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

    DEFF Research Database (Denmark)

    Ono, Hajime; Rewitz, Kim; Shinoda, Tetsu

    2006-01-01

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

  12. Alkaloid production by callous tissue cultures of Cereus peruvianus (Cactaceae).

    Science.gov (United States)

    de Oliveira, Arildo José Braz; Machado, Maria Fátima Pires da Silva

    2003-02-01

    The morphologically undifferentiated cells of nonregenerant callous tissue of Cereus peruvianus cultured in the original medium and in medium supplemented with tyrosine were used as an alkaloid source. Comparison of alkaloid production by C. peruvianus plants and by callous tissues indicated that alkaloid levels were almost twice as high in callous tissues as in shoots of C. peruvianus plants. The ratio of alkaloid concentration between mature plant and morphologically undifferentiated cells of callous tissue was 1:1.7. A relationship between culture medium containing tyrosine and alkaloid production was also observed in the callous tissues of C. peruvianus. Since increased alkaloid production may be induced by additional factors such as tyrosine, increasing levels of tyrosine or other conditions of the culture medium may be considered factors for inducing higher alkaloid production by C. peruvianus callous tissues.

  13. Therapeutic Potential of Steroidal Alkaloids in Cancer and Other Diseases.

    Science.gov (United States)

    Jiang, Qi-Wei; Chen, Mei-Wan; Cheng, Ke-Jun; Yu, Pei-Zhong; Wei, Xing; Shi, Zhi

    2016-01-01

    Steroidal alkaloids are a class of secondary metabolites isolated from plants, amphibians, and marine invertebrates. Evidence accumulated in the recent two decades demonstrates that steroidal alkaloids have a wide range of bioactivities including anticancer, antimicrobial, anti-inflammatory, antinociceptive, etc., suggesting their great potential for application. It is therefore necessary to comprehensively summarize the bioactivities, especially anticancer activities and mechanisms of steroidal alkaloids. Here we systematically highlight the anticancer profiles both in vitro and in vivo of steroidal alkaloids such as dendrogenin, solanidine, solasodine, tomatidine, cyclopamine, and their derivatives. Furthermore, other bioactivities of steroidal alkaloids are also discussed. The integrated molecular mechanisms in this review can increase our understanding on the utilization of steroidal alkaloids and contribute to the development of new drug candidates. Although the therapeutic potentials of steroidal alkaloids look promising in the preclinical and clinical studies, further pharmacokinetic and clinical studies are mandated to define their efficacy and safety in cancer and other diseases.

  14. 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.

  15. Honatisine, a novel diterpenoid alkaloid, and six known alkaloids from Delphinium honanense and their cytotoxic activity.

    Science.gov (United States)

    He, Yang Qing; Ma, Zhan Ying; Wei, Xiao Mei; Liu, Dong Jie; Du, Bao Zhong; Yao, Bing Hua; Gao, Li Ming

    2011-11-01

    A novel diterpene alkaloid named honatisine (1) has been isolated from the whole plants of Delphinium honanense, along with six known alkaloids, siwanine E (2), isoatisine (3), atisine (4), delcorinine (5), uraphine (6), and nordhagenine A (7). Their structures were deduced on the basis of their spectral data. All of them were evaluated by a SRB assay for their cytotoxicity, and compound 1 showed a significant cytotoxic activity (IC(50) =3.16 μM) against the MCF-7 cell line.

  16. Localization of ergot alkaloids in sclerotia of Claviceps purpurea by matrix-assisted laser desorption/ionization mass spectrometry imaging.

