Science.gov

Sample records for legionaminic acid biosynthesis

  1. Biosynthesis of Legionaminic Acid and Its Incorporation Into Glycoconjugates.

    PubMed

    Schoenhofen, Ian C; Young, N Martin; Gilbert, Michel

    2017-01-01

    Legionaminic acids are analogs of sialic acid that occur in cell surface glycoconjugates of several bacteria. Because legionaminic acids share the same stereochemistry as sialic acid but differ at C7 and C9, they are interesting analogs to probe the impact of varying exocyclic moieties (C7-C9) on biological activities such as susceptibilities to sialidases, interactions with Siglecs and immunogenicity. There are currently no reports on the bacterial enzymes that transfer legionaminic acids to these cell surface glycoconjugates, but some mammalian and bacterial sialyltransferases display donor promiscuity and can use CMP-Leg5,7Ac2 efficiently enough to transfer Leg5,7Ac2 to their natural acceptor glycans. When the natural activity with CMP-Leg5,7Ac2 is significant but relatively low, an alternate strategy has been to engineer versions with improved activity to transfer Leg5,7Ac2. Importantly, we have found that some bacterial sialyltransferases are very efficient for transferring Leg5,7Ac2 to small synthetic glycans with various aglycones. The two mammalian sialyltransferases that have been tested so far (porcine ST3Gal1 and human ST6Gal1) were found to be more efficient than the bacterial sialyltransferases for the modification of glycoproteins. We provide a review of the sialyltransferases selected to modify different types of glycoconjugates with Leg5,7Ac2, including small synthetic acceptors, glycolipids, and glycoproteins. In the first part, we also propose an optimized biosynthetic pathway for in vitro preparation of the donor CMP-Leg5,7Ac2, based on enzymes selected from two bacteria that naturally produce legionaminic acid. © 2017 Elsevier Inc. All rights reserved.

  2. A pseudaminic acid or a legionaminic acid derivative transferase is strain-specifically implicated in the general protein O-glycosylation system of the periodontal pathogen Tannerella forsythia.

    PubMed

    Tomek, Markus B; Janesch, Bettina; Maresch, Daniel; Windwarder, Markus; Altmann, Friedrich; Messner, Paul; Schäffer, Christina

    2017-03-16

    The occurrence of nonulosonic acids in bacteria is wide-spread and linked to pathogenicity. However, the knowledge of cognate nonulosonic acid transferases is scarce. In the periodontopathogen Tannerella forsythia, several proposed virulence factors carry strain-specifically either a pseudaminic or a legionaminic acid derivative as terminal sugar on an otherwise structurally identical, protein-bound oligosaccharide. This study aims to shed light on the transfer of either nonulosonic acid derivative on a proximal N-acetylmannosaminuronic acid residue within the O-glycan structure, exemplified with the bacterium's abundant S-layer glycoproteins. Bioinformatic analyses provided the candidate genes Tanf_01245 (strain ATCC 43037) and TFUB4_00887 (strain UB4), encoding a putative pseudaminic and a legionaminic acid derivative transferase, respectively. These transferases have identical C-termini and contain motifs typical of glycosyltransferases (DXD) and bacterial sialyltransferases (D/E-D/E-G and HP). They share homology to type B glycosyltransferases and TagB, an enzyme catalyzing glycerol transfer to an N-acetylmannosamine residue in teichoic acid biosynthesis. Analysis of a cellular pool of nucleotide-activated sugars confirmed the presence of the CMP-activated nonulosonic acid derivatives, which are most likely serving as substrates for the corresponding transferase. Single gene knock-out mutants targeted at either transferase were analyzed for S-layer O-glycan composition by ESI-MS, confirming the loss of the nonulosonic acid derivative. Cross-complementation of the mutants with the nonnative nonulosonic acid transferase was not successful indicating high stringency of the enzymes. This study identified plausible candidates for a pseudaminic and a legionaminic acid derivative transferase; these may serve as valuable tools for engineering of novel sialoglycoconjugates.

  3. Diversity in prokaryotic glycosylation: an archaeal-derived N-linked glycan contains legionaminic acid.

    PubMed

    Kandiba, Lina; Aitio, Olli; Helin, Jari; Guan, Ziqiang; Permi, Perttu; Bamford, Dennis H; Eichler, Jerry; Roine, Elina

    2012-05-01

    VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV-1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by β-elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy. Such analyses showed that the major VP4-derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5-N-formyl-legionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid-like sugar, in an archaeal-derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with N-acetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC-ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4-derived peptides bearing the N-glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N-glycosylation apparatus, as determined by LC-ESI/MS of heterologously expressed VP4. Here, however, the N-linked pentasaccharide was the same as shown to decorate the S-layer glycoprotein in this species. Hence, N-glycosylation of the haloarchaeal viral protein, VP4, is host-specific. These results thus present additional examples of archaeal N-glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.

  4. Innovations in host and microbial sialic acid biosynthesis revealed by phylogenomic prediction of nonulosonic acid structure

    PubMed Central

    Lewis, Amanda L.; Desa, Nolan; Hansen, Elizabeth E.; Knirel, Yuriy A.; Gordon, Jeffrey I.; Gagneux, Pascal; Nizet, Victor; Varki, Ajit

    2009-01-01

    Sialic acids (Sias) are nonulosonic acid (NulO) sugars prominently displayed on vertebrate cells and occasionally mimicked by bacterial pathogens using homologous biosynthetic pathways. It has been suggested that Sias were an animal innovation and later emerged in pathogens by convergent evolution or horizontal gene transfer. To better illuminate the evolutionary processes underlying the phenomenon of Sia molecular mimicry, we performed phylogenomic analyses of biosynthetic pathways for Sias and related higher sugars derived from 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids. Examination of ≈1,000 sequenced microbial genomes indicated that such biosynthetic pathways are far more widely distributed than previously realized. Phylogenetic analysis, validated by targeted biochemistry, was used to predict NulO types (i.e., neuraminic, legionaminic, or pseudaminic acids) expressed by various organisms. This approach uncovered previously unreported occurrences of Sia pathways in pathogenic and symbiotic bacteria and identified at least one instance in which a human archaeal symbiont tentatively reported to express Sias in fact expressed the related pseudaminic acid structure. Evaluation of targeted phylogenies and protein domain organization revealed that the “unique” Sia biosynthetic pathway of animals was instead a much more ancient innovation. Pathway phylogenies suggest that bacterial pathogens may have acquired Sia expression via adaptation of pathways for legionaminic acid biosynthesis, one of at least 3 evolutionary paths for de novo Sia synthesis. Together, these data indicate that some of the long-standing paradigms in Sia biology should be reconsidered in a wider evolutionary context of the extended family of NulO sugars. PMID:19666579

  5. Analysis of putative nonulosonic acid biosynthesis pathways in Archaea reveals a complex evolutionary history.

    PubMed

    Kandiba, Lina; Eichler, Jerry

    2013-08-01

    Sialic acids and the other nonulosonic acid sugars, legionaminic acid and pseudaminic acid, are nine carbon-containing sugars that can be detected as components of the glycans decorating proteins and other molecules in Eukarya and Bacteria. Yet, despite the prevalence of N-glycosylation in Archaea and the variety of sugars recruited for the archaeal version of this post-translational modification, only a single report of a nonulosonic acid sugar in an archaeal N-linked glycan has appeared. Hence, to obtain a clearer picture of nonulosonic acid sugar biosynthesis capability in Archaea, 122 sequenced genomes were scanned for the presence of genes involved in the biogenesis of these sugars. The results reveal that while Archaea and Bacteria share a common route of sialic acid biosynthesis, numerous archaeal nonulosonic acid sugar biosynthesis pathway components were acquired from elsewhere via various routes. Still, the limited number of Archaea encoding components involved in the synthesis of nonulosonic acid sugars implies that such saccharides are not major components of glycans in this domain.

  6. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration.

  7. [Biosynthesis of adipic acid].

    PubMed

    Han, Li; Chen, Wujiu; Yuan, Fei; Zhang, Yuanyuan; Wang, Qinhong; Ma, Yanhe

    2013-10-01

    Adipic acid is a six-carbon dicarboxylic acid, mainly for the production of polymers such as nylon, chemical fiber and engineering plastics. Its annual demand is close to 3 million tons worldwide. Currently, the industrial production of adipic acid is based on the oxidation of aromatics from non-renewable petroleum resources by chemo-catalytic processes. It is heavily polluted and unsustainable, and the possible alternative method for adipic acid production should be developed. In the past years, with the development of synthetic biology and metabolic engineering, green and clean biotechnological methods for adipic acid production attracted more attention. In this study, the research advances of adipic acid and its precursor production are reviewed, followed by addressing the perspective of the possible new pathways for adipic acid production.

  8. Fatty acid biosynthesis in actinomycetes

    PubMed Central

    Gago, Gabriela; Diacovich, Lautaro; Arabolaza, Ana; Tsai, Shiou-Chuan; Gramajo, Hugo

    2011-01-01

    All organisms that produce fatty acids do so via a repeated cycle of reactions. In mammals and other animals, these reactions are catalyzed by a type I fatty acid synthase (FAS), a large multifunctional protein to which the growing chain is covalently attached. In contrast, most bacteria (and plants) contain a type II system in which each reaction is catalyzed by a discrete protein. The pathway of fatty acid biosynthesis in Escherichia coli is well established and has provided a foundation for elucidating the type II FAS pathways in other bacteria (White et al., 2005). However, fatty acid biosynthesis is more diverse in the phylum Actinobacteria: Mycobacterium, possess both FAS systems while Streptomyces species have only the multi-enzyme FAS II system and Corynebacterium species exclusively FAS I. In this review we present an overview of the genome organization, biochemical properties and physiological relevance of the two FAS systems in the three genera of actinomycetes mentioned above. We also address in detail the biochemical and structural properties of the acyl-CoA carboxylases (ACCases) that catalyzes the first committed step of fatty acid synthesis in actinomycetes, and discuss the molecular bases of their substrate specificity and the structure-based identification of new ACCase inhibitors with anti-mycobacterial properties. PMID:21204864

  9. Abscisic acid biosynthesis and catabolism.

    PubMed

    Nambara, Eiji; Marion-Poll, Annie

    2005-01-01

    The level of abscisic acid (ABA) in any particular tissue in a plant is determined by the rate of biosynthesis and catabolism of the hormone. Therefore, identifying all the genes involved in the metabolism is essential for a complete understanding of how this hormone directs plant growth and development. To date, almost all the biosynthetic genes have been identified through the isolation of auxotrophic mutants. On the other hand, among several ABA catabolic pathways, current genomic approaches revealed that Arabidopsis CYP707A genes encode ABA 8'-hydroxylases, which catalyze the first committed step in the predominant ABA catabolic pathway. Identification of ABA metabolic genes has revealed that multiple metabolic steps are differentially regulated to fine-tune the ABA level at both transcriptional and post-transcriptional levels. Furthermore, recent ongoing studies have given new insights into the regulation and site of ABA metabolism in relation to its physiological roles.

  10. Antibacterial Targets in Fatty Acid Biosynthesis

    PubMed Central

    Wright, H. Tonie; Reynolds, Kevin A.

    2008-01-01

    Summary The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for development of new anti-bacterial agents. The extended use of the anti-tuberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for anti-bacterial development. Differences in subcellular organization of the bacterial and eukaryotic multi-enzyme fatty acid synthase systems offer the prospect of inhibitors with host vs. target specificity. Platensimycin, platencin, and phomallenic acids, newly discovered natural product inhibitors of the condensation steps in fatty acid biosynthesis, represent new classes of compounds with antibiotic potential. An almost complete catalogue of crystal structures for the enzymes of the type II fatty acid biosynthesis pathway can now be exploited in the rational design of new inhibitors, as well as the recently published crystal structures of type I FAS complexes. PMID:17707686

  11. Antibacterial targets in fatty acid biosynthesis.

    PubMed

    Wright, H Tonie; Reynolds, Kevin A

    2007-10-01

    The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for the development of new antibacterial agents. The extended use of the antituberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for antibacterial development. Differences in subcellular organization of the bacterial and eukaryotic multienzyme fatty acid synthase systems offer the prospect of inhibitors with host versus target specificity. Platensimycin, platencin, and phomallenic acids, newly discovered natural product inhibitors of the condensation steps in fatty acid biosynthesis, represent new classes of compounds with antibiotic potential. An almost complete catalog of crystal structures for the enzymes of the type II fatty acid biosynthesis pathway can now be exploited in the rational design of new inhibitors, as well as the recently published crystal structures of type I FAS complexes.

  12. Salicylic Acid Biosynthesis and Metabolism

    PubMed Central

    Dempsey, D'Maris Amick; Vlot, A. Corina; Wildermuth, Mary C.; Klessig, Daniel F.

    2011-01-01

    Salicylic acid (SA) has been shown to regulate various aspects of growth and development; it also serves as a critical signal for activating disease resistance in Arabidopsis thaliana and other plant species. This review surveys the mechanisms involved in the biosynthesis and metabolism of this critical plant hormone. While a complete biosynthetic route has yet to be established, stressed Arabidopsis appear to synthesize SA primarily via an isochorismate-utilizing pathway in the chloroplast. A distinct pathway utilizing phenylalanine as the substrate also may contribute to SA accumulation, although to a much lesser extent. Once synthesized, free SA levels can be regulated by a variety of chemical modifications. Many of these modifications inactivate SA; however, some confer novel properties that may aid in long distance SA transport or the activation of stress responses complementary to those induced by free SA. In addition, a number of factors that directly or indirectly regulate the expression of SA biosynthetic genes or that influence the rate of SA catabolism have been identified. An integrated model, encompassing current knowledge of SA metabolism in Arabidopsis, as well as the influence other plant hormones exert on SA metabolism, is presented. PMID:22303280

  13. Biosynthesis and metabolism of salicylic acid

    SciTech Connect

    Lee, H.; Leon, J.; Raskin, I.

    1995-05-09

    Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-{beta}-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance. 62 refs., 1 fig.

  14. Biosynthesis and metabolism of salicylic acid.

    PubMed Central

    Lee, H I; León, J; Raskin, I

    1995-01-01

    Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-beta-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance. PMID:11607533

  15. Phenol biosynthesis in higher plants. Gallic acid

    PubMed Central

    Dewick, P. M.; Haslam, E.

    1969-01-01

    The biosynthesis of gallic acid in a number of higher plants was investigated by using l-[U-14C]phenylalanine, (−)-[G-14C]shikimic acid, d-[1-14C]glucose and d-[6-14C]glucose as tracers. The results are compared with those obtained similarly for caffeic acid and are interpreted in terms of the dehydrogenation of 5-dehydroshikimic acid as a normal route of metabolism for gallic acid. PMID:5807212

  16. Cyclopiazonic acid biosynthesis by Aspergillus flavus

    USDA-ARS?s Scientific Manuscript database

    Cyclopiazonic acid (CPA) is an indole-tetramic acid mycotoxin produced by some strains of Aspergillus flavus. Characterization of the CPA biosynthesis gene cluster confirmed that formation of CPA is via a three-enzyme pathway. This review examines the structure and organization of the CPA genes, elu...

  17. Pantothenic acid biosynthesis in zymomonas

    DOEpatents

    Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V.

    2014-07-01

    Zymomonas is unable to synthesize pantothenic acid and requires this essential vitamin in growth medium. Zymomonas strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pantothenic acid supplementation in seed culture and fermentation media.

  18. Amino Acid Biosynthesis Pathways in Diatoms

    PubMed Central

    Bromke, Mariusz A.

    2013-01-01

    Amino acids are not only building blocks for proteins but serve as precursors for the synthesis of many metabolites with multiple functions in growth and other biological processes of a living organism. The biosynthesis of amino acids is tightly connected with central carbon, nitrogen and sulfur metabolism. Recent publication of genome sequences for two diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum created an opportunity for extensive studies on the structure of these metabolic pathways. Based on sequence homology found in the analyzed diatomal genes, the biosynthesis of amino acids in diatoms seems to be similar to higher plants. However, one of the most striking differences between the pathways in plants and in diatomas is that the latter possess and utilize the urea cycle. It serves as an important anaplerotic pathway for carbon fixation into amino acids and other N-containing compounds, which are essential for diatom growth and contribute to their high productivity. PMID:24957993

  19. Abscisic acid: biosynthesis, inactivation, homoeostasis and signalling.

    PubMed

    Dong, Ting; Park, Youngmin; Hwang, Inhwan

    2015-01-01

    The phytohormone abscisic acid (ABA) plays crucial roles in numerous physiological processes during plant growth and abiotic stress responses. The endogenous ABA level is controlled by complex regulatory mechanisms involving biosynthesis, catabolism, transport and signal transduction pathways. This complex regulatory network may target multiple levels, including transcription, translation and post-translational regulation of genes involved in ABA responses. Most of the genes involved in ABA biosynthesis, catabolism and transport have been characterized. The local ABA concentration is critical for initiating ABA-mediated signalling during plant development and in response to environmental changes. In this chapter we discuss the mechanisms that regulate ABA biosynthesis, catabolism, transport and homoeostasis. We also present the findings of recent research on ABA perception by cellular receptors, and ABA signalling in response to cellular and environmental conditions. © 2015 Authors; published by Portland Press Limited.

  20. Teichoic acid biosynthesis as an antibiotic target.

    PubMed

    Pasquina, Lincoln W; Santa Maria, John P; Walker, Suzanne

    2013-10-01

    The relentless spread of antibiotic-resistant pathogens makes it imperative to develop new chemotherapeutic strategies to overcome infection. The bacterial cell wall has served as a rich source for both validated and unexploited pathways that are essential for virulence and survival. Lipoteichoic acids (LTAs) and wall teichoic acids (WTAs) are cell wall polymers that play fundamental roles in Gram-positive bacterial physiology and pathogenesis, and both have been proposed as novel antibacterial targets. Here we describe recent progress toward the discovery of teichoic acid biosynthesis inhibitors and their potential as antibiotics to combat Staphylococcus aureus infections.

  1. Enzymology of retinoic acid biosynthesis and degradation

    PubMed Central

    Kedishvili, Natalia Y.

    2013-01-01

    All-trans-retinoic acid is a biologically active derivative of vitamin A that regulates numerous physiological processes. The concentration of retinoic acid in the cells is tightly regulated, but the exact mechanisms responsible for this regulation are not completely understood, largely because the enzymes involved in the biosynthesis of retinoic acid have not been fully defined. Recent studies using in vitro and in vivo models suggest that several members of the short-chain dehydrogenase/reductase superfamily of proteins are essential for retinoic acid biosynthesis and the maintenance of retinoic acid homeostasis. However, the exact roles of some of these recently identified enzymes are yet to be characterized. The properties of the known contributors to retinoid metabolism have now been better defined and allow for more detailed understanding of their interactions with retinoid-binding proteins and other retinoid enzymes. At the same time, further studies are needed to clarify the interactions between the cytoplasmic and membrane-bound proteins involved in the processing of hydrophobic retinoid metabolites. This review summarizes current knowledge about the roles of various biosynthetic and catabolic enzymes in the regulation of retinoic acid homeostasis and outlines the remaining questions in the field. PMID:23630397

  2. Fatty acid biosynthesis in pea root plastids

    SciTech Connect

    Stahl, R.J.; Sparace, S.A. )

    1989-04-01

    Fatty acid biosynthesis from (1-{sup 14}C)acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at approximately 80 nmoles/hr/mg protein in the presence of 200 {mu}M acetate, 0.5 mM each of NADH, NADPH and CoA, 6 mM each of ATP and MgCl{sub 2}, 1 mM each of the MnCl{sub 2} and glycerol-3-phosphate, 15 mM KHCO{sub 3}, and 0.1M Bis-tris-propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear from up to 6 hours with 80 to 100 {mu}g/mL plastid protein. ATP and CoA were absolute requirements, whereas KHCO{sub 3}, divalent cations and reduced nucleotides all improved activity by 80 to 85%. Mg{sup 2+} and NADH were the preferred cation and nucleotide, respectively. Dithiothreitol and detergents were generally inhibitory. The radioactive products of fatty acid biosynthesis were approximately 33% 16:0, 10% 18:0 and 56% 18:1 and generally did not vary with increasing concentrations of each cofactor.

  3. Cyclopiazonic acid biosynthesis gene cluster gene cpaM is required for speradine A biosynthesis.

    PubMed

    Tokuoka, Masafumi; Kikuchi, Tomoki; Shinohara, Yasutomo; Koyama, Akifumi; Iio, Shin-Ichiro; Kubota, Takaaki; Kobayashi, Jun'ichi; Koyama, Yasuji; Totsuka, Akira; Shindo, Hitoshi; Sato, Kazuo

    2015-01-01

    Speradine A is a derivative of cyclopiazonic acid (CPA) found in culture of an Aspergillus tamarii isolate. Heterologous expression of a predicted methyltransferase gene, cpaM, in the cpa biosynthesis gene cluster of A. tamarii resulted in the speradine A production in a 2-oxoCPA producing A. oryzae strain, indicating cpaM is involved in the speradine A biosynthesis.

  4. Retinoic acid: its biosynthesis and metabolism.

    PubMed

    Napoli, J L

    1999-01-01

    This article presents a model that integrates the functions of retinoid-binding proteins with retinoid metabolism. One of these proteins, the widely expressed (throughout retinoid target tissues and in all vertebrates) and highly conserved cellular retinol-binding protein (CRBP), sequesters retinol in an internal binding pocket that segregates it from the intracellular milieu. The CRBP-retinol complex appears to be the quantitatively major form of retinol in vivo, and may protect the promiscuous substrate from nonenzymatic degradation and/or non-specific enzymes. For example, at least seven types of dehydrogenases catalyze retinal synthesis from unbound retinol in vitro (NAD+ vs. NADP+ dependent, cytosolic vs. microsomal, short-chain dehydrogenases/reductases vs. medium-chain alcohol dehydrogenases). But only a fraction of these (some of the short-chain de-hydrogenases/reductases) have the fascinating additional ability of catalyzing retinal synthesis from CRBP-bound retinol as well. Similarly, CRBP and/or other retinoid-binding proteins function in the synthesis of retinal esters, the reduction of retinal generated from intestinal beta-carotene metabolism, and retinoic acid metabolism. The discussion details the evidence supporting an integrated model of retinoid-binding protein/metabolism. Also addressed are retinoid-androgen interactions and evidence incompatible with ethanol causing fetal alcohol syndrome by competing directly with retinol dehydrogenation to impair retinoic acid biosynthesis.

  5. Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae

    USDA-ARS?s Scientific Manuscript database

    Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by some of the same strains of A. flavus that produce aflatoxins and by some Aspergillus oryzae strains. Despite its discovery 40 years ago, few reviews of its toxicity and biosynthesis have been reported. This review examines w...

  6. Biosynthesis of resorcylic acid lactone lasiodiplodin in Lasiodiplodia theobromae.

    PubMed

    Kashima, Takasumi; Takahashi, Kosaku; Matsuura, Hideyuki; Nabeta, Kensuke

    2009-05-01

    The biosynthesis of lasiodiplodin (1) and its (5S)-5-hydroxylated derivative (2) were investigated by the administration of (13)C-labeled acetates to Lasiodiplodia theobromae. The labeling patterns of biosynthetically (13)C-labeled 1 and 2 were determined by (13)C-NMR and INADEQUATE spectra, demonstrating the octaketide origins of 1 and 2. Taking into account the biosynthetic study of resorcylic acid lactones, the involvement of highly reduced acyl intermediates in the biosynthesis of lasiodiplodins was presumed; thus, we synthesized (2)H-labeled hypothetical acyl intermediates of 1, 9-hydroxydecanoic acid (4) and its N-acetylcysteamine thioester (SNAC, 5). When L. theobromae was incubated with 5 mM of a (2)H-labeled intermediate, the (2)H-label from the intermediate was incorporated at the expected position of 1. These incorporation studies revealed that 1 was produced via a pathway which closely resembles that of resorcylic acid lactone biosynthesis.

  7. Inhibition of Abscisic Acid Biosynthesis in Cercospora rosicola by Inhibitors of Gibberellin Biosynthesis and Plant Growth Retardants

    PubMed Central

    Norman, Shirley M.; Poling, Stephen M.; Maier, Vincent P.; Orme, Edward D.

    1983-01-01

    The fungus Cercospora rosicola produces abscisic acid (ABA) as a secondary metabolite. We developed a convenient system using this fungus to determine the effects of compounds on the biosynthesis of ABA. Inasmuch as ABA and the gibberellins (GAs) both arise via the isoprenoid pathway, it was of interest to determine if inhibitors of GA biosynthesis affect ABA biosynthesis. All five putative inhibitors of GA biosynthesis tested inhibited ABA biosynthesis. Several plant growth retardants with poorly understood actions in plants were also tested; of these, six inhibited ABA biosynthesis to varying degrees and two had no effect. Effects of plant growth retardants on various branches of the isoprenoid biosynthetic pathway may help to explain some of the diverse and unexpected results reported for these compounds. Knowledge that certain inhibitors of GA biosynthesis also have the ability to inhibit ABA biosynthesis in C. rosicola indicates the need for further studies in plants on the mode of action of these compounds. PMID:16662775

  8. Aspartate-Derived Amino Acid Biosynthesis in Arabidopsis thaliana.

    PubMed

    Jander, Georg; Joshi, Vijay

    2009-01-01

    The aspartate-derived amino acid pathway in plants leads to the biosynthesis of lysine, methionine, threonine, and isoleucine. These four amino acids are essential in the diets of humans and other animals, but are present in growth-limiting quantities in some of the world's major food crops. Genetic and biochemical approaches have been used for the functional analysis of almost all Arabidopsis thaliana enzymes involved in aspartate-derived amino acid biosynthesis. The branch-point enzymes aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase, cystathionine gamma synthase, threonine synthase, and threonine deaminase contain well-studied sites for allosteric regulation by pathway products and other plant metabolites. In contrast, relatively little is known about the transcriptional regulation of amino acid biosynthesis and the mechanisms that are used to balance aspartate-derived amino acid biosynthesis with other plant metabolic needs. The aspartate-derived amino acid pathway provides excellent examples of basic research conducted with A. thaliana that has been used to improve the nutritional quality of crop plants, in particular to increase the accumulation of lysine in maize and methionine in potatoes.

  9. Engineering fatty acid biosynthesis in microalgae for sustainable biodiesel.

    PubMed

    Blatti, Jillian L; Michaud, Jennifer; Burkart, Michael D

    2013-06-01

    Microalgae are a promising feedstock for biodiesel and other liquid fuels due to their fast growth rate, high lipid yields, and ability to grow in a broad range of environments. However, many microalgae achieve maximal lipid yields only under stress conditions hindering growth and providing compositions not ideal for biofuel applications. Metabolic engineering of algal fatty acid biosynthesis promises to create strains capable of economically producing fungible and sustainable biofuels. The algal fatty acid biosynthetic pathway has been deduced by homology to bacterial and plant systems, and much of our understanding is gleaned from basic studies in these systems. However, successful engineering of lipid metabolism in algae will necessitate a thorough characterization of the algal fatty acid synthase (FAS) including protein-protein interactions and regulation. This review describes recent efforts to engineer fatty acid biosynthesis toward optimizing microalgae as a biodiesel feedstock.

  10. Protein biosynthesis with conformationally restricted amino acids

    SciTech Connect

    Mendel, D. Lawrence Berkeley Lab., CA ); Ellman, J.; Schultz, P.G. )

    1993-05-19

    The incorporation of conformationally constrained amino acids into peptides is a powerful approach for generating structurally defined peptides as conformational probes and bioactive agents. The ability to site-specifically introduce constrained amino acids into large polypeptide chains would provide a similar opportunity to probe the flexibility, conformation, folding and stability of proteins. To this end, we have examined the competence of the Escherichia coli protein biosynthetic machinery to incorporate a number of these unnatural amino acids into the 164 residue protein T4 lysozyme (T4L). Results clearly demonstrate that the protein biosynthetic machinery can accommodate a wide variety of conformationally constrained amino acids. The expansion of structural motifs that can be biosynthetically incorporated into proteins to include a large number of conformationally constrained amino acids significantly increases the power of mutagenesis methods as probes of protein structure and function and provides additional insights into the steric requirements of the translational machinery. 13 refs., 2 figs.

  11. Regulation of collagen biosynthesis by ascorbic acid: a review.

    PubMed Central

    Pinnell, S. R.

    1985-01-01

    L-ascorbic acid is an essential cofactor for lysyl hydroxylase and prolyl hydroxylase, enzymes essential for collagen biosynthesis. In addition, L-ascorbic acid preferentially stimulates collagen synthesis in a manner which appears unrelated to the effect of L-ascorbic acid on hydroxylation reactions. This reaction is stereospecific and unrelated to intracellular degradation of collagen. The effect apparently occurs at a transcriptional or translational level, since L-ascorbic acid preferentially stimulates collagen-specific mRNA. In addition, it stimulates lysyl hydroxylase activity but inhibits prolyl hydroxylase activity in human skin fibroblasts in culture. PMID:3008449

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

    PubMed Central

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

    2016-01-01

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

  13. Amino Acid Biosynthesis in the Halophilic Archaeon Haloarcula hispanica

    PubMed Central

    Hochuli, Michel; Patzelt, Heiko; Oesterhelt, Dieter; Wüthrich, Kurt; Szyperski, Thomas

    1999-01-01

    Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya. PMID:10322026

  14. Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

    PubMed

    Long, Michael C; Nagegowda, Dinesh A; Kaminaga, Yasuhisa; Ho, Kwok Ki; Kish, Christine M; Schnepp, Jennifer; Sherman, Debra; Weiner, Henry; Rhodes, David; Dudareva, Natalia

    2009-07-01

    Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

  15. Abscisic acid modulates salicylic acid biosynthesis for systemic acquired resistance in tomato.

    PubMed

    Kusajima, Miyuki; Okumura, Yasuko; Fujita, Moeka; Nakashita, Hideo

    2017-09-01

    Among the regulatory mechanisms of systemic acquired resistance (SAR) in tomato, antagonistic interaction between salicylic acid (SA) and abscisic acid (ABA) signaling pathways was investigated. Treatment with 1,2-benzisothiazol-3(2H)-one1,1-dioxide (BIT) induced SAR in tomato thorough SA biosynthesis. Pretreatment of ABA suppressed BIT-induced SAR including SA accumulation, suggesting that ABA suppressed SAR by inhibiting SA biosynthesis.

  16. GENETIC ANALYSIS OF ABSCISIC ACID BIOSYNTHESIS

    SciTech Connect

    MCCARTY D R

    2012-01-10

    The carotenoid cleavage dioxygenases (CCD) catalyze synthesis of a variety of apo-carotenoid secondary metabolites in plants, animals and bacteria. In plants, the reaction catalyzed by the 11, 12, 9-cis-epoxy carotenoid dioxygenase (NCED) is the first committed and key regulated step in synthesis of the plant hormone, abscisic acid (ABA). ABA is a key regulator of plant stress responses and has critical functions in normal root and seed development. The molecular mechanisms responsible for developmental control of ABA synthesis in plant tissues are poorly understood. Five of the nine CCD genes present in the Arabidopsis genome encode NCED's involved in control of ABA synthesis in the plant. This project is focused on functional analysis of these five AtNCED genes as a key to understanding developmental regulation of ABA synthesis and dissecting the role of ABA in plant development. For this purpose, the project developed a comprehensive set of gene knockouts in the AtNCED genes that facilitate genetic dissection of ABA synthesis. These mutants were used in combination with key molecular tools to address the following specific objectives: (1) the role of ABA synthesis in root development; (2) developmental control of ABA synthesis in seeds; (3) analysis of ATNCED over-expressers; (4) preliminary crystallography of the maize VP14 protein.

  17. Curcumin improves alcoholic fatty liver by inhibiting fatty acid biosynthesis.

    PubMed

    Guo, Chang; Ma, Jingfan; Zhong, Qionghong; Zhao, Mengyuan; Hu, Tianxing; Chen, Tong; Qiu, Longxin; Wen, Longping

    2017-08-01

    Alcoholic fatty liver is a threat to human health. It has been long known that abstinence from alcohol is the most effective therapy, other effective therapies are not available for the treatment in humans. Curcumin has a great potential for anti-oxidation and anti-inflammation, but the effect on metabolic reconstruction remains little known. Here we performed metabolomic analysis by gas chromatography/mass spectrometry and explored ethanol pathogenic insight as well as curcumin action pattern. We identified seventy-one metabolites in mouse liver. Carbohydrates and lipids were characteristic categories. Pathway analysis results revealed that ethanol-induced pathways including biosynthesis of unsaturated fatty acids, fatty acid biosynthesis and pentose and glucuronate interconversions were suppressed by curcumin. Additionally, ethanol enhanced galactose metabolism and pentose phosphate pathway. Glyoxylate and dicarboxylate metabolism and pyruvate metabolism were inhibited in mice fed ethanol diet plus curcumin. Stearic acid, oleic acid and linoleic acid were disease biomarkers and therapical biomarkers. These results reflect the landscape of hepatic metabolism regulation. Our findings illustrate ethanol pathological pathway and metabolic mechanism of curcumin therapy. Copyright © 2017. Published by Elsevier Inc.

  18. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    SciTech Connect

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  19. Plant amino acid-derived vitamins: biosynthesis and function.

    PubMed

    Miret, Javier A; Munné-Bosch, Sergi

    2014-04-01

    Vitamins are essential organic compounds for humans, having lost the ability to de novo synthesize them. Hence, they represent dietary requirements, which are covered by plants as the main dietary source of most vitamins (through food or livestock's feed). Most vitamins synthesized by plants present amino acids as precursors (B1, B2, B3, B5, B7, B9 and E) and are therefore linked to plant nitrogen metabolism. Amino acids play different roles in their biosynthesis and metabolism, either incorporated into the backbone of the vitamin or as amino, sulfur or one-carbon group donors. There is a high natural variation in vitamin contents in crops and its exploitation through breeding, metabolic engineering and agronomic practices can enhance their nutritional quality. While the underlying biochemical roles of vitamins as cosubstrates or cofactors are usually common for most eukaryotes, the impact of vitamins B and E in metabolism and physiology can be quite different on plants and animals. Here, we first aim at giving an overview of the biosynthesis of amino acid-derived vitamins in plants, with a particular focus on how this knowledge can be exploited to increase vitamin contents in crops. Second, we will focus on the functions of these vitamins in both plants and animals (and humans in particular), to unravel common and specific roles for vitamins in evolutionary distant organisms, in which these amino acid-derived vitamins play, however, an essential role.

  20. Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum.

    PubMed

    Regueira, Torsten Bak; Kildegaard, Kanchana Rueksomtawin; Hansen, Bjarne Gram; Mortensen, Uffe H; Hertweck, Christian; Nielsen, Jens

    2011-05-01

    Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering.

  1. Lipoteichoic acid biosynthesis: two steps forwards, one step sideways?

    PubMed

    Rahman, Obaidur; Dover, Lynn G; Sutcliffe, Iain C

    2009-06-01

    Lipoteichoic acids (LTAs) are membrane-anchored molecules in the cell envelopes of Gram-positive bacteria. Until recently, they were considered to be restricted to the Firmicutes, which include important pathogens such as Staphylococcus aureus and Streptococcus pneumoniae. Polyanionic LTAs have fundamentally important roles in divalent cation retention within the Gram-positive cell envelope and thereby influence bacterial cell division. Thus, LTA biosynthesis provides an attractive target for the development of novel antimicrobial interventions. Recent studies, notably two investigations of S. aureus and another of Bacillus subtilis, have greatly improved our understanding of the genetic basis of LTA biosynthesis. In addition, reports have revealed that at least some members of the Actinobacteria (another phylum of Gram-positive bacteria) produce LTAs, rather than the lipoglycans previously assumed to be typical of this taxon. The availability of whole bacterial genome sequences has enabled us to perform comparative analyses to shed light on the distribution of putative LTA biosynthetic genes among bacteria. Here, we discuss the results of these genomic analyses, together with the current literature, and propose that LTA biosynthesis in Actinobacteria might be fundamentally different to that in most Firmicutes.

  2. Kinetic Modeling of Sunflower Grain Filling and Fatty Acid Biosynthesis.

    PubMed

    Durruty, Ignacio; Aguirrezábal, Luis A N; Echarte, María M

    2016-01-01

    Grain growth and oil biosynthesis are complex processes that involve various enzymes placed in different sub-cellular compartments of the grain. In order to understand the mechanisms controlling grain weight and composition, we need mathematical models capable of simulating the dynamic behavior of the main components of the grain during the grain filling stage. In this paper, we present a non-structured mechanistic kinetic model developed for sunflower grains. The model was first calibrated for sunflower hybrid ACA855. The calibrated model was able to predict the theoretical amount of carbohydrate equivalents allocated to the grain, grain growth and the dynamics of the oil and non-oil fraction, while considering maintenance requirements and leaf senescence. Incorporating into the model the serial-parallel nature of fatty acid biosynthesis permitted a good representation of the kinetics of palmitic, stearic, oleic, and linoleic acids production. A sensitivity analysis showed that the relative influence of input parameters changed along grain development. Grain growth was mostly affected by the specific growth parameter (μ') while fatty acid composition strongly depended on their own maximum specific rate parameters. The model was successfully applied to two additional hybrids (MG2 and DK3820). The proposed model can be the first building block toward the development of a more sophisticated model, capable of predicting the effects of environmental conditions on grain weight and composition, in a comprehensive and quantitative way.

  3. Kinetic Modeling of Sunflower Grain Filling and Fatty Acid Biosynthesis

    PubMed Central

    Durruty, Ignacio; Aguirrezábal, Luis A. N.; Echarte, María M.

    2016-01-01

    Grain growth and oil biosynthesis are complex processes that involve various enzymes placed in different sub-cellular compartments of the grain. In order to understand the mechanisms controlling grain weight and composition, we need mathematical models capable of simulating the dynamic behavior of the main components of the grain during the grain filling stage. In this paper, we present a non-structured mechanistic kinetic model developed for sunflower grains. The model was first calibrated for sunflower hybrid ACA855. The calibrated model was able to predict the theoretical amount of carbohydrate equivalents allocated to the grain, grain growth and the dynamics of the oil and non-oil fraction, while considering maintenance requirements and leaf senescence. Incorporating into the model the serial-parallel nature of fatty acid biosynthesis permitted a good representation of the kinetics of palmitic, stearic, oleic, and linoleic acids production. A sensitivity analysis showed that the relative influence of input parameters changed along grain development. Grain growth was mostly affected by the specific growth parameter (μ′) while fatty acid composition strongly depended on their own maximum specific rate parameters. The model was successfully applied to two additional hybrids (MG2 and DK3820). The proposed model can be the first building block toward the development of a more sophisticated model, capable of predicting the effects of environmental conditions on grain weight and composition, in a comprehensive and quantitative way. PMID:27242809

  4. Biosynthesis of myristic acid in luminescent bacteria. [Vibrio harveyi

    SciTech Connect

    Byers, D.M.

    1987-05-01

    In vivo pulse-label studies have demonstrated that luminescent bacteria can provide myritic acid (14:0) required for the synthesis of the luciferase substrate myristyl aldehyde. Luminescent wild type Vibrio harveyi incubated with (/sup 14/C) acetate in a nutrient-depleted medium accumulated substantial tree (/sup 14/C)fatty acid (up to 20% of the total lipid label). Radio-gas chromatography revealed that > 75% of the labeled fatty acid is 14:0. No free fatty acid was detected in wild type cells labeled prior to the development of bioluminescence in the exponential growth phase, or in a dark mutant of V. harveyi (mutant M17) that requires exogenous 14:0 for light emission. The preferential accumulation of 14:0 was not observed when wild type cells were labeled with (/sup 14/C)acetate in regular growth medium. Moreover, all V. harveyi strains exhibited similar fatty acid mass compositions regardless of the state of bioluminescence. Since earlier work has shown that a luminescence-related acyltransferase (defective in the M17 mutant) can catalyze the deacylation of fatty acyl-acyl carrier protein in vitro, the present results are consistent with a model in which this enzyme diverts 14:0 to the luminescence system during fatty acid biosynthesis. Under normal conditions, the supply of 14:0 by this pathway is tightly regulated such that bioluminescence development does not significantly alter the total fatty acid composition.

  5. Fatty acid biosynthesis as a drug target in apicomplexan parasites.

    PubMed

    Goodman, C D; McFadden, G I

    2007-01-01

    Apicomplexan parasitic diseases impose devastating impacts on much of the world's population. The increasing prevalence of drug resistant parasites and the growing number of immuno-compromised individuals are exacerbating the problem to the point that the need for novel, inexpensive drugs is greater now than ever. Discovery of a prokaryotic, Type II fatty acid synthesis (FAS) pathway associated with the plastid-like organelle (apicoplast) of Plasmodium and Toxoplasma has provided a wealth of novel drug targets. Since this pathway is both essential and fundamentally different from the cytosolic Type I pathway of the human host, apicoplast FAS has tremendous potential for the development of parasite-specific inhibitors. Many components of this pathway are already the target for existing antibiotics and herbicides, which should significantly reduce the time and cost of drug development. Continuing interest--both in the pharmaceutical and herbicide industries--in fatty acid synthesis inhibitors proffers an ongoing stream of potential new anti-parasitic compounds. It has now emerged that not all apicomplexan parasites have retained the Type II fatty acid biosynthesis pathway. No fatty acid biosynthesis enzymes are encoded in the genome of Theileria annulata or T. parva, suggesting that fatty acid synthesis is lacking in these parasites. The human intestinal parasite Cryptosporidium parvum appears to have lost the apicoplast entirely; instead relying on an unusual cytosolic Type I FAS. Nevertheless, newly developed anti-cancer and anti-obesity drugs targeting human Type I FAS may yet prove efficacious against Cryptosporidium and other apicomplexans that rely on this Type I FAS pathway.

  6. Effect of low temperature on highly unsaturated fatty acid biosynthesis in activated sludge.

    PubMed

    He, Su; Ding, Li-Li; Xu, Ke; Geng, Jin-Ju; Ren, Hong-Qiang

    2016-07-01

    Low temperature is a limiting factor for the microbial activity of activated sludge for sewage treatment plant in winter. Highly unsaturated fatty acid (UFA) biosynthesis, phospholipid fatty acid (PLFA) constituents and microbial structure in activated sludge at low temperature were investigated. Over 12 gigabases of metagenomic sequence data were generated with the Illumina HiSeq 2000 platform. The result showed 43.11% of phospholipid fatty acid (PLFA) in the activated sludge participated in UFA biosynthesis, and γ-Linolenic could be converted to Arachidonic acid at low temperature. The highly UFA biosynthesis in activated sludge was n-6 highly UFA biosynthesis, rather than n-3 highly UFA biosynthesis. The microbial community structures of activated sludge were analyzed by PLFA and high-throughput sequencing (HiSeq) simultaneously. Acidovorax, Pseudomonas, Flavobacterium and Polaromonas occupied higher percentage at 5°C, and genetic changes of highly UFA biosynthesis derived from microbial community structures change.

  7. Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium.

    PubMed

    Creelman, R A; Gage, D A; Stults, J T; Zeevaart, J A

    1987-11-01

    RESEARCH ON THE BIOSYNTHESIS OF ABSCISIC ACID (ABA) HAS FOCUSED PRIMARILY ON TWO PATHWAYS: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in (18)O(2). It was found that in stressed leaves three atoms of (18)O from (18)O(2) are incorporated into the ABA molecule, and that the amount of (18)O incorporated increases with time. One (18)O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in (18)O(2) shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more (18)O into the tertiary hydroxyl group at C-1' after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, (18)O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied (14)C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional (18)O incorporated during 8'-hydroxylation of ABA to phaseic acid.

  8. Carnosic acid biosynthesis elucidated by a synthetic biology platform

    PubMed Central

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

    2016-01-01

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

  9. Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA) from renewable carbon

    PubMed Central

    2010-01-01

    Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA) as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert-butyl ether (MTBE) in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB). This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source. PMID:20184738

  10. Biosynthesis of Ascorbic Acid in Legume Root Nodules1

    PubMed Central

    Matamoros, Manuel A.; Loscos, Jorge; Coronado, Maria J.; Ramos, Javier; Sato, Shusei; Testillano, Pilar S.; Tabata, Satoshi; Becana, Manuel

    2006-01-01

    Ascorbic acid (vitamin C) is a major antioxidant and redox buffer, but is also involved in other critical processes of plants. Recently, the hypothesis has been proposed that legume nodules are unable to synthesize ascorbate and have to import it from the shoot or root, thus providing a means by which the plant regulates nodule senescence. The last step of ascorbate biosynthesis in plants is catalyzed by l-galactono-1,4-lactone dehydrogenase (GalLDH). The mRNAs encoding GalLDH and three other enzymes involved in ascorbate biosynthesis are clearly detectable in nodules. Furthermore, an active membrane-bound GalLDH enzyme is present in nodule mitochondria. Biochemical assays on dissected nodules reveal that GalLDH activity and ascorbate are correlated in nodule tissues and predominantly localized in the infected zone, with lower levels of both parameters (relative to the infected tissues) in the apex (87%) and senescent region (43%) of indeterminate nodules and in the peripheral tissues (65%) of determinate nodules. In situ RNA hybridization showed that the GalLDH mRNA is particularly abundant in the infected zone of indeterminate and determinate nodules. Thus, our results refute the hypothesis that ascorbate is not synthesized in nodules and lend support to a previous conclusion that ascorbate in the infected zone is primarily involved in the protection of host cells against peroxide damage. Likewise, the high ascorbate and GalLDH activity levels found in the apex of indeterminate nodules strongly suggest a participation of ascorbate in additional functions during symbiosis, possibly related to cell growth and division and to molecular signaling. PMID:16766673

  11. Genetic Dissection of Tropodithietic Acid Biosynthesis by Marine Roseobacters▿ ‡

    PubMed Central

    Geng, Haifeng; Bruhn, Jesper Bartholin; Nielsen, Kristian F.; Gram, Lone; Belas, Robert

    2008-01-01

    The symbiotic association between the roseobacter Silicibacter sp. strain TM1040 and the dinoflagellate Pfiesteria piscicida involves bacterial chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP demethylation, and ultimately a biofilm on the surface of the host. Biofilm formation is coincident with the production of an antibiotic and a yellow-brown pigment. In this report, we demonstrate that the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in a loss of antibiotic activity (Tda−) and pigment production. Unexpectedly, six of the genes, referred to as tdaA-F, could not be found on the annotated TM1040 genome and were instead located on a previously unidentified plasmid (ca. 130 kb; pSTM3) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp. strain TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter sp. strain 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were found by DNA-DNA hybridization in 88% of a diverse collection of nine roseobacters with known antibiotic activity. These data suggest that roseobacters may use a common pathway for TDA biosynthesis that involves plasmid-encoded proteins. Using metagenomic library databases and a bioinformatics approach, differences in the biogeographical distribution between the critical TDA synthesis genes were observed. The implications of these results to roseobacter survival and the interaction between TM1040 and its dinoflagellate host are discussed. PMID:18192410

  12. Proteolytic Pathways Induced by Herbicides That Inhibit Amino Acid Biosynthesis

    PubMed Central

    Zulet, Amaia; Gil-Monreal, Miriam; Villamor, Joji Grace; Zabalza, Ana; van der Hoorn, Renier A. L.; Royuela, Mercedes

    2013-01-01

    Background The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling. Results Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment. Conclusion These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined. PMID:24040092

  13. Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1)

    PubMed Central

    Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; Allen, Eric E.; Kalyuzhnaya, Marina G.

    2017-01-01

    Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph. PMID:28119683

  14. Phosphate limitation promotes unsaturated fatty acids and arachidonic acid biosynthesis by microalgae Porphyridium purpureum.

    PubMed

    Su, Gaomin; Jiao, Kailin; Li, Zheng; Guo, Xiaoyi; Chang, Jingyu; Ndikubwimana, Theoneste; Sun, Yong; Zeng, Xianhai; Lu, Yinghua; Lin, Lu

    2016-07-01

    Polyunsaturated fatty acids (PUFAs) are highly appreciated on their nutritive value for human health and aquaculture. P. purpureum, one of the red microalgae acknowledged as a promising accumulator of ARA, was chosen as the target algae in the present research. Effects of sodium bicarbonate (0.04-1.2 g/L), temperature (25, 30 and 33 °C) and phosphate (0.00-0.14 g/L) on biomass yield, total fatty acids (TFA) and arachidonic acid (ARA) accumulation were investigated systemically. NaHCO3 dose of 0.8 g/L and moderate temperature of 30 °C were preferred. In addition, TFA and ARA production were significantly enhanced by an appropriate concentration of phosphate, and the highest TFA yield of 666.38 mg/L and ARA yield of 159.74 mg/L were obtained at a phosphate concentration of 0.035 g/L. Interestingly, with phosphate concentration continuing to fall, UFA/TFA and ARA/EPA ratios were increased accordingly, suggesting that phosphate limitation promoted unsaturated fatty acids and arachidonic acid biosynthesis. Low concentration of phosphate may be favored to increase the enzymatic activities of ∆6-desaturase, which played a key role in catalyzing the conversion of C16:0 to C18:2, and thus the selectivity of UFA increased. Meanwhile, the increase of ARA selectivity could be attributed to ω6 pathway promotion and ∆17-desaturase activity inhibition with phosphate limitation. Phosphate limitation strategy enhanced unsaturated fatty acids and ARA biosynthesis in P. purpureum, and can be applied in commercial scale manufacturing and commercialization of ARA.

  15. Evolution of the biosynthesis of the branched-chain amino acids

    NASA Astrophysics Data System (ADS)

    Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

    1995-06-01

    The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from α-ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

  16. Evolution of the biosynthesis of the branched-chain amino acids

    NASA Technical Reports Server (NTRS)

    Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

    1995-01-01

    The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

  17. Evolution of the biosynthesis of the branched-chain amino acids

    NASA Technical Reports Server (NTRS)

    Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

    1995-01-01

    The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

  18. Fatty acid biosynthesis pathways in Methylomicrobium buryatense 5G(B1)

    DOE PAGES

    Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; ...

    2017-01-10

    Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fattymore » acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of FA transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for FA-biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the FA profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. As a result, the gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.« less

  19. Oligomeric structure of proclavaminic acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic acid biosynthesis.

    PubMed Central

    Elkins, Jonathan M; Clifton, Ian J; Hernández, Helena; Doan, Linh X; Robinson, Carol V; Schofield, Christopher J; Hewitson, Kirsty S

    2002-01-01

    During biosynthesis of the clinically used beta-lactamase inhibitor clavulanic acid, one of the three steps catalysed by clavaminic acid synthase is separated from the other two by a step catalysed by proclavaminic acid amidino hydrolase (PAH), in which the guanidino group of an intermediate is hydrolysed to give proclavaminic acid and urea. PAH shows considerable sequence homology with the primary metabolic arginases, which hydrolyse arginine to ornithine and urea, but does not accept arginine as a substrate. Like other members of the bacterial sub-family of arginases, PAH is hexameric in solution and requires Mn2+ ions for activity. Other metal ions, including Co2+, can substitute for Mn2+. Two new substrates for PAH were identified, N-acetyl-(L)-arginine and (3R)-hydroxy-N-acetyl-(L)-arginine. Crystal structures of PAH from Streptomyces clavuligerus (at 1.75 A and 2.45 A resolution, where 1 A=0.1 nm) imply how it binds beta-lactams rather than the amino acid substrate of the arginases from which it evolved. The structures also suggest how PAH selects for a particular alcohol intermediate in the clavam biosynthesis pathway. As observed for the arginases, each PAH monomer consists of a core of beta-strands surrounded by alpha-helices, and its active site contains a di-Mn2+ centre with a bridging water molecule responsible for hydrolytic attack on to the guanidino group of the substrate. Comparison of structures obtained under different conditions reveals different conformations of a flexible loop, which must move to allow substrate binding. PMID:12020346

  20. Oxalic acid biosynthesis is encoded by an operon in Burkholderia glumae

    USDA-ARS?s Scientific Manuscript database

    Although the biosynthesis of oxalic acid is known to occur in a number of bacteria, the mechanism(s) regulating its production remains largely unknown. To date, there is no report on the identification of an oxalic acid biosynthetic pathway gene from bacteria. In an attempt to identify such a gene...

  1. Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis

    SciTech Connect

    Leon, J.; Shulaev, V.; Yalpani, N.

    1995-10-24

    Benzoic acid 2-hydroxylase (BA2H) catalyzes the biosynthesis of salicylic acid from benzoic acid. The enzyme has been partially purified and characterized as a soluble protein of 160 kDa. High-efficiency in vivo labeling of salicyclic acid with {sup 18}O{sub 2} suggested that BA2H is an oxygenase that specifically hydroxylates the ortho position of benzoic acid. The enzyme was strongly induced by either tobacco mosaic virus inoculation of benzoic acid infiltration of tobacco leaves and it was inhibited by CO and other inhibitors of cytochrome P450 hydroxylases. The BA2H activity was immunodepleted by antibodies raised against SU2, a soluble cytochrome P450 from Streptomyces griseolus. The anti-SU2 antibodies immunoprecipitated a radiolabeled polypeptide of around 160 kDa from the soluble protein extracts of L-[{sup 35}S]-methionine-fed tobacco leaves. Purified BA2H showed CO-difference spectra with a maximum at 457 nm. These data suggest that BA2H belongs to a novel class of soluble, high molecular weight cytochrome P450 enzymes. 21 refs., 6 figs., 1 tab.

  2. Microbial biosynthesis and secretion of l-malic acid and its applications.

    PubMed

    Chi, Zhe; Wang, Zhi-Peng; Wang, Guang-Yuan; Khan, Ibrar; Chi, Zhen-Ming

    2016-01-01

    l-Malic acid has many uses in food, beverage, pharmaceutical, chemical and medical industries. It can be produced by one-step fermentation, enzymatic transformation of fumaric acid to l-malate and acid hydrolysis of polymalic acid. However, the process for one-step fermentation is preferred as it has many advantages over any other process. The pathways of l-malic acid biosynthesis in microorganisms are partially clear and three metabolic pathways including non-oxidative pathway, oxidative pathway and glyoxylate cycle for the production of l-malic acid from glucose have been identified. Usually, high levels of l-malate are produced under the nitrogen starvation conditions, l-malate, as a calcium salt, is secreted from microbial cells and CaCO3 can play an important role in calcium malate biosynthesis and regulation. However, it is still unclear how it is secreted into the medium. To enhance l-malate biosynthesis and secretion by microbial cells, it is very important to study the mechanisms of l-malic acid biosynthesis and secretion at enzymatic and molecular levels.

  3. Autoxidated linolenic acid inhibits aflatoxin biosynthesis in Aspergillus flavus via oxylipin species.

    PubMed

    Yan, Shijuan; Liang, Yating; Zhang, Jindan; Chen, Zhuang; Liu, Chun-Ming

    2015-08-01

    Aflatoxins produced by Aspergillus species are among the most toxic and carcinogenic compounds in nature. Although it has been known for a long time that seeds with high oil content are more susceptible to aflatoxin contamination, the role of fatty acids in aflatoxin biosynthesis remains controversial. Here we demonstrate in A. flavus that both the saturated stearic acid (C18:0) and the polyunsaturated linolenic acid (C18:3) promoted aflatoxin production, while C18:3, but not C18:0, inhibited aflatoxin biosynthesis after exposure to air for several hours. Further experiments showed that autoxidated C18:3 promoted mycelial growth, sporulation, and kojic acid production, but inhibited the expression of genes in the AF biosynthetic gene cluster. Mass spectrometry analyses of autoxidated C18:3 fractions that were able to inhibit aflatoxin biosynthesis led to the identification of multiple oxylipin species. These results may help to clarify the role of fatty acids in aflatoxin biosynthesis, and may explain why controversial results have been obtained for fatty acids in the past.

  4. Foreign gene recruitment to the fatty acid biosynthesis pathway in diatoms.

    PubMed

    Chan, Cheong Xin; Baglivi, Francesca L; Jenkins, Christina E; Bhattacharya, Debashish

    2013-09-01

    Diatoms are highly successful marine and freshwater algae that contribute up to 20% of global carbon fixation. These species are leading candidates for biofuel production owing to ease of culturing and high fatty acid content. To assist in strain improvement and downstream applications for potential use as a biofuel, it is important to understand the evolution of lipid biosynthesis in diatoms. The evolutionary history of diatoms is however complicated by likely multiple endosymbioses involving the capture of foreign cells and horizontal gene transfer into the host genome. Using a phylogenomic approach, we assessed the evolutionary history of 12 diatom genes putatively encoding functions related to lipid biosynthesis. We found evidence of gene transfer likely from a green algal source for seven of these genes, with the remaining showing either vertical inheritance or evolutionary histories too complicated to interpret given current genome data. The functions of horizontally transferred genes encompass all aspects of lipid biosynthesis (initiation, biosynthesis, and desaturation of fatty acids) as well as fatty acid elongation, and are not restricted to plastid-targeted proteins. Our findings demonstrate that the transfer, duplication, and subfunctionalization of genes were key steps in the evolution of lipid biosynthesis in diatoms and other photosynthetic eukaryotes. This target pathway for biofuel research is highly chimeric and surprisingly, our results suggest that research done on related genes in green algae may have application to diatom models.

  5. Indole-3-Acetic Acid Biosynthesis in Colletotrichum gloeosporioides f. sp. aeschynomene

    PubMed Central

    Robinson, M.; Riov, J.; Sharon, A.

    1998-01-01

    We characterized the biosynthesis of indole-3-acetic acid by the mycoherbicide Colletotrichum gloeosporioides f. sp. aeschynomene. Auxin production was tryptophan dependent. Compounds from the indole-3-acetamide and indole-3-pyruvic acid pathways were detected in culture filtrates. Feeding experiments and in vitro assay confirmed the presence of both pathways. Indole-3-acetamide was the major pathway utilized by the fungus to produce indole-3-acetic acid in culture. PMID:9835603

  6. Dietary Polyunsaturated Fatty Acids and Inflammation: The Role of Phospholipid Biosynthesis

    PubMed Central

    Raphael, William; Sordillo, Lorraine M.

    2013-01-01

    The composition of fatty acids in the diets of both human and domestic animal species can regulate inflammation through the biosynthesis of potent lipid mediators. The substrates for lipid mediator biosynthesis are derived primarily from membrane phospholipids and reflect dietary fatty acid intake. Inflammation can be exacerbated with intake of certain dietary fatty acids, such as some ω-6 polyunsaturated fatty acids (PUFA), and subsequent incorporation into membrane phospholipids. Inflammation, however, can be resolved with ingestion of other fatty acids, such as ω-3 PUFA. The influence of dietary PUFA on phospholipid composition is influenced by factors that control phospholipid biosynthesis within cellular membranes, such as preferential incorporation of some fatty acids, competition between newly ingested PUFA and fatty acids released from stores such as adipose, and the impacts of carbohydrate metabolism and physiological state. The objective of this review is to explain these factors as potential obstacles to manipulating PUFA composition of tissue phospholipids by specific dietary fatty acids. A better understanding of the factors that influence how dietary fatty acids can be incorporated into phospholipids may lead to nutritional intervention strategies that optimize health. PMID:24152446

  7. Polyunsaturated fatty acids influence differential biosynthesis of oxylipids and other lipid mediators during bovine coliform mastitis.

