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Sample records for acids biosynthesis pathways

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

    PubMed

    Cook, Sam D; Nichols, David S; Smith, Jason; Chourey, Prem S; McAdam, Erin L; Quittenden, Laura; Ross, John J

    2016-06-01

    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

  2. Amino acid biosynthesis in the spirochete Leptospira: evidence for a novel pathway of isoleucine biosynthesis.

    PubMed

    Charon, N W; Johnson, R C; Peterson, D

    1974-01-01

    Radioactive carbon dioxide was incubated with growing cells of Leptospira interrogans serotypes semaranga and tarassovi, and the specific activities and distribution of the label within the cellular amino acids were determined. The origins of the carbon skeletons of all the acid-stable amino acids except isoleucine were found to be consistent with known biosynthetic pathways for these amino acids. Experiments using radioactive carbon dioxide and other tracers indicated that most of the isoleucine was synthesized by a pathway not involving threonine. The origin of the carbon skeleton of isoleucine consisted of two residues of pyruvate (carbons 2 and 3) and acetate of acetyl-coenzyme A by this pathway. Isotope competition studies indicated that the pathway was regulated by isoleucine. The results are discussed in relation to two proposed pathways of isoleucine biosynthesis involving citramalate as an intermediate. PMID:4808901

  3. 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

  4. 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

  5. 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. PMID:24404416

  6. 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

  7. 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

  8. Metabolic engineering of a novel muconic acid biosynthesis pathway via 4-hydroxybenzoic acid in Escherichia coli.

    PubMed

    Sengupta, Sudeshna; Jonnalagadda, Sudhakar; Goonewardena, Lakshani; Juturu, Veeresh

    2015-12-01

    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, aroF(FBR), 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

  9. Genetic analysis of pathway regulation for enhancing branched-chain amino acid biosynthesis in plants.

    PubMed

    Chen, Hao; Saksa, Kristen; Zhao, Feiyi; Qiu, Joyce; Xiong, Liming

    2010-08-01

    The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over-expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE-TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map-based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant-containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed. PMID:20497381

  10. Quantitative importance of the 25-hydroxylation pathway for bile acid biosynthesis in the rat

    SciTech Connect

    Duane, W.C.; Bjoerkhem, I.H.; Hamilton, J.N.; Mueller, S.M.

    1988-05-01

    During biosynthesis of bile acid, carbons 25-26-27 are removed from the cholesterol side chain. Side-chain oxidation begins either with hydroxylation at the 26-position, in which case the three-carbon fragment is released as propionic acid, or with hydroxylation at the 25-position, in which case the three-carbon fragment is released as acetone. In the present study, we have quantitated the relative importance of these two pathways in vivo by measuring production of (14C) acetone from (14C)-26-cholesterol. Four days after intraperitoneal injection of 20 to 40 muCi (14C)-26-cholesterol and 1 day after beginning a constant intravenous infusion of unlabeled acetone at 25 mumoles per kg per min, 6 male and 2 female Sprague-Dawley rats underwent breath collections. Expired acetone was trapped and purified as the 2,4-dinitrophenylhydrazine derivative. 14CO2 was trapped quantitatively using phenethylamine. Specific activity of breath acetone was multiplied times the acetone infusion rate to calculate production of (14C)acetone. (14C) Acetone production averaged 1.7% of total release of 14C from (14C)-26-cholesterol, estimated by 14CO2 output. The method was validated by showing that (14C) acetone production from (14C)isopropanol averaged 111% of the (14C)isopropanol infusion rate. We conclude that, in the normal rat, the 25-hydroxylation pathway accounts for less than 2% of bile acid synthesis.

  11. Genetic dissection of the polyoxin building block-carbamoylpolyoxamic acid biosynthesis revealing the “pathway redundancy” in metabolic networks

    PubMed Central

    2013-01-01

    Background Polyoxin, a peptidyl nucleoside antibiotic, consists of three building blocks including a nucleoside skeleton, polyoximic acid (POIA), and carbamoylpolyoxamic acid (CPOAA), however, little is known about the “pathway redundancy” of the metabolic networks directing the CPOAA biosynthesis in the cell factories of the polyoxin producer. Results Here we report the genetic characterization of CPOAA biosynthesis with revealing a “pathway redundancy” in metabolic networks. Independent mutation of the four genes (polL-N and polP) directly resulted in the accumulation of polyoxin I, suggesting their positive roles for CPOAA biosynthesis. Moreover, the individual mutant of polN and polP also partially retains polyoxin production, suggesting the existence of the alternative homologs substituting their functional roles. Conclusions It is unveiled that argA and argB in L-arginine biosynthetic pathway contributed to the “pathway redundancy”, more interestingly, argB in S. cacaoi is indispensible for both polyoxin production and L-arginine biosynthesis. These data should provide an example for the research on the “pathway redundancy” in metabolic networks, and lay a solid foundation for targeted enhancement of polyoxin production with synthetic biology strategies. PMID:24314013

  12. 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. PMID:26453466

  13. O-Methyltransferase is shared between the pentose phosphate and shikimate pathways and is essential for mycosporine-like amino acid biosynthesis in Anabaena variabilis ATCC 29413.

    PubMed

    Pope, Matthew A; Spence, Edward; Seralvo, Valentina; Gacesa, Ranko; Heidelberger, Sibylle; Weston, Andrew J; Dunlap, Walter C; Shick, J Malcolm; Long, Paul F

    2015-01-19

    The parent core structure of mycosporine-like amino acids (MAAs) is 4-deoxygadusol, which, in cyanobacteria, is derived from conversion of the pentose phosphate pathway intermediate sedoheptulose 7-phosphate by the enzymes 2-epi-5-epivaliolone synthase (EVS) and O-methyltransferase (OMT). Yet, deletion of the EVS gene from Anabaena variabilis ATCC 29413 was shown to have little effect on MAA production, thus suggesting that its biosynthesis is not exclusive to the pentose phosphate pathway. Herein, we report how, using pathway-specific inhibitors, we demonstrated unequivocally that MAA biosynthesis occurs also via the shikimate pathway. In addition, complete in-frame gene deletion of the OMT gene from A. variabilis ATCC 29413 reveals that, although biochemically distinct, the pentose phosphate and shikimate pathways are inextricably linked to MAA biosynthesis in this cyanobacterium. Furthermore, proteomic data reveal that the shikimate pathway is the predominate route for UV-induced MAA biosynthesis. PMID:25487723

  14. 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. PMID:26853081

  15. 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.

  16. New approaches to target the mycolic acid biosynthesis pathway for the development of tuberculosis therapeutics.

    PubMed

    North, E Jeffrey; Jackson, Mary; Lee, Richard E

    2014-01-01

    Mycolic acids are the major lipid components 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

  17. 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

  18. 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. PMID:19560175

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

    PubMed Central

    2014-01-01

    Background Sterols are vital structural and regulatory components in eukaryotic cells; however, their biosynthetic pathways and functional roles in microalgae remain poorly understood. Results In the oleaginous microalga Nannochloropsis oceanica, the sterol biosynthetic pathway produces phytosterols as minor products and cholesterol as the major product. The evidence together with their deduced biosynthetic pathways suggests that N. oceanica exhibits features of both higher plants and mammals. Temporal tracking of sterol profiles and sterol-biosynthetic transcripts in response to changes in light intensity and nitrogen supply reveal that sterols play roles in cell proliferation, chloroplast differentiation, and photosynthesis. Furthermore, the dynamics of fatty acid (FA) and FA-biosynthetic transcripts upon chemical inhibitor-induced sterol depletion reveal possible co-regulation of sterol production and FA synthesis, in that the squalene epoxidase inhibitor terbinafine reduces sterol content yet significantly elevates free FA production. Thus, a feedback regulation of sterol and FA homeostasis is proposed, with the 1-deoxy-D-xylulose 5-phosphate synthase (DXS, the committed enzyme in isoprenoid and sterol biosynthesis) gene potentially subject to feedback regulation by sterols. Conclusion These findings reveal features of sterol function and biosynthesis in microalgae and suggest new genetic engineering or chemical biology approaches for enhanced oil production in microalgae. PMID:24920959

  20. 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

  1. Two separate key enzymes and two pathway-specific transcription factors are involved in fusaric acid biosynthesis in Fusarium fujikuroi.