    Science.gov (United States)

    Dopstadt, Julian; Vens-Cappell, Simeon; Neubauer, Lisa; Tudzynski, Paul; Cramer, Benedikt; Dreisewerd, Klaus; Humpf, Hans-Ulrich

    2017-02-01

    The fungus Claviceps purpurea produces highly toxic ergot alkaloids and accumulates these in the hardened bodies of fungal mycelium. These so-called sclerotia, or ergot bodies, replace the crop seed of infected plants, which can include numerous important food- and feedstuff such as rye and wheat. While several studies have explored details of the infection process and development of ergot bodies, little information is available on the spatial distribution of the mycotoxins in the sclerotia. Here we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at a lateral resolution of 35 μm to visualize the distribution of two representative alkaloids, ergocristine and ergometrine, produced by Ecc93 and Gal 310 variants of C. purpurea, respectively, after infection of rye. To improve cryosectioning of this fragile biological material tissue with complex texture, we developed a practical embedding protocol based on cellulose polymers. The MALDI-MS images recorded from the so produced intact tissues sections revealed that ergometrine exhibited a relatively homogeneous distribution throughout the ergot body, whereas ergocristine was found to be enriched in the proximal region. This finding can be correlated to the morphological development of sclerotia as ergot alkaloids are only produced in the sphacelial stage. The ability to localize toxins and other secondary metabolites in intact sections of crop-infecting fungi with high lateral resolution renders MALDI-MSI a powerful tool for investigating biosynthetic pathways and for obtaining a deeper understanding of the parasite-host interaction. Graphical abstract Workflow for identification and spatial localization of ergot alkaloids in infected rye grains.

  17. 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

  18. 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...

  19. 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.

  20. Dehydropyrrolizidine alkaloid toxicity, cytotoxicity, and carcinogenicity

    Science.gov (United States)

    Dehyro-pyrrolizidine alkaloid (PA)-containing plants compose about 5% of the world’s flowering plants and they commonly poison livestock, wildlife and humans. Previous work has produced considerable understanding of PA toxicity, species susceptibility, conditions and routes of exposure, toxin metab...

  1. A New Alkaloid from Isatis costata

    OpenAIRE

    Fatima, Itrat; Anis, Itrat; Ahmad, Ijaz; Malik, Abdul; Afza, Nighat

    2014-01-01

    A new alkaloid has been isolated from Isatis costata C.A.Mey. and assigned structure 1 on the basis of spectroscopic data including 1D and 2D NMR techniques. Methyl 2-acetoamidobenzoate (2), b-sitosterol (3), and ursolic acid (4) were also isolated for the first time from this species.

  2. A Novel Alkaloid from Stapelia hirsuta

    OpenAIRE

    SHABANA, Marwan; GONAID, Mariam; SALAMA, Maha Mahmoud; ABDEL-SATTAR, Essam

    2006-01-01

    A novel alkaloid (1,8,8-trimethyl-5,8-dihydro-1H-pyrano[3,4-b]pyridine-4,6-dione) was isolated from the chloroformic traction obtained from the total alcoholic extract of the aerial parts of Stapelia hirsuta L. In addition, apigenin, luteolin and b-sitosterol-3-O-b-D- glucopyranoside were also isolated.

  3. Alkaloids from Oriciopsis glaberrima Engl. (Rutaceae).

    Science.gov (United States)

    Wansi, Jean Duplex; Wandji, Jean; Kamdem Waffo, Alain François; Ngeufa, Happi Emmanuel; Ndom, Jean Claude; Fotso, Serge; Maskey, Rajendra Prasad; Njamen, Dieudonné; Fomum, Tanee Zacharias; Laatsch, Harmut

    2006-03-01

    Two alkaloid derivatives, oriciacridone A and B, were isolated from the stem bark of Oriciopsis glaberrima (Rutaceae). The structures were elucidated by a detailed spectroscopic analysis. The extract exhibited in vitro significant antimicrobial activity against a range of micro-organisms.

  4. Ergot alkaloids decrease rumen epithelial blood flow

    Science.gov (United States)

    Two experiments were conducted to determine if ergot alkaloids affect blood flow to the absorptive surface of the rumen of steers. Steers (n=8 total) were pair-fed alfalfa cubes at 1.5× NEM and received ground endophyte-infected tall fescue seed (E+) or endophyte-free tall fescue seed (E-) via rumen...