    PubMed

    Mavangira, Vengai; Gandy, Jeffery C; Zhang, Chen; Ryman, Valerie E; Daniel Jones, A; Sordillo, Lorraine M

    2015-09-01

    Coliform mastitis is a severe and sometimes fatal disease characterized by an unregulated inflammatory response. The initiation, progression, and resolution of inflammatory responses are regulated, in part, by potent oxylipid metabolites derived from polyunsaturated fatty acids. The purpose of this study was to characterize the biosynthesis and diversity of oxylipid metabolites during acute bovine coliform mastitis. Eleven cows diagnosed with naturally occurring acute systemic coliform mastitis and 13 healthy control cows, matched for lactation number and days in milk, were selected for comparison of oxylipid and free fatty acid concentrations in both milk and plasma. Oxylipids and free fatty acids were quantified using liquid chromatography-tandem mass spectrometry. All polyunsaturated fatty acids quantified in milk were elevated during coliform mastitis with linoleic acid being the most abundant. Oxylipids synthesized through the lipoxygenase and cytochrome P450 pathways accounted for the majority of the oxylipid biosynthesis. This study demonstrated a complex and diverse oxylipid network, most pronounced at the level of the mammary gland. Substrate availability, biosynthetic pathways, and degree of metabolism influence the biosynthesis of oxylipids during bovine coliform mastitis. Further studies are required to identify targets for novel interventions that modulate oxylipid biosynthesis during coliform mastitis to optimize inflammation. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  8. Isolated etioplasts as test system for inhibitors of fatty acid biosynthesis

    SciTech Connect

    Lichtenthaler, H.K.; Kobek, K. )

    1989-04-01

    Isolated intact chloroplasts of mono- and dicotyledonous plants possess the capacity for de novo fatty acid biosynthesis, starting from {sup 14}C-acetate. These can be taken as test system for herbicides affecting fatty acid biosynthesis as shown earlier in our laboratory. The incorporation rates of acetate into the total fatty acids depend on the photosynthetic cofactors ATP and NADPH and amount in the light to 33 kBq (oat) and 39 kBq (pea) per mg chlorophyll x h, whereas in the dark only ca. 10% of these rates are obtained. In order to establish a test system, which is fully independent of light, we isolated and characterized etioplast fractions from oat and pea seedlings with a very high capacity of de novo fatty acid biosynthesis (500 and 400 kBq per mg carotenoids in a 20 min period). This activity was blocked by herbicides such as cycloxydim, sethoxydim and diclofop in a dose-dependent manner. This new test system has the great advantage that one can verify whether inhibitors of photosynthesis affect fatty acid biosynthesis.

  9. Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants.

    PubMed

    de la Torre, Fernando; Cañas, Rafael A; Pascual, M Belén; Avila, Concepción; Cánovas, Francisco M

    2014-10-01

    In the chloroplasts and in non-green plastids of plants, aspartate is the precursor for the biosynthesis of different amino acids and derived metabolites that play distinct and important roles in plant growth, reproduction, development or defence. Aspartate biosynthesis is mediated by the enzyme aspartate aminotransferase (EC 2.6.1.1), which catalyses the reversible transamination between glutamate and oxaloacetate to generate aspartate and 2-oxoglutarate. Plastids contain two aspartate aminotransferases: a eukaryotic-type and a prokaryotic-type bifunctional enzyme displaying aspartate and prephenate aminotransferase activities. A general overview of the biochemistry, regulation, functional significance, and phylogenetic origin of both enzymes is presented. The roles of these plastidic aminotransferases in the biosynthesis of essential amino acids are discussed.

  10. Low levels of gibberellic acid control the biosynthesis of ajmalicine in Catharanthus roseus cell suspension cultures.

    PubMed

    Amini, Aniça; Glévarec, Gaëlle; Andreu, Françoise; Rideau, Marc; Crèche, Joël

    2009-02-01

    In periwinkle cell suspensions, amounts of gibberellic acid ranging from 10 ( - 10) M to 10 ( - 7) M significantly antagonized, in a dose-dependant manner, the stimulation of ajmalicine biosynthesis by cytokinins (CKs). This inhibitory effect was strictly correlated with the abolition of the expression of two genes encoding enzymes of the monoterpenoid indole alkaloid (MIA) biosynthetic pathway and was normally upregulated after CK treatments. Moreover, low concentrations of the gibberellin biosynthesis inhibitor paclobutrazol could reverse the inhibitory effects of low auxin levels on ajmalicine accumulation in the cells. On the other hand, gibberellic acid could not affect the expression of two type-A response regulators considered to be CK primary response genes in periwinkle cells. The antagonistic effects of gibberellins and cytokinins on MIA biosynthesis and their possible impact on elements of the signal transduction are discussed.

  11. Biosynthesis of natural products containing β-amino acids.

    PubMed

    Kudo, Fumitaka; Miyanaga, Akimasa; Eguchi, Tadashi

    2014-08-01

    Covering: up to January, 2014. We focus here on β-amino acids as components of complex natural products because the presence of β-amino acids produces structural diversity in natural products and provides characteristic architectures beyond those of ordinary α-L-amino acids, thus generating significant and unique biological functions in nature. In this review, we first survey the known bioactive β-amino acid-containing natural products including nonribosomal peptides, macrolactam polyketides, and nucleoside-β-amino acid hybrids. Next, the biosynthetic enzymes that form β-amino acids from α-amino acids and the de novo synthesis of β-amino acids are summarized. Then, the mechanisms of β-amino acid incorporation into natural products are reviewed. Because it is anticipated that the rational swapping of the β-amino acid moieties with various side chains and stereochemistries by biosynthetic engineering should lead to the creation of novel architectures and bioactive compounds, the accumulation of knowledge regarding β-amino acid-containing natural product biosynthetic machinery could have a significant impact in this field. In addition, genome mining of characteristic β-amino acid biosynthetic genes and unique β-amino acid incorporation machinery could lead to the discovery of new β-amino acid-containing natural products.

  12. Direct biosynthesis of adipic acid from a synthetic pathway in recombinant Escherichia coli.

    PubMed

    Yu, Jia-Le; Xia, Xiao-Xia; Zhong, Jian-Jiang; Qian, Zhi-Gang

    2014-12-01

    The C6 dicarboxylic acid, adipic acid, is an important platform chemical in industry. Biobased production of adipic acid is a promising alternative to the current petrochemical route. Here, we report biosynthesis of adipic acid using an artificial pathway inspired by the reversal of beta-oxidation of dicarboxylic acids. The biosynthetic pathway comprises condensation of acetyl-CoA and succinyl-CoA to form the C6 backbone and subsequent reduction, dehydration, hydrogenation, and release of adipic acid from its thioester. The pathway was first tested in vitro with reconstituted pathway enzymes and then functionally introduced into Escherichia coli for the biosynthesis and excretion of adipic acid into the culture medium. The production titer was increased by approximately 20-fold through the combination of recruiting enzymes that were more suitable to catalyze the synthetic reactions and increasing availability of the condensation substrates. This work demonstrates direct biosynthesis of adipic acid via non-natural synthetic pathway, which may enable its renewable production.

  13. Molecular mechanisms of the coordination between astaxanthin and fatty acid biosynthesis in Haematococcus pluvialis (Chlorophyceae).

    PubMed

    Chen, Guanqun; Wang, Baobei; Han, Danxiang; Sommerfeld, Milton; Lu, Yinghua; Chen, Feng; Hu, Qiang

    2015-01-01

    Astaxanthin, a red ketocarotenoid with strong antioxidant activity and high commercial value, possesses important physiological functions in astaxanthin-producing microalgae. The green microalga Haematococcus pluvialis accumulates up to 4% fatty acid-esterified astaxanthin (by dry weight), and is used as a model species for exploring astaxanthin biosynthesis in unicellular photosynthetic organisms. Although coordination of astaxanthin and fatty acid biosynthesis in a stoichiometric fashion was observed in H. pluvialis, the interaction mechanism is unclear. Here we dissected the molecular mechanism underlying coordination between the two pathways in H. pluvialis. Our results eliminated possible coordination of this inter-dependence at the transcriptional level, and showed that this interaction was feedback-coordinated at the metabolite level. In vivo and in vitro experiments indicated that astaxanthin esterification drove the formation and accumulation of astaxanthin. We further showed that both free astaxanthin biosynthesis and esterification occurred in the endoplasmic reticulum, and that certain diacylglycerol acyltransferases may be the candidate enzymes catalyzing astaxanthin esterification. A model of astaxanthin biosynthesis in H. pluvialis was subsequently proposed. These findings provide further insights into astaxanthin biosynthesis in H. pluvialis.

  14. Fatty acid biosynthesis revisited: Structure elucidation and metabolic engineering

    SciTech Connect

    Beld, Joris; Lee, D. John; Burkart, Michael D.

    2014-10-20

    Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. Lastly, in this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.

  15. Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering.

    PubMed

    Beld, Joris; Lee, D John; Burkart, Michael D

    2015-01-01

    Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. In this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.

  16. Fatty Acid Biosynthesis Revisited: Structure Elucidation and Metabolic Engineering

    PubMed Central

    Beld, Joris; Lee, D. John

    2014-01-01

    Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases’ many intricate structural and regulatory elements. In this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field. PMID:25360565

  17. The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

    SciTech Connect

    Hayashi, H.; Miwa, A. )

    1989-11-01

    The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using (1-{sup 14}C)butyric acid and (1-{sup 14}C)lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of ({sup 14}C)lignoceric acid into primary bile acids was approximately four times higher than that of ({sup 14}C)butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both ({sup 14}C)lignoceric acid and ({sup 14}C)butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.

  18. Regional biosynthesis of prostaglandins and hydroxyeicosatetraenoic acids from arachidonic acid in the rat stomach tissue.

    PubMed

    Park, S; Choue, R W; Cho, Y; Ziboh, V A

    2003-01-01

    The present study was conducted to determine regional differences in the biosynthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs) in the rat stomach tissue (fundus, corpus and pyloric antrum) from radioactive arachidonic acid (AA). The radioactive metabolites were validated by RP-HPLC using non-radioactive AA as substrate. PGE(2) was the major prostanoid in the tissue(.) The relative ratio of PGE(2):PGF(2)alpha:PGD(2) in the whole stomach was 1:0.5:0.1. Regionally, the fundus biosynthesized the largest amount of all three cyclo-oxygenase products. Among the lipoxygenase metabolites, 15S-HETE was the predominant product, while 12S-HETE was found to be the lowest. The relative ratio of 15S-HETE:5S-HETE:12S-HETE in the whole stomach was 1:0.6:0.4. Interestingly, the generation of lipoxygenase products was the highest in the pyloric antrum when compared to fundus or corpus. Thus, the regional differences in the biosyntheses of gastric PGs and monohydroxy fatty acids may be relevant to our understanding of corresponding differences in mucosal resistance or susceptibility to gastric disease.

  19. Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis.

    PubMed

    Meyer, Astrid; Kirsch, Helene; Domergue, Frédéric; Abbadi, Amine; Sperling, Petra; Bauer, Jörg; Cirpus, Petra; Zank, Thorsten K; Moreau, Hervé; Roscoe, Thomas J; Zähringer, Ulrich; Heinz, Ernst

    2004-10-01

    In algae, the biosynthesis of docosahexaenoic acid (22:6omega3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5omega3; EPA) to 22:5omega3, which is required as a substrate for the final Delta4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana, from the fish Oncorhynchus mykiss, from the frog Xenopus laevis, and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens. The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana: one specific for the elongation of (Delta6-)C18-PUFAs and one specific for (Delta5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss, X. laevis, and C. intestinalis accepted both C18- and C20-PUFAs. By coexpression of the Delta6- and Delta5-elongases from T. pseudonana and O. tauri, respectively, with the Delta5- and Delta4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops.

  20. Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis.

    PubMed Central

    Rajgarhia, V B; Strohl, W R

    1997-01-01

    The structure of the Streptomyces sp. strain C5 daunorubicin type II polyketide synthase (PKS) gene region is different from that of other known type II PKS gene clusters. Directly downstream of the genes encoding ketoacylsynthase alpha and beta (KS alpha, KS beta) are two genes (dpsC, dpsD) encoding proteins of unproven function, both absent from other type II PKS gene clusters. Also in contrast to other type II PKS clusters, the gene encoding the acyl carrier protein (ACP), dpsG, is located about 6.8 kbp upstream of the genes encoding the daunorubicin KS alpha and KS beta. In this work, we demonstrate that the minimal genes required to produce aklanonic acid in heterologous hosts are dpsG (ACP), dauI (regulatory activator), dpsA (KS alpha), dpsB (KS beta), dpsF (aromatase), dpsE (polyketide reductase), and dauG (putative deoxyaklanonic acid oxygenase). The two unusual open reading frames, dpsC (KASIII homolog lacking a known active site) and dpsD (acyltransferase homolog), are not required to synthesize aklanonic acid. Additionally, replacement of dpsD or dpsCD in Streptomyces sp. strain C5 with a neomycin resistance gene (aphI) results in mutant strains that still produced anthracyclines. PMID:9098068

  1. Effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungus Aureobasidium pullulans.

    PubMed

    Wang, Yongkang; Song, Xiaodan; Zhang, Yongjun; Wang, Bochu; Zou, Xiang

    2016-08-22

    Polymalic acid (PMA) is a novel polyester polymer that has been broadly used in the medical and food industries. Its monomer, L-malic acid, is also a potential C4 platform chemical. However, little is known about the mechanism of PMA biosynthesis in the yeast-like fungus, Aureobasidium pullulans. In this study, the effects of different nitrogen concentration on cell growth and PMA biosynthesis were investigated via comparative transcriptomics and proteomics analyses, and a related signaling pathway was also evaluated. A high final PMA titer of 44.00 ± 3.65 g/L (49.9 ± 4.14 g/L of malic acid after hydrolysis) was achieved in a 5-L fermentor under low nitrogen concentration (2 g/L of NH4NO3), which was 18.3 % higher yield than that obtained under high nitrogen concentration (10 g/L of NH4NO3). Comparative transcriptomics profiling revealed that a set of genes, related to the ribosome, ribosome biogenesis, proteasome, and nitrogen metabolism, were significantly up- or down-regulated under nitrogen sufficient conditions, which could be regulated by the TOR signaling pathway. Fourteen protein spots were identified via proteomics analysis, and were found to be associated with cell division and growth, energy metabolism, and the glycolytic pathway. qRT-PCR further confirmed that the expression levels of key genes involved in the PMA biosynthetic pathway (GLK, CS, FUM, DAT, and MCL) and the TOR signaling pathway (GS, TOR1, Tap42, and Gat1) were upregulated due to nitrogen limitation. Under rapamycin stress, PMA biosynthesis was obviously inhibited in a dose-dependent manner, and the transcription levels of TOR1, MCL, and DAT were also downregulated. The level of nitrogen could regulate cell growth and PMA biosynthesis. Low concentration of nitrogen was beneficial for PMA biosynthesis, which could upregulate the expression of key genes involved in the PMA biosynthesis pathway. Cell growth and PMA biosynthesis might be mediated by the TOR signaling pathway in

  2. Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

    PubMed

    Goodrich-Tanrikulu, M; Stafford, A E; Lin, J T; Makapugay, M I; Fuller, G; McKeon, T A

    1994-10-01

    New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

  3. Limited Effects of Bile Acids and Small Heterodimer Partner on Hepatitis B Virus Biosynthesis In Vivo

    PubMed Central

    Reese, Vanessa C.; Moore, David D.

    2012-01-01

    Multiple nuclear receptors, including hepatocyte nuclear factor 4α (HNF4α), retinoid X receptor α (RXRα) plus peroxisome proliferator-activated receptor α (PPARα), RXRα plus farnesoid X receptor α (FXRα), liver receptor homolog 1 (LRH1), and estrogen-related receptors (ERRs), have been shown to support efficient viral biosynthesis in nonhepatoma cells in the absence of additional liver-enriched transcription factors. Although HNF4α has been shown to be critical for the developmental expression of hepatitis B virus (HBV) biosynthesis in the liver, the relative importance of the various nuclear receptors capable of supporting viral transcription and replication in the adult in vivo has not been clearly established. To investigate the role of the nuclear receptor FXR and the corepressor small heterodimer partner (SHP) in viral biosynthesis in vivo, SHP-expressing and SHP-null HBV transgenic mice were fed a bile acid-supplemented diet. The increased FXR activity and SHP expression levels resulting from bile acid treatment did not greatly modulate HBV RNA and DNA synthesis. Therefore, FXR and SHP appear to play a limited role in modulating HBV biosynthesis, suggesting that alternative nuclear receptors are more critical determinants of viral transcription in the HBV transgenic mouse model of chronic viral infection. These observations suggest that hepatic bile acid levels or therapeutic agents targeting FXR may not greatly modulate viremia during natural infection. PMID:22171277

  4. Co-expression analysis identifies CRC and AP1 the regulator of Arabidopsis fatty acid biosynthesis.

    PubMed

    Han, Xinxin; Yin, Linlin; Xue, Hongwei

    2012-07-01

    Fatty acids (FAs) play crucial rules in signal transduction and plant development, however, the regulation of FA metabolism is still poorly understood. To study the relevant regulatory network, fifty-eight FA biosynthesis genes including de novo synthases, desaturases and elongases were selected as "guide genes" to construct the co-expression network. Calculation of the correlation between all Arabidopsis thaliana (L.) genes with each guide gene by Arabidopsis co-expression dating mining tools (ACT) identifies 797 candidate FA-correlated genes. Gene ontology (GO) analysis of these co-expressed genes showed they are tightly correlated to photosynthesis and carbohydrate metabolism, and function in many processes. Interestingly, 63 transcription factors (TFs) were identified as candidate FA biosynthesis regulators and 8 TF families are enriched. Two TF genes, CRC and AP1, both correlating with 8 FA guide genes, were further characterized. Analyses of the ap1 and crc mutant showed the altered total FA composition of mature seeds. The contents of palmitoleic acid, stearic acid, arachidic acid and eicosadienoic acid are decreased, whereas that of oleic acid is increased in ap1 and crc seeds, which is consistent with the qRT-PCR analysis revealing the suppressed expression of the corresponding guide genes. In addition, yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) revealed that CRC can bind to the promoter regions of KCS7 and KCS15, indicating that CRC may directly regulate FA biosynthesis. © 2012 Institute of Botany, Chinese Academy of Sciences.

  5. Fatty acid biosynthesis revisited: Structure elucidation and metabolic engineering

    DOE PAGES

    Beld, Joris; Lee, D. John; Burkart, Michael D.

    2014-10-20

    Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understandingmore » of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. Lastly, in this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.« less

  6. Seed development and hydroxy fatty acid biosynthesis in physaria lindheimeri

    USDA-ARS?s Scientific Manuscript database

    Hydroxy fatty acids (HFAs) are valuable industrial raw materials used in many industries. Physaria lindheimeri (Pl) accumulates over 80% HFA, in the form of lesquerolic acid (20:1OH), in its seed oil. Understanding the seed development of Pl is an important step to utilizing this unique wild specie...

  7. Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Głuszuk, Katarzyna; Surażyński, Arkadiusz

    2014-01-01

    Aim The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process. Materials and methods Collagen, [3H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase). Results Caffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis. Conclusion Caffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts. PMID:25342885

  8. Mechanism of Lysergic Acid Diethylamide Interference with Rabbit Antibody Biosynthesis

    PubMed Central

    Voss, Edward W.; Winkelhake, Jeffrey L.

    1974-01-01

    Lymphoid cells from hyperimmune rabbits producing antibodies to a hapten, incubated in the presence of d-lysergic acid diethylamide, continued to synthesize protein at a normal rate. Isoelectric focusing analysis of the low-molecular-weight protein secreted by the cells incubated with lysergic acid diethylamide indicated two components, with pI's of 4.9 and 5.2. Immune cells not exposed to lysergic acid diethylamide secreted only 7S IgG molecules with an average pI of approximately 7.0. PMID:4524614

  9. Phorbic Acid Biosynthesis in the Latex Vessel System of Euphorbia

    PubMed Central

    Nordal, Arnold; Benson, A. A.

    1969-01-01

    Evidence is presented that phorbic acid is formed in the latex producing cell system, rather than in photosynthetic or chlorophyll-free tissues of Euphorbia resinifera Berg. When a branch of the plant was kept first in a 14CO2 atmosphere with 12 hr light-dark periods for 2 days and then left under natural conditions in the air outside for at least 2 to 3 days, radioactive phorbic acid was found in the latex. Phorbic acid synthesis appeared to be independent of the photosynthetic and respiratory activities of the plant. Besides phorbic acid 2 other major radioactive compounds were recognized in the latex, a glycoside or oligosaccharide, and a lipid belonging to the group of triterpenoid compounds characteristic of the latex in several species of Euphorbia. Images PMID:16657036

  10. Mycolic Acid-Containing Bacteria Induce Natural-Product Biosynthesis in Streptomyces Species▿ †

    PubMed Central

    Onaka, Hiroyasu; Mori, Yukiko; Igarashi, Yasuhiro; Furumai, Tamotsu

    2011-01-01

    Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products. PMID:21097597

  11. Mycolic acid-containing bacteria induce natural-product biosynthesis in Streptomyces species.

    PubMed

    Onaka, Hiroyasu; Mori, Yukiko; Igarashi, Yasuhiro; Furumai, Tamotsu

    2011-01-01

    Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.

  12. Biosynthesis of pyrrolidone carboxylic acid in hairless mouse epidermis.

    PubMed

    DeLapp, N W; Dieckman, D K

    1977-05-01

    [3H]Glutamic acid (PCA) was followed with time after a single subcutaneous injection. PCA specific activity increased slowly, reaching a peak at 3 to 4 days after injection of the labeled amino acid, after which it slowly decline. Incorporation of [3H]glutamic acid into epidermal PCA was markedly inhibited by a single topical application of cycloheximide. Topical application of cycloheximide 2 hr prior to [3H]glutamate injection caused a significantly greater reduction in PCA specific activity (determined 3 days after injection) than cycloheximide treatment 3 hr after administration of the labeled amino acid. Ninety-seven percent of the PCA content of hairless mouse epidermis was shown to reside in the stratum corneum. These observations indicate the involvement of protein synthesis in the formation of PCA from glutamic acid rather than a direct conversion of the amino acid. The high level of PCA in mammalian epidermis appears to be caused by its accumulation in the stratum corneum accompainied by a relatively slow rate of PCA turnover in comparison to other tissues.

  13. Biosynthesis of pyruvic acid from glucose by Blastobotrys adeninivorans.

    PubMed

    Kamzolova, Svetlana V; Morgunov, Igor G

    2016-09-01

    The ability of taxonomically different yeasts to synthesize pyruvic acid (PA) from glucose was studied. The study showed that many yeasts are able to produce PA from glucose under the condition of growth limitation by thiamine. This ability was found in the yeast Blastobotrys adeninivorans for the first time. The production (oversynthesis) of PA in this yeast can be explained by disturbance in the function of thiamine-dependent pyruvate dehydrogenase. Namely, the partial inhibition of this enzyme brings about the excretion of PA from the yeast cells. Due to incomplete inhibition of pyruvate dehydrogenase, the formation of acetyl-CoA continues, although at a lower level, maintaining the synthesis of α-ketoglutaric acid (KGA) in the tricarboxylic acid (TCA) cycle. KGA is no longer oxidized in the TCA cycle, because thiamine limitation inhibits α-ketoglutarate dehydrogenase. As a result, KGA is excreted from the yeast cells as a byproduct of PA oversynthesis. Furthermore, the increased level of KGA in the yeast cells inhibits NAD-dependent isocitrate dehydrogenase in the TCA cycle and enhances the production and excretion of citric acid, another byproduct of PA oversynthesis. During cultivation in a fermentor, the strain Blastobotrys adeninivorans VKM Y-2677 produced 43.2 g l(-1) PA from glucose with a product yield (YPA) of 0.77 g PA/g glucose. The proportion of PA to byproducts was 18:1 for KGA and 8:1 for citric acid.

  14. Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus)

    PubMed Central

    2013-01-01

    Background Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial

  15. Enzymes for fatty acid-based hydrocarbon biosynthesis.

    PubMed

    Herman, Nicolaus A; Zhang, Wenjun

    2016-12-01

    Surging energy consumption and environmental concerns have stimulated interest in the production of chemicals and fuels through sustainable and renewable approaches. Fatty acid-based hydrocarbons, such as alkanes and alkenes, are of particular interest to directly replace fossil fuels. Towards this effort, understanding of hydrocarbon-producing enzymes is the first indispensable step to bio-production of hydrocarbons. Here, we review recent advances in the discovery and mechanistic study of enzymes capable of converting fatty acid precursors into hydrocarbons, and provide perspectives on the future of this rapidly growing field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. The Biosynthesis of Erucic Acid in Developing Embryos of Brassica rapa1

    PubMed Central

    Bao, Xiaoming; Pollard, Mike; Ohlrogge, John

    1998-01-01

    The prevailing hypothesis on the biosynthesis of erucic acid in developing seeds is that oleic acid, produced in the plastid, is activated to oleoyl-coenzyme A (CoA) for malonyl-CoA-dependent elongation to erucic acid in the cytosol. Several in vivo-labeling experiments designed to probe and extend this hypothesis are reported here. To examine whether newly synthesized oleic acid is directly elongated to erucic acid in developing seeds of Brassica rapa L., embryos were labeled with [14C]acetate, and the ratio of radioactivity of carbon atoms C-5 to C-22 (de novo fatty acid synthesis portion) to carbon atoms C-1 to C-4 (elongated portion) of erucic acid was monitored with time. If newly synthesized 18:1 (oleate) immediately becomes a substrate for elongation to erucic acid, this ratio would be expected to remain constant with incubation time. However, if erucic acid is produced from a pool of preexisting oleic acid, the ratio of 14C in the 4 elongation carbons to 14C in the methyl-terminal 18 carbons would be expected to decrease with time. This labeling ratio decreased with time and, therefore, suggests the existence of an intermediate pool of 18:1, which contributes at least part of the oleoyl precursor for the production of erucic acid. The addition of 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy] propanoic acid, which inhibits the homodimeric acetyl-CoA carboxylase, severely inhibited the synthesis of [14C]erucic acid, indicating that essentially all malonyl-CoA for elongation of 18:1 to erucate was produced by homodimeric acetyl-CoA carboxylase. Both light and 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy]-propanoic acid increased the accumulation of [14C]18:1 and the parallel accumulation of [14C]phosphatidylcholine. Taken together, these results show an additional level of complexity in the biosynthesis of erucic acid. PMID:9733537

  17. Salicylic acid induces mitochondrial injury by inhibiting ferrochelatase heme biosynthesis activity.

    PubMed

    Gupta, Vipul; Liu, Shujie; Ando, Hideki; Ishii, Ryohei; Tateno, Shumpei; Kaneko, Yuki; Yugami, Masato; Sakamoto, Satoshi; Yamaguchi, Yuki; Nureki, Osamu; Handa, Hiroshi

    2013-12-01

    Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECH-salicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.

  18. Ambuic acid inhibits the biosynthesis of cyclic peptide quormones in gram-positive bacteria.

    PubMed

    Nakayama, Jiro; Uemura, Yumi; Nishiguchi, Kenzo; Yoshimura, Norito; Igarashi, Yasuhiro; Sonomoto, Kenji

    2009-02-01

    Quorum sensing is a cell-density-dependent regulatory system in gram-positive bacteria and is often regulated by cyclic peptides called "quormones," which function as extracellular communication signals. With an aim to discover an antipathogenic agent targeting quorum sensing in gram-positive bacteria, we screened 153 samples of fungal butanol extracts with the guidance of the inhibition of quorum-sensing-mediated gelatinase production in Enterococcus faecalis. Following the screenings, we found that ambuic acid, a known secondary fungal metabolite, inhibited the quorum-sensing-mediated gelatinase production without influencing the growth of E. faecalis. We further demonstrated that ambuic acid targeted the biosynthesis of a cyclic peptide quormone called gelatinase biosynthesis-activating pheromone. Furthermore, ambuic acid also inhibited the biosynthesis of the cyclic peptide quormones of Staphylococcus aureus and Listeria innocua. These results suggest the potential use of ambuic acid as a lead compound of antipathogenic drugs that target the quorum-sensing-mediated virulence expression of gram-positive bacteria.

  19. Branched chain amino acid metabolism in the biosynthesis of Lycopersicon pennellii glucose esters

    SciTech Connect

    Walters, D.S.; Steffens, J.C. )

    1990-08-01

    Lycopersicon pennellii Corr. (D'Arcy) an insect-resistant, wild tomato possesses high densities of glandular trichomes which exude a mixture of 2,3,4-tri-O-acylated glucose esters that function as a physical impediment and feeding deterrent to small arthropod pests. The acyl moieties are branched C{sub 4} and C{sub 5} acids, and branched and straight chain C{sub 10}, C{sub 11}, and C{sub 12} acids. The structure of the branched acyl constituents suggests that the branched chain amino acid biosynthetic pathway participates in their biosynthesis. ({sup 14}C)Valine and deuterated branched chain amino acids (and their oxo-acid derivatives) were incorporated into branched C{sub 4} and C{sub 5} acid groups of glucose esters by a process of transamination, oxidative decarboxylation and subsequent acylation. C{sub 4} and C{sub 5} branched acids were elongated by two carbon units to produce the branched C{sub 10}-C{sub 12} groups. Norvaline, norleucine, allylglycine, and methionine also were processed into acyl moieties and secreted from the trichomes as glucose esters. Changes in the acyl composition of the glucose esters following sulfonylurea herbicide administration support the participation of acetohydroxyacid synthetase and the other enzymes of branched amino acid biosynthesis in the production of glucose esters.

  20. Whitefly genome expression reveals host-symbiont interaction in amino acid biosynthesis.

    PubMed

    Upadhyay, Santosh Kumar; Sharma, Shailesh; Singh, Harpal; Dixit, Sameer; Kumar, Jitesh; Verma, Praveen C; Chandrashekar, K

    2015-01-01

    Whitefly (Bemisia tabaci) complex is a serious insect pest of several crop plants worldwide. It comprises several morphologically indistinguishable species, however very little is known about their genetic divergence and biosynthetic pathways. In the present study, we performed transcriptome sequencing of Asia 1 species of B. tabaci complex and analyzed the interaction of host-symbiont genes in amino acid biosynthetic pathways. We obtained about 83 million reads using Illumina sequencing that assembled into 72716 unitigs. A total of 21129 unitigs were annotated at stringent parameters. Annotated unitigs were mapped to 52847 gene ontology (GO) terms and 131 Kyoto encyclopedia of genes and genomes (KEGG) pathways. Expression analysis of the genes involved in amino acid biosynthesis pathways revealed the complementation between whitefly and its symbiont partner Candidatus Portiera aleyrodidarum. Most of the non-essential amino acids and intermediates of essential amino acid pathways were supplied by the host insect to its symbiont. The symbiont expressed the pathways for the essential amino acids arginine, threonine and tryptophan and the immediate precursors of valine, leucine, isoleucine and phenyl-alanine. High level expression of the amino acid transporters in the whitefly suggested the molecular mechanisms for the exchange of amino acids between the host and the symbiont. Our study provides a comprehensive transcriptome data for Asia 1 species of B. tabaci complex that focusses light on integration of host and symbiont genes in amino acid biosynthesis pathways.

  1. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds.

    PubMed

    Tagashira, Yusuke; Shimizu, Tomoe; Miyamoto, Masanobu; Nishida, Sho; Yoshida, Kaoru T

    2015-04-24

    The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP₆) biosynthesis-related genes, as InsP₆ is a major storage form of P in seeds. The rice (Oryza sativa L.) low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP₆ biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP₆ and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP₆ biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP₆ biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  2. The role of cis-carotenoids in abscisic acid biosynthesis.

    PubMed

    Parry, A D; Babiano, M J; Horgan, R

    1990-08-01

    Evidence has been obtained which is consistent with 9'-cis-neoxanthin being a major precursor of abscisic acid (ABA) in higher plants. A mild, rapid procedure was developed for the extraction and analysis of carotenoids from a range of tissues. Once purified the carotenoids were identified from their light-absorbance properties, reactions with dilute acid, high-performance liquid chromatography Rts, mass spectra and the quasiequilibria resulting from iodine-catalysed or chlorophyllsensitised photoisomerisation. Two possible ABA precursors, 9'-cis-neoxanthin and 9-cis-violaxanthin, were identified in extracts of light-grown and etiolated leaves (of Lycopersicon esculentum, Phaseolus vulgaris, Vicia faba, Pisum sativum, Cicer arietinum, Zea mays, Nicotiana plumbaginifolia, Plantago lanceolata and Digitalis purpurea), and roots of light-grown and etiolated plants (Lycopersicon, Phaseolus and Zea). The 9,9'-di-cisisomer of violaxanthin was synthesised but its presence was not detected in any extracts. Levels of 9'-cis-neoxanthin and all-trans-violaxanthin were between 20- to 100-fold greater than those of ABA in light-grown leaves. The levels of 9-cis-violaxanthin were similar to those of ABA but unaffected by water stress. Etiolated Phaseolus leaves contained reduced amounts of carotenoids (15-20% compared with light-grown leaves) but retained the ability to synthesise large amounts of ABA. The amounts of ABA synthesised, measured as increases in ABA and its metabolites phaseic acid and dihydrophaseic acid, were closely matched by decreases in the levels of 9'-cis-neoxanthin and all-trans-violaxanthin. In etiolated seedlings grown on 50% D2O, deuterium incorporation into ABA was similar to that into the xanthophylls. Relative levels of carotenoids in roots and light-grown and etiolated leaves of the ABA-deficient mutants, notabilis, flacca and sitiens were the same as those found in wild-type tomato tissues.

  3. Effect of oxidoreduction potential on aroma biosynthesis by lactic acid bacteria in nonfat yogurt.

    PubMed

    Martin, F; Cachon, R; Pernin, K; De Coninck, J; Gervais, P; Guichard, E; Cayot, N

    2011-02-01

    The aim of this study was to investigate the effect of oxidoreduction potential (Eh) on the biosynthesis of aroma compounds by lactic acid bacteria in non-fat yogurt. The study was done with yogurts fermented by Lactobacillus bulgaricus and Streptococcus thermophilus. The Eh was modified by the application of different gaseous conditions (air, nitrogen, and nitrogen/hydrogen). Acetaldehyde, dimethyl sulfide, diacetyl, and pentane-2,3-dione, as the major endogenous odorant compounds of yogurt, were chosen as tracers for the biosynthesis of aroma compounds by lactic acid bacteria. Oxidative conditions favored the production of acetaldehyde, dimethyl sulfide, and diketones (diacetyl and pentane-2,3-dione). The Eh of the medium influences aroma production in yogurt by modifying the metabolic pathways of Lb. bulgaricus and Strep. thermophilus. The use of Eh as a control parameter during yogurt production could permit the control of aroma formation.

  4. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68.

    PubMed

    Samadlouie, Hamid-Reza; Hamidi-Esfahani, Zohreh; Alavi, Seyed-Mehdi; Varastegani, Boshra

    2014-01-01

    The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

  5. Histidine Regulates Seed Oil Deposition through Abscisic Acid Biosynthesis and β-Oxidation.

    PubMed

    Ma, Huimin; Wang, Shui

    2016-10-01

    The storage compounds are deposited into plant seeds during maturation. As the model oilseed species, Arabidopsis (Arabidopsis thaliana) has long been studied for seed oil deposition. However, the regulation of this process remains unclear. Through genetic screen with a seed oil body-specific reporter, we isolated low oil1 (loo1) mutant. LOO1 was mapped to HISTIDINE BIOSYNTHESIS NUMBER 1A (HISN1A). HISN1A catalyzes the first step of His biosynthesis. Oil significantly decreased, and conversely proteins markedly increased in hisn1a mutants, indicating that HISN1A regulates both oil accumulation and the oil-protein balance. HISN1A was predominantly expressed in embryos and root tips. Accordingly, the hisn1a mutants exhibited developmental phenotype especially of seeds and roots. Transcriptional profiling displayed that β-oxidation was the major metabolic pathway downstream of HISN1A β-Oxidation was induced in hisn1a mutants, whereas it was reduced in 35S:HISN1A-transgenic plants. In plants, seed storage oil is broken-down by β-oxidation, which is controlled by abscisic acid (ABA). We found that His activated genes of ABA biosynthesis and correspondingly advanced ABA accumulation. Exogenous ABA rescued the defects of hisn1a mutants, whereas mutation of ABA DEFICIENT2, a key enzyme in ABA biosynthesis, blocked the effect of His on β-oxidation, indicating that ABA mediates His regulation in β-oxidation. Intriguingly, structural analysis showed that a potential His-binding domain was present in the general amino acid sensors GENERAL CONTROL NON-DEREPRESSIBLE2 and PII, suggesting that His may serve as a signal molecule. Taken together, our study reveals that His promotes plant seed oil deposition through ABA biosynthesis and β-oxidation.

  6. 2-Keto acids based biosynthesis pathways for renewable fuels and chemicals.

    PubMed

    Tashiro, Yohei; Rodriguez, Gabriel M; Atsumi, Shota

    2015-03-01

    Global energy and environmental concerns have driven the development of biological chemical production from renewable sources. Biological processes using microorganisms are efficient and have been traditionally utilized to convert biomass (i.e., glucose) to useful chemicals such as amino acids. To produce desired fuels and chemicals with high yield and rate, metabolic pathways have been enhanced and expanded with metabolic engineering and synthetic biology approaches. 2-Keto acids, which are key intermediates in amino acid biosynthesis, can be converted to a wide range of chemicals. 2-Keto acid pathways were engineered in previous research efforts and these studies demonstrated that 2-keto acid pathways have high potential for novel metabolic routes with high productivity. In this review, we discuss recently developed 2-keto acid-based pathways.

  7. Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system.

    PubMed

    Doi, Yoshio; Ohtsuki, Takashi; Shimizu, Yoshihiro; Ueda, Takuya; Sisido, Masahiko

    2007-11-21

    Nonnatural amino acids have been introduced into proteins using expanded protein biosynthesis systems. However, some nonnatural amino acids, especially those containing large aromatic groups, are not efficiently incorporated into proteins. Reduced binding efficiency of aminoacylated tRNAs to elongation factor Tu (EF-Tu) is likely to limit incorporation of large amino acids. Our previous studies suggested that tRNAs carrying large nonnatural amino acids are bound less tightly to EF-Tu than natural amino acids. To expand the availability of nonnatural mutagenesis, EF-Tu from the E. coli translation system was improved to accept such large amino acids. We synthesized EF-Tu mutants, in which the binding pocket of the aminoacyl moiety of aminoacyl-tRNA was enlarged. L-1-Pyrenylalanine, L-2-pyrenylalanine, and DL-2-anthraquinonylalanine, which are hardly or only slightly incorporated with the wild-type EF-Tu, were successfully incorporated into a protein using these EF-Tu mutants.

  8. Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis.

    PubMed

    Joo, Hyoe-Jin; Yim, Yong-Hyeon; Jeong, Pan-Young; Jin, You-Xun; Lee, Jeong-Eui; Kim, Heekyeong; Jeong, Seul-Ki; Chitwood, David J; Paik, Young-Ki

    2009-07-29

    Caenorhabditis elegans excretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, the perception of which enables worms to enter the dauer state for long-term survival in an adverse environment. During the course of elucidation of the daumone biosynthetic pathway in which DHS-28 and DAF-22 are involved in peroxisomal beta-oxidation of VLCFAs (very long-chain fatty acids), we sought to investigate the physiological consequences of a deficiency in daumone biosynthesis in C. elegans. Our results revealed that two mutants, dhs-28(tm2581) and daf-22(ok693), lacked daumones and thus were dauer defective; this coincided with massive accumulation of fatty acyl-CoAs (up to 100-fold) inside worm bodies compared with levels in wild-type N2 worms. Furthermore, the deficiency in daumone biosynthesis and the massive accumulation of fatty acids and their acyl-CoAs caused severe developmental defects with reduced life spans (up to 30%), suggesting that daumone biosynthesis is be an essential part of C. elegans homoeostasis, affecting survival and maintenance of optimal physiological conditions by metabolizing some of the toxic non-permissible peroxisomal VLCFAs from the worm body in the form of readily excretable daumones.

  9. Traumatic Acid Reduces Oxidative Stress and Enhances Collagen Biosynthesis in Cultured Human Skin Fibroblasts.

    PubMed

    Jabłońska-Trypuć, Agata; Pankiewicz, Walentyn; Czerpak, Romuald

    2016-09-01

    Traumatic acid (TA) is a plant hormone (cytokinin) that in terms of chemical structure belongs to the group of fatty acids derivatives. It was isolated from Phaseolus vulgaris. TA activity and its influence on human cells and organism has not previously been the subject of research. The aim of this study was to examine the effects of TA on collagen content and basic oxidative stress parameters, such as antioxidative enzyme activity, reduced glutathione, thiol group content, and lipid peroxidation in physiological conditions. The results show a stimulatory effect of TA on tested parameters. TA caused a decrease in membrane phospholipid peroxidation and exhibited protective properties against ROS production. It also increases protein and collagen biosynthesis and its secretion into the culture medium. The present findings reveal that TA exhibits multiple and complex activity in fibroblast cells in vitro. TA, with its activity similar to unsaturated fatty acids, shows antioxidant and stimulatory effects on collagen biosynthesis. It is a potentially powerful agent with applications in the treatment of many skin diseases connected with oxidative stress and collagen biosynthesis disorders.

  10. Formula feeding potentiates docosahexaenoic and arachidonic acid biosynthesis in term and preterm baboon neonates.

    PubMed

    Sarkadi-Nagy, Eszter; Wijendran, Vasuki; Diau, Guan Yeu; Chao, Angela Chueh; Hsieh, Andrea T; Turpeinen, Anu; Lawrence, Peter; Nathanielsz, Peter W; Brenna, J Thomas

    2004-01-01

    Infant formulas supplemented with docosahexaenoic acid (DHA) and arachidonic acid (ARA) are now available in the United States; however, little is known about the factors that affect biosynthesis. Baboon neonates were assigned to one of four treatments: term, breast-fed; term, formula-fed; preterm (155 of 182 days gestation), formula-fed; and preterm, formula+DHA/ARA-fed. Standard formula had no DHA/ARA; supplemented formula had 0.61%wt DHA (0.3% of calories) and 1.21%wt ARA (0.6% of calories), and baboon breast milk contained 0.68 +/- 0.22%wt DHA and 0.62 +/- 0.12%wt ARA. At 14 days adjusted age, neonates received a combined oral dose of [U-13C]alpha-linolenic acid (LNA*) and [U-13C]linoleic acid (LA*), and tissues were analyzed 14 days after dose. Brain accretion of linolenic acid-derived DHA was approximately 3-fold greater for the formula groups than for the breast-fed group, and dietary DHA partially attenuated excess DHA synthesis among preterms. A similar, significant pattern was found in other organs. Brain linoleic acid-derived ARA accretion was significantly greater in the unsupplemented term group but not in the preterm groups compared with the breast-fed group. These data show that formula potentiates the biosynthesis/accretion of DHA/ARA in term and preterm neonates compared with breast-fed neonates and that the inclusion of DHA/ARA in preterm formula partially restores DHA/ARA biosynthesis to lower, breast-fed levels. Current formula DHA concentrations are inadequate to normalize long-chain polyunsaturated fatty acids synthesis to that of breast-fed levels.

  11. Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants.

    PubMed

    Rogalski, Marcelo; Carrer, Helaine

    2011-06-01

    The ability to manipulate plant fatty acid biosynthesis by using new biotechnological approaches has allowed the production of transgenic plants with unusual fatty acid profile and increased oil content. This review focuses on the production of very long chain polyunsaturated fatty acids (VLCPUFAs) and the increase in oil content in plants using molecular biology tools. Evidences suggest that regular consumption of food rich in VLCPUFAs has multiple positive health benefits. Alternative sources of these nutritional fatty acids are found in cold-water fishes. However, fish stocks are in severe decline because of decades of overfishing, and also fish oils can be contaminated by the accumulation of toxic compounds. Recently, there is also an increase in oilseed use for the production of biofuels. This tendency is partly associated with the rapidly rising costs of petroleum, increased concern about the environmental impact of fossil oil and the attractive need to develop renewable sources of fuel. In contrast to this scenario, oil derived from crop plants is normally contaminant free and less environmentally aggressive. Genetic engineering of the plastid genome (plastome) offers a number of attractive advantages, including high-level foreign protein expression, marker-gene excision and transgene containment because of maternal inheritance of plastid genome in most crops. Here, we describe the possibility to improve fatty acid biosynthesis in plastids, production of new fatty acids and increase their content in plants by genetic engineering of plastid fatty acid biosynthesis via plastid transformation. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  12. Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis

    PubMed Central

    Chen, Hsi-Chuan; Li, Quanzi; Shuford, Christopher M.; Liu, Jie; Muddiman, David C.; Sederoff, Ronald R.; Chiang, Vincent L.

    2011-01-01

    The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (Vmax/km) for any of the complexes is 70–6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex–mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis. PMID:22160716

  13. BnWRI1 coordinates fatty acid biosynthesis and photosynthesis pathways during oil accumulation in rapeseed.

    PubMed

    Wu, Xue-Long; Liu, Zhi-Hong; Hu, Zhang-Hua; Huang, Rui-Zhi

    2014-06-01

    Photosynthesis in "green" seeds, such as rapeseed, soybean, and Arabidopsis, plays a substantial role in the improved efficiency of oil accumulation. However, the molecular mechanism underpinning the coordinated expression of fatty acid (FA) biosynthesis- and photosynthesis-related genes in such developing seeds remains to be elucidated. Here, we found that seed-specific overexpression of BnWRI1, a WRI1 homolog from rapeseed (Brassica napus cv. ZGY2), results in enhanced chlorophyll content in developing seeds and increased oil content and seed mass in matured seeds. BnWRI1 was co-expressed with BnBCCP and BnCAB, two marker genes of FA biosynthesis and photosynthesis during seed development, respectively. Overexpression of BnWRI1 increased expression of both marker genes. Further, the nuclear-localized BnWRI1 protein was found to act as a transcription activator. It could bind to the GT1-element and/or GCC-box, which are widespread in the upstream regions of genes involved in FA biosynthesis and photosynthesis pathways. Accordingly, BnWRI1 could interact with promoters of BCCP2 and LHB1B2 in vivo. These results suggested that BnWRI1 may coordinate FA biosynthesis and photosynthesis pathways in developing seeds via directly stimulating expression of GT1-element and/or GCC-box containing genes.

  14. Biosynthesis of car1ssol and carissic Acid.

    PubMed

    Nizami, S S; Khan, M A; Naim, Z; Islam, M N; Azeem, S W

    1993-01-01

    Carissa carandas belongs to family apocynaceae which consists of 300 genera and 1000 species. It is a large shrub with simple thorn and commonly cultivated throughout Pakistan for hedges and is called "Kakronda". The different parts of this plant have been used for various systems of medicine (Kirtikar et al., 1993). Cardiotonic activity was found in root of this plant (Rastogi et al., 1966; Rastogi et al., 1967; Vohra et al., 1963). This plant has been mentioned in the old chemical literature as purgative, stomachic, antihelmintics and antidote for snake-bite (Kirtikar., 1933). The physical characteristics of oil from the fruits of Carissa carandas were determined by using standard methods. In addition to this a study of sugars and amino acids from the fruits of this plant was also undertaken by the present authors (Naim et al., 1986). Our studies in the chemical investigation on this plant had led to the isolation of two new triterpene carissol Ia (Naim et al., 1985) and carissic acid lb (Naim et al., 1988).

  15. A novel bile acid biosynthesis defect due to a deficiency of peroxisomal ABCD3.

    PubMed

    Ferdinandusse, Sacha; Jimenez-Sanchez, Gerardo; Koster, Janet; Denis, Simone; Van Roermund, Carlo W; Silva-Zolezzi, Irma; Moser, Ann B; Visser, Wouter F; Gulluoglu, Mine; Durmaz, Ozlem; Demirkol, Mubeccel; Waterham, Hans R; Gökcay, Gülden; Wanders, Ronald J A; Valle, David

    2015-01-15

    ABCD3 is one of three ATP-binding cassette (ABC) transporters present in the peroxisomal membrane catalyzing ATP-dependent transport of substrates for metabolic pathways localized in peroxisomes. So far, the precise function of ABCD3 is not known. Here, we report the identification of the first patient with a defect of ABCD3. The patient presented with hepatosplenomegaly and severe liver disease and showed a striking accumulation of peroxisomal C27-bile acid intermediates in plasma. Investigation of peroxisomal parameters in skin fibroblasts revealed a reduced number of enlarged import-competent peroxisomes. Peroxisomal beta-oxidation of C26:0 was normal, but beta-oxidation of pristanic acid was reduced. Genetic analysis revealed a homozygous deletion at the DNA level of 1758bp, predicted to result in a truncated ABCD3 protein lacking the C-terminal 24 amino acids (p.Y635NfsX1). Liver disease progressed and the patient required liver transplantation at 4 years of age but expired shortly after transplantation. To corroborate our findings in the patient, we studied a previously generated Abcd3 knockout mouse model. Abcd3-/- mice accumulated the branched chain fatty acid phytanic acid after phytol loading. In addition, analysis of bile acids revealed a reduction of C24 bile acids, whereas C27-bile acid intermediates were significantly increased in liver, bile and intestine of Abcd3-/- mice. Thus, both in the patient and in Abcd3-/- mice, there was evidence of a bile acid biosynthesis defect. In conclusion, our studies show that ABCD3 is involved in transport of branched-chain fatty acids and C27 bile acids into the peroxisome and that this is a crucial step in bile acid biosynthesis. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Gene-Enzyme Relationships of Aromatic Amino Acid Biosynthesis in Higher Plants

    SciTech Connect

    2002-08-12

    Inhibition studies of amino acids in Nicotiana silvestris suspension cells gave clues to the difficulties for obtaining mutants deficient in post prephenate pathway proteins of aromatic amino acid biosynthesis (prephenate aminotransferase, arogenate dehydrogenase and arogenate dehydratase). Such mutants, if successfully obtained, would allow gene-enzyme relationships of aromatic amino acid proteins to be studied. We found that amino acids were inhibitory toward plant cell growth, and thus were unable to rescue analog resistant mutants. Toxicity of all amino acids toward exponentially dividing Nicotiana silvestris suspension cultured cells was monitored by following growth rates. Except for L-glutamine, all 19 protein amino acids inhibited cell growth. Inhibition of growth progressed to cell deterioration. Electron microscopy showed that amino acids triggered a state of cell shrinkage that eventually degenerated to total cellular disorganization. L-glutamine was not only an effective agent for prevention of amino acid toxicity, but enhanced the final growth yield. L-glutamine also was able to completely reverse inhibition effects in cells that had been in the slowed exponential phase. Two types of inhibition occurred and we have proposed that any amino acid inhibition that can be completely antagonized by L-glutamine be called ''general amino acid inhibition''. ''Specific amino acid inhibition'' resulting from particular pathway imbalances caused by certain exogenous amino acids, can be recognized and studied in the presence of L-glutamine which can abolishes the complication effects of general amino acid inhibition.

  17. Genome-Wide Association Study of Genetic Control of Seed Fatty Acid Biosynthesis in Brassica napus

    PubMed Central

    Gacek, Katarzyna; Bayer, Philipp E.; Bartkowiak-Broda, Iwona; Szala, Laurencja; Bocianowski, Jan; Edwards, David; Batley, Jacqueline

    2017-01-01

    Fatty acids and their composition in seeds determine oil value for nutritional or industrial purposes and also affect seed germination as well as seedling establishment. To better understand the genetic basis of seed fatty acid biosynthesis in oilseed rape (Brassica napus L.) we applied a genome-wide association study, using 91,205 single nucleotide polymorphisms (SNPs) characterized across a mapping population with high-resolution skim genotyping by sequencing (SkimGBS). We identified a cluster of loci on chromosome A05 associated with oleic and linoleic seed fatty acids. The delineated genomic region contained orthologs of the Arabidopsis thaliana genes known to play a role in regulation of seed fatty acid biosynthesis such as Fatty acyl-ACP thioesterase B (FATB) and Fatty Acid Desaturase (FAD5). This approach allowed us to identify potential functional genes regulating fatty acid composition in this important oil producing crop and demonstrates that this approach can be used as a powerful tool for dissecting complex traits for B. napus improvement programs. PMID:28163710

  18. Genome-Wide Association Study of Genetic Control of Seed Fatty Acid Biosynthesis in Brassica napus.

    PubMed

    Gacek, Katarzyna; Bayer, Philipp E; Bartkowiak-Broda, Iwona; Szala, Laurencja; Bocianowski, Jan; Edwards, David; Batley, Jacqueline

    2016-01-01

    Fatty acids and their composition in seeds determine oil value for nutritional or industrial purposes and also affect seed germination as well as seedling establishment. To better understand the genetic basis of seed fatty acid biosynthesis in oilseed rape (Brassica napus L.) we applied a genome-wide association study, using 91,205 single nucleotide polymorphisms (SNPs) characterized across a mapping population with high-resolution skim genotyping by sequencing (SkimGBS). We identified a cluster of loci on chromosome A05 associated with oleic and linoleic seed fatty acids. The delineated genomic region contained orthologs of the Arabidopsis thaliana genes known to play a role in regulation of seed fatty acid biosynthesis such as Fatty acyl-ACP thioesterase B (FATB) and Fatty Acid Desaturase (FAD5). This approach allowed us to identify potential functional genes regulating fatty acid composition in this important oil producing crop and demonstrates that this approach can be used as a powerful tool for dissecting complex traits for B. napus improvement programs.

  19. Ferulic acid, an efficient inhibitor of type B trichothecene biosynthesis and Tri gene expression in Fusarium liquid cultures.

    PubMed

    Boutigny, Anne-Laure; Barreau, Christian; Atanasova-Penichon, Vessela; Verdal-Bonnin, Marie-Noëlle; Pinson-Gadais, Laëtitia; Richard-Forget, Florence

    2009-01-01

    The effect of ferulic acid, the most abundant phenolic acid in wheat bran, was studied in vitro on type B trichothecene biosynthesis by Fusarium. It was demonstrated that ferulic acid is an efficient inhibitor of mycotoxin production by all strains of Fusarium tested, including different chemotypes and species. To analyse the mechanism of toxin biosynthesis inhibition by ferulic acid, expression of representative Tri genes, involved in the trichothecene biosynthesis pathway, was monitored by real-time RT-PCR. A decrease in the level of Tri gene expression was measured, suggesting that inhibition of toxin synthesis by ferulic acid could be regulated at the transcriptional level. Moreover, toxin production was shown to be reduced proportionally to the initial amount of ferulic acid added in the culture medium. Addition of ferulic acid either at the spore germination step or to a mycelial culture resulted in the same final inhibitory effect on mycotoxin accumulation. A cumulative inhibitory effect on trichothecene biosynthesis was even observed with successive supplementation of ferulic acid. Ferulic acid, which content varies among wheat varieties, could then play an important role in modulating trichothecene biosynthesis by Fusarium in some wheat varieties.

  20. Abscisic acid biosynthesis in water-stressed leaves

    SciTech Connect

    Li, Yi.

    1989-01-01

    Although abscisic acid (ABA) was discovered 30 years ago, very little is known about its biosynthetic pathway in higher plants. Two hypotheses have been proposed: (i) a direct pathway involving only C-15 intermediates like farnesyl pyrophosphate, (ii) an indirect pathway involving C-40 intermediates like the xanthophylls. When {sup 14}CO{sub 2} was fed into greened bean plants, the {sup 14}C specific activity of ABA was always lower than those in xanthophylls, such as violaxanthin and lutein, regardless of {sup 12}CO{sub 2} chase periods. The ABA accumulation in green leaves was not affected by fluridone when plants were stressed once, but the {sup 14}C incorporation into ABA was inhibited to the same extent as those of xanthophylls. The incorporation of {sup 18}O into the ABA ring when violaxanthin was labeled by {sup 18}O in vivo via the violaxanthin cycle indicates that at least a portion of ABA was derived from {sup 18}O-labeled violaxanthin during water stress.

  1. The shikimate pathway and aromatic amino Acid biosynthesis in plants.

    PubMed

    Maeda, Hiroshi; Dudareva, Natalia

    2012-01-01

    L-tryptophan, L-phenylalanine, and L-tyrosine are aromatic amino acids (AAAs) that are used for the synthesis of proteins and that in plants also serve as precursors of numerous natural products, such as pigments, alkaloids, hormones, and cell wall components. All three AAAs are derived from the shikimate pathway, to which ≥30% of photosynthetically fixed carbon is directed in vascular plants. Because their biosynthetic pathways have been lost in animal lineages, the AAAs are essential components of the diets of humans, and the enzymes required for their synthesis have been targeted for the development of herbicides. This review highlights recent molecular identification of enzymes of the pathway and summarizes the pathway organization and the transcriptional/posttranscriptional regulation of the AAA biosynthetic network. It also identifies the current limited knowledge of the subcellular compartmentalization and the metabolite transport involved in the plant AAA pathways and discusses metabolic engineering efforts aimed at improving production of the AAA-derived plant natural products.

  2. Steroid hormones control on nucleic acid biosynthesis in skeletal muscle.

    PubMed

    Burtea, C; Lefter, C; Butnaru, F; David, A

    1992-01-01

    The medical, agricultural and scientific interest of the muscular tissue is well established in the literature. Its formation and function are essential for survival. It is also known that steroid hormones are involved in the growth, development and maturation of skeletal muscles (sexual hormones) as well as in the process of body adjustment to the stress factors in the environment (glucocorticoid hormones). Starting from these considerations, our experiment has made an attempt to clarify part of the mechanisms involved in the action of steroid hormones at muscle level. On this purpose, 3H thymidine and 3H uridine incorporation was followed up in various types of skeletal muscles (femoral biceps, diaphragmatic, psoas) from rats treated with steroid hormones. Though known as anabolic hormones, sexual hormones did not induce significant and persistent changes in the nucleic acid synthesis (NAS), except for testosterone which enhanced RNA synthesis only in the level femoral muscle after 21 days of administration. Progesterone and the glucocorticoid hormones are known as hormones of proteic catabolism but it seems that this effect is more marked in muscles whose structure predominantly consists of white fibres (rapid muscles), as confirmed by our experiment.