    PubMed

    Studt, Lena; Janevska, Slavica; Niehaus, Eva-Maria; Burkhardt, Immo; Arndt, Birgit; Sieber, Christian M K; Humpf, Hans-Ulrich; Dickschat, Jeroen S; Tudzynski, Bettina

    2016-03-01

    Fusaric acid (FSA) is a mycotoxin produced by several fusaria, including the rice pathogen Fusarium fujikuroi. Genes involved in FSA biosynthesis were previously identified as a cluster containing a polyketide synthase (PKS)-encoding (FUB1) and four additional genes (FUB2-FUB5). However, the biosynthetic steps leading to FSA as well as the origin of the nitrogen atom, which is incorporated into the polyketide backbone, remained unknown. In this study, seven additional cluster genes (FUB6-FUB12) were identified via manipulation of the global regulator FfSge1. The extended FUB gene cluster encodes two Zn(II)2 Cys6 transcription factors: Fub10 positively regulates expression of all FUB genes, whereas Fub12 is involved in the formation of the two FSA derivatives, i.e. dehydrofusaric acid and fusarinolic acid, serving as a detoxification mechanism. The major facilitator superfamily transporter Fub11 functions in the export of FSA out of the cell and is essential when FSA levels become critical. Next to Fub1, a second key enzyme was identified, the non-canonical non-ribosomal peptide synthetase Fub8. Chemical analyses of generated mutant strains allowed for the identification of a triketide as PKS product and the proposition of an FSA biosynthetic pathway, thereby unravelling the unique formation of a hybrid metabolite consisting of this triketide and an amino acid moiety. PMID:26662839

  2. 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

  3. Alkamides Activate Jasmonic Acid Biosynthesis and Signaling Pathways and Confer Resistance to Botrytis cinerea in Arabidopsis thaliana

    PubMed Central

    Méndez-Bravo, Alfonso; Calderón-Vázquez, Carlos; Ibarra-Laclette, Enrique; Raya-González, Javier; Ramírez-Chávez, Enrique; Molina-Torres, Jorge; Guevara-García, Angel A.; López-Bucio, José; Herrera-Estrella, Luis

    2011-01-01

    Alkamides are fatty acid amides of wide distribution in plants, structurally related to N-acyl-L-homoserine lactones (AHLs) from Gram-negative bacteria and to N- acylethanolamines (NAEs) from plants and mammals. Global analysis of gene expression changes in Arabidopsis thaliana in response to N-isobutyl decanamide, the most highly active alkamide identified to date, revealed an overrepresentation of defense-responsive transcriptional networks. In particular, genes encoding enzymes for jasmonic acid (JA) biosynthesis increased their expression, which occurred in parallel with JA, nitric oxide (NO) and H2O2 accumulation. The activity of the alkamide to confer resistance against the necrotizing fungus Botrytis cinerea was tested by inoculating Arabidopsis detached leaves with conidiospores and evaluating disease symptoms and fungal proliferation. N-isobutyl decanamide application significantly reduced necrosis caused by the pathogen and inhibited fungal proliferation. Arabidopsis mutants jar1 and coi1 altered in JA signaling and a MAP kinase mutant (mpk6), unlike salicylic acid- (SA) related mutant eds16/sid2-1, were unable to defend from fungal attack even when N-isobutyl decanamide was supplied, indicating that alkamides could modulate some necrotrophic-associated defense responses through JA-dependent and MPK6-regulated signaling pathways. Our results suggest a role of alkamides in plant immunity induction. PMID:22076141

  4. Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra).

    PubMed

    Badejo, Adebanjo A; Fujikawa, Yukichi; Esaka, Muneharu

    2009-04-01

    The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of l-ascorbic acid (AsA; vitamin C) in the seedlings of the model plant Arabidopsis thaliana. It is yet uncertain whether the same pathway holds for all other plants and their various organs as AsA may also be synthesized through alternative pathways. In this study, we have cloned some of the genes involved in the SW-pathway from acerola (Malpighia glabra), a plant containing enormous amount of AsA, and examined the expression patterns of these genes in the plant. The AsA contents of acerola leaves were about 8-fold more than that of Arabidopsis with 5-700-fold higher mRNA abundance in AsA-biosynthesizing genes. The unripe fruits have the highest AsA content but the accumulation was substantially repressed as the fruit transitions to maturation. The mRNAs encoding these genes showed correlation in their expression with the AsA contents of the fruits. Although very little AsA was recorded in the seeds the mRNAs encoding all the genes, with the exception of the mitochondrially located L-galactono-1,4-lactone dehydrogenase, were clearly detected in the seeds of the unripe fruits. In young leaves of acerola, the expression of most genes were repressed by the dark and induced by light. However, the expression of GDP-D-mannose pyrophosphorylase similar to that encoded by A. thaliana VTC1 was induced in the dark. The expressions of all the genes surged after 24h following wounding stress on the young leaves. These findings will advance the investigation into the molecular factors regulating the biosynthesis of abundant AsA in acerola. PMID:18952318

  5. mitochondrial pathway for biosynthesis of lipid mediators

    PubMed Central

    Tyurina, Yulia Y.; Poloyac, Samuel M.; Tyurin, Vladimir A.; Kapralov, Alexander A.; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I.; Vikulina, Anna S.; Jung, Mi-Yeon; Epperly, Michael W.; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C.; Kochanek, Patrick M.; Pitt, Bruce R.; Greenberger, Joel S.; Vladimirov, Yury A.; Bayır, Hülya; Kagan, Valerian E.

    2014-01-01

    The central role of mitochondria in metabolic pathways and in cell death mechanisms requires sophisticated signaling systems. Essential in this signaling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin, are oxidized by the intermembrane space hemoprotein, cytochrome c. We show that an assortment of oxygenated cardiolipin species undergoes phospholipase A2-catalyzed hydrolysis thus generating multiple oxygenated fatty acids, including well known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway including oxidation of polyunsaturated cardiolipins and accumulation of their hydrolysis products – oxygenated linoleic, arachidonic acids and monolyso-cardiolipins – is activated in vivo after acute tissue injury. PMID:24848241

  6. 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.

  7. Cyclopiazonic acid biosynthesis by Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. Evidence for a universal pathway of abscisic acid biosynthesis in higher plants from sup 18 O incorporation patterns

    SciTech Connect

    Zeevaart, J.A.D.; Heath, T.G.; Gage, D.A. )

    1989-12-01

    Previous labeling studies of abscisic acid (ABA) with {sup 18}O{sub 2} have been mainly conducted with water-stressed leaves. In this study, {sup 18}O incorporation into ABA of stressed leaves of various species was compared with {sup 18}O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), {sup 18}O was most abundant in the carboxyl group, whereas incorporation of a second and third {sup 18}O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in {sup 18}O{sub 2}. ABA from turgid bean leaves showed significant {sup 18}O incorporation, again with highest {sup 18}O enrichment in the carboxyl group. On the basis of {sup 18}O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precusor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.

  9. Oleic acid biosynthesis in cyanobacteria

    SciTech Connect

    VanDusen, W.J.; Jaworski, J.G.

    1986-05-01

    The biosynthesis of fatty acids in cyanobacteria is very similar to the well characterized system found in green plants. However, the initial desaturation of stearic acid in cyanobacteria appears to represent a significant departure from plant systems in which stearoyl-ACP is the exclusive substrate for desaturation. In Anabaena variabilis, the substrate appears to be monoglucosyldiacylglycerol, a lipid not found in plants. The authors examined five different cyanobacteria to determine if the pathway in A. variabilis was generally present in other cyanobacteria. The cyanobacteria studied were A. variabilis, Chlorogloeopsis sp., Schizothrix calcicola, Anacystis marina, and Anacystis nidulans. Each were grown in liquid culture, harvested, and examined for stearoyl-ACP desaturase activity or incubated with /sup 14/CO/sub 2/. None of the cyanobacteria contained any stearoyl-ACP desaturase activity in whole homogenates or 105,000g supernatants. All were capable of incorporating /sup 14/CO/sub 2/ into monoglucosyldiacylglycerol and results from incubations of 20 min, 1 hr, 1 hr + 10 hr chase were consistent with monoglucosyldiacylglycerol serving as precursor for monogalctosyldiacylglycerol. Thus, initial evidence is consistent with oleic acid biosynthesis occurring by desaturation of stearoyl-monoglucosyldiacylglycerol in all cyanobacteria.

  10. Redundant pathways of sunscreen biosynthesis in a cyanobacterium.

    PubMed

    Spence, Edward; Dunlap, Walter C; Shick, J Malcolm; Long, Paul F

    2012-03-01

    Route of the sun block: according to empirical evidence, sun-screening mycosporine-like amino acids (MAAs) in Eukarya originate from the shikimic acid pathway, whereas in cyanobacteria, biosynthesis of the MAA shinorine reportedly occurs through the pentose phosphate pathway. However, gene deletion shows that the cyanobacterium Anabaena variabilis ATCC 29143 does not biosynthesise shinorine exclusively by this route. PMID:22278966

  11. Carotenoid Biosynthesis in Arabidopsis: A Colorful Pathway

    PubMed Central

    Ruiz-Sola, M. Águila; Rodríguez-Concepción, Manuel

    2012-01-01

    Plant carotenoids are a family of pigments that participate in light harvesting and are essential for photoprotection against excess light. Furthermore, they act as precursors for the production of apocarotenoid hormones such as abscisic acid and strigolactones. In this review, we summarize the current knowledge on the genes and enzymes of the carotenoid biosynthetic pathway (which is now almost completely elucidated) and on the regulation of carotenoid biosynthesis at both transcriptional and post-transcriptional levels. We also discuss the relevance of Arabidopsis as a model system for the study of carotenogenesis and how metabolic engineering approaches in this plant have taught important lessons for carotenoid biotechnology. PMID:22582030

  12. BIOCHEMICAL AND GENETIC CHARACTERIZATION OF AN EARLY STEP IN A NOVEL PATHWAY FOR THE BIOSYNTHESIS OF AROMATIC AMINO ACIDS AND P-AMINOBENZOIC ACID IN THE ARCHAEON METHANOCOCCUS MARIPALUDIS

    EPA Science Inventory

    Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis ...

  13. 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

  14. Broad substrate specificity of phosphotransbutyrylase from Listeria monocytogenes: A potential participant in an alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis.

