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Sample records for saxitoxin biosynthetic genes

  1. Origin of saxitoxin biosynthetic genes in cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Ahmed Moustafa

    2009-06-01

    Full Text Available 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.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.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 circinalis strains.

  2. Evolution and Distribution of Saxitoxin Biosynthesis in Dinoflagellates

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    Kjetill S. Jakobsen

    2013-08-01

    Full Text Available Numerous species of marine dinoflagellates synthesize the potent environmental neurotoxic alkaloid, saxitoxin, the agent of the human illness, paralytic shellfish poisoning. In addition, certain freshwater species of cyanobacteria also synthesize the same toxic compound, with the biosynthetic pathway and genes responsible being recently reported. Three theories have been postulated to explain the origin of saxitoxin in dinoflagellates: The production of saxitoxin by co-cultured bacteria rather than the dinoflagellates themselves, convergent evolution within both dinoflagellates and bacteria and horizontal gene transfer between dinoflagellates and bacteria. The discovery of cyanobacterial saxitoxin homologs in dinoflagellates has enabled us for the first time to evaluate these theories. Here, we review the distribution of saxitoxin within the dinoflagellates and our knowledge of its genetic basis to determine the likely evolutionary origins of this potent neurotoxin.

  3. Anthocyanin biosynthetic genes in Brassica rapa.

    Science.gov (United States)

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

    2014-06-04

    Anthocyanins are a group of flavonoid compounds. As a group of important secondary metabolites, they perform several key biological functions in plants. Anthocyanins also play beneficial health roles as potentially protective factors against cancer and heart disease. To elucidate the anthocyanin biosynthetic pathway in Brassica rapa, we conducted comparative genomic analyses between Arabidopsis thaliana and B. rapa on a genome-wide level. In total, we identified 73 genes in B. rapa as orthologs of 41 anthocyanin biosynthetic genes in A. thaliana. In B. rapa, the anthocyanin biosynthetic genes (ABGs) have expanded and most genes exist in more than one copy. The anthocyanin biosynthetic structural genes have expanded through whole genome and tandem duplication in B. rapa. More structural genes located upstream of the anthocyanin biosynthetic pathway have been retained than downstream. More negative regulatory genes are retained in the anthocyanin biosynthesis regulatory system of B. rapa. These results will promote an understanding of the genetic mechanism of anthocyanin biosynthesis, as well as help the improvement of the nutritional quality of B. rapa through the breeding of high anthocyanin content varieties.

  4. Transcriptional profiling of Saccharomyces cerevisiae upon exposure to saxitoxin.

    Science.gov (United States)

    Cusick, Kathleen D; Boyer, Gregory L; Wilhelm, Steven W; Sayler, Gary S

    2009-08-01

    Saxitoxin is a potent neurotoxin produced by several species of dinoflagellates and cyanobacteria. The molecular target of saxitoxin in higher eukaryotes is the voltage-gated sodium channel; however, its target in lower eukaryotic organisms remains unknown. The goal of this study was to obtain the transcriptional fingerprint of the model lower eukaryote Saccharomyces cerevisiae upon exposure to saxitoxin to identify potential genes suitable for biomarker development. Microarray analyses identified multiple genes associated with copper and iron homeostasis and sulfur metabolism as significantly differentially expressed upon exposure to saxitoxin; these results were verified with quantitative reverse-transcriptase PCR (qRT-PCR). Additionally, the qRT-PCR assays were used to generate expression profiles in a subset of the differentially regulated genes across multiple exposure times and concentrations, the results of which demonstrated that overall, genes tended to respond in a consistent manner to the toxin. In general, the genes encoding the metallothioneins CUP1 and CRS5 were induced following exposure to saxitoxin, while those encoding the ferric/ cupric reductase FRE1 and the copper uptake transporter CTR1 were repressed. The gene encoding the multicopper ferroxidase FET3, part of the high-affinity iron uptake system, was also induced in all treatments, along with the STR3 gene, which codes for the cystathionine beta-lyase found in the methionine biosynthetic pathway.

  5. 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

  6. Cylindrospermopsin and saxitoxin synthetase genes in Cylindrospermopsis raciborskii strains from Brazilian freshwater.

    Directory of Open Access Journals (Sweden)

    Caroline Hoff-Risseti

    Full Text Available The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX, while cylindrospermopsin (CYN, which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.

  7. Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates.

    Science.gov (United States)

    Murray, Shauna A; Diwan, Rutuja; Orr, Russell J S; Kohli, Gurjeet S; John, Uwe

    2015-11-01

    A group of marine dinoflagellates (Alveolata, Eukaryota), consisting of ∼10 species of the genus Alexandrium, Gymnodinium catenatum and Pyrodinium bahamense, produce the toxin saxitoxin and its analogues (STX), which can accumulate in shellfish, leading to ecosystem and human health impacts. The genes, sxt, putatively involved in STX biosynthesis, have recently been identified, however, the evolution of these genes within dinoflagellates is not clear. There are two reasons for this: uncertainty over the phylogeny of dinoflagellates; and that the sxt genes of many species of Alexandrium and other dinoflagellate genera are not known. Here, we determined the phylogeny of STX-producing and other dinoflagellates based on a concatenated eight-gene alignment. We determined the presence, diversity and phylogeny of sxtA, domains A1 and A4 and sxtG in 52 strains of Alexandrium, and a further 43 species of dinoflagellates and thirteen other alveolates. We confirmed the presence and high sequence conservation of sxtA, domain A4, in 40 strains (35 Alexandrium, 1 Pyrodinium, 4 Gymnodinium) of 8 species of STX-producing dinoflagellates, and absence from non-producing species. We found three paralogs of sxtA, domain A1, and a widespread distribution of sxtA1 in non-STX producing dinoflagellates, indicating duplication events in the evolution of this gene. One paralog, clade 2, of sxtA1 may be particularly related to STX biosynthesis. Similarly, sxtG appears to be generally restricted to STX-producing species, while three amidinotransferase gene paralogs were found in dinoflagellates. We investigated the role of positive (diversifying) selection following duplication in sxtA1 and sxtG, and found negative selection in clades of sxtG and sxtA1, clade 2, suggesting they were functionally constrained. Significant episodic diversifying selection was found in some strains in clade 3 of sxtA1, a clade that may not be involved in STX biosynthesis, indicating pressure for diversification

  8. Neurotoxic Alkaloids: Saxitoxin and Its Analogs

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    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.

  9. 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

    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.

  10. Expression profile of genes coding for carotenoid biosynthetic ...

    Indian Academy of Sciences (India)

    Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits. Shuchi Smita, Ravi Rajwanshi, Sangram Keshari Lenka, Amit Katiyar, Viswanathan Chinnusamy and. Kailash Chander Bansal. J. Genet. 92, 363–368. Table 1.

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

    NARCIS (Netherlands)

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

    2010-01-01

    Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the beta-lactam biosynthetic

  12. PEGylated cationic polylactides for hybrid biosynthetic gene delivery.

    Science.gov (United States)

    Jones, Charles H; Chen, Chih-Kuang; Chen, Mingfu; Ravikrishnan, Anitha; Zhang, Hanguang; Gollakota, Akhila; Chung, Taichun; Cheng, Chong; Pfeifer, Blaine A

    2015-03-02

    Genetic vaccination is predicated on the underlying principle that diseases can be prevented by the controlled introduction of genetic material encoding antigenic proteins from pathogenic organisms to elicit the formation of protective immune responses. Driving this process is the choice of carrier that is responsible for navigating the obstacles associated with gene delivery. In this work, we expand upon a novel class of hybrid biosynthetic gene delivery vectors that are composed of a biomaterial outer coating and a bacterial (Escherichia coli) inner core. Specifically, a series of newly developed biodegradable cationic polylactides (CPLAs) and their PEGylated variants were selected to investigate the role of low polydispersity index (PDI), charge density, and PEGylation upon hybrid vector assembly and gene delivery efficacy. Upon assembly, hybrid vectors mediated increased gene delivery beyond that of the individual bacterial vector in isolation, including assays with increasing medium protein content to highlight shielding properties afforded by the PEG-functionalized CPLA component. Furthermore, after extensive characterization of surface deposition of the polymer, results prompted a new model for describing hybrid vector assembly that includes cellular coating and penetration of the CPLA component. In summary, these results provide new options and insight toward the assembly and application of next-generation hybrid biosynthetic gene delivery vectors.

  13. Draft Genome Sequence of Cylindrospermopsis raciborskii (Cyanobacteria) Strain ITEP-A1 Isolated from a Brazilian Semiarid Freshwater Body: Evidence of Saxitoxin and Cylindrospermopsin Synthetase Genes

    Science.gov (United States)

    Lorenzi, Adriana Sturion; Silva, Genivaldo Gueiros Z.; Lopes, Fabyano Alvares Cardoso; Chia, Mathias Ahii; Edwards, Robert A.

    2016-01-01

    Cylindrospermopsis raciborskii ITEP-A1 is a saxitoxin-producing cyanobacterium. We report the draft genome sequence of ITEP-A1, which comprised 195 contigs that were assembled with SPAdes and annotated with Rapid Annotation using Subsystem Technology. The identified genome sequence had 3,605,836 bp, 40.1% G+C, and predicted 3,553 coding sequences (including the synthetase genes). PMID:27151783

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

    Science.gov (United States)

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

    2015-07-03

    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. We identified 67 carotenoid biosynthetic genes in B. rapa, which were orthologs of the 47 carotenoid genes in A. thaliana. A high level of synteny was observed for carotenoid biosynthetic genes between A. thaliana and B. rapa. Out of 47 carotenoid biosynthetic genes in A. thaliana, 46 were successfully mapped to the 10 B. rapa chromosomes, and most of the genes retained more than one copy in B. rapa. The gene expansion was caused by the whole-genome triplication (WGT) event experienced by Brassica species. An expression analysis of the carotenoid biosynthetic genes suggested that their expression levels differed in root, stem, leaf, flower, callus, and silique tissues. Additionally, the paralogs of each carotenoid biosynthetic gene, which were generated from the WGT in B. rapa, showed significantly different expression levels among tissues, suggesting differentiated functions for these multi-copy genes in the carotenoid pathway. This first systematic study of carotenoid biosynthetic genes in B. rapa provides insights into the carotenoid metabolic mechanisms of Brassica crops. In addition, a better understanding of carotenoid biosynthetic genes in B. rapa will contribute to the development of conventional and transgenic B. rapa cultivars with enriched carotenoid levels in the future.

  15. Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster.

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    Oksana Bilyk

    Full Text Available Transformation-associated recombination (TAR in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the "capture" vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties.

  16. Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

    Science.gov (United States)

    Baumann, Sascha; Sander, Adrianne; Gurnon, James R.; Yanai-Balser, Giane; VanEtten, James L.; Piotrowski, Markus

    2007-01-01

    Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+- binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses. PMID:17101165

  17. Detection of photoactive siderophore biosynthetic genes in the marine environment.

    Science.gov (United States)

    Gärdes, Astrid; Triana, Christopher; Amin, Shady A; Green, David H; Romano, Ariel; Trimble, Lyndsay; Carrano, Carl J

    2013-06-01

    Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). While it has been hypothesized that the global production of siderophores by heterotrophic bacteria and some cyanobacteria constitutes the bulk of organic ligands binding iron in the ocean because stability constants of siderophores and these organic ligands are similar, and because ligand concentrations rise sharply in response to iron fertilization events, direct evidence for this proposal is lacking. This lack is due to the difficulty in characterizing these ligands due both to their extremely low concentrations and their highly heterogeneous nature. The situation for characterizing photoactive siderophores in situ is more problematic because of their expected short lifetimes in the photic zone. An alternative approach is to make use of high sensitivity molecular technology (qPCR) to search for siderophore biosynthesis genes related to the production of photoactive siderophores. In this way one can access their "biochemical potential" and utilize this information as a proxy for the presence of these siderophores in the marine environment. Here we show, using qPCR primers designed to detect biosynthetic genes for the siderophores vibrioferrin, petrobactin and aerobactin that such genes are widespread and based on their abundance, the "biochemical potential" for photoactive siderophore production is significant. Concurrently we also briefly examine the microbial biodiversity responsible for such production as a function of depth and location across a North Atlantic transect.

  18. 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...

  19. A systematic computational analysis of biosynthetic gene cluster evolution: lessons for engineering biosynthesis

    NARCIS (Netherlands)

    Medema, Marnix; Cimermancic, P.; Sali, A.; Takano, Eriko; Fischbach, M.A.

    2014-01-01

    Bacterial secondary metabolites are widely used as antibiotics, anticancer drugs, insecticides and food additives. Attempts to engineer their biosynthetic gene clusters (BGCs) to produce unnatural metabolites with improved properties are often frustrated by the unpredictability and complexity of the

  20. 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.

  1. Identification of Nocobactin NA Biosynthetic Gene Clusters in Nocardia farcinica▿ §

    OpenAIRE

    Hoshino, Yasutaka; Chiba, Kazuhiro; Ishino, Keiko; Fukai, Toshio; Igarashi, Yasuhiro; Yazawa, Katsukiyo; Mikami, Yuzuru; Ishikawa, Jun

    2010-01-01

    We identified the biosynthetic gene clusters of the siderophore nocobactin NA. The nbt clusters, which were discovered as genes highly homologous to the mycobactin biosynthesis genes by the genomic sequencing of Nocardia farcinica IFM 10152, consist of 10 genes separately located at two genomic regions. The gene organization of the nbt clusters and the predicted functions of the nbt genes, particularly the cyclization and epimerization domains, were in good agreement with the chemical structu...

  2. Reassembly of functionally intact environmental DNA-derived biosynthetic gene clusters.

    Science.gov (United States)

    Kallifidas, Dimitris; Brady, Sean F

    2012-01-01

    Only a small fraction of the bacterial diversity present in natural microbial communities is regularly cultured in the laboratory. Those bacteria that remain recalcitrant to culturing cannot be examined for the production of bioactive secondary metabolites using standard pure-culture approaches. The screening of genomic DNA libraries containing DNA isolated directly from environmental samples (environmental DNA (eDNA)) provides an alternative approach for studying the biosynthetic capacities of these organisms. One drawback of this approach has been that most eDNA isolation procedures do not permit the cloning of DNA fragments of sufficient length to capture large natural product biosynthetic gene clusters in their entirety. Although the construction of eDNA libraries with inserts big enough to capture biosynthetic gene clusters larger than ∼40kb remains challenging, it is possible to access large gene clusters by reassembling them from sets of smaller overlapping fragments using transformation-associated recombination in Saccharomyces cerevisiae. Here, we outline a method for the reassembly of large biosynthetic gene clusters from captured sets of overlapping soil eDNA cosmid clones. Natural product biosynthetic gene clusters reassembled using this approach can then be used directly for functional heterologous expression studies. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Functional analysis of a melanin biosynthetic gene using RNAi-mediated gene silencing in Rosellinia necatrix.

    Science.gov (United States)

    Shimizu, Takeo; Ito, Tsutae; Kanematsu, Satoko

    2014-04-01

    Rosellinia necatrix causes white root rot in a wide range of fruit trees and persists for extended periods as pseudosclerotia on root debris. However, the pathogenesis of this disease has yet to be clarified. The functions of endogeneous target genes have not been determined because of the inefficiency in genetic transformation. In this study, the function of a melanin biosynthetic gene was determined to examine its role in morphology and virulence. A polyketide synthase gene (termed as RnPKS1) in the R. necatrix genome is homologous to the 1,8-dihydroxynaphthalene (DHN) melanin biosynthetic gene of Colletotrichum lagenarium. Melanin-deficient strains of R. necatrix were obtained by RNA interference-mediated knockdown of RnPKS1. The virulence of these strains was not significantly reduced compared with the parental melanin-producing strain. However, knockdown strains failed to develop pseudosclerotia and were degraded sooner in soil than the parental strain. Microscopic observations of albino conidiomata produced by knockdown strains revealed that melanization is involved in synnema integrity. These results suggest that melanin is not necessary for R. necatrix pathogenesis but is involved in survival through morphogenesis. This is the first report on the functional analysis of an endogenous target gene in R. necatrix. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  4. cDNA cloning and expression of anthocyanin biosynthetic genes in ...

    African Journals Online (AJOL)

    ... stolons and terminal buds, and their transcripts could not detected in roots except 3GT. All the genes were induced expression in tuber skins by white light, and followed by anthocyanin accumulation. This is the first report cloning and expression of anthocyanin biosynthetic genes in wild potato (Solanum pinnatisectum).

  5. plantiSMASH: automated identification, annotation and expression analysis of plant biosynthetic gene clusters

    DEFF Research Database (Denmark)

    Kautsar, Satria A.; Suarez Duran, Hernando G.; Blin, Kai

    2017-01-01

    in specific genomic loci: biosynthetic gene clusters (BGCs). Here, we introduce plantiSMASH, a versatile online analysis platform that automates the identification of candidate plant BGCs. Moreover, it allows integration of transcriptomic data to prioritize candidate BGCs based on the coexpression patterns...... of predicted biosynthetic enzyme-coding genes, and facilitates comparative genomic analysis to study the evolutionary conservation of each cluster. Applied on 48 high-quality plant genomes, plantiSMASH identifies a rich diversity of candidate plant BGCs. These results will guide further experimental......Plant specialized metabolites are chemically highly diverse, play key roles in host-microbe interactions, have important nutritional value in crops and are frequently applied as medicines. It has recently become clear that plant biosynthetic pathway-encoding genes are sometimes densely clustered...

  6. [Advances on biosynthetic gene clusters of natural product from marine symbiotic microbe--a review].

    Science.gov (United States)

    Xu, Jing; Xu, Jun

    2008-07-01

    Previous research has suggested that the true producers of numerous natural products isolated from marine invertebrates were the microbial epibiont and symbiont which are deemed as not-yet-cultivated microbe. Cloning of the biosynthetic genes responsible for a specific nature product not only provides direct genetic evidence of the origin of the compounds but also establishes the feasibility of mass production of the compounds by heterologous expression. This paper reviews the progresses on the biosynthetic gene clusters of nature products from the symbiotic bacteria including marine sponge, ascidian, bryozoan, deep-sea tube worm and deep-sea sediments.

  7. Characterization of the biosynthetic gene cluster for cryptic phthoxazolin A in Streptomyces avermitilis.

    Directory of Open Access Journals (Sweden)

    Dian Anggraini Suroto

    Full Text Available Phthoxazolin A, an oxazole-containing polyketide, has a broad spectrum of anti-oomycete activity and herbicidal activity. We recently identified phthoxazolin A as a cryptic metabolite of Streptomyces avermitilis that produces the important anthelmintic agent avermectin. Even though genome data of S. avermitilis is publicly available, no plausible biosynthetic gene cluster for phthoxazolin A is apparent in the sequence data. Here, we identified and characterized the phthoxazolin A (ptx biosynthetic gene cluster through genome sequencing, comparative genomic analysis, and gene disruption. Sequence analysis uncovered that the putative ptx biosynthetic genes are laid on an extra genomic region that is not found in the public database, and 8 open reading frames in the extra genomic region could be assigned roles in the biosynthesis of the oxazole ring, triene polyketide and carbamoyl moieties. Disruption of the ptxA gene encoding a discrete acyltransferase resulted in a complete loss of phthoxazolin A production, confirming that the trans-AT type I PKS system is responsible for the phthoxazolin A biosynthesis. Based on the predicted functional domains in the ptx assembly line, we propose the biosynthetic pathway of phthoxazolin A.

  8. Quantification of trichothecene biosynthetic genes during the growth cycle of Fusarium sporotrichioides in culture

    Science.gov (United States)

    Trichothecene mycotoxins are secondary metabolites produced by several species of phytopathogenic fungi, and are potent inhibitors of protein biosynthesis. The genes involved in the biosynthetic pathway of T-2 toxin in Fusarium sporotrichioides have been characterized and are located in four identi...

  9. Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters

    NARCIS (Netherlands)

    Cimermancic, P.; Medema, Marnix; Claesen, J.; Kurika, K.; Wieland Brown, L.C.; Mavrommatis, K.; Pati, A.; Godfrey, P.A.; Koehrsen, M.; Clardy, J.; Birren, B. W.; Takano, Eriko; Sali, A.; Linington, R.G.; Fischbach, M.A.

    2014-01-01

    Although biosynthetic gene clusters (BGCs) have been discovered for hundreds of bacterial metabolites, our knowledge of their diversity remains limited. Here, we used a novel algorithm to systematically identify BGCs in the extensive extant microbial sequencing data. Network analysis of the

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

    Science.gov (United States)

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

    2014-01-01

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

  11. Functional Conservation of Coenzyme Q Biosynthetic Genes among Yeasts, Plants, and Humans

    Science.gov (United States)

    Hayashi, Kazuhiro; Ogiyama, Yuki; Yokomi, Kazumasa; Nakagawa, Tsuyoshi; Kaino, Tomohiro; Kawamukai, Makoto

    2014-01-01

    Coenzyme Q (CoQ) is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3–9) that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana) to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants. PMID:24911838

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

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Amycolatopsis balhimycina produces the vancomycin-analogue balhimycin. The strain therefore serves as a model strain for glycopeptide antibiotic production. Previous characterisation of the balhimycin biosynthetic cluster had shown that the border sequences contained both, a putative 3-deoxy......-d-arabino-heptulosonate 7-phosphate synthase (dahp), and a prephenate dehydrogenase (pdh) gene. In a metabolic engineering approach for increasing the precursor supply for balhimycin production, the dahp and pdh genes from the biosynthetic cluster were overexpressed both individually and together and the resulting strains...... production levels similar to the parent strain. Based on these results the relation between primary and secondary metabolism with regards to Dahp and Pdh is discussed....

  14. Sequencing rare marine actinomycete genomes reveals high density of unique natural product biosynthetic gene clusters

    Science.gov (United States)

    Schorn, Michelle A.; Alanjary, Mohammad M.; Aguinaldo, Kristen; Korobeynikov, Anton; Podell, Sheila; Patin, Nastassia; Lincecum, Tommie; Jensen, Paul R.; Ziemert, Nadine

    2016-01-01

    Traditional natural product discovery methods have nearly exhausted the accessible diversity of microbial chemicals, making new sources and techniques paramount in the search for new molecules. Marine actinomycete bacteria have recently come into the spotlight as fruitful producers of structurally diverse secondary metabolites, and remain relatively untapped. In this study, we sequenced 21 marine-derived actinomycete strains, rarely studied for their secondary metabolite potential and under-represented in current genomic databases. We found that genome size and phylogeny were good predictors of biosynthetic gene cluster diversity, with larger genomes rivalling the well-known marine producers in the Streptomyces and Salinispora genera. Genomes in the Micrococcineae suborder, however, had consistently the lowest number of biosynthetic gene clusters. By networking individual gene clusters into gene cluster families, we were able to computationally estimate the degree of novelty each genus contributed to the current sequence databases. Based on the similarity measures between all actinobacteria in the Joint Genome Institute's Atlas of Biosynthetic gene Clusters database, rare marine genera show a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia genera representing the highest gene cluster diversity. This research validates that rare marine actinomycetes are important candidates for exploration, as they are relatively unstudied, and their relatives are historically rich in secondary metabolites. PMID:27902408

  15. Identification of biosynthetic gene clusters from metagenomic libraries using PPTase complementation in a Streptomyces host.

    Science.gov (United States)

    Bitok, J Kipchirchir; Lemetre, Christophe; Ternei, Melinda A; Brady, Sean F

    2017-09-01

    The majority of environmental bacteria are not readily cultured in the lab, leaving the natural products they make inaccessible using culture-dependent discovery methods. Cloning and heterologous expression of DNA extracted from environmental samples (environmental DNA, eDNA) provides a means of circumventing this discovery bottleneck. To facilitate the identification of clones containing biosynthetic gene clusters, we developed a model heterologous expression reporter strain Streptomyces albus::bpsA ΔPPTase. This strain carries a 4΄-phosphopantetheinyl transferase (PPTase)-dependent blue pigment synthase A gene, bpsA, in a PPTase deletion background. eDNA clones that express a functional PPTase restore production of the blue pigment, indigoidine. As PPTase genes often occur in biosynthetic gene clusters (BGCs), indigoidine production can be used to identify eDNA clones containing BGCs. We screened a soil eDNA library hosted in S. albus::bpsA ΔPPTase and identified clones containing non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and mixed NRPS/PKS biosynthetic gene clusters. One NRPS gene cluster was shown to confer the production of myxochelin A to S. albus::bpsA ΔPPTase. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Sequencing rare marine actinomycete genomes reveals high density of unique natural product biosynthetic gene clusters.

    Science.gov (United States)

    Schorn, Michelle A; Alanjary, Mohammad M; Aguinaldo, Kristen; Korobeynikov, Anton; Podell, Sheila; Patin, Nastassia; Lincecum, Tommie; Jensen, Paul R; Ziemert, Nadine; Moore, Bradley S

    2016-12-01

    Traditional natural product discovery methods have nearly exhausted the accessible diversity of microbial chemicals, making new sources and techniques paramount in the search for new molecules. Marine actinomycete bacteria have recently come into the spotlight as fruitful producers of structurally diverse secondary metabolites, and remain relatively untapped. In this study, we sequenced 21 marine-derived actinomycete strains, rarely studied for their secondary metabolite potential and under-represented in current genomic databases. We found that genome size and phylogeny were good predictors of biosynthetic gene cluster diversity, with larger genomes rivalling the well-known marine producers in the Streptomyces and Salinispora genera. Genomes in the Micrococcineae suborder, however, had consistently the lowest number of biosynthetic gene clusters. By networking individual gene clusters into gene cluster families, we were able to computationally estimate the degree of novelty each genus contributed to the current sequence databases. Based on the similarity measures between all actinobacteria in the Joint Genome Institute's Atlas of Biosynthetic gene Clusters database, rare marine genera show a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia genera representing the highest gene cluster diversity. This research validates that rare marine actinomycetes are important candidates for exploration, as they are relatively unstudied, and their relatives are historically rich in secondary metabolites.

  17. 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.

  18. Linking Biosynthetic Gene Clusters to their Metabolites via Pathway-Targeted Molecular Networking

    Science.gov (United States)

    Trautman, Eric P.; Crawford, Jason M.

    2016-01-01

    The connection of microbial biosynthetic gene clusters to the small molecule metabolites they encode is central to the discovery and characterization of new metabolic pathways with ecological and pharmacological potential. With increasing microbial genome sequence information being deposited into publicly available databases, it is clear that microbes have the coding capacity for many more biologically active small molecules than previously realized. Of increasing interest are the small molecules encoded by the human microbiome, as these metabolites likely mediate a variety of currently uncharacterized human-microbe interactions that influence health and disease. In this mini-review, we describe the ongoing biosynthetic, structural, and functional characterizations of the genotoxic colibactin pathway in gut bacteria as a thematic example of linking biosynthetic gene clusters to their metabolites. We also highlight other natural products that are produced through analogous biosynthetic logic and comment on some current disconnects between bioinformatics predictions and experimental structural characterizations. Lastly, we describe the use of pathway-targeted molecular networking as a tool to characterize secondary metabolic pathways within complex metabolomes and to aid in downstream metabolite structural elucidation efforts. PMID:26456470

  19. Cloning and heterologous expression of the penicillin biosynthetic gene cluster from penicillum chrysogenum.

    Science.gov (United States)

    Smith, D J; Burnham, M K; Edwards, J; Earl, A J; Turner, G

    1990-01-01

    A cosmid clone containing the putative penicillin biosynthetic gene cluster from Penicillium chrysogenum was used to transform the related filamentous fungi Neurospora crassa and Aspergillus niger, which do not produce beta-lactam antibiotics. Both of the transformed hosts contained intact P. chrysogenum DNA derived from the cosmid clone and produced authentic penicillin V. Assays of penicillin biosynthetic enzyme activity additionally demonstrated that they possessed delta-(L-alpha-amino-adipyl)-L-cysteinyl-D-valine synthetase (ACVS), isopenicillin N synthetase (IPNS) and acyl coenzyme A:6-aminopenicillanic acid acyltransferase (ACT) activity. The data suggests that genes encoding all the enzymes necessary for the biosynthesis of penicillin from amino acid precursors are closely linked in P. chrysogenum and constitute a gene cluster.

  20. A Genomics Based Discovery of Secondary Metabolite Biosynthetic Gene Clusters in Aspergillus ustus

    Science.gov (United States)

    Pi, Borui; Yu, Dongliang; Dai, Fangwei; Song, Xiaoming; Zhu, Congyi; Li, Hongye; Yu, Yunsong

    2015-01-01

    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. PMID:25706180

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

    Directory of Open Access Journals (Sweden)

    Borui Pi

    Full Text Available Secondary metabolites (SMs produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

  2. Transcriptomic analyses reveal biosynthetic genes related to rosmarinic acid in Dracocephalum tanguticum.

    Science.gov (United States)

    Li, Huie; Fu, Yaru; Sun, Hao; Zhang, Yanfu; Lan, Xiaozhong

    2017-03-06

    Dracocephalum tanguticum Maxim, a Lamiaceae species endemic to the Qinghai-Tibetan Plateau and adjacent regions, is an important ornamental, medicinal and aromatic herb. In this study, a comprehensive transcriptome of 18 libraries from six organs namely, roots, stems, leaves, sepals, flowers and seeds of D. tanguticum were generated. More than 100 Gb of sequence data were obtained and assembled de novo into 187,447 transcripts, including 151,463 unigenes, among which the six organs shared 17.7% (26,841). In addition, all unigenes were assigned to 362 pathways, in which 'biosynthesis of secondary metabolites' is the second enriched pathway. Furthermore, rosmarinic acid (RA) is one of the multifunctional phenolic bioactive compounds produced in some Lamiaceae species. The six organs of D. tanguticum were confirmed to produce RA. A total of 22 predicted biosynthetic genes related to RA from the transcriptome were further isolated. Two of these genes were identified as candidates by evaluating the correlation coefficient between the RA contents and the expression of the predicted biosynthetic genes in the six organs. The new sequence information will improve the knowledge of D. tanguticum, as well as provide a reference tool for future studies of biosynthetic genes related to RA in this species.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. High Specificity of a Quantitative PCR Assay Targeting a Saxitoxin Gene for Monitoring Toxic Algae Associated with Paralytic Shellfish Toxins in the Yellow Sea.

    Science.gov (United States)

    Gao, Yan; Yu, Ren-Cheng; Murray, Shauna A; Chen, Jian-Hua; Kang, Zhen-Jun; Zhang, Qing-Chun; Kong, Fan-Zhou; Zhou, Ming-Jiang

    2015-10-01

    The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r=0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r=0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Cloning of the biosynthetic gene cluster for naphthoxanthene antibiotic FD-594 from Streptomyces sp. TA-0256.

    Science.gov (United States)

    Kudo, Fumitaka; Yonezawa, Takanori; Komatsubara, Akiko; Mizoue, Kazutoshi; Eguchi, Tadashi

    2011-01-01

    FD-594 is an unique pyrano[4',3':6,7]naphtho[1,2-b]xanthene polyketide with a trisaccharide of 2,6-dideoxysugars. In this study, we cloned the FD-594 biosynthetic gene cluster from the producer strain Streptomyces sp. TA-0256 to investigate its biosynthesis. The identified pnx gene cluster was 38143 bp, consisting of 40 open reading frames, including a minimal PKS gene, TDP-olivose biosynthetic genes, two glycosyltransferase genes, two methyltransferase genes and many oxygenase/reductase genes. Most of these enzymes coded in the pnx cluster were reasonably assigned to a plausible biosynthetic pathway for FD-594, in which an unique ring opening process via Baeyer-Villiger-type oxidation catalyzed by a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase, is speculated to lead to the unique xanthene structure. To clarify the involvement of pnx genes in the FD-594 biosynthesis, a glycosyltransferase, PnxGT2, and a methyltransferase, PnxMT2, were characterized enzymatically with the recombinant proteins expressed in Escherichia coli. As a result, PnxGT2 catalyzed the triple olivose transfers to the FD-594 aglycon with TDP-olivose as the glycosyl donor to afford triolivoside. Surprisingly, in the PnxGT2 enzymatic reaction, tetraolivoside and pentaolivoside were significantly detected along with the expected triolivoside. To our knowledge, PnxGT2 is the first contiguous oligosaccharide-forming glycosyltransferase in secondary metabolism. Furthermore, addition of PnxMT2 and S-adenosyl-L-methionine into the PnxGT2 reaction mixture afforded natural FD-594 to confirm that the PnxGT2 reaction product was the expected regiospecifically glycosylated compound. Consequently, the identified pnx gene cluster appears to be involved in FD-594 biosynthesis.

  6. Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species

    DEFF Research Database (Denmark)

    Nielsen, Jens Christian; Grijseels, Sietske; Prigent, Sylvain

    2017-01-01

    sequenced the genomes of 9 Penicillium species and, together with 15 published genomes, we investigated the secondary metabolism of Penicillium and identified an immense, unexploited potential for producing secondary metabolites by this genus. A total of 1,317 putative biosynthetic gene clusters (BGCs) were...... identified, and polyketide synthase and non-ribosomal peptide synthetase based BGCs were grouped into gene cluster families and mapped to known pathways. The grouping of BGCs allowed us to study the evolutionary trajectory of pathways based on 6-methylsalicylic acid (6-MSA) synthases. Finally, we cross...... diversity of Penicillia and highlights the potential of these species as a source of new antibiotics and other pharmaceuticals....

  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. 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.

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

    OpenAIRE

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

    2017-01-01

    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 Transc...

  11. 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.

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

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    Dionicia Gloria León-Martínez

    2012-06-01

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

  13. Genetic engineering and heterologous expression of the disorazol biosynthetic gene cluster via Red/ET recombineering.

    Science.gov (United States)

    Tu, Qiang; Herrmann, Jennifer; Hu, Shengbiao; Raju, Ritesh; Bian, Xiaoying; Zhang, Youming; Müller, Rolf

    2016-02-15

    Disorazol, a macrocyclic polykitide produced by the myxobacterium Sorangium cellulosum So ce12 and it is reported to have potential cytotoxic activity towards several cancer cell lines, including multi-drug resistant cells. The disorazol biosynthetic gene cluster (dis) from Sorangium cellulosum (So ce12) was identified by transposon mutagenesis and cloned in a bacterial artificial chromosome (BAC) library. The 58-kb dis core gene cluster was reconstituted from BACs via Red/ET recombineering and expressed in Myxococcus xanthus DK1622. For the first time ever, a myxobacterial trans-AT polyketide synthase has been expressed heterologously in this study. Expression in M. xanthus allowed us to optimize the yield of several biosynthetic products using promoter engineering. The insertion of an artificial synthetic promoter upstream of the disD gene encoding a discrete acyl transferase (AT), together with an oxidoreductase (Or), resulted in 7-fold increase in disorazol production. The successful reconstitution and expression of the genetic sequences encoding for these promising cytotoxic compounds will allow combinatorial biosynthesis to generate novel disorazol derivatives for further bioactivity evaluation.

  14. Identification of flavonoids and expression of flavonoid biosynthetic genes in two coloured tree peony flowers.

    Science.gov (United States)

    Zhao, Daqiu; Tang, Wenhui; Hao, Zhaojun; Tao, Jun

    2015-04-10

    Tree peony (Paeonia suffruticosa Andr.) has been named the "king of flowers" because of its elegant and gorgeous flower colour. Among these colours, the molecular mechanisms of white formation and how white turned to red in P. suffruticosa is little known. In this study, flower colour variables, flavonoid accumulation and expression of flavonoid biosynthetic genes of white ('Xueta') and red ('Caihui') P. suffruticosa were investigated. The results showed that the flower colours of both cultivars were gradually deepened with the development of flowers. Moreover, two anthoxanthin compositions apigenin 7-O-glucoside together with apigenin deoxyheso-hexoside were identified in 'Xueta' and 'Caihui', but one main anthocyanin composition peonidin 3,5-di-O-glucoside (Pn3G5G) was only found in 'Caihui'. Total contents of anthocyanins in 'Caihui' was increased during flower development, and the same trend was presented in anthoxanthins and flavonoids of these two cultivars, but the contents of these two category flavonoid in 'Caihui' were always higher than those in 'Xueta'. Furthermore, nine structural genes in flavonoid biosynthetic pathway were isolated including the full-length cDNAs of phenylalanine ammonialyase gene (PAL), chalcone synthase gene (CHS) and chalcone isomerase gene (CHI), together with the partial-length cDNAs of flavanone 3-hydroxylase gene (F3H), flavonoid 3'-hydroxylase gene (F3'H), dihydroflavonol 4-reductase gene (DFR), anthocyanidin synthase gene (ANS), UDP-glucose: flavonoid 3-O-glucosyltransferase gene (UF3GT) and UDP-glucose: flavonoid 5-O-glucosyltransferase gene (UF5GT), and PAL, UF3GT and UF5GT were reported in P. suffruticosa for the first time. Their expression patterns showed that transcription levels of downstream genes in 'Caihui' were basically higher than those in 'Xueta', especially PsDFR and PsANS, suggesting that these two genes may play a key role in the anthocyanin biosynthesis which resulted in the shift from white to red in

  15. 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

    2016-10-05

    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.

  16. 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.

  17. 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.

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

    Science.gov (United States)

    Park, Yun Ji; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Lim, Soon Sung; Kim, Yeon Bok; Lee, Sang Won; Park, Sang Un

    2016-05-27

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

  19. 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

  20. Nicotine infusion modulates immobilization stress-triggered induction of gene expression of rat catecholamine biosynthetic enzymes.

    Science.gov (United States)

    Serova, L; Danailov, E; Chamas, F; Sabban, E L

    1999-11-01

    The relationship between nicotine and stress is complex and paradoxical. Although people claim they smoke because it relaxes them, nicotine can trigger some of the effects observed with stress, including the release and synthesis of the catecholamines and their biosynthetic enzymes. This study examined one aspect of this confusing relationship between nicotine and stress. Multiple injections of nicotine bitartrate (5 mg/kg) elevated mRNA levels for the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase, and of preproneuropeptide Y in rat adrenal medulla more than did 1 mg/kg of nicotine bitartrate. In the locus ceruleus, substantia nigra, and ventral tegmental area both doses equally induced TH mRNA levels. Nicotine infusion (15 mg/kg/day) did not affect adrenal mRNA levels for any of the genes of interest and did not increase plasma corticosterone levels. However, in rats pre-exposed to nicotinic infusions, the response to a single immobilization (IMO) stress was markedly attenuated with respect to changes in adrenomedullary TH, DBH, and phenylethanolamine N-methyltransferase mRNA levels and in c-Fos protein levels. In the central nervous system, the chronic infusion of nicotine prevented the induction of TH mRNA by repeated IMO stress in the ventral tegmental area (but not in substantia nigra) and of DBH mRNA by single IMO in the locus ceruleus. These findings may explain some of the complex interactions between stress and exposure to nicotine.

  1. A systematic computational analysis of biosynthetic gene cluster evolution: lessons for engineering biosynthesis.

    Directory of Open Access Journals (Sweden)

    Marnix H Medema

    2014-12-01

    Full Text Available Bacterial secondary metabolites are widely used as antibiotics, anticancer drugs, insecticides and food additives. Attempts to engineer their biosynthetic gene clusters (BGCs to produce unnatural metabolites with improved properties are often frustrated by the unpredictability and complexity of the enzymes that synthesize these molecules, suggesting that genetic changes within BGCs are limited by specific constraints. Here, by performing a systematic computational analysis of BGC evolution, we derive evidence for three findings that shed light on the ways in which, despite these constraints, nature successfully invents new molecules: 1 BGCs for complex molecules often evolve through the successive merger of smaller sub-clusters, which function as independent evolutionary entities. 2 An important subset of polyketide synthases and nonribosomal peptide synthetases evolve by concerted evolution, which generates sets of sequence-homogenized domains that may hold promise for engineering efforts since they exhibit a high degree of functional interoperability, 3 Individual BGC families evolve in distinct ways, suggesting that design strategies should take into account family-specific functional constraints. These findings suggest novel strategies for using synthetic biology to rationally engineer biosynthetic pathways.

  2. A systematic computational analysis of biosynthetic gene cluster evolution: lessons for engineering biosynthesis.

    Science.gov (United States)

    Medema, Marnix H; Cimermancic, Peter; Sali, Andrej; Takano, Eriko; Fischbach, Michael A

    2014-12-01

    Bacterial secondary metabolites are widely used as antibiotics, anticancer drugs, insecticides and food additives. Attempts to engineer their biosynthetic gene clusters (BGCs) to produce unnatural metabolites with improved properties are often frustrated by the unpredictability and complexity of the enzymes that synthesize these molecules, suggesting that genetic changes within BGCs are limited by specific constraints. Here, by performing a systematic computational analysis of BGC evolution, we derive evidence for three findings that shed light on the ways in which, despite these constraints, nature successfully invents new molecules: 1) BGCs for complex molecules often evolve through the successive merger of smaller sub-clusters, which function as independent evolutionary entities. 2) An important subset of polyketide synthases and nonribosomal peptide synthetases evolve by concerted evolution, which generates sets of sequence-homogenized domains that may hold promise for engineering efforts since they exhibit a high degree of functional interoperability, 3) Individual BGC families evolve in distinct ways, suggesting that design strategies should take into account family-specific functional constraints. These findings suggest novel strategies for using synthetic biology to rationally engineer biosynthetic pathways.

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

    Science.gov (United States)

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

    2013-03-20

    Cyanosporasides are marine bacterial natural products containing a chlorinated cyclopenta[a]indene core of suspected enediyne polyketide biosynthetic origin. Herein, we report the isolation and characterization of novel cyanosporasides C-F (3-6) from the marine actinomycetes Salinispora pacifica CNS-143 and Streptomyces sp. CNT-179, highlighted by the unprecedented C-2' N-acetylcysteamine functionalized hexose group of 6. Cloning, sequencing, and mutagenesis of homologous ~50 kb cyanosporaside biosynthetic gene clusters from both bacteria afforded the first genetic evidence supporting cyanosporaside's enediyne, and thereby p-benzyne biradical, biosynthetic origin and revealed the molecular basis for nitrile and glycosyl functionalization. This study provides new opportunities for bioengineering of enediyne derivatives and expands the structural diversity afforded by enediyne gene clusters.

  4. Description of an orthologous cluster of ochratoxin A biosynthetic genes in Aspergillus and Penicillium species. A comparative analysis.

    Science.gov (United States)

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

    2018-03-02

    Ochratoxin A (OTA) is one of the most important mycotoxins due to its toxic properties and worldwide distribution which is produced by several Aspergillus and Penicillium species. The knowledge of OTA biosynthetic genes and understanding of the mechanisms involved in their regulation are essential. In this work, we obtained a clear picture of biosynthetic genes organization in the main OTA-producing Aspergillus and Penicillium species (A. steynii, A. westerdijkiae, A. niger, A. carbonarius and P. nordicum) using complete genome sequences obtained in this work or previously available on databases. The results revealed a region containing five ORFs which predicted five proteins: halogenase, bZIP transcription factor, cytochrome P450 monooxygenase, non-ribosomal peptide synthetase and polyketide synthase in all the five species. Genetic synteny was conserved in both Penicillium and Aspergillus species although genomic location seemed to be different since the clusters presented different flanking regions (except for A. steynii and A. westerdijkiae); these observations support the hypothesis of the orthology of this genomic region and that it might have been acquired by horizontal transfer. New real-time RT-PCR assays for quantification of the expression of these OTA biosynthetic genes were developed. In all species, the five genes were consistently expressed in OTA-producing strains in permissive conditions. These protocols might favour futures studies on the regulation of biosynthetic genes in order to develop new efficient control methods to avoid OTA entering the food chain. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. A Relational Database for the Discovery of Genes Encoding Amino Acid Biosynthetic Enzymes in Pathogenic Fungi

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    Nicholas J. Talbot

    2006-04-01

    Full Text Available Fungal phytopathogens continue to cause major economic impact, either directly, through crop losses, or due to the costs of fungicide application. Attempts to understand these organisms are hampered by a lack of fungal genome sequence data. A need exists, however, to develop specific bioinformatics tools to collate and analyse the sequence data that currently is available. A web-accessible gene discovery database (http://cogeme.ex.ac.uk/biosynthesis.html was developed as a demonstration tool for the analysis of metabolic and signal transduction pathways in pathogenic fungi using incomplete gene inventories. Using Bayesian probability to analyse the currently available gene information from pathogenic fungi, we provide evidence that the obligate pathogen Blumeria graminis possesses all amino acid biosynthetic pathways found in free-living fungi, such as Saccharomyces cerevisiae. Phylogenetic analysis was also used to deduce a gene history of succinate-semialdehyde dehydrogenase, an enzyme in the glutamate and lysine biosynthesis pathways. The database provides a tool and methodology to researchers to direct experimentation towards predicting pathway conservation in pathogenic microorganisms.