  5. Eremophilanes and Pyrrolizidine Alkaloids of Senecioneae Species

    OpenAIRE

    2014-01-01

    The chemical study of two species of the tribe Senecioneae afforded two eremophilanes and two pyrrolizidine alkaloids from Se - necio subauriculatus and four modified eremophilanes from Roldana oaxacana . The chemistry of these species is in accord with that re - ported for species of Senecio and Roldana studied so far, and therefore, with the already described for the tribe Senecioneae.

  6. 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.

  7. 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.

  8. 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

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

    Science.gov (United States)

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

    2016-03-21

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

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Carbazole alkaloids from Murraya koenigii trigger apoptosis and autophagic flux inhibition in human oral squamous cell carcinoma cells.

    Science.gov (United States)

    Utaipan, Tanyarath; Athipornchai, Anan; Suksamrarn, Apichart; Jirachotikoon, Canussanun; Yuan, Xiaohong; Lertcanawanichakul, Monthon; Chunglok, Warangkana

    2017-01-01

    Carbazole alkaloids, a major constituent of Murraya koenigii (L.) Sprengel (Rutaceae), exhibit biological effects such as anticancer activity via the induction of apoptosis, and they represent candidate chemotherapeutic agents. Oral squamous cell carcinoma (OSCC) is the most prevalent cancer of the oral cavity and a growing and serious health problem worldwide. In this study, we investigated the anticancer properties and mechanisms of action of two carbazole alkaloids derived from M. koenigii leaves, mahanine and isomahanine, in the OSCC cell line CLS-354. At 15 μM, mahanine and isomahanine were cytotoxic to CLS-354 cells, triggering apoptosis via caspase-dependent and -independent mechanisms. Autophagosomes, visualised using monodansylcadaverine (MDC) labelling, were numerous in carbazole alkaloid-treated cells. Mahanine and isomahanine markedly induced the expression of the autophagosome marker microtubule-associated protein 1 light chain 3, type II (LC3B-II). Genetic and chemical inhibition of autophagy via silencing of the Autophagy protein 5 gene and exposure to bafilomycin A1 (BafA1), respectively, did not arrest carbazole alkaloid-induced apoptosis, indicating that it occurs independently of autophagic activation. Surprisingly, both carbazole alkaloids caused increased accumulation of p62/sequestosome1 (p62/SQSTM1), with coordinated expression of LC3B-II and cleaved caspase-3, suggesting inhibition of autophagic flux. Our results suggest that inhibition of autophagic flux is associated with carbazole alkaloid-induced apoptosis. Our findings provide evidence of a novel cytotoxic action of natural carbazole alkaloids and support their use as candidate chemotherapeutic agents for the treatment of OSCC.

  12. Insecticidal Constituents and Activity of Alkaloids from Cynanchum mongolicum.

    Science.gov (United States)

    Ge, Yang; Liu, Pingping; Yang, Rui; Zhang, Liu; Chen, Hongxing; Camara, Ibrahima; Liu, Yiqing; Shi, Wangpeng

    2015-09-21

    Based on MS and NMR data and bioassay-guided tracing, three insecticidal alkaloids I, II and III from Cynanchum mongolicum were identified to be antofine N-oxide, antofine and tylophorine. Alkaloid I was more toxic than alkaloids II and III, but they were less active against Spodoptera litura than total alkaloids. The contact toxicity from these alkaloids against the aphid Lipaphis erysimi was significant, as the 24 h-LC50 values of alkaloids I, II, III and total alkaloids were 292.48, 367.21, 487.791 and 163.52 mg/L, respectively. The development disruption of S. litura larvae was tested, the pupation and emergence rates of S. litura decreased and the acute mortality of S. litura increased significantly by day 3 after being injected in their body cavity with 10-40 mg/L of total alkaloid. The ecdysone titer of treated S. litura larvae and prepupae declined with increasing alkaloid concentration. The alkaloids of Cynanchum mongolicum are potential insect growth inhibitors.