    PubMed

    Sirobhushanam, Sirisha; Galva, Charitha; Sen, Suranjana; Wilkinson, Brian J; Gatto, Craig

    2016-09-01

    Listeria monocytogenes, the causative organism of the serious food-borne disease listeriosis, has a membrane abundant in branched-chain fatty acids (BCFAs). BCFAs are normally biosynthesized from branched-chain amino acids via the activity of branched chain α-keto acid dehydrogenase (Bkd), and disruption of this pathway results in reduced BCFA content in the membrane. Short branched-chain carboxylic acids (BCCAs) added as media supplements result in incorporation of BCFAs arising from the supplemented BCCAs in the membrane of L. monocytogenes bkd mutant MOR401. High concentrations of the supplements also effect similar changes in the membrane of the wild type organism with intact bkd. Such carboxylic acids clearly act as fatty acid precursors, and there must be an alternative pathway resulting in the formation of their CoA thioester derivatives. Candidates for this are the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), the products of the first two genes of the bkd operon. Ptb from L. monocytogenes exhibited broad substrate specificity, a strong preference for branched-chain substrates, a lack of activity with acetyl CoA and hexanoyl CoA, and strict chain length preference (C3-C5). Ptb catalysis involved ternary complex formation. Additionally, Ptb could utilize unnatural branched-chain substrates such as 2-ethylbutyryl CoA, albeit with lower efficiency, consistent with a potential involvement of this enzyme in the conversion of the carboxylic acid additives into CoA primers for BCFA biosynthesis. PMID:27320015

  15. Regulatory Cross-Talks and Cascades in Rice Hormone Biosynthesis Pathways Contribute to Stress Signaling

    PubMed Central

    Deb, Arindam; Grewal, Rumdeep K.; Kundu, Sudip

    2016-01-01

    Crosstalk among different hormone signaling pathways play an important role in modulating plant response to both biotic and abiotic stress. Hormone activity is controlled by its bio-availability, which is again influenced by its biosynthesis. Thus, independent hormone biosynthesis pathways must be regulated and co-ordinated to mount an integrated response. One of the possibilities is to use cis-regulatory elements to orchestrate expression of hormone biosynthesis genes. Analysis of CREs, associated with differentially expressed hormone biosynthesis related genes in rice leaf under Magnaporthe oryzae attack and drought stress enabled us to obtain insights about cross-talk among hormone biosynthesis pathways at the transcriptional level. We identified some master transcription regulators that co-ordinate different hormone biosynthesis pathways under stress. We found that Abscisic acid and Brassinosteroid regulate Cytokinin conjugation; conversely Brassinosteroid biosynthesis is affected by both Abscisic acid and Cytokinin. Jasmonic acid and Ethylene biosynthesis may be modulated by Abscisic acid through DREB transcription factors. Jasmonic acid or Salicylic acid biosynthesis pathways are co-regulated but they are unlikely to influence each others production directly. Thus, multiple hormones may modulate hormone biosynthesis pathways through a complex regulatory network, where biosynthesis of one hormone is affected by several other contributing hormones. PMID:27617021

  16. Regulatory Cross-Talks and Cascades in Rice Hormone Biosynthesis Pathways Contribute to Stress Signaling.

    PubMed

    Deb, Arindam; Grewal, Rumdeep K; Kundu, Sudip

    2016-01-01

    Crosstalk among different hormone signaling pathways play an important role in modulating plant response to both biotic and abiotic stress. Hormone activity is controlled by its bio-availability, which is again influenced by its biosynthesis. Thus, independent hormone biosynthesis pathways must be regulated and co-ordinated to mount an integrated response. One of the possibilities is to use cis-regulatory elements to orchestrate expression of hormone biosynthesis genes. Analysis of CREs, associated with differentially expressed hormone biosynthesis related genes in rice leaf under Magnaporthe oryzae attack and drought stress enabled us to obtain insights about cross-talk among hormone biosynthesis pathways at the transcriptional level. We identified some master transcription regulators that co-ordinate different hormone biosynthesis pathways under stress. We found that Abscisic acid and Brassinosteroid regulate Cytokinin conjugation; conversely Brassinosteroid biosynthesis is affected by both Abscisic acid and Cytokinin. Jasmonic acid and Ethylene biosynthesis may be modulated by Abscisic acid through DREB transcription factors. Jasmonic acid or Salicylic acid biosynthesis pathways are co-regulated but they are unlikely to influence each others production directly. Thus, multiple hormones may modulate hormone biosynthesis pathways through a complex regulatory network, where biosynthesis of one hormone is affected by several other contributing hormones. PMID:27617021

  17. Inhibitors targeting on cell wall biosynthesis pathway of MRSA.

    PubMed

    Hao, Haihong; Cheng, Guyue; Dai, Menghong; Wu, Qinghua; Yuan, Zonghui

    2012-11-01

    Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA. PMID:22898792

  18. Effect of gibberellic acid and calliterpenone on plant growth attributes, trichomes, essential oil biosynthesis and pathway gene expression in differential manner in Mentha arvensis L.

    PubMed

    Bose, Subir K; Yadav, Ritesh Kumar; Mishra, Smrati; Sangwan, Rajender S; Singh, A K; Mishra, B; Srivastava, A K; Sangwan, Neelam S

    2013-05-01

    Extensive research is going on throughout the world to find out new molecules from natural sources to be used as plant growth promoter. Mentha arvensis L. is the main source of menthol rich essential oil used commercially in various food, pharmaceutical and other preparations. Experiments were conducted on field grown plants for understanding the effect of calliterpenone (CA), a stereo-isomer of abbeokutone, in comparison to gibberellic acid (GA3) on growth attributes, trichomes, essential oil biosynthesis and expression of some oil biosynthetic pathway genes. The exogenous application of CA (1 μM, 10 μM and 100 μM) was found to be better in improving plant biomass and stolon yield, leaf area, branching and leaf stem ratio than with counterpart GA3 at the same concentrations. CA treated plants showed higher glandular trichome number, density and diameter and also correlated with enhanced oil biogenetic capacity as revealed by feeding labeled (14)C-sucrose for 72 h to excised shoots. Semi-quantitative PCR analysis of key pathway genes revealed differential up regulation under CA treatments. Transcript level of menthol dehydrogenase/menthone reductase was found highly up regulated in CA treated plants with increased content of menthone and menthol in oil. These findings demonstrate that CA positively regulated the yields by enhanced branching and higher density of trichomes resulting into higher accumulation of essential oil. The results suggest CA as a novel plant derived diterpenoid with growth promoting action and opens up new possibilities for improving the crop yields and essential oil biosynthesis in qualitative and quantitative manner. PMID:23514759

  19. Bacterial exopolysaccharides: biosynthesis pathways and engineering strategies

    PubMed Central

    Schmid, Jochen; Sieber, Volker; Rehm, Bernd

    2015-01-01

    Bacteria produce a wide range of exopolysaccharides which are synthesized via different biosynthesis pathways. The genes responsible for synthesis are often clustered within the genome of the respective production organism. A better understanding of the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is critical toward genetic, metabolic and protein-engineering approaches to produce tailor-made polymers. These designer polymers will exhibit superior material properties targeting medical and industrial applications. Exploiting the natural design space for production of a variety of biopolymer will open up a range of new applications. Here, we summarize the key aspects of microbial exopolysaccharide biosynthesis and highlight the latest engineering approaches toward the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications. PMID:26074894

  20. Identification and characterization of a novel C20-elongase gene from the marine microalgae, Pavlova viridis, and its use for the reconstitution of two pathways of long-chain polyunsatured fatty acids biosynthesis in Saccharomyces cerevisiae.

    PubMed

    Shi, Tonglei; Yu, Aiqun; Li, Ming; Zhang, Meng; Xing, Laijun; Li, Mingchun

    2013-08-01

    The marine microalga, Pavlova viridis, contains long-chain polyunsatured fatty acids including eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3). A full-length cDNA sequence, pvelo5, was isolated from P. viridis. From sequence alignment, the gene was homologous to fatty acyl elongases from other organisms. Heterologous expression of pvelo5 in Saccharomyces cerevisiae confirmed that it encoded a specific C20-elongase within the n-3 and n-6 pathways. Elongation activity was confined exclusively to EPA and arachidonic acid (20:4n-6). GC analysis indicated that pvelo5 could co-express with other genes for biosynthesis to reconstitute the Δ8 and Δ6 pathways. Real-time PCR results and fatty acid analysis demonstrated that long-chain polyunsatured fatty acids production by the Δ8 pathway might be more effective than that by the Δ6 pathway. PMID:23546943

  1. An engineered pathway for the biosynthesis of renewable propane

    PubMed Central

    Kallio, Pauli; Pásztor, András; Thiel, Kati; Akhtar, M. Kalim; Jones, Patrik R.

    2014-01-01

    The deployment of next-generation renewable biofuels can be enhanced by improving their compatibility with the current infrastructure for transportation, storage and utilization. Propane, the bulk component of liquid petroleum gas, is an appealing target as it already has a global market. In addition, it is a gas under standard conditions, but can easily be liquefied. This allows the fuel to immediately separate from the biocatalytic process after synthesis, yet does not preclude energy-dense storage as a liquid. Here we report, for the first time, a synthetic metabolic pathway for producing renewable propane. The pathway is based on a thioesterase specific for butyryl-acyl carrier protein (ACP), which allows native fatty acid biosynthesis of the Escherichia coli host to be redirected towards a synthetic alkane pathway. Propane biosynthesis is markedly stimulated by the introduction of an electron-donating module, optimizing the balance of O2 supply and removal of native aldehyde reductases. PMID:25181600

  2. An engineered pathway for the biosynthesis of renewable propane.

    PubMed

    Kallio, Pauli; Pásztor, András; Thiel, Kati; Akhtar, M Kalim; Jones, Patrik R

    2014-01-01

    The deployment of next-generation renewable biofuels can be enhanced by improving their compatibility with the current infrastructure for transportation, storage and utilization. Propane, the bulk component of liquid petroleum gas, is an appealing target as it already has a global market. In addition, it is a gas under standard conditions, but can easily be liquefied. This allows the fuel to immediately separate from the biocatalytic process after synthesis, yet does not preclude energy-dense storage as a liquid. Here we report, for the first time, a synthetic metabolic pathway for producing renewable propane. The pathway is based on a thioesterase specific for butyryl-acyl carrier protein (ACP), which allows native fatty acid biosynthesis of the Escherichia coli host to be redirected towards a synthetic alkane pathway. Propane biosynthesis is markedly stimulated by the introduction of an electron-donating module, optimizing the balance of O2 supply and removal of native aldehyde reductases. PMID:25181600

  3. Biosynthesis of platelet-activating factor in glandular gastric mucosa. Evidence for the involvement of the 'de novo' pathway and modulation by fatty acids.