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

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    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.

  7. 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.

  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. 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...

  10. The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter.

    Science.gov (United States)

    Darbani, Behrooz; Motawia, Mohammed Saddik; Olsen, Carl Erik; Nour-Eldin, Hussam H; Møller, Birger Lindberg; Rook, Fred

    2016-11-14

    Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.

  11. 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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. 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. Copyright

  13. 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

  14. Mutational studies of putative biosynthetic genes for the cyanobacterial sunscreen scytonemin in Nostoc punctiforme ATCC 29133

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    Daniela eFerreira

    2016-05-01

    Full Text Available The heterocyclic indole-alkaloid scytonemin is a sunscreen found exclusively among cyanobacteria. An 18-gene cluster is responsible for scytonemin production in Nostoc punctiforme ATCC 29133. The upstream genes scyABCDEF in the cluster are proposed to be responsible for scytonemin biosynthesis from aromatic amino acid substrates. In vitro studies of ScyA, ScyB and ScyC proved that these enzymes indeed catalyze initial pathway reactions. Here we characterize the role of ScyD, ScyE and ScyF, which were logically predicted to be responsible for late biosynthetic steps, in the biological context of N. punctiforme. In-frame deletion mutants of each were constructed (∆scyD, ∆scyE and ∆scyF and their phenotypes studied. Expectedly, ∆scyE presents a scytoneminless phenotype, but no accumulation of the predicted intermediaries. Surprisingly, ∆scyD retains scytonemin production, implying that it is not required for biosynthesis. Indeed, scyD presents an interesting evolutionary paradox: it likely originated in a duplication event from scyE, and unlike other genes in the operon, it has not been subjected to purifying selection. This would suggest that it is a pseudogene, and yet scyD is highly conserved in the scytonemin operon of cyanobacteria. ∆scyF also retains scytonemin production, albeit exhibiting a reduction of the production yield compared with the wild-type. This indicates that ScyF is not essential but may play an adjuvant role for scytonemin synthesis. Altogether, our findings suggest that these downstream genes are not responsible, as expected, for the late steps of scytonemin synthesis and we must look for those functions elsewhere. These findings are particularly important for biotechnological production of this sunscreen through heterologous expression of its genes in more tractable organisms.

  15. Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties.

    Science.gov (United States)

    Hoang, Van L T; Innes, David J; Shaw, P Nicholas; Monteith, Gregory R; Gidley, Michael J; Dietzgen, Ralf G

    2015-07-30

    Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles. A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316 bp. Variety IW had the highest SNP frequency (one SNP every 258 bp) while KP and NDM had similar frequencies (one SNP every 369 bp and 360 bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3'-hydroxylase (F3'H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties. The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and

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

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    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. 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.

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

    Science.gov (United States)

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

    2016-06-01

    Tomato (Solanum lycopersicum L.) is one of the model plant to study carotenoid biosynthesis. In the present study, the fruit carotenoid content were quantified at different developmental stages for two contrasting genotypes, viz. IIHR-249-1 and IIHR-2866 by UPLC. Lycopene content was high in IIHR-249-1 (19.45 mg/100 g fresh weight) compared to IIHR-2866 (1.88 mg/100 g 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 14-fold from immature green stage to ripe stage in IIHR-249-1. The expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta 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 beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) .The F2 hybrids derived from IIHR-249-1 and IIHR-2866 were analysed at the ripe stage for lycopene content. The gene expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-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 beta-cyclases can be used in marker-assisted breeding.

  19. Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

    Science.gov (United States)

    Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

    2015-12-01

    In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Identification and characterization of a biosynthetic gene cluster for tryptophan dimers in deep sea-derived Streptomyces sp. SCSIO 03032.

    Science.gov (United States)

    Ma, Liang; Zhang, Wenjun; Zhu, Yiguang; Zhang, Guangtao; Zhang, Haibo; Zhang, Qingbo; Zhang, Liping; Yuan, Chengshan; Zhang, Changsheng

    2017-08-01

    Tryptophan dimers (TDs) are an important class of natural products with diverse bioactivities and share conserved biosynthetic pathways. We report the identification of a partial gene cluster (spm) responsible for the biosynthesis of a class of unusual TDs with non-planar skeletons including spiroindimicins (SPMs), indimicins (IDMs), and lynamicins (LNMs) from the deep-sea derived Streptomyces sp. SCSIO 03032. Bioinformatics analysis, targeted gene disruptions, and heterologous expression studies confirmed the involvement of the spm gene cluster in the biosynthesis of SPM/IDM/LNMs, and revealed the indispensable roles for the halogenase/reductase pair SpmHF, the amino acid oxidase SpmO, and the chromopyrrolic acid (CPA) synthase SpmD, as well as the positive regulator SpmR and the putative transporter SpmA. However, the spm gene cluster was unable to confer a heterologous host the ability to produce SPM/IDM/LNMs. In addition, the P450 enzyme SpmP and the monooxygenase SpmX2 were found to be non-relevant to the biosynthesis of SPM/IDM/LNMs. Sequence alignment and structure modeling suggested the lack of key conserved amino acid residues in the substrate-binding pocket of SpmP. Furthermore, feeding experiments in the non-producing ΔspmO mutant revealed several biosynthetic precursors en route to SPMs, indicating that key enzymes responsible for the biosynthesis of SPMs should be encoded by genes outside of the identified spm gene cluster. Finally, the biosynthetic pathways of SPM/IDM/LNMs are proposed to lay a basis for further insights into their intriguing biosynthetic machinery.

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

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    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.

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

    Science.gov (United States)

    García-Estrada, Carlos; Martín, Juan-Francisco

    2016-10-01

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

  3. Bacillus cereus-type polyhydroxyalkanoate biosynthetic gene cluster contains R-specific enoyl-CoA hydratase gene.

    Science.gov (United States)

    Kihara, Takahiro; Hiroe, Ayaka; Ishii-Hyakutake, Manami; Mizuno, Kouhei; Tsuge, Takeharu

    2017-08-01

    Bacillus cereus and Bacillus megaterium both accumulate polyhydroxyalkanoate (PHA) but their PHA biosynthetic gene (pha) clusters that code for proteins involved in PHA biosynthesis are different. Namely, a gene encoding MaoC-like protein exists in the B. cereus-type pha cluster but not in the B. megaterium-type pha cluster. MaoC-like protein has an R-specific enoyl-CoA hydratase (R-hydratase) activity and is referred to as PhaJ when involved in PHA metabolism. In this study, the pha cluster of B. cereus YB-4 was characterized in terms of PhaJ's function. In an in vitro assay, PhaJ from B. cereus YB-4 (PhaJYB4) exhibited hydration activity toward crotonyl-CoA. In an in vivo assay using Escherichia coli as a host for PHA accumulation, the recombinant strain expressing PhaJYB4 and PHA synthase led to increased PHA accumulation, suggesting that PhaJYB4 functioned as a monomer supplier. The monomer composition of the accumulated PHA reflected the substrate specificity of PhaJYB4, which appeared to prefer short chain-length substrates. The pha cluster from B. cereus YB-4 functioned to accumulate PHA in E. coli; however, it did not function when the phaJYB4 gene was deleted. The B. cereus-type pha cluster represents a new example of a pha cluster that contains the gene encoding PhaJ.

  4. Deletion and gene expression analyses define the paxilline biosynthetic gene cluster in Penicillium paxilli.

    Science.gov (United States)

    Scott, Barry; Young, Carolyn A; Saikia, Sanjay; McMillan, Lisa K; Monahan, Brendon J; Koulman, Albert; Astin, Jonathan; Eaton, Carla J; Bryant, Andrea; Wrenn, Ruth E; Finch, Sarah C; Tapper, Brian A; Parker, Emily J; Jameson, Geoffrey B

    2013-08-14

    The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.

  5. A gene expression analysis of cell wall biosynthetic genes in Malus x domestica infected by 'Candidatus Phytoplasma mali'.

    Science.gov (United States)

    Guerriero, Gea; Giorno, Filomena; Ciccotti, Anna Maria; Schmidt, Silvia; Baric, Sanja

    2012-11-01

    Apple proliferation (AP) represents a serious threat to several fruit-growing areas and is responsible for great economic losses. Several studies have highlighted the key role played by the cell wall in response to pathogen attack. The existence of a cell wall integrity signaling pathway which senses perturbations in the cell wall architecture upon abiotic/biotic stresses and activates specific defence responses has been widely demonstrated in plants. More recently a role played by cell wall-related genes has also been reported in plants infected by phytoplasmas. With the aim of shedding light on the cell wall response to AP disease in the economically relevant fruit-tree Malus × domestica Borkh., we investigated the expression of the cellulose (CesA) and callose synthase (CalS) genes in different organs (i.e., leaves, roots and branch phloem) of healthy and infected symptomatic outdoor-grown trees, sampled over the course of two time points (i.e., spring and autumn 2011), as well as in in vitro micropropagated control and infected plantlets. A strong up-regulation in the expression of cell wall biosynthetic genes was recorded in roots from infected trees. Secondary cell wall CesAs showed up-regulation in the phloem tissue from branches of infected plants, while either a down-regulation of some genes or no major changes were observed in the leaves. Micropropagated plantlets also showed an increase in cell wall-related genes and constitute a useful system for a general assessment of gene expression analysis upon phytoplasma infection. Finally, we also report the presence of several 'knot'-like structures along the roots of infected apple trees and discuss the occurrence of this interesting phenotype in relation to the gene expression results and the modalities of phytoplasma diffusion.

  6. A gene expression analysis of cell wall biosynthetic genes in Malus × domestica infected by ‘Candidatus Phytoplasma mali’

    Science.gov (United States)

    Guerriero, Gea; Giorno, Filomena; Ciccotti, Anna Maria; Schmidt, Silvia; Baric, Sanja

    2016-01-01

    Apple proliferation (AP) represents a serious threat to several fruit-growing areas and is responsible for great economic losses. Several studies have highlighted the key role played by the cell wall in response to pathogen attack. The existence of a cell wall integrity signaling pathway which senses perturbations in the cell wall architecture upon abiotic/biotic stresses and activates specific defence responses has been widely demonstrated in plants. More recently a role played by cell wall-related genes has also been reported in plants infected by phytoplasmas. With the aim of shedding light on the cell wall response to AP disease in the economically relevant fruit-tree Malus × domestica Borkh., we investigated the expression of the cellulose (CesA) and callose synthase (CalS) genes in different organs (i.e., leaves, roots and branch phloem) of healthy and infected symptomatic outdoor-grown trees, sampled over the course of two time points (i.e., spring and autumn 2011), as well as in in vitro micropropagated control and infected plantlets. A strong up-regulation in the expression of cell wall biosynthetic genes was recorded in roots from infected trees. Secondary cell wall CesAs showed up-regulation in the phloem tissue from branches of infected plants, while either a down-regulation of some genes or no major changes were observed in the leaves. Micropropagated plantlets also showed an increase in cell wall-related genes and constitute a useful system for a general assessment of gene expression analysis upon phytoplasma infection. Finally, we also report the presence of several ‘knot’-like structures along the roots of infected apple trees and discuss the occurrence of this interesting phenotype in relation to the gene expression results and the modalities of phytoplasma diffusion. PMID:23086810

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

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    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.

  8. 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.

  9. 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

  10. 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.

  11. The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Motawie, Mohammed Saddik; Olsen, Carl Erik

    2016-01-01

    for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated...... experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O...

  12. Gln3-Gcn4 hybrid transcriptional activator determines catabolic and biosynthetic gene expression in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Hernández, Hugo; Aranda, Cristina; Riego, Lina; González, Alicia

    2011-01-21

    The yeast Saccharomyces cerevisiae is able to sense the availability and quality of nitrogen sources and the intrinsic variation of amino acid disponibility for protein synthesis. When this yeast is provided with secondary nitrogen sources, transcription of genes encoding enzymes involved in their catabolism is elicited through the action of Gln3, which constitutes the main activator of the Nitrogen Catabolite Repression network (NCR). Activation of genes encoding enzymes involved in the amino acid biosynthetic pathways is achieved through the action of the GCN4-encoded transcriptional modulator whose transcriptional activation is induced at the translational level by limitation for any amino acid. Thus the role of each one of these activators had been secluded to either catabolic or biosynthetic pathways. However, some observations have suggested that under peculiar physiological conditions, Gln3 and Gcn4 could act simultaneously in order to contemporaneously increase expression of both sets of genes. This paper addresses the question of whether Gln3 and Gcn4 cooperatively determine expression of their target genes. Results presented herein show that induced expression of catabolic and biosynthetic genes when cells are grown under nitrogen derepressive conditions and amino acid deprivation is dependent on the concurrent action of Gln3 and Gcn4, which form part of a unique transcriptional complex. We propose that the combination of Gln3 and Gcn4 results in the constitution of a hybrid modulator which elicits a novel transcriptional response, not evoked when these modulators act in a non-combinatorial fashion. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. Differential expression of anthocyanin biosynthetic genes and transcription factor PcMYB10 in pears (Pyrus communis L.).

    Science.gov (United States)

    Li, Li; Ban, Zhao-Jun; Li, Xi-Hong; Wu, Mao-Yu; Wang, Ai-Li; Jiang, Yu-Qian; Jiang, Yun-Hong

    2012-01-01

    Anthocyanin biosynthesis in various plants is affected by environmental conditions and controlled by the transcription level of the corresponding genes. In pears (Pyrus communis cv. 'Wujiuxiang'), anthocyanin biosynthesis is significantly induced during low temperature storage compared with that at room temperature. We further examined the transcriptional levels of anthocyanin biosynthetic genes in 'Wujiuxiang' pears during developmental ripening and temperature-induced storage. The expression of genes that encode flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, UDP-glucose: flavonoid 3-O-glucosyltransferase, and R2R3 MYB transcription factor (PcMYB10) was strongly positively correlated with anthocyanin accumulation in 'Wujiuxiang' pears in response to both developmental and cold-temperature induction. Hierarchical clustering analysis revealed the expression patterns of the set of target genes, of which PcMYB10 and most anthocyanin biosynthetic genes were related to the same cluster. The present work may help explore the molecular mechanism that regulates anthocyanin biosynthesis and its response to abiotic stress at the transcriptional level in plants.

  14. Differential expression of anthocyanin biosynthetic genes and transcription factor PcMYB10 in pears (Pyrus communis L..

    Directory of Open Access Journals (Sweden)

    Li Li

    Full Text Available Anthocyanin biosynthesis in various plants is affected by environmental conditions and controlled by the transcription level of the corresponding genes. In pears (Pyrus communis cv. 'Wujiuxiang', anthocyanin biosynthesis is significantly induced during low temperature storage compared with that at room temperature. We further examined the transcriptional levels of anthocyanin biosynthetic genes in 'Wujiuxiang' pears during developmental ripening and temperature-induced storage. The expression of genes that encode flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, UDP-glucose: flavonoid 3-O-glucosyltransferase, and R2R3 MYB transcription factor (PcMYB10 was strongly positively correlated with anthocyanin accumulation in 'Wujiuxiang' pears in response to both developmental and cold-temperature induction. Hierarchical clustering analysis revealed the expression patterns of the set of target genes, of which PcMYB10 and most anthocyanin biosynthetic genes were related to the same cluster. The present work may help explore the molecular mechanism that regulates anthocyanin biosynthesis and its response to abiotic stress at the transcriptional level in plants.

  15. IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites.

    Science.gov (United States)

    Hadjithomas, Michalis; Chen, I-Min Amy; Chu, Ken; Ratner, Anna; Palaniappan, Krishna; Szeto, Ernest; Huang, Jinghua; Reddy, T B K; Cimermančič, Peter; Fischbach, Michael A; Ivanova, Natalia N; Markowitz, Victor M; Kyrpides, Nikos C; Pati, Amrita

    2015-07-14

    In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of "big" genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in Alphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules. IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to

  16. Unique actinomycetes from marine caves and coral reef sediments provide novel PKS and NRPS biosynthetic gene clusters.

    Science.gov (United States)

    Hodges, Tyler W; Slattery, Marc; Olson, Julie B

    2012-06-01

    In the ever-expanding search for novel bioactive molecules and enzymes, marine actinomycetes have proven to be a productive source. While open reef sediment and sponge-associated actinomycetes have been extensively examined, their marine cave counterparts remain unevaluated. Anchialine cave systems in the Bahamas offered an ideal setting to evaluate the occurrence and variation within sediment-associated actinomycete communities. While in close geographical proximity to open reef environments, these systems provide a specialized environmental niche devoid of light and direct exposure to nutrient input. In the present study, selective isolation techniques and molecular methods were used to test the hypothesis that variable distribution of actinomycetes and secondary metabolite gene clusters occur between open reef and marine cave systems. The results indicated that differences exist within the culturable sediment-associated actinomycete communities between marine caves and open reef systems, with members of the genus Streptomyces dominating cultures from open reef sediments and a more diverse suite of actinomycetes isolated from marine cave sediment samples. Within the cave isolates, members of the proposed genus Solwaraspora were the most represented. Based on PKS- and NRPS-gene-targeted PCR amplification and sequencing, geographic variation in the occurrence of these biosynthetic pathways was also observed. These findings indicate that marine cave systems are a lucrative source in the search for novel secondary metabolite producers with biotechnological applications and that environmental and geographic factors likely affect the occurrence of these biosynthetic pathways.

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

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    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.

  18. Expression of ethylene biosynthetic and receptor genes in rose floral tissues during ethylene-enhanced flower opening.

    Science.gov (United States)

    Xue, Jingqi; Li, Yunhui; Tan, Hui; Yang, Feng; Ma, Nan; Gao, Junping

    2008-01-01

    Ethylene production, as well as the expression of ethylene biosynthetic (Rh-ACS1-4 and Rh-ACO1) and receptor (Rh-ETR1-5) genes, was determined in five different floral tissues (sepals, petals, stamens, gynoecia, and receptacles) of cut rose (Rosa hybrida cv. Samantha upon treatment with ethylene or the ethylene inhibitor 1-methylcyclopropene (1-MCP). Ethylene-enhanced ethylene production occurred only in gynoecia, petals, and receptacles, with gynoecia showing the greatest enhancement in the early stage of ethylene treatment. However, 1-MCP did not suppress ethylene production in these three tissues. In sepals, ethylene production was highly decreased by ethylene treatment, and increased dramatically by 1-MCP. Ethylene production in stamens remained unchanged after ethylene or 1-MCP treatment. Induction of certain ethylene biosynthetic genes by ethylene in different floral tissues was positively correlated with the ethylene production, and this induction was also not suppressed by 1-MCP. The expression of Rh-ACS2 and Rh-ACS3 was quickly induced by ethylene in gynoecia, but neither Rh-ACS1 nor Rh-ACS4 was induced by ethylene in any of the five tissues. In addition, Rh-ACO1 was induced by ethylene in all floral tissues except sepals. The induced expression of ethylene receptor genes by ethylene was much faster in gynoecia than in petals, and the expression of Rh-ETR3 was strongly suppressed by 1-MCP in all floral tissues. These results indicate that ethylene biosynthesis in gynoecia is regulated developmentally, rather than autocatalytically. The response of rose flowers to ethylene occurs initially in gynoecia, and ethylene may regulate flower opening mainly through the Rh-ETR3 gene in gynoecia.

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

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    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.

  20. The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

    OpenAIRE

    Behrooz Darbani; Mohammed Saddik Motawia; Carl Erik Olsen; Nour-Eldin, Hussam H.; Birger Lindberg Møller; Fred Rook

    2016-01-01

    Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expresse...

  1. Genetic interrelations in the actinomycin biosynthetic gene clusters of Streptomyces antibioticus IMRU 3720 and Streptomyces chrysomallus ATCC11523, producers of actinomycin X and actinomycin C

    Directory of Open Access Journals (Sweden)

    Crnovčić I

    2017-04-01

    Full Text Available Ivana Crnovčić,1 Christian Rückert,2 Siamak Semsary,1 Manuel Lang,1 Jörn Kalinowski,2 Ullrich Keller1 1Institut für Chemie, Technische Universität Berlin, Berlin-Charlottenburg, 2Technology Platform Genomics, Center for Biotechnology, Bielefeld University, Bielefeld, Germany Abstract: Sequencing the actinomycin (acm biosynthetic gene cluster of Streptomyces antibioticus IMRU 3720, which produces actinomycin X (Acm X, revealed 20 genes organized into a highly similar framework as in the bi-armed acm C biosynthetic gene cluster of Streptomyces chrysomallus but without an attached additional extra arm of orthologues as in the latter. Curiously, the extra arm of the S. chrysomallus gene cluster turned out to perfectly match the single arm of the S. antibioticus gene cluster in the same order of orthologues including the the presence of two pseudogenes, scacmM and scacmN, encoding a cytochrome P450 and its ferredoxin, respectively. Orthologues of the latter genes were both missing in the principal arm of the S. chrysomallus acm C gene cluster. All orthologues of the extra arm showed a G +C-contents different from that of their counterparts in the principal arm. Moreover, the similarities of translation products from the extra arm were all higher to the corresponding translation products of orthologue genes from the S. antibioticus acm X gene cluster than to those encoded by the principal arm of their own gene cluster. This suggests that the duplicated structure of the S. chrysomallus acm C biosynthetic gene cluster evolved from previous fusion between two one-armed acm gene clusters each from a different genetic background. However, while scacmM and scacmN in the extra arm of the S. chrysomallus acm C gene cluster are mutated and therefore are non-functional, their orthologues saacmM and saacmN in the S. antibioticus acm C gene cluster show no defects seemingly encoding active enzymes with functions specific for Acm X biosynthesis. Both acm

  2. Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family.

    Science.gov (United States)

    Pearce, Stephen; Huttly, Alison K; Prosser, Ian M; Li, Yi-dan; Vaughan, Simon P; Gallova, Barbora; Patil, Archana; Coghill, Jane A; Dubcovsky, Jorge; Hedden, Peter; Phillips, Andrew L

    2015-06-05

    The gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis. The wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1β-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp. The comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD

  3. Characterization of biosynthetic genes of ascamycin/dealanylascamycin featuring a 5'-O-sulfonamide moiety in Streptomyces sp. JCM9888.

    Directory of Open Access Journals (Sweden)

    Chunhua Zhao

    Full Text Available Ascamycin (ACM and dealanylascamycin (DACM are nucleoside antibiotics elaborated by Streptomyces sp. JCM9888. The later shows broad spectrum inhibition activity to various gram-positive and gram-negative bacteria, eukaryotic Trypanosoma and is also toxic to mice, while ascamycin is active against very limited microorganisms, such as Xanthomonas. Both compounds share an unusual 5'-O-sulfonamide moiety which is attached to an adenosine nucleoside. In this paper, we first report on the 30 kb gene cluster (23 genes, acmA to acmW involved in the biosynthesis of these two antibiotics and a biosynthetic assembly line was proposed. Of them, six genes (AcmABGKIW are hypothetical genes involved in 5'-O-sulfonamide formation. Two flavin adenine dinucleotide (FAD-dependent chlorinase genes acmX and acmY were characterized which are significantly remote from acmA-W and postulated to be required for adenine C2-halogenation. Notably gene disruption of acmE resulted in a mutant which could only produce dealanylascamycin but was blocked in its ability to biosynthesize ascamycin, revealing its key role of conversion of dealanylascamycin to ascamycin.

  4. Genetic localization and heterologous expression of validamycin biosynthetic gene cluster isolated from Streptomyces hygroscopicus var. limoneus KCCM 11405 (IFO 12704).

    Science.gov (United States)

    Singh, Deepak; Seo, Myung-Ji; Kwon, Hyung-Jin; Rajkarnikar, Arishma; Kim, Kyoung-Rok; Kim, Soon-Ok; Suh, Joo-Won

    2006-07-05

    The validamycin biosynthetic gene cluster was isolated from Streptomyces hygroscopicus var. limoneus KTCC 1715 (IFO 12704) using a pair of degenerated PCR primers designed from the sequence of AcbC, 2-epi-5-epi-valiolone synthase in the acarbose biosynthesis. The nucleotide sequence analysis of the 37-kb DNA region revealed 22 complete ORFs including vldA, the acbC ortholog. Located around vldA, vldB to K were predicted to encode adenyltransferase, kinase, ketoreductase (or epimerase/dehydratase), glycosyltransferase, aminotransferase, dehydrogenase, phosphatase/phosphomutase, glycosyl hydrolase, transport protein, and glycosyltransferase, respectively. Apparently absent were any regulatory components within the sequenced region. The disruption of vldA abolished the validamycin biosynthesis and the plasmid-based complementation with vldABC restored production to the vldA-mutant; this substantiated that vldABC are essential to validamycin biosynthesis. This finding enabled us to discover the complete validamycin biosynthetic cluster. The cosmid clone of pJWS3001 harboring the 37-kb DNA region conferred validamycin-accumulation to Streptomyces lividans, indicating that the entire gene cluster of validamycin biosynthesis had been isolated. Additionally, Streptomyces albus, transformed with pJWS3001, produced a high level of alpha-glucosidase inhibitory activity in a R2YE liquid culture, which highlights the portability of the cluster within Streptomyces. The product of vldI was characterized as a glucoamylase (kcat, 32 s(-1); K(m), 5 mg/ml of starch) that does not play any apparent role in the validamycin biosynthesis. In order to characterize the upstream region, a vldW knockout was achieved via gene-replacement. A phenotypic study of the resulting mutant revealed that vldW is not essential for the host's ability to control Pellicularia filamentosa growth. The current information suggests that vldA to vldH is the genetic region essential to validamycin biosynthesis

  5. Inhibition of copper uptake in yeast reveals the copper transporter Ctr1p as a potential molecular target of saxitoxin.

    Science.gov (United States)

    Cusick, Kathleen D; Minkin, Steven C; Dodani, Sheel C; Chang, Christopher J; Wilhelm, Steven W; Sayler, Gary S

    2012-03-06

    Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria which targets voltage-gated sodium and potassium channels in higher vertebrates. However, its molecular target in planktonic aquatic community members that co-occur with the toxin producers remains unknown. Previous microarray analysis with yeast identified copper and iron-homeostasis genes as being differentially regulated in response to saxitoxin. This study sought to identify the molecular target in microbial cells by comparing the transcriptional profiles of key copper and iron homeostasis genes (CTR1, FRE1, FET3, CUP1, CRS5) in cells exposed to saxitoxin, excess copper, excess iron, an extracellular Cu(I) chelator, or an intracellular Cu(I) chelator. Protein expression and localization of Ctr1p (copper transporter), Fet3p (multicopper oxidase involved in high-affinity iron uptake), and Aft1p (iron regulator) were also compared among treatments. Combined transcript and protein profiles suggested saxitoxin inhibited copper uptake. This hypothesis was confirmed by intracellular Cu(I) imaging with a selective fluorescent probe for labile copper. On the basis of the combined molecular and physiological results, a model is presented in which the copper transporter Ctr1p serves as a molecular target of saxitoxin and these observations are couched in the context of the eco-evolutionary role this toxin may serve for species that produce it.

  6. Genomic characterization of a new endophytic Streptomyces kebangsaanensis identifies biosynthetic pathway gene clusters for novel phenazine antibiotic production

    Directory of Open Access Journals (Sweden)

    Juwairiah Remali

    2017-11-01

    Full Text Available Background Streptomyces are well known for their capability to produce many bioactive secondary metabolites with medical and industrial importance. Here we report a novel bioactive phenazine compound, 6-((2-hydroxy-4-methoxyphenoxy carbonyl phenazine-1-carboxylic acid (HCPCA extracted from Streptomyces kebangsaanensis, an endophyte isolated from the ethnomedicinal Portulaca oleracea. Methods The HCPCA chemical structure was determined using nuclear magnetic resonance spectroscopy. We conducted whole genome sequencing for the identification of the gene cluster(s believed to be responsible for phenazine biosynthesis in order to map its corresponding pathway, in addition to bioinformatics analysis to assess the potential of S. kebangsaanensis in producing other useful secondary metabolites. Results The S. kebangsaanensis genome comprises an 8,328,719 bp linear chromosome with high GC content (71.35% consisting of 12 rRNA operons, 81 tRNA, and 7,558 protein coding genes. We identified 24 gene clusters involved in polyketide, nonribosomal peptide, terpene, bacteriocin, and siderophore biosynthesis, as well as a gene cluster predicted to be responsible for phenazine biosynthesis. Discussion The HCPCA phenazine structure was hypothesized to derive from the combination of two biosynthetic pathways, phenazine-1,6-dicarboxylic acid and 4-methoxybenzene-1,2-diol, originated from the shikimic acid pathway. The identification of a biosynthesis pathway gene cluster for phenazine antibiotics might facilitate future genetic engineering design of new synthetic phenazine antibiotics. Additionally, these findings confirm the potential of S. kebangsaanensis for producing various antibiotics and secondary metabolites.

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

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    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.

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

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    Samiha Sioud

    2007-01-01

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

  9. Cell wall composition and lignin biosynthetic gene expression along a developmental gradient in an Australian sugarcane cultivar

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    William P. Bewg

    2017-12-01

    Full Text Available Sugarcane bagasse is an abundant source of lignocellulosic material for bioethanol production. Utilisation of bagasse for biofuel production would be environmentally and economically beneficial, but the recalcitrance of lignin continues to provide a challenge. Further understanding of lignin production in specific cultivars will provide a basis for modification of genomes for the production of phenotypes with improved processing characteristics. Here we evaluated the expression profile of lignin biosynthetic genes and the cell wall composition along a developmental gradient in KQ228 sugarcane. The expression levels of nine lignin biosynthesis genes were quantified in five stem sections of increasing maturity and in root tissue. Two distinct expression patterns were seen. The first saw highest gene expression in the youngest tissue, with expression decreasing as tissue matured. The second pattern saw little to no change in transcription levels across the developmental gradient. Cell wall compositional analysis of the stem sections showed total lignin content to be significantly higher in more mature tissue than in the youngest section assessed. There were no changes in structural carbohydrates across developmental sections. These gene expression and cell wall compositional patterns can be used, along with other work in grasses, to inform biotechnological approaches to crop improvement for lignocellulosic biofuel production.

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

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    Royah Vaezi

    2013-12-01

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

  11. 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.

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

    Science.gov (United States)

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

    2015-01-01

    Plant growth and secondary metabolism are commonly regulated by external cues such as light, temperature and water availability. In this study, the influences of low and high temperatures, dehydration, photoperiods, and different growing stages on the changes of steviol glycosides (SGs) contents and transcription levels of fifteen genes involved in SGs biosynthesis of Stevia rebaudiana Bertoni were examined using HPLC and RT-PCR. The observations showed that the transcript levels of all the fifteen genes were maximum under 25 °C treatment, and the transcription of SrDXS, SrDXR, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI, SrGGDPS, SrCPPS1, SrUGT85C2 and SrUGT76G1 were restrained both in low temperature (15 °C) and high temperature (35 °C). Most genes in SGs biosynthesis pathway exhibited down-regulation in dehydration. To elucidate the effect of photoperiods, the plants were treated by different simulated photoperiods (8 L/16 D, 1 0L/14 D, 14 L/10 D and 16 L/8 D), but no significant transcription changes were observed. In the study of growing stages, there were evident changes of SGs contents, and the transcript levels of all the fifteen genes were minimal in fast growing period, and exhibited evident increase both in flower-bud appearing stage and flowering stage. The obtained results strongly suggest that the effect of environmental cues on steviol glycosides contents and transcription of corresponding biosynthetic genes in S. rebaudiana is significant. It is worth to study deeply. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  13. 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.

  14. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway

    Science.gov (United States)

    Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

  15. 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.

  16. Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis.

    Science.gov (United States)

    Baud, Sébastien; Wuillème, Sylvie; To, Alexandra; Rochat, Christine; Lepiniec, Loïc

    2009-12-01

    The WRINKLED1 (WRI1) protein is an important regulator of oil accumulation in maturing Arabidopsis seeds. WRI1 is a member of a plant-specific family of transcription factors (AP2/EREBP) that share either one or two copies of a DNA-binding domain called the AP2 domain. Here, it is shown that WRI1 acts as a transcriptional enhancer of genes involved in carbon metabolism in transgenic seeds overexpressing this transcription factor. PKp-beta1 and BCCP2, two genes encoding enzymes of the glycolysis and fatty acid biosynthetic pathway, respectively, have been chosen to investigate the regulatory action exerted by WRI1 over these pathways. Using the reporter gene uidA, it was possible to demonstrate in planta that WRI1 regulates the activity of both PKp-beta1 and BCCP2 promoters. Electrophoretic mobility-shift assays and yeast one-hybrid experiments showed that WRI1 was able to interact with the BCCP2 promoter. To further elucidate the regulatory mechanism controlling the transcription of these genes, functional dissections of PKp-beta1 and BCCP2 promoters were performed. Two enhancers, of 54 and 79 bp, respectively, have thus been isolated that are essential to direct the activity of these promoters in oil-accumulating tissues of the embryo. A consensus site is present in these enhancers as well as in other putative target promoters of WRI1. Loss of this consensus sequence in the BCPP2 promoter decreases both the strength of the interaction between WRI1 and this promoter in yeast and the activity of the promoter in planta.

  17. 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. © 2015 Institute of Food Technologists®

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

    Science.gov (United States)

    Jensen, Jacob K.; Johnson, Nathan; Wilkerson, Curtis G.

    2013-01-01

    The exact biochemical steps of xylan backbone synthesis remain elusive. In Arabidopsis, three non-redundant genes from two glycosyltransferase (GT) families, IRX9 and IRX14 from GT43 and IRX10 from GT47, are candidates for forming the xylan backbone. In other plants, evidence exists that different tissues express these three genes at widely different levels, which suggests that diversity in the makeup of the xylan synthase complex exists. Recently we have profiled the transcripts present in the developing mucilaginous tissue of psyllium (Plantago ovata Forsk). This tissue was found to have high expression levels of an IRX10 homolog, but very low levels of the two GT43 family members. This contrasts with recent wheat endosperm tissue profiling that found a relatively high abundance of the GT43 family members. We have performed an in-depth analysis of all GTs genes expressed in four developmental stages of the psyllium mucilagenous layer and in a single stage of the psyllium stem using RNA-Seq. This analysis revealed several IRX10 homologs, an expansion in GT61 (homologs of At3g18170/At3g18180), and several GTs from other GT families that are highly abundant and specifically expressed in the mucilaginous tissue. Our current hypothesis is that the four IRX10 genes present in the mucilagenous tissues have evolved to function without the GT43 genes. These four genes represent some of the most divergent IRX10 genes identified to date. Conversely, those present in the psyllium stem are very similar to those in other eudicots. This suggests these genes are under selective pressure, likely due to the synthesis of the various xylan structures present in mucilage that has a different biochemical role than that present in secondary walls. The numerous GT61 family members also show a wide sequence diversity and may be responsible for the larger number of side chain structures present in the psyllium mucilage. PMID:23761806

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

    Directory of Open Access Journals (Sweden)

    Jacob Kruger Jensen

    2013-06-01

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

  20. Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties

    OpenAIRE

    Hoang, L.T.; Innes, David J.; Shaw, P. Nicholas; Monteith, Gregory R; Gidley, Michael J; Dietzgen, Ralf G.

    2015-01-01

    Background Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and man...

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

    Directory of Open Access Journals (Sweden)

    L. Gavrilovic

    2009-12-01

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

  2. The Unique Biosynthetic Route from Lupinus β-Conglutin Gene to Blad

    Science.gov (United States)

    Monteiro, Sara; Freitas, Regina; Rajasekhar, Baru T.; Teixeira, Artur R.; Ferreira, Ricardo B.

    2010-01-01

    Background During seed germination, β-conglutin undergoes a major cycle of limited proteolysis in which many of its constituent subunits are processed into a 20 kDa polypeptide termed blad. Blad is the main component of a glycooligomer, accumulating exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Principal Findings The sequence of the gene encoding β-conglutin precursor (1791 nucleotides) is reported. This gene, which shares 44 to 57% similarity and 20 to 37% identity with other vicilin-like protein genes, includes several features in common with these globulins, but also specific hallmarks. Most notable is the presence of an ubiquitin interacting motif (UIM), which possibly links the unique catabolic route of β-conglutin to the ubiquitin/proteasome proteolytic pathway. Significance Blad forms through a unique route from and is a stable intermediary product of its precursor, β-conglutin, the major Lupinus seed storage protein. It is composed of 173 amino acid residues, is encoded by an intron-containing, internal fragment of the gene that codes for β-conglutin precursor (nucleotides 394 to 913) and exhibits an isoelectric point of 9.6 and a molecular mass of 20,404.85 Da. Consistent with its role as a storage protein, blad contains an extremely high proportion of the nitrogen-rich amino acids. PMID:20066045

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

    Directory of Open Access Journals (Sweden)

    Jason King

    2010-06-01

    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.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT...

  5. Analysis of the Dendrobium officinale transcriptome reveals putative alkaloid biosynthetic genes and genetic markers.

    Science.gov (United States)

    Guo, Xu; Li, Ying; Li, Chunfang; Luo, Hongmei; Wang, Lizhi; Qian, Jun; Luo, Xiang; Xiang, Li; Song, Jingyuan; Sun, Chao; Xu, Haibin; Yao, Hui; Chen, Shilin

    2013-09-15

    Dendrobium officinale Kimura et Migo (Orchidaceae) is a traditional Chinese medicinal plant. The stem contains an alkaloid that is the primary bioactive component. However, the details of alkaloid biosynthesis have not been effectively explored because of the limited number of expressed sequence tags (ESTs) available in GenBank. In this study, we analyzed RNA isolated from the stem of D. officinale using a single half-run on the Roche 454 GS FLX Titanium platform to generate 553,084 ESTs with an average length of 417 bases. The ESTs were assembled into 36,407 unique putative transcripts. A total of 69.97% of the unique sequences were annotated, and a detailed view of alkaloid biosynthesis was obtained. Functional assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) terms revealed 69 unique sequences representing 25 genes involved in alkaloid backbone biosynthesis. A series of qRT-PCR experiments confirmed that the expression levels of 5 key enzyme-encoding genes involved in alkaloid biosynthesis are greater in the leaves of D. officinale than in the stems. Cytochrome P450s, aminotransferases, methyltransferases, multidrug resistance protein (MDR) transporters and transcription factors were screened for possible involvement in alkaloid biosynthesis. Furthermore, a total of 1061 simple sequence repeat motifs (SSR) were detected from 36,407 unigenes. Dinucleotide repeats were the most abundant repeat type. Of these, 179 genes were associated with a metabolic pathway in KEGG. This study is the first to produce a large volume of transcriptome data from D. officinale. It extends the foundation to facilitate gene discovery in D. officinale and provides an important resource for the molecular genetic and functional genomic studies in this species. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. 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

    2017-01-04

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

    Zheng, Xiasheng; Xu, Hui; Ma, Xinye; Zhan, Ruoting; Chen, Weiwen

    2014-01-01

    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. PMID:24722569

  8. The thiazolidinedione pioglitazone increases cholesterol biosynthetic gene expression in primary cortical neurons by a PPARgamma-independent mechanism.

    Science.gov (United States)

    Cocks, Graham; Wilde, Jonathan I; Graham, Simon J; Bousgouni, Vicky; Virley, David; Lovestone, Simon; Richardson, Jill

    2010-01-01

    In a recent clinical study, the thiazolidinedione (TZD) pioglitazone (Actos was reported to preserve cognitive function in patients with mild to moderate Alzheimer's disease and type II diabetes mellitus. TZDs are agonists of the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARgamma), are peripheral insulin sensitizers, and have recently been reported to increase mitochondrial biogenesis in the central nervous system and dendritic spine density. We report a transcriptional profile of the TZD pioglitazone and the non-TZD PPARgamma agonist GW347845 in primary cortical culture. We observed that pioglitazone, but not GW347845, increased cholesterol biosynthetic and lipogenic gene expression after 6 h, and the expression of the cholesterol efflux transporters Abca1 and Abcg1 after 24 h. Co-treatment of pioglitazone with the PPARgamma antagonist GW9662 did not significantly reduce these effects, suggesting a PPARgamma-independent mechanism. These findings suggest a novel effect of TZDs in neurons that may be of relevance as a novel approach against Alzheimer's disease.

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

    Directory of Open Access Journals (Sweden)

    Jungsuwadee Paiboon

    2011-02-01

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

  10. [Analysis of transicriptomes and exploring flavonoid biosynthetic pathway genes in Lithocarpus polystachyus].

    Science.gov (United States)

    Song, Ju; Huang, Jian; Li, Zhi-Dong; Long, Yue-Hong; Xing, Zhao-Bin

    2017-02-01

    The sweet taste and health effect of Lithocarpus polystachyus are mainly related flavonoid. To obtain Lithocarpus transcriptome database and flavonoid biosynthesis-related genes, the RNA-Seq techology (Illumina HiSeq 4000) was used to sequence its transcriptome. Six Gb database was assembled after assembly steps, and 41 043 of L. polystachyus unigenes were obtained. With blasting them with 7 data banks, all unigenes were involved in 51 GO-terms and 237 metabolic pathways. And furthermore 28 genes of the flavonoid biosynthesis-related were found. After using the MicroSatallite, 18 161 SSR were obtained, the single-nucleotide-repeated was the richest at 7 346. These data represent abundant messages about transcripts and provide valuable genome data sources in molecular biology of L. polystachyus. Copyright© by the Chinese Pharmaceutical Association.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Accumulation of tilianin and rosmarinic acid and expression of phenylpropanoid biosynthetic genes in Agastache rugosa.

    Science.gov (United States)

    Tuan, Pham Anh; Park, Woo Tae; Xu, Hui; Park, Nam Il; Park, Sang Un

    2012-06-13

    Korean mint (Agastache rugosa), a perennial, medicinal plant of the Labiatae family, has many useful constituents, including monoterpenes and phenylpropanoids. Among these, tilianin and rosmarinic acid, 2 well-known natural products, have many pharmacologically useful properties. Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first and second committed steps in the phenylpropanoid pathway of plants, leading to the production of tilianin. In this study, cDNAs encoding CHS (ArCHS) and CHI (ArCHI) were isolated from A. rugosa using rapid amplification of cDNA ends (RACE)-PCR. Amino acid sequence alignments showed that ArCHS and ArCHI shared high sequence identity and active sites with their respective orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of genes involved in tilianin and rosmarinic acid biosyntheses in the flowers, leaves, stems, and roots of A. rugosa. High-performance liquid chromatography (HPLC) revealed that the accumulation pattern of tilianin matched the expression patterns of ArCHS and ArCHI in different organs of A. rugosa. Moreover, acacetin, the precursor of tilianin, also demonstrated an accumulation pattern congruent with the expression of these 2 genes. The transcription levels of ArPAL, ArC4H, and Ar4CL were the highest in the leaves or flowers of the plant, which also contained a relatively high amount of rosmarinic acid. However, the roots showed a significant content of rosmarinic acid, although the transcription of ArPAL, ArC4H, and Ar4CL were low. The findings of our study support the medicinal usefulness of A. rugosa and indicate targets for increasing tilianin and rosmarinic acid production in this plant.

  13. The hedgehog pathway gene shifted functions together with the hmgcr-dependent isoprenoid biosynthetic pathway to orchestrate germ cell migration.