  3. Biosynthesis of very long chain fatty acids in Trypanosoma cruzi.

    PubMed

    Livore, Verónica I; Uttaro, Antonio D

    2015-01-01

    Trypanosoma brucei and Trypanosoma cruzi showed similar fatty acid (FA) compositions, having a high proportion of unsaturated FAs, mainly 18:2Δ9,12 (23-39%) and 18:1Δ9 (11-17%). C22 polyunsaturated FAs are in significant amounts only in T. brucei (12-20%) but represent a mere 2% of total FAs in T. cruzi. Both species have also similar profiles of medium- and long-chain saturated FAs, from 14:0 to 20:0. Interestingly, procyclic and bloodstream forms of T. brucei lack very long chain FAs (VLCFAs), whereas epimastigotes and trypomastigotes of T. cruzi contain 22:0 (0.1-0.2%), 24:0 (1.5-2%), and 26:0 (0.1-0.2%). This is in agreement with the presence of an additional FA elongase gene (TcELO4) in T. cruzi. TcELO4 was expressed in a Saccharomyces cerevisiae mutant lacking the endogenous ScELO3, rescuing the synthesis of saturated and hydroxylated C26 FAs in the yeast. Expression of TcELO4 also rescued the synthetic lethality of a ScELO2, ScELO3 double mutation, and the VLCFA profile of the transformed yeast was similar to that found in T. cruzi. By identifying TcELO4 as the enzyme responsible for the elongation of FA from 16:0 and 18:0 up to 26:0, with 24:0 being the preferred product, this work completed the characterization of FA elongases in Trypanosoma spp.

  4. D-Lactic acid biosynthesis from biomass-derived sugars via Lactobacillus delbrueckii fermentation.

    PubMed

    Zhang, Yixing; Vadlani, Praveen V

    2013-12-01

    Poly-lactic acid (PLA) derived from renewable resources is considered to be a good substitute for petroleum-based plastics. The number of poly L-lactic acid applications is increased by the introduction of a stereocomplex PLA, which consists of both poly-L and D-lactic acid and has a higher melting temperature. To date, several studies have explored the production of L-lactic acid, but information on biosynthesis of D-lactic acid is limited. Pulp and corn stover are abundant, renewable lignocellulosic materials that can be hydrolyzed to sugars and used in biosynthesis of D-lactic acid. In our study, saccharification of pulp and corn stover was done by cellulase CTec2 and sugars generated from hydrolysis were converted to D-lactic acid by a homofermentative strain, L. delbrueckii, through a sequential hydrolysis and fermentation process (SHF) and a simultaneous saccharification and fermentation process (SSF). 36.3 g L(-1) of D-lactic acid with 99.8 % optical purity was obtained in the batch fermentation of pulp and attained highest yield and productivity of 0.83 g g(-1) and 1.01 g L(-1) h(-1), respectively. Luedeking-Piret model described the mixed growth-associated production of D-lactic acid with a maximum specific growth rate 0.2 h(-1) and product formation rate 0.026 h(-1), obtained for this strain. The efficient synthesis of D-lactic acid having high optical purity and melting point will lead to unique stereocomplex PLA with innovative applications in polymer industry.

  5. Characterization of a Pipecolic Acid Biosynthesis Pathway Required for Systemic Acquired Resistance.

    PubMed

    Ding, Pingtao; Rekhter, Dmitrij; Ding, Yuli; Feussner, Kirstin; Busta, Lucas; Haroth, Sven; Xu, Shaohua; Li, Xin; Jetter, Reinhard; Feussner, Ivo; Zhang, Yuelin

    2016-10-01

    Systemic acquired resistance (SAR) is an immune response induced in the distal parts of plants following defense activation in local tissue. Pipecolic acid (Pip) accumulation orchestrates SAR and local resistance responses. Here, we report the identification and characterization of SAR-DEFICIENT4 (SARD4), which encodes a critical enzyme for Pip biosynthesis in Arabidopsis thaliana Loss of function of SARD4 leads to reduced Pip levels and accumulation of a Pip precursor, Δ(1)-piperideine-2-carboxylic acid (P2C). In Escherichia coli, expression of the aminotransferase ALD1 leads to production of P2C and addition of SARD4 results in Pip production, suggesting that a Pip biosynthesis pathway can be reconstituted in bacteria by coexpression of ALD1 and SARD4. In vitro experiments showed that ALD1 can use l-lysine as a substrate to produce P2C and P2C is converted to Pip by SARD4. Analysis of sard4 mutant plants showed that SARD4 is required for SAR as well as enhanced pathogen resistance conditioned by overexpression of the SAR regulator FLAVIN-DEPENDENT MONOOXYGENASE1. Compared with the wild type, pathogen-induced Pip accumulation is only modestly reduced in the local tissue of sard4 mutant plants, but it is below detection in distal leaves, suggesting that Pip is synthesized in systemic tissue by SARD4-mediated reduction of P2C and biosynthesis of Pip in systemic tissue contributes to SAR establishment.

  6. Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops.

    PubMed

    Tan, S; Evans, R; Singh, B

    2006-03-01

    Acetohydroxyacid synthase (AHAS) inhibitors interfere with branched-chain amino acid biosynthesis by inhibiting AHAS. Glyphosate affects aromatic amino acid biosynthesis by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glufosinate inhibits glutamine synthetase and blocks biosynthesis of glutamine. AHAS gene variants that confer tolerance to AHAS inhibitors have been discovered in plants through selection or mutagenesis. Imidazolinone-tolerant crops have been commercialized based on these AHAS gene variants. A modified maize EPSPS gene and CP4-EPSPS gene from Agrobacterium sp. have been used to transform plants for target-based tolerance to glyphosate. A gox gene isolated from Ochrobactrum anthropi has also been employed to encode glyphosate oxidoreductase to detoxify glyphosate in plants. Glyphosate-tolerant crops with EPSPS transgene alone or both EPSPS and gox transgenes have been commercialized. Similarly, bar and pat genes isolated from Streptomyces hygroscopicus and S. viridochromogenes, respectively, have been inserted into plants to encode phosphinothricin N-acetyltransferase to detoxify glufosinate. Glufosinate-tolerant crops have been commercialized using one of these two transgenes.

  7. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    PubMed Central

    Braga, Daniel; Hoffmeister, Dirk

    2016-01-01

    Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis. PMID:28144348

  8. PGC-1alpha activates CYP7A1 and bile acid biosynthesis.

    PubMed

    Shin, Dong-Ju; Campos, Jose A; Gil, Gregorio; Osborne, Timothy F

    2003-12-12

    Cholesterol 7-alpha-hydroxylase (CYP7A1) is the key enzyme that commits cholesterol to the neutral bile acid biosynthesis pathway and is highly regulated. In the current studies, we have uncovered a role for the transcriptional co-activator PGC-1alpha in CYP7A1 gene transcription. PGC-1alpha plays a vital role in adaptive thermogenesis in brown adipose tissue and stimulates genes important to mitochondrial function and oxidative metabolism. It is also involved in the activation of hepatic gluconeogenesic gene expression during fasting. Because the mRNA for CYP7A1 was also induced in mouse liver by fasting, we reasoned that PGC-1alpha might be an important co-activator for CYP7A1. Here we show that PGC-1alpha and CYP7A1 are also co-induced in livers of mice in response to streptozotocin induced diabetes. Additionally, infection of cultured HepG2 cells with a recombinant adenovirus expressing PGC-1alpha directly activates CYP7A1 gene expression and increases bile acid biosynthesis as well. Furthermore, we show that PGC-1alpha activates the CYP7A1 promoter directly in transient transfection assays in cultured cells. Thus, PGC-1alpha is a key activator of CYP7A1 and bile acid biosynthesis and is likely responsible for the fasting and diabetes dependent induction of CYP7A1. PGC-1alpha has already been shown to be a critical activator of several other oxidative processes including adaptive thermogenesis and fatty acid oxidation. Our studies provide further evidence of the fundamental role played by PGC-1alpha in oxidative metabolism and define PGC-1alpha as a link between diabetes and bile acid metabolism.

  9. Signal Biosynthesis Inhibition with Ambuic Acid as a Strategy To Target Antibiotic-Resistant Infections.

    PubMed

    Todd, Daniel A; Parlet, Corey P; Crosby, Heidi A; Malone, Cheryl L; Heilmann, Kristopher P; Horswill, Alexander R; Cech, Nadja B

    2017-08-01

    There has been major interest by the scientific community in antivirulence approaches against bacterial infections. However, partly due to a lack of viable lead compounds, antivirulence therapeutics have yet to reach the clinic. Here we investigate the development of an antivirulence lead targeting quorum sensing signal biosynthesis, a process that is conserved in Gram-positive bacterial pathogens. Some preliminary studies suggest that the small molecule ambuic acid is a signal biosynthesis inhibitor. To confirm this, we constructed a methicillin-resistant Staphylococcus aureus (MRSA) strain that decouples autoinducing peptide (AIP) production from regulation and demonstrate that AIP production is inhibited in this mutant. Quantitative mass spectrometric measurements show that ambuic acid inhibits signal biosynthesis (50% inhibitory concentration [IC50] of 2.5 ± 0.1 μM) against a clinically relevant USA300 MRSA strain. Quantitative real-time PCR confirms that this compound selectively targets the quorum sensing regulon. We show that a 5-μg dose of ambuic acid reduces MRSA-induced abscess formation in a mouse model and verify its quorum sensing inhibitory activity in vivo Finally, we employed mass spectrometry to identify or confirm the structure of quorum sensing signaling peptides in three strains each of S. aureus and Staphylococcus epidermidis and single strains of Enterococcus faecalis, Listeria monocytogenes, Staphylococcus saprophyticus, and Staphylococcus lugdunensis By measuring AIP production by these strains, we show that ambuic acid possesses broad-spectrum efficacy against multiple Gram-positive bacterial pathogens but does not inhibit quorum sensing in some commensal bacteria. Collectively, these findings demonstrate the promise of ambuic acid as a lead for the development of antivirulence therapeutics. Copyright © 2017 American Society for Microbiology.

  10. [Gene cloning and bioinformatics analysis of new gene for chlorogenic acid biosynthesis of Lonicera hypoglauca].

    PubMed

    Yu, Shu-lin; Huang, Lu-qi; Yuan, Yuan; Qi, Lin-jie; Liu, Da-hui

    2015-03-01

    To obtain the key genes for chlorogenic acid biosynthesis of Lonicera hypoglauca, four new genes ware obtained from the our dataset of L. hypoglauca. And we also predicted the structure and function of LHPAL4, LHHCT1 , LHHCT2 and LHHCT3 proteins. The phylogenetic tree showed that LHPAL4 was closely related with LHPAL1, LHHCT1 was closely related with LHHCT3, LHHCT2 clustered into a single group. By Real-time PCR to detect the gene expressed level in different organs of L. hypoglauca, we found that the transcripted level of LHPAL4, LHHCT1 and LHHCT3 was the highest in defeat flowers, and the transcripted level of LHHCT2 was the highest in leaves. These result provided a basis to further analysis the mechanism of active ingredients in different organs, as well as the element for in vitro biosynthesis of active ingredients.

  11. Mechanism of biosynthesis of unsaturated fatty acids in Pseudomonas sp. strain E-3, a psychrotrophic bacterium.

    PubMed Central

    Wada, M; Fukunaga, N; Sasaki, S

    1989-01-01

    Biosynthesis of palmitic, palmitoleic, and cis-vaccenic acids in Pseudomonas sp. strain E-3 was investigated with in vitro and in vivo systems. [1-14C]palmitic acid was aerobically converted to palmitoleate and cis-vaccenate, and the radioactivities on their carboxyl carbons were 100 and 43%, respectively, of the total radioactivity in the fatty acids. Palmitoyl coenzyme A desaturase activity was found in the membrane fraction. [1-14C]stearic acid was converted to octadecenoate and C16 fatty acids. The octadecenoate contained oleate and cis-vaccenate, but only oleate was produced in the presence of cerulenin. [1-14C]lauric acid was aerobically converted to palmitate, palmitoleate, and cis-vaccenate. Under anaerobic conditions, palmitate (62%), palmitoleate (4%), and cis-vaccenate (34%) were produced from [1-14C]acetic acid, while they amounted to 48, 39, and 14%, respectively, under aerobic conditions. In these incorporation experiments, 3 to 19% of the added radioactivity was detected in released 14CO2, indicating that part of the added fatty acids were oxidatively decomposed. Partially purified fatty acid synthetase produced saturated and unsaturated fatty acids with chain lengths of C10 to C18. These results indicated that both aerobic and anaerobic mechanisms for the synthesis of unsaturated fatty acid are operating in this bacterium. PMID:2753856

  12. Biosynthesis of the 1,3,4,6-hexanetetracarboxylic acid subunit of methanofuran

    SciTech Connect

    White, R.H.

    1987-06-02

    /sup 2/H- and /sup 13/C-labeled precursors were used to establish the pathway for the biosynthesis of the 1,3,4,6-hexanetetracarboxylic acid (TCA) component of methanofuran, which is found in some methanogenic bacteria. The extent and position of incorporation of label into TCA were measured from the mass spectrum of the tetramethyl ester of TCA that was prepared from methanofuran present in cells grown in the presence of labeled acetate. (2,2,2-/sup 2/H/sub 3/)Acetate was found to incorporate deuterium into two separate sites of the TCA molecule, with one on each side of the symmetrical molecule. One site was found to be labeled 37% with deuterium, the same as for the glutamic acid present in the cells; the other site was labeled 77% with deuterium, the same as for the malonate-derived compounds in the cells. An analog of TCA, 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid, found in methanofuran isolated from Methanobrevibacter smithii, was found to incorporate /sup 13/C/sub 2/ units from (1,2-/sup 13/C/sub 2/)acetate into three positions of the molecule. One of the acetate /sup 13/C/sub 2/ units was incorporated into the non-hydroxyl-containing side of the molecule (carbons 4, 5, and 6 and the C-6 carboxylic acid group), and two acetate units were incorporated into the hydroxyl-containing side of the molecule (carbons 1, 2, and 3 and the C-1 carboxylic acid group). On the basis of this and additional information, it is concluded that TCA is biosynthesized by the condensation of ..cap alpha..-ketoglutaric acid with malonic acid to form 1,1,2,4-butanetetracarboxylic acid, which is further condensed with a second molecule of malonate, in a series of reactions analogous to those observed during fatty acid biosynthesis, to form TCA.

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

    PubMed Central

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

    2015-01-01

    Phytic acid (PA) biosynthesis pathway genes were reported from multiple crop species. PA accumulation was enhanced during grain filling and at that time, hormones like Abscisic acid (ABA) and Gibberellic acid (GA3) interplay to control the process of seed development. Regulation of wheat PA pathway genes has not yet been reported in seeds. In an attempt to find the clues for the regulation by hormones, the promoter region of wheat PA pathway genes was analyzed for the presence of cis-elements. Multiple cis-elements of those known to be involved for ABA, GA3, salicylic acid (SA), and cAMP sensing were identified in the promoters of PA pathway genes. Eight genes (TaIMP, TaITPK1-4, TaPLC1, TaIPK2 and TaIPK1) involved in the wheat PA biosynthesis pathway were selected for the expression studies. The temporal expression response was studied in seeds treated with ABA and GA3 using quantitative real time PCR. Our results suggested that exogenous application of ABA induces few PA pathway genes in wheat grains. Comparison of expression profiles for PA pathway for GA3 and ABA suggested the antagonistic regulation of certain genes. Additionally, to reveal stress responses of wheat PA pathway genes, expression was also studied in the presence of SA and cAMP. Results suggested SA specific differential expression of few genes, whereas, overall repression of genes was observed in cAMP treated samples. This study is an effort to understand the regulation of PA biosynthesis genes in wheat. PMID:27135330

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

    PubMed

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

    2015-06-11

    Phytic acid (PA) biosynthesis pathway genes were reported from multiple crop species. PA accumulation was enhanced during grain filling and at that time, hormones like Abscisic acid (ABA) and Gibberellic acid (GA₃) interplay to control the process of seed development. Regulation of wheat PA pathway genes has not yet been reported in seeds. In an attempt to find the clues for the regulation by hormones, the promoter region of wheat PA pathway genes was analyzed for the presence of cis-elements. Multiple cis-elements of those known to be involved for ABA, GA₃, salicylic acid (SA), and cAMP sensing were identified in the promoters of PA pathway genes. Eight genes (TaIMP, TaITPK1-4, TaPLC1, TaIPK2 and TaIPK1) involved in the wheat PA biosynthesis pathway were selected for the expression studies. The temporal expression response was studied in seeds treated with ABA and GA₃ using quantitative real time PCR. Our results suggested that exogenous application of ABA induces few PA pathway genes in wheat grains. Comparison of expression profiles for PA pathway for GA₃ and ABA suggested the antagonistic regulation of certain genes. Additionally, to reveal stress responses of wheat PA pathway genes, expression was also studied in the presence of SA and cAMP. Results suggested SA specific differential expression of few genes, whereas, overall repression of genes was observed in cAMP treated samples. This study is an effort to understand the regulation of PA biosynthesis genes in wheat.

  15. An allene oxide and 12-oxophytodienoic acid are key intermediates in jasmonic acid biosynthesis by Fusarium oxysporum.

    PubMed

    Oliw, Ernst H; Hamberg, Mats

    2017-08-01

    Fungi can produce jasmonic acid (JA) and its isoleucine conjugate in large quantities, but little is known about the biosynthesis. Plants form JA from 18:3n-3 by 13S-lipoxygenase (LOX), allene oxide synthase, and allene oxide cyclase. Shaking cultures of Fusarium oxysporum f. sp. tulipae released over 200 mg of jasmonates per liter. Nitrogen powder of the mycelia expressed 10R-dioxygenase-epoxy alcohol synthase activities, which was confirmed by comparison with the recombinant enzyme. The 13S-LOX of F. oxysporum could not be detected in the cell-free preparations. Incubation of mycelia in phosphate buffer with [17,17,18,18,18-(2)H5]18:3n-3 led to biosynthesis of a [(2)H5]12-oxo-13-hydroxy-9Z,15Z-octadecadienoic acid (α-ketol), [(2)H5]12-oxo-10,15Z-phytodienoic acid (12-OPDA), and [(2)H5]13-keto- and [(2)H5]13S-hydroxyoctadecatrienoic acids. The α-ketol consisted of 90% of the 13R stereoisomer, suggesting its formation by nonenzymatic hydrolysis of an allene oxide with 13S configuration. Labeled and unlabeled 12-OPDA were observed following incubation with 0.1 mM [(2)H5]18:3n-3 in a ratio from 0.4:1 up to 47:1 by mycelia of liquid cultures of different ages, whereas 10 times higher concentration of [(2)H5]13S-hydroperoxyoctadecatrienoic acid was required to detect biosynthesis of [(2)H5]12-OPDA. The allene oxide is likely formed by a cytochrome P450 or catalase-related hydroperoxidase. We conclude that F. oxysporum, like plants, forms jasmonates with an allene oxide and 12-OPDA as intermediates. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  16. Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis.

    PubMed

    Creelman, R A; Bell, E; Mullet, J E

    1992-07-01

    Several lines of evidence indicate that abscisic acid (ABA) is derived from 9'-cis-neoxanthin or 9'-cis-violaxanthin with xanthoxin as an intermediate. (18)O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11', 12') double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.

  17. De novo amino acid biosynthesis in potato tubers is regulated by sucrose levels.

    PubMed

    Roessner-Tunali, Ute; Urbanczyk-Wochniak, Ewa; Czechowski, Tomasz; Kolbe, Anna; Willmitzer, Lothar; Fernie, Alisdair R

    2003-10-01

    Plant growth and development are strongly dependent on sink-source interactions. In the majority of plants, sucrose (Suc) is the dominant form in which photo-assimilate is transported from source to sinks. Although the effects of Suc on photosynthetic metabolism have been intensively studied, the effect of Suc supply on metabolism in sink organs has received relatively little attention. For this reason, we performed a detailed characterization of the metabolism of potato (Solanum tuberosum) plants in which the Suc supply to the tuber was restricted by genetic or environmental perturbation. These characterizations revealed a clear inverse relationship between the levels of Suc and free amino acids. When data obtained from this study were considered alongside our previous work, a negative correlation between tuber Suc and amino acid content became apparent. Analysis of the transcript levels of key enzymes involved in amino acid biosynthesis revealed that several of these were increased under these conditions. Taken together, these data strongly suggest that Suc regulates amino acid biosynthesis in storage tissues such as potato tubers, most probably at the level of transcription.

  18. Aerobic biosynthesis of hydrocinnamic acids in Escherichia coli with a strictly oxygen-sensitive enoate reductase.

    PubMed

    Sun, Jing; Lin, Yuheng; Shen, Xiaolin; Jain, Rachit; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

    2016-05-01

    3-Phenylpropionic acid (3PPA) and 3-(4-hydroxyphenyl) propionic acid (HPPA) are important commodity aromatic acids widely used in food, pharmaceutical and chemical industries. Currently, 3PPA and HPPA are mainly manufactured through chemical synthesis, which contains multiple steps involving toxic solvents and catalysts harmful to environment. Therefore, replacement of such existing petroleum-derived approaches with simple and environmentally friendly biological processes is highly desirable for manufacture of these chemicals. Here, for the first time we demonstrated the de novo biosynthesis of 3PPA and HPPA using simple carbon sources in E. coli by extending the cinnamic acids biosynthesis pathways through biological hydrogenation. We first screened 11 2-enoate reductases (ER) from nine microorganisms, leading to efficient conversion of cinnamic acid and p-coumaric acid to 3PPA and HPPA, respectively. Surprisingly, we found a strictly oxygen-sensitive Clostridia ER capable of functioning efficiently in E. coli even under aerobic conditions. On this basis, reconstitution of the full pathways led to the de novo production of 3PPA and HPPA and the accumulation of the intermediates (cinnamic acid and p-coumaric acid) with cell toxicity. To address this problem, different expression strategies were attempted to optimize individual enzyme׳s expression level and minimize intermediates accumulation. Finally, the titers of 3PPA and HPPA reached 366.77mg/L and 225.10mg/L in shake flasks, respectively. This study not only demonstrated the potential of microbial approach as an alternative to chemical process, but also proved the possibility of using oxygen-sensitive enzymes under aerobic conditions.

  19. Caffeic Acid Phenethyl Ester and Its Amide Analogue Are Potent Inhibitors of Leukotriene Biosynthesis in Human Polymorphonuclear Leukocytes

    PubMed Central

    Boudreau, Luc H.; Maillet, Jacques; LeBlanc, Luc M.; Jean-François, Jacques; Touaibia, Mohamed; Flamand, Nicolas; Surette, Marc E.

    2012-01-01

    Background 5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases. Methodology/Principal Findings Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC50 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC50 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC50 values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis. Conclusions CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis. PMID:22347509

  20. Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.

    PubMed

    Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

    2015-02-01

    Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications.

  1. Role of pipecolic acid in the biosynthesis of lysine in Rhodotorula glutinis.

    PubMed

    Kinzel, J J; Bhattacharjee, J K

    1979-05-01

    The role of pipecolic acid in the biosynthesis of lysine was investigated in Rhodotorula glutinis, an aerobic red yeast. Supplementation of pipecolic acid in the minimal medium supported the growth of mutants lys2, lys3, and lys5; alpha-aminoadipic acid supported the growth of lys5; but neither alpha-aminoadipic acid nor pipecolic acid supported the growth of mutants MNNG42 and MNNG37. During the growth of the appropriate mutants, pipecolic acid was removed from the growth medium and the intracellular pool. In tracer experiments, radioactivity from [(14)C]pipecolic acid was selectively incorporated into the cellular lysine of lys5 and the wild-type strain. l-Pipecolic acid-dependent enzyme activity did not require any cofactor and was inhibited by mercuric chloride and potassium cyanide. This activity was present in the wild-type strain and all of the mutants tested and was repressed in mutant lys5 when grown in the presence of higher concentration of lysine. The reaction product of pipecolic acid was converted to saccharopine by lys5 enzyme in the presence of glutamate and reduced nicotin-amide adenine dinucleotide phosphate. Mutant MNNG37 lacked the saccharopine dehydrogenase activity, indicating that this step is involved in the conversion of alpha-aminoadipic acid and pipecolic acid to lysine. Mutants MNNG37 and MNNG42 accumulated a p-dimethylaminobenzaldehyde-reacting product in the culture supernatant and in the intracellular pool. Chromatographic properties of the p-dimethylaminobenzaldehyde adduct and that of the pipecolic acid-dependent reaction product were similar. The reaction product and the accumulation product were characterized on the basis of mass and absorption spectra as alpha-aminoadipic-semialdehyde, which in solution remains in equilibrium with Delta(1)-piperideine-6-carboxylic acid. Since alpha-aminoadipic-semialdehyde is a known intermediate of the alpha-aminoadipic acid pathway for the biosynthesis of lysine, it is concluded that pipecolic

  2. COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

    PubMed Central

    Tejera, Noemi; Boeglin, William E.; Suzuki, Takashi; Schneider, Claus

    2012-01-01

    Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/106 cells (mean ± SEM, n = 6), reaching about half the level of LTB4 (1.3 ± 0.5 ng/106 cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/106 cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/106 cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A4-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes. PMID:22068350

  3. Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Miltyk, Wojciech; Galicka, Elżbieta; Przylipiak, Jerzy; Zaręba, Ilona; Surazynski, Arkadiusz

    2015-01-01

    Introduction The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes. Materials and methods Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2). Results Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take

  4. Biosynthesis of Polyunsaturated Fatty Acids in Marine Invertebrates: Recent Advances in Molecular Mechanisms

    PubMed Central

    Monroig, Óscar; Tocher, Douglas R.; Navarro, Juan C.

    2013-01-01

    Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading, de novo synthesis of PUFA is possible in some lower animals. The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs. PMID:24152561

  5. Biosynthesis of polyunsaturated fatty acids in marine invertebrates: recent advances in molecular mechanisms.

    PubMed

    Monroig, Óscar; Tocher, Douglas R; Navarro, Juan C

    2013-10-21

    Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading, de novo synthesis of PUFA is possible in some lower animals. The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs.

  6. Drought Stress Causes a Reduction in the Biosynthesis of Ascorbic Acid in Soybean Plants

    PubMed Central

    Seminario, Amaia; Song, Li; Zulet, Amaia; Nguyen, Henry T.; González, Esther M.; Larrainzar, Estíbaliz

    2017-01-01

    Drought provokes a number of physiological changes in plants including oxidative damage. Ascorbic acid (AsA), also known as vitamin C, is one of the most abundant water-soluble antioxidant compound present in plant tissues. However, little is known on the regulation of AsA biosynthesis under drought stress conditions. In the current work we analyze the effects of water deficit on the biosynthesis of AsA by measuring its content, in vivo biosynthesis and the expression level of genes in the Smirnoff-Wheeler pathway in one of the major legume crop, soybean (Glycine max L. Merr). Since the pathway has not been described in legumes, we first searched for the putative orthologous genes in the soybean genome. We observed a significant genetic redundancy, with multiple genes encoding each step in the pathway. Based on RNA-seq analysis, expression of the complete pathway was detected not only in leaves but also in root tissue. Putative paralogous genes presented differential expression patterns in response to drought, suggesting the existence of functional specialization mechanisms. We found a correlation between the levels of AsA and GalLDH biosynthetic rates in leaves of drought-stressed soybean plants. However, the levels of GalLDH transcripts did not show significant differences under water deficit conditions. Among the other known regulators of the pathway, only the expression of VTC1 genes correlated with the observed decline in AsA in leaves. PMID:28663755

  7. Modulation of endogenous indole-3-acetic acid biosynthesis in bacteroids within Medicago sativa nodules.

    PubMed

    Bianco, C; Senatore, B; Arbucci, S; Pieraccini, G; Defez, R

    2014-07-01

    To evaluate the dose-response effects of endogenous indole-3-acetic acid (IAA) on Medicago plant growth and dry weight production, we increased the synthesis of IAA in both free-living and symbiosis-stage rhizobial bacteroids during Rhizobium-legume symbiosis. For this purpose, site-directed mutagenesis was applied to modify an 85-bp promoter sequence, driving the expression of iaaM and tms2 genes for IAA biosynthesis. A positive correlation was found between the higher expression of IAA biosynthetic genes in free-living bacteria and the increased production of IAA under both free-living and symbiotic conditions. Plants nodulated by RD65 and RD66 strains, synthetizing the highest IAA concentration, showed a significant (up to 73%) increase in the shoot fresh weight and upregulation of nitrogenase gene, nifH, compared to plants nodulated by the wild-type strain. When these plants were analyzed by confocal microscopy, using an anti-IAA antibody, the strongest signal was observed in bacteroids of Medicago sativa RD66 (Ms-RD66) plants, even when they were located in the senescent nodule zone. We show here a simple system to modulate endogenous IAA biosynthesis in bacteria nodulating legumes suitable to investigate which is the maximum level of IAA biosynthesis, resulting in the maximal increase of plant growth.

  8. Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli

    PubMed Central

    Zhang, Junli; Kang, Zhen; Chen, Jian; Du, Guocheng

    2015-01-01

    5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthesis pathway, shows significant promise for cancer treatment. Here, we identified that in addition to hemA and hemL, hemB, hemD, hemF, hemG and hemH are also the major regulatory targets of the heme biosynthesis pathway. Interestingly, up-regulation of hemD and hemF benefited ALA accumulation whereas overexpression of hemB, hemG and hemH diminished ALA accumulation. Accordingly, by combinatorial overexpression of the hemA, hemL, hemD and hemF with different copy-number plasmids, the titer of ALA was improved to 3.25 g l−1. Furthermore, in combination with transcriptional and enzymatic analysis, we demonstrated that ALA dehydratase (HemB) encoded by hemB is feedback inhibited by the downstream intermediate protoporphyrinogen IX. This work has great potential to be scaled-up for microbial production of ALA and provides new important insights into the regulatory mechanism of the heme biosynthesis pathway. PMID:25716896

  9. Molecular Basis for Mycophenolic Acid Biosynthesis in Penicillium brevicompactum▿†

    PubMed Central

    Regueira, Torsten Bak; Kildegaard, Kanchana Rueksomtawin; Hansen, Bjarne Gram; Mortensen, Uffe H.; Hertweck, Christian; Nielsen, Jens

    2011-01-01

    Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering. PMID:21398490

  10. Nitric oxide metabolism and indole acetic acid biosynthesis cross-talk in Azospirillum brasilense SM.

    PubMed

    Koul, Vatsala; Tripathi, Chandrakant; Adholeya, Alok; Kochar, Mandira

    2015-04-01

    Production of nitric oxide (NO) and the presence of NO metabolism genes, nitrous oxide reductase (nosZ), nitrous oxide reductase regulator (nosR) and nitric oxide reductase (norB) were identified in the plant-associated bacterium (PAB) Azospirillum brasilense SM. NO presence was confirmed in all overexpressing strains, while improvement in the plant growth response of these strains was mediated by increased NO and indole-3-acetic acid (IAA) levels in the strains. Electron microscopy showed random distribution to biofilm, with surface colonization of pleiomorphic Azospirilla. Quantitative IAA estimation highlighted a crucial role of nosR and norBC in regulating IAA biosynthesis. The NO quencher and donor reduced/blocked IAA biosynthesis by all strains, indicating their common regulatory role in IAA biosynthesis. Tryptophan (Trp) and l-Arginine (Arg) showed higher expression of NO genes tested, while in the case of ipdC, only Trp and IAA increased expression, while Arg had no significant effect. The highest nosR expression in SMnosR in the presence of IAA and Trp, along with its 2-fold IAA level, confirmed the relationship of nosR overexpression with Trp in increasing IAA. These results indicate a strong correlation between IAA and NO in A. brasilense SM and suggest the existence of cross-talk or shared signaling mechanisms in these two growth regulators. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  11. Expanding the modular ester fermentative pathways for combinatorial biosynthesis of esters from volatile organic acids.

    PubMed

    Layton, Donovan S; Trinh, Cong T

    2016-08-01

    Volatile organic acids are byproducts of fermentative metabolism, for example, anaerobic digestion of lignocellulosic biomass or organic wastes, and are often times undesired inhibiting cell growth and reducing directed formation of the desired products. Here, we devised a general framework for upgrading these volatile organic acids to high-value esters that can be used as flavors, fragrances, solvents, and biofuels. This framework employs the acid-to-ester modules, consisting of an AAT (alcohol acyltransferase) plus ACT (acyl CoA transferase) submodule and an alcohol submodule, for co-fermentation of sugars and organic acids to acyl CoAs and alcohols to form a combinatorial library of esters. By assembling these modules with the engineered Escherichia coli modular chassis cell, we developed microbial manufacturing platforms to perform the following functions: (i) rapid in vivo screening of novel AATs for their catalytic activities; (ii) expanding combinatorial biosynthesis of unique fermentative esters; and (iii) upgrading volatile organic acids to esters using single or mixed cell cultures. To demonstrate this framework, we screened for a set of five unique and divergent AATs from multiple species, and were able to determine their novel activities as well as produce a library of 12 out of the 13 expected esters from co-fermentation of sugars and (C2-C6) volatile organic acids. We envision the developed framework to be valuable for in vivo characterization of a repertoire of not-well-characterized natural AATs, expanding the combinatorial biosynthesis of fermentative esters, and upgrading volatile organic acids to high-value esters. Biotechnol. Bioeng. 2016;113: 1764-1776. © 2016 Wiley Periodicals, Inc.

  12. Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis.

    PubMed

    Jamet, Stevie; Quentin, Yves; Coudray, Coralie; Texier, Pauline; Laval, Françoise; Daffé, Mamadou; Fichant, Gwennaele; Cam, Kaymeuang

    2015-12-01

    Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to

  13. Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

    PubMed Central

    Jamet, Stevie; Quentin, Yves; Coudray, Coralie; Texier, Pauline; Laval, Françoise; Daffé, Mamadou

    2015-01-01

    ABSTRACT Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many

  14. A physiological role for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris

    PubMed Central

    Munir, Erman; Yoon, Jeong Jun; Tokimatsu, Toshiaki; Hattori, Takefumi; Shimada, Mikio

    2001-01-01

    A metabolic mechanism for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris has been proposed on the basis of biochemical analyses of glucose metabolism. There was a strong correlation between glucose consumption and oxalate production. Oxalic acid was found to accumulate in the culture fluid in about 80% of the theoretical yield or about 5-fold, on the basis of the fungal biomass harvested. The results clearly indicate that glucose was not completely oxidized to CO2 by the tricarboxylic acid (TCA) cycle but converted mainly to oxalate. The determination of the 12 enzymes concerned has revealed the occurrence of the unprecedented metabolic coupling of the TCA and glyoxylate cycles that support oxalate biosynthesis. In this metabolic system, isocitrate lyase (EC 4.1.3.1), together with oxaloacetase (EC 3.7.1.1), was found to play a pivotal role in yielding oxalate from oxaloacetate via the acetate-recycling routes. Moreover, malate dehydrogenase (EC 1.1.1.37), with an extraordinarily high activity among the enzymes tested, was shown to play an important role in generating NADH by oxidation of malate to oxaloacetate. Thus, it is proposed that the wood-rotting basidiomycete acquires biochemical energy by oxidizing glucose to oxalate. PMID:11553780

  15. Biosynthesis of Polyunsaturated Fatty Acids in the Oleaginous Marine Diatom Fistulifera sp. Strain JPCC DA0580

    PubMed Central

    Liang, Yue; Maeda, Yoshiaki; Sunaga, Yoshihiko; Muto, Masaki; Matsumoto, Mitsufumi; Yoshino, Tomoko; Tanaka, Tsuyoshi

    2013-01-01

    Studies of polyunsaturated fatty acid (PUFA) biosynthesis in microalgae are of great importance for many reasons, including the production of biofuel and variable omega 3-long chain PUFAs. The elucidation of the PUFA biosynthesis pathway is necessary for bioengineering to increase or decrease PUFA content in certain microalgae. In this study, we identified the PUFA synthesis pathway in the oleaginous marine diatom, Fistulifera sp. strain JPCC DA0580, a promising candidate for biodiesel production. The data revealed not only the presence of the desaturases and elongases involved in eicosapentaenoic acid (EPA) synthesis, but also the unexpected localization of ω3-desaturase expression in the chloroplast. This suggests that this microalga might perform the final step of EPA synthesis in the chloroplast and not in the endoplasmic reticulum (ER) like other diatoms. The detailed fatty acid profile suggests that the EPA was synthesized only through the ω6-pathway in this strain, which was also different from other diatoms. Finally, the transcriptome analysis demonstrated an overall down-regulation of desaturases and elongases over incubation time. These genetic features might explain the decrease of PUFA percentage over incubation time in this strain. The important insights into metabolite synthesis acquired here will be useful for future metabolic engineering to control PUFA content in this diatom. PMID:24335525

  16. Systematic unravelling of the biosynthesis of poly (L-diaminopropionic acid) in Streptomyces albulus PD-1

    PubMed Central

    Xu, Zhaoxian; Sun, Zhuzhen; Li, Sha; Xu, Zheng; Cao, Changhong; Xu, Zongqi; Feng, Xiaohai; Xu, Hong

    2015-01-01

    Poly(L-diaminopropionic acid) (PDAP) is one of the four homopoly(amino acid)s that have been discovered in nature. However, the molecular mechanism of PDAP biosynthesis has yet to be described. In this work, the general layout of the PDAP biosynthetic pathway is characterised in Streptomyces albulus PD-1 by genome mining, gene disruption, heterologous expression and in vitro feeding experiments. As a result, L-diaminopropionic acid (L-DAP), which is the monomer of PDAP, is shown to be jointly synthesised by two protein homologues of cysteine synthetase and ornithine cyclodeaminase. Then, L-DAP is assembled into PDAP by a novel nonribosomal peptide synthetase (NRPS) with classical adenylation and peptidyl carrier protein domains. However, instead of the traditional condensation or thioesterase domain of NRPSs, this NRPS has seven transmembrane domains surrounding three tandem soluble domains at the C-terminus. As far as we know, this novel single-module NRPS structure has only been reported in poly(ε-L-lysine) synthetase. The similar NRPS structure of PDAP synthetase and poly(ε-L-lysine) synthetase may be a common characteristic of homopoly(amino acid)s synthetases. In this case, we may discover and/or design more homopoly(amino acid)s by mining this kind of novel NRPS structure in the future. PMID:26632244

  17. Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco

    SciTech Connect

    Yalpani, N.; Leon, J.; Lawton, M.A.; Raskin, I. )

    1993-10-01

    Salicylic acid (SA) is a likely endogenous regulator of localized and systemic disease resistance in plants. During the hypersensitive response of Nicotiana tabacum L. cv Xanthi-nc to tobacco mosaic virus (TMV), SA levels rise dramatically. We studied Sa biosynthesis in healthy and TMV-inoculated tobacco by monitoring the levels of SA and its likely precursors in extracts of leaves and cell suspensions. In TMV-inoculated leaves, stimulation of Sa accumulation is accompanied by a corresponding increase in the levels of benzoic acid. [sup 14]C-Tracer studies with cell suspensions and mock- or TMV-inoculated leaves indicate that the label moves from trans-cinnamic acid to SA via benzoic acid. In healthy and TMV-inoculated tobacco leaves, benzoic acid induced SA accumulation. o-Coumaric acid, which was previously reported as a possible precursor of SA in other species, did not increase SA levels in tobacco. In healthy tobacco tissue, the specific activity of newly formed SA was equal to that of the supplied [[sup 14]C] benzoic acid, whereas in TMV-inoculated leaves some isotope dilution was observed, presumably because of the increase in the pool of endogenous benzoic acid. We observed accumulation of pathogenesis-related-1 proteins and increased resistance to TMV in benzoic acid but no in 0-coumaric acid-treated tobacco leaves. This is consistent with benzoic acid being the immediate precursor of SA. We conclude that in healthy and virus-inoculated tobacco, SA is formed from cinnamic acid via benzoic acid. 27 refs., 7 figs., 1 tab.

  18. Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

    PubMed Central

    Geisler, Christoph; Jarvis, Donald L.

    2012-01-01

    The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2′-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems. PMID:23022569

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

    PubMed Central

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

    2017-01-01

    Tannerella forsythia is an anaerobic, Gram-negative periodontal pathogen. A unique O-linked oligosaccharide decorates the bacterium’s cell surface proteins and was shown to modulate the host immune response. In our study, we investigated the biosynthesis of the nonulosonic acid (NulO) present at the terminal position of this glycan. A bioinformatic analysis of T. forsythia genomes revealed a gene locus for the synthesis of pseudaminic acid (Pse) in the type strain ATCC 43037 while strains FDC 92A2 and UB4 possess a locus for the synthesis of legionaminic acid (Leg) instead. In contrast to the NulO in ATCC 43037, which has been previously identified as a Pse derivative (5-N-acetimidoyl-7-N-glyceroyl-3,5,7,9-tetradeoxy-l-glycero-l-manno-NulO), glycan analysis of strain UB4 performed in this study indicated a 350-Da, possibly N-glycolyl Leg (3,5,7,9-tetradeoxy-d-glycero-d-galacto-NulO) derivative with unknown C5,7 N-acyl moieties. We have expressed, purified and characterized enzymes of both NulO pathways to confirm these genes’ functions. Using capillary electrophoresis (CE), CE–mass spectrometry and NMR spectroscopy, our studies revealed that Pse biosynthesis in ATCC 43037 essentially follows the UDP-sugar route described in Helicobacter pylori, while the pathway in strain FDC 92A2 corresponds to Leg biosynthesis in Campylobacter jejuni involving GDP-sugar intermediates. To demonstrate that the NulO biosynthesis enzymes are functional in vivo, we created knockout mutants resulting in glycans lacking the respective NulO. Compared to the wild-type strains, the mutants exhibited significantly reduced biofilm formation on mucin-coated surfaces, suggestive of their involvement in host-pathogen interactions or host survival. This study contributes to understanding possible biological roles of bacterial NulOs. PMID:27986835

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

    PubMed

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

    2016-12-16

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

  1. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

    USDA-ARS?s Scientific Manuscript database

    Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

  2. Auxin-Induced Ethylene Triggers Abscisic Acid Biosynthesis and Growth Inhibition1

    PubMed Central

    Hansen, Hauke; Grossmann, Klaus

    2000-01-01

    The growth-inhibiting effects of indole-3-acetic acid (IAA) at high concentration and the synthetic auxins 7-chloro-3-methyl-8-quinolinecarboxylic acid (quinmerac), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), 4-amino-3,6,6-trichloropicolinic acid (picloram), and naphthalene acetic acid, were investigated in cleavers (Galium aparine). When plants were root treated with 0.5 mm IAA, shoot epinasty and inhibition of root and shoot growth developed during 24 h. Concomitantly, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, and ACC and ethylene production were transiently stimulated in the shoot tissue within 2 h, followed by increases in immunoreactive (+)-abscisic acid (ABA) and its precursor xanthoxal (xanthoxin) after 5 h. After 24 h of treatment, levels of xanthoxal and ABA were elevated up to 2- and 24-fold, relative to control, respectively. In plants treated with IAA, 7-chloro-3-methyl-8-quinolinecarboxylic acid, naphthalene acetic acid, 2-methoxy-3,6-dichlorobenzoic acid, and 4-amino-3,6,6-trichloropicolinic acid, levels of ethylene, ACC, and ABA increased in close correlation with inhibition of shoot growth. Aminoethoxyvinyl-glycine and cobalt ions, which inhibit ethylene synthesis, decreased ABA accumulation and growth inhibition, whereas the ethylene-releasing ethephon promoted ABA levels and growth inhibition. In accordance, tomato mutants defective in ethylene perception (never ripe) did not produce the xanthoxal and ABA increases and growth inhibition induced by auxins in wild-type plants. This suggests that auxin-stimulated ethylene triggers ABA accumulation and the consequent growth inhibition. Reduced catabolism most probably did not contribute to ABA increase, as indicated by immunoanalyses of ABA degradation and conjugation products in shoot tissue and by pulse experiments with [3H]-ABA in cell suspensions of G. aparine. In contrast, studies using inhibitors of ABA biosynthesis (fluridone, naproxen, and tungstate), ABA

  3. 7-deoxyloganetic acid synthase catalyzes a key 3 step oxidation to form 7-deoxyloganetic acid in Catharanthus roseus iridoid biosynthesis.

    PubMed

    Salim, Vonny; Wiens, Brent; Masada-Atsumi, Sayaka; Yu, Fang; De Luca, Vincenzo

    2014-05-01

    Iridoids are key intermediates required for the biosynthesis of monoterpenoid indole alkaloids (MIAs), as well as quinoline alkaloids. Although most iridoid biosynthetic genes have been identified, one remaining three step oxidation required to form the carboxyl group of 7-deoxyloganetic acid has yet to be characterized. Here, it is reported that virus-induced gene silencing of 7-deoxyloganetic acid synthase (7DLS, CYP76A26) in Catharanthus roseus greatly decreased levels of secologanin and the major MIAs, catharanthine and vindoline in silenced leaves. Functional expression of this gene in Saccharomyces cerevisiae confirmed its function as an authentic 7DLS that catalyzes the 3 step oxidation of iridodial-nepetalactol to form 7-deoxyloganetic acid. The identification of CYP76A26 removes a key bottleneck for expression of iridoid and related MIA pathways in various biological backgrounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis1[OPEN

    PubMed Central

    Wang, Zhen-Yu; Gehring, Chris; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

    2015-01-01

    Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1. PMID:25416474

  5. The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis.

    PubMed

    Wang, Zhen-Yu; Xiong, Liming; Li, Wenbo; Zhu, Jian-Kang; Zhu, Jianhua

    2011-05-01

    Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 gene expression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxygenase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol) treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly that CED1 encodes a putative α/β hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cutin biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling.

  6. A Novel Two-Gene Requirement for the Octanoyltransfer Reaction of Bacillus subtilis Lipoic Acid Biosynthesis

    PubMed Central

    Martin, Natalia; Christensen, Quin H.; Mansilla, María C.; Cronan, John E.; de Mendoza, Diego

    2011-01-01

    SUMMARY The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM, and ywfL which we have renamed lplJ, lipM and lipL, respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to E. coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulfur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilis ΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coli ΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion paper (Christensen et al., 2011) provide evidence that LipM and LipL catalyze sequential reactions in a novel pathway for lipoic acid biosynthesis. PMID:21338420

  7. Apicoplast and Endoplasmic Reticulum Cooperate in Fatty Acid Biosynthesis in Apicomplexan Parasite Toxoplasma gondii*

    PubMed Central

    Ramakrishnan, Srinivasan; Docampo, Melissa D.; MacRae, James I.; Pujol, François M.; Brooks, Carrie F.; van Dooren, Giel G.; Hiltunen, J. Kalervo; Kastaniotis, Alexander J.; McConville, Malcolm J.; Striepen, Boris

    2012-01-01

    Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here, we use a combination of genetic, biochemical, and metabolomic approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with [13C]glucose showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0–26:1). Genetic disruption of the apicoplast-localized type II fatty-acid synthase resulted in greatly reduced synthesis of saturated fatty acids up to 18 carbons long. Ablation of type II fatty-acid synthase activity resulted in reduced intracellular growth that was partially restored by addition of long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases, and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host. PMID:22179608

  8. Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis

    PubMed Central

    Wilson, Regina; Kumar, Pradeep; Parashar, Vijay; Vilchèze, Catherine; Veyron-Churlet, Romain; Freundlich, Joel S.; Barnes, S. Whitney; Walker, John R.; Szymonifka, Michael J.; Marchiano, Emily; Shenai, Shubhada; Colangeli, Roberto; Jacobs, William R.; Neiditch, Matthew B.; Kremer, Laurent

    2013-01-01

    We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis (Mtb) by the novel mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser-55 site in Pks13 conferred TP-resistance in Mtb. Over-expression of wild-type pks13 resulted in TP-resistance and over-expression of the F79S pks13 mutant conferred high-level resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type Mtb, but to a much lesser extent in TP-resistant Mtb. TP treatment was bactericidal and equivalent to the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment exhibited sterilizing activity. Computational-docking identified a possible TP-binding groove within the Pks13 ACP domain. This study confirms that Mtb Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway. PMID:23770708

  9. Conservation of the 2-keto-3-deoxymanno-octulosonic acid (Kdo) biosynthesis pathway between plants and bacteria.

    PubMed

    Smyth, Kevin M; Marchant, Alan

    2013-10-18

    The increasing prevalence of multi-drug resistant bacteria is driving efforts in the development of new antibacterial agents. This includes a resurgence of interest in the Gram-negative bacteria lipopolysaccharide (LPS) biosynthesis enzymes as drug targets. The six carbon acidic sugar 2-keto-3-deoxymanno-octulosonic acid (Kdo) is a component of the lipid A moiety of the LPS in Gram-negative bacteria. In most cases the lipid A substituted by Kdo is the minimum requirement for cell growth, thus presenting the possibility of targeting either the synthesis or incorporation of Kdo for the development of antibacterial agents. Indeed, potent in vitro inhibitors of Kdo biosynthesis enzymes have been reported but have so far failed to show sufficient in vivo action against Gram-negative bacteria. As part of an effort to design more potent antibacterial agents targeting Kdo biosynthesis, the crystal structures of the key Kdo biosynthesis enzymes from Escherichia coli have been solved and their structure based mechanisms characterized. In eukaryotes, Kdo is found as a component of the pectic polysaccharide rhamnogalacturonan II in the plant primary cell wall. Interestingly, despite incorporating Kdo into very different macromolecules the Kdo biosynthesis and activation pathway is almost completely conserved between plants and bacteria. This raises the possibility for plant research to exploit the increasingly detailed knowledge and resources being generated by the microbiology community. Likewise, insights into Kdo biosynthesis in plants will be potentially useful in efforts to produce new antimicrobial compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Construction of a Chimeric Biosynthetic Pathway for the de novo Biosynthesis of Rosmarinic Acid in Escherichia coli

    PubMed Central

    Bloch, Sarah E.; Schmidt-Dannert, Claudia

    2014-01-01

    Hydroxycinnamic acid esters (HCEs) are widely-distributed phenylpropanoid-derived plant natural products. Rosmarinic acid (RA 7), the most well-known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, a microbial production platform for HCEs could provide a sustainable, controlled means of production. Through the overexpression of a six-enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA 7 and the related HCE isorinic acid (IA 8) in E. coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We show HCE biosynthesis using three plant RAS orthologs exhibiting different levels of HCE biosynthesis, but the same ratio of IA 8 to RA 7 produced. This work serves as a proof of concept for a microbial production platform for HCEs using a modular biosynthetic approach to access diverse natural and non-natural HCEs. PMID:25205019

  11. Identification of a Desaturase Involved in Mycolic Acid Biosynthesis in Mycobacterium smegmatis

    PubMed Central

    Singh, Albel; Varela, Cristian; Bhatt, Kiranmai; Veerapen, Natacha; Lee, Oona Y. C.; Wu, Houdini H. T.; Besra, Gurdyal S.; Minnikin, David E.; Fujiwara, Nagatoshi; Teramoto, Kanae; Bhatt, Apoorva

    2016-01-01

    Mycolic acids are unique long chain fatty acids found in the cell walls of mycobacteria including the tubercle bacillus, Mycobacterium tuberculosis. The introduction of double bonds in mycolic acids remains poorly understood, however, genes encoding two potential aerobic desaturases have been proposed to be involved in this process. Here we show that one of these genes, desA1, is essential for growth of the saprophytic Mycobacterium smegmatis. Depletion of desA1 in a M. smegmatis conditional mutant led to reduction of mycolic acid biosynthesis and loss of viability. The DesA1-depleted cells exhibited two other phenotypes: using 14[C]-labelling, we detected the accumulation of minor mycolic acid-related species that migrated faster in a silver TLC plate. Spiral Time of Flight Mass Spectroscopic analysis suggested the presence of species with sizes corresponding to what were likely monoenoic derivatives of α-mycolic acids. Additionally, conditional depletion led to the presence of free fatty acyl species of lengths ~C26-C48 in the lysing cells. Cell viability could be rescued in the conditional mutant by Mycobacterium tuberculosis desA1, highlighting the potential of desA1 as a new drug target in pathogenic mycobacteria. PMID:27741286

  12. Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.

    PubMed

    Blatti, Jillian L; Beld, Joris; Behnke, Craig A; Mendez, Michael; Mayfield, Stephen P; Burkart, Michael D

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

  13. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  14. Oxalic acid biosynthesis is encoded by an operon in Burkholderia glumae.

    PubMed

    Nakata, Paul A; He, Cixin

    2010-03-01

    Although the biosynthesis of oxalic acid is known to occur in a number of bacteria, the mechanism(s) regulating its production remains largely unknown. To date, there is no report on the identification of an oxalic acid biosynthetic pathway gene from bacteria. In an attempt to identify such a gene(s), a mutant screen was conducted using the simple oxalic acid-producing phytopathogenic bacterium, Burkholderia glumae. Four mutants that failed to produce oxalic acid were isolated from a transposon-mutagenized B. glumae library and named Burkholderia oxalate defective (Bod)1. DNA sequence analysis revealed that each mutant contained an insertion event at different sites in the same ORF, which we referred to as the oxalate biosynthetic component (obc)A locus. Complementation of the Bod1 mutant with the obcA gene, however, resulted only in a partial restoration of the oxalic acid-producing phenotype. Further complementation studies utilizing a larger DNA fragment encompassing the obcA locus coupled with deletion mutagenesis led to the identification of another ORF that we named the obcB locus, which was essential for higher levels of oxalic acid production. Transcript analysis indicated that both obcA and obcB are coexpressed and encoded on a single polycistron message.

  15. The Catalytic Machinery of a Key Enzyme in Amino Acid Biosynthesis

    SciTech Connect

    Viola, Ronald E.; Faehnle, Christopher R.; Blanco, Julio; Moore, Roger A.; Liu, Xuying; Arachea, Buenafe T.; Pavlovsky, Alexander G.

    2013-02-28

    The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate {beta}-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

  16. The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 Gene Encodes an Aldehyde Dehydrogenase Involved in Ferulic Acid and Sinapic Acid Biosynthesis

    PubMed Central

    Nair, Ramesh B.; Bastress, Kristen L.; Ruegger, Max O.; Denault, Jeff W.; Chapple, Clint

    2004-01-01

    Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP+-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall–esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes. PMID:14729911

  17. Reduced macrophage selenoprotein expression alters oxidized lipid metabolite biosynthesis from arachidonic and linoleic acid.

    PubMed

    Mattmiller, Sarah A; Carlson, Bradley A; Gandy, Jeff C; Sordillo, Lorraine M

    2014-06-01

    Uncontrolled inflammation is an underlying etiology for multiple diseases and macrophages orchestrate inflammation largely through the production of oxidized fatty acids known as oxylipids. Previous studies showed that selenium (Se) status altered the expression of oxylipids and magnitude of inflammatory responses. Although selenoproteins are thought to mediate many of the biological effects of Se, the direct effect of selenoproteins on the production of oxylipids is unknown. Therefore, the role of decreased selenoprotein activity in modulating the production of biologically active oxylipids from macrophages was investigated. Thioglycollate-elicited peritoneal macrophages were collected from wild-type and myeloid-cell-specific selenoprotein knockout mice to analyze oxylipid production by liquid chromatography/mass spectrometry as well as oxylipid biosynthetic enzyme and inflammatory marker gene expression by quantitative real-time polymerase chain reaction. Decreased selenoprotein activity resulted in the accumulation of reactive oxygen species, enhanced cyclooxygenase and lipoxygenase expression and decreased oxylipids with known anti-inflammatory properties such as arachidonic acid-derived lipoxin A₄ (LXA₄) and linoleic acid-derived 9-​oxo-octadecadienoic acid (9-oxoODE). Treating RAW 264.7 macrophages with LXA₄ or 9-oxoODE diminished oxidant-induced macrophage inflammatory response as indicated by decreased production of TNFα. The results show for the first time that selenoproteins are important for the balanced biosynthesis of pro- and anti-inflammatory oxylipids during inflammation. A better understanding of the Se-dependent control mechanisms governing oxylipid biosynthesis may uncover nutritional intervention strategies to counteract the harmful effects of uncontrolled inflammation due to oxylipids. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Biochemical Principles and Functional Aspects of Pipecolic Acid Biosynthesis in Plant Immunity1[OPEN

    PubMed Central

    Kim, Denis; Schreiber, Stefan; Zeier, Tatyana; Schuck, Stefan; Reichel-Deland, Vanessa

    2017-01-01

    The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/μ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae. Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction. PMID:28330936

  19. Biosynthesis of cyclopropyl long-chain fatty acids from cyclopropanecarboxylic acid by mammalian tissues in vitro

    PubMed Central

    Duncombe, W. G.; Rising, T. J.

    1968-01-01

    1. Radioactivity from cyclopropane[14C]carboxylic acid is incorporated into fatty acids in vitro by rat and guinea-pig adipose tissue, by rat liver slices and by the supernatant fraction of rat liver homogenate. 2. The labelled acids are different from endogenous straight-chain fatty acids, and evidence is produced that they consist of a cyclopropyl ring in the ω-position, the remainder of the chain being built up from C2 units (not derived from cyclopropanecarboxylic acid) in the normal way via the malonate pathway. 3. It is suggested that these unnatural acids have some metabolic effect related to the hypoglycaemic action of cyclopropanecarboxylic acid. PMID:5685874

  20. Biosynthesis of the Unique Wall Teichoic Acid of Staphylococcus aureus Lineage ST395

    PubMed Central

    Winstel, Volker; Sanchez-Carballo, Patricia; Holst, Otto; Xia, Guoqing; Peschel, Andreas

    2014-01-01

    ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an α-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage Φ187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to Φ187 and enabled Φ187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. PMID:24713320

  1. Insights into the Biosynthesis of 12-Membered Resorcylic Acid Lactones from Heterologous Production in Saccharomyces cerevisiae

    PubMed Central

    2015-01-01

    The phytotoxic fungal polyketides lasiodiplodin and resorcylide inhibit human blood coagulation factor XIIIa, mineralocorticoid receptors, and prostaglandin biosynthesis. These secondary metabolites belong to the 12-membered resorcylic acid lactone (RAL12) subclass of the benzenediol lactone (BDL) family. Identification of genomic loci for the biosynthesis of lasiodiplodin from Lasiodiplodia theobromae and resorcylide from Acremonium zeae revealed collaborating iterative polyketide synthase (iPKS) pairs whose efficient heterologous expression in Saccharomyces cerevisiae provided a convenient access to the RAL12 scaffolds desmethyl-lasiodiplodin and trans-resorcylide, respectively. Lasiodiplodin production was reconstituted in the heterologous host by co-expressing an O-methyltransferase also encoded in the lasiodiplodin cluster, while a glutathione-S-transferase was found not to be necessary for heterologous production. Clarification of the biogenesis of known resorcylide congeners in the heterologous host helped to disentangle the roles that biosynthetic irregularities and chemical interconversions play in generating chemical diversity. Observation of 14-membered RAL homologues during in vivo heterologous biosynthesis of RAL12 metabolites revealed “stuttering” by fungal iPKSs. The close global and domain-level sequence similarities of the orthologous BDL synthases across different structural subclasses implicate repeated horizontal gene transfers and/or cluster losses in different fungal lineages. The absence of straightforward correlations between enzyme sequences and product structural features (the size of the macrocycle, the conformation of the exocyclic methyl group, or the extent of reduction by the hrPKS) suggest that BDL structural variety is the result of a select few mutations in key active site cavity positions. PMID:24597618

  2. Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast

    NASA Astrophysics Data System (ADS)

    Scheler, Ulschan; Brandt, Wolfgang; Porzel, Andrea; Rothe, Kathleen; Manzano, David; Božić, Dragana; Papaefthimiou, Dimitra; Balcke, Gerd Ulrich; Henning, Anja; Lohse, Swanhild; Marillonnet, Sylvestre; Kanellis, Angelos K.; Ferrer, Albert; Tissier, Alain

    2016-10-01

    Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.