    PubMed

    Fernandez-Gallardo, S; Gijon, M A; Garcia, M C; Cano, E; Sanchez Crespo, M

    1988-09-15

    The biosynthesis of platelet-activating factor (PAF), a phospholipid autocoid with potent ulcerogenic properties that is produced in secretory exocrine glands by physiological secretagogues, was assessed in microsomal preparations of glandular gastric mucosa. For this purpose, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase (EC 2.3.1.67); the enzymes of the 'de novo' pathway: 1-O-alkyl-2-lyso-sn-glycero-3-phosphate (alkyl-lyso-GP):acetyl-CoA acetyltransferase and 1-O-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-choline cholinephosphotransferase (EC 2.7.8.16); and some enzymes involved in the catabolism of PAF and lyso-PAF were assayed. Only the enzymes of the 'de novo' pathway and small amounts of PAF acetylhydrolase, phospholipase A2 and a lysophospholipase D acting on either lipids could be detected in the gastric preparations, whereas lyso-PAF:acetyl-CoA acetyltransferase activity was undetectable. The specific activity of alkyl-lyso-GP:acetyl-CoA acetyltransferase in the gastric mucosa was about one-tenth of that found in spleen microsomes and its apparent Km for acetyl-CoA was 454 microM compared with 277 microM in spleen microsomes. Glandular mucosa homogenates contained preformed PAF at a concentration of 2.7 +/- 0.7 ng equivalents of PAF (hexadecyl)/mg of protein. When gastric microsomes were incubated with micromolar concentrations of fatty acids (arachidonic, palmitic and oleic) prior to the assay of dithiothreitol (DTT)-insensitive cholinephosphotransferase, a dose-dependent reduction in the formation of PAF was observed, arachidonic acid being the most potent inhibitor, followed by linoleic acid (only tested on spleen microsomes) and oleic acid. By contrast, 1,2-diolein and phosphatidylcholine (dipalmitoyl) showed no or little effect. These results indicate that glandular gastric mucosa can produce PAF through the 'de novo' pathway, and that fatty acids, especially unsaturated, can reduce that synthesis by

  4. A mitochondrial pathway for biosynthesis of lipid mediators

    NASA Astrophysics Data System (ADS)

    Tyurina, Yulia Y.; Poloyac, Samuel M.; Tyurin, Vladimir A.; Kapralov, Alexander A.; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I.; Vikulina, Anna S.; Jung, Mi-Yeon; Epperly, Michael W.; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C.; Kochanek, Patrick M.; Pitt, Bruce R.; Greenberger, Joel S.; Vladimirov, Yury A.; Bayır, Hülya; Kagan, Valerian E.

    2014-06-01

    The central role of mitochondria in metabolic pathways and in cell-death mechanisms requires sophisticated signalling systems. Essential in this signalling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin (CL), is oxidized by the intermembrane-space haemoprotein, cytochrome c. We show that a number of oxygenated CL species undergo phospholipase A2-catalysed hydrolysis and thus generate multiple oxygenated fatty acids, including well-known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway, which includes the oxidation of polyunsaturated CLs and accumulation of their hydrolysis products (oxygenated linoleic, arachidonic acids and monolysocardiolipins), is activated in vivo after acute tissue injury.

  5. A mitochondrial pathway for biosynthesis of lipid mediators.

    PubMed

    Tyurina, Yulia Y; Poloyac, Samuel M; Tyurin, Vladimir A; Kapralov, Alexander A; Jiang, Jianfei; Anthonymuthu, Tamil Selvan; Kapralova, Valentina I; Vikulina, Anna S; Jung, Mi-Yeon; Epperly, Michael W; Mohammadyani, Dariush; Klein-Seetharaman, Judith; Jackson, Travis C; Kochanek, Patrick M; Pitt, Bruce R; Greenberger, Joel S; Vladimirov, Yury A; Bayır, Hülya; Kagan, Valerian E

    2014-06-01

    The central role of mitochondria in metabolic pathways and in cell-death mechanisms requires sophisticated signalling systems. Essential in this signalling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin (CL), is oxidized by the intermembrane-space haemoprotein, cytochrome c. We show that a number of oxygenated CL species undergo phospholipase A2-catalysed hydrolysis and thus generate multiple oxygenated fatty acids, including well-known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway, which includes the oxidation of polyunsaturated CLs and accumulation of their hydrolysis products (oxygenated linoleic, arachidonic acids and monolysocardiolipins), is activated in vivo after acute tissue injury. PMID:24848241

  6. Polyketides in Aspergillus terreus: biosynthesis pathway discovery and application.

    PubMed

    Yin, Ying; Cai, Menghao; Zhou, Xiangshan; Li, Zhiyong; Zhang, Yuanxing

    2016-09-01

    The knowledge of biosynthesis gene clusters, production improving methods, and bioactivity mechanisms is very important for the development of filamentous fungi metabolites. Metabolic engineering and heterologous expression methods can be applied to improve desired metabolite production, when their biosynthesis pathways have been revealed. And, stable supplement is a necessary basis of bioactivity mechanism discovery and following clinical trial. Aspergillus terreus is an outstanding producer of many bioactive agents, and a large part of them are polyketides. In this review, we took polyketides from A. terreus as examples, focusing on 13 polyketide synthase (PKS) genes in A. terreus NIH 2624 genome. The biosynthesis pathways of nine PKS genes have been reported, and their downstream metabolites are lovastatin, terreic acid, terrein, geodin, terretonin, citreoviridin, and asperfuranone, respectively. Among them, lovastatin is a well-known hypolipidemic agent. Terreic acid, terrein, citreoviridin, and asperfuranone show good bioactivities, especially anticancer activities. On the other hand, geodin and terretonin are mycotoxins. So, biosynthesis gene cluster information is important for the production or elimination of them. We also predicted three possible gene clusters that contain four PKS genes by homologous gene alignment with other Aspergillus strains. We think that this is an effective way to mine secondary metabolic gene clusters. PMID:27455860

  7. Engineering the MEP pathway enhanced ajmalicine biosynthesis.

    PubMed

    Chang, Kai; Qiu, Fei; Chen, Min; Zeng, Lingjiang; Liu, Xiaoqiang; Yang, Chunxian; Lan, Xiaozhong; Wang, Qiang; Liao, Zhihua

    2014-01-01

    The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes encoding DXR and MECS from Taxus species and STR from Catharanthus roseus were used to genetically modify the ajmalicine biosynthetic pathway in hairy root cultures of C. roseus. As expected, the STR-overexpressed root cultures showed twofold higher accumulation of ajmalicine than the control. It was important to discover that overexpression of the single DXR or MECS gene from the MEP pathway also remarkably enhanced ajmalicine biosynthesis in transgenic hairy root cultures, and this suggested that engineering the MEP pathway by overexpression of DXR or MECS promoted the metabolic flux into ajmalicine biosynthesis. The transgenic hairy root cultures with co-overexpression of DXR and STR or MECS and STR had higher levels of ajmalicine than those with overexpression of a single gene alone such as DXR, MECS, and STR. It could be concluded that transgenic hairy root cultures harboring both DXR/MECS and STR possessed an increased flux in the terpenoid indole alkaloid biosynthetic pathway that enhanced ajmalicine yield, which was more efficient than cultures harboring only one of the three genes. PMID:24237015

  8. Salicylic acid-induced changes in physiological parameters and genes of the flavonoid biosynthesis pathway in Artemisia vulgaris and Dendranthema nankingense during aphid feeding.

    PubMed

    Sun, Y; Xia, X L; Jiang, J F; Chen, S M; Chen, F D; Lv, G S

    2016-01-01

    Phloem-feeding aphids cause serious damage to plants. The mechanisms of plant-aphid interactions are only partially understood and involve multiple pathways, including phytohormones. In order to investigate whether salicylic acid (SA) is involved and how it plays a part in the defense response to the aphid Macrosiphoniella sanbourni, physiological changes and gene expression profiles in response to aphid inoculation with or without SA pretreatment were compared between the aphid-resistant Artemisia vulgaris 'Variegata' and the susceptible chrysanthemum, Dendranthema nankingense. Changes in levels of reactive oxygen species, malondialdehyde (MDA), and flavonoids, and in the expression of genes involved in flavonoid biosynthesis, including PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), CHI (chalcone isomerase), F3H (flavanone 3-hydroxylase), F3'H (flavanone 3'-hydroxylase), and DFR (dihydroflavonol reductase), were investigated. Levels of hydrogen peroxide, superoxide anions, MDA, and flavonoids, and their related gene expression, increased after aphid infestation and SA pretreatment followed by aphid infestation; the aphid-resistant A. vulgaris exhibited a more rapid response than the aphid-susceptible D. nankingense to SA treatment and aphid infestation. Taken together, our results suggest that SA could be used to increase aphid resistance in the chrysanthemum. PMID:26909993

  9. A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate

    PubMed Central

    Sun, Xinxiao; Lin, Yuheng; Huang, Qin; Yuan, Qipeng

    2013-01-01

    Muconic acid is the synthetic precursor of adipic acid, and the latter is an important platform chemical that can be used for the production of nylon-6,6 and polyurethane. Currently, the production of adipic acid relies mainly on chemical processes utilizing petrochemicals, such as benzene, which are generally considered environmentally unfriendly and nonrenewable, as starting materials. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway in Escherichia coli for the biosynthesis of muconic acid, in which anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially and respectively. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-per-liter level muconic acid from supplemented anthranilate in 5 h. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by overexpressing the key enzymes in the shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by overexpressing glutamine synthase significantly improved anthranilate production. Finally, the engineered E. coli strain carrying the full pathway produced 389.96 ± 12.46 mg/liter muconic acid from simple carbon sources in shake flask experiments, a result which demonstrates scale-up potential for microbial production of muconic acid. PMID:23603682

  10. Coexpression of multiple genes reconstitutes two pathways of very long-chain polyunsaturated fatty acid biosynthesis in Pichia pastoris.