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    Girish Deshpande

    Full Text Available The Drosophila embryonic gonad is assembled from two distinct cell types, the Primordial Germ Cells (PGCs and the Somatic Gonadal Precursor cells (SGPs. The PGCs form at the posterior of blastoderm stage embryos and are subsequently carried inside the embryo during gastrulation. To reach the SGPs, the PGCs must traverse the midgut wall and then migrate through the mesoderm. A combination of local repulsive cues and attractive signals emanating from the SGPs guide migration. We have investigated the role of the hedgehog (hh pathway gene shifted (shf in directing PGC migration. shf encodes a secreted protein that facilitates the long distance transmission of Hh through the proteoglycan matrix after it is released from basolateral membranes of Hh expressing cells in the wing imaginal disc. shf is expressed in the gonadal mesoderm, and loss- and gain-of-function experiments demonstrate that it is required for PGC migration. Previous studies have established that the hmgcr-dependent isoprenoid biosynthetic pathway plays a pivotal role in generating the PGC attractant both by the SGPs and by other tissues when hmgcr is ectopically expressed. We show that production of this PGC attractant depends upon shf as well as a second hh pathway gene gγ1. Further linking the PGC attractant to Hh, we present evidence indicating that ectopic expression of hmgcr in the nervous system promotes the release/transmission of the Hh ligand from these cells into and through the underlying mesodermal cell layer, where Hh can contact migrating PGCs. Finally, potentiation of Hh by hmgcr appears to depend upon cholesterol modification.

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

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    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

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

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    Luo Hongmei

    2011-12-01

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

  16. 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.

  17. Transcript profiling of a bitter variety of narrow-leafed lupin to discover alkaloid biosynthetic genes.

    Science.gov (United States)

    Yang, Ting; Nagy, Istvan; Mancinotti, Davide; Otterbach, Sophie Lisa; Andersen, Trine Bundgaard; Motawia, Mohammed Saddik; Asp, Torben; Geu-Flores, Fernando

    2017-11-28

    Lupins (Lupinus spp.) are nitrogen-fixing legumes that accumulate toxic alkaloids in their protein-rich beans. These anti-nutritional compounds belong to the family of quinolizidine alkaloids (QAs), which are of interest to the pharmaceutical and chemical industries. To unleash the potential of lupins as protein crops and as sources of QAs, a thorough understanding of the QA pathway is needed. However, only the first enzyme in the pathway, lysine decarboxylase (LDC), is known. Here, we report the transcriptome of a high-QA variety of narrow-leafed lupin (L. angustifolius), obtained using eight different tissues and two different sequencing technologies. In addition, we present a list of 33 genes that are closely co-expressed with LDC and that represent strong candidates for involvement in lupin alkaloid biosynthesis. One of these genes encodes a copper amine oxidase able to convert the product of LDC, cadaverine, into 1-piperideine, as shown by heterologous expression and enzyme assays. Kinetic analysis revealed a low KM value for cadaverine, supporting a role as the second enzyme in the QA pathway. Our transcriptomic data set represents a crucial step towards the discovery of enzymes, transporters, and regulators involved in lupin alkaloid biosynthesis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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

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    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.

  19. The bialaphos biosynthetic genes of Streptomyces viridochromogenes : cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus

    NARCIS (Netherlands)

    Hara, Osamu; Murakami, Takeshi; Imai, Satoshi; Anzai, Hiroyuki; Itoh, Reiko; Kumada, Yoichi; Takano, Eriko; Satoh, Eriko; Satoh, Atsuyuki; Nagaoka, Kozo; Thompson, Charles

    1991-01-01

    The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive

  20. Key biosynthetic gene subfamily recruited for pheromone production prior to the extensive radiation of Lepidoptera

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    Johansson Tomas

    2008-10-01

    Full Text Available Abstract Background Moths have evolved highly successful mating systems, relying on species-specific mixtures of sex pheromone components for long-distance mate communication. Acyl-CoA desaturases are key enzymes in the biosynthesis of these compounds and to a large extent they account for the great diversity of pheromone structures in Lepidoptera. A novel desaturase gene subfamily that displays Δ11 catalytic activities has been highlighted to account for most of the unique pheromone signatures of the taxonomically advanced ditrysian species. To assess the mechanisms driving pheromone evolution, information is needed about the signalling machinery of primitive moths. The currant shoot borer, Lampronia capitella, is the sole reported primitive non-ditrysian moth known to use unsaturated fatty-acid derivatives as sex-pheromone. By combining biochemical and molecular approaches we elucidated the biosynthesis paths of its main pheromone component, the (Z,Z-9,11-tetradecadien-1-ol and bring new insights into the time point of the recruitment of the key Δ11-desaturase gene subfamily in moth pheromone biosynthesis. Results The reconstructed evolutionary tree of desaturases evidenced two ditrysian-specific lineages (the Δ11 and Δ9 (18C>16C to have orthologs in the primitive moth L. capitella despite being absent in Diptera and other insect genomes. Four acyl-CoA desaturase cDNAs were isolated from the pheromone gland, three of which are related to Δ9-desaturases whereas the fourth cDNA clusters with Δ11-desaturases. We demonstrated that this transcript (Lca-KPVQ exclusively accounts for both steps of desaturation involved in pheromone biosynthesis. This enzyme possesses a Z11-desaturase activity that allows transforming the palmitate precursor (C16:0 into (Z-11-hexadecenoic acid and the (Z-9-tetradecenoic acid into the conjugated intermediate (Z,Z-9,11-tetradecadienoic acid. Conclusion The involvement of a single Z11-desaturase in pheromone

  1. Impacts of environmental stress on growth, secondary metabolite biosynthetic gene clusters and metabolite production of xerotolerant/xerophilic fungi.

    Science.gov (United States)

    Medina, Angel; Schmidt-Heydt, Markus; Rodríguez, Alicia; Parra, Roberto; Geisen, Rolf; Magan, Naresh

    2015-08-01

    This paper examines the impact that single and interacting environmental stress factors have on tolerance mechanisms, molecular ecology and the relationship with secondary metabolite production by a group of mycotoxigenic species of economic importance. Growth of these fungi (Aspergillus flavus, A.ochraceus, A.carbonarius, Penicillium nordicum and P. verrucosum) is influenced by water and temperature interactions and type of solute used to induce water stress. Such abiotic stresses are overcome by the synthesis of increased amounts of low molecular weight sugar alcohols, especially glycerol and erythritol, to enable them to remain active under abiotic stress. This is accompanied by increased expression of sugar transporter genes, e.g., in A. flavus, which provides the nutritional means of tolerating such stress. The optimum conditions of water activity (a w) × temperature stress for growth are often different from those for secondary metabolite production. The genes for toxin production are clustered together and their relative expression is influenced by abiotic interacting stress factors. For example., A. flavus synthesises aflatoxins under water stress in non-ionic solutes. In contrast, P. nordicum specifically occupies a high salt (0.87 a w = 22% NaCl) niche such as cured meats, and produces ochratoxin A (OTA). There is differential and temporal expression of the genes in the secondary metabolite clusters in response to a w × temperature stress. We have used a microarray and integrated data on growth, relative expression of key genes in the biosynthetic pathways for secondary metabolite production and toxin production using a mixed growth model. This was used to correlate these factors and predict the toxin levels produced under different abiotic stress conditions. This system approach to integrate these different data sets and model the relationships could be a powerful tool for predicting the relative toxin production under extreme stress conditions

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

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    Laura J Searle

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

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

    Science.gov (United States)

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

    2002-02-01

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

  4. Redirection of tryptophan metabolism in tobacco by ectopic expression of an Arabidopsis indolic glucosinolate biosynthetic gene.

    Science.gov (United States)

    Nonhebel, Heather; Yuan, Youxi; Al-Amier, Hussein; Pieck, Michael; Akor, Enne; Ahamed, Arifa; Cohen, Jerry D; Celenza, John L; Normanly, Jennifer

    2011-01-01

    Indole-3-acetaldoxime (IAOx) is a branch point compound of tryptophan (Trp) metabolism in glucosinolate-producing species such as Arabidopsis, serving as a precursor to indole-glucosinolates (IGs), the defense compound camalexin, indole-3-acetonitrile (IAN) and indole-3-acetic acid (IAA). We synthesized [(2)H(5)] and [(13)C(10)(15)N(2)]IAOx and [(13)C(6)], [(2)H(5)] and [2',2'-(2)H(2)]IAN in order to quantify endogenous IAOx and IAN in Arabidopsis and tobacco, a non-IG producing species. We found that side chain-labeled [2',2'-(2)H(2)]IAN overestimated the amount of IAN by 2-fold compared to when [(2)H(5)]IAN was used as internal standard, presumably due to protium-deuterium exchange within the internal standard during extraction of plant tissue. We also determined that [(13)C(1)]IAN underestimated the amount of IAN when the ratio of [(13)C(1)]IAN standard to endogenous IAN was greater than five to one, whereas either [(2)H(5)]IAN or [(13)C(6)]IAN showed a linear relationship with endogenous IAN over a broader range of concentrations. Transgenic tobacco vector control lines did not have detectable levels of IAOx or IAN (limit of detection∼100 pg/gfr.wt), while lines expressing either the IAOx-producing CYP79B2 or CYP79B3 genes from Arabidopsis under CaMV 35S promoter control accumulated IAOx in the range of 1-9 μg/gfr.wt. IAN levels in these lines ranged from 0.6 to 6.7 μg/gfr.wt, and IAA levels were ∼9-14-fold above levels in control lines. An Arabidopsis line expressing the same CYP79B2 overexpression construct accumulated IAOx in two of three lines measured (∼200 and 400 ng/gfr.wt) and accumulated IAN in all three lines. IAN is proposed to be a metabolite of IAOx or an enzymatic breakdown product of IGs induced upon tissue damage. Since tobacco does not produce detectable IGs, the tobacco data are consistent with IAN being a metabolite of IAOx. IAOx and IAN were also examined in the Arabidopsis activation tagged yucca mutant, and no accumulation of IAOx

  5. Genetic organization of the putative salbostatin biosynthetic gene cluster including the 2-epi-5-epi-valiolone synthase gene in Streptomyces albus ATCC 21838.

    Science.gov (United States)

    Choi, Woo Sik; Wu, Xiumei; Choeng, Yong-Hoon; Mahmud, Taifo; Jeong, Byeong Chul; Lee, Sang Hee; Chang, Yong Keun; Kim, Chang-Joon; Hong, Soon-Kwang

    2008-09-01

    The cyclization of sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone, catalyzed by the 2-epi-5-epi-valiolone synthases, is the first committed step in the biosynthesis of C( 7 )N-aminocyclitol-containing natural products, such as validamycin and acarbose. These natural products contain in their structures a valienamine unit, which is important for their biological activity. The same core unit is also found in salbostatin, a related pseudodisaccharide that has strong trehalase inhibitory activity. In silico analysis of the putative biosynthetic gene cluster of salbostatin from Streptomyces albus ATCC 21838 revealed 20 open reading frames, including an acbC homolog gene (salQ), which is believed to be involved in the biosynthesis of salbostatin. The salQ gene was overexpressed in Escherichia coli and the catalytic function of the recombinant protein was confirmed to be a 2-epi-5-epi-valiolone synthase. In addition, SalF, SalL, SalM, SalN, SalO, and SalR were found to be homologous to AcbR, AcbM, AcbL, AcbN, AcbO, and AcbP from the acarbose pathway, respectively, which suggests that the biosynthesis of C(7)N-aminocyclitol moiety of salbostatin may be very similar to that of acarbose.

  6. Molecular basis for chloronium-mediated meroterpene cyclization: cloning, sequencing, and heterologous expression of the napyradiomycin biosynthetic gene cluster.

    Science.gov (United States)

    Winter, Jaclyn M; Moffitt, Michelle C; Zazopoulos, Emmanuel; McAlpine, James B; Dorrestein, Pieter C; Moore, Bradley S

    2007-06-01

    Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.

  7. 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...

  8. Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae

    Science.gov (United States)

    2013-01-01

    Background Secondary metabolite production, a hallmark of filamentous fungi, is an expanding area of research for the Aspergilli. These compounds are potent chemicals, ranging from deadly toxins to therapeutic antibiotics to potential anti-cancer drugs. The genome sequences for multiple Aspergilli have been determined, and provide a wealth of predictive information about secondary metabolite production. Sequence analysis and gene overexpression strategies have enabled the discovery of novel secondary metabolites and the genes involved in their biosynthesis. The Aspergillus Genome Database (AspGD) provides a central repository for gene annotation and protein information for Aspergillus species. These annotations include Gene Ontology (GO) terms, phenotype data, gene names and descriptions and they are crucial for interpreting both small- and large-scale data and for aiding in the design of new experiments that further Aspergillus research. Results We have manually curated Biological Process GO annotations for all genes in AspGD with recorded functions in secondary metabolite production, adding new GO terms that specifically describe each secondary metabolite. We then leveraged these new annotations to predict roles in secondary metabolism for genes lacking experimental characterization. As a starting point for manually annotating Aspergillus secondary metabolite gene clusters, we used antiSMASH (antibiotics and Secondary Metabolite Analysis SHell) and SMURF (Secondary Metabolite Unknown Regions Finder) algorithms to identify potential clusters in A. nidulans, A. fumigatus, A. niger and A. oryzae, which we subsequently refined through manual curation. Conclusions This set of 266 manually curated secondary metabolite gene clusters will facilitate the investigation of novel Aspergillus secondary metabolites. PMID:23617571

  9. 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.

  10. Comparative study of withanolide production and the related transcriptional responses of biosynthetic genes in fungi elicited cell suspension culture of Withania somnifera in shake flask and bioreactor.

    Science.gov (United States)

    Ahlawat, Seema; Saxena, Parul; Ali, Athar; Khan, Shazia; Abdin, Malik Z

    2017-05-01

    Ashwagandha (Withania somnifera) is one of the most reputed medicinal plants in the traditional medicinal system. In this study, cell suspension culture of W. somnifera was elicited with cell homogenates of fungi (A. alternata, F. solani, V. dahliae and P. indica) in shake flask and the major withanolides like withanolide A, withaferin A and withanone were analysed. Simultaneously expression levels of key pathway genes from withanolides biosynthetic pathways were also checked via quantitative PCR in shake flask as well as in bioreactor. The results show that highest gene expression of 10.8, 5.8, 4.9, and 3.3 folds were observed with HMGR among all the expressed genes in cell suspension cultures with cell homogenates of 3% P. indica, 5% V. dahliae, 3% A. alternata and 3% F. solani, respectively, in comparison to the control in shake flask. Optimized concentration of cell homogenate of P. indica (3% v/v) was added to the growing culture in 5.0-l bioreactor under optimized up-scaling conditions and harvested after 22 days. The genes of MVA, MEP and withanolides biosynthetic pathways like HMGR, SS, SE, CAS, FPPS, DXR and DXS were up-regulated by 12.5, 4.9, 2.18, 4.65, 2.34, 1.89 and 1.4 folds, respectively in bioreactor. The enhancement of biomass (1.13 fold) and withanolides [withanolide A (1.7), withaferin A (1.5), and withanone (1.5) folds] in bioreactor in comparison to shake flask was also found to be in line with the up-regulation of genes of withanolide biosynthetic pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  11. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Contributions of vertical descent, horizontal transfer and gene loss to the distribution of mycotoxin biosynthetic gene clusters in Fusarium

    Science.gov (United States)

    The genus Fusarium produces a diverse array of mycotoxins and other secondary metabolites, but individual species contribute to only a small fraction of this diversity. Here, we employed comparative genomic and phylogenetic analyses to investigate the distribution and evolution of gene clusters resp...

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

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    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

  14. Detection of biosynthetic gene and phytohormone production by endophytic actinobacteria associated with Solanum lycopersicum and their plant-growth-promoting effect.

    Science.gov (United States)

    Passari, Ajit Kumar; Chandra, Preeti; Zothanpuia; Mishra, Vineet Kumar; Leo, Vincent Vineeth; Gupta, Vijai Kumar; Kumar, Brijesh; Singh, Bhim Pratap

    2016-10-01

    In the present study, fifteen endophytic actinobacterial isolates recovered from Solanum lycopersicum were studied for their antagonistic potential and plant-growth-promoting (PGP) traits. Among them, eight isolates showed significant antagonistic and PGP traits, identified by amplification of the 16S rRNA gene. Isolate number DBT204, identified as Streptomyces sp., showed multiple PGP traits tested in planta and improved a range of growth parameters in seedlings of chili (Capsicum annuum L.) and tomato (S. lycopersicum L.). Further, genes of indole acetic acid (iaaM) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) were successively amplified from five strains. Six antibiotics (trimethoprim, fluconazole, chloramphenicol, nalidixic acid, rifampicin and streptomycin) and two phytohormones [indole acetic acid (IAA) and kinetin (KI)] were detected and quantified in Streptomyces sp. strain DBT204 using UPLC-ESI-MS/MS. The study indicates the potential of these PGP strains for production of phytohormones and shows the presence of biosynthetic genes responsible for production of secondary metabolites. It is the first report showing production of phytohormones (IAA and KI) by endophytic actinobacteria having PGP and biosynthetic potential. We propose Streptomyces sp. strain DBT204 for inoculums production and development of biofertilizers for enhancing growth of chili and tomato seedlings. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  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. The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family.

    Science.gov (United States)

    Carroll, Cassandra S; Grieve, Clark L; Murugathasan, Indu; Bennet, Andrew J; Czekster, Clarissa M; Liu, Huanting; Naismith, James; Moore, Margo M

    2017-08-01

    Iron is essential for growth and in low iron environments such as serum many bacteria and fungi secrete ferric iron-chelating molecules called siderophores. All fungi produce hydroxamate siderophores with the exception of Mucorales fungi, which secrete rhizoferrin, a polycarboxylate siderophore. Here we investigated the biosynthesis of rhizoferrin by the opportunistic human pathogen, Rhizopus delemar. We searched the genome of R. delemar 99-880 for a homologue of the bacterial NRPS-independent siderophore (NIS) protein, SfnaD, that is involved in biosynthesis of staphyloferrin A in Staphylococcus aureus. A protein was identified in R. delemar with 22% identity and 37% similarity with SfnaD, containing an N-terminal IucA/IucC family domain, and a C-terminal conserved ferric iron reductase FhuF-like transporter domain. Expression of the putative fungal rhizoferrin synthetase (rfs) gene was repressed by iron. The rfs gene was cloned and expressed in E.coli and siderophore biosynthesis from citrate and diaminobutane was confirmed using high resolution LC-MS. Substrate specificity was investigated showing that Rfs produced AMP when oxaloacetic acid, tricarballylic acid, ornithine, hydroxylamine, diaminopentane and diaminopropane were employed as substrates. Based on the production of AMP and the presence of a mono-substituted rhizoferrin, we suggest that Rfs is a member of the superfamily of adenylating enzymes. We used site-directed mutagenesis to mutate selected conserved residues predicted to be in the Rfs active site. These studies revealed that H484 is essential for Rfs activity and L544 may play a role in amine recognition by the enzyme. This study on Rfs is the first characterization of a fungal NIS enzyme. Future work will determine if rhizoferrin biosynthesis is required for virulence in Mucorales fungi. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Nowroozi, Jamileh; Akhavan Sepahi, Abbas; Rashnonejad, Afrooz

    2012-01-01

    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. PMID:23626932

  18. Banana fruit VQ motif-containing protein5 represses cold-responsive transcription factor MaWRKY26 involved in the regulation of JA biosynthetic genes.

    Science.gov (United States)

    Ye, Yu-Jie; Xiao, Yun-Yi; Han, Yan-Chao; Shan, Wei; Fan, Zhong-Qi; Xu, Qun-Gang; Kuang, Jian-Fei; Lu, Wang-Jin; Lakshmanan, Prakash; Chen, Jian-Ye

    2016-03-23

    Most harvested fruits and vegetables are stored at low temperature but many of them are highly sensitive to chilling injury. Jasmonic acid (JA), a plant hormone associated with various stress responses, is known to reduce chilling injury in fruits. However, little is known about the transcriptional regulation of JA biosynthesis in relation to cold response of fruits. Here, we show the involvement of a Group I WRKY transcription factor (TF) from banana fruit, MaWRKY26, in regulating JA biosynthesis. MaWRKY26 was found to be nuclear-localized with transcriptional activation property. MaWRKY26 was induced by cold stress or by methyl jasmonate (MeJA), which enhances cold tolerance in banana fruit. More importantly, MaWRKY26 transactivated JA biosynthetic genes MaLOX2, MaAOS3 and MaOPR3 via binding to their promoters. Further, MaWRKY26 physically interacted with a VQ motif-containing protein MaVQ5, and the interaction attenuated MaWRKY26-induced transactivation of JA biosynthetic genes. These results strongly suggest that MaVQ5 might act as a repressor of MaWRKY26 in activating JA biosynthesis. Taken together, our findings provide new insights into the transcriptional regulation of JA biosynthesis in response to cold stress and a better understanding of the molecular aspects of chilling injury in banana fruit.

  19. 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

  20. Transcription factor VdCmr1 is required for pigment production, protection from UV irradiation, and regulates expression of melanin biosynthetic genes in Verticillium dahliae.

    Science.gov (United States)

    Wang, Yonglin; Hu, Xiaoping; Fang, Yulin; Anchieta, Amy; Goldman, Polly H; Hernandez, Gustavo; Klosterman, Steven J

    2018-02-27

    Verticillium dahliae is a soilborne fungus that causes vascular wilt diseases on numerous plant species worldwide. The production of darkly melanized microsclerotia is crucial in the disease cycle of V. dahliae, as these structures allow for long-term survival in soil. Previously, transcriptomic and genomic analysis identified a cluster of genes in V. dahliae that encodes some dihydroxynaphthalene (DHN) melanin biosynthetic pathway homologues found in related fungi. In this study, we explored the roles of cluster-specific transcription factor VdCmr1, as well as two other genes within the cluster encoding a polyketide synthase (VdPKS1) and a laccase (VdLac1), enzymes at initial and endpoint steps in DHN melanin production. The results revealed that VdCmr1 and VdPKS1 are required for melanin production, but neither is required for microsclerotia production. None of the three genes were required for pathogenesis on tobacco and lettuce. Exposure of ΔVdCmr1 and wild-type strains to UV irradiation, or to high temperature (40 °C), revealed an approx. 50 % reduction of survival in the ΔVdCmr1 strain, relative to the wild-type strain, in response to either condition. Expression profiles revealed that expression of some melanin biosynthetic genes are in part dependent on VdCmr1. Combined data indicate VdCmr1 is a key regulator of melanin biosynthesis, and that via regulation of melanogenesis, VdCmr1 affects survival of V. dahliae in response to abiotic threats. We conclude with a model showing regulation of VdCmr1 by a high osmolarity glycerol response (Hog)-type MAP kinase pathway.

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

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    Dandan Chen

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

  2. Cloning and characterization of the biosynthetic gene cluster of 16-membered macrolide antibiotic FD-891: involvement of a dual functional cytochrome P450 monooxygenase catalyzing epoxidation and hydroxylation.

    Science.gov (United States)

    Kudo, Fumitaka; Motegi, Atsushi; Mizoue, Kazutoshi; Eguchi, Tadashi

    2010-07-26

    FD-891 is a 16-membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL-60 and Jurkat cells. We identified the FD-891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A-8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD-891 including the oxidation states and alpha-alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD-891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD-891 production. Instead, the gfsF gene-disrupted mutant accumulated a novel FD-891 analogue 25-O-methyl-FD-892, which lacked the epoxide and the hydroxyl group of FD-891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25-O-methyl-FD-892 into FD-891. In the course of the GfsF reaction, 10-deoxy-FD-891 was isolated as an enzymatic reaction intermediate, which was also converted into FD-891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD-891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD-891 in S. graminofaciens.

  3. Starch biosynthetic genes and enzymes are expressed and active in the absence of starch accumulation in sugar beet tap-root

    Science.gov (United States)

    2014-01-01

    Background Starch is the predominant storage compound in underground plant tissues like roots and tubers. An exception is sugar beet tap-root (Beta vulgaris ssp altissima) which exclusively stores sucrose. The underlying mechanism behind this divergent storage accumulation in sugar beet is currently not fully known. From the general presence of starch in roots and tubers it could be speculated that the lack in sugar beet tap-roots would originate from deficiency in pathways leading to starch. Therefore with emphasis on starch accumulation, we studied tap-roots of sugar beet using parsnip (Pastinaca sativa) as a comparator. Results Metabolic and structural analyses of sugar beet tap-root confirmed sucrose as the exclusive storage component. No starch granules could be detected in tap-roots of sugar beet or the wild ancestor sea beet (Beta vulgaris ssp. maritima). Analyses of parsnip showed that the main storage component was starch but tap-root tissue was also found to contain significant levels of sugars. Surprisingly, activities of four main starch biosynthetic enzymes, phosphoglucomutase, ADP-glucose pyrophosphorylase, starch synthase and starch branching enzyme, were similar in sugar beet and parsnip tap-roots. Transcriptional analysis confirmed expression of corresponding genes. Additionally, expression of genes involved in starch accumulation such as for plastidial hexose transportation and starch tuning functions could be determined in tap-roots of both plant species. Conclusion Considering underground storage organs, sugar beet tap-root upholds a unique property in exclusively storing sucrose. Lack of starch also in the ancestor sea beet indicates an evolved trait of biological importance. Our findings in this study show that gene expression and enzymatic activity of main starch biosynthetic functions are present in sugar beet tap-root during storage accumulation. In view of this, the complete lack of starch in sugar beet tap-roots is enigmatic. PMID

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2015-05-01

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

  6. Network pharmacological mechanisms of Vernonia anthelmintica (L.) in the treatment of vitiligo: Isorhamnetin induction of melanogenesis via up-regulation of melanin-biosynthetic genes.

    Science.gov (United States)

    Wang, Ji Ye; Chen, Hong; Wang, Yin Yin; Wang, Xiao Qin; Chen, Han Ying; Zhang, Mei; Tang, Yun; Zhang, Bo

    2017-11-16

    Vitiligo is a long-term skin disease characterized by the loss of pigment in the skin. The current therapeutic approaches are limited. Although the anti-vitiligo mechanisms of Vernonia anthelmintica (L.) remain ambiguous, the herb has been broadly used in Uyghur hospitals to treat vitiligo. The overall objective of the present study aims to identify the potential lead compounds from Vernonia anthelmintica (L.) in the treatment of vitiligo via an oral route as well as the melanogenic mechanisms in the systematic approaches in silico of admetSAR and substructure-drug-target network-based inference (SDTNBI). The results showed that the top 5 active compounds with a relatively higher bioavailability that interacted with 23 therapeutic targets were identified in Vernonia anthelmintica (L.) using admetSAR and SDTNBI methods. Among these compounds, Isorhamnetin and Kaempferide, which are methyl-flavonoids, performed 1st and 2nd. Isorhamnetin and Kaempferide significantly increased the expression of melanin-biosynthetic genes (MC1R, MITF, TYR, TYRP1 and DCT) and the tyrosinase activity in B16F10 cells. Isorhamnetin and Kaempferide significantly increased the mRNA-expression of melanin-biosynthetic genes (MC1R, MITF, TYR, TYRP1 and DCT), the protein level of MITF and the tyrosinase activity. Based on the SDTNBI method and experimental verification, Isorhamnetin and Kaempferide effectively increased melanogenesis by targeting the MC1R-MITF signaling pathway, MAPK signaling pathway, PPAR signaling pathway (PPARA, PPARD, PPARG), arachidonic acid metabolism pathway (ALOX12, ALOX15, CBR1) and serotonergic synapses (ALOX12, ALOX15) in the treatment of vitiligo from a network perspective. We identified the melanogenic activity of the methyl-flavonoids Isorhamnetin and Kaempferide, which were successfully predicted in a network pharmacological analysis of Vernonia anthelmintica (L.) by admetSAR and SDTNBI methods.

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

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    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

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

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    Woo Tae Park

    2016-03-01

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

  9. 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

    2016-03-29

    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.

  10. The Jasmonate-Activated Transcription Factor MdMYC2 Regulates ETHYLENE RESPONSE FACTOR and Ethylene Biosynthetic Genes to Promote Ethylene Biosynthesis during Apple Fruit Ripening[OPEN

    Science.gov (United States)

    Xu, Yaxiu; Zhang, Lichao; Ji, Yinglin; Tan, Dongmei; Yuan, Hui

    2017-01-01

    The plant hormone ethylene is critical for ripening in climacteric fruits, including apple (Malus domestica). Jasmonate (JA) promotes ethylene biosynthesis in apple fruit, but the underlying molecular mechanism is unclear. Here, we found that JA-induced ethylene production in apple fruit is dependent on the expression of MdACS1, an ACC synthase gene involved in ethylene biosynthesis. The expression of MdMYC2, encoding a transcription factor involved in the JA signaling pathway, was enhanced by MeJA treatment in apple fruits, and MdMYC2 directly bound to the promoters of both MdACS1 and the ACC oxidase gene MdACO1 and enhanced their transcription. Furthermore, MdMYC2 bound to the promoter of MdERF3, encoding a transcription factor involved in the ethylene-signaling pathway, thereby activating MdACS1 transcription. We also found that MdMYC2 interacted with MdERF2, a suppressor of MdERF3 and MdACS1. This protein interaction prevented MdERF2 from interacting with MdERF3 and from binding to the MdACS1 promoter, leading to increased transcription of MdACS1. Collectively, these results indicate that JA promotes ethylene biosynthesis through the regulation of MdERFs and ethylene biosynthetic genes by MdMYC2. PMID:28550149

  11. The Jasmonate-Activated Transcription Factor MdMYC2 Regulates ETHYLENE RESPONSE FACTOR and Ethylene Biosynthetic Genes to Promote Ethylene Biosynthesis during Apple Fruit Ripening.

    Science.gov (United States)

    Li, Tong; Xu, Yaxiu; Zhang, Lichao; Ji, Yinglin; Tan, Dongmei; Yuan, Hui; Wang, Aide

    2017-06-01

    The plant hormone ethylene is critical for ripening in climacteric fruits, including apple (Malus domestica). Jasmonate (JA) promotes ethylene biosynthesis in apple fruit, but the underlying molecular mechanism is unclear. Here, we found that JA-induced ethylene production in apple fruit is dependent on the expression of MdACS1, an ACC synthase gene involved in ethylene biosynthesis. The expression of MdMYC2, encoding a transcription factor involved in the JA signaling pathway, was enhanced by MeJA treatment in apple fruits, and MdMYC2 directly bound to the promoters of both MdACS1 and the ACC oxidase gene MdACO1 and enhanced their transcription. Furthermore, MdMYC2 bound to the promoter of MdERF3, encoding a transcription factor involved in the ethylene-signaling pathway, thereby activating MdACS1 transcription. We also found that MdMYC2 interacted with MdERF2, a suppressor of MdERF3 and MdACS1 This protein interaction prevented MdERF2 from interacting with MdERF3 and from binding to the MdACS1 promoter, leading to increased transcription of MdACS1 Collectively, these results indicate that JA promotes ethylene biosynthesis through the regulation of MdERFs and ethylene biosynthetic genes by MdMYC2. © 2017 American Society of Plant Biologists. All rights reserved.

  12. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway and their regulation during reproduction

    Science.gov (United States)

    Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

  13. Acyl-Acyl carrier protein regulates transcription of fatty acid biosynthetic genes via the FabT repressor in Streptococcus pneumoniae.

    Science.gov (United States)

    Jerga, Agoston; Rock, Charles O

    2009-06-05

    Long-chain acyl-acyl carrier proteins (acyl-ACP) are established biochemical regulators of bacterial type II fatty acid synthases due to their ability to feedback-inhibit the early steps in the biosynthetic pathway. In Streptococcus pneumoniae, the expression of the fatty acid synthase (fab) genes is controlled by a helix-turn-helix transcriptional repressor called FabT. A screen of pathway intermediates identified acyl-ACP as a ligand that increased the affinity of FabT for DNA. FabT bound to a wide range of acyl-ACP chain lengths in the absence of DNA, but only the long-chain acyl-ACPs increase the affinity of FabT for DNA. FabT affinity for DNA increased with increasing acyl-ACP chain length with cis-vaccenoyl-ACP being the most effective ligand. Thus, FabT is a new ACP-interacting partner that acts as a transcriptional rheostat to fine tune the expression of the fab genes based on the demand for fatty acids.

  14. Acyl-Acyl Carrier Protein Regulates Transcription of Fatty Acid Biosynthetic Genes via the FabT Repressor in Streptococcus pneumoniae*

    Science.gov (United States)

    Jerga, Agoston; Rock, Charles O.

    2009-01-01

    Long-chain acyl-acyl carrier proteins (acyl-ACP) are established biochemical regulators of bacterial type II fatty acid synthases due to their ability to feedback-inhibit the early steps in the biosynthetic pathway. In Streptococcus pneumoniae, the expression of the fatty acid synthase (fab) genes is controlled by a helix-turn-helix transcriptional repressor called FabT. A screen of pathway intermediates identified acyl-ACP as a ligand that increased the affinity of FabT for DNA. FabT bound to a wide range of acyl-ACP chain lengths in the absence of DNA, but only the long-chain acyl-ACPs increase the affinity of FabT for DNA. FabT affinity for DNA increased with increasing acyl-ACP chain length with cis-vaccenoyl-ACP being the most effective ligand. Thus, FabT is a new ACP-interacting partner that acts as a transcriptional rheostat to fine tune the expression of the fab genes based on the demand for fatty acids. PMID:19376778

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

    Science.gov (United States)

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

    2015-07-20

    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.

  16. Isolation and diversity of natural product biosynthetic genes of cultivable bacteria associated with marine sponge Mycale sp. from the coast of Fujian, China.

    Science.gov (United States)

    Su, Pei; Wang, De-Xiang; Ding, Shao-Xiong; Zhao, Jing

    2014-04-01

    The marine sponge Mycale sp., a potential source of natural bioactive products, is widely distributed along the coast of Fujian, China. The cultivable bacterial community associated with Mycale sp., the antibacterial activities, and the PKS (polyketide synthase) and NRPS (nonribosomal peptide synthetase) gene diversity of these bacteria were investigated. Phylogenetic analysis of the 16S rRNA gene showed that the 51 isolates from Mycale sp. belonged to Actinobacteria, Bacteroidetes, Gammaproteobacteria, Alphaproteobacteria, and Firmicutes. Among them, some bacteria were first isolated from marine sponge. The 20 isolates with antimicrobial activities were primarily clustered within the groups Actinobacteria, Gammaproteobacteria, and Bacillus. Strain HNS054, which showed 99% similarity to Streptomyces labedae, exhibited the strongest antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus MTCC 1430, Bacillus subtilis MTCC 441) and Vibrio species. The screening of natural product biosynthetic genes revealed that 8 Actinobacteria species with antimicrobial activities possessed PKS-KS (ketosynthase) or NRPS-A domains, and the Nocardiopsis species contained a hybrid or mixed PKS-NRPS system. The phylogenetic analysis of the amino acid sequences indicated that the identified KS domains clustered with those from diverse bacterial groups, including Actinobacteria, Alphaproteobacteria, Cyanobacteria, and Firmicutes. Most KS domain sequences had high homology (>80%) to type I KSs, but the KS domain of Nocardiopsis sp. strain HNS048 had 77% similarity to the type II KS domain of Burkholderia gladioli. The NRPS-A domains of the 8 isolates were grouped into the Gammaproteobacteria, Actinobacteria, and Firmicutes groups. The NRPS-A gene of strain HNS052, identified as Nocardiopsis cyriacigeorgica, showed only 54% similarity to Rhodococcus opacus. All results suggested that Mycale sp. harboured diverse bacteria that could contribute to the production of novel

  17. 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...

  18. 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

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

    Directory of Open Access Journals (Sweden)

    Jinxin Liu

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

  20. De novo sequencing analysis of the Rosa roxburghii fruit transcriptome reveals putative ascorbate biosynthetic genes and EST-SSR markers.

    Science.gov (United States)

    Yan, Xiuqin; Zhang, Xue; Lu, Min; He, Yong; An, Huaming

    2015-04-25

    Rosa roxburghii Tratt. is a well-known ornamental rose species native to China. In addition, the fruits of this species are valued for their nutritional and medicinal characteristics, especially their high ascorbic acid (AsA) levels. Nevertheless, AsA biosynthesis in R. roxburghii fruit has not been explored in detail because of a lack of genomic resources for this species. High-throughput transcriptomic sequencing generating large volumes of transcript sequence data can aid in gene discovery and molecular marker development. In this study, we generated more than 53 million clean reads using Illumina paired-end sequencing technology. De novo assembly yielded 106,590 unigenes, with an average length of 343 bp. On the basis of sequence similarity to known proteins, 9301 and 2393 unigenes were classified into Gene Ontology and Clusters of Orthologous Group categories, respectively. There were 7480 unigenes assigned to 124 pathways in the Kyoto Encyclopedia of Gene and Genome pathway database. BLASTx searches identified 498 unique putative transcripts encoding various transcription factors, some known to regulate fruit development. qRT-PCR validated the expressions of most of the genes encoding the main enzymes involved in ascorbate biosynthesis. In addition, 9131 potential simple sequence repeat (SSR) loci were identified among the unigenes. One hundred and two primer pairs were synthesized and 71 pairs produced an amplification product during initial screening. Among the amplified products, 30 were polymorphic in the 16 R. roxburghii germplasms tested. Our study was the first to produce a large volume of transcriptome data from R. roxburghii. The resulting sequence collection is a valuable resource for gene discovery and marker-assisted selective breeding in this rose species. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. 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.

  2. Streptomyces venezuelae ISP5230 Maintains Excretion of Jadomycin upon Disruption of the MFS Transporter JadL Located within the Natural Product Biosynthetic Gene Cluster.

    Science.gov (United States)

    Forget, Stephanie M; McVey, Jennifer; Vining, Leo C; Jakeman, David L

    2017-01-01

    JadL was identified as a Major Facilitator Superfamily (MFS) transporter (T.C. 2.A.1) through sequence homology. The protein is encoded by jadL, situated within the jadomycin biosynthetic gene cluster. JadL has, therefore, been assigned a putative role in host defense by exporting its probable substrates, the jadomycins, a family of secondary metabolites produced by Streptomyces venezuelae ISP5230. Herein, we evaluate this assumption through the construction and analysis of a jadL disrupted mutant, S. venezuelae VS678 (ΔjadL::aac(3)IV). Quantitative determination of jadomycin production with the jadL disrupted mutant did not show a significant decrease in production in comparison to the wildtype strain, as determined by HPLC and by tandem mass spectrometry. These results suggest that efflux of jadomycin occurs upon disruption of jadL, or that JadL is not involved in jadomycin efflux. Potentially, other transporters within S. venezuelae ISP5230 may adopt this role upon inactivation of JadL to export jadomycins.

  3. Seasonal shifts in accumulation of glycerol biosynthetic gene transcripts in mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae, larvae

    Directory of Open Access Journals (Sweden)

    Jordie D. Fraser

    2017-06-01

    Full Text Available Winter mortality is a major factor regulating population size of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae. Glycerol is the major cryoprotectant in this freeze intolerant insect. We report findings from a gene expression study on an overwintering mountain pine beetle population over the course of 35 weeks. mRNA transcript levels suggest glycerol production in the mountain pine beetle occurs through glycogenolytic, gluconeogenic and potentially glyceroneogenic pathways, but not from metabolism of lipids. A two-week lag period between fall glycogen phosphorylase transcript and phosphoenolpyruvate carboxykinase transcript up-regulation suggests that gluconeogenesis serves as a secondary glycerol-production process, subsequent to exhaustion of the primary glycogenolytic source. These results provide a first look at the details of seasonal gene expression related to the production of glycerol in the mountain pine beetle.

  4. Seasonal shifts in accumulation of glycerol biosynthetic gene transcripts in mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), larvae.

    Science.gov (United States)

    Fraser, Jordie D; Bonnett, Tiffany R; Keeling, Christopher I; Huber, Dezene P W

    2017-01-01

    Winter mortality is a major factor regulating population size of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Glycerol is the major cryoprotectant in this freeze intolerant insect. We report findings from a gene expression study on an overwintering mountain pine beetle population over the course of 35 weeks. mRNA transcript levels suggest glycerol production in the mountain pine beetle occurs through glycogenolytic, gluconeogenic and potentially glyceroneogenic pathways, but not from metabolism of lipids. A two-week lag period between fall glycogen phosphorylase transcript and phosphoenolpyruvate carboxykinase transcript up-regulation suggests that gluconeogenesis serves as a secondary glycerol-production process, subsequent to exhaustion of the primary glycogenolytic source. These results provide a first look at the details of seasonal gene expression related to the production of glycerol in the mountain pine beetle.

  5. 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 biosynth...... of a lanthipeptide, a carotenoid, five terpenoid compounds, an ectoine, a siderophore and a spore pigment-associated gene cluster to their respective biosynthesis products....

  6. 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. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  7. 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.

  8. 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. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Ling Yin

    2015-11-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Ni-Hao Jiang

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

  12. Molecular Cloning and Characterization of Three Genes Encoding Dihydroflavonol-4-Reductase from Ginkgo biloba in Anthocyanin Biosynthetic Pathway

    Science.gov (United States)

    Hua, Cheng; Linling, Li; Shuiyuan, Cheng; Fuliang, Cao; Feng, Xu; Honghui, Yuan; Conghua, Wu

    2013-01-01

    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. PMID:23991027

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

    Directory of Open Access Journals (Sweden)

    Cheng Hua

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

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

    Science.gov (United States)

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

    2005-09-01

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

  15. Plasma Catecholamines (CA) and Gene Expression of CA Biosynthetic Enzymes in Adrenal Medulla and Sympathetic Ganglia of Rats Exposed to Single or Repeated Hypergravity

    Science.gov (United States)

    Petrak, J.; Jurani, M.; Baranovska, M.; Hapala, I.; Frollo, I.; Kvetnansky, R.

    2008-06-01

    The aim of this study was to evaluate plasma epinephrine (EPI) and norepinephrine (NE) levels in blood collected directly during a single or 8-times repeated centrifugation at hypergravity 4G, using remote controlled equipment. Plasma EPI levels showed a huge hypergravity-induced increase. After the last blood collection during hypergravity, the centrifuge was turned off and another blood sampling was performed immediately after the centrifuge decelerated and stopped (10 min). In these samples plasma EPI showed significantly lower levels compared to centrifugation intervals. Plasma NE levels showed none or small changes. Repeated exposure to hypergravity 4G (8 days for 60 min) eliminated the increase in plasma EPI levels at the 15 min interval but did not markedly affect plasma NE levels. To explain these findings we measured mRNA levels of CA biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla (AM) and stellate ganglia (SG) of rats exposed to continuous hypergravity (2G) up to 6 days. In AM, TH, DBH and PNMT mRNA levels were significantly increased in intervals up to 3 days, however, after 6 day hypergravity exposure, no significant elevation was found. In SG, no significant changes in gene expression of CA enzymes were seen both after a single or repeated hypergravity. Thus, our data show that hypergravity highly activates the adrenomedullary system, whereas the sympathoneural system is not significantly changed. In conclusion, our results demonstrate that during repeated or continuous exposure of the organism to hypergravity the adrenomedullary system is adapted, whereas sympathoneural system is not affected.

  16. Chromosomal location of wheat genes of the carotenoid biosynthetic pathway and evidence for a catalase gene on chromosome 7A functionally associated with flour b* colour variation.

    Science.gov (United States)

    Crawford, Allison C; Francki, Michael G

    2013-10-01

    Knowledge of molecular and genetic mechanisms controlling wheat grain quality characteristics is significant for improving flour for end-product functionality. Flour b* colour is an important quality trait for breeding wheat varieties to produce grain for specific market requirements. The degree of flour yellowness is due to the accumulation of carotenoids in grain, particularly lutein. Flour b* is under polygenic control and quantitative trait loci (QTL) have frequently been reported on chromosome 7AL. Analysis of carotenoid genes showed that phytoene synthase (PSY) co-located to the QTL on 7AL but other genes at this locus are also thought to contribute flour b* colour variation. This study used the wheat genome survey sequence and identified the chromosomal location of all wheat carotenoid genes, but none other than PSY were located on 7AL and, therefore, other genes may control flour b* colour variation including oxidative genes that degrade carotenoids. An investigation of EST bin mapped to 7AL identified a gene encoding a catalase enzyme (Cat3-A1) that was phylogenetically related to other plant class III enzymes, co-located to the QTL for flour b* colour variation on 7AL in three mapping populations and expressed during seed development. Therefore, Cat3-A1 was functionally associated with flour b* colour variation. Catalase acts upon hydrogen peroxide as a substrate and it was postulated that Cat3-A1 alleles control varying degrees of bleaching action on lutein in developing wheat grain. Markers for Cat3-A1 developed in this study can be used in conjunction with other candidate gene markers including phytoene synthase and lycopene-ε-cylase to develop a molecular signature for selecting lines with specific flour b* colour values in wheat breeding.