  3. Amino acids biosynthesis and nitrogen assimilation pathways: a great genomic deletion during eukaryotes evolution

    PubMed Central

    2011-01-01

    Background Besides being building blocks for proteins, amino acids are also key metabolic intermediates in living cells. Surprisingly a variety of organisms are incapable of synthesizing some of them, thus named Essential Amino Acids (EAAs). How certain ancestral organisms successfully competed for survival after losing key genes involved in amino acids anabolism remains an open question. Comparative genomics searches on current protein databases including sequences from both complete and incomplete genomes among diverse taxonomic groups help us to understand amino acids auxotrophy distribution. Results Here, we applied a methodology based on clustering of homologous genes to seed sequences from autotrophic organisms Saccharomyces cerevisiae (yeast) and Arabidopsis thaliana (plant). Thus we depict evidences of presence/absence of EAA biosynthetic and nitrogen assimilation enzymes at phyla level. Results show broad loss of the phenotype of EAAs biosynthesis in several groups of eukaryotes, followed by multiple secondary gene losses. A subsequent inability for nitrogen assimilation is observed in derived metazoans. Conclusions A Great Deletion model is proposed here as a broad phenomenon generating the phenotype of amino acids essentiality followed, in metazoans, by organic nitrogen dependency. This phenomenon is probably associated to a relaxed selective pressure conferred by heterotrophy and, taking advantage of available homologous clustering tools, a complete and updated picture of it is provided. PMID:22369087

  4. N-glycolylneuraminic acid biosynthesis in rat mammary tumor ascites cells

    SciTech Connect

    Sherblom, A.

    1986-05-01

    N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGl) are two of the most common forms of sialic acid, a compound found at the cell surface of all animal cells. The expression of NeuGl, which is thought to be synthesized from NeuAc, appears to be species-dependent, and the amounts of NeuGl change with differentiation and during malignancy. Hydroxylation of the acetyl group to glycolyl was monitored in 13762 rat mammary tumor ascites cells in which NeuGl accounts for 47% of the total sialic acid. N-acetylmannosamine-(/sup 14/C-acetyl) was incubated with a crude extract of MAT-Cl cells and formation of /sup 14/C-glycolate was determined by thin-layer chromatography following methanolic HCl hydrolysis and hydroxamate derivation. Conversion of /sup 14/C-acetate to /sup 14/C-glycolate increased with time of incubation and with protein concentration, and the specific activity of MAT-Cl crude extract for the conversion was 940 + 160 pmol/h/mg protein. By ion exchange chromatography, the /sup 14/C-glycolate product was predominantly neutral, with less than 10% associated with the sialic acid fraction. The results suggest that N-acetylmannosamine is a substrate for the hydroxylase under the conditions of the assay and that N-glycolylmannosamine may be an intermediate in the biosynthesis of NeuGl in rat mammary tumor ascites cells.

  5. Developmental changes in aspartate-family amino acid biosynthesis in pea chloroplasts

    SciTech Connect

    Mills, W.R.; Cato, L.W.; Stephens, B.W.; Reeves, M. )

    1990-05-01

    Isolated chloroplasts are known to synthesize the asp-derived amino acids (ile, hse, lys and thr) from ({sup 14}C)asp (Mills et al, 1980, Plant Physiol. 65, 1166). Now, we have studied the influence of tissue age on essential amino acid biosynthesis in pea (Pisum sativum) plastids. Chloroplasts from the younger (third and fourth) leaves of 12 day old plants, were 2-3 times more active in synthesizing lys and thr from ({sup 14}C)asp than those from older (first or second) leaves. We also examined two key pathway enzymes (aspartate kinase and homoserine dehydrogenase); with each enzyme,a activity in younger leaves was about 2 times that in plastids from older tissue. Both lys- and thr-sensitive forms of aspartate kinase are known in plants; in agreement with earlier work, we found that lys-sensitive activity was about 4 times higher in the younger tissues, while the thr-sensitive activity changed little during development (Davies and Miflin, 1977, Plant Sci. Lett. 9, 323). Recently the role of aspartate kinase and homoserine dehydrogenase in controlling asp-family amino acid synthesis has been questioned (Giovanelli et al, 1989, Plant Physiol. 90, 1584); we hope that measurements of amino acid levels in chloroplasts as well as further enzyme studies will help us to better understand the regulation of asp-family amino acid synthesis.

  6. Regulation of water-soluble phenolic acid biosynthesis in Salvia miltiorrhiza Bunge.

    PubMed

    Ma, Pengda; Liu, Jingling; Zhang, Chenlu; Liang, Zongsuo

    2013-07-01

    Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of various diseases. Phenolic acids in S. miltiorrhiza, as important effective compounds, have become a new research focus in plant secondary metabolism in recent years. This review summarizes the recent advances in the regulation of water-soluble phenolic acid biosynthesis in S. miltiorrhiza via regulators at molecular level, such as the phenylalanine ammonia-lyase gene (PAL), cinnamic acid 4-hydroxylase gene (C4H), 4-coumarate-CoA ligase gene (4CL), tyrosine aminotransferase gene (TAT), 4-hydroxyphenylpyruvate reductase gene (HPPR), 4-hydroxyphenylpyruvated dioxygenase gene (HPPD), hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase-like gene (RAS-like), and v-myb avian myeloblastosis viral oncogene homolog 4 gene (MYB4), and production of anthocyanin pigmentation 1 gene (AtPAP1), and via regulators at cell level, such as methyl jasmonate, salicylic acid, abscisic acid, polyamines, metal ions, hydrogen peroxide (H₂O₂), ultraviolet-B radiation, and yeast elicitor.

  7. Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast

    PubMed Central

    Scheler, Ulschan; Brandt, Wolfgang; Porzel, Andrea; Rothe, Kathleen; Manzano, David; Božić, Dragana; Papaefthimiou, Dimitra; Balcke, Gerd Ulrich; Henning, Anja; Lohse, Swanhild; Marillonnet, Sylvestre; Kanellis, Angelos K.; Ferrer, Albert; Tissier, Alain

    2016-01-01

    Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities. PMID:27703160

  8. Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

    PubMed

    Cao, Peng-Fei; Wu, Chen-Gao; Dang, Zhi-Hao; Shi, Liang; Jiang, Ai-Liang; Ren, Ang; Zhao, Ming-Wen

    2017-01-01

    We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 μmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 μg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 μmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 μmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

  9. Auxin controls Arabidopsis anther dehiscence by regulating endothecium lignification and jasmonic acid biosynthesis.

    PubMed

    Cecchetti, Valentina; Altamura, Maria Maddalena; Brunetti, Patrizia; Petrocelli, Valentina; Falasca, Giuseppina; Ljung, Karin; Costantino, Paolo; Cardarelli, Maura

    2013-05-01

    It has been suggested that, in Arabidopsis, auxin controls the timing of anther dehiscence, possibly by preventing premature endothecium lignification. We show here that auxin content in anthers peaks before the beginning of dehiscence and decreases when endothecium lignification occurs. We show that, in the auxin-perception mutants afb1-3 and tir1 afb2 afb3, endothecium lignification and anther dehiscence occur earlier than wild-type, and the gene encoding the transcription factor MYB26, which is required for endothecium lignification, is over-expressed specifically at early stages; in agreement, MYB26 expression is reduced in naphthalene acetic acid-treated anthers, and afb1 myb26 double mutants show no endothecial lignification, suggesting that auxin acts through MYB26. As jasmonic acid (JA) controls anther dehiscence, we analysed how auxin and JA interact. In the JA-defective opr3 mutant, indehiscent anthers show normal timing of endothecium lignification, suggesting that JA does not control this event. We show that expression of the OPR3 and DAD1 JA biosynthetic genes is enhanced in afb1-3 and tir1 afb2 afb3 flower buds, but is reduced in naphthalene acetic acid-treated flower buds, suggesting that auxin negatively regulates JA biosynthesis. The double mutant afb1 opr3 shows premature endothecium lignification, as in afb1-3, and indehiscent anthers due to lack of JA, which is required for stomium opening. By treating afb1 opr3 and opr3 inflorescences with JA, we show that a high JA content and precocious endothecium lignification both contribute to induction of early anther dehiscence. We propose that auxin controls anther dehiscence timing by negatively regulating two key events: endothecium lignification via MYB26, and stomium opening via the control of JA biosynthesis. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

  10. Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

    DOE PAGES

    Guarnieri, Michael T.; Chou, Yat -Chen; Salvachua, Davinia Rodriquez; ...

    2017-06-16

    Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways—namely, acetate and formate production—and overexpression of the key enzymesmore » in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes. Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Altogether, this work demonstrates genetic modifications that can lead to succinic

  11. Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis.

    PubMed

    Guarnieri, Michael T; Chou, Yat-Chen; Salvachúa, Davinia; Mohagheghi, Ali; St John, Peter C; Peterson, Darren J; Bomble, Yannick J; Beckham, Gregg T

    2017-09-01

    Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways-namely, acetate and formate production-and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe.IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid production

  12. Stimulatory Effects of Acibenzolar-S-Methyl on Chlorogenic Acids Biosynthesis in Centella asiatica Cells.

    PubMed

    Ncube, Efficient N; Steenkamp, Paul A; Madala, Ntakadzeni E; Dubery, Ian A

    2016-01-01

    Centella asiatica is a perrenial herb that grows in tropical regions with numerous medicinal properties mostly attributed to the presence of pentacyclic triterpenoids. Interestingly, this plant also possess a significant amount of phenylpropanoid-derived chlorogenic acids (CGAs) that have recently been reported to confer neuroprotective properties. In a biotechnological attempt to increase the biosynthesis of CGA-derivatives in cultured Centella cells, acibenzolar-S-methyl was applied as a xenobiotic inducer in combination with quinic acid and shikimic acid as precursor molecules. Applying a semi-targeted metabolomics-based approach, time and concentration studies were undertaken to evaluate the effect of the manipulation on cellular metabolism leading to CGA production. Phytochemical extracts were prepared using methanol and analyzed using a UHPLC-qTOF-MS platform. Data was processed and analyzed using multivariate data models. A total of four CGA-derivatives, annotated as trans-5-feruloylquinic acid, 3,5 di-caffeoylquinic acid, 3,5-O-dicaffeoyl-4-O-malonylquinic acid (irbic acid) and 3-caffeoyl, 5-feruloylquinic acid, were found to be upregulated by the acibenzolar-S-methyl treatment. To the best of our knowledge, this is the first report on the induction of CGA derivatives in this species. Contrary to expectations, the effects of precursor molecules on the levels of the CGAs were insignificant. However, a total of 16 metabolites, including CGA derivatives, were up-regulated by precursor treatment. Therefore, this study shows potential to biotechnologically manipulate C. asiatica cells to increase the production of these health beneficial CGAs.

  13. Stimulatory Effects of Acibenzolar-S-Methyl on Chlorogenic Acids Biosynthesis in Centella asiatica Cells

    PubMed Central

    Ncube, Efficient N.; Steenkamp, Paul A.; Madala, Ntakadzeni E.; Dubery, Ian A.

    2016-01-01

    Centella asiatica is a perrenial herb that grows in tropical regions with numerous medicinal properties mostly attributed to the presence of pentacyclic triterpenoids. Interestingly, this plant also possess a significant amount of phenylpropanoid-derived chlorogenic acids (CGAs) that have recently been reported to confer neuroprotective properties. In a biotechnological attempt to increase the biosynthesis of CGA-derivatives in cultured Centella cells, acibenzolar-S-methyl was applied as a xenobiotic inducer in combination with quinic acid and shikimic acid as precursor molecules. Applying a semi-targeted metabolomics-based approach, time and concentration studies were undertaken to evaluate the effect of the manipulation on cellular metabolism leading to CGA production. Phytochemical extracts were prepared using methanol and analyzed using a UHPLC-qTOF-MS platform. Data was processed and analyzed using multivariate data models. A total of four CGA-derivatives, annotated as trans-5-feruloylquinic acid, 3,5 di-caffeoylquinic acid, 3,5-O-dicaffeoyl-4-O-malonylquinic acid (irbic acid) and 3-caffeoyl, 5-feruloylquinic acid, were found to be upregulated by the acibenzolar-S-methyl treatment. To the best of our knowledge, this is the first report on the induction of CGA derivatives in this species. Contrary to expectations, the effects of precursor molecules on the levels of the CGAs were insignificant. However, a total of 16 metabolites, including CGA derivatives, were up-regulated by precursor treatment. Therefore, this study shows potential to biotechnologically manipulate C. asiatica cells to increase the production of these health beneficial CGAs. PMID:27733862

  14. Does the aromatic L-amino acid decarboxylase contribute to thyronamine biosynthesis?

    PubMed

    Hoefig, Carolin S; Renko, Kostja; Piehl, Susanne; Scanlan, Thomas S; Bertoldi, Mariarita; Opladen, Thomas; Hoffmann, Georg Friedrich; Klein, Jeannette; Blankenstein, Oliver; Schweizer, Ulrich; Köhrle, Josef

    2012-02-26

    Thyronamines (TAM), recently described endogenous signaling molecules, exert metabolic and pharmacological actions partly opposing those of the thyromimetic hormone T(3). TAM biosynthesis from thyroid hormone (TH) precursors requires decarboxylation of the L-alanine side chain and several deiodination steps to convert e.g. L-thyroxine (T(4)) into the most potent 3-T(1)AM. Aromatic L-amino acid decarboxylase (AADC) was proposed to mediate TAM biosynthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC, which actively catalyzes dopamine production from DOPA, with several TH. Under all reaction conditions tested, AADC failed to catalyze TH decarboxylation, thus challenging the initial hypothesis. These in vitro observations are supported by detection of 3-T(1)AM in plasma of patients with AADC-deficiency at levels (46 ± 18 nM, n=4) similar to those of healthy controls. Therefore, we propose that the enzymatic decarboxylation needed to form TAM from TH is catalyzed by another unique, perhaps TH-specific, decarboxylase. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  15. Role of Acid Metabolism in Streptomyces coelicolor Morphological Differentiation and Antibiotic Biosynthesis

    PubMed Central

    Viollier, Patrick H.; Minas, Wolfgang; Dale, Glenn E.; Folcher, Marc; Thompson, Charles J.

    2001-01-01

    Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent Km values of CitA for acetyl coenzyme A (acetyl-CoA) (32 μM) and oxaloacetate (17 μM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD+, NADH, ATP, ADP, isocitrate, or α-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade. PMID:11325948

  16. Analysis of Genes Involved in Biosynthesis of Coronafacic Acid, the Polyketide Component of the Phytotoxin Coronatine

    PubMed Central

    Rangaswamy, Vidhya; Mitchell, Robin; Ullrich, Matthias; Bender, Carol

    1998-01-01

    Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3′ end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated cfa8, cfa9, and tnp1; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of cfa8 and cfa9 were overproduced in Escherichia coli BL21; however, tnp1 was not translated in these experiments. Mutagenesis and complementation analysis indicated that cfa8 is required for the production of CFA and COR. Analysis of a cfa9 mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway; tnp1, however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a P. syringae strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster. PMID:9642184

  17. Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae

    PubMed Central

    Chemler, Joseph A; Yan, Yajun; Koffas, Mattheos AG

    2006-01-01

    Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties. PMID:16719921

  18. Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis.

    PubMed

    Viollier, P H; Minas, W; Dale, G E; Folcher, M; Thompson, C J

    2001-05-01

    Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K(m) values of CitA for acetyl coenzyme A (acetyl-CoA) (32 microM) and oxaloacetate (17 microM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD(+), NADH, ATP, ADP, isocitrate, or alpha-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.

  19. Alternative oxidase impacts ganoderic acid biosynthesis by regulating intracellular ROS levels in Ganoderma lucidum.

    PubMed

    Shi, Deng-Ke; Zhu, Jing; Sun, Ze-Hua; Zhang, Guang; Liu, Rui; Zhang, Tian-Jun; Wang, Sheng-Li; Ren, Ang; Zhao, Ming-Wen

    2017-09-13

    The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50 %, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44 %, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.

  20. Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor.

    PubMed

    Comba, Santiago; Menendez-Bravo, Simón; Arabolaza, Ana; Gramajo, Hugo

    2013-01-29

    Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production. The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single cell oil. Altogether, these

  1. Comprehensive profiling of amino acid response uncovers unique methionine-deprived response dependent on intact creatine biosynthesis.

    PubMed

    Tang, Xiaohu; Keenan, Melissa M; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J Will; Freedland, Stephen J; Murphy, Susan K; Chi, Jen-Tsan

    2015-04-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  2. Comprehensive Profiling of Amino Acid Response Uncovers Unique Methionine-Deprived Response Dependent on Intact Creatine Biosynthesis

    PubMed Central

    Tang, Xiaohu; Keenan, Melissa M.; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J. Will; Freedland, Stephen J.; Murphy, Susan K.; Chi, Jen-Tsan

    2015-01-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  3. Fatty acid biosynthesis is involved in the production of hepatitis B virus particles.

    PubMed

    Okamura, Hitomi; Nio, Yasunori; Akahori, Yuichi; Kim, Sulyi; Watashi, Koichi; Wakita, Takaji; Hijikata, Makoto

    2016-06-17

    Hepatitis B virus (HBV) proliferates in hepatocytes after infection, but the host factors that contribute to the HBV lifecycle are poorly understood at the molecular level. We investigated whether fatty acid biosynthesis (FABS), which was recently reported to contribute to the genomic replication of hepatitis C virus, plays a role in HBV proliferation. We examined the effects of inhibitors of the enzymes in the FABS pathway on the HBV lifecycle by using recombinant HBV-producing cultured cells and found that the extracellular HBV DNA level, reflecting HBV particle production, was decreased by treatment with inhibitors suppressed the synthesis of long-chain saturated fatty acids with little cytotoxicity. The reduced HBV DNA level was reversed when palmitic acid, which is the product of fatty acid synthase (FAS) during FABS, was used simultaneously with the inhibitor. We also observed that the amount of intracellular HBV DNA in the cells was increased by FAS inhibitor treatment, suggesting that FABS is associated with HBV particle production but not its genome replication. This suggests that FABS might be a potent target for anti-HBV drug with a mode of action different from current HBV therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Tetrahydrobiopterin Biosynthesis as a Potential Target of the Kynurenine Pathway Metabolite Xanthurenic Acid.

    PubMed

    Haruki, Hirohito; Hovius, Ruud; Pedersen, Miriam Grønlund; Johnsson, Kai

    2016-01-08

    Tryptophan metabolites in the kynurenine pathway are up-regulated by pro-inflammatory cytokines or glucocorticoids, and are linked to anti-inflammatory and immunosuppressive activities. In addition, they are up-regulated in pathologies such as cancer, autoimmune diseases, and psychiatric disorders. The molecular mechanisms of how kynurenine pathway metabolites cause these effects are incompletely understood. On the other hand, pro-inflammatory cytokines also up-regulate the amounts of tetrahydrobiopterin (BH4), an enzyme cofactor essential for the synthesis of several neurotransmitter and nitric oxide species. Here we show that xanthurenic acid is a potent inhibitor of sepiapterin reductase (SPR), the final enzyme in de novo BH4 synthesis. The crystal structure of xanthurenic acid bound to the active site of SPR reveals why among all kynurenine pathway metabolites xanthurenic acid is the most potent SPR inhibitor. Our findings suggest that increased xanthurenic acid levels resulting from up-regulation of the kynurenine pathway could attenuate BH4 biosynthesis and BH4-dependent enzymatic reactions, linking two major metabolic pathways known to be highly up-regulated in inflammation.

  5. The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival

    PubMed Central

    Fernandes, João Daniel Santos; Martho, Kevin; Tofik, Veridiana; Vallim, Marcelo A.; Pascon, Renata C.

    2015-01-01

    Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro. PMID:26162077

  6. The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival.

    PubMed

    Fernandes, João Daniel Santos; Martho, Kevin; Tofik, Veridiana; Vallim, Marcelo A; Pascon, Renata C

    2015-01-01

    Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro.

  7. Reinvestigation of the biosynthesis of 2-aminoethylphosphonic acid in Tetrahymena pyriformis

    SciTech Connect

    Barry, R.J.; Dunaway-Mariano, D.; Mariano, P.S.

    1986-05-01

    The initial step in the proposed biosynthetic pathway of 2-aminoethylphosphonate involves an intramolecular rearrangement of phosphoenolpyruvate to form the C-P compound, 3-phosphonopyruvate. A radioisotopic assay with authentic phosphonopyruvate as a cold carrier results in the formation of material chromatographically identical to phosphonopyruvate. NMR and degradation studies reveal that the assayed product is not p-pyr, but rather phosphoglyceric acid. Herein they report results from a reinvestigation of the biosynthesis of AEP in T. pyriformis. Recent study indicates that AEP is formed when radiolabeled Pi or PEP are used as precursors. P-pyr formation has yet to be demonstrated. Results from studies aimed at the direct verification or exclusion of p-pyr as an intermediate in the biosynthetic pathway leading to AEP formation is presented.

  8. An in vitro system from maize seedlings for tryptophan-independent indole-3-acetic acid biosynthesis

    SciTech Connect

    Oestin, A.; Ilic, N.; Cohen, J.D.

    1999-01-01

    The enzymatic synthesis of indole-3-acetic acid (IAA) from indole by an in vitro preparation from maize (Zea mays L.) that does not use tryptophan (Trp) as an intermediate is described. Light-grown seedlings of normal maize and the maize mutant orange pericarp were shown to contain the necessary enzymes to convert [{sup 14}C]indole to IAA. The reaction was not inhibited by unlabeled Trp and neither [{sup 14}C]Trp nor [{sup 14}C]serine substituted for [{sup 14}C]indole in this in vitro system. The reaction had a pH optimum greater than 8.0, required a reducing environment, and had an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway in plants.

  9. Resistance to herbicides inhibiting the biosynthesis of very-long-chain fatty acids.

    PubMed

    Busi, Roberto

    2014-09-01

    Herbicides that act by inhibiting the biosynthesis of very-long-chain fatty acids (VLCFAs) have been used to control grass weeds in major crops throughout the world for the past 60 years. VLCFA-inhibiting herbicides are generally highly selective in crops, induce similar symptoms in susceptible grasses and can be found within the herbicide groups classified by the HRAC as K3 and N. Even after many years of continuous use, only 12 grass weed species have evolved resistance to VLCFA-inhibiting herbicides. Here, the cases of resistance that have evolved in major grass weed species belonging to the Avena, Echinochloa and Lolium genera in three different agricultural systems are reviewed. In particular we explore the possible reasons why VLCFA herbicides have been slow to select resistant weeds, outline the herbicide mode of action and discuss the resistance mechanisms that are most likely to have been selected.

  10. In Vitro Stepwise Reconstitution of Amino Acid Derived Vinyl Isocyanide Biosynthesis: Detection of an Elusive Intermediate.

    PubMed

    Chang, Wei-Chen; Sanyal, Dev; Huang, Jhih-Liang; Ittiamornkul, Kuljira; Zhu, Qin; Liu, Xinyu

    2017-02-17

    In vitro reconstitution of a newly discovered isonitrile synthase (AmbI1 and AmbI2) and the detection of an elusive intermediate (S)-3-(1H-indol-3-yl)-2-isocyanopropanoic acid 1 in indolyl vinyl isocyanide biogenesis are reported. The characterization of iron/2-oxoglutarate (Fe/2OG) dependent desaturases IsnB and AmbI3 sheds light on the possible mechanism underlying stereoselective alkene installation to complete the biosynthesis of (E)- and (Z)-3-(2-isocyanovinyl)-1H-indole 2 and 5. Establishment of a tractable isonitrile synthase system (AmbI1 and AmbI2) paves the way to elucidate the enigmatic enzyme mechanism for isocyanide formation.

  11. An Examination of the Carbon Isotope Effects Associated with Amino Acid Biosynthesis

    NASA Astrophysics Data System (ADS)

    Scott, James H.; O'Brien, Diane M.; Emerson, David; Sun, Henry; McDonald, Gene D.; Salgado, Antonio; Fogel, Marilyn L.

    2006-12-01

    Stable carbon isotope ratios (δ13C) were determined for alanine, proline, phenylalanine, valine, leucine, isoleucine, aspartate (aspartic acid and asparagine), glutamate (glutamic acid and glutamine), lysine, serine, glycine, and threonine from metabolically diverse microorganisms. The microorganisms examined included fermenting bacteria, organotrophic, chemolithotrophic, phototrophic, methylotrophic, methanogenic, acetogenic, acetotrophic, and naturally occurring cryptoendolithic communities from the Dry Valleys of Antarctica. Here we demonstrated that reactions involved in amino acid biosynthesis can be used to distinguish amino acids formed by life from those formed by nonbiological processes. The unique patterns of δ13C imprinted by life on amino acids produced a biological bias. We also showed that, by applying discriminant function analysis to the δ13C value of a pool of amino acids formed by biological activity, it was possible to identify key aspects of intermediary carbon metabolism in the microbial world. In fact, microorganisms examined in this study could be placed within one of three metabolic groups: (1) heterotrophs that grow by oxidizing compounds containing three or more carbon-to-carbon bonds (fermenters and organotrophs), (2) autotrophs that grow by taking up carbon dioxide (chemolitotrophs and phototrophs), and (3) acetoclastic microbes that grow by assimilation of formaldehyde or acetate (methylotrophs, methanogens, acetogens, and acetotrophs). Furthermore, we demonstrated that cryptoendolithic communities from Antarctica grouped most closely with the autotrophs, which indicates that the dominant metabolic pathways in these communities are likely those utilized for CO2 fixation. We propose that this technique can be used to determine the dominant metabolic types in a community and reveal the overall flow of carbon in a complex ecosystem.

  12. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    PubMed

    Andre, Christelle M; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; Cooney, Janine M; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; Laing, William A

    2016-09-01

    Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis.

  13. Biosynthesis, structure and biological activity of hydroxyeicosatetraenoic acids in Hydra vulgaris.

    PubMed Central

    Di Marzo, V; De Petrocellis, L; Gianfrani, C; Cimino, G

    1993-01-01

    Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the control of body pattern, head and tentacle regeneration and bud formation in Hydra spp. Here we describe for the first time the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) in vitro by hydroid cytosolic extracts. Incubation of both unlabelled and tritiated AA with homogenates of Hydra vulgaris led to the conversion of up to 11% of the exogenous fatty acid into mainly two metabolites. These were characterized as 11-hydroperoxyeicosatetraenoic acid (11-HPETE) and 11-HETE by means of a combination of chromatographic, chemical, 1H-n.m.r. and electron-impact m.s. techniques. Trace amounts of 9-HETE and 12-HETE were also found. Analysis of 11-HETE by chiral-phase h.p.l.c. revealed that this metabolite was composed mainly of the R enantiomer. The production of 11-HPETE and 11-HETE was found to be: (1) associated with the cytosolic fraction of Hydra homogenates; (2) dependent on AA concentration, incubation time and protein amount in the homogenates; (3) unaffected by co-incubation with the 5- and 12-lipoxygenase inhibitors, 5,8,11-eicosatriynoic acid and nordihydroguaiaretic acid, the cyclo-oxygenase inhibitor, indomethacin, or the cytochrome P-450 inhibitors, proadifen and methoxalen. These results strongly suggest the presence of a very active (R)-11-lipoxygenase in H. vulgaris. The activity of both R and S enantiomers of synthetic 9-, 11- and 12-HETE and of 'endogenous' 11-HETE was studied on tentacle regeneration and bud formation in decapitated Hydra. Although almost all compounds tested inhibited budding, only endogenous 11-HETE and synthetic (R)-11-HETE significantly enhanced the average number of tentacles, thus suggesting that this eicosanoid might be one of the cellular regulators of regeneration in H. vulgaris. PMID:8216222

  14. Crystal structure of FadD32, an enzyme essential for mycolic acid biosynthesis in mycobacteria.

    PubMed

    Li, Wenjuan; Gu, Shoujin; Fleming, Joy; Bi, Lijun

    2015-12-02

    Fatty acid degradation protein D32 (FadD32), an enzyme required for mycolic acid biosynthesis and essential for mycobacterial growth, has recently been identified as a valid and promising target for anti-tuberculosis drug development. Here we report the crystal structures of Mycobacterium smegmatis FadD32 in the apo and ATP-bound states at 2.4 Å and 2.25 Å resolution, respectively. FadD32 consists of two globular domains connected by a flexible linker. ATP binds in a cleft at the interface between the N- and C-terminal domains and its binding induces significant local conformational changes in FadD32. The binding sites of meromycolic acid and phosphopantetheine are identified by structural comparison with other members of the adenylating enzyme superfamily. These results will improve our understanding of the catalytic mechanism of FadD32 and help in the design of inhibitors of this essential enzyme.

  15. Linoleic acid biosynthesis and characterization of the. Delta. sup 12 desaturase in insects

    SciTech Connect

    Cripps, C.

    1988-01-01

    De novo biosynthesis of linoleic acid was demonstrated in vivo in 8 of 32 insect species examined, including both holometabolous and hemimetabolous species. The incorporation of (1-{sup 14}C) acetate into linoleic acid was demonstrated by radio-gas-liquid chromatography (radio-GLC), and in selected species by radio-high-performance liquid chromatography, silver nitrate thin-layer chromatography, radio-GLC and GLC linked to mass spectrometry of ozonolysis products. Analysis of the ozonolysis products clearly demonstrated that the entire molecule was labeled and that synthesis of linoleate was de novo from acetate. The in vivo incorporation of (1-{sup 14}C)acetate into lipid was monitored during the final three stadia of both male and female house crickets, Acheta domesticus. Characterization of the {Delta}{sup 12}-desaturase showed that, in the house cricket, this enzyme is microsomal and requires a reduced pyridine dinucleotide as a cofactor, with NADPH the preferred electron donor. The optimal substrate concentration for desaturation is about 40 uM. Addition of the microsomal supernatant, MgCl{sub 2} or ATP did not enhance activity. The form of the substrate for the desaturase, oleic acid, was determined and appears to be a CoA derivative, as is true for most animal desaturases, rather than a complex lipid, as it is in plants.

  16. Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia

    PubMed Central

    Brose, Stephen A.; Golovko, Svetlana A.; Golovko, Mikhail Y.

    2016-01-01

    Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA) biosynthesis followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FA synthesis maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FA synthesis inhibition on NADH2+/NAD+ and NADPH2+/NADP+ ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FA synthesis inhibitors, TOFA (inhibits Acetyl-CoA carboxylase) and cerulenin (inhibits FA synthase), increased NADH2+/NAD+ and NADPH2+/NADP+ ratios under hypoxia. Further, FA synthesis inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke. PMID:27965531

  17. A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum

    PubMed Central

    Taura, Futoshi; Iijima, Miu; Yamanaka, Eriko; Takahashi, Hironobu; Kenmoku, Hiromichi; Saeki, Haruna; Morimoto, Satoshi; Asakawa, Yoshinori; Kurosaki, Fumiya; Morita, Hiroyuki

    2016-01-01

    Rhododendron dauricum L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of R. dauricum. The primary structure of ORS shared relatively low identities to those of PKSs from other plants, and the active site of ORS had a unique amino acid composition. The bacterially expressed, recombinant ORS accepted acetyl-CoA as the preferable starter substrate, and produced orcinol as the major reaction product, along with four minor products including OSA. The ORS identified in this study is the first plant PKS that generates acetate-derived aromatic tetraketides, such as orcinol and OSA. Interestingly, OSA production was clearly enhanced in the presence of Cannabis sativa olivetolic acid cyclase, suggesting that the ORS is involved in OSA biosynthesis together with an unidentified cyclase in R. dauricum. PMID:27729920

  18. RNA-seq based transcriptomic analysis uncovers α-linolenic acid and jasmonic acid biosynthesis pathways respond to cold acclimation in Camellia japonica

    PubMed Central

    Li, Qingyuan; Lei, Sheng; Du, Kebing; Li, Lizhi; Pang, Xufeng; Wang, Zhanchang; Wei, Ming; Fu, Shao; Hu, Limin; Xu, Lin

    2016-01-01

    Camellia is a well-known ornamental flower native to Southeast of Asia, including regions such as Japan, Korea and South China. However, most species in the genus Camellia are cold sensitive. To elucidate the cold stress responses in camellia plants, we carried out deep transcriptome sequencing of ‘Jiangxue’, a cold-tolerant cultivar of Camellia japonica, and approximately 1,006 million clean reads were generated using Illumina sequencing technology. The assembly of the clean reads produced 367,620 transcripts, including 207,592 unigenes. Overall, 28,038 differentially expressed genes were identified during cold acclimation. Detailed elucidation of responses of transcription factors, protein kinases and plant hormone signalling-related genes described the interplay of signal that allowed the plant to fine-tune cold stress responses. On the basis of global gene regulation of unsaturated fatty acid biosynthesis- and jasmonic acid biosynthesis-related genes, unsaturated fatty acid biosynthesis and jasmonic acid biosynthesis pathways were deduced to be involved in the low temperature responses in C. japonica. These results were supported by the determination of the fatty acid composition and jasmonic acid content. Our results provide insights into the genetic and molecular basis of the responses to cold acclimation in camellia plants. PMID:27819341

  19. RNA-seq based transcriptomic analysis uncovers α-linolenic acid and jasmonic acid biosynthesis pathways respond to cold acclimation in Camellia japonica.

    PubMed

    Li, Qingyuan; Lei, Sheng; Du, Kebing; Li, Lizhi; Pang, Xufeng; Wang, Zhanchang; Wei, Ming; Fu, Shao; Hu, Limin; Xu, Lin

    2016-11-07

    Camellia is a well-known ornamental flower native to Southeast of Asia, including regions such as Japan, Korea and South China. However, most species in the genus Camellia are cold sensitive. To elucidate the cold stress responses in camellia plants, we carried out deep transcriptome sequencing of 'Jiangxue', a cold-tolerant cultivar of Camellia japonica, and approximately 1,006 million clean reads were generated using Illumina sequencing technology. The assembly of the clean reads produced 367,620 transcripts, including 207,592 unigenes. Overall, 28,038 differentially expressed genes were identified during cold acclimation. Detailed elucidation of responses of transcription factors, protein kinases and plant hormone signalling-related genes described the interplay of signal that allowed the plant to fine-tune cold stress responses. On the basis of global gene regulation of unsaturated fatty acid biosynthesis- and jasmonic acid biosynthesis-related genes, unsaturated fatty acid biosynthesis and jasmonic acid biosynthesis pathways were deduced to be involved in the low temperature responses in C. japonica. These results were supported by the determination of the fatty acid composition and jasmonic acid content. Our results provide insights into the genetic and molecular basis of the responses to cold acclimation in camellia plants.

  20. Chlorogenic Acids Biosynthesis in Centella asiatica Cells Is not Stimulated by Salicylic Acid Manipulation.

    PubMed

    Ncube, E N; Steenkamp, P A; Madala, N E; Dubery, I A

    2016-07-01

    Exogenous application of synthetic and natural elicitors of plant defence has been shown to result in mass production of secondary metabolites with nutraceuticals properties in cultured cells. In particular, salicylic acid (SA) treatment has been reported to induce the production of phenylpropanoids, including cinnamic acid derivatives bound to quinic acid (chlorogenic acids). Centella asiatica is an important medicinal plant with several therapeutic properties owing to its wide spectrum of secondary metabolites. We investigated the effect of SA on C. asiatica cells by monitoring perturbation of chlorogenic acids in particular. Different concentrations of SA were used to treat C. asiatica cells, and extracts from both treated and untreated cells were analysed using an optimised UHPLC-QTOF-MS/MS method. Semi-targeted multivariate data analyses with the aid of principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed a concentration-dependent metabolic response. Surprisingly, a range of chlorogenic acid derivatives were found to be downregulated as a consequence of SA treatment. Moreover, irbic acid (3,5-O-dicaffeoyl-4-O-malonilquinic acid) was found to be a dominant CGA in C. asiatica cells, although the SA treatment also had a negative effect on its concentration. Overall SA treatment was found to be an ineffective elicitor of CGA production in cultured C. asiatica cells.

  1. Salicylic Acid sans Aspirin in Animals and Man: Persistence in Fasting and Biosynthesis from Benzoic Acid

    PubMed Central

    2008-01-01

    Salicylic acid (SA), which is central to defense mechanisms in plants and the principal metabolite of aspirin, occurs naturally in man with higher levels of SA and its urinary metabolite salicyluric acid (SU) in vegetarians overlapping with levels in patients on low-dose aspirin regimens. SA is widely distributed in animal blood. Fasting for major colorectal surgery did not cause disappearance of SA from plasma, even in patients following total proctocolectomy. A 13C6 benzoic acid load ingested by six volunteers led, between 8 and 16 h, to a median 33.9% labeling of urinary salicyluric acid. The overall contribution of benzoic acid (and its salts) to the turnover of circulating SA thus requires further assessment. However, that SA appears to be, at least partially, an endogenous compound should lead to reassessment of its role in human (and animal) pathophysiology. PMID:19053387

  2. Salicylic acid sans aspirin in animals and man: persistence in fasting and biosynthesis from benzoic acid.

    PubMed

    Paterson, John R; Baxter, Gwendoline; Dreyer, Jacob S; Halket, John M; Flynn, Robert; Lawrence, James R

    2008-12-24

    Salicylic acid (SA), which is central to defense mechanisms in plants and the principal metabolite of aspirin, occurs naturally in man with higher levels of SA and its urinary metabolite salicyluric acid (SU) in vegetarians overlapping with levels in patients on low-dose aspirin regimens. SA is widely distributed in animal blood. Fasting for major colorectal surgery did not cause disappearance of SA from plasma, even in patients following total proctocolectomy. A (13)C(6) benzoic acid load ingested by six volunteers led, between 8 and 16 h, to a median 33.9% labeling of urinary salicyluric acid. The overall contribution of benzoic acid (and its salts) to the turnover of circulating SA thus requires further assessment. However, that SA appears to be, at least partially, an endogenous compound should lead to reassessment of its role in human (and animal) pathophysiology.

  3. myo-Inositol 1-Phosphate Synthase Inhibition and Control of Uridine Diphosphate-d-glucuronic Acid Biosynthesis in Plants 12

    PubMed Central

    Loewus, Mary W.; Loewus, Frank

    1974-01-01

    Of the eight intermediates associated with the two pathways of UDP-d-glucuronic acid biosynthesis found in plants, only d-glucuronic acid inhibited myo-inositol 1-phosphate synthase (EC 5.5.1.4), formerly referred to as d-glucose 6-phosphate cycloaldolase. Inhibition was competitive. An attempt to demonstrate over-all reversibility of the synthase indicated that it was less than 5% reversible, if at all. PMID:16658890

  4. Hardening with salicylic acid induces concentration-dependent changes in abscisic acid biosynthesis of tomato under salt stress.

    PubMed

    Horváth, Edit; Csiszár, Jolán; Gallé, Ágnes; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2015-07-01

    The role of salicylic acid (SA) in the control of abscisic acid (ABA) biosynthesis is controversial although both plant growth regulators may accumulate in tissues under abiotic and biotic stress conditions. Hardening of tomato plants to salinity stress with 10(-4)M SA ("high SA") resulted in an up-regulation of ABA biosynthesis genes, zeaxanthin epoxidase (SlZEP1), 9-cis-epoxycarotenoid dioxygenase (SlNCED1) and aldehyde oxidases (SlAO1 and SlAO2) in the roots and led to ABA accumulation both in root and leaf tissues. In plants pre-treated with lower concentration of SA (10(-7)M, "low SA"), the up-regulation of SlNCED1 in the roots promoted ABA accumulation in the root tissues but the hormone concentration remained at control level in the leaves. Salt stress induced by 100mM NaCl reduced the transcript abundance of ABA biosynthetic genes and inhibited SlAO activity in plants hardened with "high SA", but the tissues maintained root ABA level over the untreated control. The combined effect of "high SA" and ABA under salt stress led to partially recovered photosynthetic activity, reduced ethylene production in root apices, and restored root growth, which is one of the main features of salt tolerance. Unlike "high SA", hardening with "low SA" had no influence on ethylene production, and led to reduced elongation of roots in plants exposed to 100mM NaCl. The up-regulation of carotenoid cleavage dioxygenases SlCCD1A and SlCCD1B by SA, which produce apocarotenoids, may open new pathways in SA sensing and signalling processes.

  5. Fatty acid biosynthesis is involved in the production of hepatitis B virus particles

    SciTech Connect

    Okamura, Hitomi; Nio, Yasunori; Akahori, Yuichi; Kim, Sulyi; Watashi, Koichi; Wakita, Takaji; Hijikata, Makoto

    2016-06-17

    Hepatitis B virus (HBV) proliferates in hepatocytes after infection, but the host factors that contribute to the HBV lifecycle are poorly understood at the molecular level. We investigated whether fatty acid biosynthesis (FABS), which was recently reported to contribute to the genomic replication of hepatitis C virus, plays a role in HBV proliferation. We examined the effects of inhibitors of the enzymes in the FABS pathway on the HBV lifecycle by using recombinant HBV-producing cultured cells and found that the extracellular HBV DNA level, reflecting HBV particle production, was decreased by treatment with inhibitors suppressed the synthesis of long-chain saturated fatty acids with little cytotoxicity. The reduced HBV DNA level was reversed when palmitic acid, which is the product of fatty acid synthase (FAS) during FABS, was used simultaneously with the inhibitor. We also observed that the amount of intracellular HBV DNA in the cells was increased by FAS inhibitor treatment, suggesting that FABS is associated with HBV particle production but not its genome replication. This suggests that FABS might be a potent target for anti-HBV drug with a mode of action different from current HBV therapy. -- Highlights: •Inhibitors of ACC1 and FAS but not SCD1 decreased production of extracellular HBV DNA. •Products of FABS, long chain fatty acids, increased production of extracellular HBV DNA. •FAS inhibitor increased intracellular levels of HBV DNA and HBcAg. •FABS was suggested to contribute to HBV particle production without significant relation with secretory pathway of the cells.

  6. New insights into the early biochemical activation of jasmonic acid biosynthesis in leaves

    PubMed Central

    Baldwin, Ian T

    2010-01-01

    In plants, herbivore attack elicits the rapid accumulation of jasmonic acid (JA) which results from the activation of constitutively expressed biosynthetic enzymes. The molecular mechanisms controlling the activation of JA biosynthesis remain largely unknown however new research has elucidated some of the early regulatory components involved in this process. Nicotiana attenuata plants, a wild tobacco species, responds to fatty acid amino acid conjuguates (FAC) elicitors in the oral secretion of its natural herbivore, Manduca sexta, by triggering specific defense and tolerance responses against it; all of the defense responses known to date require the amplification of the wound-induced JA increase. We recently demonstrated that this FAC-elicited JA burst requires an increased flux of free linolenic acid (18:3) likely originating from the activation of a plastidial glycerolipase (GLA1) which is activated by an abundant FAC found in insect oral secretions, N-linolenoyl-glutamate (18:3-Glu). The lack of accumulation of free 18:3 after elicitation suggests a tight physical association between GLA1 and LOX3 in N. attenuata leaves. In addition, the salicylate-induced protein kinase (SIPK) and the nonexpressor of PR-1 (NPR1) participate in this activation mechanism that controls the supply of 18:3. In contrast, the wound-induced protein kinase (WIPK) does not but instead regulates the conversion of 13(S)-hydroperoxy-18:3 into 12-oxo-phytodienoic acid (OPDA). These results open new perspectives on the complex network of signals and regulatory components inducing the JA biosynthetic pathway. PMID:20037473

  7. Chlorogenic Acid Biosynthesis Appears Linked with Suberin Production in Potato Tuber (Solanum tuberosum).

    PubMed

    Valiñas, Matías Ariel; Lanteri, María Luciana; ten Have, Arjen; Andreu, Adriana Balbina

    2015-05-20

    Potato (Solanum tuberosum L.) is a good source of dietary antioxidants. Chlorogenic acid (CGA) and caffeic acid (CA) are the most abundant phenolic acid antioxidants in potato and are formed by the phenylpropanoid pathway. A number of CGA biosynthetic routes that involve hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and/or hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) have been proposed, but little is known about their path in potato. CA production requires a caffeoyl shikimate esterase (CSE), and CA serves as a substrate of lignin precursor ferulic acid via the action of caffeic/5-hydroxyferulic acid O-methyltransferase (COMT I). CGA is precursor of caffeoyl-CoA and, via caffeoyl-CoA O-methyltransferase (CCoAOMT), of feruloyl-CoA. Feruloyl-CoA is required for lignin and suberin biosynthesis, crucial for tuber development. Here, metabolite and transcript levels of the mentioned and related enzymes, such as cinnamate 4-hydroxylase (C4H), were determined in the flesh and skin of fresh and stored tubers. Metabolite and transcript levels were higher in skin than in flesh, irrespective of storage. CGA and CA production appear to occur via p-coumaroyl-CoA, using HQT and CSE, respectively. HCT is likely involved in CGA remobilization toward suberin. The strong correlation between CGA and CA, the correspondence with C4H, HQT, CCoAOMT2, and CSE, and the negative correlation of HCT and COMT I in potato tubers suggest a major flux toward suberin.

  8. Metabolomics Analysis and Biosynthesis of Rosmarinic Acid in Agastache rugosa Kuntze Treated with Methyl Jasmonate

    PubMed Central

    Uddin, Md. Romij; Xu, Hui; Park, Woo Tae; Tuan, Pham Anh; Li, Xiaohua; Chung, Eunsook; Lee, Jai-Heon; Park, Sang Un

    2013-01-01

    This study investigated the effect of methyl jasmonate (MeJA) on metabolic profiles and rosmarinic acid (RA) biosynthesis in cell cultures of Agastache rugosa Kuntze. Transcript levels of phenylpropanoid biosynthetic genes, i.e., ArPAL, Ar4CL, and ArC4H, maximally increased 4.5-fold, 3.4-fold, and 3.5-fold, respectively, compared with the untreated controls, and the culture contained relatively high amounts of RA after exposure of cells to 50 µM MeJA. RA levels were 2.1-, 4.7-, and 3.9-fold higher after exposure to 10, 50, and 100 µM MeJA, respectively, than those in untreated controls. In addition, the transcript levels of genes attained maximum levels at different time points after the initial exposure. The transcript levels of ArC4H and Ar4CL were transiently induced by MeJA, and reached a maximum of up to 8-fold at 3 hr and 6 hr, respectively. The relationships between primary metabolites and phenolic acids in cell cultures of A. rugosa treated with MeJA were analyzed by gas chromatography coupled with time-of-flight mass spectrometry. In total, 45 metabolites, including 41 primary metabolites and 4 phenolic acids, were identified from A. rugosa. Metabolite profiles were subjected to partial least square-discriminate analysis to evaluate the effects of MeJA. The results indicate that both phenolic acids and precursors for the phenylpropanoid biosynthetic pathway, such as aromatic amino acids and shikimate, were induced as a response to MeJA treatment. Therefore, MeJA appears to have an important impact on RA accumulation, and the increased RA accumulation in the treated cells might be due to activation of the phenylpropanoid genes ArPAL, ArC4H, and Ar4CL. PMID:23724034

  9. Genomic and Transcriptomic Analyses of Indole-3-Acetic Acid Biosynthesis in Diatoms

    NASA Astrophysics Data System (ADS)

    Lim, R.; Armbrust, V.

    2016-02-01

    Indole-3-acetic acid (IAA) is a major plant growth hormone and a common mediator of plant-bacterial interactions. Recently, IAA has also been found to play a role in interactions between diatoms and bacteria, with IAA production by an associated Sulfitobacter leading to increased growth rates in the marine diatom Pseudo-nitzschia multiseries. It is unclear, however, if diatoms themselves are able to synthesize IAA and whether this capability is widespread throughout Bacillariophyta. Four major tryptophan-dependent IAA biosynthesis pathways have been identified in plants and bacteria, each denoted by the first intermediate downstream of tryptophan: the indole-3-pyruvate (IPyA), tryptamine (TAM), indole-3-acetaldoxime (IAOx) and indole-3-acetamide (IAM) pathways. To investigate the possibility of IAA biosynthesis in diatoms, we first analyzed publicly available genomes of raphid pennates P. multiseries, Phaeodactylum tricornutum, Fragilariopsis cylindrus and centric Thalassiosira pseudonana for potential homologs to plant and bacterial IAA biosynthesis genes. The P. multiseries, F. cylindrus and P. tricornutum genomes encode downstream enzymes for bacterial TAM and IAM and plant IPyA pathways. The more evolutionarily ancient T. pseudonana encodes one TAM enzyme in its genome. To investigate the potential distribution of these pathways more broadly, we surveyed the transcriptomes of 11 diatom species that include representatives from all four Bacillariophyta classes. Datasets used were sequenced as part of the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) and obtained from cultures maintained axenically. Transcripts associated with the TAM pathway were most frequently detected, with potential homologs to required enzymes identified in 10 of the 11 species examined. Transcripts homologous to rate-limiting IPyA enzymes were detected in six species. Only two centric and araphid pennate species expressed transcripts associated with enzymes in the

  10. A Single Amino Acid Change Is Responsible for Evolution of Acyltransferase Specificity in Bacterial Methionine Biosynthesis

    SciTech Connect

    Zubieta, C.; Arkus, K.A.J.; Cahoon, R.E.; Jez, J.M.

    2009-05-28

    Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 {angstrom} resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.

  11. Biological Role of Aldo–Keto Reductases in Retinoic Acid Biosynthesis and Signaling

    PubMed Central

    Ruiz, F. Xavier; Porté, Sergio; Parés, Xavier; Farrés, Jaume

    2012-01-01

    Several aldo–keto reductase (AKR) enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3), as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA) biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance. PMID:22529810

  12. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

    PubMed Central

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-01-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  13. Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids.

    PubMed

    Oku, Hirosuke; Futamori, Naoya; Masuda, Kenichi; Shimabukuro, Yumiko; Omine, Tomoyo; Iwasaki, Hironori

    2003-10-01

    It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.

  14. Coordinated Regulation of Species-Specific Hydroxycinnamic Acid Degradation and Siderophore Biosynthesis Pathways in Agrobacterium fabrum

    PubMed Central

    Baude, Jessica; Vial, Ludovic; Villard, Camille; Campillo, Tony; Lavire, Céline; Nesme, Xavier

    2016-01-01

    ABSTRACT The rhizosphere-inhabiting species Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to degrade hydroxycinnamic acids (HCAs), especially ferulic acid and p-coumaric acid, via the novel A. fabrum HCA degradation pathway. Gene expression profiles of A. fabrum strain C58 were investigated in the presence of HCAs, using a C58 whole-genome oligoarray. Both ferulic acid and p-coumaric acid caused variations in the expression of more than 10% of the C58 genes. Genes of the A. fabrum HCA degradation pathway, together with the genes involved in iron acquisition, were among the most highly induced in the presence of HCAs. Two operons coding for the biosynthesis of a particular siderophore, as well as genes of the A. fabrum HCA degradation pathway, have been described as being specific to the species. We demonstrate here their coordinated expression, emphasizing the interdependence between the iron concentration in the growth medium and the rate at which ferulic acid is degraded by cells. The coordinated expression of these functions may be advantageous in HCA-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. The present results confirm that there is cooperation between the A. fabrum-specific genes, defining a particular ecological niche. IMPORTANCE We previously identified seven genomic regions in Agrobacterium fabrum that were specifically present in all of the members of this species only. Here we demonstrated that two of these regions, encoding the hydroxycinnamic acid degradation pathway and the iron acquisition pathway, were regulated in a coordinated manner. The coexpression of these functions may be advantageous in hydroxycinnamic acid-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. These data support the view that bacterial genomic species

  15. Auxin Biosynthesis

    PubMed Central

    Zhao, Yunde

    2014-01-01

    lndole-3-acetic acid (IAA), the most important natural auxin in plants, is mainly synthesized from the amino acid tryptophan (Trp). Recent genetic and biochemical studies in Arabidopsis have unambiguously established the first complete Trp-dependent auxin biosynthesis pathway. The first chemical step of auxin biosynthesis is the removal of the amino group from Trp by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family of transaminases to generate indole-3-pyruvate (IPA). IPA then undergoes oxidative decarboxylation catalyzed by the YUCCA (YUC) family of flavin monooxygenases to produce IAA. This two-step auxin biosynthesis pathway is highly conserved throughout the plant kingdom and is essential for almost all of the major developmental processes. The successful elucidation of a complete auxin biosynthesis pathway provides the necessary tools for effectively modulating auxin concentrations in plants with temporal and spatial precision. The progress in auxin biosynthesis also lays a foundation for understanding polar auxin transport and for dissecting auxin signaling mechanisms during plant development. PMID:24955076

  16. Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32

    PubMed Central

    Stanley, Sarah A.; Kawate, Tomohiko; Iwase, Noriaki; Shimizu, Motohisa; Clatworthy, Anne E.; Kazyanskaya, Edward; Sacchettini, James C.; Ioerger, Thomas R.; Siddiqi, Noman A.; Minami, Shoko; Aquadro, John A.; Schmidt Grant, Sarah; Rubin, Eric J.; Hung, Deborah T.

    2013-01-01

    Infection with the bacterial pathogen Mycobacterium tuberculosis imposes an enormous burden on global public health. New antibiotics are urgently needed to combat the global tuberculosis pandemic; however, the development of new small molecules is hindered by a lack of validated drug targets. Here, we describe the identification of a 4,6-diaryl-5,7-dimethyl coumarin series that kills M. tuberculosis by inhibiting fatty acid degradation protein D32 (FadD32), an enzyme that is required for biosynthesis of cell-wall mycolic acids. These substituted coumarin inhibitors directly inhibit the acyl-acyl carrier protein synthetase activity of FadD32. They effectively block bacterial replication both in vitro and in animal models of tuberculosis, validating FadD32 as a target for antibiotic development that works in the same pathway as the established antibiotic isoniazid. Targeting new steps in well-validated biosynthetic pathways in antitubercular therapy is a powerful strategy that removes much of the usual uncertainty surrounding new targets and in vivo clinical efficacy, while circumventing existing resistance to established targets. PMID:23798446

  17. Effect of nitrogen deficiency on ascorbic acid biosynthesis and recycling pathway in cucumber seedlings.

    PubMed

    Zhang, Xue; Yu, Hong Jun; Zhang, Xiao Meng; Yang, Xue Yong; Zhao, Wen Chao; Li, Qiang; Jiang, Wei Jie

    2016-11-01

    L-Ascorbic acid (AsA, ascorbate) is one of the most abundant natural antioxidants, and it is an important factor in the nutritional quality of cucumber. In this work, key enzymes involved in the ascorbic acid biosynthesis and recycling pathway in cucumber seedlings under nitrogen deficiency were investigated at the levels of transcription and enzyme activity. The activities of myo-inositol oxygenase (MIOX) and transcript levels of MIOXs increased dramatically, while the activities of ascorbate oxidase (AO) and glutathione reductase (GR) and transcript levels of AOs and GR2 decreased significantly in N-limited leaves, as did the ascorbate concentration, in nitrogen-deficient cucumber seedlings. The activities of other enzymes and transcript levels of other genes involved in the ascorbate recycling pathway and ascorbate synthesis pathways decreased or remained unchanged under nitrogen deficiency. These results indicate that nitrogen deficiency induced genes involved in the ascorbate-glutathione recycling and myo-inositol pathway in cucumber leaves. Thus, the AO, GR and MIOX involved in the pathways might play roles in AsA accumulation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  18. Articulation of three core metabolic processes in Arabidopsis: Fatty acid biosynthesis, leucine catabolism and starch metabolism

    PubMed Central

    Mentzen, Wieslawa I; Peng, Jianling; Ransom, Nick; Nikolau, Basil J; Wurtele, Eve Syrkin

    2008-01-01

    Background Elucidating metabolic network structures and functions in multicellular organisms is an emerging goal of functional genomics. We describe the co-expression network of three core metabolic processes in the genetic model plant Arabidopsis thaliana: fatty acid biosynthesis, starch metabolism and amino acid (leucine) catabolism. Results These co-expression networks form modules populated by genes coding for enzymes that represent the reactions generally considered to define each pathway. However, the modules also incorporate a wider set of genes that encode transporters, cofactor biosynthetic enzymes, precursor-producing enzymes, and regulatory molecules. We tested experimentally the hypothesis that one of the genes tightly co-expressed with starch metabolism module, a putative kinase AtPERK10, will have a role in this process. Indeed, knockout lines of AtPERK10 have an altered starch accumulation. In addition, the co-expression data define a novel hierarchical transcript-level structure associated with catabolism, in which genes performing smaller, more specific tasks appear to be recruited into higher-order modules with a broader catabolic function. Conclusion Each of these core metabolic pathways is structured as a module of co-expressed transcripts that co-accumulate over a wide range of environmental and genetic perturbations and developmental stages, and represent an expanded set of macromolecules associated with the common task of supporting the functionality of each metabolic pathway. As experimentally demonstrated, co-expression analysis can provide a rich approach towards understanding gene function. PMID:18616834

  19. How do background ozone concentrations affect the biosynthesis of rosmarinic acid in Melissa officinalis?

    PubMed

    Döring, Anne S; Pellegrini, Elisa; Della Batola, Michele; Nali, Cristina; Lorenzini, Giacomo; Petersen, Maike

    2014-03-01

    Lemon balm (Melissa officinalis; Lamiaceae) plants were exposed to background ozone (O3) dosages (80ppb for 5h), because high background levels of O3 are considered to be as harmful as episodic O3 peaks. Immediately at the end of fumigation the plants appeared visually symptomless, but necrotic lesions were observed later. The biosynthesis of rosmarinic acid (RA) comprises eight enzymes, among them phenylalanine ammonia-lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), tyrosine aminotransferase (TAT) and rosmarinic acid synthase (RAS). The transcript levels of these genes have been investigated by quantitative RT-PCR. There was a quick up-regulation of all genes at 3h of O3 exposure, but at 24h from beginning of exposure (FBE) only RAS and PAL were up-regulated. The specific activity of RAS was closely correlated with a decrease of RA concentration in lemon balm leaves. The specific activity of PAL increased at 12h FBE to 163% in comparison to control levels. This work provides insight into the effect of O3 stress on the formation of the main phenolic ingredient of the pharmaceutically important plant M. officinalis.