    PubMed

    Kim, Sun Hee; Roh, Kyung Hee; Kim, Kwang-Soo; Kim, Hyun Uk; Lee, Kyeong-Ryeol; Kang, Han-Chul; Kim, Jong-Bum

    2014-09-01

    The introduction of novel traits to cells often requires the stable coexpression of multiple genes within the same cell. Herein, we report that C22 very long-chain polyunsaturated fatty acids (VLC-PUFAs) were synthesized from C18 precursors by reactions catalyzed by delta 6-desaturase, an ELOVL5 involved in VLC-PUFA elongation, and delta 5-desaturase. The coexpression of McD6DES, AsELOVL5, and PtD5DES encoding the corresponding enzymes, produced docosatetraenoic acid (C22:4 n-6) and docosapentaenoic acid (C22:5 n-3), as well as arachidonic acid (C20:4 n-6) and eicosapentaenoic acid (C20:5 n-3) in the methylotrophic yeast Pichia pastoris. The expression of each gene increased within 24 h, with high transcript levels after induction with 0.5 or 1 % methanol. High levels of the newly expressed VLC-PUFAs occurred after 144 h. This expression system exemplifies the recent progress and future possibilities of the metabolic engineering of VLC-PUFAs in oilseed crops. PMID:24863294

  11. Functional analysis of the role of glutathione peroxidase (GPx) in the ROS signaling pathway, hyphal branching and the regulation of ganoderic acid biosynthesis in Ganoderma lucidum.

    PubMed

    Li, Chenyang; Shi, Liang; Chen, Dongdong; Ren, Ang; Gao, Tan; Zhao, Mingwen

    2015-09-01

    Ganoderma lucidum, a hallmark of traditional Chinese medicine, has been widely used as a pharmacologically active compound. Although numerous research studies have focused on the pharmacological mechanism, fewer studies have explored the basic biological features of this species, restricting the further development and application of this important mushroom. Because of the ability of this mushroom to reduce and detoxify the compounds produced by various metabolic pathways, glutathione peroxidase (GPx) is one of the most important antioxidant enzymes with respect to ROS. Although studies in both animals and plants have suggested many important physiological functions of GPx, there are few systematic research studies concerning the role of this enzyme in fungi, particularly in large basidiomycetes. In the present study, we cloned the GPx gene and created GPx-silenced strains by the down-regulation of GPx gene expression using RNA interference. The results indicated an essential role for GPx in controlling the intracellular H2O2 content, hyphal branching, antioxidant stress tolerance, cytosolic Ca(2+) content and ganoderic acid biosynthesis. Further mechanistic investigation revealed that GPx is regulated by intracellular H2O2 levels and suggested that crosstalk occurs between GPx and intracellular H2O2. Moreover, evidence was obtained indicating that GPx regulation of hyphal branching via ROS might occur independently of the cytosolic Ca(2+) content. Further mechanistic investigation also revealed that the effects of GPx on ganoderic acid synthesis via ROS are regulated by the cytosolic Ca(2+) content. Taken together, these findings indicate that ROS have a complex influence on growth, development and secondary metabolism in fungi and that GPx serves an important function. The present study provides an excellent framework to identify GPx functions and highlights a role for this enzyme in ROS regulation. PMID:26216672

  12. Biosynthesis of the Aromatic Amino Acids.

    PubMed

    Pittard, James; Yang, Ji

    2008-09-01

    This chapter describes in detail the genes and proteins of Escherichia coli involved in the biosynthesis and transport of the three aromatic amino acids tyrosine, phenylalanine, and tryptophan. It provides a historical perspective on the elaboration of the various reactions of the common pathway converting erythrose-4-phosphate and phosphoenolpyruvate to chorismate and those of the three terminal pathways converting chorismate to phenylalanine, tyrosine, and tryptophan. The regulation of key reactions by feedback inhibition, attenuation, repression, and activation are also discussed. Two regulatory proteins, TrpR (108 amino acids) and TyrR (513 amino acids), play a major role in transcriptional regulation. The TrpR protein functions only as a dimer which, in the presence of tryptophan, represses the expression of trp operon plus four other genes (the TrpR regulon). The TyrR protein, which can function both as a dimer and as a hexamer, regulates the expression of nine genes constituting the TyrR regulon. TyrR can bind each of the three aromatic amino acids and ATP and under their influence can act as a repressor or activator of gene expression. The various domains of this protein involved in binding the aromatic amino acids and ATP, recognizing DNA binding sites, interacting with the alpha subunit of RNA polymerase, and changing from a monomer to a dimer or a hexamer are all described. There is also an analysis of the various strategies which allow TyrR in conjunction with particular amino acids to differentially affect the expression of individual genes of the TyrR regulon. PMID:26443741

  13. Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis

    PubMed Central

    Božić, Dragana; Papaefthimiou, Dimitra; Brückner, Kathleen; de Vos, Ric C. H.; Tsoleridis, Constantinos A.; Katsarou, Dimitra; Papanikolaou, Antigoni; Pateraki, Irini; Chatzopoulou, Fani M.; Dimitriadou, Eleni; Kostas, Stefanos; Manzano, David; Scheler, Ulschan; Ferrer, Albert; Tissier, Alain; Makris, Antonios M.; Kampranis, Sotirios C.; Kanellis, Angelos K.

    2015-01-01

    Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera. PMID:26020634

  14. Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis.

    PubMed

    Božić, Dragana; Papaefthimiou, Dimitra; Brückner, Kathleen; de Vos, Ric C H; Tsoleridis, Constantinos A; Katsarou, Dimitra; Papanikolaou, Antigoni; Pateraki, Irini; Chatzopoulou, Fani M; Dimitriadou, Eleni; Kostas, Stefanos; Manzano, David; Scheler, Ulschan; Ferrer, Albert; Tissier, Alain; Makris, Antonios M; Kampranis, Sotirios C; Kanellis, Angelos K

    2015-01-01

    Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera. PMID:26020634

  15. A mutation in the indole-3-acetic acid biosynthesis pathway of Pseudomonas syringae pv. syringae affects growth in Phaseolus vulgaris and syringomycin production.

    PubMed Central

    Mazzola, M; White, F F

    1994-01-01

    Homologs of the genes for indole-3-acetic acid (IAA) biosynthesis from Pseudomonas syringae pv. savastanoi were retrieved from a genomic library of P. syringae pv. syringae, and their nucleotide sequences were determined. Sequence relatedness between the P. syringae pv. syringae and P. syringae pv. savastanoi iaa operons is greater than 90% within the iaaM and iaaH loci but declines dramatically at a position approximately 200 bp 5' of the iaaM translation initiation codon. A third open reading frame was detected downstream of iaaH. Production of IAA was undetectable in mutant strain Y30-53.29, which was generated by transposition of Tn5 into the iaaM gene of P. syringae pv. syringae Y30. The IAA-deficient (IAA-) mutant retained the ability to colonize the bean phylloplane and induced disease symptoms on bean which were similar to those produced by the parental strain. However, the population dynamics of the IAA- strain during the parasitic phase in leaves differed from those of both the parental strain and the mutant genetically restored for IAA biosynthesis. The mutant was capable of inducing disease symptoms when established in bean tissues at a lower initial cell density than either IAA-producing strain. Syringomycin biosynthesis by the IAA- strain was diminished in comparison with the parental strain or the mutant genetically restored for IAA production. The results indicate that bacterially derived IAA, or its biosynthesis, is involved in the regulation of in planta growth and in the expression of other factors that affect the host-pathogen interaction. Images PMID:8113177

  16. Metabolic engineering of cottonseed oil biosynthesis pathway via RNA interference.

    PubMed

    Xu, Zhongping; Li, Jingwen; Guo, Xiaoping; Jin, Shuangxia; Zhang, Xianlong

    2016-01-01

    Cottonseed oil is recognized as an important oil in food industry for its unique characters: low flavor reversion and the high level of antioxidants (VitaminE) as well as unsaturated fatty acid. However, the cottonseed oil content of cultivated cotton (Gossypium hirsutum) is only around 20%. In this study, we modified the accumulation of oils by the down-regulation of phosphoenolpyruvate carboxylase 1 (GhPEPC1) via RNA interference in transgenic cotton plants. The qRT-PCR and enzyme activity assay revealed that the transcription and expression of GhPEPC1 was dramatically down-regulated in transgenic lines. Consequently, the cottonseed oil content in several transgenic lines showed a significant (P < 0.01) increase (up to 16.7%) without obvious phenotypic changes under filed condition when compared to the control plants. In order to elucidate the molecular mechanism of GhPEPC1 in the regulation of seed oil content, we quantified the expression of the carbon metabolism related genes of transgenic GhPEPC1 RNAi lines by transcriptome analysis. This analysis revealed the decrease of GhPEPC1 expression led to the increase expression of triacylglycerol biosynthesis-related genes, which eventually contributed to the lipid biosynthesis in cotton. This result provides a valuable information for cottonseed oil biosynthesis pathway and shows the potential of creating high cottonseed oil germplasm by RNAi strategy for cotton breeding. PMID:27620452