  17. DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers

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    Cloeckaert Axel

    2009-05-01

    Full Text Available Abstract Background The lipopolysaccharide is a major antigen and virulence factor of Brucella, an important bacterial pathogen. In smooth brucellae, lipopolysaccharide is made of lipid A-core oligosaccharide and N-formylperosamine O-polysaccharide. B. ovis and B. canis (rough species lack the O-polysaccharide. Results The polymorphism of O-polysaccharide genes wbkE, manAO-Ag, manBO-Ag, manCO-Ag, wbkF and wbkD and wbo (wboA and wboB, and core genes manBcore and wa** was analyzed. Although most genes were highly conserved, species- and biovar-specific restriction patterns were found. There were no significant differences in putative N-formylperosamyl transferase genes, suggesting that Brucella A and M serotypes are not related to specific genes. In B. pinnipedialis and B. ceti (both smooth, manBO-Ag carried an IS711, confirming its dispensability for perosamine synthesis. Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization. B. ovis wbkF carried a frame-shift and B. canis had a long deletion partially encompassing both genes. In smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism. Conclusion The results define species and biovar markers, confirm the dispensability of manBO-Ag for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

  18. 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.

  19. Post-repolarization block of cardiac sodium channels by saxitoxin.

    Science.gov (United States)

    Makielski, J C; Satin, J; Fan, Z

    1993-08-01

    Phasic block of rat cardiac Na+ current by saxitoxin was assessed using pulse trains and two-pulse voltage clamp protocols, and the results were fit to several kinetic models. For brief depolarizations (5 to 50 ms) the depolarization duration did not affect the rate of development or the amplitude of phasic block for pulse trains. The pulse train data were well described by a recurrence relation based upon the guarded receptor model, and it provided rate constants that accurately predicted first-pulse block as well as recovery time constants in response to two-pulse protocols. However, the amplitudes and rates of phasic block development at rapid rates (> 5 Hz) were less than the model predicted. For two pulse protocols with a short (10 ms) conditioning step to -30 mV, block developed only after repolarization to -150 mV and then recovered as the interpulse interval was increased. This suggested that phasic block under these conditions was caused by binding with increased affinity to a state that exists transiently after repolarization to -150 mV. This "post-repolarization block" was fit to a three-state model consisting of a transient state with high affinity for the toxin, the toxin bound state, and the ultimate resting state of the channel. This model accounted for the biphasic post-repolarization block development and recovery observed in two-pulse protocols, and it more accurately described phasic block in pulse trains. The transient state after repolarization was predicted to have a dwell time of 570 ms, an on rate for saxitoxin of 16 s-1 micro M-1, and an off rate of 0.2 s-1 (KD = 12 nM). These results and the proposed model suggest a novel variation on phasic block mechanisms and suggest a long-lived transient Na+ channel conformation during recovery.

  20. Saxitoxin Level Comparation in Bali Sardine (Sardinella Lemuru in Bali Strait in Different Monsoons

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    Endang Yuli Herawati

    2017-12-01

    Full Text Available Sardinella fish (Sardinella lemuru is a species of important pelagic fish from the family of Clupeidae found in Bali Strait. The dynamic condition of Bali Strait makes it fertile which is identical with plankton blooming. The objective of this research was to understand the potential of saxitoxin in sardinella fish because of dinoflagellates consumption in Bali Strait. The research was conducted in Southeast Monsoon (June - August 2015 and Northwest Monsoon (December 2015 – February 2016. The method used was in situ plankton sampling, counting and ELISA test to determine the level of saxitoxin in the fish. Hydro-oceanographic parameters measured were temperatures, salinity, DO (dissolved oxygen, pH level, phosphate level, nitrate level and transparency. They showed average optimum value for phytoplankton growth especially during Southeast Monsoon which its temperature lower and the nutrients (phosphate nitrate higher than the Northwest Monsoon. This condition caused high richness of phytoplankton in the water and followed with dinoflagellates richness so they accumulated in sardinella fish. It was supported by the analysis of the fish gastric which showed positive correlation between dinoflagellates richness in the water and the fish gastric. ELISA test also showed that saxitoxin level of fish caught in Southeast Monsoon was higher than the one caught in Northwest Monsoon even though the level of saxitoxin was still in the safe range (less than 80 μg STXeq. per 100 g but the danger of saxitoxin accumulation should be watched out for.

  1. Reduction in the endogenous arginine decarboxylase transcript levels in rice leads to depletion of the putrescine and spermidine pools with no concomitant changes in the expression of downstream genes in the polyamine biosynthetic pathway.

    Science.gov (United States)

    Trung-Nghia, Pham; Bassie, Ludovic; Safwat, Gehan; Thu-Hang, Pham; Lepri, Olivia; Rocha, Pedro; Christou, Paul; Capell, Teresa

    2003-11-01

    We investigated whether down-regulation of arginine decarboxylase (ADC) activity and concomitant changes in polyamine levels result in changes in the expression of downstream genes in the polyamine pathway. We generated transgenic rice (Oryza sativa L.) plants in which the rice adc gene was down-regulated by expression of its antisense oat (Avena sativa L.) ortholog. Plants expressed the oat mRNA adc transcript at different levels. The endogenous transcript was down-regulated in five out of eight plant lineages we studied in detail. Reduction in the steady-state rice adc mRNA levels resulted in a concomitant decrease in ADC activity. The putrescine and spermidine pool was significantly reduced in plants with lower ADC activity. Expression of the rice ornithine decarboxylase (odc), S-adenosylmethionine decarboxylase (samdc) and spermidine synthase (spd syn) transcripts was not affected. We demonstrate that even though levels of the key metabolites in the pathway were compromised, this did not influence steady-state transcription levels of the other genes involved in the pathway. Our results provide an insight into the different regulatory mechanisms that control gene expression in the polyamine biosynthetic pathway in plants by demonstrating that the endogenous pathway is uncoupled from manipulations that modulate polyamine levels by expression of orthologous transgenes.

  2. Biosynthetic infochemical communication.

    Science.gov (United States)

    Olsson, S B; Challiss, R A J; Cole, M; Gardeniers, J G E; Gardner, J W; Guerrero, A; Hansson, B S; Pearce, T C

    2015-07-09

    There is an ever-increasing demand for data to be embedded in our environment at ever-decreasing temporal and spatial scales. Whilst current communication and storage technologies generally exploit the electromagnetic properties of media, chemistry offers us a new alternative for nanoscale signaling using molecules as messengers with high information content. Biological systems effectively overcome the challenges of chemical communication using highly specific biosynthetic pathways for signal generation together with specialized protein receptors and nervous systems. Here we consider a new approach for information transmission based upon nature's quintessential example of infochemical communication, the moth pheromone system. To approach the sensitivity, specificity and versatility of infochemical communication seen in nature, we describe an array of biologically-inspired technologies for the production, transmission, detection, and processing of molecular signals. We show how it is possible to implement each step of the moth pheromone pathway for biosynthesis, transmission, receptor protein binding/transduction, and antennal lobe processing of monomolecular and multimolecular signals. For each implemented step, we discuss the value, current limitations, and challenges for the future development and integration of infochemical communication technologies. Together, these building blocks provide a starting point for future technologies that can utilize programmable emission and detection of multimolecular information for a new and robust means of communicating chemical information.

  3. 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.

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    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.

  4. 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. PMID:23626761

  5. The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II)2Cys6 transcription factor BcBot6.

    Science.gov (United States)

    Porquier, Antoine; Morgant, Guillaume; Moraga, Javier; Dalmais, Bérengère; Luyten, Isabelle; Simon, Adeline; Pradier, Jean-Marc; Amselem, Joëlle; Collado, Isidro González; Viaud, Muriel

    2016-11-01

    Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G+C/A+T-equilibrated regions that contain the Bcbot genes alternate with A+T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)2Cys6 putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the ΔBcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BOT synthesis and of its ecological role in the B. cinerea life cycle. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Two transcription factors, CabA and CabR, are independently involved in multilevel regulation of the biosynthetic gene cluster encoding the novel aminocoumarin, cacibiocin.

    Science.gov (United States)

    Wolański, Marcin; Łebkowski, Tomasz; Kois-Ostrowska, Agnieszka; Zettler, Judith; Apel, Alexander K; Jakimowicz, Dagmara; Zakrzewska-Czerwińska, Jolanta

    2016-04-01

    Aminocoumarins are potent antibiotics belonging to a relatively small group of secondary metabolites produced by actinomycetes. Genome mining of Catenulispora acidiphila has recently led to the discovery of a gene cluster responsible for biosynthesis of novel aminocoumarins, cacibiocins. However, regulation of the expression of this novel gene cluster has not yet been analyzed. In this study, we identify transcriptional regulators of the cacibiocin gene cluster. Using a heterologous expression system, we show that the CabA and CabR proteins encoded by cabA and cabR genes in the cacibiocin gene cluster control the expression of genes involved in the biosynthesis, modification, regulation, and potentially, efflux/resistance of cacibiocins. CabA positively regulates the expression of cabH (the first gene in the cabHIYJKL operon) and cabhal genes encoding key enzymes responsible for the biosynthesis and halogenation of the aminocoumarin moiety, respectively. We provide evidence that CabA is a direct inducer of cacibiocin production, whereas the second transcriptional factor, CabR, is involved in the negative regulation of its own gene and cabT-the latter of which encodes a putative cacibiocin transporter. We also demonstrate that CabR activity is negatively regulated in vitro by aminocoumarin compounds, suggesting the existence of analogous regulation in vivo. Finally, we propose a model of multilevel regulation of gene transcription in the cacibiocin gene cluster by CabA and CabR.

  7. 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. Copyright © 2015. Published by Elsevier Ltd.

  8. Bioengineering natural product biosynthetic pathways for therapeutic applications.

    Science.gov (United States)

    Wu, Ming-Cheng; Law, Brian; Wilkinson, Barrie; Micklefield, Jason

    2012-12-01

    With the advent of next-generation DNA sequencing technologies, the number of microbial genome sequences has increased dramatically, revealing a vast array of new biosynthetic gene clusters. Genomics data provide a tremendous opportunity to discover new natural products, and also to guide the bioengineering of new and existing natural product scaffolds for therapeutic applications. Notably, it is apparent that the vast majority of biosynthetic gene clusters are either silent or produce very low quantities of the corresponding natural products. It is imperative therefore to devise methods for activating unproductive biosynthetic pathways to provide the quantities of natural products needed for further development. Moreover, on the basis of our expanding mechanistic and structural knowledge of biosynthetic assembly-line enzymes, new strategies for re-programming biosynthetic pathways have emerged, resulting in focused libraries of modified products with potentially improved biological properties. In this review we will focus on the latest bioengineering approaches that have been utilised to optimise yields and increase the structural diversity of natural product scaffolds for future clinical applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. An overview on the marine neurotoxin, saxitoxin: genetics, molecular targets, methods of detection and ecological functions.

    Science.gov (United States)

    Cusick, Kathleen D; Sayler, Gary S

    2013-03-27

    Marine neurotoxins are natural products produced by phytoplankton and select species of invertebrates and fish. These compounds interact with voltage-gated sodium, potassium and calcium channels and modulate the flux of these ions into various cell types. This review provides a summary of marine neurotoxins, including their structures, molecular targets and pharmacologies. Saxitoxin and its derivatives, collectively referred to as paralytic shellfish toxins (PSTs), are unique among neurotoxins in that they are found in both marine and freshwater environments by organisms inhabiting two kingdoms of life. Prokaryotic cyanobacteria are responsible for PST production in freshwater systems, while eukaryotic dinoflagellates are the main producers in marine waters. Bioaccumulation by filter-feeding bivalves and fish and subsequent transfer through the food web results in the potentially fatal human illnesses, paralytic shellfish poisoning and saxitoxin pufferfish poisoning. These illnesses are a result of saxitoxin's ability to bind to the voltage-gated sodium channel, blocking the passage of nerve impulses and leading to death via respiratory paralysis. Recent advances in saxitoxin research are discussed, including the molecular biology of toxin synthesis, new protein targets, association with metal-binding motifs and methods of detection. The eco-evolutionary role(s) PSTs may serve for phytoplankton species that produce them are also discussed.

  10. 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

  11. Paralytic shellfish poison reference materials: an intercomparison of methods for the determination of saxitoxin.

    Science.gov (United States)

    van Egmond, H P; van den Top, H J; Paulsch, W E; Goenaga, X; Vieytes, M R

    1994-01-01

    Within the framework of the European Commission's Measurements and Testing Programme (BCR) a project has been undertaken to develop shellfish reference materials for Paralytic Shellfish Poisons (PSP). In a preliminary phase of the project, an intercomparison study of methods was undertaken. In this exercise 18 laboratories were asked to analyse solutions of saxitoxin and PSP-containing shellfish extracts with a method of their choice. The study revealed that: all the methods considered (four HPLC methods, one ELISA method) were in principle adequate for the quantification of saxitoxin in solution in the absence of interfering substances (Coefficient of variation (CV) 33% at a concentration of 0.5 microgram/ml); three of the HPLC methods used were able to quantify saxitoxin in PSP-positive mussel extract, the fourth method gave significant overestimation; the CV of all HPLC results was 53% at a mean saxitoxin mass fraction of 2.06 mg/kg mussel meat, the recoveries varied from 59-173%; and the ELISA method grossly overestimated the saxitoxin content in mussel extract, probably due to cross reactions of the antibodies with other PSP. The feasibility of preparing a homogeneous batch of ampouled mussel extracts (CV 3.5% at a saxitoxin concentration of approximately 1.5 mg/kg shellfish), sufficiently stable for at least 4 months storage both at 4 degrees C and approximately 20 degrees C, was demonstrated. The performance of the different methods for the analysis of PSP other than saxitoxin has not yet been evaluated, due to the current lack of PSP standards. Some of the problems observed in the intercomparison study were partly due to the nature of the chromatographic columns used, the composition of the HPLC mobile phase (pH, ion strength), non-optimal conditions for derivatization and matrix interference. Following the outcome of this study, a three year multistage project involving 15-20 European laboratories has been initiated, aimed at improving the accuracy and

  12. Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on heavy metal-contaminated soil, and this is associated with upregulation of foliar metallothionein and polyamine biosynthetic gene expression

    Science.gov (United States)

    Cicatelli, Angela; Lingua, Guido; Todeschini, Valeria; Biondi, Stefania; Torrigiani, Patrizia; Castiglione, Stefano

    2010-01-01

    Background and Aims It is increasingly evident that plant tolerance to stress is improved by mycorrhiza. Thus, suitable plant–fungus combinations may also contribute to the success of phytoremediation of heavy metal (HM)-polluted soil. Metallothioneins (MTs) and polyamines (PAs) are implicated in the response to HM stress in several plant species, but whether the response is modulated by arbuscular mycorrhizal fungi (AMF) remains to be clarified. The aim of the present study was to check whether colonization by AMF could modify growth, metal uptake/translocation, and MT and PA gene expression levels in white poplar cuttings grown on HM-contaminated soil, and to compare this with plants grown on non-contaminated soil. Methods In this greenhouse study, plants of a Populus alba clone were pre-inoculated, or not, with either Glomus mosseae or G. intraradices and then grown in pots containing either soil collected from a multimetal- (Cu and Zn) polluted site or non-polluted soil. The expression of MT and PA biosynthetic genes was analysed in leaves using quantitative reverse transcription–PCR. Free and conjugated foliar PA concentrations were determined in parallel. Results On polluted soil, AMF restored plant biomass despite higher Cu and Zn accumulation in plant organs, especially roots. Inoculation with the AMF caused an overall induction of PaMT1, PaMT2, PaMT3, PaSPDS1, PaSPDS2 and PaADC gene expression, together with increased free and conjugated PA levels, in plants grown on polluted soil, but not in those grown on non-polluted soil. Conclusions Mycorrhizal plants of P. alba clone AL35 exhibit increased capacity for stabilization of soil HMs, together with improved growth. Their enhanced stress tolerance may derive from the transcriptional upregulation of several stress-related genes, and the protective role of PAs. PMID:20810743

  13. Characterization of the Genes Encoding d-Amino Acid Transaminase and Glutamate Racemase, Two d-Glutamate Biosynthetic Enzymes of Bacillus sphaericus ATCC 10208

    Science.gov (United States)

    Fotheringham, Ian G.; Bledig, Stefan A.; Taylor, Paul P.

    1998-01-01

    In Bacillus sphaericus and other Bacillus spp., d-amino acid transaminase has been considered solely responsible for biosynthesis of d-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding d-amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glr gene encodes a 28.8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus, Pediococcus, and Staphylococcus species. The dat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the d-amino acid transaminase of the thermophilic Bacillus sp. strain YM1. PMID:9696787

  14. Overexpression of a three-gene conidial pigment biosynthetic pathway in Aspergillus nidulans reveals the first NRPS known to acetylate tryptophan.

    Science.gov (United States)

    Sung, Calvin T; Chang, Shu-Lin; Entwistle, Ruth; Ahn, Green; Lin, Tzu-Shyang; Petrova, Vessela; Yeh, Hsu-Hua; Praseuth, Mike B; Chiang, Yi-Ming; Oakley, Berl R; Wang, Clay C C

    2017-04-01

    Fungal nonribosomal peptide synthetases (NRPSs) are megasynthetases that produce cyclic and acyclic peptides. In Aspergillus nidulans, the NRPS ivoA (AN10576) has been associated with the biosynthesis of grey-brown conidiophore pigments. Another gene, ivoB (AN0231), has been demonstrated to be an N-acetyl-6-hydroxytryptophan oxidase that putatively acts downstream of IvoA. A third gene, ivoC, has also been predicted to be involved in pigment biosynthesis based on publicly available genomic and transcriptomic information. In this paper, we report the replacement of the promoters of the ivoA, ivoB, and ivoC genes with the inducible promoter alcA in a single cotransformation. Co-overexpression of the three genes resulted in the production of a dark-brown pigment in hyphae. In addition, overexpression of each of the Ivo genes, ivoA-C, individually or in combination, allowed us to isolate intermediates and confirm the function of each gene. IvoA was found to be the first known NRPS to carry out the acetylation of the amino acid, tryptophan. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. 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 tha...

  16. 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

  17. 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 ...

  18. Effect of clay mineralogy on iron bioavailability and rhizosphere transcription of 2,4-diacetylphloroglucinol biosynthetic genes in biocontrol Pseudomonas protegens.

    Science.gov (United States)

    Almario, Juliana; Prigent-Combaret, Claire; Muller, Daniel; Moënne-Loccoz, Yvan

    2013-05-01

    Pseudomonas strains producing 2,4-diacetylphloroglucinol (DAPG) can protect plants from soilborne phytopathogens and are considered the primary reason for suppressiveness of morainic Swiss soils to Thielaviopsis basicola-mediated black root-rot disease of tobacco, even though they also occur nearby in conducive sandstone soils. The underlying molecular mechanisms accounting for this discrepancy are not understood. In this study, we assessed the hypothesis that the presence of iron-rich vermiculite clay (dominant in suppressive soils) instead of illite (dominant in neighboring conducive soils) translates into higher levels of iron bioavailability and transcription of Pseudomonas DAPG synthetic genes in the tobacco rhizosphere. Rhizosphere monitoring of reporter gene systems pvd-inaZ and phlA-gfp in Pseudomonas protegens indicated that the level of iron bioavailability and the number of cells expressing phl genes (DAPG synthesis), respectively, were higher in vermiculitic than in illitic artificial soils. This was in accordance with the effect of iron on phlA-gfp expression in vitro and, indeed, iron addition to the illitic soil increased the number of cells expressing phlA-gfp. Similar findings were made in the presence of the pathogen T. basicola. Altogether, results substantiate the hypothesis that iron-releasing minerals may confer disease suppressiveness by modulating iron bioavailability in the rhizosphere and expression of biocontrol-relevant genes in antagonistic P. protegens.

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Michael D Barton

    2010-08-01

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

  1. The DapA gene encoding the lysine biosynthetic enzyme dihydrodipicolinate synthase from Coix lacryma-jobi: cloning, characterization, and expression analysis.

    Science.gov (United States)

    Dante, R A; Neto, G C; Leite, A; Yunes, J A; Arruda, P

    1999-11-01

    Dihydrodipicolinate synthase (DHPS) is the main enzyme of a specific branch of the aspartate pathway leading to lysine biosynthesis in higher plants. We have cloned and characterized the DHPS-encoding Dap)A gene from the maize-related grass Coix lacryiana-jobi. The DapA open reading frame is interrupted by two introns and encodes the 326 amino acid-long Coix DHPS protein, which is 95% identical to the maize DHPS protein. Coix DNA gel blot analysis with maize DHPS cDNA as a probe showed a single strongly hybridizing band along with faint bands. RNA gel blot analysis showed that DHPS transcripts are present in coleoptiles, embryos, endosperms, and roots but are almost undetectable in blades of young leaves of both Coix and maize. The 5'-flanking region of the DapA gene contains a TGACTC GCN4-like element located 372 bp upstream the putative translation start codon. Steady-state levels of DHPS mRNA were slightly reduced in the endosperms and embryos of the maize lysine-rich opaque2 mutants when compared with those in normal kernels. Selective binding assay with the maize Opaque2 protein (O2) showed that the GCN4-like element is not an O2 binding site, suggesting that the DHPS gene is not under the control of O2.

  2. De novo genetic engineering of the camalexin biosynthetic pathway.

    Science.gov (United States)

    Møldrup, Morten E; Salomonsen, Bo; Geu-Flores, Fernando; Olsen, Carl E; Halkier, Barbara A

    2013-09-10

    Camalexin is a tryptophan-derived phytoalexin that is induced in the model plant Arabidopsis thaliana upon pathogen attack. Only few genes in the biosynthetic pathway of camalexin remain unidentified, however, investigation of candidate genes for these steps has proven particularly difficult partly because of redundancy in the genome of Arabidopsis. Here we describe metabolic engineering of the camalexin biosynthetic pathway in the transient Nicotiana benthamiana expression system. Camalexin accumulated in levels corresponding to what is seen in induced Arabidopsis thaliana. We have used this system to evaluate candidate genes suggested to be involved in the camalexin pathway. This has provided biochemical evidence for CYP71A12 conducting same reaction as CYP71A13 in the pathway. We discuss the prospects of using metabolic engineering of camalexin, both with respect to engineering plant defense and as a tool for screening yet unidentified candidate genes in the camalexin pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. 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.

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the...... as observed in T0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.......In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance...... the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8...

  5. 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....

  6. Rapid reconstitution of biosynthetic machinery for fungal metabolites in Aspergillus oryzae: total biosynthesis of aflatrem.

    Science.gov (United States)

    Tagami, Koichi; Minami, Atsushi; Fujii, Ryuya; Liu, Chengwei; Tanaka, Mizuki; Gomi, Katsuya; Dairi, Tohru; Oikawa, Hideaki

    2014-09-22

    Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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; hide

    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.

  8. Rhizophagus intraradices or its associated bacteria affect gene expression of key enzymes involved in the rosmarinic acid biosynthetic pathway of basil.

    Science.gov (United States)

    Battini, Fabio; Bernardi, Rodolfo; Turrini, Alessandra; Agnolucci, Monica; Giovannetti, Manuela

    2016-10-01

    In recent years, arbuscular mycorrhizal fungi (AMF) have been reported to enhance plant biosynthesis of secondary metabolites with health-promoting activities, such as polyphenols, carotenoids, vitamins, anthocyanins, flavonoids and lycopene. In addition, plant growth-promoting (PGP) bacteria were shown to modulate the concentration of nutraceutical compounds in different plant species. This study investigated for the first time whether genes encoding key enzymes of the biochemical pathways leading to the production of rosmarinic acid (RA), a bioactive compound showing antioxidant, antibacterial, antiviral and anti-inflammatory properties, were differentially expressed in Ocimum basilicum (sweet basil) inoculated with AMF or selected PGP bacteria, by using quantitative real-time reverse transcription PCR. O. basilicum plants were inoculated with either the AMF species Rhizophagus intraradices or a combination of two PGP bacteria isolated from its sporosphere, Sinorhizobium meliloti TSA41 and Streptomyces sp. W43N. Present data show that the selected PGP bacteria were able to trigger the overexpression of tyrosine amino-transferase (TAT), hydroxyphenylpyruvate reductase (HPPR) and p-coumaroyl shikimate 3'-hydroxylase isoform 1 (CS3'H iso1) genes, 5.7-fold, 2-fold and 2.4-fold, respectively, in O. basilicum leaves. By contrast, inoculation with R. intraradices triggered TAT upregulation and HPPR and CS3'H iso1 downregulation. Our data suggest that inoculation with the two selected strains of PGP bacteria utilised here could represent a suitable biotechnological tool to be implemented for the production of O. basilicum plants with increased levels of key enzymes for the biosynthesis of RA, a compound showing important functional properties as related to human health.

  9. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

    Directory of Open Access Journals (Sweden)

    Shinji Kishimoto

    2016-08-01

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

  10. The proportion of non-aflatoxigenic strains of the Aspergillus flavus/oryzae complex from meju by analyses of the aflatoxin biosynthetic genes.

    Science.gov (United States)

    Hong, Seung-Beom; Lee, Mina; Kim, Dae-Ho; Chung, Soo-Hyun; Shin, Hyeon-Dong; Samson, Robert A

    2013-12-01

    Strains of the Aspergillus flavus/oryzae complex are frequently isolated from meju, a fermented soybean product, that is used as the starting material for ganjang (soy sauce) and doenjang (soybean paste) production. In this study, we examined the aflatoxin producing capacity of A. flavus/oryzae strains isolated from meju. 192 strains of A. flavus/oryzae were isolated from more than 100 meju samples collected from diverse regions of Korea from 2008 to 2011, and the norB-cypA, omtA, and aflR genes in the aflatoxin biosynthesis gene cluster were analyzed. We found that 178 strains (92.7%) belonged to non-aflatoxigenic group (Type I of norB-cypA, IB-L-B-, IC-AO, or IA-L-B- of omtA, and AO type of aflR), and 14 strains (7.3%) belonged to aflatoxin-producible group (Type II of norB-cypA, IC-L-B+/B- or IC-L-B+ of omtA, and AF type of aflR). Only 7 strains (3.6%) in the aflatoxin-producible group produced aflatoxins on Czapek yeast-extract medium. The aflatoxin-producing capability of A. flavus/oryzae strains from other sources in Korea were also investigated, and 92.9% (52/56) strains from air, 93.9% (31/33) strains from rice straw, 91.7% (11/12) strains from soybean, 81.3% (13/16) strains from corn, 82% (41/50) strains from peanut, and 73.2% (41/56) strains from arable soil were included in the non-aflatoxigenic group. The proportion of non-aflatoxigenicity of meju strains was similar to that of strains from soybean, air and rice straw, all of which have an effect on the fermentation of meju. The data suggest that meju does not have a preference for non-aflatoxigenic or aflatoxin-producible strains of A. flavus/oryzae from the environment of meju. The non-aflatoxigenic meju strains are proposed to be named A. oryzae, while the meju strains that can produce aflatoxins should be referred to A. flavus in this study.

  11. An Overview on the Marine Neurotoxin, Saxitoxin: Genetics, Molecular Targets, Methods of Detection and Ecological Functions

    Directory of Open Access Journals (Sweden)

    Gary S. Sayler

    2013-03-01

    Full Text Available Marine neurotoxins are natural products produced by phytoplankton and select species of invertebrates and fish. These compounds interact with voltage-gated sodium, potassium and calcium channels and modulate the flux of these ions into various cell types. This review provides a summary of marine neurotoxins, including their structures, molecular targets and pharmacologies. Saxitoxin and its derivatives, collectively referred to as paralytic shellfish toxins (PSTs, are unique among neurotoxins in that they are found in both marine and freshwater environments by organisms inhabiting two kingdoms of life. Prokaryotic cyanobacteria are responsible for PST production in freshwater systems, while eukaryotic dinoflagellates are the main producers in marine waters. Bioaccumulation by filter-feeding bivalves and fish and subsequent transfer through the food web results in the potentially fatal human illnesses, paralytic shellfish poisoning and saxitoxin pufferfish poisoning. These illnesses are a result of saxitoxin’s ability to bind to the voltage-gated sodium channel, blocking the passage of nerve impulses and leading to death via respiratory paralysis. Recent advances in saxitoxin research are discussed, including the molecular biology of toxin synthesis, new protein targets, association with metal-binding motifs and methods of detection. The eco-evolutionary role(s PSTs may serve for phytoplankton species that produce them are also discussed.

  12. A rapid bioassay for detecting saxitoxins using a Daphnia acute toxicity test

    Energy Technology Data Exchange (ETDEWEB)

    Ferrao-Filho, Aloysio da S., E-mail: aloysio@ioc.fiocruz.b [Laboratorio de Avaliacao e Promocao da Saude Ambiental, Departamento de Biologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900 (Brazil); Soares, Maria Carolina S., E-mail: mcarolsoares@gmail.co [Departamento de Engenharia Sanitaria e Ambiental Faculdade de Engenharia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 36036-900 (Brazil); Freitas de Magalhaes, Valeria, E-mail: valeria@biof.ufrj.b [Laboratorio de Ecofisiologia e Toxicologia de Cianobacterias, Instituto de Biofisica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundao, Rio de Janeiro, RJ 21949-900 (Brazil); Azevedo, Sandra M.F.O., E-mail: sazevedo@biof.ufrj.b [Laboratorio de Ecofisiologia e Toxicologia de Cianobacterias, Instituto de Biofisica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundao, Rio de Janeiro, RJ 21949-900 (Brazil)

    2010-06-15

    Bioassays using Daphnia pulex and Moina micrura were designed to detect cyanobacterial neurotoxins in raw water samples. Phytoplankton and cyanotoxins from seston were analyzed during 15 months in a eutrophic reservoir. Effective time to immobilize 50% of the exposed individuals (ET{sub 50}) was adopted as the endpoint. Paralysis of swimming movements was observed between approx0.5-3 h of exposure to lake water containing toxic cyanobacteria, followed by an almost complete recovery of the swimming activity within 24 h after being placed in control water. The same effects were observed in bioassays with a saxitoxin-producer strain of Cylindrospermopsis raciborskii isolated from the reservoir. Regression analysis showed significant relationships between ET{sub 50}vs. cell density, biomass and saxitoxins content, suggesting that the paralysis of Daphnia in lake water samples was caused by saxitoxins found in C. raciborskii. Daphnia bioassay was found to be a sensitive method for detecting fast-acting neurotoxins in natural samples, with important advantages over mouse bioassays. - A new Daphnia bioassay, as an alternative to the mouse bioassay, is able to detect effects of fast-acting, potent neurotoxins in raw water.

  13. Biosynthetic pathway for poly(3-hydroxypropionate) in recombinant Escherichia coli.

    Science.gov (United States)

    Wang, Qi; Liu, Changshui; Xian, Mo; Zhang, Yongguang; Zhao, Guang

    2012-08-01

    Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, we engineered a P3HP biosynthetic pathway in recombinant Escherichia coli. The genes for malonyl-CoA reductase (mcr, from Chloroflexus aurantiacus), propionyl-CoA synthetase (prpE, from E. coli), and polyhydroxyalkanoate synthase (phaC1, from Ralstonia eutropha) were cloned and expressed in E. coli. The E. coli genes accABCD encoding acetyl-CoA carboxylase were used to channel the carbon into the P3HP pathway. Using glucose as a sole carbon source, the cell yield and P3HP content were 1.32 g/L and 0.98% (wt/wt [cell dry weight]), respectively. Although the yield is relatively low, our study shows the feasibility of engineering a P3HP biosynthetic pathway using a structurally unrelated carbon source in bacteria.

  14. 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.

  15. Spatial organization of the vindoline biosynthetic pathway in Catharanthus roseus.

    Science.gov (United States)

    Guirimand, Grégory; Guihur, Anthony; Poutrain, Pierre; Héricourt, François; Mahroug, Samira; St-Pierre, Benoit; Burlat, Vincent; Courdavault, Vincent

    2011-04-15

    Vindoline constitutes the main terpenoid indole alkaloid accumulated in leaves of Catharanthus roseus, and four genes involved in its biosynthesis have been identified. However, the spatial organization of the tabersonine-to-vindoline biosynthetic pathway is still incomplete. To pursue the characterization of this six-step conversion, we illustrated, with in situ hybridization, that the transcripts of the second biosynthetic enzyme, 16-hydroxytabersonine 16-O-methyltransferase (16OMT), are specifically localized to the aerial organ epidermis. At the subcellular level, by combining GFP imaging, bimolecular fluorescence complementation assays and yeast two-hybrid analysis, we established that the first biosynthetic enzyme, tabersonine 16-hydroxylase (T16H), is anchored to the ER as a monomer via a putative N-terminal helix that we cloned using a PCR approach. We also showed that 16OMT homodimerizes in the cytoplasm, allowing its exclusion from the nucleus and thus facilitating the uptake of T16H conversion product, although no T16H/16OMT interactions occur. Moreover, the two last biosynthetic enzymes, desacetoxyvindoline-4-hydroxylase (D4H) and deacetylvindoline-4-O-acetyltransferase (DAT), were shown to operate as monomers that reside in the nucleocytoplasmic compartment following passive diffusion to the nucleus allowed by the protein size. No D4H/DAT interactions were detected, suggesting the absence of metabolic channeling in the vindoline biosynthetic pathway. Finally, these results highlight the importance of the inter- and intracellular translocations of intermediates during the vindoline biosynthesis and their potential regulatory role. Copyright © 2010 Elsevier GmbH. All rights reserved.

  16. Facile and Cost-Effective Detection of Saxitoxin Exploiting Aptamer Structural Switching

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    Karol Alfaro

    2015-01-01

    Full Text Available A simple method to detect saxitoxin (STX, one of the main components of the paralytic shellfish poison from red tide, has been developed. By using a next generation dye for double-stranded DNA we were able to differentiate fluorescence from STX-binding aptamers when exposed to different concentrations of STX, suggesting a change in aptamer folding upon target binding. The developed method is extremely rapid, only requiring small sample volumes, with quantitative results in the concentration range of 15 ng/mL to 3 μg/mL of STX, with a detection limit of 7.5 ng/mL.

  17. Biosynthetic Modularity Rules in the Bisintercalator Family of Antitumor Compounds

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    Javier Fernández

    2014-05-01

    Full Text Available Diverse actinomycetes produce a family of structurally and biosynthetically related non-ribosomal peptide compounds which belong to the chromodepsipeptide family. These compounds act as bisintercalators into the DNA helix. They give rise to antitumor, antiparasitic, antibacterial and antiviral bioactivities. These compounds show a high degree of conserved modularity (chromophores, number and type of amino acids. This modularity and their high sequence similarities at the genetic level imply a common biosynthetic origin for these pathways. Here, we describe insights about rules governing this modular biosynthesis, taking advantage of the fact that nowadays five of these gene clusters have been made public (thiocoraline, triostin, SW-163 and echinomycin/quinomycin. This modularity has potential application for designing and producing novel genetic engineered derivatives, as well as for developing new chemical synthesis strategies. These would facilitate their clinical development.

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

    Science.gov (United States)

    Nakata, Paul A

    2011-10-20

    Although it is known that oxalic acid provides a selective advantage to the secreting microbe our understanding of how this acid is biosynthesized remains incomplete. This study reports the identification, cloning, and partial characterization of the oxalic acid biosynthetic enzyme from the animal bacterial pathogen, Burkholderia mallei. The discovered gene was named oxalate biosynthetic component (obc)1. Complementation of Burkholderia oxalate defective (Bod)1, a Burkholderia glumae mutant that lacks expression of a functional oxalic acid biosynthetic operon, revealed that the obc1 was able to rescue the no oxalate mutant phenotype. This single gene rescue is in contrast to the situation found in B. glumae which required the expression of two genes, obcA and obcB, to achieve complementation. Enzyme assays showed that even though the two Burkholderia species differed in the number of genes required to encode a functional enzyme, both catalyzed the same acyl-CoA dependent biosynthetic reaction. In addition, mutagenesis studies suggested a similar domain structure of the assembled oxalate biosynthetic enzymes whether encoded by one or two genes. Published by Elsevier GmbH.

  19. Biosynthetic pathway and health benefits of fucoxanthin, an algae-specific xanthophyll in brown seaweeds.

    Science.gov (United States)

    Mikami, Koji; Hosokawa, Masashi

    2013-07-02

    Fucoxanthin is the main carotenoid produced in brown algae as a component of the light-harvesting complex for photosynthesis and photoprotection. In contrast to the complete elucidation of the carotenoid biosynthetic pathways in red and green algae, the biosynthetic pathway of fucoxanthin in brown algae is not fully understood. Recently, two models for the fucoxanthin biosynthetic pathway have been proposed in unicellular diatoms; however, there is no such information for the pathway in brown seaweeds to date. Here, we propose a biosynthetic pathway for fucoxanthin in the brown seaweed, Ectocarpus siliculosus, derived from comparison of carotenogenic genes in its sequenced genome with those in the genomes of two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum. Currently, fucoxanthin is receiving attention, due to its potential benefits for human health. Therefore, new knowledge regarding the medical and nutraceutical properties of fucoxanthin from brown seaweeds is also summarized here.

  20. Biosynthetic Pathway and Health Benefits of Fucoxanthin, an Algae-Specific Xanthophyll in Brown Seaweeds

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    Masashi Hosokawa

    2013-07-01

    Full Text Available Fucoxanthin is the main carotenoid produced in brown algae as a component of the light-harvesting complex for photosynthesis and photoprotection. In contrast to the complete elucidation of the carotenoid biosynthetic pathways in red and green algae, the biosynthetic pathway of fucoxanthin in brown algae is not fully understood. Recently, two models for the fucoxanthin biosynthetic pathway have been proposed in unicellular diatoms; however, there is no such information for the pathway in brown seaweeds to date. Here, we propose a biosynthetic pathway for fucoxanthin in the brown seaweed, Ectocarpus siliculosus, derived from comparison of carotenogenic genes in its sequenced genome with those in the genomes of two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum. Currently, fucoxanthin is receiving attention, due to its potential benefits for human health. Therefore, new knowledge regarding the medical and nutraceutical properties of fucoxanthin from brown seaweeds is also summarized here.

  1. Uncovering the Complex Transcriptome Response of Mytilus chilensis against Saxitoxin: Implications of Harmful Algal Blooms on Mussel Populations.

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    Camille Detree

    Full Text Available Saxitoxin (STX, a principal phycotoxin contributing to paralytic shellfish poisoning, is largely produced by marine microalgae of the genus Alexandrium. This toxin affects a wide range of species, inducing massive deaths in fish and other marine species. However, marine bivalves can resist and accumulate paralytic shellfish poisons. Despite numerous studies on the impact of STX in marine bivalves, knowledge regarding STX recognition at molecular level by benthic species remains scarce. Therefore, the aim of this study was to identify novel genes that interact with STX in the Chilean mussel Mytilus chilensis. For this, RNA-seq and RT-qPCR approaches were used to evaluate the transcriptomic response of M. chilensis to a purified STX as well as in vivo Alexandrium catenella exposure. Approximately 800 million reads were assembled, generating 138,883 contigs that were blasted against the UniProt Mollusca database. Pattern Recognition Receptors (PRRs involved in mussel immunity, such as Toll-like receptors, tumor necrosis factor receptors, and scavenger-like receptors were found to be strongly upregulated at 8 and 16 h post-STX injection. These results suggest an involvement of PRRs in the response to STX, as well as identifying potential, novel STX-interacting receptors in this Chilean mussel. This study is the first transcriptomic overview of the STX-response in the edible species M. chilensis. However, the most significant contribution of this work is the identification of immune receptors and pathways potentially involved in the recognition and defense against STX's toxicity and its impact of harmful algae blooms on wild and cultivated mussel populations.

  2. Reconstitution of highly purified saxitoxin-sensitive Na+-channels into planar lipid bilayers.

    Science.gov (United States)

    Hanke, W; Boheim, G; Barhanin, J; Pauron, D; Lazdunski, M

    1984-01-01

    Highly purified Na+-channels isolated from rat brain have been reconstituted into virtually solvent-free planar lipid bilayer membranes. Two different types of electrically excitable channels were detected in the absence of any neurotoxins. The activity of both channels was blocked by saxitoxin. The first channel type is highly selective for Na+ over K+ (approximately 10:1), it shows a bursting behavior, a conductance of 25 pS in Na+-Ringer and undergoes continuous opening and closing events for periods of minutes within a defined range of negative membranes voltages. The second channel type has a conductance of 150 pS and a lower selectivity for Na+ and K+ (2.2:1); only a few opening and closing events are observed with this channel after one voltage jump. The latter type of channel is also found with highly purified Na+-channel from Electrophorus electricus electroplax. A qualitative analysis of the physicochemical and pharmacological properties of the high conductance channel has been carried out. Channel properties are affected not only by saxitoxin but also by a scorpion (Centruroides suffusus suffusus) toxin and a sea anemone (Anemonia sulcata) toxin both known to be selective for the Na+-channel. The spontaneous transformation of the large conductance channel type into the small one has been considered; the two channel types may represent the expression of activity of different conformational states of the same protein. Images Fig. 1. PMID:6325173

  3. A biosynthetic pathway for anandamide.

    Science.gov (United States)

    Liu, Jie; Wang, Lei; Harvey-White, Judith; Osei-Hyiaman, Douglas; Razdan, Raj; Gong, Qian; Chan, Andrew C; Zhou, Zhifeng; Huang, Bill X; Kim, Hee-Yong; Kunos, George

    2006-09-05

    The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released "on demand" by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE-PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC/phosphatase pathway, which is up-regulated by LPS, whereas NAPE-PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC/phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC/phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target.

  4. Fungal peroxisomes as biosynthetic organelles.

    Science.gov (United States)

    Stehlik, Thorsten; Sandrock, Björn; Ast, Julia; Freitag, Johannes

    2014-12-01

    Peroxisomes are nearly ubiquitous single-membrane organelles harboring multiple metabolic pathways beside their prominent role in the β-oxidation of fatty acids. Here we review the diverse metabolic functions of peroxisomes in fungi. A variety of fungal metabolites are at least partially synthesized inside peroxisomes. These include the essential co-factor biotin but also different types of secondary metabolites. Peroxisomal metabolites are often derived from acyl-CoA esters for example β-oxidation intermediates. In several ascomycetes a subtype of peroxisomes has been identified that is metabolically inactive but is required to plug the septal pores of wounded hyphae. Thus, peroxisomes are versatile organelles that can adapt their function to the life style of an organism. This remarkable variability suggests that the full extent of the biosynthetic capacity of peroxisomes is still elusive. Moreover, in fungi peroxisomes are non-essential under laboratory conditions making them attractive organelles for biotechnological approaches and the design of novel metabolic pathways in customized peroxisomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

    Science.gov (United States)

    Nozaki, T; Asai, T; Sanchez, L B; Kobayashi, S; Nakazawa, M; Takeuchi, T

    1999-11-05

    The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.

  6. On the Chemistry, Toxicology and Genetics of the Cyanobacterial Toxins, Microcystin, Nodularin, Saxitoxin and Cylindrospermopsin

    Directory of Open Access Journals (Sweden)

    Leanne Pearson

    2010-05-01

    Full Text Available The cyanobacteria or “blue-green algae”, as they are commonly termed, comprise a diverse group of oxygenic photosynthetic bacteria that inhabit a wide range of aquatic and terrestrial environments, and display incredible morphological diversity. Many aquatic, bloom-forming species of cyanobacteria are capable of producing biologically active secondary metabolites, which are highly toxic to humans and other animals. From a toxicological viewpoint, the cyanotoxins span four major classes: the neurotoxins, hepatotoxins, cytotoxins, and dermatoxins (irritant toxins. However, structurally they are quite diverse. Over the past decade, the biosynthesis pathways of the four major cyanotoxins: microcystin, nodularin, saxitoxin and cylindrospermopsin, have been genetically and biochemically elucidated. This review provides an overview of these biosynthesis pathways and additionally summarizes the chemistry and toxicology of these remarkable secondary metabolites.