  13. Alkaloids modulate motility, biofilm formation and antibiotic susceptibility of uropathogenic Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Devendra H Dusane

    Full Text Available Alkaloid-containing natural compounds have shown promise in the treatment of microbial infections. However, practical application of many of these compounds is pending a mechanistic understanding of their mode of action. We investigated the effect of two alkaloids, piperine (found in black pepper and reserpine (found in Indian snakeroot, on the ability of the uropathogenic bacterium Escherichia coli CFT073 to colonize abiotic surfaces. Sub-inhibitory concentrations of both compounds (0.5 to 10 µg/mL decreased bacterial swarming and swimming motilities and increased biofilm formation. qRT-PCR revealed a decrease in the expression of the flagellar gene (fliC and motility genes (motA and motB along with an increased expression of adhesin genes (fimA, papA, uvrY. Interestingly, piperine increased penetration of the antibiotics ciprofloxacin and azithromycin into E. coli CFT073 biofilms and consequently enhanced the ability of these antibiotics to disperse pre-established biofilms. The findings suggest that these alkaloids can potentially affect bacterial colonization by hampering bacterial motility and may aid in the treatment of infection by increasing antibiotic penetration in biofilms.

  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. 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.

  17. 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.

  18. 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.

  19. Recent developments in the chemistry of quinazolinone alkaloids.

    Science.gov (United States)

    Kshirsagar, U A

    2015-09-28

    Quinazolinones, an important class of fused heterocyclic alkaloids has attracted high attention in organic and medicinal chemistry due to their significant and wide range of biological activities. There are approximately 150 naturally occurring quinazolinone alkaloids known till 2005. Several new quinazolinone alkaloids (∼55) have been isolated in the last decade. Natural quinazolinones with exotic structural features and remarkable biological activities have incited a lot of activities in the synthetic community towards the development of new synthetic strategies and approaches for the total synthesis of quinazolinone alkaloids. This review is focused on these advances in the chemistry of quinazolinone alkaloids in the last decade. This article covers the newly isolated quinazolinone natural products with their biological activities and the recently reported total syntheses of quinazolinone alkaloids from 2006 to 2015.

  20. 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.

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

    Science.gov (United States)

    Diaz, Gonzalo J.

    2015-01-01

    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. PMID:26690479

  2. 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.

  3. Chlorinated alkaloids in Menispermum dauricum DC: root culture.

    Science.gov (United States)

    Sugimoto, Y; Babiker, H A; Saisho, T; Furumoto, T; Inanaga, S; Kato, M

    2001-05-18

    Feeding experiments using (36)Cl showed that Menispermum dauricum root culture produces four alkaloids containing chlorine. They included the novel alkaloids dauricumine and dauricumidine as well as the known alkaloids acutumine and acutumidine. The structures of novel alkaloids were established by spectroscopic, crystallographic, and chemical methods. These four alkaloids were labeled with (36)Cl, isolated, and fed independently to root cultures. Mutual conversion between acutumine and acutumidine, and between dauricumine and dauricumidine by N-methylation and N-demethylation, was demonstrated. Moreover, dauricumine was converted to acutumine and acutumidine. Epimerization of acutumidine to dauricumidine or vice versa was not observed. These results suggest that dauricumine is the first chlorinated alkaloid formed in cultured M. dauricum roots. Skewed distribution of radioactivity derived from labeled dauricumine is proof that epimerization at C-1 proceeds at a lower rate than N-demethylation.

  4. 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.

  5. 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.

  6. 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.

  7. Total synthesis of the Daphniphyllum alkaloid daphenylline

    Science.gov (United States)

    Lu, Zhaoyong; Li, Yong; Deng, Jun; Li, Ang

    2013-08-01

    The Daphniphyllum alkaloids are a large class of natural products isolated from a genus of evergreen plants widely used in Chinese herbal medicine. They display a remarkable range of biological activities, including anticancer, antioxidant, and vasorelaxation properties as well as elevation of nerve growth factor. Daphenylline is a structurally unique member among the predominately aliphatic Daphniphyllum alkaloids, and contains a tetrasubstituted arene moiety mounted on a sterically compact hexacyclic scaffold. Herein, we describe the first total synthesis of daphenylline. A gold-catalysed 6-exo-dig cyclization reaction and a subsequent intramolecular Michael addition reaction, inspired by Dixon's seminal work, were exploited to construct the bridged 6,6,5-tricyclic motif of the natural product at an early stage, and the aromatic moiety was forged through a photoinduced olefin isomerization/6π-electrocyclization cascade followed by an oxidative aromatization process.