  17. Heterologous biosynthesis of artemisinic acid in Saccharomyces cerevisiae.

    PubMed

    Li, C; Li, J; Wang, G; Li, X

    2016-06-01

    Artemisinic acid is a precursor of antimalarial compound artemisinin. The titre of biosynthesis of artemisinic acid using Saccharomyces cerevisiae platform has been achieved up to 25 g l(-1) ; however, the performance of platform cells is still industrial unsatisfied. Many strategies have been proposed to improve the titre of artemisinic acid. The traditional strategies mainly focused on partial target sites, simple up-regulation key genes or repression competing pathways in the total synthesis route. However, this may result in unbalance of carbon fluxes and dysfunction of metabolism. In this review, the recent advances on the promising methods in silico and in vivo for biosynthesis of artemisinic acid have been discussed. The bioinformatics and omics techniques have brought a great prospect for improving production of artemisinin and other pharmacal compounds in heterologous platform. PMID:26743771

  18. Metabolic flux analysis of diterpene biosynthesis pathway in rice.

    PubMed

    Chang, Yung-Jin; Kim, Bo-Ra; Kim, Soo-Un

    2005-09-01

    Relative transcript levels of eight rice diterpene cyclases at the branch points of gibberellins and phytoalexins biosynthesis pathway were measured by reverse transcription quantitative PCR. Metabolic flux analysis by the distribution ratio of common substrate showed that UV-irradiation of etiolated rice seedlings decreased the flux for primary metabolism of gibberellins biosynthesis by half (from 62 to 27%) and 41% of geranylgeranyl pyrophosphate was used for induction of pimaradiene intermediate as the major phytoalexin. In comparison, light-illumination used almost all geranylgeranyl pyrophosphate (96%) for gibberellin biosynthesis to stimulate the plant growth and strongly repressed the metabolic flux for phytoalexins biosynthesis. PMID:16215852

  19. A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

    SciTech Connect

    Fan, J.; Xu, C.; Andre, C.

    2011-06-23

    Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.

  20. “Teichoic acid biosynthesis as an antibiotic target”

    PubMed Central

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

    2013-01-01

    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. PMID:23916223

  1. Pantothenic acid biosynthesis in zymomonas

    SciTech Connect

    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.

  2. 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

  3. In vivo kinetic analysis of the penicillin biosynthesis pathway using PAA stimulus response experiments.

    PubMed

    Deshmukh, Amit T; Verheijen, Peter J T; Maleki Seifar, Reza; Heijnen, Joseph J; van Gulik, Walter M

    2015-11-01

    In this study we combined experimentation with mathematical modeling to unravel the in vivo kinetic properties of the enzymes and transporters of the penicillin biosynthesis pathway in a high yielding Penicillium chrysogenum strain. The experiment consisted of a step response experiment with the side chain precursor phenyl acetic acid (PAA) in a glucose-limited chemostat. The metabolite data showed that in the absence of PAA all penicillin pathway enzymes were expressed, leading to the production of a significant amount of 6-aminopenicillanic acid (6APA) as end product. After the stepwise perturbation with PAA, the pathway produced PenG within seconds. From the extra- and intracellular metabolite measurements, hypotheses for the secretion mechanisms of penicillin pathway metabolites were derived. A dynamic model of the penicillin biosynthesis pathway was then constructed that included the formation and transport over the cytoplasmic membrane of pathway intermediates, PAA and the product penicillin-G (PenG). The model parameters and changes in the enzyme levels of the penicillin biosynthesis pathway under in vivo conditions were simultaneously estimated using experimental data obtained at three different timescales (seconds, minutes, hours). The model was applied to determine changes in the penicillin pathway enzymes in time, calculate fluxes and analyze the flux control of the pathway. This led to a reassessment of the in vivo behavior of the pathway enzymes and in particular Acyl-CoA:Isopenicillin N Acyltransferase (AT). PMID:26476338

  4. 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

  5. Effects of modulation of pentose-phosphate pathway on biosynthesis of ansamitocins in Actinosynnema pretiosum.

    PubMed

    Fan, Yuxiang; Hu, Fengxian; Wei, Liujing; Bai, Linquan; Hua, Qiang

    2016-07-20

    Ansamitocins, produced by Actinosynnema pretiosum, are a group of maytansinoid antibiotics that block the assembly of tubulin into functional microtubules. The precursors of ansamitocin biosynthesis are generally derived from the Embden-Meyerhof-Parnas (EMP) pathway and the tricarboxylic acid cycle. In this study, central carbon flux distributions were analyzed by (13)C-based flux analysis to reveal the contribution of individual central carbon metabolism pathways. To direct more carbon flux into ansamitocin biosynthesis, pentose phosphate (PP) pathway only and the combination of PP pathway and Entner-Doudoroff (ED) pathway were weakened, respectively. Ansamitocin P-3 (AP-3) productions by both kinds of pathways weakened mutant strains were significantly enhanced in chemically defined medium. In order to draw metabolic flux to the biosynthesis of ansamitocins more efficiently, heterologous phosphoglucomutase was subsequently overexpressed based on a mutant strain with combinational regulation of PP pathway and ED pathway. More fluxes were successfully directed into the UDP-glucose synthetic pathway and the AP-3 production was further improved in this case, reaching approximately 185mg/L in fermentation medium. It was demonstrated that eliminating the bypass pathways and favoring the precursor synthetic pathway could effectively improve ansamitocin production by A. pretiosum, suggesting a promising role of metabolic strategy in improving secondary metabolite production. PMID:27173582

  6. Stress-induced biosynthesis of dicaffeoylquinic acids in globe artichoke.

    PubMed

    Moglia, Andrea; Lanteri, Sergio; Comino, Cinzia; Acquadro, Alberto; de Vos, Ric; Beekwilder, Jules

    2008-09-24

    Leaf extracts from globe artichoke ( Cynara cardunculus L. var. scolymus) have been widely used in medicine as hepatoprotectant and choleretic agents. Globe artichoke leaves represent a natural source of phenolic acids with dicaffeoylquinic acids, such as cynarin (1,3-dicaffeoylquinic acid), along with its biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the most abundant molecules. This paper reports the development of an experimental system to induce caffeoylquinic acids. This system may serve to study the regulation of the biosynthesis of (poly)phenolic compounds in globe artichoke and the genetic basis of this metabolic regulation. By means of HPLC-PDA and accurate mass LC-QTOF MS and MS/MS analyses, the major phenolic compounds in globe artichoke leaves were identified: four isomers of dicaffeoylquinic acid, three isomers of caffeoylquinic acid, and the flavone luteolin 7-glucoside. Next, plant material was identified in which the concentration of phenolic compounds was comparable in the absence of particular treatments, with the aim to use this material to test the effect of stress application on the regulation of biosynthesis of caffeoylquinic acids. Using this material, the effect of UV-C, methyl jasmonate, and salicylic acid treatments on (poly)phenolic compounds was tested in different globe artichoke genotypes. UV-C exposure consistently increased the levels of dicaffeoylquinic acids in all genotypes, whereas the effect on compounds from the same biosynthetic pathway, for example, chlorogenic acid and luteolin-7-glucoside, was much less pronounced and was not statistically significant. No effect of methyl jasmonate or salicylic acid was found. Time-response experiments indicated that the level of dicaffeoylquinic acids reached a maximum at 24 h after UV radiation. On the basis of these results a role of dicaffeoylquinic acids in UV protection in globe artichoke is hypothesized. PMID:18710252

  7. Biosynthesis of the halogenated auxin, 4-chloroindole-3-acetic acid.

    PubMed

    Tivendale, Nathan D; Davidson, Sandra E; Davies, Noel W; Smith, Jason A; Dalmais, Marion; Bendahmane, Abdelhafid I; Quittenden, Laura J; Sutton, Lily; Bala, Raj K; Le Signor, Christine; Thompson, Richard; Horne, James; Reid, James B; Ross, John J

    2012-07-01

    Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea. PMID:22573801

  8. Biosynthesis of Phosphonic and Phosphinic Acid Natural Products

    PubMed Central

    Metcalf, William W.; van der Donk, Wilfred A.

    2009-01-01

    Natural products containing carbon-phosphorus bonds (phosphonic and phosphinic acids) have found widespread use in medicine and agriculture. Recent years have seen a renewed interest in the biochemistry and biology of these compounds with the cloning of the biosynthetic gene clusters for several family members. This review discusses the commonalities and differences in the molecular logic that lies behind the biosynthesis of these compounds. The current knowledge regarding the metabolic pathways and enzymes involved in the production of a number of natural products, including the approved antibiotic fosfomycin, the widely used herbicide phosphinothricin, and the clinical candidate for treatment of malaria FR900098, is presented. Many of the enzymes involved in the biosynthesis of these compounds catalyze chemically and biologically unprecedented transformations and a wealth of new biochemistry has been revealed through their study. These studies have also suggested new strategies for natural product discovery. PMID:19489722