  20. Completion of the core β-oxidative pathway of benzoic acid biosynthesis in plants

    PubMed Central

    Qualley, Anthony V.; Widhalm, Joshua R.; Adebesin, Funmilayo; Kish, Christine M.; Dudareva, Natalia

    2012-01-01

    Despite the importance of benzoic acid (BA) as a precursor for a wide array of primary and secondary metabolites, its biosynthesis in plants has not been fully elucidated. BA formation from phenylalanine requires shortening of the C3 side chain by two carbon units, which can occur by a non–β-oxidative route and/or a β-oxidative pathway analogous to the catabolism of fatty acids. Enzymes responsible for the first and last reactions of the core BA β-oxidative pathway (cinnamic acid → cinnamoyl-CoA → 3-hydroxy-3-phenylpropanoyl-CoA → 3-oxo-3-phenylpropanoyl-CoA → BA-CoA) have previously been characterized in petunia, a plant with flowers rich in phenylpropanoid/benzenoid volatile compounds. Using a functional genomics approach, we have identified a petunia gene encoding cinnamoyl-CoA hydratase-dehydrogenase (PhCHD), a bifunctional peroxisomal enzyme responsible for two consecutively occurring unexplored intermediate steps in the core BA β-oxidative pathway. PhCHD spatially, developmentally, and temporally coexpresses with known genes in the BA β-oxidative pathway, and correlates with emission of benzenoid volatiles. Kinetic analysis of recombinant PhCHD revealed it most efficiently converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA, thus forming the substrate for the final step in the pathway. Down-regulation of PhCHD expression in petunia flowers resulted in reduced CHD enzyme activity, as well as decreased formation of BA-CoA, BA and their derived volatiles. Moreover, transgenic lines accumulated the PhCHD substrate cinnamoyl-CoA and the upstream pathway intermediate cinnamic acid. Discovery of PhCHD completes the elucidation of the core BA β-oxidative route in plants, and together with the previously characterized CoA-ligase and thiolase enzymes, provides evidence that the whole pathway occurs in peroxisomes. PMID:22988098

  1. A structural basis for the biosynthesis of the major chlorogenic acids found in coffee.

    PubMed

    Lallemand, Laura A; Zubieta, Chloe; Lee, Soon Goo; Wang, Yechun; Acajjaoui, Samira; Timmins, Joanna; McSweeney, Sean; Jez, Joseph M; McCarthy, James G; McCarthy, Andrew A

    2012-09-01

    Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.

  2. Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

    PubMed Central

    Sengupta, Sudeshna; Goonewardena, Lakshani; Juturu, Veeresh

    2015-01-01

    cis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain of Escherichia coli K-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes, pobA, aroY, and catA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed in E. coli to establish the MA biosynthetic pathway. E. coli native genes ubiC, aroFFBR, aroE, and aroL were overexpressed and the genes ptsH, ptsI, crr, and pykF were deleted from the E. coli genome in order to increase the precursors of the proposed MA pathway. The final engineered E. coli strain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars. PMID:26362984

  3. Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-Acid-Derived Biofuels and Chemicals with Relieved Side-Pathway Competition.

    PubMed

    Zhou, Yongjin J; Buijs, Nicolaas A; Zhu, Zhiwei; Gómez, Diego Orol; Boonsombuti, Akarin; Siewers, Verena; Nielsen, Jens

    2016-11-30

    Establishing efficient synthetic pathways for microbial production of biochemicals is often hampered by competing pathways and/or insufficient precursor supply. Compartmentalization in cellular organelles can isolate synthetic pathways from competing pathways, and provide a compact and suitable environment for biosynthesis. Peroxisomes are cellular organelles where fatty acids are degraded, a process that is inhibited under typical fermentation conditions making them an interesting workhouse for production of fatty-acid-derived molecules. Here, we show that targeting synthetic pathways to peroxisomes can increase the production of fatty-acid-derived fatty alcohols, alkanes and olefins up to 700%. In addition, we demonstrate that biosynthesis of these chemicals in the peroxisomes results in significantly decreased accumulation of byproducts formed by competing enzymes. We further demonstrate that production can be enhanced up to 3-fold by increasing the peroxisome population. The strategies described here could be used for production of other chemicals, especially acyl-CoA-derived molecules.

  4. Retroconversion of docosapentaenoic acid (n-6): an alternative pathway for biosynthesis of arachidonic acid in Daphnia magna.

    PubMed

    Strandberg, Ursula; Taipale, Sami J; Kainz, Martin J; Brett, Michael T

    2014-06-01

    The aim of this study was to assess metabolic pathways for arachidonic acid (20:4n-6) biosynthesis in Daphnia magna. Neonates of D. magna were maintained on [(13)C] enriched Scenedesmus obliquus and supplemented with liposomes that contained separate treatments of unlabeled docosapentaenoic acid (22:5n-6), 20:4n-6, linoleic acid (18:2n-6) or oleic acid (18:1n-9). Daphnia in the control treatment, without any supplementary fatty acids (FA) containing only trace amounts of 20:4n-6 (~0.3% of all FA). As expected, the highest proportion of 20:4n-6 (~6.3%) was detected in Daphnia that received liposomes supplemented with this FA. Higher availability of 18:2n-6 in the diet increased the proportion of 18:2n-6 in Daphnia, but the proportion of 20:4n-6 was not affected. Daphnia supplemented with 22:5n-6 contained ~3.5% 20:4n-6 in the lipids and FA specific stable isotope analyses validated that the increase in the proportion of 20:4n-6 was due to retroconversion of unlabeled 22:5n-6. These results suggest that chain shortening of 22:5n-6 is a more efficient pathway to synthesize 20:4n-6 in D. magna than elongation and desaturation of 18:2n-6. These results may at least partially explain the discrepancies noticed between phytoplankton FA composition and the expected FA composition in freshwater cladocerans. Finally, retroconversion of dietary 22:5n-6 to 20:4n-6 indicates Daphnia efficiently retain long chain n-6 FA in lake food webs, which might be important for the nutritional ecology of fish.

  5. Comprehensive transcriptome profiling of Salvia miltiorrhiza for discovery of genes associated with the biosynthesis of tanshinones and phenolic acids.

    PubMed

    Zhou, Wei; Huang, Qiang; Wu, Xiao; Zhou, Zewen; Ding, Mingquan; Shi, Min; Huang, Fenfen; Li, Shen; Wang, Yao; Kai, Guoyin

    2017-09-05

    Tanshinones and phenolic acids are crucial bioactive compounds biosynthesized in Salvia miltiorrhiza. Methyl jasmonate (MeJA) is an effective elicitor to enhance the production of phenolic acids and tanshinones simultaneously, while yeast extract (YE) is used as a biotic elicitor that only induce tanshinones accumulation. However, little was known about the different molecular mechanism. To identify the downstream and regulatory genes involved in tanshinone and phenolic acid biosynthesis, we conducted comprehensive transcriptome profiling of S. miltiorrhiza hairy roots treated with either MeJA or YE. Total 55588 unigenes were assembled from about 1.72 billion clean reads, of which 42458 unigenes (76.4%) were successfully annotated. The expression patterns of 19 selected genes in the significantly upregulated unigenes were verified by quantitative real-time PCR. The candidate downstream genes and other cytochrome P450s involved in the late steps of tanshinone and phenolic acid biosynthesis pathways were screened from the RNA-seq dataset based on co-expression pattern analysis with specific biosynthetic genes. Additionally, 375 transcription factors were identified to exhibit a significant up-regulated expression pattern in response to induction. This study can provide us a valuable gene resource for elucidating the molecular mechanism of tanshinones and phenolic acids biosynthesis in hairy roots of S. miltiorrhiza.

  6. Characterization of the formation of branched short-chain fatty acid:CoAs for bitter acid biosynthesis in hop glandular trichomes.

    PubMed

    Xu, Haiyang; Zhang, Fengxia; Liu, Baoxiu; Huhman, David V; Sumner, Lloyd W; Dixon, Richard A; Wang, Guodong

    2013-07-01

    Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop (Humulus lupulus) glandular trichomes. Branched short-chain acyl-CoAs (e.g. isobutyryl-CoA, isovaleryl-CoA and 2-methylbutyryl-CoA), derived from the degradation of branched-chain amino acids (BCAAs), are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyl-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HlCCL2 (carboxyl CoA ligase) protein had high specific activity for isovaleric acid as a substrate (K cat /K m = 4100 s(-1) M(-1)), whereas recombinant HlCCL4 specifically utilized isobutyric acid (Kcat/K m = 1800 s(-1) M(-1)) and 2-methylbutyric acid (Kcat/K m = 6900 s(-1) M(-1)) as substrates. Both HlCCLs, like hop valerophenone synthase (HlVPS), were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HlCCL2 and HlCCL4 with HlVPS in yeast led to significant production of acylphloroglucinols (the direct precursors for bitter acid biosynthesis), which further confirmed the biochemical function of these two HlCCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid:CoA ligases in planta are also discussed.

  7. Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene.

    PubMed

    Buch, Aditi D; Archana, G; Kumar, G Naresh

    2009-08-01

    Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant approximately 2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525

  8. Expression of sialic acids and other nonulosonic acids in Leptospira

    PubMed Central

    2012-01-01

    Background Sialic acids are negatively charged nine carbon backbone sugars expressed on mammalian cell surfaces. Sialic acids are part of a larger family of nonulosonic acid (NulO) molecules that includes pseudaminic and legionaminic acids. Microbial expression of sialic acids and other nonulosonic acids has been shown to contribute to host-microbe interactions in a variety of contexts, including participation in colonization, immune subversion, and behaviors such as biofilm formation, autoagglutination and motility. Previous research has suggested that some spirochetes may also express these molecules. Results Here we use a combination of molecular tools to investigate the presence of NulO biosynthetic gene clusters among clinical and saprophytic isolates of the genus Leptospira. Polymerase chain reaction and Southern blotting suggested that a variety of leptospires encoded NulO biosynthetic pathways. High performance liquid chromatography and mass spectrometry analyses provided biochemical evidence that di-N-acetylated NulO molecules are expressed at relatively high levels by L. interrogans serovar Lai strain 55601, and at lower levels by L. alexanderi serovar Manhao and L. fainei serovar Hurstbridge. Endogenous expression of N-acetylneuraminic acid (Neu5Ac, the most common sialic acid) was documented in L. interrogans serovar Copenhageni strain L1-130. Neu5Ac biosynthesis is also supported by a unique gene fusion event resulting in an enzyme with an N-terminal N-acetylneuraminic acid synthase domain and a C-terminal phosphatase domain. This gene fusion suggests that L. interrogans uses a Neu5Ac biosynthetic pathway more similar to animals than to other bacteria. Analysis of the composition and phylogeny of putative NulO biosynthetic gene clusters in L. interrogans serovar Lai and serovar Copenhageni revealed that both strains have complete biosynthetic pathways for legionamimic acid synthesis, a molecule with the same stereochemistry as sialic acid. Lectin

  9. Biosynthesis of Indoleacetic Acid from Tryptophan-14C in Cell-free Extracts of Pea Shoot Tips 1

    PubMed Central

    Moore, Thomas C.; Shaner, Coralie A.

    1967-01-01

    A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-14C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a 14C-product as IAA was by (a) co-chromatography of authentic IAA and 14C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of α-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall ≅ etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA. PMID:16656720

  10. Mutations in the Prokaryotic Pathway Rescue the fatty acid biosynthesis1 Mutant in the Cold.

    PubMed

    Gao, Jinpeng; Wallis, James G; Browse, John

    2015-09-01

    The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0 due to decreased activity of 3-ketoacyl-acyl carrier protein (ACP) synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains up to 45% high-melting-point molecular species (molecules that contain only 16:0, 16:1-trans, and 18:0), a trait associated with chilling-sensitive plants, compared with less than 10% in wild-type Arabidopsis. Although they do not exhibit typical chilling sensitivity, when exposed to low temperatures (2°C-6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. A screen for suppressors of this low-temperature phenotype has identified 11 lines, some of which contain additional alterations in leaf-lipid composition relative to fab1. Here, we report the identification of two suppressor mutations, one in act1, which encodes the chloroplast acyl-ACP:glycerol-3-phosphate acyltransferase, and one in lpat1, which encodes the chloroplast acyl-ACP:lysophosphatidic acid acyltransferase. These enzymes catalyze the first two steps of the prokaryotic pathway for glycerolipid synthesis, so we investigated whether other mutations in this pathway would rescue the fab1 phenotype. Both the gly1 mutation, which reduces glycerol-3-phosphate supply to the prokaryotic pathway, and fad6, which is deficient in the chloroplast 16:1/18:1 fatty acyl desaturase, were discovered to be suppressors. Analyses of leaf-lipid compositions revealed that mutations at all four of the suppressor loci result in reductions in the proportion of high-melting-point molecular species of phosphatidylglycerol relative to fab1. We conclude that these reductions are likely the basis for the suppressor phenotypes. © 2015 American Society of Plant Biologists. All Rights Reserved.

  11. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae

    PubMed Central

    Mohedano, Maria L.; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M.; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation. PMID:27610104

  12. The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae.

    PubMed

    Mohedano, Maria L; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M; López, Paloma

    2016-01-01

    The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

  13. Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase

    PubMed Central

    2004-01-01

    Isoprenoid biosynthesis via the methylerythritol phosphate pathway is a target against pathogenic bacteria and the malaria parasite Plasmodium falciparum. 4-(Hydroxyamino)-4-oxobutylphosphonic acid and 4-[hydroxy(methyl)amino]-4-oxobutyl phosphonic acid, two novel inhibitors of DXR (1-deoxy-D-xylulose 5-phosphate reducto-isomerase), the second enzyme of the pathway, have been synthesized and compared with fosmidomycin, the best known inhibitor of this enzyme. The latter phosphonohydroxamic acid showed a high inhibitory activity towards DXR, much like fosmidomycin, as well as significant antibacterial activity against Escherichia coli in tests on Petri dishes. PMID:15473867

  14. Isolation and characterisation of 8-hydroxy-3Z,5Z-tetradecadienoic acid, a putative intermediate in Pichia guilliermondii gamma-decalactone biosynthesis from ricinoleic acid.

    PubMed

    Iacazio, G; Martini, D; Faure, B; N'Guyen, M H

    2002-03-19

    During a screening procedure for the discovery of a strong gamma-decalactone producer from ricinoleic acid, we observed that the yeast Pichia guilliermondii accumulated transiently 8-hydroxy-3Z,5Z-tetradecadienoic acid 1 during gamma-decalactone biosynthesis in the stationary phase of growth. The structural elucidation of 1 was based on nuclear magnetic resonance, infrared, ultraviolet and gas chromatography-mass spectrometry experiments. The occurrence of 1 is discussed in relation with previously proposed gamma-decalactone biosynthetic pathways.

  15. Pantothenic Acid Biosynthesis in the Parasite Toxoplasma gondii: a Target for Chemotherapy

    PubMed Central

    Mageed, Sarmad N.; Cunningham, Fraser; Hung, Alvin Wei; Silvestre, Hernani Leonardo; Wen, Shijun; Blundell, Tom L.; Abell, Chris

    2014-01-01

    Toxoplasma gondii is a major food pathogen and neglected parasitic infection that causes eye disease, birth defects, and fetal abortion and plays a role as an opportunistic infection in AIDS. In this study, we investigated pantothenic acid (vitamin B5) biosynthesis in T. gondii. Genes encoding the full repertoire of enzymes for pantothenate synthesis and subsequent metabolism to coenzyme A were identified and are expressed in T. gondii. A panel of inhibitors developed to target Mycobacterium tuberculosis pantothenate synthetase were tested and found to exhibit a range of values for inhibition of T. gondii growth. Two inhibitors exhibited lower effective concentrations than the currently used toxoplasmosis drug pyrimethamine. The inhibition was specific for the pantothenate pathway, as the effect of the pantothenate synthetase inhibitors was abrogated by supplementation with pantothenate. Hence, T. gondii encodes and expresses the enzymes for pantothenate synthesis, and this pathway is essential for parasite growth. These promising findings increase our understanding of growth and metabolism in this important parasite and highlight pantothenate synthetase as a new drug target. PMID:25049241

  16. Differently Localized Lysophosphatidic Acid Acyltransferases Crucial for Triacylglycerol Biosynthesis in the Oleaginous Alga Nannochloropsis.

    PubMed

    Nobusawa, Takashi; Hori, Koichi; Mori, Hiroshi; Kurokawa, Ken; Ohta, Hiroyuki

    2017-02-20

    Production of renewable bioenergy will be necessary to meet rising global fossil fuel demands. Members of the marine microalgae genus Nannochloropsis produce large amounts of oils (triacylglycerols; TAGs), and this genus is regarded as one of the most promising for biodiesel production. Recent genome sequencing and transcriptomic studies on Nannochloropsis have provided a foundation for understanding its oleaginous trait, but the mechanism underlying oil accumulation remains to be clarified. Here we report Nannochloropsis knockout strains of four extraplastidic lysophosphatidic acid acyltransferases (LPAT1-4), which catalyze a major de novo biosynthetic step of TAGs and membrane lipids. We found that the four LPATs are differently involved in lipid metabolic flow in Nannochloropsis. Double knockouts among the LPATs revealed the pivotal LPATs for TAG biosynthesis, and localization analysis indicated that the stramenopile-specific LPATs (LPAT3 and LPAT4) associated with TAG synthesis reside at the perimeter of lipid droplets. However, no homologous region has been found with other lipid droplet-associated proteins. Lipid droplets are an organelle found in nearly all organisms, and recently they were shown to play important roles in cellular metabolism and signaling. Our results provide direct evidence for the importance of the perimeter of lipid droplet in TAG synthesis in addition to its known role in maintaining TAG stability, and these findings suggest that the oleaginous trait of Nannochloropsis is enabled by acquisition of LPATs at the perimeter of lipid droplets. This article is protected by copyright. All rights reserved.

  17. Regulation of ascorbic acid biosynthesis and recycling during root development in carrot (Daucus carota L.).

    PubMed

    Wang, Guang-Long; Xu, Zhi-Sheng; Wang, Feng; Li, Meng-Yao; Tan, Guo-Fei; Xiong, Ai-Sheng

    2015-09-01

    Ascorbic acid (AsA), also known as vitamin C, is an essential nutrient in fruits and vegetables. The fleshy root of carrot (Daucus carota L.) is a good source of AsA for humans. However, the metabolic pathways and molecular mechanisms involved in the control of AsA content during root development in carrot have not been elucidated. To gain insights into the regulation of AsA accumulation and to identify the key genes involved in the AsA metabolism, we cloned and analyzed the expression of 21 related genes during carrot root development. The results indicate that AsA accumulation in the carrot root is regulated by intricate pathways, of which the l-galactose pathway may be the major pathway for AsA biosynthesis. Transcript levels of the genes encoding l-galactose-1-phosphate phosphatase and l-galactono-1,4-lactone dehydrogenase were strongly correlated with AsA levels during root development. Data from this research may be used to assist breeding for improved nutrition, quality, and stress tolerance in carrots. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  18. Co-culture engineering for microbial biosynthesis of 3-amino-benzoic acid in Escherichia coli.

    PubMed

    Zhang, Haoran; Stephanopoulos, Gregory

    2016-07-01

    3-amino-benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli-E. coli co-culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co-culture system was found to improve 3AB production by 15 fold, compared to the mono-culture approach. Further engineering of the co-culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co-culture engineering can be a powerful new approach in the broad field of metabolic engineering. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. [Antitoxic properties of pantothenic acid derivatives, precursors of coenzyme A biosynthesis, with regard to kanamycin].

    PubMed

    Moĭseenok, A G; Dorofeev, B F; Sheĭbak, V M; Khomich, T I

    1984-11-01

    The effect of calcium pantothenate (CPN)B 4'-phospho-CPN (PCP), pantetheine (PT) and calcium S-sulfopantetheine (SPN) on acute toxicity of kanamycin sulfate was studied on albino mice. The above derivatives of pantothenic acid except PT lowered the antibiotic toxicity. The coefficient of the antitoxic effect (LD50/ED50) of SPN and PCP was 1.3-1.4 times higher than that of CPN. The combined use of kanamycin (1/5 of the LD50) with CPN, PCP or PT (30 mg/kg bw was equivalent to CPN) for 15 days prevented the increase in the total content of CoA and in the content of the fraction of free CoA and the precursors of its biosynthesis participating in the reaction of N-acetylation in the liver and brain. The contents of these substances were within the normal during the whole experiment. A certain increase in the activity of pantothenate kinase in the liver cytosol due to the use of kanamycin was eliminated by the simultaneous use of PCP and PT. The vitamin-containing compounds PCP and SPN were recommended for the clinical trials as agents preventing complications of kanamycin therapy.

  20. Transcriptomic Analysis of the Regulation of Lipid Fraction Migration and Fatty Acid Biosynthesis in Schizochytrium sp.

    PubMed

    Ren, Lujing; Hu, Xuechao; Zhao, Xiaoyan; Chen, Shenglan; Wu, Yi; Li, Dan; Yu, Yadong; Geng, Lingjun; Ji, Xiaojun; Huang, He

    2017-06-15

    Schizochytrium sp. is the main source of docosahexaenoic acid-rich oil, which is widely used in food additive and pharmaceutical industry. In this study, using RNA-seq, comparative transcriptomic analyses were performed at four stages of DHA fermentation by Schizochytrium sp to get potential genes related to cell transition from cell growth to lipid accumulation and then to lipid turnover. 1406, 385, 1384 differently expressed genes were identified by comparisons in pairs of S2 vs S1, S3 vs S2 and S4 vs S3. Functional analysis revealed that binding and single-organism process might be involve in the cell transition from cell growth to lipid accumulation while oxidation-reduction process played an important role in the transition from lipid accumulation to lipid turnover. pfaC in the PKS pathway showed higher sensitivity to the environmental change, which might be the key regulator for enhancing PUFA biosynthesis in the future. Some other genes in signal transduction and cell transport were revealed to be related to lipid turnover, which would enrich the current knowledge regarding lipid metabolism and help to enhance the DHA production and enrich different lipid fractions by Schizochytrium in the future.

  1. Dissociation of cephamycin C and clavulanic acid biosynthesis by 1,3-diaminopropane in Streptomyces clavuligerus.

    PubMed

    Leite, Carla A; Cavallieri, André P; Baptista, Amanda S; Araujo, Maria L G C

    2016-01-01

    Streptomyces clavuligerus produces simultaneously cephamycin C (CephC) and clavulanic acid (CA). Adding 1,3-diaminopropane to culture medium stimulates production of beta-lactam antibiotics. However, there are no studies on the influence of this diamine on coordinated production of CephC and CA. This study indicates that 1,3-diaminopropane can dissociate CephC and CA productions. Results indicated that low diamine concentrations (below 1.25 g l(-1)) in culture medium increased CA production by 200%, but not that of CephC. Conversely, CephC production increased by 300% when 10 g l(-1) 1,3-diaminopropane was added to culture medium. Addition of just L-lysine (18.3 g l(-1)) to culture medium increased both biocompounds. On the other hand, while L-lysine plus 7.5 g l(-1) 1,3-diaminopropane increased volumetric production of CephC by 1100%, its impact on CA production was insignificant. The combined results suggest that extracellular concentration of 1,3-diaminopropane may trigger the dissociation of CephC and CA biosynthesis in S. clavuligerus.

  2. Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis.

    PubMed

    Vaillancourt, Frédéric H; Yeh, Ellen; Vosburg, David A; O'Connor, Sarah E; Walsh, Christopher T

    2005-08-25

    Enzymatic incorporation of chlorine, bromine or iodine atoms occurs during the biosynthesis of more than 4,000 natural products. Halogenation can have significant consequences for the bioactivity of these products so there is great interest in understanding the biological catalysts that perform these reactions. Enzymes that halogenate unactivated aliphatic groups have not previously been characterized. Here we report the activity of five proteins-CmaA, CmaB, CmaC, CmaD and CmaE-in the construction of coronamic acid (CMA; 1-amino-1-carboxy-2-ethylcyclopropane), a constituent of the phytotoxin coronatine synthesized by the phytopathogenic bacterium Pseudomonas syringae. CMA derives from l-allo-isoleucine, which is covalently attached to CmaD through the actions of CmaA, a non-ribosomal peptide synthetase module, and CmaE, an unusual acyltransferase. We show that CmaB, a member of the non-haem Fe(2+), alpha-ketoglutarate-dependent enzyme superfamily, is the first of its class to show halogenase activity, chlorinating the gamma-position of l-allo-isoleucine. Another previously undescribed enzyme, CmaC, catalyses the formation of the cyclopropyl ring from the gamma-Cl-l-allo-isoleucine product of the CmaB reaction. Together, CmaB and CmaC execute gamma-halogenation followed by intramolecular gamma-elimination, in which biological chlorination is a cryptic strategy for cyclopropyl ring formation.

  3. Mutant characterization and in vivo conditional repression identify aromatic amino acid biosynthesis to be essential for Aspergillus fumigatus virulence

    PubMed Central

    Sasse, Anna; Hamer, Stefanie N; Amich, Jorge; Binder, Jasmin; Krappmann, Sven

    2016-01-01

    Pathogenicity of the saprobe Aspergillus fumigatus strictly depends on nutrient acquisition during infection, as fungal growth determines colonisation and invasion of a susceptible host. Primary metabolism has to be considered as a valid target for antimycotic therapy, based on the fact that several fungal anabolic pathways are not conserved in higher eukaryotes. To test whether fungal proliferation during invasive aspergillosis relies on endogenous biosynthesis of aromatic amino acids, defined auxotrophic mutants of A. fumigatus were generated and assessed for their infectious capacities in neutropenic mice and found to be strongly attenuated in virulence. Moreover, essentiality of the complete biosynthetic pathway could be demonstrated, corroborated by conditional gene expression in infected animals and inhibitor studies. This brief report not only validates the aromatic amino acid biosynthesis pathway of A. fumigatus to be a promising antifungal target but furthermore demonstrates feasibility of conditional gene expression in a murine infection model of aspergillosis. PMID:26605426

  4. PhzA, the shunt switch of phenazine-1,6-dicarboxylic acid biosynthesis in Pseudomonas chlororaphis HT66.

    PubMed

    Guo, Shuqi; Wang, Yining; Dai, Bona; Wang, Wei; Hu, Hongbo; Huang, Xianqing; Zhang, Xuehong

    2017-09-05

    Natural phenazines are versatile secondary metabolites that are mainly produced by Pseudomonas and Streptomyces. All phenazine-type metabolites originate from two precursors: phenazine-1-carboxylic acid (PCA) in Pseudomonas or phenazine-1,6-dicarboxylic acid (PDC) in Streptomyces and other bacteria. Although the biosynthesis of PCA in Pseudomonas has been extensively studied, the origin of PDC still remains unclear. Comparing the phenazine biosynthesis operons of different species, we found that the phzA gene was restricted to Pseudomonas in which PCA is produced. By generating phzA-inactivated mutant, we found a new compound obviously accumulated; it was then isolated and identified as PDC. Protein sequence alignment showed that PhzA proteins from Pseudomonas form a separate group that is recognized by H73L and S77L mutations. Generating mutations of L(73) into H(73) and L(77) into S(77) resulted in a significant increase in PDC production. These findings suggest that phzA may act as a shunt switch of PDC biosynthesis in Pseudomonas and distinguish the pathway producing only PCA from the pathway forming PCA plus PDC. Using real-time PCR analysis, we suggested that the phzA, phzB, and phzG genes either directly or indirectly regulate the production of PDC, and phzA plays the most significant regulatory role. This is the first description of phzA in the biosynthesis of PDC, and the first-time substantial PDC was obtained in Pseudomonas. Therefore, this study not only provides valuable clues to better understand the biosynthesis of PCA and PDC in Pseudomonas but also introduces a method to produce PDC derivatives by genetically engineered strains.

  5. Molecular cloning and characterization of genes involved in rosmarinic acid biosynthesis from Prunella vulgaris.

    PubMed

    Kim, Yeon Bok; Shin, YouJin; Tuan, Pham Anh; Li, Xiaohua; Park, Yunji; Park, Nam-il; Park, Sang Un

    2014-01-01

    Prunella vulgaris L., commonly known as "self-heal" or "heal-all," is a perennial herb with a long history of medicinal use. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme-A (CoA) ligase (4CL) are important enzymes in the phenylpropanoid pathway and in the accumulation of rosmarinic acid (RA), which is a major secondary metabolite in P. vulgaris. In this study, we isolated cDNAs encoding PvPAL, PvC4H, and Pv4CL from P. vulgaris using rapid amplification of cDNA ends polymerase chain reaction (PCR). The amino acid sequence alignments of PvPAL, PvC4H, and Pv4CL showed high sequence identity to those of other plants. Quantitative real-time PCR analysis was used to determine the transcript levels of genes involved in RA biosynthesis in the flowers, leaves, stems, and roots of P. vulgaris. The transcript levels of PvPAL, PvC4H, and Pv4CL1 were the highest in flowers, whereas Pv4CL2 was the highest in roots. High-performance liquid chromatography analysis also showed the highest RA content in the flowers (3.71 mg/g dry weight). We suggest that the expression of the PvPAL, PvC4H, and Pv4CL1 genes is correlated with the accumulation of RA. Our results revealed that P. vulgaris flowers are appropriate for medicinal usage, and our findings provide support for increasing RA production in this plant.

  6. Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi.

    PubMed

    Zhang, De-Huai; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2017-02-01

    Ganoderic acids (GAs) in Ganoderma lingzhi exhibit anticancer and antimetastatic activities. GA yields can be potentially improved by manipulating G. lingzhi through genetic engineering. In this study, a putative lanosterol synthase (LS) gene was cloned and overexpressed in G. lingzhi. Results showed that its overexpression (OE) increased the ganoderic acid (GA) content and the accumulation of lanosterol and ergosterol in a submerged G. lingzhi culture. The maximum contents of GA-O, GA-Mk, GA-T, GA-S, GA-Mf, and GA-Me in transgenic strains were 46.6 ± 4.8, 24.3 ± 3.5, 69.8 ± 8.2, 28.9 ± 1.4, 15.4 ± 1.2, and 26.7 ± 3.1 μg/100 mg dry weight, respectively, these values being 6.1-, 2.2-, 3.2-, 4.8-, 2.0-, and 1.9-times higher than those in wild-type strains. In addition, accumulated amounts of lanosterol and ergosterol in transgenic strains were 2.3 and 1.4-fold higher than those in the control strains, respectively. The transcription level of LS was also increased by more than five times in the presence of the G. lingzhi glyceraldehyde-3-phosphate dehydrogenase gene promoter, whereas transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A enzyme and squalene synthase did not change significantly in transgenic strains. This study demonstrated that OE of the homologous LS gene can enhance lanosterol accumulation. A large precursor supply promotes GA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis*

    PubMed Central

    Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Cohen-Gonsaud, Martin; Molle, Virginie; Kremer, Laurent

    2010-01-01

    Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential β-ketoacyl-acyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr191 being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr191 alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid

  8. P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination

    PubMed Central

    Jiang, Jishan; Chen, Zhihong; Ban, Liping; Wu, Yudi; Huang, Jianping; Chu, Jinfang; Fang, Shuang; Wang, Zan; Gao, Hongwen; Wang, Xuemin

    2017-01-01

    P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling. PMID:28084442

  9. Abscisic acid induces biosynthesis of bisbibenzyls and tolerance to UV-C in the liverwort Marchantia polymorpha.

    PubMed

    Kageyama, Akito; Ishizaki, Kimitsune; Kohchi, Takayuki; Matsuura, Hideyuki; Takahashi, Kosaku

    2015-09-01

    Environmental stresses are effective triggers for the biosynthesis of various secondary metabolites in plants, and phytohormones such as jasmonic acid and abscisic acid are known to mediate such responses in flowering plants. However, the detailed mechanism underlying the regulation of secondary metabolism in bryophytes remains unclear. In this study, the induction mechanism of secondary metabolites in the model liverwort Marchantia polymorpha was investigated. Abscisic acid (ABA) and ultraviolet irradiation (UV-C) were found to induce the biosynthesis of isoriccardin C, marchantin C, and riccardin F, which are categorized as bisbibenzyls, characteristic metabolites of liverworts. UV-C led to the significant accumulation of ABA. Overexpression of MpABI1, which encodes protein phosphatase 2C (PP2C) as a negative regulator of ABA signaling, suppressed accumulation of bisbibenzyls in response to ABA and UV-C irradiation and conferred susceptibility to UV-C irradiation. These data show that ABA plays a significant role in the induction of bisbibenzyl biosynthesis, which might confer tolerance against UV-C irradiation in M. polymorpha. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Deuterium NMR used to indicate a common mechanism for the biosynthesis of ricinoleic acid by Ricinus communis and Claviceps purpurea.

    PubMed

    Billault, Isabelle; Mantle, Peter G; Robins, Richard J

    2004-03-17

    Previous studies have shown that ricinoleic acid from castor bean oil of Ricinus communis is synthesized by the direct hydroxyl substitution of oleate, while it has been proposed that ricinoleate is formed by hydration of linoleate in the ergot fungus Claviceps purpurea. The mechanism of the enzymes specific to ricinoleate synthesis has not yet been established, but hydroxylation and desaturation of fatty acids in plants apparently involve closely related mechanisms. As mechanistic differences in the enzymes involved in the biosynthesis of natural products can lead to different isotopic distributions in the product, we could expect ricinoleate isolated from castor or ergot oil to show distinct (2)H distribution patterns. To obtain information concerning the substrate and isotope effects that occur during the biosynthesis of ricinoleate, the site-specific natural deuterium distributions in methyl ricinoleate isolated from castor oil and in methyl ricinoleate and methyl linoleate isolated from ergot oils have been measured by quantitative (2)H NMR. First, the deuterium profiles for methyl ricinoleate from the plant and fungus are equivalent. Second, the deuterium profile for methyl linoleate from ergot is incompatible with this chemical species being the precursor of methyl ricinoleate. Hence, it is apparent that 12-hydroxylation in C. purpurea is consistent with the biosynthetic mechanisms proposed for R. communis and is compatible with the general fundamental mechanistic similarities between hydroxylation and desaturation previously proposed for plant fatty acid biosynthesis.

  11. Metabolic engineering of Escherichia coli for 1-butanol biosynthesis through the inverted aerobic fatty acid β-oxidation pathway.

    PubMed

    Gulevich, Andrey Yu; Skorokhodova, Alexandra Yu; Sukhozhenko, Alexey V; Shakulov, Rustem S; Debabov, Vladimir G

    2012-03-01

    The basic reactions of the clostridial 1-butanol biosynthesis pathway can be regarded to be the inverted reactions of the fatty acid β-oxidation pathway. A pathway for the biosynthesis of fuels and chemicals was recently engineered by combining enzymes from both aerobic and anaerobic fatty acid β-oxidation as well as enzymes from other metabolic pathways. In the current study, we demonstrate the inversion of the entire aerobic fatty acid β-oxidation cycle for 1-butanol biosynthesis. The constructed markerless and plasmidless Escherichia coli strain BOX-3 (MG1655 lacI(Q) attB-P(trc-ideal-4)-SD(φ10)-adhE(Glu568Lys) attB-P(trc-ideal-4)-SD(φ10)-atoB attB-P(trc-ideal-4)-SD(φ10)-fadB attB-P(trc-ideal-4)-SD(φ10)-fadE) synthesises 0.3-1 mg 1-butanol/l in the presence of the specific inducer. No 1-butanol production was detected in the absence of the inducer.

  12. Molecular characterization of a mutation affecting abscisic acid biosynthesis and consequently stomatal responses to humidity in an agriculturally important species

    PubMed Central

    McAdam, Scott A. M.; Sussmilch, Frances C.; Brodribb, Timothy J.; Ross, John J.

    2015-01-01

    Mutants deficient in the phytohormone abscisic acid (ABA) have been instrumental in determining not only the biosynthetic pathway for this hormone, but also its physiological role in land plants. The wilty mutant of Pisum sativum is one of the classical, well-studied ABA-deficient mutants; however, this mutant remains uncharacterized at a molecular level. Using a candidate gene approach, we show that the wilty mutation affects the xanthoxin dehydrogenase step in ABA biosynthesis. To date, this step has only been represented by mutants in the ABA2 gene of Arabidopsis thaliana. Functional ABA biosynthesis appears to be critical for normal stomatal responses to changes in humidity in angiosperms, with wilty mutant plants having no increase in foliar ABA levels in response to a doubling in vapour pressure deficit, and no closure of stomata. Phylogenetic analysis of the ABA2 gene family from diverse land plants indicates that an ABA-biosynthesis-specific short-chain dehydrogenase (ABA2) evolved in the earliest angiosperms. The relatively recent origin of specificity in this step has important implications for both the evolution of ABA biosynthesis and action in land plants. PMID:26216469

  13. Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis

    PubMed Central

    2009-01-01

    Background The original anaerobic unsaturated fatty acid biosynthesis pathway proposed by Goldfine and Bloch was based on in vivo labeling studies in Clostridium butyricum ATCC 6015 (now C. beijerinckii) but to date no dedicated unsaturated fatty acid biosynthetic enzyme has been identified in Clostridia. C. acetobutylicium synthesizes the same species of unsaturated fatty acids as E. coli, but lacks all of the known unsaturated fatty acid synthetic genes identified in E. coli and other bacteria. A possible explanation was that two enzymes of saturated fatty acid synthesis of C. acetobutylicium, FabZ and FabF might also function in the unsaturated arm of the pathway (a FabZ homologue is known to be an unsaturated fatty acid synthetic enzyme in enterococci). Results We report that the FabF homologue located within the fatty acid biosynthetic gene cluster of C. acetobutylicium functions in synthesis of both unsaturated fatty acids and saturated fatty acids. Expression of this protein in E. coli functionally replaced both the FabB and FabF proteins of the host in vivo and replaced E. coli FabB in a defined in vitro fatty acid synthesis system. In contrast the single C. acetobutylicium FabZ homologue, although able to functionally replace E. coli FabZ in vivo and in vitro, was unable to replace FabA, the key dehydratase-isomerase of E. coli unsaturated fatty acid biosynthesis in vivo and lacked isomerase activity in vitro. Conclusion Thus, C. acetobutylicium introduces the double of unsaturated fatty acids by use of a novel and unknown enzyme. PMID:19493359

  14. Salmonella survival and differential expression of fatty acid biosynthesis-associated genes in a low-water-activity food.

    PubMed

    Chen, W; Golden, D A; Critzer, F J

    2014-08-01

    The purpose of this study was to investigate the difference in expression of fatty acid biosynthesis genes and survival of different serotypes of Salmonella when incubated in a low-water-activity (aw ) food over a 14-day period. Stationary cells of five strains of Salmonella enterica belonging to 3 different serovars (Typhimurium ATCC 2486, Enteritidis H4267, Tennessee ARI-33, Tennessee S13952 and Tennessee K4643) were inoculated into granular sugar (aW   = 0·50) and held aerobically over a 14-day period at 25°C. Survival was determined by enumerating colonies on TSA and XLT-4 plates at 0, 1, 3, 5, 7 and 14 days. Correspondingly, gene expression was evaluated for three selected genes involved in fatty acid biosynthesis and modification (fabA, fabD and cfa). After 14 days of incubation, the population was reduced from 2·29 to 3·36 log for all five strains. Salmonella Tennessee ARI-33 and Salm. Tennessee K4643 displayed greater survival than Salm. Typhimurium and Salm. Enteritidis. The increased expression of the cfa gene (involved in cyclopropane fatty acid biosynthesis) over 14 days was found associated with strains with a lower survival rate. The fabA gene (involved in unsaturated fatty acid biosynthesis) was observed up-regulated for all strains for at least one sampling time and for Salm. Tennessee ARI-33 for all time points tested, suggesting its potential role in enhancing Salmonella survival in low aw foods. Numerous outbreaks of salmonellosis associated with low-water-activity foods have been reported. Therefore, the adaptive mechanisms utilized by Salmonella to survive in low-water-activity foods for prolonged periods of time need to be better understood. The results in this study showed that low-water-activity environments increase expression of gene fabA, which is involved in unsaturated fatty acid biosynthesis of Salmonella, while the increased expression of cfa, associated with cyclopropane fatty acid synthesis, was associated with decreased

  15. Biosynthesis and metabolism of retinoic acid: roles of CRBP and CRABP in retinoic acid: roles of CRBP and CRABP in retinoic acid homeostasis.

    PubMed

    Napoli, J L

    1993-02-01

    The enzymes that constitute the pathway of retinoic acid biosynthesis and metabolism may recognize retinoid binding proteins as effectors and substrates. Apocellular retinol-binding protein (CRBP) stimulates a bile-salt independent membrane-bound retinyl ester hydrolase resulting in the hydrolysis of endogenous retinyl esters and the formation of holoCRBP. HoloCRBP delivers retinol to a microsomal nicotin-amide-adenine dinucleotide phosphate-dependent dehydrogenase, protects it from artifactual oxidation and denies enzymes that cannot recognize the binding protein access to retinol. The retinal synthesized may be transferred from the microsomes to the cytosol by CRBP. A cytosolic retinal dehydrogenase has been purified that produces retinoic acid from retinal generated by microsomes in the presence of CRBP and from the complex CRBP-retinal itself. Thus, CRBP(type I) seems to channel retinoids through the reactions of retinoic acid synthesis via a series of protein-protein interactions. Cellular retinoic acid-binding protein (type I) facilitates retinoic acid metabolism by sequestering it and by acting as a low Km substrate, thereby also modulating the steady-state concentrations of retinoic acid.

  16. Biosynthesis of 1-aminocyclopropane-1-carboxylic acid and ethylene from delta-aminolevulinic acid in ripening tomato fruits

    SciTech Connect

    El-Rayes, D.E.D.A.

    1987-01-01

    A new pathway for ethylene (C/sub 2/H/sub 4/) biosynthesis, which utilizes delta-aminolevulinic acid (ALA) as a precursor of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of C/sub 2/H/sub 4/, is presented. ALA enhanced ACC accumulation to 410% and C/sub 2/H/sub 4/ production to 232% of the control. The C/sub 2/H/sub 4/ production rate varied with the ALA concentration and the stage of tomato fruit development. As the ALA concentration increased from zero to 40 mM, the C/sub 2/H/sub 4/ production rate increased. Both treated and untreated pericarp discs from fruits at the pink stage of development yielded the largest C/sub 2/H/sub 4/ production rate. Radioactivity from (2,3-/sup 3/H)ALA was detected in both ACC and C/sub 2/H/sub 4/, and radioactivity from (4-/sup 14/C)ALA was detected in ACC and CO/sub 2/ but not in C/sub 2/H/sub 4/. However, radioactivity from (5-/sup 14/C)ALA was detected in CO/sub 2/, and its amount was greater than that obtained from (4-/sup 14/C)ALA. Neither ACC nor C/sub 2/H/sub 4/ showed any radioactivity when (5-/sup 14/C)ALA was supplied to the fruit discs. In addition, when (2,3-/sup 3/H)ALA or (4-/sup 14/C)ALA was supplied to the fruit discs, radioactivity was detected in other metabolites such as fumarate, succinate, malate, glutamate, glutamine, ..cap alpha..-ketoglutarate, and methionine, but the amount of radioactivity was insignificant as compared with the amount of radioactivity found in C/sub 2/H/sub 4/ and ACC.

  17. Functional characterization of MpaG', the O-methyltransferase involved in the biosynthesis of mycophenolic acid.

    PubMed

    Zhang, Wei; Cao, Shaona; Qiu, Li; Qi, Fengxia; Li, Zhong; Yang, Ying; Huang, Shaohua; Bai, Fali; Liu, Changning; Wan, Xiaobo; Li, Shengying

    2015-03-02

    Mycophenolic acid (MPA, 1) is a clinically important immunosuppressant. In this report, a gene cluster mpa' responsible for the biosynthesis of 1 was identified from Penicillium brevicompactum NRRL 864. The S-adenosyl-L-methionine-dependent (SAM-dependent) O-methyltransferase encoded by the mpaG' gene was functionally and kinetically characterized in vitro. MpaG' catalyzes the methylation of demethylmycophenolic acid (DMMPA, 6) to form 1. It also showed significant substrate flexibility by methylating two structural derivatives of 6 prepared by organic synthesis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

    PubMed

    Lee, Su-Jin; Kim, Chul Ho; Seo, Pil-Soo; Kwon, Ohsuk; Hur, Byung-Ki; Seo, Jeong-Woo

    2008-12-01

    To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

  19. Unsaturated macrocyclic dihydroxamic acid siderophores produced by Shewanella putrefaciens using precursor-directed biosynthesis.

    PubMed

    Soe, Cho Z; Codd, Rachel

    2014-04-18

    To acquire iron essential for growth, the bacterium Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (pbH2; [M + H(+)](+), m/zcalc 373.2) as its native siderophore. The assembly of pbH2 requires endogenous 1,4-diaminobutane (DB), which is produced from the ornithine decarboxylase (ODC)-catalyzed decarboxylation of l-ornithine. In this work, levels of endogenous DB were attenuated in S. putrefaciens cultures by augmenting the medium with the ODC inhibitor 1,4-diamino-2-butanone (DBO). The presence in the medium of DBO together with alternative exogenous non-native diamine substrates, (15)N2-1,4-diaminobutane ((15)N2-DB) or 1,4-diamino-2(E)-butene (E-DBE), resulted in the respective biosynthesis of (15)N-labeled pbH2 ((15)N4-pbH2; [M + H(+)](+), m/zcalc 377.2, m/zobs 377.2) or the unsaturated pbH2 variant, named here: E,E-putrebactene (E,E-pbeH2; [M + H(+)](+), m/zcalc 369.2, m/zobs 369.2). In the latter system, remaining endogenous DB resulted in the parallel biosynthesis of the monounsaturated DB-E-DBE hybrid, E-putrebactene (E-pbxH2; [M + H(+)](+), m/zcalc 371.2, m/zobs 371.2). These are the first identified unsaturated macrocyclic dihydroxamic acid siderophores. LC-MS measurements showed 1:1 complexes formed between Fe(III) and pbH2 ([Fe(pb)](+); [M](+), m/zcalc 426.1, m/zobs 426.2), (15)N4-pbH2 ([Fe((15)N4-pb)](+); [M](+), m/zcalc 430.1, m/zobs 430.1), E,E-pbeH2 ([Fe(E,E-pbe)](+); [M](+), m/zcalc 422.1, m/zobs 422.0), or E-pbxH2 ([Fe(E-pbx)](+); [M](+), m/zcalc 424.1, m/zobs 424.2). The order of the gain in siderophore-mediated Fe(III) solubility, as defined by the difference in retention time between the free ligand and the Fe(III)-loaded complex, was pbH2 (ΔtR = 8.77 min) > E-pbxH2 (ΔtR = 6.95 min) > E,E-pbeH2 (ΔtR = 6.16 min), which suggests one possible reason why nature has selected for saturated rather than unsaturated siderophores as Fe(III) solubilization agents. The potential to conduct multiple types of ex situ chemical

  20. Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels.

    PubMed

    Mandal, Shantanu; Upadhyay, Shivangi; Wajid, Saima; Ram, Mauji; Jain, Dharam Chand; Singh, Ved Pal; Abdin, Malik Zainul; Kapoor, Rupam

    2015-07-01

    It is becoming increasingly evident that the formation of arbuscular mycorrhiza (AM) enhances secondary metabolite production in shoots. Despite mounting evidence, relatively little is known about the underlying mechanisms. This study suggests that increase in artemisinin concentration in Artemisia annua colonized by Rhizophagus intraradices is due to altered trichome density as well as transcriptional patterns that are mediated via enhanced jasmonic acid (JA) levels. Mycorrhizal (M) plants had higher JA levels in leaf tissue that may be due to induction of an allene oxidase synthase gene (AOS), encoding one of the key enzymes for JA production. Non-mycorrhizal (NM) plants were exogenously supplied with a range of methyl jasmonic acid concentrations. When leaves of NM and M plants with similar levels of endogenous JA were compared, these matched closely in terms of shoot trichome density, artemisinin concentration, and transcript profile of artemisinin biosynthesis genes. Mycorrhization increased artemisinin levels by increasing glandular trichome density and transcriptional activation of artemisinin biosynthesis genes. Transcriptional analysis of some rate-limiting enzymes of mevalonate and methyl erythritol phosphate (MEP) pathways revealed that AM increases isoprenoids by induction of the MEP pathway. A decline in artemisinin concentration in shoots of NM and M plants treated with ibuprofen (an inhibitor of JA biosynthesis) further confirmed the implication of JA in the mechanism of artemisinin production.

  1. The 5-lipoxygenase inhibitor, zileuton, suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages

    PubMed Central

    Rossi, A; Pergola, C; Koeberle, A; Hoffmann, M; Dehm, F; Bramanti, P; Cuzzocrea, S; Werz, O; Sautebin, L

    2010-01-01

    BACKGROUND AND PURPOSE Zileuton is the only 5-lipoxygenase (5-LOX) inhibitor marketed as a treatment for asthma, and is often utilized as a selective tool to evaluate the role of 5-LOX and leukotrienes. The aim of this study was to investigate the effect of zileuton on prostaglandin (PG) production in vitro and in vivo. EXPERIMENTAL APPROACH Peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon γ (LPS/IFNγ), J774 macrophages and human whole blood stimulated with LPS were used as in vitro models and rat carrageenan-induced pleurisy as an in vivo model. KEY RESULTS Zileuton suppressed PG biosynthesis by interference with arachidonic acid (AA) release in macrophages. We found that zileuton significantly reduced PGE2 and 6-keto prostaglandin F1α (PGF1α) levels in activated mouse peritoneal macrophages and in J774 macrophages. This effect was not related to 5-LOX inhibition, because it was also observed in macrophages from 5-LOX knockout mice. Notably, zileuton inhibited PGE2 production in LPS-stimulated human whole blood and suppressed PGE2 and 6-keto PGF1α pleural levels in rat carrageenan-induced pleurisy. Interestingly, zileuton failed to inhibit the activity of microsomal PGE2 synthase1 and of cyclooxygenase (COX)-2 and did not affect COX-2 expression. However, zileuton significantly decreased AA release in macrophages accompanied by inhibition of phospholipase A2 translocation to cellular membranes. CONCLUSIONS AND IMPLICATION Zileuton inhibited PG production by interfering at the level of AA release. Its mechanism of action, as well as its use as a pharmacological tool, in experimental models of inflammation should be reassessed. PMID:20880396

  2. 5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway.

    PubMed

    Ramzi, Ahmad Bazli; Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2015-12-01

    ALA (5-aminolevulinic acid) is an important intermediate in the synthesis of tetrapyrroles and the use of ALA has been gradually increasing in many fields, including medicine and agriculture. In this study, improved biological production of ALA in Corynebacterium glutamicum was achieved by overexpressing glutamate-initiated C5 pathway. For this purpose, copies of the glutamyl t-RNA reductase HemA from several bacteria were mutated by site-directed mutagenesis of which a HemA version from Salmonella typhimurium exhibited the highest ALA production. Cultivation of the HemA-expressing strain produced approximately 204 mg/L of ALA, while co-expression with HemL (glutamate-1-semialdehyde aminotransferase) increased ALA concentration to 457 mg/L, representing 11.6- and 25.9-fold increases over the control strain (17 mg/L of ALA). Further effects of metabolic perturbation were investigated, leading to penicillin addition that further improves ALA production to 584 mg/L. In an optimized flask fermentation, engineered C. glutamicum strains expressing the HemA and hemAL operon produced up to 1.1 and 2.2g/L ALA, respectively, under glutamate-producing conditions. The final yields represent 10.7- and 22.0-fold increases over the control strain (0.1g/L of ALA). From these findings, ALA biosynthesis from glucose was successfully demonstrated and this study is the first to report ALA overproduction in C. glutamicum via metabolic engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Novel Type II Fatty Acid Biosynthesis (FAS II) Inhibitors as Multistage Antimalarial Agents

    PubMed Central

    Schrader, Florian C.; Glinca, Serghei; Sattler, Julia M.; Dahse, Hans-Martin; Afanador, Gustavo A.; Prigge, Sean T.; Lanzer, Michael; Mueller, Ann-Kristin; Klebe, Gerhard; Schlitzer, Martin

    2013-01-01

    Malaria is a potentially fatal disease caused by Plasmodium parasites and poses a major medical risk in large parts of the world. The development of new, affordable antimalarial drugs is of vital importance as there are increasing reports of resistance to the currently available therapeutics. In addition, most of the current drugs used for chemoprophylaxis merely act on parasites already replicating in the blood. At this point, a patient might already be suffering from the symptoms associated with the disease and could additionally be infectious to an Anopheles mosquito. These insects act as a vector, subsequently spreading the disease to other humans. In order to cure not only malaria but prevent transmission as well, a drug must target both the blood- and pre-erythrocytic liver stages of the parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) reductase (ENR) is a key enzyme of plasmodial type II fatty acid biosynthesis (FAS II). It has been shown to be essential for liver-stage development of Plasmodium berghei and is therefore qualified as a target for true causal chemoprophylaxis. Using virtual screening based on two crystal structures of PfENR, we identified a structurally novel class of FAS inhibitors. Subsequent chemical optimization yielded two compounds that are effective against multiple stages of the malaria parasite. These two most promising derivatives were found to inhibit blood-stage parasite growth with IC50 values of 1.7 and 3.0 µm and lead to a more prominent developmental attenuation of liver-stage parasites than the gold-standard drug, primaquine. PMID:23341167

  4. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro.

    PubMed

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-04-01

    Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell-cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase.

  5. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro

    PubMed Central

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-01-01

    Abstract Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase. PMID:19382912

  6. Inhibition of carnitine biosynthesis by valproic acid in rats--the biochemical mechanism of inhibition.

    PubMed

    Farkas, V; Bock, I; Cseko, J; Sandor, A

    1996-11-08

    The anticonvulsive drug, valproic acid (VPA), inhibits the biosynthesis of carnitine, and may contribute in this way to carnitine deficiency associated with VPA therapy. The conversion of [3H]-butyrobetaine into [3H]-carnitine was determined 60 min following a single intraperitoneal (i.p.) dose of 1.2 mmol/kg VPA in rats. The fraction of radioactivity found in [3H]-carnitine in the liver decreased from 63.2 +/- 1.50% to 39.2 +/- 1.11% (mean +/- SEM). Total carnitine in the liver also decreased, whereas the precursor butyrobetaine increased from 5.01 +/- 0.71 nmol/g to 8.22 +/- 0.82 nmol/g (mean +/- SEM). VPA also exhibited a dramatic effect on the conversion of an unlabeled loading amount of butyrobetaine. The increment in total carnitine caused by butyrobetaine in liver was reduced from 161 +/- 15.4 nmol/g to 53.2 +/- 5.11 nmol/g (mean +/- SEM). These data prove that VPA reduces the flux through butyrobetaine hydroxylase (EC 1.14.11.1.). The drug in vitro, however, did not inhibit the enzyme directly. Searching for the mechanism of action, we found that VPA decreased the level of alpha-ketoglutarate (alpha-KG; a cofactor of butyrobetaine hydroxylase) from 73.5 +/- 2.90 nmol/g to 52.9 +/- 2.2 nmol/g (mean +/- SEM) in the liver. The level of 1-glutamate showed a rather dramatic decrease in the liver. Moreover, alpha-KG proved to have a protective role against VPA in the [3H]-butyrobetaine conversion experiment.

  7. Cloning and characterization of a cDNA coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis.

    PubMed

    Liu, Ying; Xu, Qiao-Xian; Xi, Pei-Yu; Chen, Hong-Hao; Liu, Chun-Sheng

    2013-05-01

    The roots of Glycyrrhiza uralensis are widely used in Chinese medicine for their action of clearing heat, detoxicating, relieving cough, dispelling sputum and tonifying spleen and stomach. The reason why Glycyrrhiza uralensis has potent and significant actions is that it contains various active secondary metabolites, especially glycyrrhizic acid. In the present study, we cloned the cDNA coding 3-hydroxy-3-methylglutary CoA reductase (HMGR) involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis. The corresponding cDNA was expressed in Escherichia coli as fusion proteins. Recombinant HMGR exhibited catalysis activity in reduction of HMG-CoA to mevalonic acid (MVA) just as HMGR isolated from other species. Because HMGR gene is very important in the biosynthesis of glycyrrhizic acid in Glycyrrhiza uralensis, this work is significant for further studies concerned with strengthening the efficacy of Glycyrrhiza uralensis by means of increasing glycyrrhizic acid content and exploring the biosynthesis of glycyrrhizic acid in vitro.

  8. Salt-Related MYB1 Coordinates Abscisic Acid Biosynthesis and Signaling during Salt Stress in Arabidopsis1

    PubMed Central

    Wang, Ting; Tohge, Takayuki; Ivakov, Alexander; Mueller-Roeber, Bernd; Fernie, Alisdair R.; Mutwil, Marek; Schippers, Jos H.M.; Persson, Staffan

    2015-01-01

    Abiotic stresses, such as salinity, cause global yield loss of all major crop plants. Factors and mechanisms that can aid in plant breeding for salt stress tolerance are therefore of great importance for food and feed production. Here, we identified a MYB-like transcription factor, Salt-Related MYB1 (SRM1), that negatively affects Arabidopsis (Arabidopsis thaliana) seed germination under saline conditions by regulating the levels of the stress hormone abscisic acid (ABA). Accordingly, several ABA biosynthesis and signaling genes act directly downstream of SRM1, including SALT TOLERANT1/NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, RESPONSIVE TO DESICCATION26, and Arabidopsis NAC DOMAIN CONTAINING PROTEIN19. Furthermore, SRM1 impacts vegetative growth and leaf shape. We show that SRM1 is an important transcriptional regulator that directly targets ABA biosynthesis and signaling-related genes and therefore may be regarded as an important regulator of ABA-mediated salt stress tolerance. PMID:26243618

  9. trans-Cinnamic and Chlorogenic Acids Affect the Secondary Metabolic Profiles and Ergosterol Biosynthesis by Fusarium culmorum and F. graminearum Sensu Stricto.

    PubMed

    Kulik, Tomasz; Stuper-Szablewska, Kinga; Bilska, Katarzyna; Buśko, Maciej; Ostrowska-Kołodziejczak, Anna; Załuski, Dariusz; Perkowski, Juliusz

    2017-06-22

    Plant-derived compounds limiting mycotoxin contamination are currently of major interest in food and feed production. However, their potential application requires an evaluation of their effects on fungal secondary metabolism and membrane effects. In this study, different strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to trans-cinnamic and chlorogenic acids on solid YES media. Fusaria produced phenolic acids, whose accumulation was lowered by exogenous phenolic compounds. In addition, fungi reduced exogenous phenolic acids, leading either to their conversion or degradation. trans-Cinnamic acid was converted to caffeic and ferulic acids, while chlorogenic acid was degraded to caffeic acid. The latter underwent further degradation to protocatechuic acid. Fungal-derived trans-cinnamic acid, as the first intermediate of the shikimate pathway, increased after chlorogenic acid treatment, presumably due to the further inhibition of the conversion of trans-cinnamic acid. Exogenous trans-cinnamic and chlorogenic acid displayed the inhibition of mycotoxin production by Fusaria, which appeared to be largely dependent on the phenolic compound and its concentration and the assayed strain. Exogenous phenolic acids showed different effects on ergosterol biosynthesis by fungi. It was found that the production of this membrane sterol was stimulated by trans-cinnamic acid, while chlorogenic acid negatively impacted ergosterol biosynthesis, suggesting that phenolic acids with stronger antifungal activities may upregulate ergosterol biosynthesis by Fusaria. This paper reports on the production of phenolic acids by Fusaria for the first time.