  7. Cyanobacterial occurrence and detection of microcystins and saxitoxins in reservoirs of the Brazilian semi-arid

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    Jessica Roberts Fonseca

    2015-03-01

    Full Text Available Aim:The rapid spread of cyanobacteria in water sources and reservoirs has caused serious environmental damage and public health problems, and consists in a problem that challenges the institutions responsible for providing water to the population. In this study, the quantification of microcystin, saxitoxins and cyanobacteria levels was performed over 3 years in the semi-arid reservoirs of Rio Grande do Norte (Brazil. In addition, we analyzed the seasonal distribution of cyanotoxins and the percentage of cyanobacteria and cyanotoxins which were above the limit established by Brazilian law.MethodsThe study was conducted between 2009 and 2011 in four dams with six sites: Armando Ribeiro Gonçalves (ARG in Itajá, San Rafael (SR and Jucurutu; Passagem das Traíras (PT; Itans and Gargalheiras (GARG. Cyanobacteria presence were quantified and identified and the presence of microcystins (MCYs and saxitoxins (STXs was investigated by ELISA.ResultsThe densities of cyanobacteria were found to be above the permitted in 76% of cases. The ELISA results showed that of the 128 samples analyzed, 27% were above the maximum allowed by the Brazilian Ministry of Health Order 2914/2011. A seasonal pattern for the presence of MCYs was found (0.00227 to 24.1954 µg.L–1, with the highest values in the rainy season. There was no clear seasonal pattern for STXs (0.003 to 0.766 µg.L–1.ConclusionsThis study showed the importance of establishing a water quality monitoring for human consumption and its potability standards since the concentration of MCYs in some samples was above the maximum limit allowed by Brazilian law, thus posing a risk to public health since the conventional water treatment is not able to eliminate these potent hepatotoxins.

  8. 4-Aminopyridine antagonizes saxitoxin-and tetrodotoxin-induced cardiorespiratory depression.

    Science.gov (United States)

    Chang, F C; Bauer, R M; Benton, B J; Keller, S A; Capacio, B R

    1996-06-01

    Antagonism of saxitoxin-and tetrodotoxin-induced lethality by 4-aminopyridine was studied in urethane-anesthetized guinea pigs instrumented for the concurrent recordings of medullary respiratory-related unit activities (Bötzinger complex and Nu. para-Ambiguus), diaphragmatic electromyogram, electrocorticogram, Lead II electrocardiogram, blood pressure, end-tidal CO2 and arterial O2/CO2/pH. The toxin (either saxitoxin or tetrodotoxin) was infused at a dose rate of 0.3 microgram/kg/min (i.v.) to produce a state of progressive cardiorespiratory depression. The animals were artificially ventilated when the magnitude of integrated diaphragm activities was reduced to 50% of control. Immediately after the disappearance of the diaphragm electromyogram, the toxin infusion was terminated, and 4-aminopyridine (2 mg/kg, i.v.) was administered. The therapeutic effect of 4-aminopyridine was striking in that the toxin-induced blockade of diaphragmatic neurotransmission, vascular hypotension, myocardial anomalies, bradycardia and aberrant discharge patterns of medullary respiratory-related neurons could all be promptly restored to a level comparable to that of control condition. The animals were typically able to breathe spontaneously within minutes after 4-aminopyridine. At the dose level used to achieve the desired therapeutic responses, 4-aminopyridine produced no sign of seizure and convulsion. Although less serious side-effects such as cortical excitant/arousal and transient periods of fascicular twitch could be observed, these events were of minor concern, in our opinion, particularly in view of the remarkable therapeutic effects of 4-aminopyridine.

  9. Recovery from the lethal effects of saxitoxin: a therapeutic window for 4-aminopyridine (4-AP).

    Science.gov (United States)

    Benton, B J; Keller, S A; Spriggs, D L; Capacio, B R; Chang, F C

    1998-04-01

    We have shown that saxitoxin (STX) induced lethality can be reversed by 4-AP when it is administered at the time of respiratory arrest [Benton, B. J., Spriggs, D. L., Capacio, B. R. and Chang, F.-C. T. (1995) 4-Aminopyridine antagonizes the lethal effects of saxitoxin (STX) and tetrodotoxin (TTX). International Society of Toxicology, 5th Pan American Symposium on Animal, Plant and Microbial Toxins, Frederick, MD. July/August 1995, p. 217]. The purpose of this study was to determine whether 4-AP's efficacy could be enhanced further when administered at different times relative to STX intoxication. The animals used in this study were chronically instrumented for concurrent recordings of diaphragm electromyogram (DEMG), neck skeletal muscle electromyogram, Lead II electrocardiogram, and electrocorticogram (ECoG). There were five groups of unanesthetized guinea pigs. The first group served as 4-AP controls and received a 2 mg/kg i.m. dose of 4-AP. The four remaining groups were given a lethal dose of STX (5 microg/kg i.m.); the second group, STX controls, received no 4-AP; the third group, the 4-AP treatment group, received 4-AP immediately following cardiorespiratory collapse; the fourth group was the 4-AP/STX co-administration group and 4-AP was given concurrently with STX; and the fifth group was the 4-AP pretreatment group in which 4-AP was given 10 min before STX. At the point of STX-induced cardiorespiratory collapse, the guinea pigs were ventilated and given an i.p. injection of sodium bicarbonate. Results showed that 4-AP prevented cardiorespiratory collapse in 3/7 animals in the 4-AP pretreatment group. Also, 4-AP in conjunction with artificial ventilation and sodium bicarbonate accelerated recovery from STX-induced cardiorespiratory collapse in all the treatment groups compared to the STX controls.

  10. Biosynthetic studies on terpenoids produced by Streptomyces.

    Science.gov (United States)

    Kuzuyama, Tomohisa

    2017-07-01

    Terpenoids are a large and highly diverse group of natural products. All terpenoids are biosynthesized from isoprenyl diphosphate formed by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer dimethylallyl diphosphate (DMAPP). Two distinct biosynthetic pathways produce the essential primary metabolites IPP and DMAPP: the 2-C-methylerythritol 4-phosphate pathway and the mevalonate pathway. The isoprenyl substrates can be cyclized by terpene cyclase into single-ring or multi-ring products, which can be further diversified by subsequent modification reactions, such as hydroxylation and glycosylation. This review article describes the biosynthetic pathways of terpenoids produced by Streptomyces and their related novel enzymes.

  11. Gustatory Detection of Tetrodotoxin and Saxitoxin, and Its Competitive Inhibition by Quinine and Strychnine in Freshwater Fishes

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    Toshiaki J. Hara

    2011-11-01

    Full Text Available Fish detect extremely low levels of marine toxins tetrodotoxin (TTX and saxitoxin (STX via the specialized gustatory receptor(s. Physiological and pharmacological studies show that receptor(s for TTX and STX are distinct from those which detect feeding stimulant amino acids and bile acids, and that TTX and STX do not share the same receptor populations, while interacting with quinine and strychnine in a competitive fashion suggestive of an antidotal relationship.

  12. Evolution of the isoprene biosynthetic pathway in kudzu.

    Science.gov (United States)

    Sharkey, Thomas D; Yeh, Sansun; Wiberley, Amy E; Falbel, Tanya G; Gong, Deming; Fernandez, Donna E

    2005-02-01

    Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.

  13. Evolution of the Isoprene Biosynthetic Pathway in Kudzu1[w

    Science.gov (United States)

    Sharkey, Thomas D.; Yeh, Sansun; Wiberley, Amy E.; Falbel, Tanya G.; Gong, Deming; Fernandez, Donna E.

    2005-01-01

    Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate. PMID:15653811

  14. Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

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    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.

  15. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-05-20

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

  17. 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.

  18. Results of a Saxitoxin Proficiency Test Including Characterization of Reference Material and Stability Studies

    Directory of Open Access Journals (Sweden)

    Kirsi Harju

    2015-11-01

    Full Text Available A saxitoxin (STX proficiency test (PT was organized as part of the Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox project. The aim of this PT was to provide an evaluation of existing methods and the European laboratories’ capabilities for the analysis of STX and some of its analogues in real samples. Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning (PSP toxins were produced as reference sample matrices. The reference material was characterized using various analytical methods. Acidified algal extract samples at two concentration levels were prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose. Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries. The PT offered the participating laboratories the possibility to assess their performance regarding the qualitative and quantitative detection of PSP toxins. Various techniques such as official Association of Official Analytical Chemists (AOAC methods, immunoassays, and liquid chromatography-mass spectrometry were used for sample analyses.

  19. Results of a Saxitoxin Proficiency Test Including Characterization of Reference Material and Stability Studies.

    Science.gov (United States)

    Harju, Kirsi; Rapinoja, Marja-Leena; Avondet, Marc-André; Arnold, Werner; Schär, Martin; Luginbühl, Werner; Kremp, Anke; Suikkanen, Sanna; Kankaanpää, Harri; Burrell, Stephen; Söderström, Martin; Vanninen, Paula

    2015-11-25

    A saxitoxin (STX) proficiency test (PT) was organized as part of the Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox) project. The aim of this PT was to provide an evaluation of existing methods and the European laboratories' capabilities for the analysis of STX and some of its analogues in real samples. Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning (PSP) toxins were produced as reference sample matrices. The reference material was characterized using various analytical methods. Acidified algal extract samples at two concentration levels were prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose. Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries. The PT offered the participating laboratories the possibility to assess their performance regarding the qualitative and quantitative detection of PSP toxins. Various techniques such as official Association of Official Analytical Chemists (AOAC) methods, immunoassays, and liquid chromatography-mass spectrometry were used for sample analyses.

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  1. Metabolic modeling of Rosmarinic acid biosynthetic pathway

    OpenAIRE

    Sundaram, Shanthy; Tripathi, Ashutosh; Gupta, Deepak K

    2010-01-01

    Rosmarinic acid (RA) is an ester of caffeic acid and 3, 4‐dihydroxyphenyllacticacid. It is commonly found in Coleus blumei, Salvia officinalis, Melissa officinalis and Rosmarinus officinalis. The biosynthesis of RA starts with precursor molecules L‐phenylalanine and L‐tyrosine. Simulation of RA biosynthetic pathway was done using Gepasi Software, includes the reaction kinetics of each step of the pathway and different integration methods such as Euler's method. Optimization of the significant...

  2. The Biosynthetic Pathways of Tanshinones and Phenolic Acids in Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Xiao-Hui Ma

    2015-09-01

    Full Text Available Secondary metabolites from plants play key roles in human medicine and chemical industries. Due to limited accumulation of secondary metabolites in plants and their important roles, characterization of key enzymes involved in biosynthetic pathway will enable metabolic engineering or synthetic biology to improve or produce the compounds in plants or microorganisms, which provides an alternative for production of these valuable compounds. Salvia miltiorrhiza, containing tanshinones and phenolic acids as its active compounds, has been widely used for the treatment of cardiovascular and cerebrovascular diseases. The biosynthetic analysis of secondary metabolites in S. miltiorrhiza has made great progress due to the successful genetic transformation system, simplified hairy roots system, and high-throughput sequencing. The cloned genes in S. miltiorrhiza had provided references for functional characterization of the post-modification steps involved in biosynthesis of tanshinones and phenolic acids, and further utilization of these steps in metabolic engineering. The strategies used in these studies could provide solid foundation for elucidation of biosynthetic pathways of diterpenoids and phenolic acids in other species. The present review systematically summarizes recent advances in biosynthetic pathway analysis of tanshinones and phenolic acids as well as synthetic biology and metabolic engineering applications of the rate-limiting genes involved in the secondary metabolism in S. miltiorrhiza.

  3. Intertidal marine sediment harbours Actinobacteria with promising bioactive and biosynthetic potential.

    Science.gov (United States)

    Jose, Polpass Arul; Jha, Bhavanath

    2017-08-30

    Actinobacteria are the major source of bioactive natural products that find their value in research and drug discovery programmes. Antimicrobial resistance and the resulting high demand for novel antibiotics underscore the need for exploring novel sources of these bacteria endowed with biosynthetic potential. Intertidal ecosystems endure regular periods of immersion and emersion, and represent an untapped source of Actinobacteria. In this study, we studied the diversity and biosynthetic potential of cultivable Actinobacteria from intertidal sediments of Diu Island in the Arabian Sea. A total of 148 Actinobacteria were selectively isolated using a stamping method with eight isolation media. Isolates were grouped into OTUs based on their 16S rRNA gene sequence, and categorized within actinobacterial families such as Glycomycetaceae, Micromonosporaceae, Nocardiaceae, Nocardiopsaceae, Pseudonocardiaceae, Streptomycetaceae, and Thermomonosporaceae. The biosynthetic potential of the Actinobacteria, necessary for secondary metabolite biosynthesis, was screened and confirmed by extensive fingerprinting approaches based on genes coding for polyketide synthases and nonribosomal peptide synthetases. The observed biosynthetic potential was correlated with the antibacterial activity exhibited by these isolates in laboratory conditions. Ultimately, the results demonstrate that intertidal sediment is a rich source of diverse cultivable Actinobacteria with high potential to synthesize novel bioactive compounds in their genomes.

  4. 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.

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

    Science.gov (United States)

    Zuiter, Afnan Saeid; Sawwan, Jammal; Al Abdallat, Ayed

    2012-08-10

    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. 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. 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.

  6. CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production.

    Science.gov (United States)

    Kim, Seong Keun; Han, Gui Hwan; Seong, Wonjae; Kim, Haseong; Kim, Seon-Won; Lee, Dae-Hee; Lee, Seung-Goo

    2016-11-01

    Methods for simple and efficient regulation of metabolic pathway genes are essential for maximizing product titers and conversion yields, and for minimizing the metabolic burden caused by heterologous expression of multiple genes often in the operon context. Clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) is emerging as a promising tool for transcriptional modulation. In this study, we developed a regulatable CRISPRi system for fine-tuning biosynthetic pathways and thus directing carbon flux toward target product synthesis. By exploiting engineered Escherichia coli harboring a biosynthetic mevalonate (MVA) pathway and plant-derived terpenoid synthases, the CRISPRi system successfully modulated the expression of all the MVA pathway genes in the context of operon and blocked the transcription of the acetoacetyl-CoA thiolase enzyme that catalyzes the first step in the MVA pathway. This CRISPRi-guided balancing of expression of MVA pathway genes led to enhanced production of (-)-α-bisabolol (C15) and lycopene (C40) and alleviation of cell growth inhibition that may be caused by expression of multiple enzymes or production of toxic intermediate metabolites in the MVA pathway. Coupling CRISPRi to cell growth by regulating an endogenous essential gene (ispA) increased isoprene (C5) production. The regulatable CRISPRi system proved to be a robust platform for systematic modulation of biosynthetic and endogenous gene expression, and can be used to tune biosynthetic metabolic pathways. Its application can enable the development of microbial 'smart cell' factories that can produce other industrially valuable products in the future. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  7. Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhai, Ying; Bai, Silei; Liu, Jingjing; Yang, Liyuan [National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Han, Li; Huang, Xueshi [Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University, Shenyang 110819 (China); He, Jing, E-mail: hejingjj@mail.hzau.edu.cn [National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China)

    2016-04-22

    Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-frame gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis. - Highlights: • Cloning of the aureothricin biosynthetic gene cluster from Streptomyces thioluteus DSM 40027. • Identification of the aureothricin gene cluster by heterologous expression and in-frame gene deletion. • The heterogenetic thioesterase HlmK significantly improved dithiolopyrrolones production of the aureothricin gene cluster. • Identification of HlmK as an unusual type II thioesterase.

  8. 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

  9. The Biosynthetic Basis of Cell Size Control.

    Science.gov (United States)

    Schmoller, Kurt M; Skotheim, Jan M

    2015-12-01

    Cell size is an important physiological trait that sets the scale of all biosynthetic processes. Although physiological studies have revealed that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained unclear. Here we review recent progress in identifying the molecular mechanisms of cell size control. We focus on budding yeast, where cell growth dilutes a cell cycle inhibitor to couple growth and division. We discuss a new model for size control based on the titration of activator and inhibitor molecules whose synthesis rates are differentially dependent on cell size. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. 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.

  11. 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

  12. Effects of crude extracts of a saxitoxin-producer strain of the cyanobacterium Cylindrospermopsis raciborskii on the swimming behavior of wild and laboratory reared guppy Poecilia vivipara.

    Science.gov (United States)

    Lopes, Karina C; Ferrão-Filho, Aloysio da S; Dos Santos, Everton G N; Cunha, Rodolfo A; Santos, Cláudia P

    2017-04-01

    The cyanobacterium Cylindrospermopsis raciborskii is an invasive species in water supply reservoirs worldwide, which can produces cylindrospermopsins and saxitoxins. In the wild, guppy (Poecilia vivipara) can be exposed to cyanotoxins, but those born and reared in laboratory are free of this contact. The aim of this paper was to comparatively measure the locomotor activity of 'wild' and 'lab' P. vivipara before and after exposure to crude extracts of two different cultures of C. raciborskii (CYRF-01), a saxitoxin-procucer strain. The movement of each fish was recorded using an image monitoring system (Videomex V(®)) before and after 48 h exposure to cyanobacterial extracts. Each experiment was performed during 4 h, with 1 h acclimation and 3 h recording period of the parameters Distance performed (DP), Swimming time (SwT), Stereotypic time (StT), Resting time (RT) and Average speed (AS). The quantification of saxitoxin in the solutions was performed by the enzyme-linked immunosorbent assay (ELISA). The weight or the total length did not influence the locomotor activity of fish in any of the experiments. The saxitoxin value was similar for both cultures (Culture 1: 7.3 μg L(-1) and Culture 2: 8.6 μg L(-1)). However, in experiments with Culture 1 an increased activity in most parameters was observed, while in Culture 2, a decreased activity was observed only in 'lab' fish. Wild fish was less affected, showing higher resistance to both cyanobacterial crude extracts. This study showed that different cultures of the same strain of C. raciborskii and with similar contents of saxitoxin are able to change the locomotor activity of P. vivipara, contributing to the validation of the use of behavioral parameters to the evaluation of sublethal effects of toxic cyanobacteria on fish. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

    Ono, Hajime; Rewitz, Kim; Shinoda, Tetsu

    2006-01-01

    Ecdysteroids regulate many key developmental events in arthropods including molting and metamorphosis. Recently, members of the Drosophila Halloween group of genes, that are required for embryonic viability and cuticle deposition, have been shown to code for several cytochrome P450 enzymes...... of the ecdysone biosynthetic machinery. This hypothesis comes from the observation that Cyp307A1 is encoded by the Halloween gene spook (spo), but unlike other Halloween class genes, Dmspo is not expressed during the larval stages. In contrast, Cyp307a2, dubbed spookier (spok), is expressed primarily during...

  14. Regulation of the cholesterol biosynthetic pathway and its integration with fatty acid biosynthesis in the oleaginous microalga Nannochloropsis oceanica.

    Science.gov (United States)

    Lu, Yandu; Zhou, Wenxu; Wei, Li; Li, Jing; Jia, Jing; Li, Fei; Smith, Steven M; Xu, Jian

    2014-01-01

    Sterols are vital structural and regulatory components in eukaryotic cells; however, their biosynthetic pathways and functional roles in microalgae remain poorly understood. In the oleaginous microalga Nannochloropsis oceanica, the sterol biosynthetic pathway produces phytosterols as minor products and cholesterol as the major product. The evidence together with their deduced biosynthetic pathways suggests that N. oceanica exhibits features of both higher plants and mammals. Temporal tracking of sterol profiles and sterol-biosynthetic transcripts in response to changes in light intensity and nitrogen supply reveal that sterols play roles in cell proliferation, chloroplast differentiation, and photosynthesis. Furthermore, the dynamics of fatty acid (FA) and FA-biosynthetic transcripts upon chemical inhibitor-induced sterol depletion reveal possible co-regulation of sterol production and FA synthesis, in that the squalene epoxidase inhibitor terbinafine reduces sterol content yet significantly elevates free FA production. Thus, a feedback regulation of sterol and FA homeostasis is proposed, with the 1-deoxy-D-xylulose 5-phosphate synthase (DXS, the committed enzyme in isoprenoid and sterol biosynthesis) gene potentially subject to feedback regulation by sterols. These findings reveal features of sterol function and biosynthesis in microalgae and suggest new genetic engineering or chemical biology approaches for enhanced oil production in microalgae.

  15. Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

    Science.gov (United States)

    Zhang, Lei; Ding, Ruxian; Chai, Yourong; Bonfill, Mercedes; Moyano, Elisabet; Oksman-Caldentey, Kirsi-Marja; Xu, Tiefeng; Pi, Yan; Wang, Zinan; Zhang, Hanming; Kai, Guoyin; Liao, Zhihua; Sun, Xiaofen; Tang, Kexuan

    2004-01-01

    Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 β-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T3) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H11 (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors. PMID:15084741

  16. Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures.

    Science.gov (United States)

    Zhang, Lei; Ding, Ruxian; Chai, Yourong; Bonfill, Mercedes; Moyano, Elisabet; Oksman-Caldentey, Kirsi-Marja; Xu, Tiefeng; Pi, Yan; Wang, Zinan; Zhang, Hanming; Kai, Guoyin; Liao, Zhihua; Sun, Xiaofen; Tang, Kexuan

    2004-04-27

    Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 beta-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T(3)) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H(11) (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

  17. Enhancement of Nucleoside Production in Hirsutella sinensis Based on Biosynthetic Pathway Analysis

    Directory of Open Access Journals (Sweden)

    Zhi-Qiang Liu

    2017-01-01

    Full Text Available To enhance nucleoside production in Hirsutella sinensis, the biosynthetic pathways of purine and pyrimidine nucleosides were constructed and verified. The differential expression analysis showed that purine nucleoside phosphorylase, inosine monophosphate dehydrogenase, and guanosine monophosphate synthase genes involved in purine nucleotide biosynthesis were significantly upregulated 16.56-fold, 8-fold, and 5.43-fold, respectively. Moreover, dihydroorotate dehydrogenase, uridine nucleosidase, uridine/cytidine monophosphate kinase, and inosine triphosphate pyrophosphatase genes participating in pyrimidine nucleoside biosynthesis were upregulated 4.53-fold, 10.63-fold, 4.26-fold, and 5.98-fold, respectively. To enhance the nucleoside production, precursors for synthesis of nucleosides were added based on the analysis of biosynthetic pathways. Uridine and cytidine contents, respectively, reached 5.04 mg/g and 3.54 mg/g when adding 2 mg/mL of ribose, resulting in an increase of 28.6% and 296% compared with the control, respectively. Meanwhile, uridine and cytidine contents, respectively, reached 10.83 mg/g 2.12 mg/g when adding 0.3 mg/mL of uracil, leading to an increase of 176.3% and 137.1%, respectively. This report indicated that fermentation regulation was an effective way to enhance the nucleoside production in H. sinensis based on biosynthetic pathway analysis.

  18. Biosynthetic pathway for acrylic acid from glycerol in recombinant Escherichia coli.

    Science.gov (United States)

    Tong, Wenhua; Xu, Ying; Xian, Mo; Niu, Wei; Guo, Jiantao; Liu, Huizhou; Zhao, Guang

    2016-06-01

    Acrylic acid is an important industrial feedstock. In this study, a de novo acrylate biosynthetic pathway from inexpensive carbon source glycerol was constructed in Escherichia coli. The acrylic acid was produced from glycerol via 3-hydroxypropionaldehyde, 3-hydroxypropionyl-CoA, and acrylyl-CoA. The acrylate production was improved by screening and site-directed mutagenesis of key enzyme enoyl-CoA hydratase and chromosomal integration of some exogenous genes. Finally, our recombinant strain produced 37.7 mg/L acrylic acid under shaking flask conditions. Although the acrylate production is low, our study shows feasibility of engineering an acrylate biosynthetic pathway from inexpensive carbon source. Furthermore, the reasons for limited acrylate production and further strain optimization that should be performed in the future were also discussed.

  19. Total cylindrospermopsins, microcystins/nodularins, and saxitoxins data for the 2007 United States Environmental Protection Agency National Lake Assessment

    Science.gov (United States)

    Loftin, Keith A.; Dietze, Julie E.; Meyer, Michael T.; Graham, Jennifer L.; Maksimowicz, Megan M.; Toyne, Kathryn D.

    2016-05-26

    Phytoplankton communities in freshwater lakes, ponds, and reservoirs may be dominated by cyanobacteria (also called blue-green algae) under certain environmental conditions. Cyanobacteria may cause a range of water-quality impairments, including the potential for toxin production. Cyanobacteria toxins (cyanotoxins) may adversely impact human and ecological health. Microcystins are considered to be one of the most commonly found classes of cyanotoxins in freshwater ecosystems, and as such were selected as a recreational indicator of water quality for the 2007 United States Environmental Protection Agency (EPA) National Lakes Assessment. However, much less is known about the occurrence of other classes of cyanotoxins in fresh surface water such as anatoxins, cylindrospermopsins, nodularins, and saxitoxins.

  20. Saxitoxin puffer fish poisoning in the United States, with the first report of Pyrodinium bahamense as the putative toxin source.

    Science.gov (United States)

    Landsberg, Jan H; Hall, Sherwood; Johannessen, Jan N; White, Kevin D; Conrad, Stephen M; Abbott, Jay P; Flewelling, Leanne J; Richardson, R William; Dickey, Robert W; Jester, Edward L E; Etheridge, Stacey M; Deeds, Jonathan R; Van Dolah, Frances M; Leighfield, Tod A; Zou, Yinglin; Beaudry, Clarke G; Benner, Ronald A; Rogers, Patricia L; Scott, Paula S; Kawabata, Kenji; Wolny, Jennifer L; Steidinger, Karen A

    2006-10-01

    From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 microg STX equivalents (eq)/100 g tissue (action level, 80 microg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida's marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in shellfish.

  1. Biosynthetic Origin of the Ether Ring in Platensimycin.

    Science.gov (United States)

    Rudolf, Jeffrey D; Dong, Liao-Bin; Manoogian, Karina; Shen, Ben

    2016-12-28

    Platensimycin (PTM) and platencin (PTN) are highly functionalized bacterial diterpenoid natural products that target bacterial and mammalian fatty acid synthases. PTM and PTN feature varying diterpene-derived ketolides that are linked to the same 3-amino-2,4-dihydroxybenzoic acid moiety. As a result, PTM is a selective inhibitor for FabF/FabB, while PTN is a dual inhibitor of FabF/FabB and FabH. We previously determined that the PTM cassette, consisting of five genes found in the ptm, but not ptn, gene cluster, partitions the biosynthesis of the PTM and PTN diterpene-derived ketolides. We now report investigation of the PTM cassette through the construction of diterpene production systems in E. coli and genetic manipulation in the PTM-PTN dual overproducer Streptomyces platensis SB12029, revealing two genes, ptmT3 and ptmO5, that are responsible for the biosynthetic divergence between the PTM and PTN diterpene-derived ketolides. PtmT3, a type I diterpene synthase, was determined to be a (16R)-ent-kauran-16-ol synthase, the first of its kind found in bacteria. PtmO5, a cytochrome P450 monooxygenase, is proposed to catalyze the formation of the characteristic 11S,16S-ether ring found in PTM. Inactivation of ptmO5 in SB12029 afforded the ΔptmO5 mutant SB12036 that accumulated nine PTM and PTN congeners, seven of which were new, including seven 11-deoxy-16R-hydroxy-PTM congeners. The two fully processed PTM analogues showed antibacterial activities, albeit lower than that of PTM, indicating that the ether ring, or minimally the stereochemistry of the hydroxyl group at C-16, is crucial for the activity of PTM.

  2. Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Xu Guoqiang

    2012-02-01

    Full Text Available Abstract Background Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive. Results In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH and fumarase (RoFUM1 were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2 was up-regulated. The resultant yeast strain, FMME-001 ↑PYC2 + ↑RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1 than the control strain FMME-001 empty vector. Conclusions The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner.

  3. Metabolic modeling of Rosmarinic acid biosynthetic pathway

    Science.gov (United States)

    Sundaram, Shanthy; Tripathi, Ashutosh; Gupta, Deepak K

    2010-01-01

    Rosmarinic acid (RA) is an ester of caffeic acid and 3, 4‐dihydroxyphenyllacticacid. It is commonly found in Coleus blumei, Salvia officinalis, Melissa officinalis and Rosmarinus officinalis. The biosynthesis of RA starts with precursor molecules L‐phenylalanine and L‐tyrosine. Simulation of RA biosynthetic pathway was done using Gepasi Software, includes the reaction kinetics of each step of the pathway and different integration methods such as Euler's method. Optimization of the significant parameters responsible for RA biosynthesis was carried out. As the goal of the work was to increase the productivity of i.e. to maximize the concentration of the RA, the final concentration of RA ([RA]t) was selected as an objective function and selected initial concentration of the Caffeoyl‐3’‐4’hydroxyphenyllactic acid (3’C4HPLA) as parameter constraint and varied its initial concentration as: 0≤ [3’C4HPLA]i ≤ 0.025. Several optimization methods such as Simulated annealing, Evolutionary algorithms and Genetic algorithms were used to optimize the objective function. After optimization the final concentration of RA was slightly higher (4.566132e‐002 mM) than before optimization (4.047119e‐ 002 mM). On the basis of results obtained, it is clear that 4‐hydroxyphenyllactic acid and 3’C4HPLA play major role in the high productivity of the RA. PMID:21364781

  4. 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.

  5. Distribution of mycotoxin biosynthetic genes in 200 Fusarium genomes

    Science.gov (United States)

    Fusarium is a species-rich genus of fungi that causes disease on most crop plants and produces diverse secondary metabolites (SMs), including some of the mycotoxins of greatest concern to food and feed safety. To determine the potential SM diversity within Fusarium as well as the distribution and ev...

  6. Differential expression of carotenoid biosynthetic pathway genes in ...

    Indian Academy of Sciences (India)

    2016-04-08

    Apr 8, 2016 ... 1Division of Biotechnology, 2Division of Plant Physiology and Biochemistry and 3Division of Vegetable Crops,. ICAR–Indian Institute of .... For immature green stage, mature green stage and breaker stage fruits hexane extract was further subjected to saponification using 15%. KOH. The carotenoids in the ...

  7. Expression profile of genes coding for carotenoid biosynthetic ...

    Indian Academy of Sciences (India)

    In this study, we quantified the β-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato ... At the green stage of fruit ripening, lutein accumulates in. ∗For correspondence. E-mail: ..... Liu Y., Roof S., Ye Z., Barry C., van Tuinen A., Vrebalov J. et al. 2004 Manipulation of light signal ...

  8. Expression profile of genes coding for carotenoid biosynthetic ...

    Indian Academy of Sciences (India)

    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 ...

  9. Saxitoxin Puffer Fish Poisoning in the United States, with the First Report of Pyrodinium bahamense as the Putative Toxin Source

    Science.gov (United States)

    Landsberg, Jan H.; Hall, Sherwood; Johannessen, Jan N.; White, Kevin D.; Conrad, Stephen M.; Abbott, Jay P.; Flewelling, Leanne J.; Richardson, R. William; Dickey, Robert W.; Jester, Edward L.E.; Etheridge, Stacey M.; Deeds, Jonathan R.; Van Dolah, Frances M.; Leighfield, Tod A.; Zou, Yinglin; Beaudry, Clarke G.; Benner, Ronald A.; Rogers, Patricia L.; Scott, Paula S.; Kawabata, Kenji; Wolny, Jennifer L.; Steidinger, Karen A.

    2006-01-01

    Background From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. Methods We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. Results We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 μg STX equivalents (eq)/100 g tissue (action level, 80 μg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida’s marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. Conclusions Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in

  10. Heritability of thromboxane A2 and prostaglandin E2 biosynthetic machinery in a Spanish population.

    Science.gov (United States)

    Vila, Luis; Martinez-Perez, Angel; Camacho, Mercedes; Buil, Alfonso; Alcolea, Sonia; Pujol-Moix, Nuria; Soler, Marta; Antón, Rosa; Souto, Juan-Carlos; Fontcuberta, Jordi; Soria, José-Manuel

    2010-01-01

    Prostanoids play a critical role in clinical areas such as inflammation, thrombosis, immune response, and cancer. Although some studies suggest that there are genes that determine variability of some prostanoid-related phenotypes, the genetic influence on these traits has not been evaluated. The relative contributions of genetic and environmental influences to the prostanoid biosynthetic pathway-related phenotypes, cyclooxygenase isoenzymes, microsomal-PGE-synthase-1 and TxA-synthase expression, and thromboxane-A(2) and prostaglandin-E(2) production by stimulated whole blood, were assessed in a sample of 308 individuals in 15 extended families. The effects of measured covariates (such as sex, age, and smoking), genes, and environmental variables shared by members of a household were quantified. Heritabilities ranging from 0.406 to 0.634 for enzyme expression and from 0.283 to 0. 751 for prostanoid production were found. These results demonstrate clearly the importance of genetic factors in determining variation in phenotypes that are components of the prostanoid biosynthetic pathways. The presence of such strong genetic effects suggest that it will be possible to localize previously unknown genes that influence quantitative variation in these phenotypes, some of which affect multiple aspects of cell biology, with important clinical implications.

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

    OpenAIRE

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

    2014-01-01

    Monascorubrin and its derivatives are polyketides used as natural colorants for a wide range of food for more than one thousand years. Since the biosynthetic pathway for this ancient chemical compound is unknown and genome sequence unavailable for any Monascus species, monascorubrin production has relied on extraction from fungal cultures of Monascus species. In vitro synthesis and genetic manipulation are not possible. Here we report the polyketide gene cluster and pathway for monascorubrin ...

  12. 4-Aminopyridine reverses saxitoxin (STX)- and tetrodotoxin (TTX)-induced cardiorespiratory depression in chronically instrumented guinea pigs.

    Science.gov (United States)

    Chang, F C; Spriggs, D L; Benton, B J; Keller, S A; Capacio, B R

    1997-07-01

    The extent to which cardiorespiratory infirmity and other sublethal effects of saxitoxin (STX) and tetrodotoxin (TTX) can be reversed by 4-aminopyridine (4-AP) was investigated in guinea pigs chronically instrumented for the concurrent electrophysiological recordings of electrocorticogram (ECoG), diaphragmatic electromyogram (DEMG), Lead II electrocardiogram, and neck skeletal muscle electromyogram. Animals were intoxicated with either STX or TTX (2 and 3 microg/kg, im) to produce a state of progressive cardiorespiratory depression (depicted by decreasing DEMG amplitude, bradypnea, and bradycardia). At the point where cardiorespiratory performance was most seriously compromised (approximately 30 min posttoxin), 4-AP (1 or 2 mg/kg, im) was administered. The therapeutic effect of 4-AP was striking in that, within minutes, the toxin-induced diaphragmatic blockade, bradypnea, bradycardia, and depressed cortical activity were all restored to a level either comparable to, or surpassing, that of control. The optimal 4-AP dose level was determined to be 2 mg/kg (im) based on analyses of cardiorespiratory activity profiles throughout the course of intoxication and 4-AP treatment. At the dose levels (either 1 or 2 mg/kg) used to restore ventilatory function and cardiovascular performance, 4-AP produced no sign of seizures and convulsions. Although less serious secondary effects such as cortical excitant/arousal effect (indicated by ECoG power spectral analysis) and transient periods of skeletal muscle fasciculation were observed, these events were of minor concern particularly in view of the remarkable therapeutic effects of 4-AP.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2016-03-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Nori Satoh

    2013-02-01

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

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

    Science.gov (United States)

    Shoguchi, Eiichi; Tanaka, Makiko; Takeuchi, Takeshi; Shinzato, Chuya; Satoh, Nori

    2013-01-01

    Genome sequences of the reef-building coral, Acropora digitifera, have been decoded. Acropora inhabits an environment with intense ultraviolet exposure and hosts the photosynthetic endosymbiont, Symbiodinium. Acropora homologs of all four genes necessary for biosynthesis of the photoprotective cyanobacterial compound, shinorine, are present. Among metazoans, these genes are found only in anthozoans. To gain further evolutionary insights into biosynthesis of photoprotective compounds and associated coral proteins, we surveyed the Acropora genome for 18 clustered genes involved in cyanobacterial synthesis of the anti-UV compound, scytonemin, even though it had not previously been detected in corals. We identified candidates for only 6 of the 18 genes, including tyrP, scyA, and scyB. Therefore, it does not appear that Acropora digitifera can synthesize scytonemin independently. On the other hand, molecular phylogenetic analysis showed that one tyrosinase gene is an ortholog of vertebrate tyrosinase genes and that the coral homologs, scyA and scyB, are similar to bacterial metabolic genes, phosphonopyruvate (ppyr) decarboxylase and glutamate dehydrogenase (GDH), respectively. Further genomic searches for ppyr gene-related biosynthetic components indicate that the coral possesses a metabolic pathway similar to the bacterial 2-aminoethylphosphonate (AEP) biosynthetic pathway. The results suggest that de novo synthesis of carbon-phosphorus compounds is performed in corals. PMID:23434798

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

    Directory of Open Access Journals (Sweden)

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

    Science.gov (United States)

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

    2015-09-10

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

  19. 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.

  20. Analysis of Polygala tenuifolia Transcriptome and Description of Secondary Metabolite Biosynthetic Pathways by Illumina Sequencing

    Directory of Open Access Journals (Sweden)

    Hongling Tian

    2015-01-01

    Full Text Available Radix polygalae, the dried roots of Polygala tenuifolia and P. sibirica, is one of the most well-known traditional Chinese medicinal plants. Radix polygalae contains various saponins, xanthones, and oligosaccharide esters and these compounds are responsible for several pharmacological properties. To provide basic breeding information, enhance molecular biological analysis, and determine secondary metabolite biosynthetic pathways of P. tenuifolia, we applied Illumina sequencing technology and de novo assembly. We also applied this technique to gain an overview of P. tenuifolia transcriptome from samples with different years. Using Illumina sequencing, approximately 67.2% of unique sequences were annotated by basic local alignment search tool similarity searches against public sequence databases. We classified the annotated unigenes by using Nr, Nt, GO, COG, and KEGG databases compared with NCBI. We also obtained many candidates CYP450s and UGTs by the analysis of genes in the secondary metabolite biosynthetic pathways, including putative terpenoid backbone and phenylpropanoid biosynthesis pathway. With this transcriptome sequencing, future genetic and genomics studies related to the molecular mechanisms associated with the chemical composition of P. tenuifolia may be improved. Genes involved in the enrichment of secondary metabolite biosynthesis-related pathways could enhance the potential applications of P. tenuifolia in pharmaceutical industries.

  1. Verification and quantification of saxitoxin from algal samples using fast and validated hydrophilic interaction liquid chromatography-tandem mass spectrometry method.

    Science.gov (United States)

    Halme, Mia; Rapinoja, Marja-Leena; Karjalainen, Maaret; Vanninen, Paula

    2012-01-01

    Hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was validated with algal samples for verification and quantification of saxitoxin (STX), a potent neurotoxin which is listed in the Chemical Weapons Convention (CWC) in Schedule 1A. Isocratic elution, conventional bore HILIC column and high flow rate together with accurate post-column splitter provided detection of STX in 6.5 min with total analysis time of 9 min per sample. STX analogue, gonyautoxin 1 (GTX 1) was used as an internal standard. Sample preparation of freeze-dried algae included liquid extraction and centrifugal filtering with mean recovery of 99.9% at concentration level of 10 ng/ml (n=3). Retention times for STX and GTX 1 were 6.47±0.03 min and 4.44±0.01 min (n=45), respectively. Four diagnostic product ions were used for reliable verification of saxitoxin. Method was found to be precise and linear (R(2)=0.9714 and R(2)=0.9768) in concentration ranges of 5-50 ng/ml and 25-200 ng/ml, respectively. For saxitoxin, calculated LOD was 3 ng/ml and LLOQ 11 ng/ml. Validation was conducted using spiked algal matrix since this method is not only needed for verification analysis for the CWC but also for safety analysis of other environmental samples for presence of STX. Identification criteria for verification of STX with HILIC-MS/MS method are discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Dual responsive physical networks from asymmetric biosynthetic triblock copolymers

    NARCIS (Netherlands)

    Pham, T.H.T.

    2013-01-01

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

  3. 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.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2015-09-15

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

  6. 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. © 2015 John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2016-08-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. © 2016 American Society of Plant Biologists. All Rights Reserved.

  8. 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.

  9. Elucidating the biosynthetic and regulatory mechanisms of flavonoid-derived bioactive components in Epimedium sagittatum

    Directory of Open Access Journals (Sweden)

    Wenjun eHuang

    2015-09-01

    Full Text Available Herba epimedii (Epimedium, a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. In Epimedium, flavonoids have been demonstrated to be the main bioactive components (BCs. However, the molecular biosynthetic and regulatory mechanisms of flavonoid-derived BCs remain obscure. In this study, we isolated twelve structural genes and two putative transcription factors (TFs in the flavonoid pathway. Phytochemical analysis showed that the total content of four representative BCs (epimedin A, B, C and icariin decreased slightly or dramatically in two lines of E. sagittatum during leaf development. Transcriptional analysis revealed that two R2R3-MYB TFs (EsMYBA1 and EsMYBF1, together with a bHLH TF (EsGL3 and WD40 protein (EsTTG1, were supposed to coordinately regulate the anthocyanin and flavonol-derived BCs biosynthesis in leaves. Overexpression of EsFLS (flavonol synthase in tobacco resulted in increased flavonols content and decreased anthocyanins content in flowers. Moreover, EsMYB12 negatively correlated with the accumulation of the four BCs, and might act as a transcriptional repressor in the flavonoid pathway. Therefore, the anthocyanin pathway may coordinate with the flavonol-derived BCs pathway in Epimedium leaves. A better understanding of the flavonoid biosynthetic and regulatory mechanisms in E. sagittatum will facilitate functional characterization, metabolic engineering and molecular breeding studies of Epimedium species.

  10. Comprehensive Analysis of the Triterpenoid Saponins Biosynthetic Pathway in Anemone flaccida by Transcriptome and Proteome Profiling.

    Science.gov (United States)

    Zhan, Chuansong; Li, Xiaohua; Zhao, Zeying; Yang, Tewu; Wang, Xuekui; Luo, Biaobiao; Zhang, Qiyun; Hu, Yanru; Hu, Xuebo

    2016-01-01

    Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as 'Di Wu' in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida. In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared. A combination of the de novo transcriptome and proteome profiling based on the Illumina HiSeq 2000 sequencing platform and iTRAQ technique was shown to be a powerful method for the discovery of candidate genes, which encoded enzymes that were responsible for the biosynthesis of novel secondary metabolites in a non-model plant. The transcriptome data of our study provides a very important resource for the understanding of the triterpenoid saponins biosynthesis of A. flaccida.

  11. Functional Characterization of a Novel R2R3-MYB Transcription Factor Modulating the Flavonoid Biosynthetic Pathway from Epimedium sagittatum

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    2017-07-01

    Full Text Available Epimedium species have been widely used both as traditional Chinese medicinal plants and ornamental perennials. Both flavonols, acting as the major bioactive components (BCs and anthocyanins, predominantly contributing to the color diversity of Epimedium flowers belong to different classes of flavonoids. It is well-acknowledged that flavonoid biosynthetic pathway is predominantly regulated by R2R3-MYB transcription factor (TF as well as bHLH TF and WD40 protein at the transcriptional level. MYB TFs specifically regulating anthocyanin or flavonol biosynthetic pathway have been already isolated and functionally characterized from Epimedium sagittatum, but a R2R3-MYB TF involved in regulating both these two pathways has not been functionally characterized to date in Epimedium plants. In this study, we report the functional characterization of EsMYB9, a R2R3-MYB TF previously isolated from E. sagittatum. The previous study indicated that EsMYB9 belongs to a small subfamily of R2R3-MYB TFs containing grape VvMYB5a and VvMYB5b TFs, which regulate flavonoid biosynthetic pathway. The present studies show that overexpression of EsMYB9 in tobacco leads to increased transcript levels of flavonoid pathway genes and increased contents of anthocyanins and flavonols. Yeast two-hybrid assay indicates that the C-terminal region of EsMYB9 contributes to the autoactivation activity, and EsMYB9 interacts with EsTT8 or AtTT8 bHLH regulator. Transient reporter assay shows that EsMYB9 slightly activates the expression of EsCHS (chalcone synthase promoter in transiently transformed leaves of Nicotiana benthamiana, but the addition of AtTT8 or EsTT8 bHLH regulator strongly enhances the transcriptional activation of EsMYB9 against five promoters of the flavonoid pathway genes except EsFLS (flavonol synthase. In addition, co-transformation of EsMYB9 and EsTT8 in transiently transfected tobacco leaves strongly induces the expressions of flavonoid biosynthetic genes. The

  12. Functional Characterization of a Novel R2R3-MYB Transcription Factor Modulating the Flavonoid Biosynthetic Pathway from Epimedium sagittatum.