  9. Inhibition of the isoprenoid biosynthesis pathway; detection of intermediates by UPLC-MS/MS.

    PubMed

    Henneman, Linda; van Cruchten, Arno G; Kulik, Willem; Waterham, Hans R

    2011-04-01

    The isoprenoid biosynthesis pathway provides the cell with a variety of compounds which are involved in multiple cellular processes. Inhibition of this pathway with statins and bisphosphonates is widely applied in the treatment of hypercholesterolemia and metabolic bone disease, respectively. In addition, since isoprenylation of proteins is an important therapeutic target in cancer research there is interest in interfering with isoprenoid biosynthesis, for which new inhibitors to block farnesylation and geranylgeranylation of small GTPases are being developed. We recently developed a sensitive method using UPLC-MS/MS that allows the direct detection and quantification of all intermediates of the mevalonate pathway from MVA to GGPP which can be used to verify the specificity of inhibitors of the isoprenoid biosynthesis pathway. We here investigated the specificity of several inhibitors of the isoprenoid biosynthesis pathway in HepG2 cells, fibroblasts and lymphoblasts. The nitrogen-containing bisphosphonates pamidronate and zoledronate specifically inhibit farnesyl pyrophosphate synthase indicated by the accumulation of IPP/DMAPP. However, zaragozic acid A, a squalene synthase inhibitor, causes an increase of MVA in addition to the expected increase of FPP. Analysis of isoprenoid intermediate profiles after incubation with 6-fluoromevalonate showed a very nonspecific result with an increase in MVA, MVAP, MVAPP and IPP/DMAPP. These results show that inhibitors of a particular enzyme of the isoprenoid biosynthesis pathway can have additional effects on other enzymes of the pathway either direct or indirect through accumulation of isoprenoid intermediates. Our method can be used to test new inhibitors and their effect on overall isoprenoid biosynthesis. PMID:21237288

  10. Pathways associated with lignin biosynthesis in lignomaniac jute fibres.

    PubMed

    Chakraborty, Avrajit; Sarkar, Debabrata; Satya, Pratik; Karmakar, Pran Gobinda; Singh, Nagendra Kumar

    2015-08-01

    We generated the bast transcriptomes of a deficient lignified phloem fibre mutant and its wild-type jute (Corchorus capsularis) using Illumina paired-end sequencing. A total of 34,163 wild-type and 29,463 mutant unigenes, with average lengths of 1442 and 1136 bp, respectively, were assembled de novo, ~77-79 % of which were functionally annotated. These annotated unigenes were assigned to COG (~37-40 %) and GO (~22-28 %) classifications and mapped to 189 KEGG pathways (~19-21 %). We discovered 38 and 43 isoforms of 16 and 10 genes of the upstream shikimate-aromatic amino acid and downstream monolignol biosynthetic pathways, respectively, rendered their sequence similarities, confirmed the identities of 22 of these candidate gene families by phylogenetic analyses and reconstructed the pathway leading to lignin biosynthesis in jute fibres. We also identified major genes and bast-related transcription factors involved in secondary cell wall (SCW) formation. The quantitative RT-PCRs revealed that phenylalanine ammonia-lyase 1 (CcPAL1) was co-down-regulated with several genes of the upstream shikimate pathway in mutant bast tissues at an early growth stage, although its expression relapsed to the normal level at the later growth stage. However, cinnamyl alcohol dehydrogenase 7 (CcCAD7) was strongly down-regulated in mutant bast tissues irrespective of growth stages. CcCAD7 disruption at an early growth stage was accompanied by co-up-regulation of SCW-specific genes cellulose synthase A7 (CcCesA7) and fasciclin-like arabinogalactan 6 (CcFLA6), which was predicted to be involved in coordinating the S-layers' deposition in the xylan-type jute fibres. Our results identified CAD as a promising target for developing low-lignin jute fibres using genomics-assisted molecular approaches. PMID:25724692

  11. 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.

  12. Biosynthesis of the labdane diterpene marrubiin in Marrubium vulgare via a non-mevalonate pathway.

    PubMed

    Knöss, W; Reuter, B; Zapp, J

    1997-09-01

    The biosynthesis of the furanic labdane diterpene marrubiin has been studied in plantlets and shoot cultures of Marrubium vulgare (Lamiaceae). The use of [2-14C]acetate, [2-14C]pyruvate, [2-14C]mevalonic acid and [U-14C]glucose incorporation experiments showed that the labelling of sterols in etiolated shoot cultures of M. vulgare was in accordance with their biosynthesis via the acetate-mevalonate pathway. In contrast, the incorporation rates of these precursors into the diterpene marrubiin could not be explained by biosynthesis of this compound via the acetate-mevalonate pathway. Cultivation of etiolated shoot cultures of M. vulgare on medium containing [1-13C]glucose and subsequent 13C-NMR spectroscopy of marrubiin led to the conclusion that the biosynthesis of marrubiin follows a non-mevalonate pathway. All isoprenic units of 13C-labelled marrubiin were enriched in those carbons that correspond to positions 1 and 5 of a putative precursor isopentenyl diphosphate. This labelling pattern from [1-13C]glucose is consistent with an alternative pathway via trioses, which has already been shown to occur in Eubacteria and Gymnospermae. The labdane skeleton is a precursor of many other skeletal types of diterpenes. Therefore it becomes obvious that in connection with the few known examples of a non-mevalonate pathway to isoprenoids the formation of some isoprenoids in plants via a non-mevalonate pathway might be quite common. PMID:9291117

  13. Carnosic acid biosynthesis elucidated by a synthetic biology platform.

    PubMed

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

    2016-03-29

    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

  14. Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs

    PubMed Central

    de Souza, Wanderley; Rodrigues, Juliany Cola Fernandes

    2009-01-01

    Sterols are constituents of the cellular membranes that are essential for their normal structure and function. In mammalian cells, cholesterol is the main sterol found in the various membranes. However, other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. It is now well established that an important metabolic pathway in fungi and in members of the Trypanosomatidae family is one that produces a special class of sterols, including ergosterol, and other 24-methyl sterols, which are required for parasitic growth and viability, but are absent from mammalian host cells. Currently, there are several drugs that interfere with sterol biosynthesis (SB) that are in use to treat diseases such as high cholesterol in humans and fungal infections. In this review, we analyze the effects of drugs such as (a) statins, which act on the mevalonate pathway by inhibiting HMG-CoA reductase, (b) bisphosphonates, which interfere with the isoprenoid pathway in the step catalyzed by farnesyl diphosphate synthase, (c) zaragozic acids and quinuclidines, inhibitors of squalene synthase (SQS), which catalyzes the first committed step in sterol biosynthesis, (d) allylamines, inhibitors of squalene epoxidase, (e) azoles, which inhibit C14α-demethylase, and (f) azasterols, which inhibit Δ24(25)-sterol methyltransferase (SMT). Inhibition of this last step appears to have high selectivity for fungi and trypanosomatids, since this enzyme is not found in mammalian cells. We review here the IC50 values of these various inhibitors, their effects on the growth of trypanosomatids (both in axenic cultures and in cell cultures), and their effects on protozoan structural organization (as evaluted by light and electron microscopy) and lipid composition. The results show that the mitochondrial membrane as well as the membrane lining the protozoan cell body and flagellum are the main targets. Probably as a consequence of these primary effects, other important changes take place in

  15. Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs.

    PubMed

    de Souza, Wanderley; Rodrigues, Juliany Cola Fernandes

    2009-01-01

    Sterols are constituents of the cellular membranes that are essential for their normal structure and function. In mammalian cells, cholesterol is the main sterol found in the various membranes. However, other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. It is now well established that an important metabolic pathway in fungi and in members of the Trypanosomatidae family is one that produces a special class of sterols, including ergosterol, and other 24-methyl sterols, which are required for parasitic growth and viability, but are absent from mammalian host cells. Currently, there are several drugs that interfere with sterol biosynthesis (SB) that are in use to treat diseases such as high cholesterol in humans and fungal infections. In this review, we analyze the effects of drugs such as (a) statins, which act on the mevalonate pathway by inhibiting HMG-CoA reductase, (b) bisphosphonates, which interfere with the isoprenoid pathway in the step catalyzed by farnesyl diphosphate synthase, (c) zaragozic acids and quinuclidines, inhibitors of squalene synthase (SQS), which catalyzes the first committed step in sterol biosynthesis, (d) allylamines, inhibitors of squalene epoxidase, (e) azoles, which inhibit C14alpha-demethylase, and (f) azasterols, which inhibit Delta(24(25))-sterol methyltransferase (SMT). Inhibition of this last step appears to have high selectivity for fungi and trypanosomatids, since this enzyme is not found in mammalian cells. We review here the IC50 values of these various inhibitors, their effects on the growth of trypanosomatids (both in axenic cultures and in cell cultures), and their effects on protozoan structural organization (as evaluted by light and electron microscopy) and lipid composition. The results show that the mitochondrial membrane as well as the membrane lining the protozoan cell body and flagellum are the main targets. Probably as a consequence of these primary effects, other important changes take

  16. 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.

  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. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis.