  10. trans-Cinnamic and Chlorogenic Acids Affect the Secondary Metabolic Profiles and Ergosterol Biosynthesis by Fusarium culmorum and F. graminearum Sensu Stricto

    PubMed Central

    Kulik, Tomasz; Stuper-Szablewska, Kinga; Bilska, Katarzyna; Buśko, Maciej; Ostrowska-Kołodziejczak, Anna; Załuski, Dariusz; Perkowski, Juliusz

    2017-01-01

    Plant-derived compounds limiting mycotoxin contamination are currently of major interest in food and feed production. However, their potential application requires an evaluation of their effects on fungal secondary metabolism and membrane effects. In this study, different strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to trans-cinnamic and chlorogenic acids on solid YES media. Fusaria produced phenolic acids, whose accumulation was lowered by exogenous phenolic compounds. In addition, fungi reduced exogenous phenolic acids, leading either to their conversion or degradation. trans-Cinnamic acid was converted to caffeic and ferulic acids, while chlorogenic acid was degraded to caffeic acid. The latter underwent further degradation to protocatechuic acid. Fungal-derived trans-cinnamic acid, as the first intermediate of the shikimate pathway, increased after chlorogenic acid treatment, presumably due to the further inhibition of the conversion of trans-cinnamic acid. Exogenous trans-cinnamic and chlorogenic acid displayed the inhibition of mycotoxin production by Fusaria, which appeared to be largely dependent on the phenolic compound and its concentration and the assayed strain. Exogenous phenolic acids showed different effects on ergosterol biosynthesis by fungi. It was found that the production of this membrane sterol was stimulated by trans-cinnamic acid, while chlorogenic acid negatively impacted ergosterol biosynthesis, suggesting that phenolic acids with stronger antifungal activities may upregulate ergosterol biosynthesis by Fusaria. This paper reports on the production of phenolic acids by Fusaria for the first time. PMID:28640190

  11. Synthesis and identification in bacterial lipopolysaccharides of 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto- and -D-glycero-D-talo-non-2-ulosonic acids.

    PubMed

    Tsvetkov, Y E; Shashkov, A S; Knirel, Y A; Zähringer, U

    2001-04-12

    5,7-Diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto- and -D-glycero-D-talo-non-2-ulosonic acids were synthesized by condensation of 2,4-diacetamido-2,4,6-trideoxy-D-mannose with oxalacetic acid. Comparison of the 1H and 13C NMR data and the specific optical rotation values of these monosaccharides and the corresponding L-glycero-D-galacto and L-glycero-D-talo isomers synthesized earlier [Tsvetkov, Y. E.; Shashkov, A. S.; Knirel, Y. A.; Backinowsky, L. V.; Zähringer, U. Mendeleev Commun. 2000, 90-92] with data of the natural compounds enabled the identification in bacterial lipopolysaccharides of derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid and epimers of legionaminic acid at C-4 and C-8.

  12. The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis.

    PubMed

    Milborrow, B V

    2001-06-01

    The pathway of biosynthesis of abscisic acid (ABA) can be considered to comprise three stages: (i) early reactions in which small phosphorylated intermediates are assembled as precursors of (ii) intermediate reactions which begin with the formation of the uncyclized C40 carotenoid phytoene and end with the cleavage of 9'-cis-neoxanthin (iii) to form xanthoxal, the C15 skeleton of ABA. The final phase comprising C15 intermediates is not yet completely defined, but the evidence suggests that xanthoxal is first oxidized to xanthoxic acid by a molybdenum-containing aldehyde oxidase and this is defective in the aba3 mutant of Arabidopsis and present in a 1-fold acetone precipitate of bean leaf proteins. This oxidation precludes the involvement of AB-aldehyde as an intermediate. The oxidation of the 4'-hydroxyl group to the ketone and the isomerization of the 1',2'-epoxy group to the 1'-hydroxy-2'-ene may be brought about by one enzyme which is defective in the aba2 mutant and is present in the 3-fold acetone fraction of bean leaves. Isopentenyl diphosphate (IPP) is now known to be derived by the pyruvate-triose (Methyl Erythritol Phosphate, MEP) pathway in chloroplasts. (14C)IPP is incorporated into ABA by washed, intact chloroplasts of spinach leaves, but (14C)mevalonate is not, consequently, all three phases of biosynthesis of ABA occur within chloroplasts. The incorporation of labelled mevalonate into ABA by avocado fruit and orange peel is interpreted as uptake of IPP made in the cytoplasm, where it is the normal precursor of sterols, and incorporated into carotenoids after uptake by a carrier in the chloroplast envelope. An alternative bypass pathway becomes more important in aldehyde oxidase mutants, which may explain why so many wilty mutants have been found with this defect. The C-1 alcohol group is oxidized, possibly by a mono-oxygenase, to give the C-1 carboxyl of ABA. The 2-cis double bond of ABA is essential for its biological activity but it is not known

  13. The biosynthesis of N-arachidonoyl dopamine (NADA), a putative endocannabinoid and endovanilloid, via conjugation of arachidonic acid with dopamine

    PubMed Central

    Hu, Sherry Shu-Jung; Bradshaw, Heather B.; Benton, Valery M.; Chen, Jay Shih-Chieh; Huang, Susan M.; Minassi, Alberto; Bisogno, Tiziana; Masuda, Kim; Tan, Bo; Roskoski, Robert; Cravatt, Benjamin F.; Di Marzo, Vincenzo

    2009-01-01

    Summary N-arachidonoyl dopamine (NADA) is an endogenous ligand that activates the cannabinoid type 1 receptor and the transient receptor potential vanilloid type 1 channel. Two potential biosynthetic pathways for NADA have been proposed, though no conclusive evidence exists for either. The first is the direct conjugation of arachidonic acid with dopamine; the other is via metabolism of a putative N-arachidonoyl tyrosine (NA-tyrosine). In the present study we investigated these biosynthetic mechanisms and report that NADA synthesis requires TH in dopaminergic terminals, however, NA-tyrosine, which we identify here as an endogenous lipid, is not an intermediate. We show that NADA biosynthesis primarily occurs through an enzyme-mediated conjugation of arachidonic acid with dopamine. While this conjugation likely involves a complex of enzymes, our data suggest a direct involvement of fatty acid amide hydrolase in NADA biosynthesis as either a rate-limiting enzyme that liberates arachidonic acid from AEA, as a conjugation enzyme, or both. PMID:19570666

  14. Methyl jasmonate stimulates biosynthesis of 2-phenylethylamine, phenylacetic acid and 2-phenylethanol in seedlings of common buckwheat.

    PubMed

    Horbowicz, Marcin; Wiczkowski, Wiesław; Sawicki, Tomasz; Szawara-Nowak, Dorota; Sytykiewicz, Hubert; Mitrus, Joanna

    2015-01-01

    Methyl jasmonate has a strong effect on secondary metabolizm in plants, by stimulating the biosynthesis a number of phenolic compounds and alkaloids. Common buckwheat (Fagopyrum esculentum Moench) is an important source of biologically active compounds. This research focuses on the detection and quantification of 2-phenylethylamine and its possible metabolites in the cotyledons, hypocotyl and roots of common buckwheat seedlings treated with methyl jasmonate. In cotyledons of buckwheat sprouts, only traces of 2-phenylethylamine were found, while in the hypocotyl and roots its concentration was about 150 and 1000-times higher, respectively. Treatment with methyl jasmonate resulted in a 4-fold increase of the 2-phenylethylamine level in the cotyledons of 7-day buckwheat seedlings, and an 11-fold and 5-fold increase in hypocotyl and roots, respectively. Methyl jasmonate treatment led also to about 4-fold increase of phenylacetic acid content in all examined seedling organs, but did not affect the 2-phenylethanol level in cotyledons, and slightly enhanced in hypocotyl and roots. It has been suggested that 2-phenylethylamine is a substrate for the biosynthesis of phenylacetic acid and 2-phenylethanol, as well as cinnamoyl 2-phenethylamide. In organs of buckwheat seedling treated with methyl jasmonate, higher amounts of aromatic amino acid transaminase mRNA were found. The enzyme can be involved in the synthesis of phenylpyruvic acid, but the presence of this compound could not be confirmed in any of the examined organs of common buckwheat seedling.

  15. The biosynthesis of N-arachidonoyl dopamine (NADA), a putative endocannabinoid and endovanilloid, via conjugation of arachidonic acid with dopamine.

    PubMed

    Hu, Sherry Shu-Jung; Bradshaw, Heather B; Benton, Valery M; Chen, Jay Shih-Chieh; Huang, Susan M; Minassi, Alberto; Bisogno, Tiziana; Masuda, Kim; Tan, Bo; Roskoski, Robert; Cravatt, Benjamin F; Di Marzo, Vincenzo; Walker, J Michael

    2009-10-01

    N-arachidonoyl dopamine (NADA) is an endogenous ligand that activates the cannabinoid type 1 receptor and the transient receptor potential vanilloid type 1 channel. Two potential biosynthetic pathways for NADA have been proposed, though no conclusive evidence exists for either. The first is the direct conjugation of arachidonic acid with dopamine and the other is via metabolism of a putative N-arachidonoyl tyrosine (NA-tyrosine). In the present study we investigated these biosynthetic mechanisms and report that NADA synthesis requires TH in dopaminergic terminals; however, NA-tyrosine, which we identify here as an endogenous lipid, is not an intermediate. We show that NADA biosynthesis primarily occurs through an enzyme-mediated conjugation of arachidonic acid with dopamine. While this conjugation likely involves a complex of enzymes, our data suggest a direct involvement of fatty acid amide hydrolase in NADA biosynthesis either as a rate-limiting enzyme that liberates arachidonic acid from AEA, or as a conjugation enzyme, or both.

  16. From Amino Acid to Glucosinolate Biosynthesis: Protein Sequence Changes in the Evolution of Methylthioalkylmalate Synthase in Arabidopsis[W][OA

    PubMed Central

    de Kraker, Jan-Willem; Gershenzon, Jonathan

    2011-01-01

    Methylthioalkylmalate synthase (MAM) catalyzes the committed step in the side chain elongation of Met, yielding important precursors for glucosinolate biosynthesis in Arabidopsis thaliana and other Brassicaceae species. MAM is believed to have evolved from isopropylmalate synthase (IPMS), an enzyme involved in Leu biosynthesis, based on phylogenetic analyses and an overlap of catalytic abilities. Here, we investigated the changes in protein structure that have occurred during the recruitment of IPMS from amino acid to glucosinolate metabolism. The major sequence difference between IPMS and MAM is the absence of 120 amino acids at the C-terminal end of MAM that constitute a regulatory domain for Leu-mediated feedback inhibition. Truncation of this domain in Arabidopsis IPMS2 results in loss of Leu feedback inhibition and quaternary structure, two features common to MAM enzymes, plus an 8.4-fold increase in the kcat/Km for a MAM substrate. Additional exchange of two amino acids in the active site resulted in a MAM-like enzyme that had little residual IPMS activity. Hence, combination of the loss of the regulatory domain and a few additional amino acid exchanges can explain the evolution of MAM from IPMS during its recruitment from primary to secondary metabolism. PMID:21205930

  17. Endurance exercise and conjugated linoleic acid (CLA) supplementation up-regulate CYP17A1 and stimulate testosterone biosynthesis.

    PubMed

    Barone, Rosario; Macaluso, Filippo; Catanese, Patrizia; Marino Gammazza, Antonella; Rizzuto, Luigi; Marozzi, Paola; Lo Giudice, Giuseppe; Stampone, Tomaso; Cappello, Francesco; Morici, Giuseppe; Zummo, Giovanni; Farina, Felicia; Di Felice, Valentina

    2013-01-01

    A new role for fat supplements, in particular conjugated linoleic acid (CLA), has been delineated in steroidogenesis, although the underlying molecular mechanisms have not yet been elucidated. The aims of the present study were to identify the pathway stimulated by CLA supplementation using a cell culture model and to determine whether this same pathway is also stimulated in vivo by CLA supplementation associated with exercise. In vitro, Leydig tumour rat cells (R2C) supplemented with different concentrations of CLA exhibited increasing testosterone biosynthesis accompanied by increasing levels of CYP17A1 mRNA and protein. In vivo, trained mice showed an increase in free plasma testosterone and an up-regulation of CYP17A1 mRNA and protein. The effect of training on CYP17A1 expression and testosterone biosynthesis was significantly higher in the trained mice supplemented with CLA compared to the placebo. The results of the present study demonstrated that CLA stimulates testosterone biosynthesis via CYP17A1, and endurance training led to the synthesis of testosterone in vivo by inducing the overexpression of CYP17A1 mRNA and protein in the Leydig cells of the testis. This effect was enhanced by CLA supplementation. Therefore, CLA-associated physical activity may be used for its steroidogenic property in different fields, such as alimentary industry, human reproductive medicine, sport science, and anti-muscle wasting.

  18. Endurance Exercise and Conjugated Linoleic Acid (CLA) Supplementation Up-Regulate CYP17A1 and Stimulate Testosterone Biosynthesis

    PubMed Central

    Catanese, Patrizia; Marino Gammazza, Antonella; Rizzuto, Luigi; Marozzi, Paola; Lo Giudice, Giuseppe; Stampone, Tomaso; Cappello, Francesco; Morici, Giuseppe; Zummo, Giovanni; Farina, Felicia; Di Felice, Valentina

    2013-01-01

    A new role for fat supplements, in particular conjugated linoleic acid (CLA), has been delineated in steroidogenesis, although the underlying molecular mechanisms have not yet been elucidated. The aims of the present study were to identify the pathway stimulated by CLA supplementation using a cell culture model and to determine whether this same pathway is also stimulated in vivo by CLA supplementation associated with exercise. In vitro, Leydig tumour rat cells (R2C) supplemented with different concentrations of CLA exhibited increasing testosterone biosynthesis accompanied by increasing levels of CYP17A1 mRNA and protein. In vivo, trained mice showed an increase in free plasma testosterone and an up-regulation of CYP17A1 mRNA and protein. The effect of training on CYP17A1 expression and testosterone biosynthesis was significantly higher in the trained mice supplemented with CLA compared to the placebo. The results of the present study demonstrated that CLA stimulates testosterone biosynthesis via CYP17A1, and endurance training led to the synthesis of testosterone in vivo by inducing the overexpression of CYP17A1 mRNA and protein in the Leydig cells of the testis. This effect was enhanced by CLA supplementation. Therefore, CLA-associated physical activity may be used for its steroidogenic property in different fields, such as alimentary industry, human reproductive medicine, sport science, and anti-muscle wasting. PMID:24223995

  19. Polyunsaturated fatty acid biosynthesis is involved in phenylephrine-mediated calcium release in vascular smooth muscle cells.

    PubMed

    Irvine, Nicola A; Lillycrop, Karen A; Fielding, Barbara; Torrens, Christopher; Hanson, Mark A; Burdge, Graham C

    2015-10-01

    Stimulation of vascular smooth muscle (VSM) α1-adrenoceptors induces myosin phosphorylation and vasoconstriction via mobilisation of intracellular calcium and production of specific eicosanoids. Polyunsaturated fatty acid (PUFA) biosynthesis in VSM cells is involved, although the precise mechanism is not known. To address this, we characterised PUFA biosynthesis in VSM cells and determined its role in intracellular calcium release and eicosanoid production. Murine VSM cells converted 18:2n-6 to longer chain PUFA including 22:5n-6. Δ6 (D6d) and Δ5 (D5d) desaturase, and elongase (Elovl) 5 were expressed. Elovl2 was not detected in human, mouse or rat VSM cells, or in rat or mouse aortae, but tit was not associated with hypermethylation of its promoter. D6d or D5d inhibition reduced 18:3n-6 and 20:4n-6 synthesis, respectively, and induced concentration-related decrease in phenylephrine-mediated calcium release, and in PGE2 and PGF2α secretion. Together these findings suggest that PUFA biosynthesis in VSM cells is involved in calcium release associated with vasoconstriction. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Biosynthesis of L-ascorbic acid and conversion of carbons 1 and 2 of L-ascorbic acid to oxalic acid occurs within individual calcium oxalate crystal idioblasts.

    PubMed

    Kostman, T A; Tarlyn, N M; Loewus, F A; Franceschi, V R

    2001-02-01

    L-Ascorbic acid (AsA) and its metabolic precursors give rise to oxalic acid (OxA) found in calcium oxalate crystals in specialized crystal idioblast cells in plants; however, it is not known if AsA and OxA are synthesized within the crystal idioblast cell or transported in from surrounding mesophyll cells. Isolated developing crystal idioblasts from Pistia stratiotes were used to study the pathway of OxA biosynthesis and to determine if idioblasts contain the entire path and are essentially independent in OxA synthesis. Idioblasts were supplied with various (14)C-labeled compounds and examined by micro-autoradiography for incorporation of (14)C into calcium oxalate crystals. [(14)C]OxA gave heavy labeling of crystals, indicating the isolated idioblasts are functional in crystal formation. Incubation with [1-(14)C]AsA also gave heavy labeling of crystals, whereas [6-(14)C]AsA gave no labeling. Labeled precursors of AsA (L-[1-(14)C]galactose; D-[1-(14)C]mannose) also resulted in crystal labeling, as did the ascorbic acid analog, D-[1-(14)C]erythorbic acid. Intensity of labeling of isolated idioblasts followed the pattern OxA > AsA (erythorbic acid) > L-galactose > D-mannose. Our results demonstrate that P. stratiotes crystal idioblasts synthesize the OxA used for crystal formation, the OxA is derived from the number 1 and 2 carbons of AsA, and the proposed pathway of ascorbic acid synthesis via D-mannose and L-galactose is operational in individual P. stratiotes crystal idioblasts. These results are discussed with respect to fine control of calcium oxalate precipitation and the concept of crystal idioblasts as independent physiological compartments.

  1. The Zinc Finger Transcription Factor SlZFP2 Negatively Regulates Abscisic Acid Biosynthesis and Fruit Ripening in Tomato1

    PubMed Central

    Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong

    2015-01-01

    Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening. PMID:25637453

  2. PgLOX6 encoding a lipoxygenase contributes to jasmonic acid biosynthesis and ginsenoside production in Panax ginseng

    PubMed Central

    Rahimi, Shadi; Kim, Yu-Jin; Sukweenadhi, Johan; Zhang, Dabing; Yang, Deok-Chun

    2016-01-01

    Ginsenosides, the valuable pharmaceutical compounds in Panax ginseng, are triterpene saponins that occur mainly in ginseng plants. It was shown that in vitro treatment with the phytohormone jasmonic acid (JA) is able to increase ginsenoside production in ginseng plants. To understand the molecular link between JA biosynthesis and ginsenoside biosynthesis, we identified a JA biosynthetic 13-lipoxygenase gene (PgLOX6) in P. ginseng that promotes ginsenoside production. The expression of PgLOX6 was high in vascular bundles, which corresponds with expression of ginsenoside biosynthetic genes. Consistent with the role of PgLOX6 in synthesizing JA and promoting ginsenoside synthesis, transgenic plants overexpressing PgLOX6 in Arabidopsis had increased amounts of JA and methyl jasmonate (MJ), increased expression of triterpene biosynthetic genes such as squalene synthase (AtSS1) and squalene epoxidase (AtSE1), and increased squalene content. Moreover, transgenic ginseng roots overexpressing PgLOX6 had around 1.4-fold increased ginsenoside content and upregulation of ginsenoside biosynthesis-related genes including PgSS1, PgSE1, and dammarenediol synthase (PgDDS), which is similar to that of treatment with MJ. However, MJ treatment of transgenic ginseng significantly enhanced JA and MJ, associated with a 2.8-fold increase of ginsenoside content compared with the non-treated, non-transgenic control plant, which was 1.4 times higher than the MJ treatment effect on non-transgenic plants. These results demonstrate that PgLOX6 is responsible for the biosynthesis of JA and promotion of the production of triterpenoid saponin through up-regulating the expression of ginsenoside biosynthetic genes. This work provides insight into the role of JA in biosynthesizing secondary metabolites and provides a molecular tool for increasing ginsenoside production. PMID:27811076

  3. Profiling and Quantifying Differential Gene Transcription Provide Insights into Ganoderic Acid Biosynthesis in Ganoderma lucidum in Response to Methyl Jasmonate

    PubMed Central

    Shi, Liang; Mu, Da-Shuai; Jiang, Ai-Liang; Han, Qin; Zhao, Ming-Wen

    2013-01-01

    Ganoderma lucidum is a mushroom with traditional medicinal properties that has been widely used in China and other countries in Eastern Asia. Ganoderic acids (GA) produced by G. lucidum exhibit important pharmacological activities. Previous studies have demonstrated that methyl jasmonate (MeJA) is a potent inducer of GA biosynthesis and the expression of genes involved in the GA biosynthesis pathway in G. lucidum. To further explore the mechanism of GA biosynthesis, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to identify genes that are differentially expressed in response to MeJA. Using 64 primer combinations, over 3910 transcriptionally derived fragments (TDFs) were obtained. Reliable sequence data were obtained for 390 of 458 selected TDFs. Ninety of these TDFs were annotated with known functions through BLASTX searching the GenBank database, and 12 annotated TDFs were assigned into secondary metabolic pathways by searching the KEGGPATHWAY database. Twenty-five TDFs were selected for qRT-PCR analysis to confirm the expression patterns observed with cDNA-AFLP. The qRT-PCR results were consistent with the altered patterns of gene expression revealed by the cDNA-AFLP technique. Additionally, the transcript levels of 10 genes were measured at the mycelium, primordia, and fruiting body developmental stages of G. lucidum. The greatest expression levels were reached during primordia for all of the genes except cytochrome b2 reached its highest expression level in the mycelium stage. This study not only identifies new candidate genes involved in the regulation of GA biosynthesis but also provides further insight into MeJA-induced gene expression and secondary metabolic response in G. lucidum. PMID:23762280

  4. The zinc finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato.

    PubMed

    Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong; Xiao, Han

    2015-03-01

    Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening. © 2015 American Society of Plant Biologists. All Rights Reserved.

  5. PgLOX6 encoding a lipoxygenase contributes to jasmonic acid biosynthesis and ginsenoside production in Panax ginseng.

    PubMed

    Rahimi, Shadi; Kim, Yu-Jin; Sukweenadhi, Johan; Zhang, Dabing; Yang, Deok-Chun

    2016-11-01

    Ginsenosides, the valuable pharmaceutical compounds in Panax ginseng, are triterpene saponins that occur mainly in ginseng plants. It was shown that in vitro treatment with the phytohormone jasmonic acid (JA) is able to increase ginsenoside production in ginseng plants. To understand the molecular link between JA biosynthesis and ginsenoside biosynthesis, we identified a JA biosynthetic 13-lipoxygenase gene (PgLOX6) in P. ginseng that promotes ginsenoside production. The expression of PgLOX6 was high in vascular bundles, which corresponds with expression of ginsenoside biosynthetic genes. Consistent with the role of PgLOX6 in synthesizing JA and promoting ginsenoside synthesis, transgenic plants overexpressing PgLOX6 in Arabidopsis had increased amounts of JA and methyl jasmonate (MJ), increased expression of triterpene biosynthetic genes such as squalene synthase (AtSS1) and squalene epoxidase (AtSE1), and increased squalene content. Moreover, transgenic ginseng roots overexpressing PgLOX6 had around 1.4-fold increased ginsenoside content and upregulation of ginsenoside biosynthesis-related genes including PgSS1, PgSE1, and dammarenediol synthase (PgDDS), which is similar to that of treatment with MJ. However, MJ treatment of transgenic ginseng significantly enhanced JA and MJ, associated with a 2.8-fold increase of ginsenoside content compared with the non-treated, non-transgenic control plant, which was 1.4 times higher than the MJ treatment effect on non-transgenic plants. These results demonstrate that PgLOX6 is responsible for the biosynthesis of JA and promotion of the production of triterpenoid saponin through up-regulating the expression of ginsenoside biosynthetic genes. This work provides insight into the role of JA in biosynthesizing secondary metabolites and provides a molecular tool for increasing ginsenoside production. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  6. Biosynthesis, biological effects, and receptors of hydroxyeicosatetraenoic acids (HETEs) and oxoeicosatetraenoic acids (oxo-ETEs) derived from arachidonic acid.

    PubMed

    Powell, William S; Rokach, Joshua

    2015-04-01

    Arachidonic acid can be oxygenated by a variety of different enzymes, including lipoxygenases, cyclooxygenases, and cytochrome P450s, and can be converted to a complex mixture of oxygenated products as a result of lipid peroxidation. The initial products in these reactions are hydroperoxyeicosatetraenoic acids (HpETEs) and hydroxyeicosatetraenoic acids (HETEs). Oxoeicosatetraenoic acids (oxo-ETEs) can be formed by the actions of various dehydrogenases on HETEs or by dehydration of HpETEs. Although a large number of different HETEs and oxo-ETEs have been identified, this review will focus principally on 5-oxo-ETE, 5S-HETE, 12S-HETE, and 15S-HETE. Other related arachidonic acid metabolites will also be discussed in less detail. 5-Oxo-ETE is synthesized by oxidation of the 5-lipoxygenase product 5S-HETE by the selective enzyme, 5-hydroxyeicosanoid dehydrogenase. It actions are mediated by the selective OXE receptor, which is highly expressed on eosinophils, suggesting that it may be important in eosinophilic diseases such as asthma. 5-Oxo-ETE also appears to stimulate tumor cell proliferation and may also be involved in cancer. Highly selective and potent OXE receptor antagonists have recently become available and could help to clarify its pathophysiological role. The 12-lipoxygenase product 12S-HETE acts by the GPR31 receptor and promotes tumor cell proliferation and metastasis and could therefore be a promising target in cancer therapy. It may also be involved as a proinflammatory mediator in diabetes. In contrast, 15S-HETE may have a protective effect in cancer. In addition to GPCRs, higher concentration of HETEs and oxo-ETEs can activate peroxisome proliferator-activated receptors (PPARs) and could potentially regulate a variety of processes by this mechanism. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

  7. Structural Characterization of the Mycobacterium tuberculosis Biotin Biosynthesis Enzymes 7,8-Diaminopelargonic Acid Synthase and Dethiobiotin Synthetase†,‡

    PubMed Central

    Dey, Sanghamitra; Lane, James M.; Lee, Richard E.; Rubin, Eric J.; Sacchettini, James C.

    2010-01-01

    Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 Å and 1.85 Å. Superimposition of the DAPAS structures bound either to the SAM analog sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 Å, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb. PMID:20565114

  8. Amalgamation of nucleosides and amino acids in antibiotic biosynthesis: discovery of an L-threonine:uridine-5'-aldehyde transaldolase.

    PubMed

    Barnard-Britson, Sandra; Chi, Xiuling; Nonaka, Koichi; Spork, Anatol P; Tibrewal, Nidhi; Goswami, Anwesha; Pahari, Pallab; Ducho, Christian; Rohr, Jurgen; Van Lanen, Steven G

    2012-11-14

    The lipopeptidyl nucleoside antibiotics represented by A-90289, caprazamycin, and muraymycin are structurally highlighted by a nucleoside core that contains a nonproteinogenic β-hydroxy-α-amino acid named 5'-C-glycyluridine (GlyU). Bioinformatic analysis of the biosynthetic gene clusters revealed a shared open reading frame encoding a protein with sequence similarity to serine hydroxymethyltransferases, resulting in the proposal that this shared enzyme catalyzes an aldol-type condensation with glycine and uridine-5'-aldehyde to furnish GlyU. Using LipK involved in A-90289 biosynthesis as a model, we now functionally assign and characterize the enzyme responsible for the C-C bond-forming event during GlyU biosynthesis as an l-threonine:uridine-5'-aldehyde transaldolase. Biochemical analysis revealed this transformation is dependent upon pyridoxal-5'-phosphate, the enzyme has no activity with alternative amino acids, such as glycine or serine, as aldol donors, and acetaldehyde is a coproduct. Structural characterization of the enzyme product is consistent with stereochemical assignment as the threo diastereomer (5'S,6'S)-GlyU. Thus this enzyme orchestrates C-C bond breaking and formation with concomitant installation of two stereocenters to make a new l-α-amino acid with a nucleoside side chain.

  9. Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis.

    PubMed

    Rodrigues, Elisete P; Soares, Cleiton de Paula; Galvão, Patrícia G; Imada, Eddie L; Simões-Araújo, Jean L; Rouws, Luc F M; de Oliveira, André L M; Vidal, Márcia S; Baldani, José I

    2016-01-01

    Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5(T) strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5(T). In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5(T). In conclusion, our study demonstrated that G. diazotrophicus PAL5(T) produces IAA via the IPyA pathway in cultures supplemented with tryptophan

  10. Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis

    PubMed Central

    Rodrigues, Elisete P.; Soares, Cleiton de Paula; Galvão, Patrícia G.; Imada, Eddie L.; Simões-Araújo, Jean L.; Rouws, Luc F. M.; de Oliveira, André L. M.; Vidal, Márcia S.; Baldani, José I.

    2016-01-01

    Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and

  11. Activation of glycerol metabolic pathway by evolutionary engineering of Rhizopus oryzae to strengthen the fumaric acid biosynthesis from crude glycerol.

    PubMed

    Huang, Di; Wang, Ru; Du, Wenjie; Wang, Guanyi; Xia, Menglei

    2015-11-01

    Rhizopus oryzae is strictly inhibited by biodiesel-based by-product crude glycerol, which results in low fumaric acid production. In this study, evolutionary engineering was employed to activate the glycerol utilization pathway for fumaric acid production. An evolved strain G80 was selected, which could tolerate and utilize high concentrations of crude glycerol to produce 14.9g/L fumaric acid with a yield of 0.248g/g glycerol. Key enzymes activity analysis revealed that the evolved strain displayed a significant upregulation in glycerol dissimilation, pyruvate consumption and reductive tricarboxylic acid pathways, compared with the parent strain. Subsequently, intracellular metabolic profiling analysis showed that amino acid biosynthesis, tricarboxylic acid cycle, fatty acid and stress response metabolites accounted for metabolic difference between two strains. Moreover, a glycerol fed-batch strategy was optimized to obtain the highest fumaric acid production of 25.5g/L, significantly increased by 20.9-fold than that of the parent strain of 1.2g/L.

  12. DNA Methylation Perturbations in Genes Involved in Polyunsaturated Fatty Acid Biosynthesis Associated with Depression and Suicide Risk

    PubMed Central

    Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-yu; Cooper, Thomas B.; Burke, Ainsley K.; Oquendo, Maria A.; Mann, J. John; Sublette, M. Elizabeth

    2015-01-01

    Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention. PMID:25972837

  13. Sphingolipid biosynthesis upregulation by TOR complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress.

    PubMed

    Guerreiro, Joana F; Muir, Alexander; Ramachandran, Subramaniam; Thorner, Jeremy; Sá-Correia, Isabel

    2016-12-01

    Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks. © 2016 The Author(s); published by Portland Press Limited on behalf of the

  14. Sphingolipid biosynthesis upregulation by TOR Complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress

    PubMed Central

    Guerreiro, Joana F.; Muir, Alexander; Ramachandran, Subramaniam; Thorner, Jeremy; Sá-Correia, Isabel

    2016-01-01

    Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus, understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for S. cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the Target of Rapamycin (TOR) Complex 2 (TORC2). We show here by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of L-serine: palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus, appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks. PMID:27671892

  15. 6-Methyl-1,2,4-benzenetriol, a new intermediate in penicillic acid biosynthesis in Penicillium cyclopium

    SciTech Connect

    Sekiguchi, J.; Katayama, S.; Yamada, Y.

    1987-07-01

    Penicillic acid-negative mutants were obtained from a color mutant derived from Penicillium cyclopium NRRL 1888 through N-methyl-N'-nitro-N-nitrosoguanidine treatment. One mutant (SK2N6) accumulated 6-methyl-1,2,4-benzenetriol, which was not previously known to be a metabolite of P. cyclopium, in addition to orsellinic acid and orcinol. The radioactivity of (1-/sup 14/C)acetic acid was rapidly incorporated into 6-methyl-1,2,4-benzenetriol in a culture of P. cyclopium SK2N6. Moreover, the radioactivity of (/sup 14/C)6-methyl-1,2,4-benzenetriol was efficiently incorporated into penicillic acid in a culture of P. cyclopium NRRL 1888. These data indicate that 6-methyl-1,2,4-benzenetriol is a precursor for penicillic acid biosynthesis. The results on the addition of 1,4-dihydroxy-6-methoxy-2-methylbenzene, 6-methoxy-2-methylbenzoquinone (1,4), and 1-O-methylorcinol to a culture of P. cyclopium SK2N6 indicated that only the former two compounds are converted to penicillic acid. Thus, a new portion of the penicillic acid biosynthetic pathway is proposed.

  16. Calcium-dependent protein kinases, CDPK4 and CDPK5, affect early steps of jasmonic acid biosynthesis in Nicotiana attenuata.

    PubMed

    Hettenhausen, Christian; Yang, Da-Hai; Baldwin, Ian T; Wu, Jianqiang

    2013-01-01

    Calcium-dependent protein kinases (CDPKs) modulate plant development and growth and are important regulators of biotic and abiotic stress responses. Recently it was found that simultaneously silencing Nicotiana attenuata NaCDPK4 and NaCDPK5 (IRcdpk4/5 plants) results in accumulation of exceptionally high JA levels after wounding or simulated herbivory treatments, which in turn induced high levels of defense metabolites that slowed the growth of Manduca sexta, a specialist insect herbivore. To investigate the mechanism by which NaCDPK4 and NaCDPK5 regulate JA accumulation, we analyzed the transcript levels of all important enzymes involved in JA biosynthesis, but these genes showed no differences between wild-type and IRcdpk4/5 plants. Moreover, the dynamics of JA were similar between these plants, excluding the possibility of decreased degradation rates in IRcdpk4/5 plants. To gain insight into the mechanism by which NaCDPK4 and NaCDPK5 regulate JA biosynthesis, free fatty acids, including C18:3, and (9S,13S)-12-oxo-phytodienoic acid (OPDA), two important precursors of JA were quantified at different times before and after wounding and simulated herbivore feeding treatments. We show that after these treatments, IRcdpk4/5 plants have decreased levels of C18:3, but have enhanced OPDA and JA levels, suggesting that NaCDPK4 and NaCDPK5 have a role in the early steps of JA biosynthesis. The possible role of NaCDPK4 and NaCDPK5 regulating AOS and AOC enzymatic activity is discussed.

  17. The Plant Cuticle Is Required for Osmotic Stress Regulation of Abscisic Acid Biosynthesis and Osmotic Stress Tolerance in Arabidopsis[W

    PubMed Central

    Wang, Zhen-Yu; Xiong, Liming; Li, Wenbo; Zhu, Jian-Kang; Zhu, Jianhua

    2011-01-01

    Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 gene expression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxygenase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol) treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly that CED1 encodes a putative α/β hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cutin biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling. PMID:21610183

  18. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis.

    PubMed

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  19. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis

    PubMed Central

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  20. X-ray crystallographic structure of a bacterial polysialyltransferase provides insight into the biosynthesis of capsular polysialic acid.

    PubMed

    Lizak, Christian; Worrall, Liam J; Baumann, Lars; Pfleiderer, Moritz M; Volkers, Gesa; Sun, Tianjun; Sim, Lyann; Wakarchuk, Warren; Withers, Stephen G; Strynadka, Natalie C J

    2017-07-19

    Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

  1. The role of aromatic L-amino acid decarboxylase in bacillamide C biosynthesis by Bacillus atrophaeus C89.

    PubMed

    Yuwen, Lei; Zhang, Feng-Li; Chen, Qi-Hua; Lin, Shuang-Jun; Zhao, Yi-Lei; Li, Zhi-Yong

    2013-01-01

    For biosynthesis of bacillamide C by Bacillus atrophaeus C89 associated with South China sea sponge Dysidea avara, it is hypothesized that decarboxylation from L-tryptophan to tryptamine could be performed before amidation by the downstream aromatic L-amino acid decarboxylase (AADC) to the non-ribosomal peptide synthetases (NRPS) gene cluster for biosynthesizing bacillamide C. The structural analysis of decarboxylases' known substrates in KEGG database and alignment analysis of amino acid sequence of AADC have suggested that L-tryptophan and L-phenylalanine are the potential substrates of AADC. The enzymatic kinetic experiment of the recombinant AADC proved that L-tryptophan is a more reactive substrate of AADC than L-phenylalanine. Meanwhile, the AADC-catalyzed conversion of L-tryptophan into tryptamine was confirmed by means of HPLC and LC/MS. Thus during bacillamide C biosynthesis, the decarboxylation of L-tryptophan to tryptamine is likely conducted first under AADC catalysis, followed by the amidation of tryptamine with the carboxylic product of NRPS gene cluster.

  2. Chlorogenic acid, anthocyanin and flavan-3-ol biosynthesis in flesh and skin of Andean potato tubers (Solanum tuberosum subsp. andigena).

    PubMed

    Valiñas, Matías Ariel; Lanteri, María Luciana; Ten Have, Arjen; Andreu, Adriana Balbina

    2017-08-15

    Natural variation of Andean potato was used to study the biosynthesis of phenolic compounds. Levels of phenolic compounds and corresponding structural gene transcripts were examined in flesh and skin of tubers. Phenolic acids, mainly chlorogenic acid (CGA), represent the major compounds, followed by anthocyanins and flavan-3-ols. High-anthocyanin varieties have high levels of CGA. Both metabolite and transcript levels were higher in skin than in flesh and showed a good correspondence. Two hydroxycinnamoyl-CoA transferases (HCT/HQT) have been involved in CGA production, of which HCT reflects CGA levels. Catechin was found in pigmented tissues whereas epicatechin was restricted to tuber skin. Transcripts of leucoanthocyanidin reductase (LCR), which generates catechin, could not be detected. Anthocyanidin reductase (ANR) transcripts, the enzyme responsible for epicatechin production, showed similar levels among samples. These data suggest that the biosynthesis of flavan-3-ols in potato tuber would require ANR but not LCR and that an epimerization process is involved. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Silencing of BnTT1 family genes affects seed flavonoid biosynthesis and alters seed fatty acid composition in Brassica napus.

    PubMed

    Lian, Jianping; Lu, Xiaochun; Yin, Nengwen; Ma, Lijuan; Lu, Jing; Liu, Xue; Li, Jiana; Lu, Jun; Lei, Bo; Wang, Rui; Chai, Yourong

    2017-01-01

    TRANSPARENT TESTA1 (TT1) is a zinc finger protein that contains a WIP domain. It plays important roles in controlling differentiation and pigmentation of the seed coat endothelium, and can affect the expression of early biosynthetic genes and late biosynthetic genes of flavonoid biosynthesis in Arabidopsis thaliana. In Brassica napus (AACC, 2n=38), the functions of BnTT1 genes remain unknown and few studies have focused on their roles in fatty acid (FA) biosynthesis. In this study, BnTT1 family genes were silenced by RNA interference, which resulted in yellow rapeseed, abnormal testa development (a much thinner testa), decreased seed weight, and altered seed FA composition in B. napus. High-throughput sequencing of genes differentially expressed between developing transgenic B. napus and wild-type seeds revealed altered expression of numerous genes involved in flavonoid and FA biosynthesis. As a consequence of this altered expression, we detected a marked decrease of oleic acid (C18:1) and notable increases of linoleic acid (C18:2) and α-linolenic acid (C18:3) in mature transgenic B. napus seeds by gas chromatography and near-infrared reflectance spectroscopy. Meanwhile, liquid chromatography-mass spectrometry showed reduced accumulation of flavonoids in transgenic seeds. Therefore, we propose that BnTT1s are involved in the regulation of flavonoid biosynthesis, and may also play a role in FA biosynthesis in B. napus. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. 4-Coumaroyl and caffeoyl shikimic acids inhibit 4-coumaric acid:coenzyme A ligases and modulate metabolic flux for 3-hydroxylation in monolignol biosynthesis of Populus trichocarpa.

    PubMed

    Lin, Chien-Yuan; Wang, Jack P; Li, Quanzi; Chen, Hsi-Chuan; Liu, Jie; Loziuk, Philip; Song, Jina; Williams, Cranos; Muddiman, David C; Sederoff, Ronald R; Chiang, Vincent L

    2015-01-01

    Downregulation of 4-coumaric acid:coenzyme A ligase (4CL) can reduce lignin content in a number of plant species. In lignin precursor (monolignol) biosynthesis during stem wood formation in Populus trichocarpa, two enzymes, Ptr4CL3 and Ptr4CL5, catalyze the coenzyme A (CoA) ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. CoA ligation of 4-coumaric acid is essential for the 3-hydroxylation of 4-coumaroyl shikimic acid. This hydroxylation results from sequential reactions of 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) and 4-coumaric acid 3-hydroxylase 3 (PtrC3H3). Alternatively, 3-hydroxylation of 4-coumaric acid to caffeic acid may occur through an enzyme complex of cinnamic acid 4-hydroxylase 1 and 2 (PtrC4H1 and PtrC4H2) and PtrC3H3. We found that 4-coumaroyl and caffeoyl shikimic acids are inhibitors of Ptr4CL3 and Ptr4CL5. 4-Coumaroyl shikimic acid strongly inhibits the formation of 4-coumaroyl-CoA and caffeoyl-CoA. Caffeoyl shikimic acid inhibits only the formation of 4-coumaroyl-CoA. 4-Coumaroyl and caffeoyl shikimic acids both act as competitive and uncompetitive inhibitors. Metabolic flux in wild-type and PtrC3H3 downregulated P. trichocarpa transgenics has been estimated by absolute protein and metabolite quantification based on liquid chromatography-tandem mass spectrometry, mass action kinetics, and inhibition equations. Inhibition by 4-coumaroyl and caffeoyl shikimic acids may play significant regulatory roles when these inhibitors accumulate.

  5. Aromatic amino acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway.

    PubMed Central

    Braus, G H

    1991-01-01

    This review focuses on the gene-enzyme relationships and the regulation of different levels of the aromatic amino acid biosynthetic pathway in a simple eukaryotic system, the unicellular yeast Saccharomyces cerevisiae. Most reactions of this branched pathway are common to all organisms which are able to synthesize tryptophan, phenylalanine, and tyrosine. The current knowledge about the two main control mechanisms of the yeast aromatic amino acid biosynthesis is reviewed. (i) At the transcriptional level, most structural genes are regulated by the transcriptional activator GCN4, the regulator of the general amino acid control network, which couples transcriptional derepression to amino acid starvation of numerous structural genes in multiple amino acid biosynthetic pathways. (ii) At the enzyme level, the carbon flow is controlled mainly by modulating the enzyme activities at the first step of the pathway and at the branch points by feedback action of the three aromatic amino acid end products. Implications of these findings for the relationship of S. cerevisiae to prokaryotic as well as to higher eukaryotic organisms and for general regulatory mechanisms occurring in a living cell such as initiation of transcription, enzyme regulation, and the regulation of a metabolic branch point are discussed. PMID:1943992

  6. Hyaluronic Acid--an "Old" Molecule with "New" Functions: Biosynthesis and Depolymerization of Hyaluronic Acid in Bacteria and Vertebrate Tissues Including during Carcinogenesis.

    PubMed

    Tsepilov, R N; Beloded, A V

    2015-09-01

    Hyaluronic acid is an evolutionarily ancient molecule commonly found in vertebrate tissues and capsules of some bacteria. Here we review modern data regarding structure, properties, and biological functions of hyaluronic acid in mammals and Streptococcus spp. bacteria. Various aspects of biogenesis and degradation of hyaluronic acid are discussed, biosynthesis and degradation metabolic pathways for glycosaminoglycan together with involved enzymes are described, and vertebrate and bacterial hyaluronan synthase genes are characterized. Special attention is given to the mechanisms underlying the biological action of hyaluronic acid as well as the interaction between polysaccharide and various proteins. In addition, all known signaling pathways involving hyaluronic acid are outlined. Impaired hyaluronic acid metabolism, changes in biopolymer molecular weight, hyaluronidase activity, and enzyme isoforms often accompany carcinogenesis. The interaction between cells and hyaluronic acid from extracellular matrix that may be important during malignant change is discussed. An expected role for high molecular weight hyaluronic acid in resistance of naked mole rat to oncologic diseases and the protective role of hyaluronic acid in bacteria are discussed.

  7. Transcriptome Profiling of Tomato Fruit Development Reveals Transcription Factors Associated with Ascorbic Acid, Carotenoid and Flavonoid Biosynthesis.

    PubMed

    Ye, Jie; Hu, Tixu; Yang, Congmei; Li, Hanxia; Yang, Mingze; Ijaz, Raina; Ye, Zhibiao; Zhang, Yuyang

    2015-01-01

    Tomato (Solanum lycopersicum) serves as a research model for fruit development; however, while it is an important dietary source of antioxidant nutrients, the transcriptional regulation of genes that determine nutrient levels remains poorly understood. Here, the transcriptomes of fruit at seven developmental stages (7, 14, 21, 28, 35, 42 and 49 days after flowering) from two tomato cultivars (Ailsa Craig and HG6-61) were evaluated using the Illumina sequencing platform. A total of 26,397 genes, which were expressed in at least one developmental stage, were detected in the two cultivars, and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis. Gene Ontology term enrichment analysis indicated that genes involved in RNA regulation, secondary metabolism, hormone metabolism and cell wall metabolism were the most highly differentially expressed genes during fruit development and ripening. A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid, carotenoid and flavonoid biosynthesis. This transcriptional correlation was confirmed by agroinfiltration mediated transient expression, which showed that most of the enzymatic genes in the ascorbic acid biosynthesis were regulated by the overexpression of each of the three transcription factors that were tested. The metabolic dynamics of ascorbic acid, carotenoid and flavonoid were investigated during fruit development and ripening, and some selected transcription factors showed transcriptional correlation with the accumulation of ascorbic acid, carotenoid and flavonoid. This transcriptome study provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism.

  8. Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance.

    PubMed

    Jenkins, Benjamin J; Seyssel, Kevin; Chiu, Sally; Pan, Pin-Ho; Lin, Shih-Yi; Stanley, Elizabeth; Ament, Zsuzsanna; West, James A; Summerhill, Keith; Griffin, Julian L; Vetter, Walter; Autio, Kaija J; Hiltunen, Kalervo; Hazebrouck, Stéphane; Stepankova, Renata; Chen, Chun-Jung; Alligier, Maud; Laville, Martine; Moore, Mary; Kraft, Guillaume; Cherrington, Alan; King, Sarah; Krauss, Ronald M; de Schryver, Evelyn; Van Veldhoven, Paul P; Ronis, Martin; Koulman, Albert

    2017-03-23

    Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1(-/-) mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1(-/-) only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.

  9. Redundant Systems of Phosphatidic Acid Biosynthesis via Acylation of Glycerol-3-Phosphate or Dihydroxyacetone Phosphate in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Athenstaedt, Karin; Weys, Sabine; Paltauf, Fritz; Daum, Günther

    1999-01-01

    In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611–7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-3H, U-14C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the gat1 mutant and the 1-acylglycerol-3-phosphate acyltransferase slc1 mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate. PMID:10049376

  10. Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance

    PubMed Central

    Jenkins, Benjamin J.; Seyssel, Kevin; Chiu, Sally; Pan, Pin-Ho; Lin, Shih-Yi; Stanley, Elizabeth; Ament, Zsuzsanna; West, James A.; Summerhill, Keith; Griffin, Julian L.; Vetter, Walter; Autio, Kaija J.; Hiltunen, Kalervo; Hazebrouck, Stéphane; Stepankova, Renata; Chen, Chun-Jung; Alligier, Maud; Laville, Martine; Moore, Mary; Kraft, Guillaume; Cherrington, Alan; King, Sarah; Krauss, Ronald M.; de Schryver, Evelyn; Van Veldhoven, Paul P.; Ronis, Martin; Koulman, Albert

    2017-01-01

    Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1−/− mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1−/− only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease. PMID:28332596

  11. Accumulation of Phenolic Compounds and Expression Profiles of Phenolic Acid Biosynthesis-Related Genes in Developing Grains of White, Purple, and Red Wheat

    PubMed Central

    Ma, Dongyun; Li, Yaoguang; Zhang, Jian; Wang, Chenyang; Qin, Haixia; Ding, Huina; Xie, Yingxin; Guo, Tiancai

    2016-01-01

    Polyphenols in whole grain wheat have potential health benefits, but little is known about the expression patterns of phenolic acid biosynthesis genes and the accumulation of phenolic acid compounds in different-colored wheat grains. We found that purple wheat varieties had the highest total phenolic content (TPC) and antioxidant activity. Among phenolic acid compounds, bound ferulic acid, vanillic, and caffeic acid levels were significantly higher in purple wheat than in white and red wheat, while total soluble phenolic acid, soluble ferulic acid, and vanillic acid levels were significantly higher in purple and red wheat than in white wheat. Ferulic acid and syringic acid levels peaked at 14 days after anthesis (DAA), whereas p-coumaric acid and caffeic acid levels peaked at 7 DAA, and vanillic acid levels gradually increased during grain filling and peaked near ripeness (35 DAA). Nine phenolic acid biosynthesis pathway genes (TaPAL1, TaPAL2, TaC3H1, TaC3H2, TaC4H, Ta4CL1, Ta4CL2, TaCOMT1, and TaCOMT2) exhibited three distinct expression patterns during grain filling, which may be related to the different phenolic acids levels. White wheat had higher phenolic acid contents and relatively high gene expression at the early stage, while purple wheat had the highest phenolic acid contents and gene expression levels at later stages. These results suggest that the expression of phenolic acid biosynthesis genes may be closely related to phenolic acids accumulation. PMID:27148345

  12. Accumulation of Phenolic Compounds and Expression Profiles of Phenolic Acid Biosynthesis-Related Genes in Developing Grains of White, Purple, and Red Wheat.

    PubMed

    Ma, Dongyun; Li, Yaoguang; Zhang, Jian; Wang, Chenyang; Qin, Haixia; Ding, Huina; Xie, Yingxin; Guo, Tiancai

    2016-01-01

    Polyphenols in whole grain wheat have potential health benefits, but little is known about the expression patterns of phenolic acid biosynthesis genes and the accumulation of phenolic acid compounds in different-colored wheat grains. We found that purple wheat varieties had the highest total phenolic content (TPC) and antioxidant activity. Among phenolic acid compounds, bound ferulic acid, vanillic, and caffeic acid levels were significantly higher in purple wheat than in white and red wheat, while total soluble phenolic acid, soluble ferulic acid, and vanillic acid levels were significantly higher in purple and red wheat than in white wheat. Ferulic acid and syringic acid levels peaked at 14 days after anthesis (DAA), whereas p-coumaric acid and caffeic acid levels peaked at 7 DAA, and vanillic acid levels gradually increased during grain filling and peaked near ripeness (35 DAA). Nine phenolic acid biosynthesis pathway genes (TaPAL1, TaPAL2, TaC3H1, TaC3H2, TaC4H, Ta4CL1, Ta4CL2, TaCOMT1, and TaCOMT2) exhibited three distinct expression patterns during grain filling, which may be related to the different phenolic acids levels. White wheat had higher phenolic acid contents and relatively high gene expression at the early stage, while purple wheat had the highest phenolic acid contents and gene expression levels at later stages. These results suggest that the expression of phenolic acid biosynthesis genes may be closely related to phenolic acids accumulation.

  13. TaOPR2 encodes a 12-oxo-phytodienoic acid reductase involved in the biosynthesis of jasmonic acid in wheat (Triticum aestivum L.).

    PubMed

    Wang, Yukun; Yuan, Guoliang; Yuan, Shaohua; Duan, Wenjing; Wang, Peng; Bai, Jianfang; Zhang, Fengting; Gao, Shiqing; Zhang, Liping; Zhao, Changping

    2016-01-29

    The 12-oxo-phytodienoic acid reductases (OPRs) are involved in the various processes of growth and development in plants, and classified into the OPRⅠ and OPRⅡ subgroups. In higher plants, only OPRⅡ subgroup genes take part in the biosynthesis of endogenous jasmonic acid. In this study, we isolated a novel OPRⅡ subgroup gene named TaOPR2 (GeneBank accession: KM216389) from the thermo-sensitive genic male sterile (TGMS) wheat cultivar BS366. TaOPR2 was predicted to encode a protein with 390 amino acids. The encoded protein contained the typical oxidored_FMN domain, the C-terminus peroxisomal-targeting signal peptide, and conserved FMN-binding sites. TaOPR2 was mapped to wheat chromosome 7B and located on peroxisome. Protein evolution analysis revealed that TaOPR2 belongs to the OPRⅡ subgroup and shares a high degree of identity with other higher plant OPR proteins. The quantitative real-time PCR results indicated that the expression of TaOPR2 is inhibited by abscisic acid (ABA), salicylic acid (SA), gibberellic acid (GA3), low temperatures and high salinity. In contrast, the expression of TaOPR2 can be induced by wounding, drought and methyl jasmonate (MeJA). Furthermore, the transcription level of TaOPR2 increased after infection with Puccinia striiformis f. sp. tritici and Puccinia recondite f. sp. tritici. TaOPR2 has NADPH-dependent oxidoreductase activity. In addition, the constitutive expression of TaOPR2 can rescue the male sterility phenotype of Arabidopsis mutant opr3. These results suggest that TaOPR2 is involved in the biosynthesis of jasmonic acid (JA) in wheat. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Fractionation of Carbon Isotopes in Biosynthesis of Fatty Acids by A Piezophilic Bacterium Moritella Japonica DSK1

    NASA Astrophysics Data System (ADS)

    Fang, J.; Uhle, M.; Bartlett, D.; Kato, C.

    2005-12-01

    We examined stable carbon isotope fractionation in biosynthesis of fatty acids of a piezophilic bacterium Moritella japonica DSK1. DSK1 was grown to stationary phase at pressures of 0.1, 10, 20, and 50 MPa in media prepared using natural seawater supplied with glucose with the sole carbon source. DSk1 synthesized typical bacterial fatty acids (C14-19 saturated, monounsaturated, and cyclopropane fatty acids) as well as long-chain polyunsaturated fatty acids (PUFA) (20:6ω3). Bacterial cell biomass and individual fatty acids exhibited consistent pressure-dependent carbon isotope fractionations relative to glucose. The observed ΔδFA-glucose (-1.0 to -11.9%) at 0.1 MPa was comparable to or slightly higher than fractionations reported on surface bacteria. However, Bulk biomass and fatty acids became more depleted in 13C with pressure. Average carbon isotope fractionation ΔδFA-glucose) at high pressures was much higher than that for surface bacteria: -15.7, -15.3, and -18.3‰ at 10, 20, and 50 MPa, respectively. PUFA were more 13C depleted than saturated and monounsaturated fatty acids at all pressures. The observed isotope effects may be ascribed to the kinetics of enzymatic reactions affected by hydrostatic pressure and to different biosynthetic pathways for short-chain and long-chain fatty acids. Our results have important implications for marine biogeochemistry. The 13C depleted fatty acids in marine sediments and water column may be derived simply from piezophilic bacteria resynthesis of organic matter, not from bacterial utilization of a 13C-depleted carbon source (i.e., methane). The interpretation of carbon isotope signatures of marine lipids must be based on principles derived from piezophilic bacteria.

  15. Biosynthesis of Chloro-β-Hydroxytyrosine, a Nonproteinogenic Amino Acid of the Peptidic Backbone of Glycopeptide Antibiotics

    PubMed Central

    Puk, Oliver; Bischoff, Daniel; Kittel, Claudia; Pelzer, Stefan; Weist, Stefan; Stegmann, Efthimia; Süssmuth, Roderich D.; Wohlleben, Wolfgang

    2004-01-01

    The role of the putative P450 monooxygenase OxyD and the chlorination time point in the biosynthesis of the glycopeptide antibiotic balhimycin produced by Amycolatopsis balhimycina were analyzed. The oxyD gene is located directly downstream of the bhp (perhydrolase) and bpsD (nonribosomal peptide synthetase D) genes, which are involved in the synthesis of the balhimycin building block β-hydroxytyrosine (β-HT). Reverse transcriptase experiments revealed that bhp, bpsD, and oxyD form an operon. oxyD was inactivated by an in-frame deletion, and the resulting mutant was unable to produce an active compound. Balhimycin production could be restored (i) by complementation with an oxyD gene, (ii) in cross-feeding studies using A. balhimycina JR1 (a null mutant with a block in the biosynthesis pathway of the building blocks hydroxy- and dihydroxyphenylglycine) as an excretor of the missing precursor, and (iii) by supplementation of β-HT in the growth medium. These data demonstrated an essential role of OxyD in the formation pathway of this amino acid. Liquid chromatography-electrospray ionization-mass spectrometry analysis indicated the biosynthesis of completely chlorinated balhimycin by the oxyD mutant when culture filtrates were supplemented with nonchlorinated β-HT. In contrast, supplementation with 3-chloro-β-HT did not restore balhimycin production. These results indicated that the chlorination time point was later than the stage of free β-HT, most likely during heptapeptide synthesis. PMID:15342578

  16. Two New Alleles of the abscisic aldehyde oxidase 3 Gene Reveal Its Role in Abscisic Acid Biosynthesis in Seeds1

    PubMed Central

    González-Guzmán, Miguel; Abia, David; Salinas, Julio; Serrano, Ramón; Rodríguez, Pedro L.

    2004-01-01

    The abscisic aldehyde oxidase 3 (AAO3) gene product of Arabidopsis catalyzes the final step in abscisic acid (ABA) biosynthesis. An aao3-1 mutant in a Landsberg erecta genetic background exhibited a wilty phenotype in rosette leaves, whereas seed dormancy was not affected (Seo et al., 2000a). Therefore, it was speculated that a different aldehyde oxidase would be the major contributor to ABA biosynthesis in seeds (Seo et al., 2000a). Through a screening based on germination under high-salt concentration, we isolated two mutants in a Columbia genetic background, initially named sre2-1 and sre2-2 (for salt resistant). Complementation tests with different ABA-deficient mutants indicated that sre2-1 and sre2-2 mutants were allelic to aao3-1, and therefore they were renamed as aao3-2 and aao3-3, respectively. Indeed, molecular characterization of the aao3-2 mutant revealed a T-DNA insertional mutation that abolished the transcription of AAO3 gene, while sequence analysis of AAO3 in aao3-3 mutant revealed a deletion of three nucleotides and several missense mutations. Physiological characterization of aao3-2 and aao3-3 mutants revealed a wilty phenotype and osmotolerance in germination assays. In contrast to aao3-1, both aao3-2 and aao3-3 mutants showed a reduced dormancy. Accordingly, ABA levels were reduced in dry seeds and rosette leaves of both aao3-2 and aao3-3. Taken together, these results indicate that AAO3 gene product plays a major role in seed ABA biosynthesis. PMID:15122034

  17. High levels of jasmonic acid antagonize the biosynthesis of gibberellins and inhibit the growth of Nicotiana attenuata stems.