  8. 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.

  9. Neurotoxic Alkaloids: Saxitoxin and Its Analogs

    OpenAIRE

    Mihali, Troco K; Moffitt, Michelle C.; Neilan, Brett A.; Maria Wiese; D’Agostino, Paul M.

    2010-01-01

    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...

  10. 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)

  11. Pyrrolizidine Alkaloids from Onosmakaheirei Teppner (Boraginaceae

    Directory of Open Access Journals (Sweden)

    Ioanna Maria Orfanou

    2016-03-01

    Full Text Available The new pyrrolizidine alkaloid (PA 3΄-O-acetylechinatine N-oxide (7, along with two more known PAs (5, 6, two known flavonoids (3, 4, one known alkannin (1, two known triterpenoids, one known sterol, and allantoin (2 were isolated from the aerial parts of Onosma kaheirei. In addition, the retention indeces of the reduced PAs 6 and 7 were determined in a DB-5 WCOT column, to aid their detection by GC/MS in the future.

  12. 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

  13. Plant alkaloids as drug leads for Alzheimer's disease.

    Science.gov (United States)

    Ng, Yu Pong; Or, Terry Cho Tsun; Ip, Nancy Y

    2015-10-01

    Alzheimer's disease (AD) is a neurodegenerative illness associated with dementia and is most prevalent among the elderly population. Current medications can only treat symptoms. Alkaloids are structurally diverse and have been an important source of therapeutics for various brain disorders. Two US Food and Drug Administration (FDA)-approved acetylcholinesterase inhibitors for AD, galantamine and rivastigmine, are in fact alkaloids. In addition, clinical trials of four other extensively studied alkaloids-huperzine A, caffeine, nicotine, and indomethacin-have been conducted but do not convincingly demonstrate their clinical efficacy for AD. Interestingly, rhynchophylline, a known neuroprotective alkaloid, was recently discovered by in silico screening as an inhibitor of EphA4, a novel target for AD. Here, we review the pathophysiological mechanisms underlying AD, current treatment strategies, and therapeutic potential of several selected plant alkaloids in AD, highlighting their various drug targets and the key supportive preclinical and clinical studies. Future research should include more rigorous clinical studies of the most promising alkaloids, the further development of recently discovered candidate alkaloids, and the continual search for new alkaloids for relevant drug targets. It remains promising that an alkaloid drug candidate could significantly affect the progression of AD in addition to providing symptomatic relief.

  14. New Perspectives in the Chemistry of Marine Pyridoacridine Alkaloids.

    Science.gov (United States)

    Plodek, Alois; Bracher, Franz

    2016-01-26

    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.

  15. TOXIC PYRROLIZIDINE ALKALOIDS OF ECHIUM AMOENUM FISCH. & MEY.

    Directory of Open Access Journals (Sweden)

    MITRA MEHRABANI

    2006-06-01

    Full Text Available Toxic pyrrolizidine alkaloids are present in some species of Echium (Boraginaceae. In this study petals of Echium amoenum Fisch. & Mey. (Gol-e-Gavzaban as a popular herbal medicine in Iran, were investigated for pyrrolizidine alkaloids (PAs. The alkaloids were separated and purified by preparative TLC and characterized by IR, one and two dimensional 1H and 13C-NMR and Mass spectroscopy. Four toxic alkaloids namely: echimidine I, echimidine isomer II, 7-angeloyl retronecine III and 7-tigloyl retronecine IV were identified.