    Science.gov (United States)

    Huang, Wenjun; Lv, Haiyan; Wang, Ying

    2017-01-01

    Epimedium species have been widely used both as traditional Chinese medicinal plants and ornamental perennials. Both flavonols, acting as the major bioactive components (BCs) and anthocyanins, predominantly contributing to the color diversity of Epimedium flowers belong to different classes of flavonoids. It is well-acknowledged that flavonoid biosynthetic pathway is predominantly regulated by R2R3-MYB transcription factor (TF) as well as bHLH TF and WD40 protein at the transcriptional level. MYB TFs specifically regulating anthocyanin or flavonol biosynthetic pathway have been already isolated and functionally characterized from Epimedium sagittatum, but a R2R3-MYB TF involved in regulating both these two pathways has not been functionally characterized to date in Epimedium plants. In this study, we report the functional characterization of EsMYB9, a R2R3-MYB TF previously isolated from E. sagittatum. The previous study indicated that EsMYB9 belongs to a small subfamily of R2R3-MYB TFs containing grape VvMYB5a and VvMYB5b TFs, which regulate flavonoid biosynthetic pathway. The present studies show that overexpression of EsMYB9 in tobacco leads to increased transcript levels of flavonoid pathway genes and increased contents of anthocyanins and flavonols. Yeast two-hybrid assay indicates that the C-terminal region of EsMYB9 contributes to the autoactivation activity, and EsMYB9 interacts with EsTT8 or AtTT8 bHLH regulator. Transient reporter assay shows that EsMYB9 slightly activates the expression of EsCHS (chalcone synthase) promoter in transiently transformed leaves of Nicotiana benthamiana, but the addition of AtTT8 or EsTT8 bHLH regulator strongly enhances the transcriptional activation of EsMYB9 against five promoters of the flavonoid pathway genes except EsFLS (flavonol synthase). In addition, co-transformation of EsMYB9 and EsTT8 in transiently transfected tobacco leaves strongly induces the expressions of flavonoid biosynthetic genes. The potential role of

  13. A proposed biosynthetic pathway of picrosides linked through the detection of biochemical intermediates in the endangered medicinal herb Picrorhiza kurroa.

    Science.gov (United States)

    Kumar, Varun; Sood, Hemant; Sharma, Manu; Chauhan, Rajinder Singh

    2013-01-01

    Picrorhiza kurroa Royle ex Benth is an important medicinal herb used in the preparation of several herbal drug formulations due to the presence of picroside-I (P-I) and picroside-II (P-II) along with other iridoid-glucosides derivatives. The endangered status of P. kurroa coupled with lack of information on biosynthesis of P-I and P-II necessitate deciphering the biosynthetic pathway for picrosides. LC with electrospray ionisation (ESI) and quadrupole time of flight combined with MS/MS was used to detect intermediates and assemble the picrosides biosynthetic pathway in P. kurroa. The presence of catalpol and aucubin, the major backbone structures of picrosides, along with intermediate metabolites boschnaloside, bartsioside and mussaenosidic acid, was confirmed in ESI negative mode with pseudomolecular ion peaks, that is, m/z 361, m/z 343, m/z 345, m/z 329 and m/z 375 ions and their fragmentation patterns. The picrosides biosynthetic pathway is expected to provide a reliable platform towards understanding the molecular components (genes/enzymes) of P-I and P-II biosynthesis in P. kurroa for their eventual utilisation in various applications. Copyright © 2013 John Wiley & Sons, Ltd.

  14. Central and peripheral cardio-respiratory effects of saxitoxin (STX) in urethane-anesthetized guinea-pigs.

    Science.gov (United States)

    Chang, F C; Benton, B J; Lenz, R A; Capacio, B R

    1993-05-01

    Effects of saxitoxin (STX; 10 micrograms/kg; i.p.) on cardio-respiratory activities were evaluated in urethane-anesthetized guinea-pigs. Concurrent recordings were made of electrocorticogram (ECoG), bulbar respiratory-related unit activities, diaphragmatic electromyogram (DEMG), electrocardiogram (Lead II ECG), blood pressure, heart rate, end-tidal CO2, arterial O2/CO2 tensions, and arterial pH. The average time to STX-induced respiratory failure was about 10 min. The most striking effect prior to apnea was a state of progressive bradypnea which emerged 5-7 min after the toxin administration. Other noteworthy responses included (i) a time-dependent decrease in ECoG amplitudes which typically began before the development of a bradypneic profile; (ii) an increasing degree of diaphragm neuromuscular blockade; (iii) a state of combined hypercapnia and uncompensated acidemia; (iv) a declining blood pressure; (v) an incrementally dysfunctional myocardial performance; and (vi) an increasingly degenerative central respiratory activity profile which ultimately culminated in a complete loss of central respiratory drive. The therapeutic effect of intratracheally administered oxygen was equivocal in that the cardio-respiratory activities, be they of central of peripheral nature, remained conspicuously dysfunctional and precarious despite 100% oxygen ventilation. What can be inferred from this study is two-fold. First, STX-induced ventilatory insufficiency can be attributed to a loss of functional integrity of both central and peripheral respiratory system components. That is, although diaphragm blockade contributes significantly to STX-induced respiratory failure, analyses of single respiratory unit activity data revealed that the central respiratory rhythmogenic mechanism also appeared to play a pivotal role in the development of a bradypneic profile which promotes, and directly causes, a complete loss of respiratory drive. Second, a state of unabating depression of central

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

    Science.gov (United States)

    Song, Jing; Zhang, Hesheng; Wu, Bingyuan; Zhang, Yingxin; Li, Hanfen; Xiao, Min; Wang, Peng George

    2003-01-01

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

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

    Science.gov (United States)

    Nawrath, C; Poirier, Y; Somerville, C

    1994-01-01

    In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plants accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-micron granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB that accumulated. We conclude that there does not appear to be any biological barrier to high-level production of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7809117

  17. Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.

    Directory of Open Access Journals (Sweden)

    Sandra Fresquet-Corrales

    Full Text Available Proanthocyanidins (PAs, or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (- catechin/g FW and 228.5 nmol (- epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloat-safe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass are discussed.

  18. Comprehensive analysis of the triterpenoid saponins biosynthetic pathway in Anemone flaccida by transcriptome and proteome profiling

    Directory of Open Access Journals (Sweden)

    Xuebo Hu

    2016-07-01

    Full Text Available In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing platform and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from proteomic analysis. Among the A. flaccida transcripts, 36,617 (77.97 % showed significant similarity (E-value<1e-5 to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 ORFs (open reading frame were predicated. By the RPKM (reads per kilobases per million reads statistics, 14,004 isoforms (unigenes were found to be upregulated, and 14,090 isoforms (unigenes were downregulated in the rhizome as compared to those in the leaf. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA and the plastidial methylerythritol pathway (MEP. Additionally, a total of 126 putative cytochrome P450 (CYP450 and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, 4 of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome sequencing, as well as quantitative RT-PCR verification, the expression level of gene and protein commit to triterpenoids biosynthesis in the leaf versus the rhizome was compared.

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

    Science.gov (United States)

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

    2015-11-01

    Secondary metabolites of filamentous fungi can be highly bioactive, ranging from antibiotic to cancerogenic properties. In this study we were able to identify a new, yet unknown metabolite produced by Fusarium fujikuroi, an ascomycetous rice pathogen. With the help of genomic engineering and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS) followed by isolation and detailed structure elucidation, the new substance could be designated as an unknown bikaverin precursor, missing two methyl- and one hydroxy group, hence named oxo-pre-bikaverin. Though the bikaverin gene cluster has been extensively studied in the past, elucidation of the biosynthetic pathway remained elusive due to a negative feedback loop that regulates the genes within the cluster. To decipher the bikaverin biosynthetic pathway and to overcome these negative regulation circuits, the structural cluster genes BIK2 and BIK3 were overexpressed independently in the ΔΔBIK2/BIK3+OE::BIK1 mutant background by using strong constitutive promoters. Using the software tool MZmine 2, the metabolite profile of the generated mutants obtained by HPLC-HRMS was compared, revealing further intermediates. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Biosynthetic Pathway and Health Benefits of Fucoxanthin, an Algae-Specific Xanthophyll in Brown Seaweeds

    OpenAIRE

    Mikami, Koji; Hosokawa, Masashi

    2013-01-01

    Fucoxanthin is the main carotenoid produced in brown algae as a component of the light-harvesting complex for photosynthesis and photoprotection. In contrast to the complete elucidation of the carotenoid biosynthetic pathways in red and green algae, the biosynthetic pathway of fucoxanthin in brown algae is not fully understood. Recently, two models for the fucoxanthin biosynthetic pathway have been proposed in unicellular diatoms; however, there is no such information for the pathway in brown...

  1. A kinetic model for the penicillin biosynthetic pathway in

    DEFF Research Database (Denmark)

    Nielsen, Jens; Jørgensen, Henrik

    1996-01-01

    A kinetic model for the first two steps in the penicillin biosynthetic pathway, i.e. the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS) is proposed. The model is based on Michaelis-Menten type kinetics with non-competitive inhibition of the ACVS by ACV, and competitive inhibition...... of the IPNS by glutathione. The model predicted flux through the pathway corresponds well with the measured rate of penicillin biosynthesis. From the kinetic model the elasticity coefficients and the flux control coefficients are calculated throughout a fed-batch cultivation, and it is found...

  2. Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms.

    Directory of Open Access Journals (Sweden)

    Sacha Coesel

    Full Text Available Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE and zeaxanthin epoxidase (ZEP enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several

  3. Redox Impact on Starch Biosynthetic Enzymes in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Skryhan, Katsiaryna

    Summary The thesis provides new insight into the influence of the plant cell redox state on the transient starch metabolism in Arabidopsis thaliana with a focus on starch biosynthetic enzymes. Two main hypotheses forms the basis of this thesis: 1) photosynthesis and starch metabolism are coordina......Summary The thesis provides new insight into the influence of the plant cell redox state on the transient starch metabolism in Arabidopsis thaliana with a focus on starch biosynthetic enzymes. Two main hypotheses forms the basis of this thesis: 1) photosynthesis and starch metabolism...... are coordinated by the redox state of the cell via post-translational modification of the starch metabolic enzymes containing redox active cysteine residues and these cysteine residues became cross-linked upon oxidation providing a conformational change leading to activity loss; 2) cysteine residues...... of chloroplast enzymes can play a role not only in enzyme activity and redox sensitivity but also in protein folding and stability upon oxidation. Several redox sensitive enzymes identified in this study can serve as potential targets to control the carbon flux to and from starch during the day and night...

  4. Substrate specificity of the sialic acid biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

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

    2001-07-18

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

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

    Science.gov (United States)

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

    2014-10-22

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

  6. Coordinated transcriptional regulation of isopentenyl diphosphate biosynthetic pathway enzymes in plastids by phytochrome-interacting factor 5.

    Science.gov (United States)

    Mannen, Kazuto; Matsumoto, Takuro; Takahashi, Seiji; Yamaguchi, Yuta; Tsukagoshi, Masanori; Sano, Ryosuke; Suzuki, Hideyuki; Sakurai, Nozomu; Shibata, Daisuke; Koyama, Tanetoshi; Nakayama, Toru

    2014-01-10

    All isoprenoids are derived from a common C5 unit, isopentenyl diphosphate (IPP). In plants, IPP is synthesized via two distinct pathways; the cytosolic mevalonate pathway and the plastidial non-mevalonate (MEP) pathway. In this study, we used a co-expression analysis to identify transcription factors that coordinately regulate the expression of multiple genes encoding enzymes in the IPP biosynthetic pathway. Some candidates showed especially strong correlations with multiple genes encoding MEP-pathway enzymes. We report here that phytochrome-interacting factor 5 (PIF5), a basic-helix-loop-helix type transcription factor, functions as a positive regulator of the MEP pathway. Its overexpression in T87 suspension cultured cells resulted in increased accumulation of chlorophylls and carotenoids. Detailed analyses of carotenoids by HPLC indicated that some carotenoid biosynthetic pathways were concomitantly up-regulated, possibly as a result of enhanced IPP metabolic flow. Our results also revealed other PIF family proteins that play different roles from that of PIF5 in IPP metabolism. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Discovery of an unusual biosynthetic origin for circular proteins in legumes.

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    Poth, Aaron G; Colgrave, Michelle L; Lyons, Russell E; Daly, Norelle L; Craik, David J

    2011-06-21

    Cyclotides are plant-derived proteins that have a unique cyclic cystine knot topology and are remarkably stable. Their natural function is host defense, but they have a diverse range of pharmaceutically important activities, including uterotonic activity and anti-HIV activity, and have also attracted recent interest as templates in drug design. Here we report an unusual biosynthetic origin of a precursor protein of a cyclotide from the butterfly pea, Clitoria ternatea, a representative member of the Fabaceae plant family. Unlike all previously reported cyclotides, the domain corresponding to the mature cyclotide from this Fabaceae plant is embedded within an albumin precursor protein. We confirmed the expression and correct processing of the cyclotide encoded by the Cter M precursor gene transcript following extraction from C. ternatea leaf and sequencing by tandem mass spectrometry. The sequence was verified by direct chemical synthesis and the peptide was found to adopt a classic knotted cyclotide fold as determined by NMR spectroscopy. Seven additional cyclotide sequences were also identified from C. ternatea leaf and flower, five of which were unique. Cter M displayed insecticidal activity against the cotton budworm Helicoverpa armigera and bound to phospholipid membranes, suggesting its activity is modulated by membrane disruption. The Fabaceae is the third largest family of flowering plants and many Fabaceous plants are of huge significance for human nutrition. Knowledge of Fabaceae cyclotide gene transcripts should enable the production of modified cyclotides in crop plants for a variety of agricultural or pharmaceutical applications, including plant-produced designer peptide drugs.

  8. Marine Actinobacteria from the Gulf of California: diversity, abundance and secondary metabolite biosynthetic potential.

    Science.gov (United States)

    Becerril-Espinosa, Amayaly; Freel, Kelle C; Jensen, Paul R; Soria-Mercado, Irma E

    2013-04-01

    The Gulf of California is a coastal marine ecosystem characterized as having abundant biological resources and a high level of endemism. In this work we report the isolation and characterization of Actinobacteria from different sites in the western Gulf of California. We collected 126 sediment samples and isolated on average 3.1-38.3 Actinobacterial strains from each sample. Phylogenetic analysis of 136 strains identified them as members of the genera Actinomadura, Micromonospora, Nocardiopsis, Nonomuraea, Saccharomonospora, Salinispora, Streptomyces and Verrucosispora. These strains were grouped into 26-56 operational taxonomic units (OTUs) based on 16S rRNA gene sequence identities of 98-100 %. At 98 % sequence identity, three OTUs appear to represent new taxa while nine (35 %) have only been reported from marine environments. Sixty-three strains required seawater for growth. These fell into two OTUs at the 98 % identity level and include one that failed to produce aerial hyphae and was only distantly related (≤95.5 % 16S identity) to any previously cultured Streptomyces sp. Phylogenetic analyses of ketosynthase domains associated with polyketide synthase genes revealed sequences that ranged from 55 to 99 % nucleotide identity to experimentally characterized biosynthetic pathways suggesting that some may be associated with the production of new secondary metabolites. These results indicate that marine sediments from the Gulf of California harbor diverse Actinobacterial taxa with the potential to produce new secondary metabolites.

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

    Science.gov (United States)

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

    2014-02-01

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

  10. In silico methods for linking genes and secondary metabolites: The way forward

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    Shradha Khater

    2016-06-01

    Full Text Available In silico methods for linking genomic space to chemical space have played a crucial role in genomics driven discovery of new natural products as well as biosynthesis of altered natural products by engineering of biosynthetic pathways. Here we give an overview of available computational tools and then briefly describe a novel computational framework, namely retro-biosynthetic enumeration of biosynthetic reactions, which can add to the repertoire of computational tools available for connecting natural products to their biosynthetic gene clusters. Most of the currently available bioinformatics tools for analysis of secondary metabolite biosynthetic gene clusters utilize the “Genes to Metabolites” approach. In contrast to the “Genes to Metabolites” approach, the “Metabolites to Genes” or retro-biosynthetic approach would involve enumerating the various biochemical transformations or enzymatic reactions which would generate the given chemical moiety starting from a set of precursor molecules and identifying enzymatic domains which can potentially catalyze the enumerated biochemical transformations. In this article, we first give a brief overview of the presently available in silico tools and approaches for analysis of secondary metabolite biosynthetic pathways. We also discuss our preliminary work on development of algorithms for retro-biosynthetic enumeration of biochemical transformations to formulate a novel computational method for identifying genes associated with biosynthesis of a given polyketide or nonribosomal peptide.

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

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    Valerio Mori

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

  12. Resorbable biosynthetic mesh for crural reinforcement during hiatal hernia repair.

    Science.gov (United States)

    Alicuben, Evan T; Worrell, Stephanie G; DeMeester, Steven R

    2014-10-01

    The use of mesh to reinforce crural closure during hiatal hernia repair is controversial. Although some studies suggest that using synthetic mesh can reduce recurrence, synthetic mesh can erode into the esophagus and in our opinion should be avoided. Studies with absorbable or biologic mesh have not proven to be of benefit for recurrence. The aim of this study was to evaluate the outcome of hiatal hernia repair with modern resorbable biosynthetic mesh in combination with adjunct tension reduction techniques. We retrospectively analyzed all patients who had crural reinforcement during repair of a sliding or paraesophageal hiatal hernia with Gore BioA resorbable mesh. Objective follow-up was by videoesophagram and/or esophagogastroduodenoscopy. There were 114 patients. The majority of operations (72%) were laparoscopic primary repairs with all patients receiving a fundoplication. The crura were closed primarily in all patients and reinforced with a BioA mesh patch. Excessive tension prompted a crural relaxing incision in four per cent and a Collis gastroplasty in 39 per cent of patients. Perioperative morbidity was minor and unrelated to the mesh. Median objective follow-up was one year, but 18 patients have objective follow-up at two or more years. A recurrent hernia was found in one patient (0.9%) three years after repair. The use of crural relaxing incisions and Collis gastroplasty in combination with crural reinforcement with resorbable biosynthetic mesh is associated with a low early hernia recurrence rate and no mesh-related complications. Long-term follow-up will define the role of these techniques for hiatal hernia repair.

  13. Isolation and Biosynthetic Analysis of Haliamide, a New PKS-NRPS Hybrid Metabolite from the Marine Myxobacterium Haliangium ochraceum

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    Yuwei Sun

    2016-01-01

    Full Text Available Myxobacteria of marine origin are rare and hard-to-culture microorganisms, but they genetically harbor high potential to produce novel antibiotics. An extensive investigation on the secondary metabolome of the unique marine myxobacterium Haliangium ochraceum SMP-2 led to the isolation of a new polyketide-nonribosomal peptide hybrid product, haliamide (1. Its structure was elucidated by spectroscopic analyses including NMR and HR-MS. Haliamide (1 showed cytotoxicity against HeLa-S3 cells with IC50 of 12 μM. Feeding experiments were performed to identify the biosynthetic building blocks of 1, revealing one benzoate, one alanine, two propionates, one acetate and one acetate-derived terminal methylene. The biosynthetic gene cluster of haliamide (hla, 21.7 kbp was characterized through the genome mining of the producer, allowing us to establish a model for the haliamide biosynthesis. The sulfotransferase (ST-thioesterase (TE domains encoded in hlaB appears to be responsible for the terminal alkene formation via decarboxylation.

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

    Science.gov (United States)

    Nakayasu, Masaru; Ohyama, Kiyoshi; Saito, Kazuki

    2016-01-01

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

  15. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

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    Peifer Susanne

    2012-10-01

    Full Text Available Abstract Background Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum. Results Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol gCDW-1. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol gCDW-1. Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol gCDW-1 derived from IMP degradation. Conclusions The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization.

  16. Different Expression Analysis in Fruit Softening and Ethylene Biosynthetic Pathways in Peaches of Different Flesh Textures

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    Yong Yang

    2016-03-01

    Full Text Available The aim of our study was to assess differences in the expression of genes involved in fruit softening and ethylene biosynthetic pathways under different temperature storage conditions. Different peach cultivars of ‘Xiacui’ and ‘Yumyeong’, which are stonyhard, ‘Yinhualu’, which is soft-melting, ‘Hujing Milu’, which is hard-melting, and ‘Baby Gold 6’, which is non-melting at 80% ripening, were collected as test materials. The results showed that only slight ethylene production was detected after harvesting of ‘Yumyeong’ and ‘Xiacui’ under either a room temperature (25 °C or low temperature (4 °C. The fruit firmness of stonyhard cultivars was retained at a high level under room temperature over time, whereas a low temperature induced ‘Yumyeong’ fruit to soften. Quantitative real-time PCR results indicated that the PpACS1 gene was highly expressed in soft-melting, hard-melting and non-melting cultivars; however, expression was extremely low in stonyhard peaches. PpACS2 or PpACS3, however, was not detected in all five cultivars. Interestingly, cold treatment significantly decreased firmness along with endo-PG expression obviously up-regulated in ‘Yumyeong’, but not in ‘Xiacui’ peaches. In conclusion, this study revealed that fruit softening of peaches with different flesh textures was closely related to ethylene biosynthesis during the storage period, which was controlled via regulating relevant gene expression levels under different storage temperatures.

  17. Interplay between siderophores and colibactin genotoxin biosynthetic pathways in Escherichia coli.

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    Patricia Martin

    Full Text Available In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin and of a genotoxin (colibactin are known to require a 4'-phosphopantetheinyl transferase (PPTase. Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti

  18. Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry

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    Kirsi Harju

    2015-11-01

    Full Text Available Saxitoxin (STX and some selected paralytic shellfish poisoning (PSP analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS. Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk. Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of ±1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD.

  19. Docking Simulation of the Binding Interactions of Saxitoxin Analogs Produced by the Marine Dinoflagellate Gymnodinium catenatum to the Voltage-Gated Sodium Channel Nav1.4

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    Lorena M. Durán-Riveroll

    2016-05-01

    Full Text Available Saxitoxin (STX and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav, impeding passage of Na+ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs.

  20. Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry.

    Science.gov (United States)

    Harju, Kirsi; Rapinoja, Marja-Leena; Avondet, Marc-André; Arnold, Werner; Schär, Martin; Burrell, Stephen; Luginbühl, Werner; Vanninen, Paula

    2015-11-25

    Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of ±1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD).

  1. The effects of elevated CO2 on the growth and toxicity of field populations and cultures of the saxitoxin-producing dinoflagellate, Alexandrium fundyense.

    Science.gov (United States)

    Hattenrath-Lehmann, Theresa K; Smith, Juliette L; Wallace, Ryan B; Merlo, Lucas; Koch, Florian; Mittelsdorf, Heidi; Goleski, Jennifer A; Anderson, Donald M; Gobler, Christopher J

    2015-01-01

    The effects of coastal acidification on the growth and toxicity of the saxitoxin-producing dinoflagellate Alexandrium fundyense were examined in culture and ecosystem studies. In culture experiments, Alexandrium strains isolated from Northport Bay NY, USA, and the Bay of Fundy, Canada, grew significantly faster (16 -190%; pchanges in toxicity were detected in the Bay of Fundy strain. The positive relationship between pCO2 enhancement and elevated growth was reproducible using natural populations from Northport; Alexandrium densities were significantly and consistently enhanced when natural populations were incubated at 1500 μatm pCO2, a value at the upper range of those recorded in Northport Bay, 390 - 1500 µatm. During natural Alexandrium blooms in Northport Bay, pCO2 concentrations increased over the course of a bloom to more than 1700μatm and were highest in regions with the greatest Alexandrium abundances, suggesting Alexandrium may be further exacerbating acidification or be especially adapted to these extreme, acidified conditions. The co-occurrence of Alexandrium blooms and elevated pCO2 represents a previously unrecognized, compounding environmental threat to coastal ecosystems. The ability of elevated pCO2 to enhance the growth and toxicity of Alexandrium indicates that acidification promoted by eutrophication or climate change can intensify these, and perhaps other, harmful algal blooms.

  2. Location, formation and biosynthetic regulation of cellulases in the gliding bacteria Cytophaga hutchinsonii

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    Elijah Johnson

    2006-01-01

    Full Text Available An analysis of the recently published genome sequence of Cytophagahutchinsonii revealed an unusual collection of genes for an organism that can attackcrystalline cellulose. Consequently, questions were being raised by cellulase scientists, as towhat mechanism this organism uses to degrade its insoluble substrates. Cellulose, being ahighly polymeric compound and insoluble in water, cannot enter the cell walls ofmicroorganisms. Cellulose-degrading enzymes have therefore to be located on the surface ofthe cell wall or released extracellularly. The location of most cellulase enzymes has beenstudied. However, basic information on C. hutchinsonii cellulases is almost non-existent. Inthe present study, the location, formation and biosynthetic regulation of cellulases in C.hutchinsonii were demonstrated on different substrates. Various fractions isolated from C.hutchinsonii after cell rupture were assayed for carboxymethyl-cellulase activity (CMC.The cellulases were found to be predominantly cell-free during active growth on solka-flok,although 30% of activity was recorded on cell-bound enzymes. Relatively little CM-cellulase was formed when cells were grown on glucose and cellobiose. Apparently glucoseor labile substrates such as cellobiose seem to repress the formation of CM-cellulase. Thesefindings should provide some insight into possible hydrolysis mechanisms by C.hutchinsonii.

  3. Evolution of the Tetrapyrrole Biosynthetic Pathway in Secondary Algae: Conservation, Redundancy and Replacement.

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    Jaromír Cihlář

    Full Text Available Tetrapyrroles such as chlorophyll and heme are indispensable for life because they are involved in energy fixation and consumption, i.e. photosynthesis and oxidative phosphorylation. In eukaryotes, the tetrapyrrole biosynthetic pathway is shaped by past endosymbioses. We investigated the origins and predicted locations of the enzymes of the heme pathway in the chlorarachniophyte Bigelowiella natans, the cryptophyte Guillardia theta, the "green" dinoflagellate Lepidodinium chlorophorum, and three dinoflagellates with diatom endosymbionts ("dinotoms": Durinskia baltica, Glenodinium foliaceum and Kryptoperidinium foliaceum. Bigelowiella natans appears to contain two separate heme pathways analogous to those found in Euglena gracilis; one is predicted to be mitochondrial-cytosolic, while the second is predicted to be plastid-located. In the remaining algae, only plastid-type tetrapyrrole synthesis is present, with a single remnant of the mitochondrial-cytosolic pathway, a ferrochelatase of G. theta putatively located in the mitochondrion. The green dinoflagellate contains a single pathway composed of mostly rhodophyte-origin enzymes, and the dinotoms hold two heme pathways of apparently plastidal origin. We suggest that heme pathway enzymes in B. natans and L. chlorophorum share a predominantly rhodophytic origin. This implies the ancient presence of a rhodophyte-derived plastid in the chlorarachniophyte alga, analogous to the green dinoflagellate, or an exceptionally massive horizontal gene transfer.

  4. A new genome-mining tool redefines the lasso peptide biosynthetic landscape.

    Science.gov (United States)

    Tietz, Jonathan I; Schwalen, Christopher J; Patel, Parth S; Maxson, Tucker; Blair, Patricia M; Tai, Hua-Chia; Zakai, Uzma I; Mitchell, Douglas A

    2017-05-01

    Ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are attractive for genome-driven discovery and re-engineering, but limitations in bioinformatic methods and exponentially increasing genomic data make large-scale mining of RiPP data difficult. We report RODEO (Rapid ORF Description and Evaluation Online), which combines hidden-Markov-model-based analysis, heuristic scoring, and machine learning to identify biosynthetic gene clusters and predict RiPP precursor peptides. We initially focused on lasso peptides, which display intriguing physicochemical properties and bioactivities, but their hypervariability renders them challenging prospects for automated mining. Our approach yielded the most comprehensive mapping to date of lasso peptide space, revealing >1,300 compounds. We characterized the structures and bioactivities of six lasso peptides, prioritized based on predicted structural novelty, including one with an unprecedented handcuff-like topology and another with a citrulline modification exceptionally rare among bacteria. These combined insights significantly expand the knowledge of lasso peptides and, more broadly, provide a framework for future genome-mining efforts.

  5. The flavonoid biosynthetic enzyme chalcone isomerase modulates terpenoid production in glandular trichomes of tomato.

    Science.gov (United States)

    Kang, Jin-Ho; McRoberts, John; Shi, Feng; Moreno, Javier E; Jones, A Daniel; Howe, Gregg A

    2014-03-01

    Flavonoids and terpenoids are derived from distinct metabolic pathways but nevertheless serve complementary roles in mediating plant interactions with the environment. Here, we show that glandular trichomes of the anthocyanin free (af) mutant of cultivated tomato (Solanum lycopersicum) fail to accumulate both flavonoids and terpenoids. This pleiotropic metabolic deficiency was associated with loss of resistance to native populations of coleopteran herbivores under field conditions. We demonstrate that Af encodes an isoform (SlCHI1) of the flavonoid biosynthetic enzyme chalcone isomerase (CHI), which catalyzes the conversion of naringenin chalcone to naringenin and is strictly required for flavonoid production in multiple tissues of tomato. Expression of the wild-type SlCHI1 gene from its native promoter complemented the anthocyanin deficiency in af. Unexpectedly, the SlCHI1 transgene also complemented the defect in terpenoid production in glandular trichomes. Our results establish a key role for SlCHI1 in flavonoid production in tomato and reveal a link between CHI1 and terpenoid production. Metabolic coordination of the flavonoid and terpenoid pathways may serve to optimize the function of trichome glands in dynamic environments.

  6. SANDPUMA: ensemble predictions of nonribosomal peptide chemistry reveal biosynthetic diversity across Actinobacteria.

    Science.gov (United States)

    Chevrette, Marc G; Aicheler, Fabian; Kohlbacher, Oliver; Currie, Cameron R; Medema, Marnix H

    2017-10-15

    Nonribosomally synthesized peptides (NRPs) are natural products with widespread applications in medicine and biotechnology. Many algorithms have been developed to predict the substrate specificities of nonribosomal peptide synthetase adenylation (A) domains from DNA sequences, which enables prioritization and dereplication, and integration with other data types in discovery efforts. However, insufficient training data and a lack of clarity regarding prediction quality have impeded optimal use. Here, we introduce prediCAT, a new phylogenetics-inspired algorithm, which quantitatively estimates the degree of predictability of each A-domain. We then systematically benchmarked all algorithms on a newly gathered, independent test set of 434 A-domain sequences, showing that active-site-motif-based algorithms outperform whole-domain-based methods. Subsequently, we developed SANDPUMA, a powerful ensemble algorithm, based on newly trained versions of all high-performing algorithms, which significantly outperforms individual methods. Finally, we deployed SANDPUMA in a systematic investigation of 7635 Actinobacteria genomes, suggesting that NRP chemical diversity is much higher than previously estimated. SANDPUMA has been integrated into the widely used antiSMASH biosynthetic gene cluster analysis pipeline and is also available as an open-source, standalone tool. SANDPUMA is freely available at https://bitbucket.org/chevrm/sandpuma and as a docker image at https://hub.docker.com/r/chevrm/sandpuma/ under the GNU Public License 3 (GPL3). chevrette@wisc.edu or marnix.medema@wur.nl. Supplementary data are available at Bioinformatics online.

  7. Evolution of a Biosynthetic Temporary Skin Substitute: A Preliminary Study.

    Science.gov (United States)

    Woodroof, Aubrey; Phipps, Richard; Woeller, Collynn; Rodeheaver, George; Naughton, Gail K; Piney, Emmett; Hickerson, William; Branski, Ludwik; Holmes, James H

    2015-01-01

    To compare PermeaDerm to first temporary biosynthetic skin substitute (Biobrane, cleared by the Food and Drug Administration in 1979). Different temporary skin substitutes (Biobrane, PermeaDerm, and PermeaDerm derivatives) were tested for physical differences, impact on healing wounds, inflammatory response, and ability to allow adequate growth of dermal fibroblasts and mesenchymal stem cells without accumulation of excessive scar-forming myofibroblasts. Proliferation of fibroblasts and stem cells on various skin substitutes was measured, and myofibroblast marker accumulation was evaluated by the expression of α-smooth muscle actin and fibronectin. Fibroblast migration was measured by tracking viable cells with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye. In vivo testing shows PermeaDerm works well as a temporary skin substitute, performing better than Biobrane with respect to inflammation and fluid accumulation. Tissue culture techniques revealed that cells on PermeaDerm grow in a more uniform fashion and migrated to a greater extent than cells on Biobrane. Furthermore, cells grown in the presence of PermeaDerm expressed lower levels of the myofibroblast markers α-smooth muscle actin and fibronectin than cells grown on Biobrane. PermeaDerm with variable porosity possesses all attributes and properties known to be important for a successful temporary skin substitute and enables the clinician to control porosity from essentially zero to what the wound requires. The ability of the clinician to minimize wound desiccation without fluid accumulation is related to the reduction of punctate scarring.

  8. Taxifolin suppresses rat and human testicular androgen biosynthetic enzymes.

    Science.gov (United States)

    Ge, Fei; Tian, Erpo; Wang, Li; Li, Xiaoheng; Zhu, Qiqi; Wang, Yiyan; Zhong, Ying; Ge, Ren-Shan

    2018-03-01

    Taxifolin is a flavonoid. It has been used as a chemopreventive agent and supplement. It may have some beneficial effects to treat prostate cancer by suppressing androgen production in Leydig cells. The objective of the present study was to study the effects of taxifolin on androgen production of rat Leydig cells isolated from immature testis and some rat and human testosterone biosynthetic enzyme activities. Rat Leydig cells were incubated with 100μM taxifolin without (basal) or with 10ng/ml luteinizing hormone (LH), 10mM 8-bromoadenosine 3',5'-cyclic monophosphate (8BR), and steroid enzyme substrates (20μM): 22R-hydroxychloesterol, pregnenolone, progesterone, and androstenedione. The medium concentrations of 5α-androstane-3α, 17β-diol (DIOL) and testosterone were measured. Taxifolin significantly suppressed basal, LH-stimulated, 8BR-stimulated, pregnenolone-mediated, and progesterone-mediated androgen production by Leydig cells. Further study demonstrated that taxifolin inhibited rat 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase/17, 20-lyase with IC 50 values of 14.55±0.013 and 16.75±0.011μM, respectively. Taxifolin also inhibited these two enzyme activities in human testis with IC 50 value of about 100μM. Taxifolin was a competitive inhibitor for these two enzymes when steroid substrates were used. In conclusion, taxifolin may have benefits for the treatment of prostate cancer. Copyright © 2018. Published by Elsevier B.V.

  9. Nanolipoprotein particles comprising a natural rubber biosynthetic enzyme complex and related products, methods and systems

    Energy Technology Data Exchange (ETDEWEB)

    Hoeprich, Paul D.; Whalen, Maureen

    2016-04-05

    Provided herein are nanolipoprotein particles that comprise a biosynthetic enzyme more particularly an enzyme capable of catalyzing rubber or other rubbers polymerization, and related assemblies, devices, methods and systems.

  10. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  11. Biosynthetic relationship among aflatoxins B1, B2, M1, and M2.

    OpenAIRE

    Dutton, M F; Ehrlich, K.; Bennett, J W

    1985-01-01

    Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medi...

  12. Intertidal marine sediment harbours Actinobacteria with promising bioactive and biosynthetic potential

    OpenAIRE

    Jose, Polpass Arul; Jha, Bhavanath

    2017-01-01

    Actinobacteria are the major source of bioactive natural products that find their value in research and drug discovery programmes. Antimicrobial resistance and the resulting high demand for novel antibiotics underscore the need for exploring novel sources of these bacteria endowed with biosynthetic potential. Intertidal ecosystems endure regular periods of immersion and emersion, and represent an untapped source of Actinobacteria. In this study, we studied the diversity and biosynthetic poten...

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

    Science.gov (United States)

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

    2013-01-01

    Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 μg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites. PMID:23898213

  14. Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction

    Directory of Open Access Journals (Sweden)

    Schulz Burkhard

    2010-12-01

    Full Text Available Abstract Background Plant growth depends on both cell division and cell expansion. Plant hormones, including brassinosteroids (BRs, are central to the control of these two cellular processes. Despite clear evidence that BRs regulate cell elongation, their roles in cell division have remained elusive. Results Here, we report results emphasizing the importance of BRs in cell division. An Arabidopsis BR biosynthetic mutant, dwarf7-1, displayed various characteristics attributable to slower cell division rates. We found that the DWARF4 gene which encodes for an enzyme catalyzing a rate-determining step in the BR biosynthetic pathways, is highly expressed in the actively dividing callus, suggesting that BR biosynthesis is necessary for dividing cells. Furthermore, dwf7-1 showed noticeably slower rates of callus growth and shoot induction relative to wild-type control. Flow cytometric analyses of the nuclei derived from either calli or intact roots revealed that the cell division index, which was represented as the ratio of cells at the G2/M vs. G1 phases, was smaller in dwf7-1 plants. Finally, we found that the expression levels of the genes involved in cell division and shoot induction, such as PROLIFERATING CELL NUCLEAR ANTIGEN2 (PCNA2 and ENHANCER OF SHOOT REGENERATION2 (ESR2, were also lower in dwf7-1 as compared with wild type. Conclusions Taken together, results of callus induction, shoot regeneration, flow cytometry, and semi-quantitative RT-PCR analysis suggest that BRs play important roles in both cell division and cell differentiation in Arabidopsis.

  15. Overexpression and biosynthetic deuterium enrichment of TEM-1 beta-lactamase for structural characterization by magnetic resonance methods.

    Science.gov (United States)

    Sosa-Peinado, A; Mustafi, D; Makinen, M W

    2000-07-01

    An expression system has been developed that allows high levels of production of TEM-1 beta-lactamase with ease of biosynthetic incorporation of nuclear isotopes. The gene for mature TEM-1 beta-lactamase fused to the leader sequence of the ompA protein was subcloned into the pET-24a(+) vector by introduction of an NdeI restriction site at the first codon of the fused genes and transformed into Escherichia coli BL21 (DE3) cells. With protein induction at 25 degrees C supported by LB medium supplemented with osmolytes (300 mM sucrose and 2.5 mM betaine), the extracellular, mature form of wild-type TEM-1 beta-lactamase was recovered at a level of 140 mg/L. The production level of E166N, E240C, E104C, and M272C mutants depended on the mutation but was invariably higher than reported by others for expression systems of the wild-type enzyme. Comparison of different carbon sources on the efficiency of biosynthetic incorporation of covalent deuterium showed maximal (90%) incorporation with minimal medium containing 99% (2)H(2)O and sodium d(3)-acetate (99 atom% (2)H). The yield of deuterium-enriched wild-type enzyme was 80 mg/L with yields for mutants proportionally reduced. The high level of protein deuteration achieved with this system allowed detection of the hyperfine coupling between the paramagnetic nitroxyl group of a spin-labeled penicillin substrate and hydrogens on the penicillin moiety in a cryokinetically isolated acylenzyme reaction intermediate because of the decrease in overlapping resonances of active site residues. The overexpression system is readily adaptable for other target proteins and facilitates studies requiring large quantities of protein in isotopically enriched forms. Copyright 2000 Academic Press.

  16. Self-protection against gliotoxin--a component of the gliotoxin biosynthetic cluster, GliT, completely protects Aspergillus fumigatus against exogenous gliotoxin.

    Directory of Open Access Journals (Sweden)

    Markus Schrettl

    Full Text Available Gliotoxin, and other related molecules, are encoded by multi-gene clusters and biosynthesized by fungi using non-ribosomal biosynthetic mechanisms. Almost universally described in terms of its toxicity towards mammalian cells, gliotoxin has come to be considered as a component of the virulence arsenal of Aspergillus fumigatus. Here we show that deletion of a single gene, gliT, in the gliotoxin biosynthetic cluster of two A. fumigatus strains, rendered the organism highly sensitive to exogenous gliotoxin and completely disrupted gliotoxin secretion. Addition of glutathione to both A. fumigatus Delta gliT strains relieved gliotoxin inhibition. Moreover, expression of gliT appears to be independently regulated compared to all other cluster components and is up-regulated by exogenous gliotoxin presence, at both the transcript and protein level. Upon gliotoxin exposure, gliT is also expressed in A. fumigatus Delta gliZ, which cannot express any other genes in the gliotoxin biosynthetic cluster, indicating that gliT is primarily responsible for protecting this strain against exogenous gliotoxin. GliT exhibits a gliotoxin reductase activity up to 9 microM gliotoxin and appears to prevent irreversible depletion of intracellular glutathione stores by reduction of the oxidized form of gliotoxin. Cross-species resistance to exogenous gliotoxin is acquired by A. nidulans and Saccharomyces cerevisiae, respectively, when transformed with gliT. We hypothesise that the primary role of gliotoxin may be as an antioxidant and that in addition to GliT functionality, gliotoxin secretion may be a component of an auto-protective mechanism, deployed by A. fumigatus to protect itself against this potent biomolecule.

  17. Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy

    Directory of Open Access Journals (Sweden)

    Ximena Escalera-Fanjul

    2017-06-01

    Full Text Available Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, ScAlt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1, alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display LkAlt1 and KlAlt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s. Furthermore, phenotypic analysis of null mutants uncovered the fact that KlAlt1 and LkAlt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that ScAlt2 conserves 64

  18. Interaction of single-walled carbon nanotubes and saxitoxin: Ab initio simulations and biological responses in hippocampal cell line HT-22.

    Science.gov (United States)

    Ramos, Patrícia; Schmitz, Marcos; Filgueira, Daza; Votto, Ana Paula; Durruthy, Michael; Gelesky, Marcos; Ruas, Caroline; Yunes, João; Tonel, Mariana; Fagan, Solange; Monserrat, José

    2017-07-01

    Saxitoxins (STXs) are potent neurotoxins that also induce cytotoxicity through the generation of reactive oxygen species. Carbon nanotubes (CNTs) are nanomaterials that can promote a Trojan horse effect, facilitating the entry of toxic molecules to cells when adsorbed to nanomaterials. The interaction of pristine single-walled (SW)CNTs and carboxylated (SWCNT-COOH) nanotubes with STX was evaluated by ab initio simulation and bioassays using the cell line HT-22. Cells (5 × 104  cells/mL) were exposed to SWCNT and SWCNT-COOH (5 μg mL-1 ), STX (200 μg L-1 ), SWCNT+STX, and SWCNT-COOH+STX for 30 min or 24 h. Results of ab initio simulation showed that the interaction between SWCNT and SWCNT-COOH with STX occurs in a physisorption. The interaction of SWCNT+STX induced a decrease in cell viability. Cell proliferation was not affected in any treatment after 30 min or 24 h of exposure (p > 0.05). Treatment with SWCNT-COOH induced high reactive oxygen species levels, an effect attenuated in SWCNT-COOH+STX treatment. In terms of cellular oxygen consumption, both CNTs when coexposed with STX antagonize the toxin effect. Based on these results, it can be concluded that the results obtained in vitro corroborate the semiempirical evidence found using density functional theory ab initio simulation. Environ Toxicol Chem 2017;36:1728-1737. © 2016 SETAC. © 2016 SETAC.