    PubMed

    Heinrich, Maria; Hettenhausen, Christian; Lange, Theo; Wünsche, Hendrik; Fang, Jingjing; Baldwin, Ian T; Wu, Jianqiang

    2013-02-01

    Hormones play pivotal roles in regulating plant development, growth, and stress responses, and cross-talk among different hormones fine-tunes various aspects of plant physiology. Jasmonic acid (JA) is important for plant defense against herbivores and necrotic fungi and also regulates flower development; in addition, Arabidopsis mutants over-producing JA usually have stunted stems and wound-induced jasmonates suppress Arabidopsis growth, suggesting that JA is also involved in stem elongation. Gibberellins (GAs) promote stem and leaf growth and modulate seed germination, flowering time, and the development of flowers, fruits, and seeds. However, little is known about the interaction between the JA and GA pathways. Two calcium-dependent protein kinases, CDPK4 and CDPK5, are important suppressors of JA accumulation in a wild tobacco species, Nicotiana attenuata. The stems of N. attenuata silenced in CDPK4 and CDPK5 (irCDPK4/5 plants) had dramatically increased levels of JA and exhibited stunted elongation and had very high contents of secondary metabolites. Genetic analysis indicated that the high JA levels in irCDPK4/5 stems accounted for the suppressed stem elongation and the accumulation of secondary metabolites. Supplementation of GA(3) to irCDPK4/5 plants largely restored normal stem growth to wild-type levels. Measures of GA levels indicated that over-accumulation of JA in irCDPK4/5 stems inhibited the biosynthesis of GAs. Finally, we show that JA antagonizes GA biosynthesis by strongly inhibiting the transcript accumulation of GA20ox and possibly GA13ox, the key genes in GA production, demonstrating that high JA levels antagonize GA biosynthesis in stems. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  18. A basic leucine zipper transcription factor, AabZIP1, connects abscisic acid signaling with artemisinin biosynthesis in Artemisia annua.

    PubMed

    Zhang, Fangyuan; Fu, Xueqing; Lv, Zongyou; Lu, Xu; Shen, Qian; Zhang, Ling; Zhu, Mengmeng; Wang, Guofeng; Sun, Xiaofen; Liao, Zhihua; Tang, Kexuan

    2015-01-01

    Artemisinin is a sesquiterpenoid especially synthesized in the Chinese herbal plant, Artemisia annua, which is widely used in the treatment of malaria. Artemisinin accumulation can be enhanced by exogenous abscisic acid (ABA) treatment. However, it is not known how ABA signaling regulates artemisinin biosynthesis. A global expression profile and phylogenetic analysis as well as the dual-LUC screening revealed that a basic leucine zipper family transcription factor from A. annua (namely AabZIP1) was involved in ABA signaling to regulate artemisinin biosynthesis. AabZIP1 had a higher expression level in the inflorescences than in other tissues; ABA treatment, drought, and salt stress strongly induced the expression of AabZIP1. Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that AabZIP1 bound to the ABA-responsive elements (ABRE) in the promoter regions of the amorpha-4,11-diene synthase (ADS) gene and CYP71AV1, which are two key structural genes of the artemisinin biosynthetic pathway. A mutagenesis assay showed that the C1 domain in the N-terminus of AabZIP1 was important for its transactivation activity. Furthermore, the activation of ADS and CYP71AV1 promoters by AabZIP1 was enhanced by ABA treatment in transient dual-LUC analysis. The AabZIP1 variant with C1 domain deletion lost the ability to activate ADS and CYP71AV1 promoters regardless of ABA treatment. Notably, overexpression of AabZIP1 in A. annua resulted in significantly increased accumulation of artemisinin. Our results indicate that ABA promotes artemisinin biosynthesis, likely through 1 activation of ADS and CYP71AV1 expression by AabZIP in A. annua. Meanwhile, our findings reveal the potential value of AabZIP1 in genetic engineering of artemisinin production. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  19. Regulation of the Bacterial Cell Wall: Analysis of a Mutant of Bacillus subtilis Defective in Biosynthesis of Teichoic Acid

    PubMed Central

    Boylan, R. J.; Mendelson, N. H.; Brooks, D.; Young, F. E.

    1972-01-01

    Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in a deficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-l-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-1) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis. PMID:4622900

  20. Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid.

    PubMed

    Hayashi, H; Huang, P; Inoue, K; Hiraoka, N; Ikeshiro, Y; Yazaki, K; Tanaka, S; Kushiro, T; Shibuya, M; Ebizuka, Y

    2001-12-01

    An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols.

  1. Characterization of three novel desaturases involved in the delta-6 desaturation pathways for polyunsaturated fatty acid biosynthesis from Phytophthora infestans.

    PubMed

    Sun, Quanxi; Liu, Jiang; Zhang, Qin; Qing, Xiaohe; Dobson, Gary; Li, Xinzheng; Qi, Baoxiu

    2013-09-01

    Phytophthora infestans is the causative agent of potato blight that resulted in the great famine in Ireland in the nineteenth century. This microbe can release large amounts of the C20 very long-chain polyunsaturated fatty acids arachidonic acid (ARA; 20:4Δ(5, 8, 11, 14)) and eicosapentaenoic acid (EPA; 20:5Δ(5, 8, 11, 14, 17)) upon invasion that is known to elicit a hypersensitive response to their host plant. In order to identify enzymes responsible for the biosynthesis of these fatty acids, we blasted the recently fully sequenced P. infestans genome and identified three novel putatively encoding desaturase sequences. These were subsequently functionally characterized by expression in Saccharomyces cerevisiae and confirmed that they encode desaturases with Δ12, Δ6 and Δ5 activity, designated here as PinDes12, PinDes6 and PinDes5, respectively. This, together with the combined fatty acid profiles and a previously identified Δ6 elongase activity, implies that the ARA and EPA are biosynthesized predominantly via the Δ6 desaturation pathways in P. infestans. Elucidation of ARA and EPA biosynthetic mechanism may provide new routes to combating this potato blight microbe directly or by means of conferring resistance to important crops.

  2. DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

    PubMed Central

    Castro, Consuelo B.; Whittock, Lucy D.; Whittock, Simon P.; Leggett, Grey; Koutoulis, Anthony

    2008-01-01

    Background The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Methods Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Results Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, ‘Symphony’ and ‘Ember’, which typically have high bitter acid levels. Conclusions In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development. PMID:18519445

  3. New tuberculostatic agents targeting nucleic acid biosynthesis: drug design using QSAR approaches.

    PubMed

    Bueno, Renata V; Braga, Rodolpho C; Segretti, Natanael D; Ferreira, Elizabeth I; Trossini, Gustavo H G; Andrade, Carolina H

    2014-01-01

    Worldwide, tuberculosis (TB) is the leading cause of death among curable infectious diseases. The emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) TB is a growing global health concern and there is an urgent need for new anti-TB drugs. Enzymes involved in DNA and ATP biosynthesis are potential targets for tuberculostatic drug design, since these enzymes are essential for Mycobacterium tuberculosis growth. This review presents the current progress and applications of structure-activity relationship analysis for the discovery of innovative tuberculostatic agents as inhibitors of ribonucleotide reductase, DNA gyrase, ATP synthase, and thymidylate kinase enzymes, highlighting present challenges and new opportunities in TB drug design.

  4. Ozone affects ascorbate and glutathione biosynthesis as well as amino acid contents in three Euramerican poplar genotypes.

    PubMed

    Dumont, Jennifer; Keski-Saari, Sarita; Keinänen, Markku; Cohen, David; Ningre, Nathalie; Kontunen-Soppela, Sari; Baldet, Pierre; Gibon, Yves; Dizengremel, Pierre; Vaultier, Marie-Noëlle; Jolivet, Yves; Oksanen, Elina; Le Thiec, Didier

    2014-03-01

    Ozone is an air pollutant that causes oxidative stress by generation of reactive oxygen species (ROS) within the leaf. The capacity to detoxify ROS and repair ROS-induced damage may contribute to ozone tolerance. Ascorbate and glutathione are known to be key players in detoxification. Ozone effects on their biosynthesis and on amino acid metabolism were investigated in three Euramerican poplar genotypes (Populus deltoides Bartr. × Populus nigra L.) differing in ozone sensitivity. Total ascorbate and glutathione contents were increased in response to ozone in all genotypes, with the most resistant genotype (Carpaccio) showing an increase of up to 70%. Reduced ascorbate (ASA) concentration at least doubled in the two most resistant genotypes (Carpaccio and Cima), whereas the most sensitive genotype (Robusta) seemed unable to regenerate ASA from oxidized ascorbate (DHA), leading to an increase of 80% of the oxidized form. Increased ascorbate (ASA + DHA) content correlated with the increase in gene expression in its biosynthetic pathway, especially the putative gene of GDP-l-galactose phosphorylase VTC2. Increased cysteine availability combined with increased expression of γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2) genes allows higher glutathione biosynthesis in response to ozone, particularly in Carpaccio. In addition, ozone caused a remobilization of amino acids with a decreased pool of total amino acids and an increase of Cys and putrescine, especially in Carpaccio. In addition, the expression of genes encoding threonine aldolase was strongly induced only in the most tolerant genotype, Carpaccio. Reduced ascorbate levels could partly explain the sensitivity to ozone for Robusta but not for Cima. Reduced ascorbate level alone is not sufficient to account for ozone tolerance in poplar, and it is necessary to consider several other factors including glutathione content.

  5. Involvement of a Natural Fusion of a Cytochrome P450 and a Hydrolase in Mycophenolic Acid Biosynthesis

    PubMed Central

    Hansen, Bjarne Gram; Mnich, Ewelina; Nielsen, Kristian Fog; Nielsen, Jakob Blæsbjerg; Nielsen, Morten Thrane; Mortensen, Uffe Hasbro

    2012-01-01

    Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway. PMID:22544261

  6. Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis.

    PubMed

    Hansen, Bjarne Gram; Mnich, Ewelina; Nielsen, Kristian Fog; Nielsen, Jakob Blæsbjerg; Nielsen, Morten Thrane; Mortensen, Uffe Hasbro; Larsen, Thomas Ostenfeld; Patil, Kiran Raosaheb

    2012-07-01

    Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.

  7. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    PubMed

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  8. Dietary ɛ-Polylysine Decreased Serum and Liver Lipid Contents by Enhancing Fecal Lipid Excretion Irrespective of Increased Hepatic Fatty Acid Biosynthesis-Related Enzymes Activities in Rats

    PubMed Central

    Hosomi, Ryota; Yamamoto, Daiki; Otsuka, Ren; Nishiyama, Toshimasa; Yoshida, Munehiro; Fukunaga, Kenji

    2015-01-01

    ɛ-Polylysine (EPL) is used as a natural preservative in food. However, few studies have been conducted to assess the beneficial functions of dietary EPL. The purpose of this study was to elucidate the mechanism underlying the inhibition of neutral and acidic sterol absorption and hepatic enzyme activity-related fatty acid biosynthesis following EPL intake. EPL digest prepared using an in vitro digestion model had lower lipase activity and micellar lipid solubility and higher bile acid binding capacity than casein digest. Male Wistar rats were fed an AIN-93G diet containing 1% (wt/wt) EPL or l-lysine. After 4 weeks of feeding these diets, the marked decrease in serum and liver triacylglycerol contents by the EPL diet was partly attributed to increased fecal fatty acid excretion. The activities of hepatic acetyl-coenzyme A carboxylase and glucose-6-phosphate dehydrogenase, which are key enzymes of fatty acid biosynthesis, were enhanced in rats fed EPL diet. The increased fatty acid biosynthesis activity due to dietary EPL may be prevented by the enhancement of fecal fatty acid excretion. The hypocholesterolemic effect of EPL was mediated by increased fecal neutral and acidic sterol excretions due to the EPL digest suppressing micellar lipid solubility and high bile acid binding capacity. These results show that dietary EPL has beneficial effects that could help prevent lifestyle-related diseases such as hyperlipidemia and atherosclerosis. PMID:25866749

  9. Characterization of the 9-Cis-Epoxycarotenoid Dioxygenase Gene Family and the Regulation of Abscisic Acid Biosynthesis in Avocado1

    PubMed Central

    Chernys, Jacqueline T.; Zeevaart, Jan A.D.

    2000-01-01

    Avocado (Persea americana Mill. cv Lula) is a climacteric fruit that exhibits a rise in ethylene as the fruit ripens. This rise in ethylene is followed by an increase in abscisic acid (ABA), with the highest level occurring just after the peak in ethylene production. ABA is synthesized from the cleavage of carotenoid precursors. The cleavage of carotenoid precursors produces xanthoxin, which can subsequently be converted into ABA via ABA-aldehyde. Indirect evidence indicates that the cleavage reaction, catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED), is the regulatory step in ABA synthesis. Three genes encoding NCED cleavage-like enzymes were cloned from avocado fruit. Two genes, PaNCED1 and PaNCED3, were strongly induced as the fruit ripened. The other gene, PaNCED2, was constitutively expressed during fruit ripening, as well as in leaves. This gene lacks a predicted chloroplast transit peptide. It is therefore unlikely to be involved in ABA biosynthesis. PaNCED1 was induced by water stress, but expression of PaNCED3 was not detectable in dehydrated leaves. Recombinant PaNCED1 and PaNCED3 were capable of in vitro cleavage of 9-cis-xanthophylls into xanthoxin and C25-apocarotenoids, but PaNCED2 was not. Taken together, the results indicate that ABA biosynthesis in avocado is regulated at the level of carotenoid cleavage. PMID:10982448

  10. Evolution of aromatic amino acid biosynthesis and application to the fine-tuned phylogenetic positioning of enteric bacteria.

    PubMed

    Ahmad, S; Weisburg, W G; Jensen, R A

    1990-02-01

    A comprehensive phylogenetic tree for virtually the entire assemblage of enteric bacteria is presented. Character states of aromatic amino acid biosynthesis are used as criteria, and the results are compared with partial trees based upon sequencing of 16S rRNA, 5S rRNA, and tryptophan leader peptide. Three major clusters are apparent. Enterocluster 1 possesses a gene fusion (trpG-trpD) encoding anthranilate synthase: anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase of tryptophan biosynthesis. This cluster includes the genera Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella, and Enterobacter. The remaining two clusters lack the trpG-trpD gene fusion, but differ in the presence (enterocluster 2) or absence (enterocluster 3) of the three-step overflow pathway to L-phenylalanine. Enterocluster 2 consists of the genera Serratia and Erwinia. Enterocluster 3 includes the genera Cedecea, Kluyvera, Edwardsiella, Hafnia, Yersinia, Proteus, Providencia, and Morganella. Within these three major clusters, a tentative hierarchy of subcluster ordering is formulated on the basis of all data available. This hierarchical framework is proposed as a general working basis for continued refinement of the phylogenetic relationships of enteric bacteria.

  11. Ethylene-Inhibited Jasmonic Acid Biosynthesis Promotes Mesocotyl/Coleoptile Elongation of Etiolated Rice Seedlings[OPEN

    PubMed Central

    Xiong, Qing; Ma, Biao; Lu, Xiang; Huang, Yi-Hua; He, Si-Jie; Yang, Chao; Yin, Cui-Cui; Zhou, Yang; Wang, Wen-Sheng; Li, Zhi-Kang; Chen, Shou-Yi

    2017-01-01

    Elongation of the mesocotyl and coleoptile facilitates the emergence of rice (Oryza sativa) seedlings from soil and is affected by various genetic and environment factors. The regulatory mechanism underlying this process remains largely unclear. Here, we examined the regulation of mesocotyl and coleoptile growth by characterizing a gaoyao1 (gy1) mutant that exhibits a longer mesocotyl and longer coleoptile than its original variety of rice. GY1 was identified through map-based cloning and encodes a PLA1-type phospholipase that localizes in chloroplasts. GY1 functions at the initial step of jasmonic acid (JA) biosynthesis to repress mesocotyl and coleoptile elongation in etiolated rice seedlings. Ethylene inhibits the expression of GY1 and other genes in the JA biosynthesis pathway to reduce JA levels and enhance mesocotyl and coleoptile growth by promoting cell elongation. Genetically, GY1 acts downstream of the OsEIN2-mediated ethylene signaling pathway to regulate mesocotyl/coleoptile growth. Through analysis of the resequencing data from 3000 rice accessions, we identified a single natural variation of the GY1 gene, GY1376T, which contributes to mesocotyl elongation in rice varieties. Our study reveals novel insights into the regulatory mechanism of mesocotyl/coleoptile elongation and should have practical applications in rice breeding programs. PMID:28465411

  12. Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate

    PubMed Central

    Song, Heng; Hu, Wen; Naowarojna, Nathchar; Her, Ampon Sae; Wang, Shu; Desai, Rushil; Qin, Li; Chen, Xiaoping; Liu, Pinghua

    2015-01-01

    Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction. PMID:26149121

  13. The ergosterol biosynthesis inhibitor zaragozic acid promotes vacuolar degradation of the tryptophan permease Tat2p in yeast.

    PubMed

    Daicho, Katsue; Maruyama, Hironori; Suzuki, Asuka; Ueno, Masaru; Uritani, Masahiro; Ushimaru, Takashi

    2007-07-01

    Ergosterol is the yeast functional equivalent of cholesterol in mammalian cells. Deletion of the ERG6 gene, which encodes an enzyme catalyzing a late step of ergosterol biosynthesis, impedes targeting of the tryptophan permease Tat2p to the plasma membrane, but does not promote vacuolar degradation. It is unknown whether similar features appear when other steps of ergosterol biogenesis are inhibited. We show herein that the ergosterol biosynthesis inhibitor zaragozic acid (ZA) evoked massive vacuolar degradation of Tat2p, accompanied by a decrease in tryptophan uptake. ZA inhibits squalene synthetase (SQS, EC 2.5.1.21), which catalyzes the first committed step in the formation of cholesterol/ergosterol. The degradation of Tat2p was dependent on the Rsp5p-mediated ubiquitination of Tat2p and was not suppressed by deletions of VPS1, VPS27, VPS45 or PEP12. We will discuss ZA-mediated Tat2p degradation in the context of lipid rafts.

  14. Biosynthesis and stereochemical configuration of N5-(1-carboxyethyl)ornithine. An unusual amino acid produced by Streptococcus lactis

    SciTech Connect

    Miller, S.P.; Thompson, J.

    1987-11-25

    In a recent communication we described the purification and characterization of N5-(1-carboxyethyl)ornithine from cells of Streptococcus lactis 133. This unusual amino acid has not previously been found in nature. Radiotracer experiments presented here reveal that exogenous (/sup 14/C)ornithine serves as the precursor for biosynthesis of (/sup 14/C)arginine, (/sup 14/C)N5-(1-carboxyethyl)ornithine, and (/sup 14/C)N5-acetylornithine by cells of S. lactis K1 during growth in a defined medium lacking arginine. In the absence of both arginine and ornithine, cells of S. lactis K1 can also generate intracellular (/sup 14/C)N5-(1-carboxyethyl)ornithine from exogenous (/sup 14/C)glutamic acid. Previously we showed that the properties of N5-(1-carboxyethyl)ornithine prepared from S. lactis were identical to one of the two diastereomers (2S, 7S) or (2S, 7R) present in a synthetic preparation of (2S, 7RS)-N5-(1-carboxyethyl)ornithine. The two diastereomers have now been unambiguously synthesized by an Abderhalden-Haase condensation between (2S)-N2-t-butoxycarbonyl-ornithine and the chiral (2S)-, and (2R)-bromopropionates. By /sup 13/C-NMR spectroscopy it has been established that the preparation from S. lactis is exclusively (2S, 7S)-N5-(1-carboxyethyl)ornithine. has been demonstrated in a cell-free extract of S. lactis 133. The requirements for ornithine, pyruvic acid, and NAD(P)H suggest that biosynthesis of N5-(1-carboxyethyl)ornithine occurs via a reductive condensation mechanism. A general survey revealed that N5-(1-carboxyethyl)ornithine was produced only by certain strains of Group N streptococci. These findings may indicate a plasmid locus for the gene(s) encoding the enzyme(s) for N5-(1-carboxyethyl)ornithine biosynthesis.

  15. Transcriptome Analysis of Genes Associated with the Artemisinin Biosynthesis by Jasmonic Acid Treatment under the Light in Artemisia annua.

    PubMed

    Hao, Xiaolong; Zhong, Yijun; Fu, Xueqing; Lv, Zongyou; Shen, Qian; Yan, Tingxiang; Shi, Pu; Ma, Yanan; Chen, Minghui; Lv, Xueying; Wu, Zhangkuanyu; Zhao, Jingya; Sun, Xiaofen; Li, Ling; Tang, Kexuan

    2017-01-01

    Artemisinin is a sesquiterpene lactone endoperoxide extracted from a traditional Chinese medicinal plant Artemisia annua. Artemisinin-based combination therapies (ACTs) are recommended as the best treatment of malaria by the World Health Organization (WHO). Both the phytohormone jasmonic acid (JA) and light promote artemisinin biosynthesis in A. annua. Interestingly, we found that the increase of artemisinin biosynthesis by JA was dependent on light. However, the relationship between the two signal pathways mediated by JA and light remains unclear. Here, we collected the A. annua seedlings of 24 h continuous light (Light), 24 h dark treatment (Dark), 4 h MeJA treatment under the continuous light conditions (Light-MeJA-4h) and 4 h MeJA treatment under the dark conditions (Dark-MeJA-4h) and performed the transcriptome sequencing using Illumina HiSeq 4000 System. A total of 266.7 million clean data were produced and assembled into 185,653 unigenes, with an average length of 537 bp. Among them, 59,490 unigenes were annotated and classified based on the public information. Differential expression analyses were performed between Light and Dark, Light and Light-MeJA-4h, Dark and Dark-MeJA-4h, Light-MeJA-4h, and Dark-MeJA-4h, respectively. Furthermore, transcription factor (TF) analysis revealed that 1588 TFs were identified and divided into 55 TF families, with 284 TFs down-regulated in the Dark relative to Light and 96 TFs up-regulated in the Light-MeJA-4h relative to Light. 8 TFs were selected as candidates for regulating the artemisinin biosynthesis and one of them was validated to be involved in artemisinin transcriptional regulation by Dual-Luciferase (Dual-LUC) assay. The transcriptome data shown in our study offered a comprehensive transcriptional expression pattern influenced by the MeJA and light in A. annua seedling, which will serve as a valuable resource for further studies on transcriptional regulation mechanisms underlying artemisinin biosynthesis.

  16. Transcriptome Analysis of Genes Associated with the Artemisinin Biosynthesis by Jasmonic Acid Treatment under the Light in Artemisia annua

    PubMed Central

    Hao, Xiaolong; Zhong, Yijun; Fu, Xueqing; Lv, Zongyou; Shen, Qian; Yan, Tingxiang; Shi, Pu; Ma, Yanan; Chen, Minghui; Lv, Xueying; Wu, Zhangkuanyu; Zhao, Jingya; Sun, Xiaofen; Li, Ling; Tang, Kexuan

    2017-01-01

    Artemisinin is a sesquiterpene lactone endoperoxide extracted from a traditional Chinese medicinal plant Artemisia annua. Artemisinin-based combination therapies (ACTs) are recommended as the best treatment of malaria by the World Health Organization (WHO). Both the phytohormone jasmonic acid (JA) and light promote artemisinin biosynthesis in A. annua. Interestingly, we found that the increase of artemisinin biosynthesis by JA was dependent on light. However, the relationship between the two signal pathways mediated by JA and light remains unclear. Here, we collected the A. annua seedlings of 24 h continuous light (Light), 24 h dark treatment (Dark), 4 h MeJA treatment under the continuous light conditions (Light-MeJA-4h) and 4 h MeJA treatment under the dark conditions (Dark-MeJA-4h) and performed the transcriptome sequencing using Illumina HiSeq 4000 System. A total of 266.7 million clean data were produced and assembled into 185,653 unigenes, with an average length of 537 bp. Among them, 59,490 unigenes were annotated and classified based on the public information. Differential expression analyses were performed between Light and Dark, Light and Light-MeJA-4h, Dark and Dark-MeJA-4h, Light-MeJA-4h, and Dark-MeJA-4h, respectively. Furthermore, transcription factor (TF) analysis revealed that 1588 TFs were identified and divided into 55 TF families, with 284 TFs down-regulated in the Dark relative to Light and 96 TFs up-regulated in the Light-MeJA-4h relative to Light. 8 TFs were selected as candidates for regulating the artemisinin biosynthesis and one of them was validated to be involved in artemisinin transcriptional regulation by Dual-Luciferase (Dual-LUC) assay. The transcriptome data shown in our study offered a comprehensive transcriptional expression pattern influenced by the MeJA and light in A. annua seedling, which will serve as a valuable resource for further studies on transcriptional regulation mechanisms underlying artemisinin biosynthesis. PMID

  17. Structure and Biosynthesis of Cuticular Lipids: Hydroxylation of Palmitic Acid and Decarboxylation of C(28), C(30), and C(32) Acids in Vicia faba Flowers.

    PubMed

    Kolattukudy, P E; Croteau, R; Brown, L

    1974-11-01

    ) alkane. Trichloroacetate inhibited the conversion of the exogenous acids into alkanes with carbon chains longer than the exogenous acid, and at the same time increased the amount of the direct decarboxylation product formed. These results clearly demonstrate direct decarboxylation as well as elongation and decarboxylation of exogenous fatty acids, and thus constitute the most direct evidence thus far obtained for an elongation-decarboxylation mechanism for the biosynthesis of alkanes.

  18. A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis.

    PubMed

    Makris, Thomas M; Chakrabarti, Mrinmoy; Münck, Eckard; Lipscomb, John D

    2010-08-31

    The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mössbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.

  19. Naturally occurring conjugated octadecatrienoic acids are strong inhibitors of prostaglandin biosynthesis.

    PubMed

    Nugteren, D H; Christ-Hazelhof, E

    1987-03-01

    Fatty acids from natural sources (mostly seed oils) were isolated and assayed for their effect on the bioconversion of arachidonic acid into prostaglandin E2, using sheep vesicular gland microsomes. Homologues and isomers of the naturally occurring fatty acids, obtained by chemical modification and/or organic synthetic methods, were also tested. Two very active cyclooxygenase inhibitors were discovered, namely jacarandic acid (8Z, 10E, 12Z-octadecatrienoic acid), isolated from Jacaranda mimosifolia, the concentration which gives 50% inhibition ([I]50) being 2.4 microM and the synthetic 8Z, 10E, 12E-octadecatrienoic acid, having an [I]50 of 1.0 microM. Under the conditions of the assay (75 microM substrate), earlier described potent inhibitors showed the following [I]50's: indomethacin: 1.3 microM; 9,12-octadecadiynoic acid: 1.3 microM, 8Z, 12E, 14Z-eicosatrienoic acid: 2.7 microM; 5,8,11,14-eicosatetraynoic acid: 4.4 microM. At a concentration of about half that of the substrate, the following naturally occurring fatty acids revealed inhibition ([I]50): columbinic acid (29 microM), calendulic acid (31 microM), liagoric acid (31 microM), ximenynic acid (39 microM), crepenynic acid (40 microM) and timnodonic acid (43 microM). Other fatty acids, and some of the above acids, were converted themselves more or less rapidly, mostly into conjugated monohydroxy fatty acids.

  20. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance.

    PubMed Central

    Kaneda, T

    1991-01-01

    Branched-chain fatty acids of the iso and anteiso series occur in many bacteria as the major acyl constituents of membrane lipids. In addition, omega-cyclohexyl and omega-cycloheptyl fatty acids are present in several bacterial species. These two types of fatty acids are synthesized by the repeated condensation of malonyl coenzyme A with one of the branched-chain and cyclic primers by the same enzyme system. The pathway of de novo branched-chain fatty acid synthesis differs only in initial steps of synthesis from that of the common straight-chain fatty acid (palmitic acid) present in most organisms. The cell membranes composed largely of iso-, anteiso-, and omega-alicyclic acids support growth of bacteria, which inhabit normal as well as extreme environments. The occurrence of these types of fatty acids as major cellular fatty acids is an important criterion used to aid identification and classification of bacteria. PMID:1886522

  1. crl mutants of Saccharomyces cerevisiae resemble both mutants affecting general control of amino acid biosynthesis and omnipotent translational suppressor mutants.

    PubMed

    McCusker, J H; Haber, J E

    1988-06-01

    Cyocloheximide resistant lethal (crl) mutants of Saccharomyces cerevisiae, defining 22 unlinked complementation groups, are unable to grow at 37 degrees. They are also highly pleiotropic at their permissive temperature of 25 degrees. The mutants are all unable to arrest at the G1 stage of the cell cycle when grown to stationary phase or when starved for a single amino acid, though they do arrest at G1 when deprived of all nitrogen. The crl mutants are also hypersensitive to various amino acid analogs and to 3-aminotriazole. These mutants also "tighten" leaky auxotrophic mutations that permit wild-type cells to grow in the absence of the appropriate amino acid. All of these phenotypes are also exhibited by gcn mutants affecting general control of amino acid biosynthesis. In addition, the crl mutants are all hypersensitive to hygromycin B, an aminoglycoside antibiotic that stimulates translational misreading. The crl mutations also suppress one nonsense mutation which is phenotypically suppressed by hygromycin B. Many crl mutants are also osmotically sensitive. These are phenotypes which the crl mutations have in common with previously isolated omnipotent suppressors. We suggest that the the crl mutations all affect the fidelity of protein translation.

  2. Function Analysis of Caffeoyl-CoA O-Methyltransferase for Biosynthesis of Lignin and Phenolic Acid in Salvia miltiorrhiza.

    PubMed

    Wang, Zhengjun; Ge, Qian; Chen, Chen; Jin, Xinxin; Cao, Xiaoyan; Wang, Zhezhi

    2017-02-01

    In this study, we cloned a full-length cDNA and the genomic DNA sequence of SmCCoAOMT (GenBank ID JQ007585) from Salvia miltiorrhiza. The 744-bp open-reading frame encodes a protein of 247 amino acids that shares 95 % similarity with one in Vitis vinifera. Real-time quantitative PCR analysis revealed that SmCCoAOMT is most highly expressed in the stems and can be induced by methyl jasmonate (MeJA) and XC-1 treatment. To evaluate its function in vivo, we generated RNA interference transgenic plants through Agrobacterium tumefaciens-mediated gene transfer. Compared with untransformed control plants, the transgenics had significantly less lignin and the expression of lignin-biosynthetic genes SmCCR and SmCOMT was depressed. In 90-day-old roots from plants of transgenic line M5, accumulations of rosmarinic acid and salvianolic acid B (Sal B) were greatly reduced by 0.89- and 0.69-fold, respectively. This low-Sal B phenotype was stable in the roots, with the level of accumulation being approximately 43.58 mg g(-1) dry weight, which was 52 % of the amount measured in the untransformed control. Our results suggest that SmCCoAOMT is involved in lignin biosynthesis and affects the accumulation of phenolic acids. This study also provides potential guidance for using lignin-related genes to genetically engineer Salvia miltiorrhiza.

  3. Characterization of a Citrus R2R3-MYB Transcription Factor that Regulates the Flavonol and Hydroxycinnamic Acid Biosynthesis

    PubMed Central

    Liu, Chaoyang; Long, Jianmei; Zhu, Kaijie; Liu, Linlin; Yang, Wei; Zhang, Hongyan; Li, Li; Xu, Qiang; Deng, Xiuxin

    2016-01-01

    Flavonols and hydroxycinnamic acids are important phenylpropanoid metabolites in plants. In this study, we isolated and characterized a citrus R2R3-MYB transcription factor CsMYBF1, encoding a protein belonging to the flavonol-specific MYB subgroup. Ectopic expression of CsMYBF1 in tomato led to an up-regulation of a series of genes involved in primary metabolism and the phenylpropanoid pathway, and induced a strong accumulation of hydroxycinnamic acid compounds but not the flavonols. The RNAi suppression of CsMYBF1 in citrus callus caused a down-regulation of many phenylpropanoid pathway genes and reduced the contents of hydroxycinnamic acids and flavonols. Transactivation assays indicated that CsMYBF1 activated several promoters of phenylpropanoid pathway genes in tomato and citrus. Interestingly, CsMYBF1 could activate the CHS gene promoter in citrus, but not in tomato. Further examinations revealed that the MYBPLANT cis-elements were essential for CsMYBF1 in activating phenylpropanoid pathway genes. In summary, our data indicated that CsMYBF1 possessed the function in controlling the flavonol and hydroxycinnamic acid biosynthesis, and the regulatory differences in the target metabolite accumulation between two species may be due to the differential activation of CHS promoters by CsMYBF1. Therefore, CsMYBF1 constitutes an important gene source for the engineering of specific phenylpropanoid components. PMID:27162196

  4. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    PubMed

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. A linoleic acid (8R)-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis. Biosynthesis of (8R)-hydroxylinoleic acid and (7S,8S)-dihydroxylinoleic acid from (8R)-hydroperoxylinoleic acid.

    PubMed

    Brodowsky, I D; Hamberg, M; Oliw, E H

    1992-07-25

    The fungus Gaeumannomyces graminis metabolized linoleic acid extensively to (8R)-hydroperoxylinoleic acid, (8R)-hydroxylinoleic acid, and threo-(7S,8S)-dihydroxylinoleic acid. When G. graminis was incubated with linoleic acid under an atmosphere of oxygen-18, the isotope was incorporated into (8R)-hydroxylinoleic acid and 7,8-dihydroxylinoleic acid. The two hydroxyls of the latter contained either two oxygen-18 or two oxygen-16 atoms, whereas a molecular species that contained both oxygen isotopes was formed in negligible amounts. Glutathione peroxidase inhibited the biosynthesis of 7,8-dihydroxylinoleic acid. These findings demonstrated that the diol was formed from (8R)-hydroperoxylinoleic acid by intramolecular hydroxylation at carbon 7, catalyzed by a hydroperoxide isomerase. The (8R)-dioxygenase appeared to metabolize substrates with a saturated carboxylic side chain and a 9Z-double bond. G. graminis also formed omega 2- and omega 3-hydroxy metabolites of the fatty acids. In addition, linoleic acid was converted to small amounts of nearly (65% R) racemic 10-hydroxy-8,12-octadecadienoic acid by incorporation of atmospheric oxygen. An unstable metabolite, 11-hydroxylinoleic acid, could also be isolated as well as (13R,13S)-hydroxy-(9E,9Z), (11E)-octadecadienoic acids and (9R,9S)-hydroxy-(10E), (12E,12Z)-octadecadienoic acids. In summary, G. graminis contains a prominent linoleic acid (8R)-dioxygenase, which differs from the lipoxygenase family of dioxygenases by catalyzing the formation of a hydroperoxide without affecting the double bonds of the substrate.

  6. DmSAS is required for sialic acid biosynthesis in cultured Drosophila third instar larvae CNS neurons.

    PubMed

    Granell, Annelise E von Bergen; Palter, Karen B; Akan, Ihan; Aich, Udayanath; Yarema, Kevin J; Betenbaugh, Michael J; Thornhill, William B; Recio-Pinto, Esperanza

    2011-11-18

    Sialylation is an important carbohydrate modification of glycoconjugates that has been shown to modulate many cellular/molecular interactions in vertebrates. In Drosophila melanogaster (Dm), using sequence homology, several enzymes of the sialylation pathway have been cloned and their function tested in expression systems. Here we investigated whether sialic acid incorporation in cultured Dm central nervous system (CNS) neurons required endogenously expressed Dm sialic acid synthase (DmSAS). We compared neurons derived from wild type Dm larvae with those containing a DmSAS mutation (148 bp deletion). The ability of these cells to produce Sia5NAz (sialic acid form) from Ac(4)ManNAz (azide-derivatized N-acetylmannosamine) and incorporate it into their glycoconjugates was measured by tagging the azide group of Sia5NAz with fluorescent agents via Click-iT chemistry. We found that most of the wild type Dm CNS neurons incorporated Sia5NAz into their glycoconjugates. Sialic acid incorporation was higher at the soma than at the neurite and could also be detected at perinuclear regions and the plasma membrane. In contrast, neurons from the DmSAS mutant did not incorporate Sia5NAz unless DmSAS was reintroduced (rescue mutant). Most of the neurons expressed α2,6-sialyltransferase. These results confirm that the mutation was a null mutation and that no redundant sialic acid biosynthetic activity exists in Dm cells, i.e., there is only one DmSAS. They also provide the strongest proof to date that DmSAS is a key enzyme in the biosynthesis of sialic acids in Dm CNS neurons, and the observed subcellular distribution of the newly synthesized sialic acids offers insights into their biological function.

  7. Cloning and Regulation of Cholesterol 7α-Hydroxylase, the Rate-limiting Enzyme in Bile Acid Biosynthesis*

    PubMed Central

    Jelinek, Diane F.; Andersson, Stefan; Slaughter, Clive A.; Russell, David W.

    2015-01-01

    The rate-limiting step in bile acid biosynthesis is catalyzed by the microsomal cytochrome P-450 cholesterol 7α-hydroxylase (7α-hydroxylase). The expression of this enzyme is subject to feedback regulation by sterols and is thought to be coordinately regulated with enzymes in the cholesterol supply pathways, including the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and synthase. Here we report the purification of rat 7α-hydroxylase and the determination of a partial amino acid sequence. Oligonucleotides derived from peptide sequence were used to clone a full-length cDNA encoding 7α-hydroxylase. DNA sequence analysis of the cDNA revealed a 7α-hydroxylase protein of 503 amino acids with a predicted molecular weight of 56,890 which represents a novel family of cytochrome P-450 enzymes. Transfection of a 7α-hydroxylase cDNA into simian COS cells resulted in the synthesis of a functional enzyme whose activity was stimulated in vitro by the addition of rat microsomal cytochrome P-450 reductase protein. RNA blot hybridization experiments indicated that the mRNA for 7α-hydroxylase is found only in the liver. The levels of this mRNA increased when bile acids were depleted by dietary cholestyramine and decreased when bile acids were consumed. Dietary cholesterol led to an increase in 7α-hydroxylase mRNA levels. The enzymatic activity of 7α-hydroxylase paralleled the observed changes in mRNA levels. These results suggest that bile acids and sterols are able to alter the transcription of the 7α-hydroxylase gene and that this control explains the previously observed feedback regulation of bile acid synthesis. PMID:2335522

  8. In Vivo Studies of the Biosynthesis of [alpha]-Eleostearic Acid in the Seed of Momordica charantia L.

    PubMed Central

    Liu, L.; Hammond, E. G.; Nikolau, B. J.

    1997-01-01

    In vivo radiotracer experiments using 14C-labeled acetate, oleate, linoleate, and linolenate were conducted to investigate the biosynthesis of [alpha]-eleostearic acid in the seeds of Momordica charantia. With the exception of [14C]linolenate, all of these precursors radioactively labeled [alpha]-eleostearate. Kinetics of the time course of metabolism of the radioactive precursors indicate that linoleate is the acyl precursor of [alpha]-eleostearate and that its conversion to [alpha]-eleostearate occurs while the acyl moiety is esterified to PC. Pulse-chase experiments with 14C-labeled acetate or linoleate provided additional corroborative evidence that linoleoyl PC is the precursor of [alpha]-eleostearoyl PC. PMID:12223677

  9. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid W

    SciTech Connect

    Messing, S.; Gabelli, S; Echeverria, I; Vogel, J; Guan, J; Tan, B; Klee, H; McCarty, D; Amzela, M

    2010-01-01

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  10. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid

    SciTech Connect

    Messing, Simon A.J.; Gabelli, Sandra B.; Echeverria, Ignacia; Vogel, Jonathan T.; Guan, Jiahn Chou; Tan, Bao Cai; Klee, Harry J.; McCarty, Donald R.; Amzel, L. Mario

    2011-09-06

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  11. Identification of microRNAs Actively Involved in Fatty Acid Biosynthesis in Developing Brassica napus Seeds Using High-Throughput Sequencing

    PubMed Central

    Wang, Jia; Jian, Hongju; Wang, Tengyue; Wei, Lijuan; Li, Jiana; Li, Chao; Liu, Liezhao

    2016-01-01

    Seed development has a critical role during the spermatophyte life cycle. In Brassica napus, a major oil crop, fatty acids are synthesized and stored in specific tissues during embryogenesis, and understanding the molecular mechanism underlying fatty acid biosynthesis during seed development is an important research goal. In this study, we constructed three small RNA libraries from early seeds at 14, 21, and 28 days after flowering (DAF) and used high-throughput sequencing to examine microRNA (miRNA) expression. A total of 85 known miRNAs from 30 families and 1160 novel miRNAs were identified, of which 24, including 5 known and 19 novel miRNAs, were found to be involved in fatty acid biosynthesis.bna-miR156b, bna-miR156c, bna-miR156g, novel_mir_1706, novel_mir_1407, novel_mir_173, and novel_mir_104 were significantly down-regulated at 21 DAF and 28 DAF, whereas bna-miR159, novel_mir_1081, novel_mir_19 and novel_mir_555 were significantly up-regulated. In addition, we found that some miRNAs regulate functional genes that are directly involved in fatty acid biosynthesis and that other miRNAs regulate the process of fatty acid biosynthesis by acting on a large number of transcription factors. The miRNAs and their corresponding predicted targets were partially validated by quantitative RT-PCR. Our data suggest that diverse and complex miRNAs are involved in the seed development process and that miRNAs play important roles in fatty acid biosynthesis during seed development. PMID:27822220

  12. Cloning and expression of a CYP720B orthologue involved in the biosynthesis of diterpene resin acids in Pinus brutia.

    PubMed

    Semiz, Asli; Sen, Alaattin

    2015-03-01

    Cytochrome P450 monooxygenases mediate a broad range of oxidative reactions involved in the biosynthesis of both primary and secondary metabolites in plants. Until now, only two P450 genes, CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, have been functionally characterised and described in the literature. The purpose of this study was to describe the cloning and expression of CYP720B from Pinus brutia due to its suggested role in the synthesis of bioactive compounds used for chemical defence against insects. A PCR product of the P. brutia CYP720B gene was cloned into the pCR8/GW/TOPO cloning vector. After optimising the sequence for codon usage in yeast, it was transferred into the inducible expression vector pYES-DEST52 and transfected into the S. cerevisiae INVSc1 strain. Sequence analysis showed that the P. brutia CYP720B gene contains an open reading frame of 1,464 nucleotides, which encodes a 53,570 Da putative protein of 487 amino acid residues. The putative protein contains the classic heme-binding sequence motif that is conserved in all P450 enzymes. It shares 99 and 61% identity with the deduced amino acid sequences of CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, respectively. Recombinant CYP720B protein expression was confirmed using western blot analysis. Furthermore, recombinant CYP720B was functionally active, showing a Soret peak at approximately 448 nm in the reduced CO difference spectra. These data suggest that the cloned gene is an orthologue of CYP720B in P. brutia and might be involved in DRA biosynthesis.

  13. Efficient biosynthesis of d-ribose using a novel co-feeding strategy in Bacillus subtilis without acid formation.

    PubMed

    Cheng, J; Zhuang, W; Li, N N; Tang, C L; Ying, H J

    2017-01-01

    Normally, low d-ribose production was identified as responsible for plenty of acid formation by Bacillus subtilis due to its carbon overflow. An approach of co-feeding glucose and sodium citrate is developed here and had been proved to be useful in d-ribose production. This strategy is critical because it affects the cell concentration, the productivity of d-ribose and, especially, the formation of by-products such as acetoin, lactate and acetate. d-ribose production was increased by 59·6% from 71·06 to 113·41 g l(-1) without acid formation by co-feeding 2·22 g l(-1)  h(-1) glucose and 0·036 g l(-1)  h(-1) sodium citrate to a 60 g l(-1) glucose reaction system. Actually, the cell density was also enhanced from 11·51 to 13·84 g l(-1) . These parameters revealed the importance of optimization and modelling of the d-ribose production process. Not only could zero acid formation was achieved over a wide range of co-feeding rate by reducing glycolytic flux drastically but also the cell density and d-ribose yield were elevated by increasing the hexose monophosphate pathway flux. Bacillus subtilis usually produce d-ribose accompanied by plenty of organic acids when glucose is used as a carbon source, which is considered to be a consequence of mismatched glycolytic and tricarboxylic acid cycle capacities. This is the first study to provide high-efficiency biosynthesis of d-ribose without organic acid formation in B. subtilis, which would be lower than the cost of separation and purification. The strain transketolase-deficient B. subtilis CGMCC 3720 can be potentially applied to the production of d-ribose in industry. © 2016 The Society for Applied Microbiology.

  14. Biosynthesis of tiglic, ethacrylic, and 2-methylbutyric acids in a carabid beetle, Pterostichus (Hypherpes) californicus.

    PubMed

    Attygalle, Athula B; Wu, Xiaogang; Will, Kipling W

    2007-05-01

    Tiglic, 2-methylbutyric, and ethacrylic acids are found in the pygidial gland defensive fluid of many carabid beetles. By injecting a deuterium-labeled precursor into the carabid beetle Pterostichus (Hypherpes) californicus, and analyzing the defensive fluid by gas chromatography/mass spectrometry, we were able to demonstrate that tiglic and ethacrylic acids are biosynthesized from isoleucine via 2-methylbutyric acid. Moreover, we observed that the injection of L-isoleucine induces an increased production of tiglic acid in P. californicus. A strong primary kinetic isotope effect was found to operate in the dehydrogenation step of 2-methylbutyric acid to tiglic and ethacrylic acids. Consequently, ethacrylic acid was found to preferentially accumulate the deuterium labeling from [2,3,4,4-(2)H(4)]isoleucine during our biosynthetic experiments.

  15. Solubilization and purification of the glucosyltransferase involved in the biosynthesis of teichuronic acid by fragments of Micrococcus luteus cell membranes

    SciTech Connect

    Hildebrandt, K.M.; Anderson, J.S.

    1987-05-01

    Enzymes involved in the biosynthesis of teichuronic acid have been demonstrated in cytoplasmic membrane fragments recovered from lysozyme treated Micrococcus luteus cells. Solubilization of the glucosyltransferase activity was effected with aqueous solutions of Triton X-100, Nonidet P-40, Tween 20, or Thesit. Thesit proved most amenable for recovery of glucosyltransferase activity as well as spectrophotometric protein determinations. Recovery of the glucosyltranferase activity was aided during purification by inclusion of 15% glycerol, 0.75% Thesit, 20 mM magnesium ion and 2 mM 2-mercaptoethanol in all buffers. Glucosyltransferase activity was monitored by the transfer of (/sup 14/C)glucose from UDP-(/sup 14/C)glucose to an artificial acceptor. Although the natural acceptor is presumed to be an undecaprenyl diphosphate-activated oligosaccharide, alternate acceptors such as isolated cell wall fractions containing teichuronic acid served equally well. Highly purified teichuronic acid devoid of peptidoglycan was the most effective alternate acceptor. The glucosyltransferase was purified by ammonium sulfate precipitation followed by ion exchange chromatography on DEAE-cellulose yielding an overall 200-fold increase in specific activity.

  16. Biosynthesis of poly(4-hydroxybutyrate) in recombinant Escherichia coli grown on glycerol is stimulated by propionic acid.

    PubMed

    Kämpf, Michael M; Thöny-Meyer, Linda; Ren, Qun

    2014-11-01

    One of the most promising polyhydroxyalkanoates (PHAs) for medical applications is poly(4-hydroxybutyrate) (P4HB) due to its biodegradability, biocompatibility and mechanical properties. Currently, the major hurdle for expanding P4HB applications is the production and recovery cost. In this study, we investigated the stimulating factors for P4HB biosynthesis with the ultimate goal of reducing production cost. We found that addition of propionic acid to the culture medium stimulates the P4HB accumulation in recombinant Escherichia coli JM109 grown on glycerol. This stimulating effect was significantly weakened by addition of exogenous methionine, whereas it was not influenced by addition of cysteine. These results suggest that propionic acid enhances P4HB synthesis by reducing the intracellular methionine pool. Utilizing these findings for P4HB production in batch cultures on glycerol, the volumetric yield of P4HB could be improved 4 fold from 0.9g/L to 3.7g/L by adding 2g/L propionic acid into the medium. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Phosphatidic acid: biosynthesis, pharmacokinetics, mechanisms of action and effect on strength and body composition in resistance-trained individuals.

    PubMed

    Bond, Peter

    2017-01-01

    The mechanistic target of rapamycin complex 1 (mTORC1) has received much attention in the field of exercise physiology as a master regulator of skeletal muscle hypertrophy. The multiprotein complex is regulated by various signals such as growth factors, energy status, amino acids and mechanical stimuli. Importantly, the glycerophospholipid phosphatidic acid (PA) appears to play an important role in mTORC1 activation by mechanical stimulation. PA has been shown to modulate mTOR activity by direct binding to its FKBP12-rapamycin binding domain. Additionally, it has been suggested that exogenous PA activates mTORC1 via extracellular conversion to lysophosphatidic acid and subsequent binding to endothelial differentiation gene receptors on the cell surface. Recent trials have therefore evaluated the effects of PA supplementation in resistance-trained individuals on strength and body composition. As research in this field is rapidly evolving, this review attempts to provide a comprehensive overview of its biosynthesis, pharmacokinetics, mechanisms of action and effect on strength and body composition in resistance-trained individuals.

  18. Tracer studies on the biosynthesis of amino acids from lactate by Peptostreptococcus elsdenii

    PubMed Central

    Somerville, H. J.; Peel, J. L.

    1967-01-01

    Peptostreptococcus elsdenii, a strict anaerobe from the rumen, was grown on a medium containing yeast extract and [1-14C]- or [2-14C]-lactate. Radioisotope from lactate was found in all cell fractions, but mainly in the protein. The label in the protein fraction was largely confined to a few amino acids: alanine, serine, aspartic acid, glutamic acid and diaminopimelic acid. The alanine, serine, aspartic acid and glutamic acid were separated, purified and degraded to establish the distribution of 14C from lactate within the amino acid molecules. The labelling patterns in alanine and serine suggested their formation from lactate without cleavage of the carbon chain. The pattern in aspartic acid suggested formation by condensation of a C3 unit derived directly from lactate with a C1 unit, probably carbon dioxide. The distribution in glutamic acid was consistent with two possible pathways of formation: (a) by the reactions of the tricarboxylic acid cycle leading from oxaloacetate to 2-oxoglutarate, followed by transamination; (b) by a pathway involving the reaction sequence 2 acetyl-CoA→crotonyl-CoA→glutaconate→glutamate. PMID:6069834

  19. Further Studies on Oxalic Acid Biosynthesis in Oxalate-accumulating Plants 1

    PubMed Central

    Nuss, Richard F.; Loewus, Frank A.

    1978-01-01

    l-Ascorbic acid functions as a precursor of oxalic acid in several oxalate-accumulating plants. The present study extends this observation to include Rumex crispus L. (curly dock), Amaranthus retroflexus L. (red root pigweed), Chenopodium album L. (lamb's-quarters), Beta vulgaris L. (sugar beet), Halogeton glomeratus M. Bieb. (halogeton), and Rheum rhabarbarum L. (rhubarb). Several species with low oxalate content are also examined. When l-[1-14C]ascorbic acid is supplied to young seedlings of R. crispus or H. glomeratus, a major portion of the 14C is released over a 24-hour period as 14CO2 and only a small portion is recovered as [14C]oxalate, unlike cuttings from 2- or 4-month-old plants which retain a large part of the 14C as [14C]oxalic acid and release very little 14CO2. Support for an intermediate role of oxalate in the release of 14CO2 from l-[1-14C]ascorbic acid is seen in the rapid release of 14CO2 by R. crispus and H. glomeratus seedlings labeled with [14C]oxalic acid. The common origin of oxalic acid carbon in the C1 and C2 fragment from l-ascorbic acid is demonstrated by comparison of 14C content of oxalic acid in several oxalate-accumulators after cuttings or seedlings are supplied equal amounts of l-[1-14C]- or l-[UL-14C]ascorbic acid. Theoretically, l-[1-14C]ascorbic acid will produce labeled oxalic acid containing three times as much 14C as l-[UL-14C]ascorbic acid when equal amounts of label are provided. Experimentally, a ratio of 2.7 ± 0.5 is obtained in duplicate experiments with six different species. PMID:16660342

  20. Further Studies on Oxalic Acid Biosynthesis in Oxalate-accumulating Plants.

    PubMed

    Nuss, R F; Loewus, F A

    1978-04-01

    l-Ascorbic acid functions as a precursor of oxalic acid in several oxalate-accumulating plants. The present study extends this observation to include Rumex crispus L. (curly dock), Amaranthus retroflexus L. (red root pigweed), Chenopodium album L. (lamb's-quarters), Beta vulgaris L. (sugar beet), Halogeton glomeratus M. Bieb. (halogeton), and Rheum rhabarbarum L. (rhubarb). Several species with low oxalate content are also examined.When l-[1-(14)C]ascorbic acid is supplied to young seedlings of R. crispus or H. glomeratus, a major portion of the (14)C is released over a 24-hour period as (14)CO(2) and only a small portion is recovered as [(14)C]oxalate, unlike cuttings from 2- or 4-month-old plants which retain a large part of the (14)C as [(14)C]oxalic acid and release very little (14)CO(2). Support for an intermediate role of oxalate in the release of (14)CO(2) from l-[1-(14)C]ascorbic acid is seen in the rapid release of (14)CO(2) by R. crispus and H. glomeratus seedlings labeled with [(14)C]oxalic acid.The common origin of oxalic acid carbon in the C1 and C2 fragment from l-ascorbic acid is demonstrated by comparison of (14)C content of oxalic acid in several oxalate-accumulators after cuttings or seedlings are supplied equal amounts of l-[1-(14)C]- or l-[UL-(14)C]ascorbic acid. Theoretically, l-[1-(14)C]ascorbic acid will produce labeled oxalic acid containing three times as much (14)C as l-[UL-(14)C]ascorbic acid when equal amounts of label are provided. Experimentally, a ratio of 2.7 +/- 0.5 is obtained in duplicate experiments with six different species.

  1. Fractionation of carbon isotopes in biosynthesis of fatty acids by a piezophilic bacterium Moritella japonica strain DSK1

    NASA Astrophysics Data System (ADS)

    Fang, Jiasong; Uhle, Maria; Billmark, Kaycie; Bartlett, Douglas H.; Kato, Chaki

    2006-04-01

    We examined stable carbon isotope fractionation in biosynthesis of fatty acids of a piezophilic bacterium Moritella japonica strain DSK1. The bacterium was grown to stationary phase at pressures of 0.1, 10, 20, and 50 MPa in media prepared using sterile-filtered natural seawater supplied with glucose as the sole carbon source. Strain DSK1 synthesized typical bacterial fatty acids (C 14-19 saturated, monounsaturated, and cyclopropane fatty acids) as well as long-chain polyunsaturated fatty acids (PUFA) (20:6 ω3). Bacterial cell biomass and individual fatty acids exhibited consistent pressure-dependent carbon isotope fractionations relative to glucose. The observed Δδ FA-glucose (-1.0‰ to -11.9‰) at 0.1 MPa was comparable to or slightly higher than fractionations reported in surface bacteria. However, bulk biomass and fatty acids became more depleted in 13C with pressure. Average carbon isotope fractionation (Δδ FA-glucose) at high pressures was much higher than that for surface bacteria: -15.7‰, -15.3‰, and -18.3‰ at 10, 20, and 50 MPa, respectively. PUFA were more 13C depleted than saturated and monounsaturated fatty acids at all pressures. The observed isotope effects may be ascribed to the kinetics of enzymatic reactions that are affected by hydrostatic pressure and to biosynthetic pathways that are different for short-chain and long-chain fatty acids. A simple quantitative calculation suggests that in situ piezophilic bacterial contribution of polyunsaturated fatty acids to marine sediments is nearly two orders of magnitude higher than that of marine phytoplankton and that the carbon isotope imprint of piezophilic bacteria can override that of surface phytoplankton. Our results have important implications for marine biogeochemistry. Depleted fatty acids reported in marine sediments and the water column may be derived simply from piezophilic bacteria resynthesis of organic matter, not from bacterial utilization of a 13C-depleted carbon source (i

  2. Salicylic Acid Induction of Flavonoid Biosynthesis Pathways in Wheat Varies by Treatment.

    PubMed

    Gondor, Orsolya K; Janda, Tibor; Soós, Vilmos; Pál, Magda; Majláth, Imre; Adak, Malay K; Balázs, Ervin; Szalai, Gabriella

    2016-01-01

    Salicylic acid is a promising compound for the reduction of stress sensitivity in plants. Although several biochemical and physiological changes have been described in plants treated with salicylic acid, the mode of action of the various treatments has not yet been clarified. The present work reports a detailed comparative study on the effects of different modes of salicylic acid application at the physiological, metabolomic, and transcriptomic levels. Seed soaking and hydroponic treatments were found to induce various changes in the protective mechanisms of wheat plants. The possible involvement of the flavonoid metabolism in salicylic acid-related stress signaling was also demonstrated. Different salicylic acid treatments were shown to induce different physiological and biochemical processes, with varying responses in the leaves and roots. Hydroponic treatment enhanced the level of oxidative stress, the expression of genes involved in the flavonoid metabolism and the amount of non-enzymatic antioxidant compounds, namely ortho-hydroxycinnamic acid and the flavonol quercetin in the leaves, while it decreased the ortho-hydroxycinnamic acid and flavonol contents and enhanced ascorbate peroxidase activity in the roots. In contrast, seed soaking only elevated the gene expression level of phenylalanine ammonia lyase in the roots and caused a slight increase in the amount of flavonols. These results draw attention to the fact that the effects of exogenous salicylic acid application cannot be generalized in different experimental systems and that the flavonoid metabolism may be an important part of the action mechanisms induced by salicylic acid.