  16. Binding of a biosynthetic intermediate to AtrA modulates the production of lidamycin by Streptomyces globisporus.

    Science.gov (United States)

    Li, Xingxing; Yu, Tengfei; He, Qing; McDowall, Kenneth J; Jiang, Bingya; Jiang, Zhibo; Wu, Linzhuan; Li, Guangwei; Li, Qinglian; Wang, Songmei; Shi, Yuanyuan; Wang, Lifei; Hong, Bin

    2015-06-01

    The control of secondary production in streptomycetes involves the funneling of environmental and physiological signals to the cluster-situated (transcriptional) regulators (CSRs) of the biosynthetic genes. For some systems, the binding of biosynthetic products to the CSR has been shown to provide negative feedback. Here we show for the production of lidamycin (C-1027), a clinically relevant antitumor agent, by Streptomyces globisporus that negative feedback can extend to a point higher in the regulatory cascade. We show that the DNA-binding activity of the S. globisporus orthologue of AtrA, which was initially described as a transcriptional activator of actinorhodin biosynthesis in S. coelicolor, is inhibited by the binding of heptaene, a biosynthetic intermediate of lidamycin. Additional experiments described here show that S. globisporus AtrA binds in vivo as well as in vitro to the promoter region of the gene encoding SgcR1, one of the CSRs of lidamycin production. The feedback to the pleiotropic regulator AtrA is likely to provide a mechanism for coordinating the production of lidamycin with that of other secondary metabolites. The activity of AtrA is also regulated by actinorhodin. As AtrA is evolutionarily conserved, negative feedback of the type described here may be widespread within the streptomycetes.

  17. Ergot Alkaloids (Regenerate New Leads as Antiparasitics.

    Directory of Open Access Journals (Sweden)

    John D Chan

    Full Text Available Praziquantel (PZQ is a key therapy for treatment of parasitic flatworm infections of humans and livestock, but the mechanism of action of this drug is unresolved. Resolving PZQ-engaged targets and effectors is important for identifying new druggable pathways that may yield novel antiparasitic agents. Here we use functional, genetic and pharmacological approaches to reveal that serotonergic signals antagonize PZQ action in vivo. Exogenous 5-hydroxytryptamine (5-HT rescued PZQ-evoked polarity and mobility defects in free-living planarian flatworms. In contrast, knockdown of a prevalently expressed planarian 5-HT receptor potentiated or phenocopied PZQ action in different functional assays. Subsequent screening of serotonergic ligands revealed that several ergot alkaloids possessed broad efficacy at modulating regenerative outcomes and the mobility of both free living and parasitic flatworms. Ergot alkaloids that phenocopied PZQ in regenerative assays to cause bipolar regeneration exhibited structural modifications consistent with serotonergic blockade. These data suggest that serotonergic activation blocks PZQ action in vivo, while serotonergic antagonists phenocopy PZQ action. Importantly these studies identify the ergot alkaloid scaffold as a promising structural framework for designing potent agents targeting parasitic bioaminergic G protein coupled receptors.

  18. 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.

  19. Dietary alkaloid sequestration in a poison frog: an experimental test of alkaloid uptake in Melanophryniscus stelzneri (Bufonidae).

    Science.gov (United States)

    Hantak, Maggie M; Grant, Taran; Reinsch, Sherri; McGinnity, Dale; Loring, Marjorie; Toyooka, Naoki; Saporito, Ralph A