  19. Identification of the Viridicatumtoxin and Griseofulvin Gene Clusters from Penicillium aethiopicum

    National Research Council Canada - National Science Library

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

    2010-01-01

    ...: the tetracycline-like viridicatumtoxin 1 and the classic antifungal agent griseofulvin 2. Here, we report the concurrent discovery of the two corresponding biosynthetic gene clusters (vrt and gsf...

  20. AntiSMASH 4.0 - improvements in chemistry prediction and gene cluster boundary identification

    NARCIS (Netherlands)

    Blin, Kai; Wolf, Thomas; Chevrette, Marc G.; Lu, Xiaowen; Schwalen, Christopher J.; Kautsar, Satria A.; Suarez Duran, Hernando G.; Los Santos, De Emmanuel L.C.; Kim, Hyun Uk; Nave, Mariana; Dickschat, Jeroen S.; Mitchell, Douglas A.; Shelest, Ekaterina; Breitling, Rainer; Takano, Eriko; Lee, Sang Yup; Weber, Tilmann; Medema, Marnix H.

    2017-01-01

    Many antibiotics, chemotherapeutics, crop protection agents and food preservatives originate from molecules produced by bacteria, fungi or plants. In recent years, genome mining methodologies have been widely adopted to identify and characterize the biosynthetic gene clusters encoding the production

  1. Biosynthetic thiolase from zoogloea ramigera. I. Preliminary characterization and analysis of proton transfer reaction

    Energy Technology Data Exchange (ETDEWEB)

    Davis, J.T.; Moore, R.N.; Imperiali, B.; Pratt, A.J.; Kobayashi, K.; Masamune, S.; Sinskey, A.J.; Walsh, C.T.; Fukui, T.; Tomita, K.

    1987-01-05

    The biosynthetic thiolase, from Zoogloea ramigera, involved in generation of acetoacetyl-CoA for poly-beta-hydroxybutyrate synthesis, has been prepared pure in quantity for initial structural characterization of this homotetrameric enzyme. Edman degradation provided the sequence of the NH2 terminal 25 residues and an active site cysteine-containing nonapeptide labeled on stoichiometric inactivation by iodoacetamide. Both sequences were used to align the encoding DNA sequence of the cloned gene as described in an accompanying paper. Synthetic analogs of acetoacetyl-S-CoA, modified in the CoA moiety, were prepared and tested, and acetoacetyl-S-pantetheine 11-pivalate 1 was shown to have a kcat/Km of 6.4 X 10(6) M-1 s-1, comparable to the kcat/Km of 2 X 10(7) M-1 s-1 for acetoacetyl-S-CoA. The pantetheine pivalate group facilitates nonaqueous synthetic manipulations and may be generally useful as a CoA replacement. We have also prepared the carba analog of 1, with CH2 replacing S, to yield a beta-diketone analog 10 of acetoacetyl-S-CoA and the corresponding methyl ketone analog 9 of acetyl-S-CoA. These analogs have been used to prove the ability of Z. ramigera thiolase to catalyze proton abstraction from the C-2 methyl group of the acetyl portion of substrate in a transition state separate from C-C bond formation. NMR studies in D2O show exchange only when condensation is possible. Further studies with (2-/sup 3/H)acetyl-CoA show there is neither pre-equilibrium washout nor detectable kH/kT expressed in turnover and provide no evidence for a discrete acetyl-CoA C-2 carbanion or a nonconcerted reaction.

  2. Biosynthetic Chlorination of the Piperazate Residue in Kutzneride Biosynthesis by KthP

    Science.gov (United States)

    2011-01-01

    Kutznerides 2 and 8 of the cyclic hexadepsipeptide family of antifungal natural products from the soil actinomycete Kutzneria sp. 744 contain two sets of chlorinated residues, a 6,7-dichlorohexahydropyrroloindole moiety derived from dichlorotryptophan and a 5-chloropiperazate moiety, as well as a methylcyclopropylglycine residue that may arise from isoleucine via a cryptic chlorination pathway. Previous studies identified KtzD, KtzQ, and KtzR as three halogenases in the kutzneride pathway but left no candidate for installing the C5 chlorine on piperazate. On the basis of analysis of the complete genome sequence of Kutzneria, we now identify a fourth halogenase in the pathway whose gene is separated from the defined kutzneride cluster by 12 open reading frames. KthP (kutzneride halogenase for piperazate) is a mononuclear nonheme iron halogenase that acts on the piperazyl ring tethered by a thioester linkage to the holo forms of thiolation domains. MS analysis of the protein-bound product confirmed chlorination of the piperazate framework from the (3S)- but not the (3R)-piperazyl-S-pantetheinyl thiolation proteins. After thioesterase-mediated release, nuclear magnetic resonance was used to assign the free imino acid as (3S,5S)-5-chloropiperazate, distinct from the 3S,5R stereoisomer reported in the mature kutznerides. These results demonstrate that a fourth halogenase, KthP, is active in the kutzneride biosynthetic pathway and suggest further processing of the (3S,5S)-5-chloropiperazate during subsequent incorporation into the kutzneride depsipeptide frameworks. PMID:21648411

  3. γ-Aminobutyric acid transporter 2 mediates the hepatic uptake of guanidinoacetate, the creatine biosynthetic precursor, in rats.

    Directory of Open Access Journals (Sweden)

    Masanori Tachikawa

    Full Text Available Guanidinoacetic acid (GAA is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [(14C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [(14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [(14C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA and nipecotinic acid, an inhibitor of GABA transporters (GATs. The characteristics of Na(+- and Cl(--dependent [(14C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 µM was close to that of GAT2-mediated GAA transport (78.9 µM. GABA caused a marked inhibition with an IC(50 value of 8.81 µM. The [(14C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine:guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver.

  4. Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

    Science.gov (United States)

    Horsman, Geoff P.; Chen, Yihua; Thorson, Jon S.; Shen, Ben

    2010-01-01

    Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: (i) all five cognate PKSE-TE pairs in Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence. PMID:20534556

  5. Early Wound Morbidity after Open Ventral Hernia Repair with Biosynthetic or Polypropylene Mesh.

    Science.gov (United States)

    Sahoo, Sambit; Haskins, Ivy N; Huang, Li-Ching; Krpata, David M; Derwin, Kathleen A; Poulose, Benjamin K; Rosen, Michael J

    2017-10-01

    Recently introduced slow-resorbing biosynthetic and non-resorbing macroporous polypropylene meshes are being used in hernias with clean-contaminated and contaminated wounds. However, information about the use of biosynthetic meshes and their outcomes compared with polypropylene meshes in clean-contaminated and contaminated cases is lacking. Here we evaluate the use of biosynthetic mesh and polypropylene mesh in elective open ventral hernia repair (OVHR) and investigate differences in early wound morbidity after OVHR within clean-contaminated and contaminated cases. All elective, OVHR with biosynthetic mesh or uncoated polypropylene mesh from January 2013 through October 2016 were identified within the Americas Hernia Society Quality Collaborative. Association of mesh type with 30-day wound events in clean-contaminated or contaminated wounds was investigated using a 1:3 propensity-matched analysis. Biosynthetic meshes were used in 8.5% (175 of 2,051) of elective OVHR, with the majority (57.1%) used in low-risk or comorbid clean cases. Propensity-matched analysis in clean-contaminated and contaminated cases showed no significant difference between biosynthetic mesh and polypropylene mesh groups for 30-day surgical site occurrences (20.7% vs 16.7%; p = 0.49) or unplanned readmission (13.8% vs 9.8%; p = 0.4). However, surgical site infections (22.4% vs 10.9%; p = 0.03), surgical site occurrences requiring procedural intervention (24.1% vs 13.2%; p = 0.049), and reoperation rates (13.8% vs 4.0%; p = 0.009) were significantly higher in the biosynthetic group. Biosynthetic mesh appears to have higher rates of 30-day wound morbidity compared with polypropylene mesh in elective OVHR with clean-contaminated or contaminated wounds. Additional post-market analysis is needed to provide evidence defining best mesh choices, location, and surgical technique for repairing contaminated ventral hernias. Copyright © 2017 American College of Surgeons. Published by Elsevier Inc

  6. Algal toxins and reverse osmosis desalination operations: Laboratory bench testing and field monitoring of domoic acid, saxitoxin, brevetoxin and okadaic acid

    KAUST Repository

    Seubert, Erica L.

    2012-12-01

    The occurrence and intensity of harmful algal blooms (HABs) have been increasing globally during the past few decades. The impact of these events on seawater desalination facilities has become an important topic in recent years due to enhanced societal interest and reliance on this technology for augmenting world water supplies. A variety of harmful bloom-forming species of microalgae occur in southern California, as well as many other locations throughout the world, and several of these species are known to produce potent neurotoxins. These algal toxins can cause a myriad of human health issues, including death, when ingested via contaminated seafood. This study was designed to investigate the impact that algal toxin presence may have on both the intake and reverse osmosis (RO) desalination process; most importantly, whether or not the naturally occurring algal toxins can pass through the RO membrane and into the desalination product. Bench-scale RO experiments were conducted to explore the potential of extracellular algal toxins contaminating the RO product. Concentrations exceeding maximal values previously reported during natural blooms were used in the laboratory experiments, with treatments comprised of 50 μg/L of domoic acid (DA), 2 μg/L of saxitoxin (STX) and 20 μg/L of brevetoxin (PbTx). None of the algal toxins used in the bench-scale experiments were detectable in the desalinated product water. Monitoring for intracellular and extracellular concentrations of DA, STX, PbTx and okadaic acid (OA) within the intake and desalinated water from a pilot RO desalination plant in El Segundo, CA, was conducted from 2005 to 2009. During the five-year monitoring period, DA and STX were detected sporadically in the intake waters but never in the desalinated water. PbTx and OA were not detected in either the intake or desalinated water. The results of this study demonstrate the potential for HAB toxins to be inducted into coastal RO intake facilities, and the

  7. In silico tools for the analysis of antibiotic biosynthetic pathways

    DEFF Research Database (Denmark)

    Weber, Tilmann

    2014-01-01

    Natural products of bacteria and fungi are the most important source for antimicrobial drug leads. For decades, such compounds were exclusively found by chemical/bioactivity-guided screening approaches. The rapid progress in sequencing technologies only recently allowed the development of novel...... and tools are crucial for genome mining. In this review, a comprehensive overview is given on programs and databases for the identification and analysis of antibiotic biosynthesis gene clusters in genomic data....

  8. Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographical origin : Proof for the existence of chemotypes

    NARCIS (Netherlands)

    Wallaart, TE; Pras, N; Beekman, AC; Quax, WJ

    The time course of the levels of artemisinin, its biosynthetic precursors and the biosynthetically related sesquiterpenes was monitored during a vegetation period of Artemisia annua plants of different geographical origin. Considerable differences in contents of artemisinin and its direct precursors

  9. HPLC-SPE-NMR for combinatorial biosynthetic investigations – expanding the landscape of diterpene structural diversity

    DEFF Research Database (Denmark)

    Kongstad, Kenneth Thermann; Andersen-Ranberg, Johan; Hamberger, Björn Robert

    In this work, the analytical technique, HPLC-HRMS-SPE-NMR was used for the first time in combination with combinatorial biosynthetic investigations in N. benthamiana. This efficient setup allowed for identification of several diterpene synthase (diTPS) combinations responsible for stereospecific ...

  10. Leveraging microbial biosynthetic pathways for the generation of 'drop-in' biofuels

    DEFF Research Database (Denmark)

    Zargar, Amin; Bailey, Constance B.; Haushalter, Robert W.

    2017-01-01

    hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical properties (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty...

  11. Phytochemical and Biosynthetic Studies of Lignans, with a Focus on Indonesian Medicinal Plants

    NARCIS (Netherlands)

    Elfahmi, [No Value

    2006-01-01

    In this thesis phytochemical and biosynthetic studies of lignans are described. The focus is on the Indonesian medicinal plants Phyllanthus niruri and Piper cubeba and on two Linum species, Linum flavum and L. leonii, native to European countries. Both Indonesian plants are used in jamu. Jamu is the

  12. Sugars as the optimal biosynthetic carbon substrate of aqueous life throughout the universe

    Science.gov (United States)

    Weber, A. L.

    2000-01-01

    Our previous analysis of the energetics of metabolism showed that both the biosynthesis of amino acids and lipids from sugars, and the fermentation of organic substrates, were energetically driven by electron transfer reactions resulting in carbon redox disproportionation (Weber, 1997). Redox disproportionation--the spontaneous (energetically favorable) direction of carbon group transformation in biosynthesis--is brought about and driven by the energetically downhill transfer of electron pairs from more oxidized carbon groups (with lower half-cell reduction potentials) to more reduced carbon groups (with higher half-cell reduction potentials). In this report, we compare the redox and kinetic properties of carbon groups in order to evaluate the relative biosynthetic capability of organic substrates, and to identify the optimal biosubstrate. This analysis revealed that sugars (monocarbonyl alditols) are the optimal biosynthetic substrate because they contain the maximum number of biosynthetically useful high energy electrons/carbon atom while still containing a single carbonyl group needed to kinetically facilitate their conversion to useful biosynthetic intermediates. This conclusion applies to aqueous life throughout the Universe because it is based on invariant aqueous carbon chemistry--primarily, the universal reduction potentials of carbon groups.

  13. Covariation and phenotypic integration in chemical communication displays: biosynthetic constraints and eco-evolutionary implications.

    Science.gov (United States)

    Junker, Robert R; Kuppler, Jonas; Amo, Luisa; Blande, James D; Borges, Renee M; van Dam, Nicole M; Dicke, Marcel; Dötterl, Stefan; Ehlers, Bodil K; Etl, Florian; Gershenzon, Jonathan; Glinwood, Robert; Gols, Rieta; Groot, Astrid T; Heil, Martin; Hoffmeister, Mathias; Holopainen, Jarmo K; Jarau, Stefan; John, Lena; Kessler, Andre; Knudsen, Jette T; Kost, Christian; Larue-Kontic, Anne-Amélie C; Leonhardt, Sara Diana; Lucas-Barbosa, Dani; Majetic, Cassie J; Menzel, Florian; Parachnowitsch, Amy L; Pasquet, Rémy S; Poelman, Erik H; Raguso, Robert A; Ruther, Joachim; Schiestl, Florian P; Schmitt, Thomas; Tholl, Dorothea; Unsicker, Sybille B; Verhulst, Niels; Visser, Marcel E; Weldegergis, Berhane T; Köllner, Tobias G

    2017-03-03

    Chemical communication is ubiquitous. The identification of conserved structural elements in visual and acoustic communication is well established, but comparable information on chemical communication displays (CCDs) is lacking. We assessed the phenotypic integration of CCDs in a meta-analysis to characterize patterns of covariation in CCDs and identified functional or biosynthetically constrained modules. Poorly integrated plant CCDs (i.e. low covariation between scent compounds) support the notion that plants often utilize one or few key compounds to repel antagonists or to attract pollinators and enemies of herbivores. Animal CCDs (mostly insect pheromones) were usually more integrated than those of plants (i.e. stronger covariation), suggesting that animals communicate via fixed proportions among compounds. Both plant and animal CCDs were composed of modules, which are groups of strongly covarying compounds. Biosynthetic similarity of compounds revealed biosynthetic constraints in the covariation patterns of plant CCDs. We provide a novel perspective on chemical communication and a basis for future investigations on structural properties of CCDs. This will facilitate identifying modules and biosynthetic constraints that may affect the outcome of selection and thus provide a predictive framework for evolutionary trajectories of CCDs in plants and animals. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  14. EcoSynther: A Customized Platform To Explore the Biosynthetic Potential in E. coli.

    Science.gov (United States)

    Ding, Shaozhen; Liao, Xiaoping; Tu, Weizhong; Wu, Ling; Tian, Yu; Sun, Qiuping; Chen, Junni; Hu, Qian-Nan

    2017-11-17

    Developing computational tools for a chassis-centered biosynthetic pathway design is very important for a productive heterologous biosynthesis system by considering enormous foreign biosynthetic reactions. For many cases, a pathway to produce a target molecule consists of both native and heterologous reactions when utilizing a microbial organism as the host organism. Due to tens of thousands of biosynthetic reactions existing in nature, it is not trivial to identify which could be served as heterologous ones to produce the target molecule in a specific organism. In the present work, we integrate more than 10,000 E. coli non-native reactions and utilize a probability-based algorithm to search pathways. Moreover, we built a user-friendly Web server named EcoSynther. It is able to explore the precursors and heterologous reactions needed to produce a target molecule in Escherichia coli K12 MG1655 and then applies flux balance analysis to calculate theoretical yields of each candidate pathway. Compared with other chassis-centered biosynthetic pathway design tools, EcoSynther has two unique features: (1) allow for automatic search without knowing a precursor in E. coli and (2) evaluate the candidate pathways under constraints from E. coli physiological states and growth conditions. EcoSynther is available at http://www.rxnfinder.org/ecosynther/ .

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

    Science.gov (United States)

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

  16. Investigation of Patulin and its Biosynthetic Precursors by Density Functional Methods

    Science.gov (United States)

    Production of the toxin patulin by fungi that contaminate fruit and vegetable products is a food safety concern, especially for decaying apples. Reducing exposure to this regulated mycotoxin can be complicated by post-harvest biosynthesis during storage and processing. These biosynthetic pathways ...

  17. Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol

    NARCIS (Netherlands)

    Cerneus, D. P.; Ueffing, E.; Posthuma, G.; Strous, G. J.; van der Ende, A.

    1993-01-01

    Alkaline phosphatase is anchored to the outer leaflet of the plasma membrane by a covalently attached glycosyl-phosphatidylinositol anchor. We have studied the biosynthetic transport and endocytosis of alkaline phosphatase in the choriocarcinoma cell line BeWo, which endogenously expresses this

  18. 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

    , such as streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has...... and cultivation of transgenic lines, and metabolite analysis of terpenoids produced in transgenic moss lines. We also provide tools for metabolic engineering through genome editing using homologous recombination....

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

    Science.gov (United States)

    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 str...

  20. The antiSMASH database, a comprehensive database of microbial secondary metabolite biosynthetic gene clusters

    DEFF Research Database (Denmark)

    Blin, Kai; Medema, Marnix H.; Kottmann, Renzo

    2017-01-01

    . Therefore, we developed the antiSMASH database, a simple but highly useful new resource to browse antiSMASH-annotated BGCs in the currently 3907 bacterial genomes in the database and perform advanced search queries combining multiple search criteria. antiSMASH-DB is available at http://antismash-db.secondarymetabolites.org/....

  1. Transcript profiling of a bitter variety of narrow-leafed lupin to discover alkaloid biosynthetic genes

    DEFF Research Database (Denmark)

    Yang, Ting; Nagy, Istvan; Mancinotti, Davide

    2017-01-01

    Lupins (Lupinus spp.) are nitrogen-fixing legumes that accumulate toxic alkaloids in their protein-rich beans. These anti-nutritional compounds belong to the family of quinolizidine alkaloids (QAs), which are of interest to the pharmaceutical and chemical industries. To unleash the potential...

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

    Directory of Open Access Journals (Sweden)

    Xiaozhong Lan

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

  3. The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence

    Science.gov (United States)

    de Gontijo, Fabiano Assis; Pascon, Renata C.; Fernandes, Larissa; Machado, Joel; Alspaugh, J. Andrew; Vallim, Marcelo A

    2015-01-01

    Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37°C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. Among several mutants unable to grow at 37°C, we explored one bearing an interruption in the URA4 gene. This gene encodes dihydroorotase (DHOase) that is involved in the de novo synthesis of pyrimidine ribonucleotides. Loss of the C. neoformans Ura4 protein, by targeted gene interruption, resulted in an expected uracil/uridine auxotrophy and an unexpected high temperature growth defect. In addition, the ura4 mutant displayed phenotypic defects in other prominent virulence factors (melanin, capsule and phospholipase) and reduced stress response compared to wild type and reconstituted strains. Accordingly, this mutant had a decreased survival rate in macrophages and attenuated virulence in a murine model of cryptococcal infection. Quantitative PCR analysis suggests that this biosynthetic pathway is induced during the transition from 30°C to 37°C, and that transcriptional regulation of de novo and salvage pyrimidine pathway are under the control of the Ura4 protein. PMID:25011011

  4. Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis.

    Science.gov (United States)

    Sechler, Aaron J; Tancos, Matthew A; Schneider, David J; King, Jonas G; Fennessey, Christine M; Schroeder, Brenda K; Murray, Timothy D; Luster, Douglas G; Schneider, William L; Rogers, Elizabeth E

    2017-01-01

    Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

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

    Science.gov (United States)

    Lan, Xiaozhong; Chang, Kai; Zeng, Lingjiang; Liu, Xiaoqiang; Qiu, Fei; Zheng, Weilie; Quan, Hong; Liao, Zhihua; Chen, Min; Huang, Wenlin; Liu, Wanhong; Wang, Qiang

    2013-01-01

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

  6. Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by Ventral veins lacking and Knirps

    DEFF Research Database (Denmark)

    Danielsen, Erik Thomas; Møller, Morten Erik; Dorry, Elad

    2014-01-01

    Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine...... cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU...

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

    Directory of Open Access Journals (Sweden)

    Bo Wu

    2009-07-01

    Full Text Available Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole can interrupt transmission predominantly by killing microfilariae (mf larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs.Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP, which targets ferrochelatase (FC, the last step. Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a approximately 600-fold difference in drug sensitivities to succinyl acetone (SA between Wolbachia and human 5

  8. Transcriptional profiling of three key genes of terpenoid indole ...

    African Journals Online (AJOL)

    The response of three key genes: strictosidine synthase (str1), tryptophan decarboxylase (tdc) and secologanin synthase (cyp72A1) of the wild plant species, Catharanthus roseus to different plant tissue culture treatments was studied. These genes encode enzymes acting early in the biosynthetic pathway of terpenoid ...

  9. A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    Full Text Available Herba epimedii (Epimedium, a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1 from Epimedium sagittatum (Sieb. Et Zucc. Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR and anthocyanidin synthase (ANS. In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.

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

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    2016-07-01

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

  11. Regulation of Salmonella typhimurium pyr Gene Expression: Effect of Changing Both Purine and Pyrimidine Nucleotide Pools

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1989-01-01

    The synthesis of the pyrimidine biosynthetic enzymes is repressed by the pyrimidine nucleotide end-products of the pathway. However, purine nucleotides also play a role. In this study, I have measured expression of the pyr genes (pyrA-E) in Salmonella typhimurium strains harbouring mutations...... of each pyr gene is discussed in relation to present knowledge on gene structure and regulatory mechanism....

  12. Effect of prolonged nicotine infusion on response of rat catecholamine biosynthetic enzymes to restraint and cold stress.

    Science.gov (United States)

    Cheng, Shu-Yuan; Glazkova, Dina; Serova, Lidia; Sabban, Esther L

    2005-11-01

    There is a paradoxical relationship between nicotine and stress. To help elucidate their relationship on catecholamine biosynthesis, rats were infused with nicotine for 7-14 days before exposure to cold or restraint stress. Nicotine (5 mg/kg/day, 14 days) did not alter basal plasma corticosterone or its elevation with 24 h cold stress, but prevented corticosterone elevation following 2 h restraint stress. In adrenal medulla (AM), response of dopamine beta-hydroxylase (DBH), but not tyrosine hydroxylase (TH) mRNA, to both stressors was attenuated in nicotine-infused rats. In locus coeruleus (LC), restraint stress elevated TH and DBH mRNA in saline-, but not in nicotine-infused rats. Cold stress triggered a similar response of TH and DBH mRNAs in LC with and without nicotine infusion. With shorter nicotine infusion (8 mg/kg/day, 7 days), TH mRNA in AM was not induced by restraint stress on one (1x) or two (2x) consecutive days nor was DBH mRNA in AM or LC by 2x. The findings demonstrate that constant release of nicotine can modulate, or even prevent, some stress responses at the level of the HPA axis and gene expression of catecholamine biosynthetic enzymes in LC and AM.

  13. The Flavonoid Biosynthetic Enzyme Chalcone Isomerase Modulates Terpenoid Production in Glandular Trichomes of Tomato1[C][W][OPEN

    Science.gov (United States)

    Kang, Jin-Ho; McRoberts, John; Shi, Feng; Moreno, Javier E.; Jones, A. Daniel; Howe, Gregg A.

    2014-01-01

    Flavonoids and terpenoids are derived from distinct metabolic pathways but nevertheless serve complementary roles in mediating plant interactions with the environment. Here, we show that glandular trichomes of the anthocyanin free (af) mutant of cultivated tomato (Solanum lycopersicum) fail to accumulate both flavonoids and terpenoids. This pleiotropic metabolic deficiency was associated with loss of resistance to native populations of coleopteran herbivores under field conditions. We demonstrate that Af encodes an isoform (SlCHI1) of the flavonoid biosynthetic enzyme chalcone isomerase (CHI), which catalyzes the conversion of naringenin chalcone to naringenin and is strictly required for flavonoid production in multiple tissues of tomato. Expression of the wild-type SlCHI1 gene from its native promoter complemented the anthocyanin deficiency in af. Unexpectedly, the SlCHI1 transgene also complemented the defect in terpenoid production in glandular trichomes. Our results establish a key role for SlCHI1 in flavonoid production in tomato and reveal a link between CHI1 and terpenoid production. Metabolic coordination of the flavonoid and terpenoid pathways may serve to optimize the function of trichome glands in dynamic environments. PMID:24424324

  14. Characterization of Cyanobacterial Hydrocarbon Composition and Distribution of Biosynthetic Pathways

    Science.gov (United States)

    Coates, R. Cameron; Podell, Sheila; Korobeynikov, Anton; Lapidus, Alla; Pevzner, Pavel; Sherman, David H.; Allen, Eric E.; Gerwick, Lena; Gerwick, William H.

    2014-01-01

    Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene) and methyl group positions (3-, 4- and 5-methylheptadecane) for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO). The second involves a polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS). Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both. PMID:24475038

  15. Biosynthetic mechanism for sunscreens of the biocontrol agent Lysobacter enzymogenes.

    Directory of Open Access Journals (Sweden)

    Yan Wang

    Full Text Available Lysobacter are ubiquitous environmental bacteria emerging as novel biocontrol agents and new sources of anti-infectives. So far, very little effort has been invested in the study of the biology of these Gram-negative gliding bacteria. Many Lysobacter species are characterized by their yellow-orange appearance. Using transposon mutagenesis, we identified a stand-alone polyketide synthase (PKS gene cluster required for the pigment production in L. enzymogenes OH11. The yellow pigments were abolished in the "white" mutants generated by target-specific deletions of ketosynthase (KS, acyl carrier protein, or ketoreductase. Spectroscopic data suggested that the pigments belong to xanthomonadin-like aryl polyenes. Polyene-type polyketides are known to be biosynthesized by modular PKS (Type I, not by stand-alone PKS (Type II which always contain the heterodimer KS-CLF (chain-length factor as the key catalytic component. Remarkably, this aryl polyene PKS complex only contains the KS (ORF17, but not the CLF. Instead, a hypothetical protein (ORF16 is located immediately next to ORF17. ORF16-17 homologs are widespread in numerous uncharacterized microbial genomes, in which an ORF17 homolog is always accompanied by an ORF16 homolog. The deletion of ORF16 eliminated pigment production, and homology modeling suggested that ORF16 shares a structural similarity to the N-terminal half of CLF. A point-mutation of glutamine (Q166A that is the conserved active site of known CLF abolished pigment production. The "white" mutants are significantly more sensitive to UV/visible light radiation or H2O2 treatment than the wild type. These results unveil the first example of Type II PKS-synthesized polyene pigments and show that the metabolites serve as Lysobacter "sunscreens" that are important for the survival of these ubiquitous environmental organisms.

  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

    in bacteria and mine the associated chemical diversity. Thirteen strains closely related to Pseudoalteromonas luteoviolacea isolated from all over the Earth were analyzed using an untargeted metabolomics strategy, and metabolomic profiles were correlated with whole-genome sequences of the strains. We found...... 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......, including the thiomarinols that were reported from P. luteoviolacea here for the first time. By comparative genomics, we identified the biosynthetic cluster responsible for the production of the antibiotic indolmycin, which could not be predicted with standard methods. In conclusion, we present an efficient...

  17. Gibberellin biosynthetic inhibitors make human malaria parasite Plasmodium falciparum cells swell and rupture to death.

    Directory of Open Access Journals (Sweden)

    Tomoko Toyama

    Full Text Available Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites.

  18. Gibberellin Biosynthetic Inhibitors Make Human Malaria Parasite Plasmodium falciparum Cells Swell and Rupture to Death

    Science.gov (United States)

    Toyama, Tomoko; Tahara, Michiru; Nagamune, Kisaburo; Arimitsu, Kenji; Hamashima, Yoshio; Palacpac, Nirianne M. Q.; Kawaide, Hiroshi; Horii, Toshihiro; Tanabe, Kazuyuki

    2012-01-01

    Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites. PMID:22412858

  19. Biosynthetic Studies on Water-Soluble Derivative 5c (DTX5c

    Directory of Open Access Journals (Sweden)

    José J. Fernández

    2012-10-01

    Full Text Available The dinoflagellate Prorocentrum belizeanum is responsible for the production of several toxins involved in the red tide phenomenon known as Diarrhetic Shellfish Poisoning (DSP. In this paper we report on the biosynthetic origin of an okadaic acid water-soluble ester derivative, DTX5c, on the basis of the spectroscopical analysis of 13C enriched samples obtained by addition of labelled sodium [l-13C], [2-13C] acetate to artificial cultures of this dinoflagellate.

  20. Biosynthetic Studies on Water-Soluble Derivative 5c (DTX5c)

    OpenAIRE

    Vilches, Tamara S.; Norte, Manuel; Daranas, Antonio Hernández; Fernández, José J.

    2012-01-01

    The dinoflagellate Prorocentrum belizeanum is responsible for the production of several toxins involved in the red tide phenomenon known as Diarrhetic Shellfish Poisoning (DSP). In this paper we report on the biosynthetic origin of an okadaic acid water-soluble ester derivative, DTX5c, on the basis of the spectroscopical analysis of 13C enriched samples obtained by addition of labelled sodium [l-13C], [2-13C] acetate to artificial cultures of this dinoflagellate.

  1. Biosynthetic studies on water-soluble derivative 5c (DTX5c).

    Science.gov (United States)

    Vilches, Tamara S; Norte, Manuel; Daranas, Antonio Hernández; Fernández, José J

    2012-10-01

    The dinoflagellate Prorocentrum belizeanum is responsible for the production of several toxins involved in the red tide phenomenon known as Diarrhetic Shellfish Poisoning (DSP). In this paper we report on the biosynthetic origin of an okadaic acid water-soluble ester derivative, DTX5c, on the basis of the spectroscopical analysis of ¹³C enriched samples obtained by addition of labelled sodium [l-¹³C], [2-¹³C] acetate to artificial cultures of this dinoflagellate.

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

    Directory of Open Access Journals (Sweden)

    Naoki eSato

    2016-02-01

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

  3. Crystal structure of the pyocyanin biosynthetic protein PhzS.

    Science.gov (United States)

    Greenhagen, Bryan T; Shi, Katherine; Robinson, Howard; Gamage, Swarna; Bera, Asim K; Ladner, Jane E; Parsons, James F

    2008-05-13

    The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-1-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS. PhzM is an adenosylmethionine dependent methyltransferase, and PhzS is a flavin dependent hydroxylase. It has been shown that PhzM is only active in the physical presence of PhzS, suggesting that a protein-protein interaction is involved in pyocyanin formation. Such a complex would prevent the release of 5-methyl-phenazine-1-carboxylate, the putative intermediate, and an apparently unstable compound. Here, we describe the three-dimensional structure of PhzS, solved by single anomalous dispersion, at a resolution of 2.4 A. The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. The PhzS flavin, however, appears to be held in a strained conformation by a combination of stacking interactions and hydrogen bonds. The structure suggests that access to the active site is gained via a tunnel on the opposite side of the protein from where the flavin is exposed. The C-terminal 23 residues are disordered as no electron density is present for these atoms. The probable location of the C-terminus, near the substrate access tunnel, suggests that it may be involved in substrate binding as has been shown for another structural homologue, RebC. This region also may be an element of a PhzM-PhzS interface. Aromatic hydroxylases have been shown to catalyze electrophilic substitution reactions on activated substrates. The putative PhzS substrate, however, is electron deficient and unlikely to act as

  4. Crystal Structure of the Pyocyanin Biosynthetic Protein PhzS

    Energy Technology Data Exchange (ETDEWEB)

    Greenhagen,B.; Shi, K.; Robinson, H.; Gamage, S.; Bera, A.; Ladner, J.; Parsons, J.

    2008-01-01

    The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-1-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS. PhzM is an adenosylmethionine dependent methyltransferase, and PhzS is a flavin dependent hydroxylase. It has been shown that PhzM is only active in the physical presence of PhzS, suggesting that a protein-protein interaction is involved in pyocyanin formation. Such a complex would prevent the release of 5-methyl-phenazine-1-carboxylate, the putative intermediate, and an apparently unstable compound. Here, we describe the three-dimensional structure of PhzS, solved by single anomalous dispersion, at a resolution of 2.4 Angstroms. The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. The PhzS flavin, however, appears to be held in a strained conformation by a combination of stacking interactions and hydrogen bonds. The structure suggests that access to the active site is gained via a tunnel on the opposite side of the protein from where the flavin is exposed. The C-terminal 23 residues are disordered as no electron density is present for these atoms. The probable location of the C-terminus, near the substrate access tunnel, suggests that it may be involved in substrate binding as has been shown for another structural homologue, RebC. This region also may be an element of a PhzM-PhzS interface. Aromatic hydroxylases have been shown to catalyze electrophilic substitution reactions on activated substrates. The putative PhzS substrate, however, is electron deficient and unlikely

  5. A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate.

    Science.gov (United States)

    Tatsis, Evangelos C; Carqueijeiro, Inês; Dugé de Bernonville, Thomas; Franke, Jakob; Dang, Thu-Thuy T; Oudin, Audrey; Lanoue, Arnaud; Lafontaine, Florent; Stavrinides, Anna K; Clastre, Marc; Courdavault, Vincent; O'Connor, Sarah E

    2017-08-22

    Monoterpene indole alkaloids comprise a diverse family of over 2000 plant-produced natural products. This pathway provides an outstanding example of how nature creates chemical diversity from a single precursor, in this case from the intermediate strictosidine. The enzymes that elicit these seemingly disparate products from strictosidine have hitherto been elusive. Here we show that the concerted action of two enzymes commonly involved in natural product metabolism-an alcohol dehydrogenase and a cytochrome P450-produces unexpected rearrangements in strictosidine when assayed simultaneously. The tetrahydro-β-carboline of strictosidine aglycone is converted into akuammicine, a Strychnos alkaloid, an elusive biosynthetic transformation that has been investigated for decades. Importantly, akuammicine arises from deformylation of preakuammicine, which is the central biosynthetic precursor for the anti-cancer agents vinblastine and vincristine, as well as other biologically active compounds. This discovery of how these enzymes can function in combination opens a gateway into a rich family of natural products.The biosynthetic pathway of preakuammicine, a monoterpene precursor of the anti-cancer agent vinblastine, has remained largely unexplored. Here, the authors provide transcriptomic and biochemical data to identify two enzymes that, in tandem, convert strictosidine to akuammicine, the stable shunt product of preakuammicine.

  6. Volatile terpenes from actinomycetes: a biosynthetic study correlating chemical analyses to genome data.

    Science.gov (United States)

    Rabe, Patrick; Citron, Christian A; Dickschat, Jeroen S

    2013-11-25

    The volatile terpenes of 24 actinomycetes whose genomes have been sequenced (or are currently being sequenced) were collected by use of a closed-loop stripping apparatus and identified by GC/MS. The analytical data were compared against a phylogenetic analysis of all 192 currently available sequences of bacterial terpene cyclases (excluding geosmin and 2-methylisoborneol synthases). In addition to the several groups of terpenes with known biosynthetic origin, selinadienes were identified as a large group of biosynthetically related sesquiterpenes that are produced by several streptomycetes. The detection of a large number of previously unrecognised side products of known terpene cyclases proved to be particularly important for an in depth understanding of biosynthetic pathways to known terpenes in actinomycetes. Interpretation of the chemical analytical data in the context of the phylogenetic tree of bacterial terpene cyclases pointed to the function of three new enzymes: (E)-β-caryophyllene synthase, selina-3,7(11)-diene synthase and aristolochene synthase. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

    Science.gov (United States)

    Petrik, Deborah L; Karlen, Steven D; Cass, Cynthia L; Padmakshan, Dharshana; Lu, Fachuang; Liu, Sarah; Le Bris, Philippe; Antelme, Sébastien; Santoro, Nicholas; Wilkerson, Curtis G; Sibout, Richard; Lapierre, Catherine; Ralph, John; Sedbrook, John C

    2014-01-01

    Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta. PMID:24372757

  8. Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins

    Science.gov (United States)

    Yabe, Kimiko; Nakamura, Miki; Hamasaki, Takashi

    1999-01-01

    We detected biosynthetic activity for aflatoxins G1 and G2 in cell extracts of Aspergillus parasiticus NIAH-26. We found that in the presence of NADPH, aflatoxins G1 and G2 were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B1, aflatoxin B2, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G2 from dihydro-O-methylsterigmatocystin was suppressed when O-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G1 was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G1 and G2. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin. PMID:10473388

  9. Evidence that a Secondary Metabolic Biosynthetic Gene Cluster has Grown by Gene Relocation During Evolution of the Filamentous Fungus Fusarium

    Science.gov (United States)

    Trichothecenes are terpene-derived secondary metabolites produced by multiple genera of filamentous fungi, including many plant pathogenic species of Fusarium. These metabolites are of medical and agricultural interest because they are toxic to animals and plants and can contribute to pathogenesis ...

  10. Novel bio-synthetic hybrid materials and coculture systems for musculoskeletal tissue engineering

    Science.gov (United States)

    Lee, Hyeseung Janice

    Tissue Engineering is a truly exciting field of this age, trying to regenerate and repair impaired tissues. Unlike the old artificial implants, tissue engineering aims at making a long-term functional biological replacement. One strategy for such tissue engineering requires the following three components: cells, scaffolds, and soluble factors. Cells are cultured in a three-dimensional (3D) scaffold with medium containing various soluble factors. Once a tissue is developed in vitro, then it is implanted in vivo. The overall goal of this thesis was to develop novel bio-synthetic hybrid scaffolds and coculture system for musculoskeletal tissue engineering. The most abundant cartilage extracellular matrix (ECM) components are collagen and glycosaminoglycan (GAG), which are the natural scaffold for chondrocytes. As two different peptides, collagen mimetic peptide (CMP) and hyaluronic acid binding peptide (HABPep) were previously shown to bind to collagen and hyaluronic acid (HA) of GAG, respectively, it was hypothesized that immobilizing CMP and HABP on 3D scaffold would results in an interaction between ECM components and synthetic scaffolds via peptide-ECM bindings. CMP or HABPep-conjugated photopolymerizable poly(ethylene oxide) diacrylate (PEODA) hydrogels were synthesized and shown to retain encapsulated collagen or HA, respectively. This result supported that conjugated CMP and HABPep can interact with collagen and HA, respectively, and can serve as biological linkers in 3D synthetic hydrogels. When chondrocytes or mesenchymal stem cells (MSCs) were seeded, cells in CMP-conjugated scaffolds produced significantly more amount of type II collagen and GAG, compared to those in control scaffolds. Moreover, MSCs cultured in CMP-conjugated scaffolds exhibited lower level of hypertrophic markers, cbfa-1 and type X collagen. These results demonstrated that enhanced interaction between collagen and scaffold via CMP improves chondrogenesis of chondrocytes and MSCs and

  11. Biosynthesis of the C(7)-cyclitol moiety of acarbose in Actinoplanes species SE50/110. 7-O-phosphorylation of the initial cyclitol precursor leads to proposal of a new biosynthetic pathway.

    Science.gov (United States)

    Zhang, Chang-Sheng; Stratmann, Ansgar; Block, Oliver; Brückner, Ralph; Podeschwa, Michael; Altenbach, Hans-Josef; Wehmeier, Udo F; Piepersberg, Wolfgang

    2002-06-21

    We have previously demonstrated that the biosynthesis of the C(7)-cyclitol, called valienol (or valienamine), of the alpha-glucosidase inhibitor acarbose starts from the cyclization of sedo-heptulose 7-phosphate to 2-epi-5-epi-valiolone (Stratmann, A., Mahmud, T., Lee, S., Distler, J., Floss, H. G., and Piepersberg, W. (1999) J. Biol. Chem. 274, 10889-10896). Synthesis of the intermediate 2-epi-5-epi-valiolone is catalyzed by the cyclase AcbC encoded in the biosynthetic (acb) gene cluster of Actinoplanes sp. SE50/110. The acbC gene lies in a possible transcription unit, acbKLMNOC, cluster encompassing putative biosynthetic genes for cyclitol conversion. All genes were heterologously expressed in strains of Streptomyces lividans 66 strains 1326, TK23, and TK64. The AcbK protein was identified as the acarbose 7-kinase, which had been described earlier (Drepper, A., and Pape, H. (1996) J. Antibiot. (Tokyo) 49, 664-668). The multistep conversion of 2-epi-5-epi-valiolone to the final cyclitol moiety was studied by testing enzymatic mechanisms such as dehydration, reduction, epimerization, and phosphorylation. Thus, a phosphotransferase activity was identified modifying 2-epi-5-epi-valiolone by ATP-dependent phosphorylation. This activity could be attributed to the AcbM protein by verifying this activity in S. lividans strain TK64/pCW4123M, expressing His-tagged AcbM. The His-tagged AcbM protein was purified and subsequently characterized as a 2-epi-5-epi-valiolone 7-kinase, presumably catalyzing the first enzyme reaction in the biosynthetic route, leading to an activated form of the intermediate 1-epi-valienol. The AcbK protein could not catalyze the same reaction nor convert any of the other C(7)-cyclitol monomers tested. The 2-epi-5-epi-valiolone 7-phosphate was further converted by the AcbO protein to another isomeric and phosphorylated intermediate, which was likely to be the 2-epimer 5-epi-valiolone 7-phosphate. The products of both enzyme reactions were

  12. Ex Uno Plura: Differential Labeling of Phospholipid Biosynthetic Pathways with a Single Bioorthogonal Alcohol.

    Science.gov (United States)

    Bumpus, Timothy W; Liang, Felice J; Baskin, Jeremy M

    2018-01-16

    Imaging approaches that track biological molecules within cells are essential tools in modern biochemistry. Lipids are particularly challenging to visualize, as they are not directly genetically encoded. Phospholipids, the most abundant subgroup of lipids, are structurally diverse and accomplish many cellular functions, acting as major structural components of membranes and as signaling molecules that regulate cell growth, division, apoptosis, cytoskeletal dynamics, and numerous other physiological processes. Cells regulate the abundance, and therefore bioactivity, of phospholipids by modulating the activities of their biosynthetic enzymes. Thus, techniques that enable monitoring of flux through individual lipid biosynthetic pathways can provide key functional information. For example, the choline analogue propargylcholine (ProCho) can report on de novo biosynthesis of phosphatidylcholine by conversion to an alkynyl lipid that can be imaged following click chemistry tagging with an azido fluorophore. We report that ProCho is also a substrate of phospholipase D enzymes-which normally hydrolyze phosphatidylcholine to generate the lipid second messenger phosphatidic acid-in a transphosphatidylation reaction, generating the identical alkynyl lipid. By controlling the activities of phosphatidylcholine biosynthesis and phospholipase D enzymes, we establish labeling conditions that enable this single probe to selectively report on two different biosynthetic pathways. Just as nature exploits the economy of common metabolic intermediates to efficiently diversify biosynthesis, so can biochemists in interrogating such pathways with careful probe design. We envision that ProCho's ability to report on multiple metabolic pathways will enable studies of membrane dynamics and improve our understanding of the myriad roles that lipids play in cellular homeostasis.