  3. Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

    PubMed Central

    Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

    2015-01-01

    The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

  4. Salicylic Acid Induction of Flavonoid Biosynthesis Pathways in Wheat Varies by Treatment

    PubMed Central

    Gondor, Orsolya K.; Janda, Tibor; Soós, Vilmos; Pál, Magda; Majláth, Imre; Adak, Malay K.; Balázs, Ervin; Szalai, Gabriella

    2016-01-01

    Salicylic acid is a promising compound for the reduction of stress sensitivity in plants. Although several biochemical and physiological changes have been described in plants treated with salicylic acid, the mode of action of the various treatments has not yet been clarified. The present work reports a detailed comparative study on the effects of different modes of salicylic acid application at the physiological, metabolomic, and transcriptomic levels. Seed soaking and hydroponic treatments were found to induce various changes in the protective mechanisms of wheat plants. The possible involvement of the flavonoid metabolism in salicylic acid-related stress signaling was also demonstrated. Different salicylic acid treatments were shown to induce different physiological and biochemical processes, with varying responses in the leaves and roots. Hydroponic treatment enhanced the level of oxidative stress, the expression of genes involved in the flavonoid metabolism and the amount of non-enzymatic antioxidant compounds, namely ortho-hydroxycinnamic acid and the flavonol quercetin in the leaves, while it decreased the ortho-hydroxycinnamic acid and flavonol contents and enhanced ascorbate peroxidase activity in the roots. In contrast, seed soaking only elevated the gene expression level of phenylalanine ammonia lyase in the roots and caused a slight increase in the amount of flavonols. These results draw attention to the fact that the effects of exogenous salicylic acid application cannot be generalized in different experimental systems and that the flavonoid metabolism may be an important part of the action mechanisms induced by salicylic acid. PMID:27733857

  5. Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

    PubMed Central

    Winter, E; Brummel, M; Schuch, R; Spener, F

    1997-01-01

    In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

  6. Dietary enhancement of selected fatty acid biosynthesis in the digestive gland of Mytilus galloprovincialis Lmk.

    PubMed

    Ventrella, Vittoria; Pagliarani, Alessandra; Nesci, Salvatore; Trombetti, Fabiana; Pirini, Maurizio

    2013-01-30

    The fatty acid composition of the digestive gland from the mussel Mytilus galloprovincialis subjected to three different dietary regimens for 30 days was analyzed. Samples were collected at the beginning and end of the trial to obtain a comprehensive picture of fatty acid dynamics. Group A was unfed; group B received a diet consisting of 100% Thalassiosira weissflogii and, thus, similar to natural food; and group C received a diet consisting of 100% wheat germ conferring a 18:2ω-6 abundance. Results indicate that fatty acid composition of lipid and phospholipid classes was affected by dietary treatments. However, adult mussel homeostatic skills minimized effects, and thus, only wheat germ diet deeply modified the fatty acid composition. Furthermore, in group C, the occurrence of the non-methylene-interrupted trienoic fatty acids was indicative of de novo fatty acid synthesis presumably because of active fatty acid elongation and Δ5 desaturation system, also supported by the general ω-3 polyunsaturated fatty acid decrease.

  7. Transcriptional regulation of chlorogenic acid biosynthesis in carrot root slices exposed to UV-B light

    USDA-ARS?s Scientific Manuscript database

    Orange carrots are well known for their nutritional value as producers of ß-carotene, a Vitamin A precursor. Lesser known, is their ability to accumulate antioxidants such as chlorogenic acid. Chlorogenic acid is produced through the same biosynthetic pathway that produces lignins, anthocyanins, f...

  8. Biosynthetic mechanism for L-Gulose in main polar lipids of Thermoplasma acidophilum and possible resemblance to plant ascorbic acid biosynthesis.

    PubMed

    Yamauchi, Noriaki; Nakayama, Yusuke

    2013-01-01

    L-Gulose is a very rare sugar, but appears as a sugar component of the main polar lipids characteristic in such a thermophilic archaeon as Thermoplasma acidophilum that lives without cell walls in a highly acidic environment. The biosynthesis of L-gulose in this thermophilic organism was investigated with deuterium-labeling experiments. L-Gulose was found to be biosynthesized from D-glucose via stepwise stereochemical inversion at C-2 and C-5. The involvement of an epimerase related to GDP-mannose 3,5-epimerase, the key enzyme of plant ascorbate biosynthesis, was also suggested in this C-5 inversion. The resemblance of L-gulose biosynthesis in archaea and plants might be suggested from these results.

  9. Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

    PubMed Central

    Serrano-Carreon, L.; Hathout, Y.; Bensoussan, M.; Belin, J.-M.

    1993-01-01

    The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-14C]linoleic acid or [5-14C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [14C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [14C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-14C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species. PMID:16349040

  10. Metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. II. Biosynthesis of higher branched-chain fatty acids and aldehydes.

    PubMed

    ALLISON, M J; BRYANT, M P; KATZ, I; KEENEY, M

    1962-05-01

    Allison, M. J. (Dairy Cattle Research Branch, USDA, Beltsville, Md.), M. P. Bryant, I. Katz, and M. Keeney. Metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. II. Biosynthesis of higher branched-chain fatty acids and aldehydes. J. Bacteriol. 83:1084-1093. 1962.-A number of strains of rumen bacteria require branched-chain volatile fatty acids for growth. A strain of Ruminococcus flavefaciens that requires either isovalerate or isobutyrate incorporates radioactive carbon from isovalerate-1-C(14) and isovalerate-3-C(14) into leucine and into the lipid fraction of the cells. Evidence obtained by both paper and gas chromatography indicated that most of the label in the lipid of cells grown in isovalerate-1-C(14) was in a branched-chain 15-carbon fatty acid, with some in a 17-carbon acid; about 7.5% of the C(14) was recovered in a branched-chain 15-carbon aldehyde. The aldehydes were in the phospholipid fraction and were presumably present as plasmalogen.A strain of R. albus was shown to require isobutyrate, 2-methyl-n-butyrate, or 2-ketoisovalerate for growth. This strain did not incorporate appreciable C(14) from isovalerate-1-C(14) or isovalerate-3-C(14). When grown in a medium containing isobutyrate-1-C(14), most of the cellular C(14) was found in the lipid fraction. Analysis of the lipid demonstrated that the label was present mainly as branched-chain 14-carbon and 16-carbon fatty acids, with 11% of the C(14) present in 14- and 16-carbon carbonyl compounds, presumably branched-chain aldehydes.Branched-chain 14-, 15-, and 16-carbon fatty acids are major components of the lipids of these rumen bacteria. The possibility that these acids and aldehydes, which are found in ruminant body and milk lipids, may be of microbial origin is discussed.

  11. Biosynthesis of Polyunsaturated Fatty Acids in Octopus vulgaris: Molecular Cloning and Functional Characterisation of a Stearoyl-CoA Desaturase and an Elongation of Very Long-Chain Fatty Acid 4 Protein.

    PubMed

    Monroig, Óscar; de Llanos, Rosa; Varó, Inmaculada; Hontoria, Francisco; Tocher, Douglas R; Puig, Sergi; Navarro, Juan C

    2017-03-21

    Polyunsaturated fatty acids (PUFAs) have been acknowledged as essential nutrients for cephalopods but the specific PUFAs that satisfy the physiological requirements are unknown. To expand our previous investigations on characterisation of desaturases and elongases involved in the biosynthesis of PUFAs and hence determine the dietary PUFA requirements in cephalopods, this study aimed to investigate the roles that a stearoyl-CoA desaturase (Scd) and an elongation of very long-chain fatty acid 4 (Elovl4) protein play in the biosynthesis of essential fatty acids (FAs). Our results confirmed the Octopus vulgaris Scd is a ∆9 desaturase with relatively high affinity towards saturated FAs with ≥ C18 chain lengths. Scd was unable to desaturate 20:1n-15 ((∆5)20:1) suggesting that its role in the biosynthesis of non-methylene interrupted FAs (NMI FAs) is limited to the introduction of the first unsaturation at ∆9 position. Interestingly, the previously characterised ∆5 fatty acyl desaturase was indeed able to convert 20:1n-9 ((∆11)20:1) to (∆5,11)20:2, an NMI FA previously detected in octopus nephridium. Additionally, Elovl4 was able to mediate the production of 24:5n-3 and thus can contribute to docosahexaenoic acid (DHA) biosynthesis through the Sprecher pathway. Moreover, the octopus Elovl4 was confirmed to play a key role in the biosynthesis of very long-chain (>C24) PUFAs.

  12. Biosynthesis of Polyunsaturated Fatty Acids in Octopus vulgaris: Molecular Cloning and Functional Characterisation of a Stearoyl-CoA Desaturase and an Elongation of Very Long-Chain Fatty Acid 4 Protein

    PubMed Central

    Monroig, Óscar; de Llanos, Rosa; Varó, Inmaculada; Hontoria, Francisco; Tocher, Douglas R.; Puig, Sergi; Navarro, Juan C.

    2017-01-01

    Polyunsaturated fatty acids (PUFAs) have been acknowledged as essential nutrients for cephalopods but the specific PUFAs that satisfy the physiological requirements are unknown. To expand our previous investigations on characterisation of desaturases and elongases involved in the biosynthesis of PUFAs and hence determine the dietary PUFA requirements in cephalopods, this study aimed to investigate the roles that a stearoyl-CoA desaturase (Scd) and an elongation of very long-chain fatty acid 4 (Elovl4) protein play in the biosynthesis of essential fatty acids (FAs). Our results confirmed the Octopus vulgaris Scd is a ∆9 desaturase with relatively high affinity towards saturated FAs with ≥ C18 chain lengths. Scd was unable to desaturate 20:1n-15 (∆520:1) suggesting that its role in the biosynthesis of non-methylene interrupted FAs (NMI FAs) is limited to the introduction of the first unsaturation at ∆9 position. Interestingly, the previously characterised ∆5 fatty acyl desaturase was indeed able to convert 20:1n-9 (∆1120:1) to ∆5,1120:2, an NMI FA previously detected in octopus nephridium. Additionally, Elovl4 was able to mediate the production of 24:5n-3 and thus can contribute to docosahexaenoic acid (DHA) biosynthesis through the Sprecher pathway. Moreover, the octopus Elovl4 was confirmed to play a key role in the biosynthesis of very long-chain (>C24) PUFAs. PMID:28335553

  13. Biosynthesis of platform chemical 3-hydroxypropionic acid (3-HP) directly from CO2 in cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Wang, Yunpeng; Sun, Tao; Gao, Xingyan; Shi, Mengliang; Wu, Lina; Chen, Lei; Zhang, Weiwen

    2016-03-01

    3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. So far large-scale production of 3-HP has been mainly through petroleum-based chemical processes, whose sustainability and environmental issues have attracted widespread attention. With the ability to fix CO2 directly, cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals. In this study, we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp. PCC 6803, and then optimized the system through the following approaches: i) increasing expression of malonyl-CoA reductase (MCR) gene using different promoters and cultivation conditions; ii) enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-CoA carboxylase and biotinilase; iii) improving NADPH supply by overexpressing the NAD(P) transhydrogenase gene; iv) directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis. Together, the efforts led to a production of 837.18 mg L(-1) (348.8 mg/g dry cell weight) 3-HP directly from CO2 in Synechocystis after 6 days cultivation, demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO2 in cyanobacteria. In addition, the results showed that overexpression of the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) gene from Anabaena sp. PCC 7120 and Synechococcus sp. PCC 7942 led to no increase of 3-HP production, suggesting CO2 fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.

  14. Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

    PubMed Central

    Mafu, Sibongile; Karunanithi, Prema Sambandaswami; Palazzo, Teresa Ann; Harrod, Bronwyn Lee; Rodriguez, Selina Marakana; Mollhoff, Iris Natalie; O’Brien, Terrence Edward; Tong, Shen; Fiehn, Oliver; Tantillo, Dean J.; Bohlmann, Jörg; Zerbe, Philipp

    2017-01-01

    The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch (Pseudolarix amabilis, Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined “pseudolaratriene.” Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P. amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono- and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7-trans-fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene. PMID:28096378

  15. Transcription of the Streptococcus pyogenes hyaluronic acid capsule biosynthesis operon is regulated by previously unknown upstream elements.

    PubMed

    Falaleeva, Marina; Zurek, Oliwia W; Watkins, Robert L; Reed, Robert W; Ali, Hadeel; Sumby, Paul; Voyich, Jovanka M; Korotkova, Natalia

    2014-12-01

    The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.

  16. Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation

    PubMed Central

    Min, Bokkee; Pelaschier, Joanne T.; Graham, David E.; Tumbula-Hansen, Debra; Söll, Dieter

    2002-01-01

    Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway [Becker, H. D. & Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837; and Curnow, A. W., Tumbula, D. L., Pelaschier, J. T., Min, B. & Söll, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12838–12843]. A genetic knockout disrupting this pathway deprives D. radiodurans of the ability to synthesize asparagine and confers asparagine auxotrophy. The organism's capacity to make asparagine could be restored by transformation with Escherichia coli asnB. This result demonstrates that in Deinococcus, the only route to asparagine is via asparaginyl-tRNA. Analysis of the completed genomes of many bacteria reveal that, barring the existence of an unknown pathway of asparagine biosynthesis, a wide spectrum of bacteria rely on the tRNA-dependent transamidation pathway as the sole route to asparagine. PMID:11880622

  17. Characterization of two key enzymes for aromatic amino acid biosynthesis in symbiotic archaea.

    PubMed

    Shlaifer, Irina; Turnbull, Joanne L

    2016-07-01

    Biosynthesis of L-tyrosine (L-Tyr) and L-phenylalanine (L-Phe) is directed by the interplay of three enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which can be either converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD) or to phenylpyruvate by prephenate dehydratase (PDT). This work reports the first characterization of a trifunctional PD-CM-PDT from the smallest hyperthermophilic archaeon Nanoarchaeum equitans and a bifunctional CM-PD from its host, the crenarchaeon Ignicoccus hospitalis. Hexa-histidine tagged proteins were expressed in Escherichia coli and purified by affinity chromatography. Specific activities determined for the trifunctional enzyme were 21, 80, and 30 U/mg for CM, PD, and PDT, respectively, and 47 and 21 U/mg for bifunctional CM and PD, respectively. Unlike most PDs, these two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP(+) to NAD(+) as a cofactor. Both the enzymes were highly thermally stable and exhibited maximal activity at 90 °C. N. equitans PDT was feedback inhibited by L-Phe (Ki = 0.8 µM) in a non-competitive fashion consistent with L-Phe's combination at a site separate from that of prephenate. Our results suggest that PD from the unique symbiotic archaeal pair encompass a distinct subfamily of prephenate dehydrogenases with regard to their regulation and co-substrate specificity.

  18. New Approaches to Target the Mycolic Acid Biosynthesis Pathway for the Development of Tuberculosis Therapeutics

    PubMed Central

    North, E. Jeffrey; Jackson, Mary; Lee, Richard E.

    2015-01-01

    Mycolic acids are the major lipid component of the unique mycobacterial cell wall responsible for the protection of the tuberculosis bacilli from many outside threats. Mycolic acids are synthesized in the cytoplasm and transported to the outer membrane as trehalose-containing glycolipids before being esterified to the arabinogalactan portion of the cell wall and outer membrane glycolipids. The large size of these unique fatty acids is a result of a huge metabolic investment that has been evolutionarily conserved, indicating the importance of these lipids to the mycobacterial cellular survival. There are many key enzymes involved in the mycolic acid biosynthetic pathway, including fatty acid synthesis (KasA, KasB, MabA, InhA, HadABC), mycolic acid modifying enzymes (SAM-dependent methyltransferases, aNAT), fatty acid activating and condensing enzymes (FadD32, Acc, Pks13), transporters (MmpL3) and tranferases (Antigen 85A-C) all of which are excellent potential drug targets. Not surprisingly, in recent years many new compounds have been reported to inhibit specific portions of this pathway, discovered through both phenotypic screening and target enzyme screening. In this review, we analyze the new and emerging inhibitors of this pathway discovered in the post-genomic era of tuberculosis drug discovery, several of which show great promise as selective tuberculosis therapeutics. PMID:24245756

  19. Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium, Oscillatoria limnetica

    NASA Technical Reports Server (NTRS)

    Jahnke, L. L.; Lee, B.; Sweeney, M. J.; Klein, H. P.

    1989-01-01

    The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C16:1) of aerobically grown O. limnetica was shown to contain both the delta 7 (79%) and delta 9 (21%) isomers, while the octadecenoic (C18:1) acid was entirely the delta 9 acid. Incorporation of [2-14C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the delta 7 and delta 9 C16:1 and the delta 9 C18:1. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both delta 7 and delta 9 C16:1 and delta 9 and delta 11 C18:1. The synthesis of these is characteristic of a bacterial-type, anaerobic pathway.

  20. Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium, Oscillatoria limnetica

    NASA Technical Reports Server (NTRS)

    Jahnke, L. L.; Lee, B.; Sweeney, M. J.; Klein, H. P.

    1989-01-01

    The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C16:1) of aerobically grown O. limnetica was shown to contain both the delta 7 (79%) and delta 9 (21%) isomers, while the octadecenoic (C18:1) acid was entirely the delta 9 acid. Incorporation of [2-14C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the delta 7 and delta 9 C16:1 and the delta 9 C18:1. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both delta 7 and delta 9 C16:1 and delta 9 and delta 11 C18:1. The synthesis of these is characteristic of a bacterial-type, anaerobic pathway.

  1. Pseudomonas aeruginosa Directly Shunts β-Oxidation Degradation Intermediates into De Novo Fatty Acid Biosynthesis

    PubMed Central

    Yuan, Yanqiu; Leeds, Jennifer A.

    2012-01-01

    We identified the fatty acid synthesis (FAS) initiation enzyme in Pseudomonas aeruginosa as FabY, a β-ketoacyl synthase KASI/II domain-containing enzyme that condenses acetyl coenzyme A (acetyl-CoA) with malonyl-acyl carrier protein (ACP) to make the FAS primer β-acetoacetyl-ACP in the accompanying article (Y. Yuan, M. Sachdeva, J. A. Leeds, and T. C. Meredith, J. Bacteriol. 194:5171-5184, 2012). Herein, we show that growth defects stemming from deletion of fabY can be suppressed by supplementation of the growth media with exogenous decanoate fatty acid, suggesting a compensatory mechanism. Fatty acids eight carbons or longer rescue growth by generating acyl coenzyme A (acyl-CoA) thioester β-oxidation degradation intermediates that are shunted into FAS downstream of FabY. Using a set of perdeuterated fatty acid feeding experiments, we show that the open reading frame PA3286 in P. aeruginosa PAO1 intercepts C8-CoA by condensation with malonyl-ACP to make the FAS intermediate β-keto decanoyl-ACP. This key intermediate can then be extended to supply all of the cellular fatty acid needs, including both unsaturated and saturated fatty acids, along with the 3-hydroxyl fatty acid acyl groups of lipopolysaccharide. Heterologous PA3286 expression in Escherichia coli likewise established the fatty acid shunt, and characterization of recombinant β-keto acyl synthase enzyme activity confirmed in vitro substrate specificity for medium-chain-length acyl CoA thioester acceptors. The potential for the PA3286 shunt in P. aeruginosa to curtail the efficacy of inhibitors targeting FabY, an enzyme required for FAS initiation in the absence of exogenous fatty acids, is discussed. PMID:22753057

  2. Biosynthesis of 5-aminopentanoic acid and 2-piperidone from cadaverine and 1-piperideine in mouse.

    PubMed

    Callery, P S; Geelhaar, L A

    1984-12-01

    1-Piperideine, 5-aminopentanoic acid, and its lactam, 2-piperidone, were identified as metabolites of cadaverine in 10,000 g mouse liver supernatants to which diamine oxidase had been added. Both metabolites were also found when the cadaverine metabolite 1-piperideine was incubated with the preparation which suggested that 1-piperideine is an intermediate in the formation of 5-aminopentanoic acid and 2-piperidone. Identification of the metabolites was based on gas chromatography-mass spectrometric analysis in comparison to authentic standards. Mouse brain homogenates converted 1-piperideine to 5-aminopentanoic acid. The results suggest that the metabolic fate of cadaverine may provide precursors of pharmacologically active analogues of GABA.

  3. Transcriptional profiling of genes involved in ascorbic acid biosynthesis, recycling, and degradation during three leaf developmental stages in celery.

    PubMed

    Huang, Wei; Wang, Guang-Long; Li, Hui; Wang, Feng; Xu, Zhi-Sheng; Xiong, Ai-Sheng

    2016-12-01

    Ascorbic acid (AsA) is an important nutrient in the human body and performs various healthy functions. With considerable medicinal properties, celery (Apium graveolens L.) could be a good source of AsA for human health. However, the biosynthetic, recycling, and degradation pathways of AsA in celery have yet to be characterized. To study the metabolic pathways involved in AsA, the genes involved in AsA biosynthesis, recycling, and degradation were isolated from celery, and their expression profiles and AsA levels were analyzed in the leaf blades and petioles of two celery varieties at three different growth stages. AsA levels were higher in 'Ventura' compared with 'Liuhehuangxinqin' in both tissues possibly because of different transcription levels of genes, such as L-galactose dehydrogenase (GalDH), L-galactono-1,4-lactone dehydrogenase (GalLDH), and glutathione reductase (GR). Results revealed that the D-mannose/L-galactose pathway may be the predominant pathway in celery, and the D-galacturonic acid pathway appeared to contribute largely to AsA accumulation in petioles than in leaf blades in 'Liuhehuangxinqin.' AsA contents are regulated by complex regulatory mechanisms and vary at different growth stages, tissues, and varieties in celery. The results provide novel insights into AsA metabolic pathways in leaf during celery growth and development.

  4. Long-Term Submergence-Induced Elongation in Rumex palustris Requires Abscisic Acid-Dependent Biosynthesis of Gibberellin11

    PubMed Central

    Benschop, Joris J.; Bou, Jordi; Peeters, Anton J.M.; Wagemaker, Niels; Gühl, Kerstin; Ward, Dennis; Hedden, Peter; Moritz, Thomas; Voesenek, Laurentius A.C.J.

    2006-01-01

    Rumex palustris (polygonceae) responds to complete submergence with enhanced elongation of its youngest petioles. This process requires the presence of gibberellin (GA) and is associated with an increase in the concentration of GA1 in elongating petioles. We have examined how GA biosynthesis was regulated in submerged plants. Therefore, cDNAs encoding GA-biosynthetic enzymes GA 20-oxidase and GA 3-oxidase, and the GA-deactivating enzyme GA 2-oxidase were cloned from R. palustris and the kinetics of transcription of the corresponding genes was determined during a 24 h submergence period. The submergence-induced elongation response could be separated into several phases: (1) during the first phase of 4 h, petiole elongation was insensitive to GA; (2) from 4 to 6 h onward growth was limited by GA; and (3) from 15 h onward underwater elongation was dependent, but not limited by GA. Submergence induced an increase of GA1 concentration, as well as enhanced transcript levels of RpGA3ox1. Exogenous abscisic acid repressed the transcript levels of RpGA20ox1 and RpGA3ox1 and thus inhibited the submergence-induced increase in GA1. Abscisic acid had no effect on the tissue responsiveness to GA. PMID:16766669

  5. A Structural Basis for the Biosynthesis of the Major Chlorogenic Acids Found in Coffee1[W][OA

    PubMed Central

    Lallemand, Laura A.; Zubieta, Chloe; Lee, Soon Goo; Wang, Yechun; Acajjaoui, Samira; Timmins, Joanna; McSweeney, Sean; Jez, Joseph M.; McCarthy, James G.; McCarthy, Andrew A.

    2012-01-01

    Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds. PMID:22822210

  6. Type II fatty acid biosynthesis is essential for Plasmodium falciparum sporozoite development in the midgut of Anopheles mosquitoes.

    PubMed

    van Schaijk, Ben C L; Kumar, T R Santha; Vos, Martijn W; Richman, Adam; van Gemert, Geert-Jan; Li, Tao; Eappen, Abraham G; Williamson, Kim C; Morahan, Belinda J; Fishbaugher, Matt; Kennedy, Mark; Camargo, Nelly; Khan, Shahid M; Janse, Chris J; Sim, Kim Lee; Hoffman, Stephen L; Kappe, Stefan H I; Sauerwein, Robert W; Fidock, David A; Vaughan, Ashley M

    2014-05-01

    The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.

  7. Ascorbic Acid Biosynthesis and Brackish Water Acclimation in the Euryhaline Freshwater White-Rimmed Stingray, Himantura signifer.

    PubMed

    Wong, Samuel Z H; Ching, Biyun; Chng, You R; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2013-01-01

    L-gulono-γ-lactone oxidase (Gulo) catalyzes the last step of ascorbic acid biosynthesis, which occurs in the kidney of elasmobranchs. This study aimed to clone and sequence gulonolactone oxidase (gulo) from the kidney of the euryhaline freshwater stingray, Himantura signifer, and to determine the effects of acclimation from freshwater to brackish water (salinity 20) on its renal gulo mRNA expression and Gulo activity. We also examined the effects of brackish water acclimation on concentrations of ascorbate, dehydroascorbate and ascorbate + dehydroascorbate in the kidney, brain and gill. The complete cDNA coding sequence of gulo from the kidney of H. signifer contained 1323 bp coding for 440 amino acids. The expression of gulo was kidney-specific, and renal gulo expression decreased significantly by 67% and 50% in fish acclimated to brackish water for 1 day and 6 days, respectively. There was also a significant decrease in renal Gulo activity after 6 days of acclimation to brackish water. Hence, brackish water acclimation led to a decrease in the ascorbic acid synthetic capacity in the kidney of H. signifer. However, there were significant increases in concentrations of ascorbate and ascorbate + dehydroascorbate in the gills (after 1 or 6 days), and a significant increase in the concentration of ascorbate and a significant decrease in the concentration of dehydroascorbate in the brain (after 1 day) of fish acclimated to brackish water. Taken together, our results indicate that H. signifer might experience greater salinity-induced oxidative stress in freshwater than in brackish water, possibly related to its short history of freshwater invasion. These results also suggest for the first time a possible relationship between the successful invasion of the freshwater environment by some euryhaline marine elasmobranchs and the ability of these elasmobranchs to increase the capacity of ascorbic acid synthesis in response to hyposalinity stress.

  8. Ascorbic Acid Biosynthesis and Brackish Water Acclimation in the Euryhaline Freshwater White-Rimmed Stingray, Himantura signifer

    PubMed Central

    Wong, Samuel Z. H.; Ching, Biyun; Chng, You R.; Wong, Wai P.; Chew, Shit F.; Ip, Yuen K.

    2013-01-01

    L-gulono-γ-lactone oxidase (Gulo) catalyzes the last step of ascorbic acid biosynthesis, which occurs in the kidney of elasmobranchs. This study aimed to clone and sequence gulonolactone oxidase (gulo) from the kidney of the euryhaline freshwater stingray, Himantura signifer, and to determine the effects of acclimation from freshwater to brackish water (salinity 20) on its renal gulo mRNA expression and Gulo activity. We also examined the effects of brackish water acclimation on concentrations of ascorbate, dehydroascorbate and ascorbate + dehydroascorbate in the kidney, brain and gill. The complete cDNA coding sequence of gulo from the kidney of H. signifer contained 1323 bp coding for 440 amino acids. The expression of gulo was kidney-specific, and renal gulo expression decreased significantly by 67% and 50% in fish acclimated to brackish water for 1 day and 6 days, respectively. There was also a significant decrease in renal Gulo activity after 6 days of acclimation to brackish water. Hence, brackish water acclimation led to a decrease in the ascorbic acid synthetic capacity in the kidney of H. signifer. However, there were significant increases in concentrations of ascorbate and ascorbate + dehydroascorbate in the gills (after 1 or 6 days), and a significant increase in the concentration of ascorbate and a significant decrease in the concentration of dehydroascorbate in the brain (after 1 day) of fish acclimated to brackish water. Taken together, our results indicate that H. signifer might experience greater salinity-induced oxidative stress in freshwater than in brackish water, possibly related to its short history of freshwater invasion. These results also suggest for the first time a possible relationship between the successful invasion of the freshwater environment by some euryhaline marine elasmobranchs and the ability of these elasmobranchs to increase the capacity of ascorbic acid synthesis in response to hyposalinity stress. PMID:23825042

  9. Genetic Adaptation of Fatty-Acid Metabolism: A Human-Specific Haplotype Increasing the Biosynthesis of Long-Chain Omega-3 and Omega-6 Fatty Acids

    PubMed Central

    Ameur, Adam; Enroth, Stefan; Johansson, Åsa; Zaboli, Ghazal; Igl, Wilmar; Johansson, Anna C.V.; Rivas, Manuel A.; Daly, Mark J.; Schmitz, Gerd; Hicks, Andrew A.; Meitinger, Thomas; Feuk, Lars; van Duijn, Cornelia; Oostra, Ben; Pramstaller, Peter P.; Rudan, Igor; Wright, Alan F.; Wilson, James F.; Campbell, Harry; Gyllensten, Ulf

    2012-01-01

    Omega-3 and omega-6 long-chain polyunsaturated fatty acids (LC-PUFAs) are essential for the development and function of the human brain. They can be obtained directly from food, e.g., fish, or synthesized from precursor molecules found in vegetable oils. To determine the importance of genetic variability to fatty-acid biosynthesis, we studied FADS1 and FADS2, which encode rate-limiting enzymes for fatty-acid conversion. We performed genome-wide genotyping (n = 5,652 individuals) and targeted resequencing (n = 960 individuals) of the FADS region in five European population cohorts. We also analyzed available genomic data from human populations, archaic hominins, and more distant primates. Our results show that present-day humans have two common FADS haplotypes—defined by 28 closely linked SNPs across 38.9 kb—that differ dramatically in their ability to generate LC-PUFAs. No independent effects on FADS activity were seen for rare SNPs detected by targeted resequencing. The more efficient, evolutionarily derived haplotype appeared after the lineage split leading to modern humans and Neanderthals and shows evidence of positive selection. This human-specific haplotype increases the efficiency of synthesizing essential long-chain fatty acids from precursors and thereby might have provided an advantage in environments with limited access to dietary LC-PUFAs. In the modern world, this haplotype has been associated with lifestyle-related diseases, such as coronary artery disease. PMID:22503634

  10. Biosynthesis of gallic acid in Rhus typhina: discrimination between alternative pathways from natural oxygen isotope abundance.

    PubMed

    Werner, Roland A; Rossmann, Andreas; Schwarz, Christine; Bacher, Adelbert; Schmidt, Hanns-Ludwig; Eisenreich, Wolfgang

    2004-10-01

    The biosynthetic pathway of gallic acid in leaves of Rhus typhina is studied by oxygen isotope ratio mass spectrometry at natural oxygen isotope abundance. The observed delta18O-values of gallic acid indicate an 18O-enrichment of the phenolic oxygen atoms of more than 30 per thousand above that of the leaf water. This enrichment implies biogenetical equivalence with oxygen atoms of carbohydrates but not with oxygen atoms introduced by monooxygenase activation of molecular oxygen. It can be concluded that all phenolic oxygen atoms of gallic acid are retained from the carbohydrate-derived precursor 5-dehydroshikimate. This supports that gallic acid is synthesized entirely or predominantly by dehydrogenation of 5-dehydroshikimate.

  11. Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis.

    PubMed

    Degu, Asfaw; Hatew, Bayissa; Nunes-Nesi, Adriano; Shlizerman, Ludmila; Zur, Naftali; Katz, Ehud; Fernie, Alisdair R; Blumwald, Eduardo; Sadka, Avi

    2011-09-01

    Citrate, a major determinant of citrus fruit quality, accumulates early in fruit development and declines towards maturation. The isomerization of citrate to isocitrate, catalyzed by aconitase is a key step in acid metabolism. Inhibition of mitochondrial aconitase activity early in fruit development contributes to acid accumulation, whereas increased cytosolic activity of aconitase causes citrate decline. It was previously hypothesized that the block in mitochondrial aconitase activity, inducing acid accumulation, is caused by citramalate. Here, we investigated the effect of citramalate and of another aconitase inhibitor, oxalomalate, on aconitase activity and regulation in callus originated from juice sacs. These compounds significantly increased citrate content and reduced the enzyme's activity, while slightly inducing its protein level. Citramalate inhibited the mitochondrial, but not cytosolic form of the enzyme. Its external application to mandarin fruits resulted in inhibition of aconitase activity, with a transient increase in fruit acidity detected a few weeks later. The endogenous level of citramalate was analyzed in five citrus varieties: its pattern of accumulation challenged the notion of its action as an endogenous inhibitor of mitochondrial aconitase. Metabolite profiling of oxalomalate-treated cells showed significant increases in a few amino acids and organic acids. The activities of alanine transaminase, aspartate transaminase and aspartate kinase, as well as these of two γ-aminobutyrate (GABA)-shunt enzymes, succinic semialdehyde reductase (SSAR) and succinic semialdehyde dehydrogenase (SSAD) were significantly induced in oxalomalate-treated cells. It is suggested that the increase in citrate, caused by aconitase inhibition, induces amino acid synthesis and the GABA shunt, in accordance with the suggested fate of citrate during the acid decline stage in citrus fruit.

  12. Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis.

    PubMed

    Rodriguez, Angelica; Kildegaard, Kanchana R; Li, Mingji; Borodina, Irina; Nielsen, Jens

    2015-09-01

    Aromatic amino acids are precursors of numerous plant secondary metabolites with diverse biological functions. Many of these secondary metabolites are already being used as active pharmaceutical or nutraceutical ingredients, and there are numerous exploratory studies of other compounds with promising applications. p-Coumaric acid is derived from aromatic amino acids and, besides being a valuable chemical building block, it serves as precursor for biosynthesis of many secondary metabolites, such as polyphenols, flavonoids, and some polyketides. Here we developed a p-coumaric acid-overproducing Saccharomyces cerevisiae platform strain. First, we reduced by-product formation by knocking out phenylpyruvate decarboxylase ARO10 and pyruvate decarboxylase PDC5. Second, different versions of feedback-resistant DAHP synthase and chorismate mutase were overexpressed. Finally, we identified shikimate kinase as another important flux-controlling step in the aromatic amino acid pathway by overexpressing enzymes from Escherichia coli, homologous to the pentafunctional enzyme Aro1p and to the bifunctional chorismate synthase-flavin reductase Aro2p. The highest titer of p-coumaric acid of 1.93 ± 0.26 g L(-1) was obtained, when overexpressing tyrosine ammonia-lyase TAL from Flavobacterium johnsoniaeu, DAHP synthase ARO4(K229L), chorismate mutase ARO7(G141S) and E. coli shikimate kinase II (aroL) in Δpdc5Δaro10 strain background. To our knowledge this is the highest reported titer of an aromatic compound produced by yeast. The developed S. cerevisiae strain represents an attractive platform host for production of p-coumaric-acid derived secondary metabolites, such as flavonoids, polyphenols, and polyketides.

  13. Methyl-branched poly(hydroxyalkanoate) biosynthesis from 13- methyltetradecanoic acid and mixed isostearic acid isomer substrates

    USDA-ARS?s Scientific Manuscript database

    Pseudomonas resinovorans, a known medium-chain-length (mcl-) poly(hydroxyalkanoate) (PHA) producer, was grown on 13-methyltetradecanoic acid (13-MTDA) and a mixture of isostearic acid (IA) isomers to produce methyl-branched mcl-PHA polymers. Shake flask experiments revealed polymer productivities (...

  14. De novo amino acid biosynthesis contributes to salmonella enterica growth in Alfalfa seedling exudates.

    PubMed

    Kwan, Grace; Pisithkul, Tippapha; Amador-Noguez, Daniel; Barak, Jeri

    2015-02-01

    Salmonella enterica is a member of the plant microbiome. Growth of S. enterica in sprouting-seed exudates is rapid; however, the active metabolic networks essential in this environment are unknown. To examine the metabolic requirements of S. enterica during growth in sprouting-seed exudates, we inoculated alfalfa seeds and identified 305 S. enterica proteins extracted 24 h postinoculation from planktonic cells. Over half the proteins had known metabolic functions, and they are involved in over one-quarter of the known metabolic reactions. Ion and metabolite transport accounted for the majority of detected reactions. Proteins involved in amino acid transport and metabolism were highly represented, suggesting that amino acid metabolic networks may be important for S. enterica growth in association with roots. Amino acid auxotroph growth phenotypes agreed with the proteomic data; auxotrophs in amino acid-biosynthetic pathways that were detected in our screen developed growth defects by 48 h. When the perceived sufficiency of each amino acid was expressed as a ratio of the calculated biomass requirement to the available concentration and compared to growth of each amino acid auxotroph, a correlation between nutrient availability and bacterial growth was found. Furthermore, glutamate transport acted as a fitness factor during S. enterica growth in association with roots. Collectively, these data suggest that S. enterica metabolism is robust in the germinating-alfalfa environment; that single-amino-acid metabolic pathways are important but not essential; and that targeting central metabolic networks, rather than dedicated pathways, may be necessary to achieve dramatic impacts on bacterial growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Discovery and characterization of de novo sialic acid biosynthesis in the phylum Fusobacterium.

    PubMed

    Lewis, Amanda L; Robinson, Lloyd S; Agarwal, Kavita; Lewis, Warren G

    2016-10-01

    Sialic acids are nine-carbon backbone carbohydrates found in prominent outermost positions of glycosylated molecules in mammals. Mimicry of sialic acid (N-acetylneuraminic acid, Neu5Ac) enables some pathogenic bacteria to evade host defenses. Fusobacterium nucleatum is a ubiquitous oral bacterium also linked with invasive infections throughout the body. We employed multidisciplinary approaches to test predictions that F. nucleatum engages in de novo synthesis of sialic acids. Here we show that F. nucleatum sbsp. polymorphum ATCC10953 NeuB (putative Neu5Ac synthase) restores Neu5Ac synthesis to an Escherichia coli neuB mutant. Moreover, purified F. nucleatum NeuB participated in synthesis of Neu5Ac from N-acetylmannosamine and phosphoenolpyruvate in vitro Further studies support the interpretation that F. nucleatum ATCC10953 NeuA encodes a functional CMP-sialic acid synthetase and suggest that it may also contain a C-terminal sialic acid O-acetylesterase. We also performed BLAST queries of F. nucleatum genomes, revealing that only 4/31 strains encode a complete pathway for de novo Neu5Ac synthesis. Biochemical studies including mass spectrometry were consistent with the bioinformatic predictions, showing that F. nucleatum ATCC10953 synthesizes high levels of Neu5Ac, whereas ATCC23726 and ATCC25586 do not express detectable levels above background. While there are a number of examples of sialic acid mimicry in other phyla, these experiments provide the first biochemical and genetic evidence that a member of the phylum Fusobacterium can engage in de novo Neu5Ac synthesis. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Discovery and characterization of de novo sialic acid biosynthesis in the phylum Fusobacterium

    PubMed Central

    Lewis, Amanda L; Robinson, Lloyd S; Agarwal, Kavita; Lewis, Warren G

    2016-01-01

    Sialic acids are nine-carbon backbone carbohydrates found in prominent outermost positions of glycosylated molecules in mammals. Mimicry of sialic acid (N-acetylneuraminic acid, Neu5Ac) enables some pathogenic bacteria to evade host defenses. Fusobacterium nucleatum is a ubiquitous oral bacterium also linked with invasive infections throughout the body. We employed multidisciplinary approaches to test predictions that F. nucleatum engages in de novo synthesis of sialic acids. Here we show that F. nucleatum sbsp. polymorphum ATCC10953 NeuB (putative Neu5Ac synthase) restores Neu5Ac synthesis to an Escherichia coli neuB mutant. Moreover, purified F. nucleatum NeuB participated in synthesis of Neu5Ac from N-acetylmannosamine and phosphoenolpyruvate in vitro. Further studies support the interpretation that F. nucleatum ATCC10953 NeuA encodes a functional CMP-sialic acid synthetase and suggest that it may also contain a C-terminal sialic acid O-acetylesterase. We also performed BLAST queries of F. nucleatum genomes, revealing that only 4/31 strains encode a complete pathway for de novo Neu5Ac synthesis. Biochemical studies including mass spectrometry were consistent with the bioinformatic predictions, showing that F. nucleatum ATCC10953 synthesizes high levels of Neu5Ac, whereas ATCC23726 and ATCC25586 do not express detectable levels above background. While there are a number of examples of sialic acid mimicry in other phyla, these experiments provide the first biochemical and genetic evidence that a member of the phylum Fusobacterium can engage in de novo Neu5Ac synthesis. PMID:27613803

  17. De Novo Amino Acid Biosynthesis Contributes to Salmonella enterica Growth in Alfalfa Seedling Exudates

    PubMed Central

    Kwan, Grace; Pisithkul, Tippapha; Amador-Noguez, Daniel

    2014-01-01

    Salmonella enterica is a member of the plant microbiome. Growth of S. enterica in sprouting-seed exudates is rapid; however, the active metabolic networks essential in this environment are unknown. To examine the metabolic requirements of S. enterica during growth in sprouting-seed exudates, we inoculated alfalfa seeds and identified 305 S. enterica proteins extracted 24 h postinoculation from planktonic cells. Over half the proteins had known metabolic functions, and they are involved in over one-quarter of the known metabolic reactions. Ion and metabolite transport accounted for the majority of detected reactions. Proteins involved in amino acid transport and metabolism were highly represented, suggesting that amino acid metabolic networks may be important for S. enterica growth in association with roots. Amino acid auxotroph growth phenotypes agreed with the proteomic data; auxotrophs in amino acid-biosynthetic pathways that were detected in our screen developed growth defects by 48 h. When the perceived sufficiency of each amino acid was expressed as a ratio of the calculated biomass requirement to the available concentration and compared to growth of each amino acid auxotroph, a correlation between nutrient availability and bacterial growth was found. Furthermore, glutamate transport acted as a fitness factor during S. enterica growth in association with roots. Collectively, these data suggest that S. enterica metabolism is robust in the germinating-alfalfa environment; that single-amino-acid metabolic pathways are important but not essential; and that targeting central metabolic networks, rather than dedicated pathways, may be necessary to achieve dramatic impacts on bacterial growth. PMID:25416761

  18. Biosynthesis: Imaging cell-wall biosynthesis live

    NASA Astrophysics Data System (ADS)

    Bugg, Timothy D. H.

    2013-01-01

    The biosynthesis of peptidoglycan is an important step in bacterial cell division and cell-wall maturation. Now it has been shown that fluorescent D-amino acids can be used to label the peptidoglycan cell wall of living bacteria, providing a new tool to study this important process.

  19. Inhibition of carotenoid accumulation and abscisic acid biosynthesis in fluridone-treated dark-grown barley.

    PubMed

    Gamble, P E; Mullet, J E

    1986-10-01

    Treatment of dark-grown barley with 0.1 mM fluridone inhibited carotenoid accumulation but did not alter plastid biogenesis. Plastids isolated from dark-grown control and dark-grown fluridone-treated plants were similar in size and protein compositions. Dehydration of dark-grown control barley caused abscisic acid levels to increase 30-40-fold in 4 h, while plants treated with 0.1 mM fluridone accumulated very little abscisic acid in response to dehydration. These results suggest that fluridone-treated plants do not accumulate abscisic acid because of carotenoid deficiency rather than plastid dysfunction. Dark-grown barley plants treated with 0.31 microM fluridone accumulated low levels of carotenoids. Dehydration of these plants resulted in a 4-8-fold increase in abscisic acid and a decrease in antheraxanthin, violaxanthin and neoxanthin, but no change in beta-carotene or lutein plus zeaxanthin levels. This result is consistent with the suggestion that xanthophylls are precursors to abscisic acid in dehydrated plants.

  20. Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification.

    PubMed Central

    Kranz, R G; Gabbert, K K; Madigan, M T

    1997-01-01

    The photosynthetic bacterium Rhodobacter capsulatus can grow with short- to long-chain fatty acids as the sole carbon source (R. G. Kranz, K. K. Gabbert, T. A. Locke, and M. T. Madigan, Appl. Environ. Microbiol. 63:3003-3009, 1997). Concomitant with growth on fatty acids is the production to high levels of the polyester storage compounds called polyhydroxyalkanoates (PHAs). Here, we describe colony screening and selection systems to analyze the production of PHAs in R. capsulatus. A screen with Nile red dissolved in acetone distinguishes between PHA producers and nonproducers. Unlike the wild type, an R. capsulatus PhaC- strain with the gene encoding PHA synthase deleted is unable to grow on solid media containing high concentrations of certain fatty acids. It is proposed that this deficiency is due to the inability of the PhaC- strain to detoxify the surrounding medium by consumption of fatty acids and their incorporation into PHAs. This fatty acid toxicity phenotype is used in selection for the cloning and characterization of heterologous phaC genes. PMID:9251190

  1. Very long chain fatty acids (policosanols) and phytosterols affect plasma lipid levels and cholesterol biosynthesis in hamsters.

    PubMed

    Wang, Yanwen; Ebine, Naoyuki; Jia, Xiaoming; Jones, Peter J H; Fairow, Clint; Jaeger, Ralf

    2005-04-01

    The aim of the current study was to examine the effects of very long chain fatty acids (VLCFA) alone at 2 dietary levels, or in combination of VLCFA at the lower level with lecithin (LT) or phytosterols (PS), on lipid profiles and cholesterol biosynthesis in hamsters. Seventy-five male Golden Syrian hamsters, weighing 100 to 120 g, were fed a regular rodent chow for 2 weeks before being randomly assigned into 5 groups of 15 animals each fed semisynthetic diets for 4 weeks. Group 1 was given a control diet that contained 0.25% cholesterol and 5% fat with a polyunsaturated to saturated fatty acids ratio of 0.4. Groups 2 to 5 were fed the control diet and given 25 mg/kg BW per day of VLCFA (Licowax) (VLCFA25), 50 mg/kg BW per day of VLCFA (VLCFA50), 25 mg/kg BW per day of VLCFA+1000 mg/kg BW per day of LT (VLCFA25/LT), and 25 mg/kg BW per day of VLCFA+1000 mg/kg BW per day of PS (Cholestatin, VLCFA25/PS), respectively. Results showed that HDL-cholesterol (HDL-C) levels were not changed by VLCFA25, although increased by VLCFA50 (P<.05) relative to control. Total cholesterol (T-C) and non-HDL-C levels were not affected by VLCFA25 and VLCFA50 as compared with control. VLCFA25/LT had higher (P<.02) T-C and HDL-C levels than any other treatments and increased (P<.05) liver weight relative to control. In contrast, VLCFA25/PS reduced T-C (P=.0004) and non-HDL-C (P=.007) without effect on HDL-C levels compared with control. Triglyceride levels were not affected by any treatment. Cholesterol biosynthesis rate was higher (P<.05) in animals fed VLCFA25 and VLCFA50 than those fed control or VLCFA25/LT or VLCFA25/PS. Results suggest that PSs can decrease total and non-HDL-C cholesterol, whereas VLCFA may increase HDL-C in hamsters.

  2. Biochemical and genetic engineering of diatoms for polyunsaturated fatty acid biosynthesis.

    PubMed

    Li, Hong-Ye; Lu, Yang; Zheng, Jian-Wei; Yang, Wei-Dong; Liu, Jie-Sheng

    2014-01-07

    The role of diatoms as a source of bioactive compounds has been recently explored. Diatom cells store a high amount of fatty acids, especially certain polyunsaturated fatty acids (PUFAs). However, many aspects of diatom metabolism and the production of PUFAs remain unclear. This review describes a number of technical strategies, such as modulation of environmental factors (temperature, light, chemical composition of culture medium) and culture methods, to influence the content of PUFAs in diatoms. Genetic engineering, a newly emerging field, also plays an important role in controlling the synthesis of fatty acids in marine microalgae. Several key points in the biosynthetic pathway of PUFAs in diatoms as well as recent progresses are also a critical part and are summarized here.

  3. Biochemical and Genetic Engineering of Diatoms for Polyunsaturated Fatty Acid Biosynthesis

    PubMed Central

    Li, Hong-Ye; Lu, Yang; Zheng, Jian-Wei; Yang, Wei-Dong; Liu, Jie-Sheng

    2014-01-01

    The role of diatoms as a source of bioactive compounds has been recently explored. Diatom cells store a high amount of fatty acids, especially certain polyunsaturated fatty acids (PUFAs). However, many aspects of diatom metabolism and the production of PUFAs remain unclear. This review describes a number of technical strategies, such as modulation of environmental factors (temperature, light, chemical composition of culture medium) and culture methods, to influence the content of PUFAs in diatoms. Genetic engineering, a newly emerging field, also plays an important role in controlling the synthesis of fatty acids in marine microalgae. Several key points in the biosynthetic pathway of PUFAs in diatoms as well as recent progresses are also a critical part and are summarized here. PMID:24402175

  4. Biosynthesis of Germacrene A Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP+-Dependent Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis

    PubMed Central

    de Kraker, Jan-Willem; Franssen, Maurice C. R.; Dalm, Marcella C. F.; de Groot, Aede; Bouwmeester, Harro J.

    2001-01-01

    Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates β-elemene with a modest degree of enantioselectivity. PMID:11299372

  5. De novo Fatty Acid Biosynthesis Contributes Significantly to Establishment of a Bioenergetically Favorable Environment for Vaccinia Virus Infection

    PubMed Central

    Greseth, Matthew D.; Traktman, Paula

    2014-01-01

    The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

  6. Mutations in the Prokaryotic Pathway Rescue the fatty acid biosynthesis1 Mutant in the Cold1[OPEN

    PubMed Central

    Gao, Jinpeng; Wallis, James G.; Browse, John

    2015-01-01

    The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0 due to decreased activity of 3-ketoacyl-acyl carrier protein (ACP) synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains up to 45% high-melting-point molecular species (molecules that contain only 16:0, 16:1-trans, and 18:0), a trait associated with chilling-sensitive plants, compared with less than 10% in wild-type Arabidopsis. Although they do not exhibit typical chilling sensitivity, when exposed to low temperatures (2°C–6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. A screen for suppressors of this low-temperature phenotype has identified 11 lines, some of which contain additional alterations in leaf-lipid composition relative to fab1. Here, we report the identification of two suppressor mutations, one in act1, which encodes the chloroplast acyl-ACP:glycerol-3-phosphate acyltransferase, and one in lpat1, which encodes the chloroplast acyl-ACP:lysophosphatidic acid acyltransferase. These enzymes catalyze the first two steps of the prokaryotic pathway for glycerolipid synthesis, so we investigated whether other mutations in this pathway would rescue the fab1 phenotype. Both the gly1 mutation, which reduces glycerol-3-phosphate supply to the prokaryotic pathway, and fad6, which is deficient in the chloroplast 16:1/18:1 fatty acyl desaturase, were discovered to be suppressors. Analyses of leaf-lipid compositions revealed that mutations at all four of the suppressor loci result in reductions in the proportion of high-melting-point molecular species of phosphatidylglycerol relative to fab1. We conclude that these reductions are likely the basis for the suppressor phenotypes. PMID:26224803

  7. Regulation of indole-3-acetic acid biosynthesis by branched-chain amino acids in Enterobacter cloacae UW5.

    PubMed

    Parsons, Cassandra V; Harris, Danielle M M; Patten, Cheryl L

    2015-09-01

    The soil bacterium Enterobacter cloacae UW5 produces the rhizosphere signaling molecule indole-3-acetic acid (IAA) via the indolepyruvate pathway. Expression of indolepyruvate decarboxylase, a key pathway enzyme encoded by ipdC, is upregulated by the transcription factor TyrR in response to aromatic amino acids. Some members of the TyrR regulon may also be controlled by branched-chain amino acids and here we show that expression from the ipdC promoter and production of IAA are downregulated by valine, leucine and isoleucine. Regulation of the IAA synthesis pathway by both aromatic and branched-chain amino acids suggests a broader role for this pathway in bacterial physiology, beyond plant interactions. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Biosynthesis of terephthalic acid, isophthalic acid and their derivatives from the corresponding dinitriles by tetrachloroterephthalonitrile-induced Rhodococcus sp.

    PubMed

    He, Yu-Cai; Wu, Ya-Dong; Pan, Xue-He; Ma, Cui-Luan

    2014-02-01

    The nitrilase from Rhodococcus sp. CCZU10-1 catalyses the hydrolysis of dinitriles to acids without the formation of amides and cyanocarboxylic acids. It was induced by benzonitrile and its analogues (tetrachloroterephthalonitrile > ε-caprolactam > benzonitrile > phenylacetonitrile), and had activity towards aromatic nitriles (terephthalonitrile > tetrachloroterephthalonitrile > isophthalonitrile > tetrachloroisophthalonitrile > tetrafluoroterephthalonitrile > benzonitrile). After the optimization, the highest nitrilase induction [311 U/(g DCW)] was achieved with tetrachloroterephthalonitrile (1 mM) in the medium after 24 h at 30 °C after optimum enzyme activity was at pH 6.8 and at 30 °C. Efficient biocatalyst recycling was achieved by cell immobilization in calcium alginate, with a product-to-biocatalyst ratios of 776 g terephthalic acid/g DCW and 630 g isophthalic acid/g DCW.

  9. The biosynthesis of dicoumarol

    PubMed Central

    Bellis, D. M.; Spring, M. S.; Stoker, J. R.

    1967-01-01

    Micro-organisms have been isolated that can utilize o-coumaric acid as a sole carbon source with the subsequent production of 4-hydroxycoumarin and dicoumarol. One of these organisms, Penicillium jenseni, has been used to examine the biosynthesis of dicoumarol. Certain thermophilic fungi have also been found that can convert o-coumaric acid into dicoumarol. PMID:6033758

  10. Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia).

    PubMed

    Muir, Ryann M; Ibáñez, Ana M; Uratsu, Sandra L; Ingham, Elizabeth S; Leslie, Charles A; McGranahan, Gale H; Batra, Neelu; Goyal, Sham; Joseph, Jorly; Jemmis, Eluvathingal D; Dandekar, Abhaya M

    2011-04-01

    Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.

  11. Desaturase and elongase limiting endogenous long chain polyunsaturated fatty acid biosynthesis

    PubMed Central

    Zhang, Ji Yao; Kothapalli, Kumar S.D.; Brenna, J. Thomas

    2016-01-01

    Purpose of Review Endogenous synthesis of the long chain polyunsaturated fatty acids (LCPUFA) is mediated by the fatty acid desaturase (FADS) gene cluster (11q12-13.1) and elongation of very long chain fatty acids 2 (ELOVL2) (6p24.2) and ELOVL5 (6p12.1). Though older biochemical work identified the product of one gene, FADS2, rate limiting for LCPUFA synthesis, recent studies suggest that polymorphisms in any of these genes can limit accumulation of product LCPUFA. Recent findings Genome-wide association study (GWAS) of Greenland Inuit show strong adaptation signals within FADS gene cluster, attributed to high omega-3 fatty acid intake, while GWAS found ELOVL2 associated with sleep duration, age and DNA methylation. ELOVL5 coding mutations cause spinocerebellar ataxia 38, and epigenetic marks were associated with depression and suicide risk. Two sterol response element binding sites were found on ELOVL5, a SREBP-1c target gene. Minor allele carriers of a 3 single nucleotide polymorphism (SNP) haplotype in ELOVL2 have decreased 22:6n-3 levels. Unequivocal molecular evidence shows mammalian FADS2 catalyzes direct Δ4-desaturation to yield 22:6n-3 and 22:5n-6. A SNP near FADS1 influences the levels of 5-lipoxygenase products and epigenetic alteration. Summary Genetic polymorphisms within FADS and ELOVL can limit LCPUFA product accumulation at any step of the biosynthetic pathway. PMID:26828581

  12. Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

    SciTech Connect

    Molle, Virginie; Gulten, Gulcin; Vilchèze, Catherine; Veyron-Churlet, Romain; Zanella-Cléon, Isabelle; Sacchettini, James C.; Jacobs, Jr, William R.; Kremer, Laurent

    2011-08-24

    The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA{_}T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.

  13. Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis

    USDA-ARS?s Scientific Manuscript database

    Caenorhabditis elegans secretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, perception of which enables worms to gauge depletion of food or a high worm population density. As a result, worms enter the dauer state, a specific developmental stage capable of surviv...

  14. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    PubMed

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-02

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  15. D27E mutation of VTC1 impairs the interaction with CSN5B and enhances ascorbic acid biosynthesis and seedling growth in Arabidopsis.

    PubMed

    Li, Shenghui; Wang, Juan; Yu, Yanwen; Wang, Fengru; Dong, Jingao; Huang, Rongfeng

    2016-11-01

    Our previous investigation revealed that GDP-Man pyrophosphorylase (VTC1), a vital ascorbic acid (AsA) biosynthesis enzyme, could be degraded through interaction with the photomorphogenic factor COP9 signalosome subunit 5B (CSN5B) in the darkness, demonstrating the posttranscriptional regulation of light signal in AsA production. Here, we further report that a point mutation in D27E of VTC1 disables the interaction with CSN5B, resulting in enhancement of AsA biosynthesis and seedling growth in Arabidopsis thaliana. To identify the interaction sites with CSN5B, we first predicted the key amino acids in VTC1 via bioinformatics analysis. And then we biochemically and genetically demonstrated that the 27th Asp was the amino acid that influenced the interaction of VTC1 with CSN5B in plants. Moreover, transgenic lines overexpressing the site-specific mutagenesis from D27 (Asp) into E27 (Glu) in VTC1 showed enhanced AsA accumulation and reduced H2O2 content in Arabidopsis seedlings, compared with the lines overexpressing the mutation from D27 into N27 (Asn) in VTC1. In addition, this regulation of VTC1 D27E mutation promoted seedling growth. Together, our data reveal that the 27th amino acid of VTC1 confers a key regulation in the interaction with CSN5B and AsA biosynthesis, as well as in Arabidopsis seedling growth.

  16. Indole-3-Acetic Acid Produced by Burkholderia heleia Acts as a Phenylacetic Acid Antagonist to Disrupt Tropolone Biosynthesis in Burkholderia plantarii

    PubMed Central

    Wang, Mengcen; Tachibana, Seiji; Murai, Yuta; Li, Li; Lau, Sharon Yu Ling; Cao, Mengchao; Zhu, Guonian; Hashimoto, Makoto; Hashidoko, Yasuyuki

    2016-01-01

    Burkholderia heleia PAK1-2 is a potent biocontrol agent isolated from rice rhizosphere, as it prevents bacterial rice seedling blight disease caused by Burkholderia plantarii. Here, we isolated a non-antibacterial metabolite from the culture fluid of B. heleia PAK1-2 that was able to suppress B. plantarii virulence and subsequently identified as indole-3-acetic acid (IAA). IAA suppressed the production of tropolone in B. plantarii in a dose-dependent manner without any antibacterial and quorum quenching activity, suggesting that IAA inhibited steps of tropolone biosynthesis. Consistent with this, supplementing cultures of B. plantarii with either L-[ring-2H5]phenylalanine or [ring-2H2~5]phenylacetic acid revealed that phenylacetic acid (PAA), which is the dominant metabolite during the early growth stage, is a direct precursor of tropolone. Exposure of B. plantarii to IAA suppressed production of both PAA and tropolone. These data particularly showed that IAA produced by B. heleia PAK1-2 disrupts tropolone production during bioconversion of PAA to tropolone via the ring-rearrangement on the phenyl group of the precursor to attenuate the virulence of B. plantarii. B. heleia PAK1-2 is thus likely a microbial community coordinating bacterium in rhizosphere ecosystems, which never eliminates phytopathogens but only represses production of phytotoxins or bacteriocidal substances. PMID:26935539

  17. Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

    PubMed

    Sun, Ruhao; Gao, Lingchao; Yu, Xiaoping; Zheng, Yusheng; Li, Dongdong; Wang, Xinguang

    2016-10-10

    Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374.