    2013-12-01

    Several lineages of brightly colored anurans independently evolved the ability to secrete alkaloid-containing defensive chemicals from granular glands in the skin. These species, collectively referred to as 'poison frogs,' form a polyphyletic assemblage that includes some species of Dendrobatidae, Mantellidae, Myobatrachidae, Bufonidae, and Eleutherodactylidae. The ability to sequester alkaloids from dietary arthropods has been demonstrated experimentally in most poison frog lineages but not in bufonid or eleutherodactylid poison frogs. As with other poison frogs, species of the genus Melanophryniscus (Bufonidae) consume large numbers of mites and ants, suggesting they might also sequester defensive alkaloids from dietary sources. To test this hypothesis, fruit flies dusted with alkaloid/nutritional supplement powder were fed to individual Melanophryniscus stelzneri in two experiments. In the first experiment, the alkaloids 5,8-disubstituted indolizidine 235B' and decahydroquinoline were administered to three individuals for 104 days. In the second experiment, the alkaloids 3,5-disubstituted indolizidine 239Q and decahydroquinoline were given to three frogs for 153 days. Control frogs were fed fruit flies dusted only with nutritional supplement. Gas chromatography/mass spectrometry analyses revealed that skin secretions of all experimental frogs contained alkaloids, whereas those of all control frogs lacked alkaloids. Uptake of decahydroquinoline was greater than uptake of 5,8-disubstituted indolizidine, and uptake of 3,5-disubstituted indolizidine was greater than uptake of decahydroquinoline, suggesting greater uptake efficiency of certain alkaloids. Frogs in the second experiment accumulated a greater amount of alkaloid, which corresponds to the longer duration and greater number of alkaloid-dusted fruit flies that were consumed. These findings provide the first experimental evidence that bufonid poison frogs sequester alkaloid-based defenses from dietary

  20. Engineering overexpression of ORCA3 and strictosidine glucosidase in Catharanthus roseus hairy roots increases alkaloid production.

    Science.gov (United States)

    Sun, Jiayi; Peebles, Christie A M

    2016-09-01

    Catharanthus roseus produces many pharmaceutically important terpenoid indole alkaloids (TIAs) such as vinblastine, vincristine, ajmalicine, and serpentine. Past metabolic engineering efforts have pointed to the tight regulation of the TIA pathway and to multiple rate-limiting reactions. Transcriptional regulator ORCA3 (octadecanoid responsive Catharanthus AP2-domain protein), activated by jasmonic acid, plays a central role in regulating the TIA pathway. In this study, overexpressing ORCA3 under the control of a glucocorticoid-inducible promoter in C. roseus hairy roots resulted in no change in the total amount of TIAs measured. RT-qPCR results showed that ORCA3 overexpression triggered the upregulation of transcripts of most of the known TIA pathway genes. One notable exception was the decrease in strictosidine glucosidase (SGD) transcripts. These results corresponded to previously published results. In this study, ORCA3 and SGD were both engineered in hairy roots under the control of a glucocorticoid-inducible promoter. Co-overexpression of ORCA3 and SGD resulted in a significant (p < 0.05) increase in serpentine by 44 %, ajmalicine by 32 %, catharanthine by 38 %, tabersonine by 40 %, lochnericine by 60 % and hörhammericine by 56 % . The total alkaloid pool was increased significantly by 47 %. Thus, combining overexpression of a positive regulator and a pathway gene which is not controlled by this regulator provided a way to enhance alkaloid production.

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

    Science.gov (United States)

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

    2008-06-01

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

  3. 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

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Science.gov (United States)

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

    2012-09-01

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

  6. Identification of a Pantoea biosynthetic cluster that directs the synthesis of an antimicrobial natural product.

    Directory of Open Access Journals (Sweden)

    Alyssa M Walterson

    Full Text Available Fire Blight is a destructive disease of apple and pear caused by the enteric bacterial pathogen, Erwinia amylovora. E. amylovora initiates infection by colonizing the stigmata of apple and pear trees, and entering the plants through natural openings. Epiphytic populations of the related enteric bacterium, Pantoea, reduce the incidence of disease through competition and antibiotic production. In this study, we identify an antibiotic from Pantoea ananatis BRT175, which is effective against E. amylovora and select species of Pantoea. We used transposon mutagenesis to create a mutant library, screened approximately 5,000 mutants for loss of antibiotic production, and recovered 29 mutants. Sequencing of the transposon insertion sites of these mutants revealed multiple independent disruptions of an 8.2 kb cluster consisting of seven genes, which appear to be coregulated. An analysis of the distribution of this cluster revealed that it was not present in any other of our 115 Pantoea isolates, or in any of the fully sequenced Pantoea genomes, and is most closely related to antibiotic biosynthetic clusters found in three different species of Pseudomonas. This identification of this biosynthetic cluster highlights the diversity of natural products produced by Pantoea.

  7. 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.

  8. 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.

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