  13. Riboflavin-UV--a crosslinking for fixation of biosynthetic corneal collagen implants.

    Science.gov (United States)

    Wand, Kerstin; Neuhann, Raphael; Ullmann, Andrea; Plank, Katharina; Baumann, Michael; Ritter, Roland; Griffith, May; Lohmann, Chris P; Kobuch, Karin

    2015-05-01

    To evaluate riboflavin-UV-A crosslinking as an alternative suture-free fixation method for biosynthetic corneal collagen implants. A range of cell-free corneal implants consisting of recombinant human collagen type III were examined. In vitro, the implants were crosslinked with different riboflavin solutions and irradiations. Ex vivo, the biosynthetic corneal implants were placed on the anterior cornea of porcine and rabbit eyes after performing deep anterior lamellar keratoplasty with a trephine, femtosecond laser, or excimer laser. UV-A crosslinking was performed with isotonic or hypotonic riboflavin at either standard or rapid procedure. The corneas were excised, fixed in PFA 4%, and embedded in paraffin. Crosslinking effects on the implants and the adhesion between implant and corneal bed were evaluated by slit-lamp biomicroscopy, optical coherence tomography (OCT) images, and histologically. After the crosslinking procedure, the implants showed different degrees of thinning. The accuracy of cutting the corneal bed was highest with the excimer laser. Good adhesion of the implant in the corneal bed could be demonstrated in OCT images. This was more accurate in porcine eyes than in rabbit eyes. Histologically, crosslinks between implant and corneal stroma were demonstrated. There was no difference between standard and rapid crosslinking procedures. Riboflavin-UV-A crosslinking as a fixation method for biosynthetic corneal collagen implants was demonstrated to be promising. It can reduce suture-related complications such as haze formation and surface irregularity. Stability of the implants, especially shrinkage after riboflavin-UV-A crosslinking, needs to be further evaluated. Biostability, integration, and long-term outcome are further evaluated in in vivo animal experiments.

  14. Integration of Fermentation and Organic Synthesis: Studies of Roquefortine C and Biosynthetic Derivatives

    Science.gov (United States)

    Gober, Claire Marie

    Roquefortine C is one of the most ubiquitous indoline alkaloids of fungal origin. It has been isolated from over 30 different species of Penicillium fungi and has garnered attention in recent years for its role as a biosynthetic precursor to the triazaspirocyclic natural products glandicoline B, meleagrin, and oxaline. The triazaspirocyclic motif, which encompasses three nitrogen atoms attached to one quaternary carbon forming a spirocyclic scaffold, is a unique chemical moiety that has been shown to impart a wide array of biological activity, from anti-bacterial activity and antiproliferative activity against cancer cell lines to anti-biofouling against marine organisms. Despite the promise of these compounds in the pharmaceutical and materials industries, few syntheses of triazaspirocycles exist in the literature. The biosynthesis of roquefortine C-derived triazaspirocycles, however, provides inspiration for the synthesis of these compounds, namely through a nitrone-promoted transannular rearrangement. This type of internal rearrangement has never been carried out synthetically and would provide an efficient stereoselective synthesis of triazaspirocycles. This work encompasses efforts towards elucidating the biosynthetic pathway of roquefortine C-derived triazaspirocycles as well as synthetic efforts towards the construction of triazaspirocycles. Chapter 1 will discuss a large-scale fermentation procedure for the production of roquefortine C from Penicillium crustosum. Chapters 2 and 3 explore (through enzymatic and synthetic means, respectively) the formation of the key indoline nitrone moiety required for the proposed transannular rearrangement. Finally, chapter 4 will discuss synthetic efforts towards the synthesis of triazaspirocycles. This work has considerably enhanced our understanding of the roquefortine C biosynthetic pathway and the unique chemistry of this natural product, and our efforts towards the synthesis of triazaspirocycles will facilitate the

  15. Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

    Energy Technology Data Exchange (ETDEWEB)

    Whicher, Jonathan R.; Florova, Galina; Sydor, Paulina K.; Singh, Renu; Alhamadsheh, Mamoun; Challis, Gregory L.; Reynolds, Kevin A.; Smith, Janet L. (Michigan); (Warwick); (Portland SU)

    2011-08-17

    Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a {beta}-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 {angstrom} resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.

  16. EPA, DHA, and Lipoic Acid Differentially Modulate the n-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

    Science.gov (United States)

    Bou, Marta; Østbye, Tone-Kari; Berge, Gerd M; Ruyter, Bente

    2017-03-01

    The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n-3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1-14C] 18:3n-3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1-14C] 18:3n-3 to 18:4n-3, 20:4n-3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n-3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n-3 by both DHA and LA. Possibly the route by 20:3n-3 and then Δ8 desaturation to 20:4n-3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health-beneficial FA in fish fed diets with low levels of EPA and/or DHA.

  17. Cobalamin production by Lactobacillus coryniformis: biochemical identification of the synthetized corrinoid and genomic analysis of the biosynthetic cluster.

    Science.gov (United States)

    Torres, Andrea Carolina; Vannini, Verónica; Bonacina, Julieta; Font, Graciela; Saavedra, Lucila; Taranto, María Pía

    2016-10-13

    Despite the fact that most vitamins are present in a variety of foods, malnutrition, unbalanced diets or insufficient intake of foods are still the cause of vitamin deficiencies in humans in some countries. Vitamin B12 (Cobalamin) is a complex compound that is only naturally produced by bacteria and archea. It has been reported that certain strains belonging to lactic acid bacteria group are capable of synthesized water-soluble vitamins such as those included in the B-group, as vitamin B12. In this context, the goal of the present paper was to evaluate and characterize the production of vitamin B12 in Lactobacillus coryniformis CRL 1001, a heterofermentative strain isolated from silage. Cell extract of L. coryniformis CRL 1001, isolated from silage, is able to correct the coenzyme B12 requirement of Salmonella enterica serovar Typhimurium AR 2680 in minimal medium. The chemical characterization of the corrinoid-like molecule isolated from CRL 1001 cell extract using HPLC and mass spectrometry is reported. The majority of the corrinoid produced by this strain has adenine like Coα-ligand instead 5,6-dimethylbenzimidazole. Genomic studies revealed the presence of the complete machinery of the anaerobic biosynthesis pathway of coenzyme B12. The detected genes encode all proteins for the corrin ring biosynthesis and for the binding of upper (β) and lower (α) ligands in one continuous stretch of the chromosome. The results here described show for the first time that L. coryniformis subsp. coryniformis CRL 1001 is able to produce pseudocobalamin containing adenine instead of 5,6-dimethlbenzimidazole in the Coα-ligand. Genomic analysis allowed the identification and characterization of the complete de novo biosynthetic pathway of the corrinoid produced by the CRL 1001 strain.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-03

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

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

    Directory of Open Access Journals (Sweden)

    Cynthia A Dick

    2011-04-01

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

  20. Molecular characterization and functional analysis of chalcone synthase from Syringa oblata Lindl. in the flavonoid biosynthetic pathway.

    Science.gov (United States)

    Wang, Yu; Dou, Ying; Wang, Rui; Guan, Xuelian; Hu, Zenghui; Zheng, Jian

    2017-11-30

    The flower color of Syringa oblata Lindl., which is often modulated by the flavonoid content, varies and is an important ornamental feature. Chalcone synthase (CHS) catalyzes the first key step in the flavonoid biosynthetic pathway. However, little is known about the role of S. oblata CHS (SoCHS) in flavonoid biosynthesis in this species. Here, we isolate and analyze the cDNA (SoCHS1) that encodes CHS in S. oblata. We also sought to analyzed the molecular characteristics and function of flavonoid metabolism by SoCHS1. We successfully isolated the CHS-encoding genomic DNA (gDNA) in S. oblata (SoCHS1), and the gene structural analysis indicated it had no intron. The opening reading frame (ORF) sequence of SoCHS1 was 1170bp long and encoded a 389-amino acid polypeptide. Multiple sequence alignment revealed that both the conserved CHS active site residues and CHS signature sequence were in the deduced amino acid sequence of SoCHS1. Crystallographic analysis revealed that the protein structure of SoCHS1 is highly similar to that of FnCHS1 in Freesia hybrida. The quantitative real-time polymerase chain reaction (PCR) performed to detect the SoCHS1 transcript expression levels in flowers, and other tissues revealed the expression was significantly correlated with anthocyanin accumulation during flower development. The ectopic expression results of Nicotiana tabacum showed that SoCHS1 overexpression in transgenic tobacco changed the flower color from pale pink to pink. In conclusion, these results suggest that SoCHS1 plays an essential role in flavonoid biosynthesis in S. oblata, and could be used to modify flavonoid components in other plant species. Copyright © 2017. Published by Elsevier B.V.

  1. Expression profiles of genes involved in tanshinone biosynthesis of ...

    Indian Academy of Sciences (India)

    tanshinone biosynthetic gene; quantitative real-time PCR; Salvia miltiorrhiza. Abstract. Author Affiliations. ZHENQIAO SONG1 2 JIANHUA WANG1 2 XINGFENG LI1 2. State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, a; Agronomy College, Shandong Agricultural University, Taian 271018, ...

  2. Computational identification of putative cytochrome P450 genes in ...

    African Journals Online (AJOL)

    Chattha

    protein identified to date (Morant et al., 2003). P450s are found in all major plant biosynthetic pathways, including ... The large scale of soybean expressed sequence tags (ESTs) database (NCBI) contains over 1,386,618 ... are still large quantities of genes to be annotated. This study used ESTs which is a good approach for.

  3. Genes encoding chimeras of Neurospora crassa erg-3 and human ...

    Indian Academy of Sciences (India)

    http://www.ias.ac.in/article/fulltext/jbsc/027/02/0105-0112. Keywords. Lamin B receptor; sterol reductase. Abstract. The human gene TM7SF2 encodes a polypeptide (SR-1) with high sequence similarity to sterol C-14 reductase, a key sterol biosynthetic enzyme in fungi, plants and mammals. In Neurospora and yeast this ...

  4. Temporal expression of genes involved in the biosynthesis of ...

    African Journals Online (AJOL)

    Jane

    2011-10-10

    Oct 10, 2011 ... 2010). Thus, the regulation of the expression of the. GA20ox, GA3ox and GA2ox plays a critical role in GA homeostasis. GA-mediated responses can be controlled through the regulation of specific biosynthetic genes. (Chen et al., 2007), including GA20ox, GA3ox and. GA2ox. Transcript levels of many GA ...

  5. Distribution of homologous proteins to puffer fish saxitoxin and tetrodotoxin binding protein in the plasma of puffer fish and among the tissues of Fugu pardalis examined by Western blot analysis.

    Science.gov (United States)

    Yotsu-Yamashita, Mari; Yamaki, Hiroe; Okoshi, Natsumi; Araki, Nao

    2010-06-01

    Puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP) is a glycoprotein (200 kDa as a dimer) that we previously isolated from the plasma of Fugu pardalis (Yotsu-Yamashita et al., 2001). For the study on functions of PSTBP, here we examined distribution of homologous proteins to PSTBP in the plasma of seven species of puffer fish, and among the tissues of F. pardalis by Western blot analysis probed with a polyclonal IgG against unglycosylated PSTBP1 expressed in Echelichia coli. One or two major positive broad bands were detected at 105-140 kDa molecular weight range in the plasma (0.5 microg protein) of all species of puffer fish tested, while no band was detected in the plasma (5 microg protein) of fish other than puffer fish. Glycopeptidase F treated plasma of all species of puffer fish tested commonly showed the bands at approximately 42 kDa that was consistent to the molecular weight of unglycosylated PSTBP. These data suggest that puffer fish commonly possess glycoproteins homologous to PSTBP, but the sizes of N-glycan are specific to the species. Among soluble protein extracts (5 microg protein) from the tissues of F. pardalis, PSTBP was detected in all tissues examined, most prominently in heart, skin, and gall. Copyright 2009 Elsevier Ltd. All rights reserved.

  6. Acute Toxicities of the Saxitoxin Congeners Gonyautoxin 5, Gonyautoxin 6, Decarbamoyl Gonyautoxin 2&3, Decarbamoyl Neosaxitoxin, C-1&2 and C-3&4 to Mice by Various Routes of Administration

    Directory of Open Access Journals (Sweden)

    Andrew I. Selwood

    2017-02-01

    Full Text Available Paralytic shellfish poisoning results from consumption of seafood naturally contaminated by saxitoxin and its congeners, the paralytic shellfish toxins (PSTs. The levels of such toxins are regulated internationally, and maximum permitted concentrations in seafood have been established in many countries. A mouse bioassay is an approved method for estimating the levels of PSTs in seafood, but this is now being superseded in many countries by instrumental methods of analysis. Such analyses provide data on the levels of many PSTs in seafood, but for risk assessment, knowledge of the relative toxicities of the congeners is required. These are expressed as “Toxicity Equivalence Factors” (TEFs. At present, TEFs are largely based on relative specific activities following intraperitoneal injection in a mouse bioassay rather than on acute toxicity determinations. A more relevant parameter for comparison would be median lethal doses via oral administration, since this is the route through which humans are exposed to PSTs. In the present study, the median lethal doses of gonyautoxin 5, gonyautoxin 6, decarbamoyl neosaxitoxin and of equilibrium mixtures of decarbamoyl gonyautoxins 2&3, C1&2 and C3&4 by oral administration to mice have been determined and compared with toxicities via intraperitoneal injection. The results indicate that the TEFs of several of these substances require revision in order to more accurately reflect the risk these toxins present to human health.

  7. Variation in sequence and location of the fumonisin mycotoxin niosynthetic gene cluster in Fusarium

    NARCIS (Netherlands)

    Proctor, R.H.; Hove, van F.; Susca, A.; Stea, A.; Busman, M.; Lee, van der T.A.J.; Waalwijk, C.; Moretti, A.

    2010-01-01

    In Fusarium, the ability to produce fumonisins is governed by a 17-gene fumonisin biosynthetic gene (FUM) cluster. Here, we examined the cluster in F. oxysporum strain O-1890 and nine other species selected to represent a wide range of the genetic diversity within the GFSC.

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

    Science.gov (United States)

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

    2015-12-01

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

  9. Genetic, metabolomic and transcriptomic analyses of the de novo L-cysteine biosynthetic pathway in the enteric protozoan parasite Entamoeba histolytica.

    Science.gov (United States)

    Jeelani, Ghulam; Sato, Dan; Soga, Tomoyoshi; Nozaki, Tomoyoshi

    2017-11-15

    The de novo L-cysteine biosynthetic pathway is critical for the growth, antioxidative stress defenses, and pathogenesis of bacterial and protozoan pathogens, such as Salmonella typhimurium and Entamoeba histolytica. This pathway involves two key enzymes, serine acetyltransferase (SAT) and cysteine synthase (CS), which are absent in mammals and therefore represent rational drug targets. The human parasite E. histolytica possesses three SAT and CS isozymes; however, the specific roles of individual isoforms and significance of such apparent redundancy remains unclear. In the present study, we generated E. histolytica cell lines in which CS and SAT expression was knocked down by transcriptional gene silencing. The strain in which CS1, 2 and 3 were simultaneously silenced and the SAT3 gene-silenced strain showed impaired growth when cultured in a cysteine lacking BI-S-33 medium, whereas silencing of SAT1 and SAT2 had no effects on growth. Combined transcriptomic and metabolomic analyses revealed that, CS and SAT3 are involved in S-methylcysteine/cysteine synthesis. Furthermore, silencing of the CS1-3 or SAT3 caused upregulation of various iron-sulfur flavoprotein genes. Taken together, these results provide the first direct evidence of the biological importance of SAT3 and CS isoforms in E. histolytica and justify the exploitation of these enzymes as potential drug targets.

  10. Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

    Science.gov (United States)

    Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

    2012-01-01

    Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can

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

    Science.gov (United States)

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey

    2006-08-16

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

  12. Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga.

    Directory of Open Access Journals (Sweden)

    Michael T Guarnieri

    Full Text Available Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal-derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae. In order to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga, Chlorella vulgaris, we have established a strategy with which to bypass the necessity for genomic sequence information by using the transcriptome as a guide. Our results indicate an upregulation of both fatty acid and triacylglycerol biosynthetic machinery under oil-accumulating conditions, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of an unsequenced microalga.

  13. An improved in vivo deuterium labeling method for measuring the biosynthetic rate of cytokinins.

    Science.gov (United States)

    Tarkowski, Petr; Floková, Kristýna; Václavíková, Kateřina; Jaworek, Pavel; Raus, Martin; Nordström, Anders; Novák, Ondřej; Doležal, Karel; Sebela, Marek; Frébortová, Jitka

    2010-12-15

    An improved method for determining the relative biosynthetic rate of isoprenoid cytokinins has been developed. A set of 11 relevant isoprenoid cytokinins, including zeatin isomers, was separated by ultra performance liquid chromatography in less than 6 min. The iP-type cytokinins were observed to give rise to a previously-unknown fragment at m/z 69; we suggest that the diagnostic (204-69) transition can be used to monitor the biosynthetic rate of isopentenyladenine. Furthermore, we found that by treating the cytokinin nucleotides with alkaline phosphatase prior to analysis, the sensitivity of the detection process could be increased. In addition, derivatization (propionylation) improved the ESI-MS response by increasing the analytes' hydrophobicity. Indeed, the ESI-MS response of propionylated isopentenyladenosine was about 34% higher than that of its underivatized counterpart. Moreover, the response of the derivatized zeatin ribosides was about 75% higher than that of underivatized zeatin ribosides. Finally, we created a web-based calculator (IZOTOP) that facilitates MS/MS data processing and offer it freely to the research community.

  14. Biosynthetic relationship among aflatoxins B1, B2, M1, and M2.

    Science.gov (United States)

    Dutton, M F; Ehrlich, K; Bennett, J W

    1985-01-01

    Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway. PMID:3925881

  15. Biosynthetic relationship among aflatoxins B1, B2, M1, and M2.

    Science.gov (United States)

    Dutton, M F; Ehrlich, K; Bennett, J W

    1985-06-01

    Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.

  16. [Biosynthetic schwertmannite as catalyst in Fenton-like reactions for degradation of methyl orange].

    Science.gov (United States)

    Wang, Kuai-Bing; Fang, Di; Xu, Zhi-Hui; Shi, Ying; Zheng, Guan-Yu; Zhou, Li-Xiang

    2015-03-01

    Biosynthesized schwertmannite was used as catalyst in photo-Fenton-like reaction to facilitate the degradation of methyl orange (MO). Schwertmannite was synthesized through the oxidation of FeSO4 by Acidithiobacillus ferrooxidans LX5 cell suspension at an initial pH 2.5 and 28 degress C for 3 days and characterized using X-ray diffraction spectroscopy and scanning electron microscope. The oxidative degradation of MO in the photo-Fenton-like reaction was studied at different initial pH values of suspension, concentrations of H2O2 and dosages of catalyst. The results suggested that the biosynthetic schwertmannite showed a good catalytic activity in the MO degradation via *OH radical mechanism. Considerable degradation efficiency of MO was still obtained in approximately neutral condition or in the presence of high concentrations of chloride, sulfate and nitrate. This work demonstrated that the heterogeneous photo-Fenton-like reaction catalyzed by the biosynthetic schwertmannite is a promising advanced oxidation technology for the treatment of wastewater containing MO.

  17. Alterations in the heme biosynthetic pathway as an index of exposure to toxins

    Energy Technology Data Exchange (ETDEWEB)

    Marks, G.S.; Zelt, D.T.; Cole, S.P.

    1982-07-01

    Under normal circumstances the heme biosynthetic pathway is carefully controlled and porphyrins are formed in only trace amounts. When control mechanisms are disturbed by xenobiotics, porphyrins may be formed and serve as a signal of the interaction between a xenobiotic and the heme biosynthetic pathway. For example, porphyrinuria was an early manifestation of a hexachlorobenzene-induced porphyria outbreak in Turkey. In humans exposed to polybrominated biphenyls and to 2,3,7,8-tetrachlorodibenzo-p-dioxin the urinary porphyrin pattern was significantly different from normal in a large number of exposed individuals. The question is raised whether measurement of urinary porphyrin profiles by improved methods will enable an estimate to be made of the extent of exposure to haloaromatic hydrocarbons in the human population. A wide variety of xenobiotics interact with the prosthetic heme of cytochrome P-450 forming novel N-alkylporphyrins. Identification of these N-alkylporphyrins in body fluids might provide a means of assessing exposure to a variety of xenobiotics in human populations.

  18. Genome Sequence of Thermofilum pendens Reveals an Exceptional Loss of Biosynthetic Pathways without Genome Reduction

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Iain [U.S. Department of Energy, Joint Genome Institute; Rodriquez, Jason [Virginia Polytechnic Institute and State University (Virginia Tech); Susanti, Dwi [Virginia Polytechnic Institute and State University (Virginia Tech); Porat, I. [University of Georgia, Athens, GA; Reich, Claudia [University of Illinois, Urbana-Champaign; Ulrich, Luke [ORNL; Elkins, James G [ORNL; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Lykidis, A [U.S. Department of Energy, Joint Genome Institute; Kim, Edwin [U.S. Department of Energy, Joint Genome Institute; Thompson, Linda S [Los Alamos National Laboratory (LANL); Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Copeland, A [U.S. Department of Energy, Joint Genome Institute; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Detter, J C [U.S. Department of Energy, Joint Genome Institute; Zhulin, Igor B [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Olsen, Gary [ORNL; Whitman, W. B. [University of Georgia, Athens, GA; Mukhopadhyay, Biswarup [Virginia Polytechnic Institute and State University (Virginia Tech); Bristow, James [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching member of class Thermoproteales of Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It 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 transporters of the phosphotransferase system. T. pendens is known to require 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. T. pendens has fewer biosynthetic enzymes than any other free-living organism. In addition to heterotrophy, 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 from a new subfamily. Predicted highly expressed proteins include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins, suggesting that defense against viruses is a high priority.

  19. Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation

    Science.gov (United States)

    Li, Fengfu; Carlsson, David; Lohmann, Chris; Suuronen, Erik; Vascotto, Sandy; Kobuch, Karin; Sheardown, Heather; Munger, Rejean; Nakamura, Masatsugu; Griffith, May

    2003-12-01

    Our objective was to determine whether key properties of extracellular matrix (ECM) macromolecules can be replicated within tissue-engineered biosynthetic matrices to influence cellular properties and behavior. To achieve this, hydrated collagen and N-isopropylacrylamide copolymer-based ECMs were fabricated and tested on a corneal model. The structural and immunological simplicity of the cornea and importance of its extensive innervation for optimal functioning makes it an ideal test model. In addition, corneal failure is a clinically significant problem. Matrices were therefore designed to have the optical clarity and the proper dimensions, curvature, and biomechanical properties for use as corneal tissue replacements in transplantation. In vitro studies demonstrated that grafting of the laminin adhesion pentapeptide motif, YIGSR, to the hydrogels promoted epithelial stratification and neurite in-growth. Implants into pigs' corneas demonstrated successful in vivo regeneration of host corneal epithelium, stroma, and nerves. In particular, functional nerves were observed to rapidly regenerate in implants. By comparison, nerve regeneration in allograft controls was too slow to be observed during the experimental period, consistent with the behavior of human cornea transplants. Other corneal substitutes have been produced and tested, but here we report an implantable matrix that performs as a physiologically functional tissue substitute and not simply as a prosthetic device. These biosynthetic ECM replacements should have applicability to many areas of tissue engineering and regenerative medicine, especially where nerve function is required. regenerative medicine | tissue engineering | cornea | implantation | innervation

  20. The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae.

    Science.gov (United States)

    Rouhbakhsh, D; Lai, C Y; von Dohlen, C D; Clark, M A; Baumann, L; Baumann, P; Moran, N A; Voegtlin, D J

    1996-04-01

    The bacterial endosymbionts (Buchnera) from the aphids Rhopalosiphum padi, R. maidis, Schizaphis graminum, and Acyrthosiphon pisum contain the genes for anthranilate synthase (trpEG) on plasmids made up of one or more 3.6-kb units. Anthranilate synthase is the first as well as the rate-limiting enzyme in the tryptophan biosynthetic pathway. The amplification of trpEG on plasmids may result in an increase of enzyme protein and overproduction of this essential amino acid, which is required by the aphid host. The nucleotide sequence of trpEG from endosymbionts of different species of aphids is highly conserved, as is an approximately 500-bp upstream DNA segment which has the characteristics of an origin of replication. Phylogenetic analyses were performed using trpE and trpG from the endosymbionts of these four aphids as well as from the endosymbiont of Schlechtendalia chinensis, in which trpEG occurs on the chromosome. The resulting phylogeny was congruent with trees derived from sequences of two chromosome-located bacterial genes (part of trpB and 16S ribosomal DNA). In turn, trees obtained from plasmid-borne and bacterial chromosome-borne sequences were congruent with the tree resulting from phylogenetic analysis of three aphid mitochondrial regions (portions of the small and large ribosomal DNA subunits, as well as cytochrome oxidase II). Congruence of trees based on genes from host mitochondria and from bacteria adds to previous support for exclusively vertical transmission of the endosymbionts within aphid lineages. Congruence with trees based on plasmid-borne genes supports the origin of the plasmid-borne trpEG from the chromosomal genes of the same lineage and the absence of subsequent plasmid exchange among endosymbionts of different species of aphids.

  1. Cloning and characterization of a putative R2R3 MYB transcriptional repressor of the rosmarinic acid biosynthetic pathway from Salvia miltiorrhiza.

    Directory of Open Access Journals (Sweden)

    Shuncang Zhang

    Full Text Available Salvia miltiorrhiza Bunge is one of the most renowned traditional medicinal plants in China. Phenolic acids that are derived from the rosmarinic acid pathway, such as rosmarinic acid and salvianolic acid B, are important bioactive components in S. miltiorrhiza. Accumulations of these compounds have been reported to be induced by various elicitors, while little is known about transcription factors that function in their biosynthetic pathways. We cloned a subgroup 4 R2R3 MYB transcription factor gene (SmMYB39 from S. miltiorrhiza and characterized its roles through overexpression and RNAi-mediated silencing. As the results showed, the content of 4-coumaric acid, rosmarinic acid, salvianolic acid B, salvianolic acid A and total phenolics was dramatically decreased in SmMYB39-overexpressing S. miltiorrhiza lines while being enhanced by folds in SmMYB39-RNAi lines. Quantitative real-time PCR and enzyme activities analyses showed that SmMYB39 negatively regulated transcripts and enzyme activities of 4-hydroxylase (C4H and tyrosine aminotransferase (TAT. These data suggest that SmMYB39 is involved in regulation of rosmarinic acid pathway and acts as a repressor through suppressing transcripts of key enzyme genes.

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

    Science.gov (United States)

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

    2016-11-01

    The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified Escherichia coli

    Science.gov (United States)

    Maric, Selma; Thygesen, Mikkel B.; Schiller, Jürgen; Marek, Magdalena; Moulin, Martine; Haertlein, Michael; Forsyth, V. Trevor; Bogdanov, Mikhail; Dowhan, William; Arleth, Lise

    2014-01-01

    Phosphatidylcholine (PC) is a major component of eukaryotic cell membranes and one of the most commonly used phospholipids for reconstitution of membrane proteins into carrier systems such as lipid vesicles, micelles and nanodiscs. Selectively deuterated versions of this lipid have many applications, especially in structural studies using techniques such as NMR, neutron reflectivity and small-angle neutron scattering. Here we present a comprehensive study of selective deuteration of phosphatidylcholine through biosynthesis in a genetically modified strain of Escherichia coli. By carefully tuning the deuteration level in E. coli growth media and varying the deuteration of supplemented carbon sources, we show that it is possible to achieve a controlled deuteration for three distinct parts of the PC lipid molecule, namely the (a) lipid head group, (b) glycerol backbone and (c) fatty acyl tail. This biosynthetic approach paves the way for the synthesis of specifically deuterated, physiologically relevant phospholipid species which remain difficult to obtain through standard chemical synthesis. PMID:25301578

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-04-01

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

  5. Water splitting-biosynthetic system with CO₂ reduction efficiencies exceeding photosynthesis.

    Science.gov (United States)

    Liu, Chong; Colón, Brendan C; Ziesack, Marika; Silver, Pamela A; Nocera, Daniel G

    2016-06-03

    Artificial photosynthetic systems can store solar energy and chemically reduce CO2 We developed a hybrid water splitting-biosynthetic system based on a biocompatible Earth-abundant inorganic catalyst system to split water into molecular hydrogen and oxygen (H2 and O2) at low driving voltages. When grown in contact with these catalysts, Ralstonia eutropha consumed the produced H2 to synthesize biomass and fuels or chemical products from low CO2 concentration in the presence of O2 This scalable system has a CO2 reduction energy efficiency of ~50% when producing bacterial biomass and liquid fusel alcohols, scrubbing 180 grams of CO2 per kilowatt-hour of electricity. Coupling this hybrid device to existing photovoltaic systems would yield a CO2 reduction energy efficiency of ~10%, exceeding that of natural photosynthetic systems. Copyright © 2016, American Association for the Advancement of Science.

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

    Science.gov (United States)

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

    2013-11-01

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

  7. Leveraging microbial biosynthetic pathways for the generation of 'drop-in' biofuels.

    Science.gov (United States)

    Zargar, Amin; Bailey, Constance B; Haushalter, Robert W; Eiben, Christopher B; Katz, Leonard; Keasling, Jay D

    2017-06-01

    Advances in retooling microorganisms have enabled bioproduction of 'drop-in' biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gas-turbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), 'drop-in' biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical properties (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin

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    Srijana Upadhyay

    2016-03-01

    Full Text Available Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs and how it is deposited as extracellular granules remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell-wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally derived melanosomes.

  9. Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

    Science.gov (United States)

    Tsuchiya, Takashi; Iwakura, Hiroshi; Minamino, Naoto; Kangawa, Kenji; Sasaki, Kazuki

    2017-09-02

    Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Volatile secondary metabolites of Micromeria dalmatica Benth. (Lamiaceae): biosynthetical and chemotaxonomical aspects.

    Science.gov (United States)

    Radulović, Niko S; Blagojević, Polina D

    2012-07-01

    Analysis by GC and GC/MS of the essential oil obtained from above-ground parts of Micromeria dalmatica Benth. allowed the identification of 116 components, comprising 93.6% of the total oil composition. The major compounds are 3-oxygenated p-menthane monoterpenes and were identified as pulegone (29.6%), menthone (11.7%), and piperitenone (10.8%). The chemical composition of this and additional 30 oils obtained from selected Micromeria Benth. taxa were compared by using multivariate statistical analysis (agglomerative hierarchical cluster analysis and principal component analysis (PCA)). The results of statistical analyses, as well as the domination of different concurrent p-menthane-skeleton-type monoterpene biosynthetical sub-branches in the compared M. dalmatica samples, implied the occurrence of at least two different chemotypes of the mentioned species. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

  11. Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

    Science.gov (United States)

    Fletcher, Sarah J; Iqbal, Mudassar; Jabbari, Sara; Stekel, Dov; Rappoport, Joshua Z

    2014-01-01

    Tight junctions (TJs) link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK) epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes). By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

  12. Determining the Biochemical Properties of the Oxalate Biosynthetic Component (Obc)1 from Burkholderia mallei.

    Science.gov (United States)

    Lambert, Peter M; Nakata, Paul A

    2016-01-01

    Oxalic acid is produced by a variety of organisms ranging from simple microbes to complex animals. This acid has been proposed to fulfill various physiological and pathological functions which vary between organisms. In bacteria from the Burkholderia genus, oxalate secretion has been shown to be quorum sensing dependent and to support pathogenicity and cell viability. In light of the critical roles of oxalate in Burkholderia as well as other organisms, it is surprising that our understanding of how this simple dicarboxylate is biosynthesized remains incomplete. Here we report the expression, purification, and partial characterization of the first intact bacterial oxalate biosynthetic enzyme, Obc1, from B. mallei. An N-terminal His-tagged Bmobc1 was cloned into pDUET, expressed in E. coli BLR (DE3), and the recombinant enzyme purified by affinity chromatography. Oxalate biosynthetic enzyme assays coupled with HPLC analysis revealed that BmObc1 catalyzed the biosynthesis of oxalate, acetoacetate, and free CoA from oxaloacetate and a short chain acyl-CoA following Michaelis-Menten kinetics. Optimal enzyme activity was measured at pH 8.0 and a temperature around 44°C. Kinetic analysis conducted under conditions of saturating acetyl-CoA and varying oxaloacetate concentrations resulted in a calculated Km value for oxaloacetate of 94.3± 9.2 μM (mean ± SE). Under conditions of saturating oxaloacetate concentration and varying acyl-CoA (acetyl- or propionyl-CoA) concentrations kinetic analysis generated a calculated Km value of 26.8 ± 2.3 μM (mean ± SE) for acetyl-CoA and 104.4 ± 12.7 μM for propionyl-CoA. The significantly lower Km for acetyl-CoA suggests that it is strongly favored as a substrate over propionyl-CoA.

  13. Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

    Directory of Open Access Journals (Sweden)

    Sarah J Fletcher

    Full Text Available Tight junctions (TJs link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes. By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

  14. Seasonal variations of artemisinin and its biosynthetic precursors in tetraploid Artemisia annua plants compared with the diploid wild-type

    NARCIS (Netherlands)

    Wallaart, T.E.; Pras, N.; Quax, Wim

    1999-01-01

    Using colchicine we induced tetraploidy in Artemisia annua L. plants. During a vegetation period we monitored the time course of the levels of artemisinin, its direct precursors, the biosynthetically related sesquiterpenes and the essential oil content in the diploid (wild-type) and tetraploid A.

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

    NARCIS (Netherlands)

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

    Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of Gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway

  16. Biosynthetic controls on the 13C-contents of organic components in the photoautotrophic bacterium Chloroflexus aurantiacus

    NARCIS (Netherlands)

    Sinninghe Damsté, J.S.; Meer, M.T.J. van der; Schouten, S.; Dongen, B.E. van; Rijpstra, W.I.C.; Fuchs, G.; Leeuw, J.W. de; Ward, D.M.

    2001-01-01

    To assess the effects related to known and proposed biosynthetic pathways on the 13C content of lipids and storage products of the photoautotrophic bacterium Chloroflexus aurantiacus, the isotopic compositions of bulk cell material, alkyl and isoprenoid lipids, and storage products such as glycogen

  17. A fast and simple GC MS method for lignan profiling in Anthriscus sylvestris and biosynthetically related plant species

    NARCIS (Netherlands)

    Koulman, A; Bos, R; Medarde, M; Pras, N; Quax, WJ

    2001-01-01

    A new GC-MS method for monitoring lignans was developed to study the variation in plants and elucidate the biosynthetic steps. A simple and fast extraction procedure for lyophilised plant material was developed, giving a lignan-rich extract. A GC-MS method was set up using an apolar WCOT fused

  18. Identification of a trichothecene gene cluster and description of the harzianum A biosynthesis pathway in the fungus Trichoderma arundinaceum

    Science.gov (United States)

    Trichothecenes are sesquiterpenes that act like mycotoxins. Their biosynthesis has been mainly studied in the fungal genera Fusarium, where most of the biosynthetic genes (tri) are grouped in a cluster regulated by ambient conditions and regulatory genes. Unexpectedly, few studies are available abou...

  19. Improved fructan accumulation in perennial ryegrass transformed with the onion fructosyltransferase genes 1-SST and 6G-FFT

    DEFF Research Database (Denmark)

    Gadegaard, Gitte; Didion, Thomas; Foiling, Marianne

    2008-01-01

    value of ryegrass (Lolium perenne) by increasing the fructan content through expression of heterologous fructan biosynthetic genes. We developed perennial ryegrass Lines expressing sucrose:sucrose 1-fructosyltransferase and fructan:fructan 6G-fructosyltransferase genes from onion (Allium cepa) which...

  20. Biosynthesis of Akaeolide and Lorneic Acids and Annotation of Type I Polyketide Synthase Gene Clusters in the Genome of Streptomyces sp. NPS554

    Directory of Open Access Journals (Sweden)

    Tao Zhou

    2015-01-01

    Full Text Available The incorporation pattern of biosynthetic precursors into two structurally unique polyketides, akaeolide and lorneic acid A, was elucidated by feeding experiments with 13C-labeled precursors. In addition, the draft genome sequence of the producer, Streptomyces sp. NPS554, was performed and the biosynthetic gene clusters for these polyketides were identified. The putative gene clusters contain all the polyketide synthase (PKS domains necessary for assembly of the carbon skeletons. Combined with the 13C-labeling results, gene function prediction enabled us to propose biosynthetic pathways involving unusual carbon-carbon bond formation reactions. Genome analysis also indicated the presence of at least ten orphan type I PKS gene clusters that might be responsible for the production of new polyketides.

  1. A genetically amenable platensimycin- and platencin-overproducer as a platform for biosynthetic explorations: a showcase of PtmO4, a long-chain acyl-CoA dehydrogenase.

    Science.gov (United States)

    Rudolf, Jeffrey D; Dong, Liao-Bin; Huang, Tingting; Shen, Ben

    2015-10-01

    Platensimycin (PTM) and platencin (PTN) are members of a new class of promising drug leads that target bacterial and mammalian fatty acid synthases. We previously cloned and sequenced the PTM and PTN gene clusters, discovered six additional PTM-PTN dual producing strains, and demonstrated the dramatic overproduction of PTM and PTN by inactivating the pathway-specific regulators ptmR1 or ptnR1 in five different strains. Our ability to utilize these PTM-PTN dual overproducing strains was limited by their lack of genetic amenability. Here we report the construction of Streptomyces platensis SB12029, a genetically amenable, in-frame ΔptmR1 dual PTM-PTN overproducing strain. To highlight the potential of this strain for future PTM and PTN biosynthetic studies, we created the ΔptmR1 ΔptmO4 double mutant S. platensis SB12030. Fourteen PTM and PTN congeners, ten of which were new, were isolated from SB12030, shedding new insights into PTM and PTN biosynthesis. PtmO4, a long-chain acyl-CoA dehydrogenase, is strongly implicated to catalyze β-oxidation of the diterpenoid intermediates into the PTM and PTN scaffolds. SB12029 sets the stage for future biosynthetic and bioengineering studies of the PTM and PTN family of natural products.

  2. Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway.

    Science.gov (United States)

    Ong, Seong Siang; Wickneswari, Ratnam

    2011-11-30

    Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem. In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in epigenetic silencing

  3. Evidence for Horizontal Gene Transfer as Origin of Putrescine Production in Oenococcus oeni RM83▿

    Science.gov (United States)

    Marcobal, Ángela; de las Rivas, Blanca; Moreno-Arribas, M. Victoria; Muñoz, Rosario

    2006-01-01

    The nucleotide sequence of a 17.2-kb chromosomal DNA fragment containing the odc gene encoding ornithine decarboxylase has been determined in the putrescine producer Oenococcus oeni RM83. This DNA fragment contains 13 open reading frames, including genes coding for five transposases and two phage proteins. This description might represent the first evidence of a horizontal gene transfer event as the origin of a biogenic amine biosynthetic locus. PMID:17056681

  4. The Bacillus subtilis TRAP protein can induce transcription termination in the leader region of the tryptophan biosynthetic (trp operon independent of the trp attenuator RNA.

    Directory of Open Access Journals (Sweden)

    Natalie M McAdams

    Full Text Available In Bacillus subtilis, transcription of the tryptophan biosynthetic operon is regulated by an attenuation mechanism. When intracellular tryptophan levels are high, the TRAP protein binds to the 5' leader region of the nascent trp mRNA and induces transcription termination prior to the structural genes. In limiting tryptophan, TRAP does not bind and the operon is transcribed. Two competing RNA secondary structures termed the antiterminator and terminator (attenuator can form in the leader region RNA. In prior attenuation models, the only role of TRAP binding was to alter the RNA secondary structure to allow formation of the attenuator, which has been thought function as an intrinsic transcription terminator. However, recent studies have shown that the attenuator is not an effective intrinsic terminator. From these studies it was not clear whether TRAP functions independently or requires the presence of the attenuator RNA structure. Hence we have further examined the role of the attenuator RNA in TRAP-mediated transcription termination. TRAP was found to cause efficient transcription termination in the trp leader region in vivo when the attenuator was mutated or deleted. However, TRAP failed to induce transcription termination at these mutant attenuators in a minimal in vitro transcription system with B. subtilis RNA polymerase. Further studies using this system showed that NusA as well as the timing of TRAP binding to RNA play a role in the observed differences in vivo and in vitro.

  5. Characterization of the endosperm starch and the pleiotropic effects of biosynthetic enzymes on their properties in novel mutant rice lines with high resistant starch and amylose content.

    Science.gov (United States)

    Itoh, Yuuki; Crofts, Naoko; Abe, Misato; Hosaka, Yuko; Fujita, Naoko

    2017-05-01

    Resistant starch (RS) is beneficial to human health. In order to reduce the current prevalence of diabetes and obesity, several transgenic and mutant crops containing high RS content are being developed. RS content of steamed rice with starch-branching enzyme (BE)IIb-deficient mutant endosperms is considerably high. To understand the mechanisms of RS synthesis and to increase RS content, we developed novel mutant rice lines by introducing the gene encoding starch synthase (SS)IIa and/or granule-bound starch synthase (GBSS)I from an indica rice cultivar into a japonica rice-based BEIIb-deficient mutant line, be2b. Introduction of SSIIa from an indica rice cultivar produced higher levels of amylopectin chains with degree of polymerization (DP) 11-18 than those in be2b; the extent of the change was slight due to the shortage of donor chains for SSIIa (DP 6-12) owing to BEIIb deficiency. The introduction of GBSSI from an indica rice cultivar significantly increased amylose content (by approximately 10%) in the endosperm starch. RS content of the new mutant lines was the same as or slightly higher than that of the be2b parent line. The relationship linking starch structure, RS content, and starch biosynthetic enzymes in the new mutant lines has also been discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Synthesis of ent-BE-43547A1 reveals a potent hypoxia-selective anticancer agent and uncovers the biosynthetic origin of the APD-CLD natural products

    Science.gov (United States)

    Villadsen, Nikolaj L.; Jacobsen, Kristian M.; Keiding, Ulrik B.; Weibel, Esben T.; Christiansen, Bjørn; Vosegaard, Thomas; Bjerring, Morten; Jensen, Frank; Johannsen, Mogens; Tørring, Thomas; Poulsen, Thomas B.

    2017-03-01

    Tumour hypoxia is speculated to be a key driver of therapeutic resistance and metastatic dissemination. Consequently, the discovery of new potent agents that selectively target the hypoxic cell population may reveal new and untapped antitumour mechanisms. Here we demonstrate that the BE-43547 subclass of the APD-CLD (amidopentadienoate-containing cyclolipodepsipeptides) natural products possesses highly hypoxia-selective growth-inhibitory activity against pancreatic cancer cells. To enable this discovery, we have developed the first synthesis of the BE-43547-macrocyclic scaffold in 16 steps (longest linear sequence), which also allowed access to the full panel of relative stereoisomers and ultimately to the assignment of stereochemical configuration. Discrepancies between the spectroscopic signatures of the synthetic compounds with that originally reported for the BE-43547 members stimulated us to re-isolate the natural product from a BE-43547-producing microorganism during which we elucidated the biosynthetic gene clusters for the BE-43547 family as well as for all other known APD-CLDs. Our studies underline the exciting possibilities for the further development of the anticancer activities of these natural products.

  7. A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model.

    Science.gov (United States)

    Gesteira, Tarsis F; Coulson-Thomas, Vivien Jane; Ogata, Fernando T; Farias, Eduardo H C; Cavalheiro, Renan P; de Lima, Marcelo A; Cunha, Gabriel L A; Nakayasu, Ernesto S; Almeida, Igor C; Toma, Leny; Nader, Helena B

    2011-12-01

    Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. High-density biosynthetic fuels: the intersection of heterogeneous catalysis and metabolic engineering.

    Science.gov (United States)

    Harvey, Benjamin G; Meylemans, Heather A; Gough, Raina V; Quintana, Roxanne L; Garrison, Michael D; Bruno, Thomas J

    2014-05-28

    Biosynthetic valencene, premnaspirodiene, and natural caryophyllene were hydrogenated and evaluated as high performance fuels. The parent sesquiterpenes were then isomerized to complex mixtures of hydrocarbons with the heterogeneous acid catalyst Nafion SAC-13. High density fuels with net heats of combustion ranging from 133-141 000 Btu gal(-1), or u