    PubMed

    Kuzuyama, Tomohisa; Seto, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP. PMID:22450534

  19. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis

    PubMed Central

    KUZUYAMA, Tomohisa; SETO, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP. PMID:22450534

  20. Elucidation of an alternate isoleucine biosynthesis pathway in Geobacter sulfurreducens.

    PubMed

    Risso, Carla; Van Dien, Stephen J; Orloff, Amber; Lovley, Derek R; Coppi, Maddalena V

    2008-04-01

    The central metabolic model for Geobacter sulfurreducens included a single pathway for the biosynthesis of isoleucine that was analogous to that of Escherichia coli, in which the isoleucine precursor 2-oxobutanoate is generated from threonine. 13C labeling studies performed in G. sulfurreducens indicated that this pathway accounted for a minor fraction of isoleucine biosynthesis and that the majority of isoleucine was instead derived from acetyl-coenzyme A and pyruvate, possibly via the citramalate pathway. Genes encoding citramalate synthase (GSU1798), which catalyzes the first dedicated step in the citramalate pathway, and threonine ammonia-lyase (GSU0486), which catalyzes the conversion of threonine to 2-oxobutanoate, were identified and knocked out. Mutants lacking both of these enzymes were auxotrophs for isoleucine, whereas single mutants were capable of growth in the absence of isoleucine. Biochemical characterization of the single mutants revealed deficiencies in citramalate synthase and threonine ammonia-lyase activity. Thus, in G. sulfurreducens, 2-oxobutanoate can be synthesized either from citramalate or threonine, with the former being the main pathway for isoleucine biosynthesis. The citramalate synthase of G. sulfurreducens constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases, which contains representatives from a wide variety of microorganisms. PMID:18245290

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

    SciTech Connect

    Sporty, J; Lin, S; Kato, M; Ognibene, T; Stewart, B; Turteltaub, K; Bench, G

    2009-02-18

    Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses on BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD

  3. Genome-Wide Analysis of the Lysine Biosynthesis Pathway Network during Maize Seed Development

    PubMed Central

    Liu, Yuwei; Xie, Shaojun; Yu, Jingjuan

    2016-01-01

    Lysine is one of the most limiting essential amino acids for humans and livestock. The nutritional value of maize (Zea mays L.) is reduced by its poor lysine content. To better understand the lysine biosynthesis pathway in maize seed, we conducted a genome-wide analysis of the genes involved in lysine biosynthesis. We identified lysine biosynthesis pathway genes (LBPGs) and investigated whether a diaminopimelate pathway variant exists in maize. We analyzed two genes encoding the key enzyme dihydrodipicolinate synthase, and determined that they contribute differently to lysine synthesis during maize seed development. A coexpression network of LBPGs was constructed using RNA-sequencing data from 21 developmental stages of B73 maize seed. We found a large set of genes encoding ribosomal proteins, elongation factors and zein proteins that were coexpressed with LBPGs. The coexpressed genes were enriched in cellular metabolism terms and protein related terms. A phylogenetic analysis of the LBPGs from different plant species revealed different relationships. Additionally, six transcription factor (TF) families containing 13 TFs were identified as the Hub TFs of the LBPGs modules. Several expression quantitative trait loci of LBPGs were also identified. Our results should help to elucidate the lysine biosynthesis pathway network in maize seed. PMID:26829553

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

    PubMed

    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. PMID:24625837

  5. 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.

  6. Inhibitors of fatty acid biosynthesis in sunflower seeds.

    PubMed

    Pleite, Rafael; Martínez-Force, Enrique; Garcés, Rafael

    2006-09-01

    During de novo fatty acid synthesis in sunflower seeds, saturated fatty acid production is influenced by the competition between the enzymes of the principal pathways and the saturated acyl-ACP thioesterases. Genetic backgrounds with more efficient saturated acyl-ACP thioesterase alleles only express their phenotypic effects when the alleles for the enzymes in the main pathway are less efficient. For this reason, we studied the incorporation of [2-(14)C]acetate into the lipids of developing sunflower seeds (Helianthus annuus L.) from several mutant lines in vivo. The labelling of different triacylglycerol fatty acids in different oilseed mutants reflects the fatty acid composition of the seed and supports the channelling theory of fatty acid biosynthesis. Incubation with methyl viologen diminished the conversion of stearoyl-ACP to oleoyl-ACP in vivo through a decrease in the available reductant power. In turn, this led to the accumulation of stearoyl-ACP to the levels detected in seeds from high stearic acid mutants. The concomitant reduction of oleoyl-ACP content inside the plastid allowed us to study the activity of acyl-ACP thioesterases on saturated fatty acids. In these mutants, we verified that the accumulation of saturated fatty acids requires efficient thioesterase activity on saturated-ACPs. By studying the effects of cerulenin on the in vivo incorporation of [2-(14)C]acetate into lipids and on the in vitro activity of beta-ketoacyl-ACP synthase II, we found that elongation to very long chain fatty acids can occur both inside and outside of the plastid in sunflower seeds. PMID:16500723

  7. Molecular Genetic Characterization of Terreic Acid Pathway in Aspergillus terreus

    SciTech Connect

    Guo, Chun-Jun; Sun, Wei-wen; Bruno, Kenneth S.; Wang, Clay C.

    2014-09-29

    Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact gene cluster for its biosynthesis was characterized. Isolation of the intermediates and shunt products from the mutant strains, in combined with bioinformatic analyses, allowed us to propose a biosynthetic pathway for terreic acid. Defining the pathway and the genes involved will facilitate the engineering of this molecule with interesting antimicrobial and antitumor bioactivities.

  8. Molecular Genetic Characterization of Terreic Acid Pathway in Aspergillus terreus

    SciTech Connect

    Guo, Chun-Jun; Sun, Wei-wen; Bruno, Kenneth S.; Wang, Clay C.

    2014-09-29

    Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact gene cluster for its biosynthesis was characterized. Isolation of the intermediates and shunt products from the mutant strains, in combined with bioinformatic analyses, allowed us to propose a biosynthetic pathway for terreic acid. Lastly, defining the pathway and the genes involved will facilitate the engineering of this molecule with interesting antimicrobial and antitumor bioactivities.

  9. Intermediates of Salicylic Acid Biosynthesis in Tobacco1

    PubMed Central

    Ribnicky, David M.; Shulaev, Vladimir; Raskin, Ilya

    1998-01-01

    Salicylic acid (SA) is an important component of systemic-acquired resistance in plants. It is synthesized from benzoic acid (BA) as part of the phenylpropanoid pathway. Benzaldehyde (BD), a potential intermediate of this pathway, was found in healthy and tobacco mosaic virus (TMV)-inoculated tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaf tissue at 100 ng/g fresh weight concentrations as measured by gas chromatography-mass spectrometry. BD was also emitted as a volatile organic compound from tobacco tissues. Application of gaseous BD to plants enclosed in jars caused a 13-fold increase in SA concentration, induced the accumulation of the pathogenesis-related transcript PR-1, and increased the resistance of tobacco to TMV inoculation. [13C6]BD and [2H5]benzyl alcohol were converted to BA and SA. Labeling experiments using [13C1]Phe in temperature-shifted plants inoculated with the TMV showed high enrichment of cinnamic acids (72%), BA (34%), and SA (55%). The endogenous BD, however, contained nondetectable enrichment, suggesting that BD was not the intermediate between cinnamic acid and BA. These results show that BD and benzyl alcohol promote SA accumulation and expression of defense responses in tobacco, and provide insight into the early steps of SA biosynthesis. PMID:9765542

  10. Molecular Evolution of Multiple-Level Control of Heme Biosynthesis Pathway in Animal Kingdom

    PubMed Central

    Tzou, Wen-Shyong; Chu, Ying; Lin, Tzung-Yi; Hu, Chin-Hwa; Pai, Tun-Wen; Liu, Hsin-Fu; Lin, Han-Jia; Cases, Ildeofonso; Rojas, Ana; Sanchez, Mayka; You, Zong-Ye; Hsu, Ming-Wei

    2014-01-01

    Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5′ untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway. PMID:24489775

  11. 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. PMID:10506831

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

    PubMed

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

    2016-01-01

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

  13. Abscisic acid biosynthesis in leaves and roots of Xanthium strumarium

    SciTech Connect

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

    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. The authors have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in /sup 18/O/sub 2/. It was found that in stressed leaves three atoms of /sup 18/O from /sup 18/O/sub 2/ are incorporated into the ABA molecule, and that the amount of /sup 18/O incorporated increases with time. One /sup 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 /sup 18/O/sub 2/ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more /sup 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 plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, /sup 18/O is incorporated into ABA to a much lesser extent that it is in stressed leaves, whereas exogenously applied /sup 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 /sup 18/O incorporated during 8'-hydroxylation of ABA to phaseic acid.

  14. Fungal Indole Alkaloid Biosynthesis: Genetic and Biochemical Investigation of Tryptoquialanine Pathway in Penicillium aethiopicum

    PubMed Central

    Gao, Xue; Chooi, Yit-Heng; Ames, Brian D.; Wang, Peng; Walsh, Christopher T.; Tang, Yi

    2011-01-01

    Tremorgenic mycotoxins are a group of indole alkaloids which include the quinazoline-containing tryptoquivaline 2 that are capable of eliciting intermittent or sustained tremors in vertebrate animals. The biosynthesis of this group of bioactive compounds, which are characterized by an acetylated quinazoline ring connected to a 6-5-5 imidazoindolone ring system via a 5-membered spirolactone, has remained uncharacterized. Here, we report the identification of a gene cluster (tqa) from P. aethiopicum that is involved in the biosynthesis of tryptoquialanine 1, which is structurally similar to 2. The pathway has been confirmed to go through an intermediate common to the fumiquinazoline pathway, fumiquinazoline F, which originates from a fungal trimodular nonribosomal peptide synthetase (NRPS). By systematically inactivating every biosynthetic gene in the cluster, followed by isolation and characterization of the intermediates, we were able to establish the biosynthetic sequence of the pathway. An unusual oxidative opening of the pyrazinone ring by an FAD-dependent berberine bridge enzyme-like oxidoreductase has been proposed based on genetic knockout studies. Notably, a 2-aminoisobutyric acid (AIB)-utilizing NRPS module has been identified and reconstituted in vitro, along with two putative enzymes of unknown functions that are involved in the synthesis of the unnatural amino acid by genetic analysis. This work provides new genetic and biochemical insights into the biosynthesis of this group of fungal alkaloids, including the tremorgens related to 2. PMID:21299212

  15. Salicylic acid biosynthesis is enhanced and contributes to increased biotrophic pathogen resistance in Arabidopsis hybrids.

    PubMed

    Yang, Li; Li, Bosheng; Zheng, Xiao-yu; Li, Jigang; Yang, Mei; Dong, Xinnian; He, Guangming; An, Chengcai; Deng, Xing Wang

    2015-01-01

    Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen. PMID:26065719

  16. Salicylic acid biosynthesis is enhanced and contributes to increased biotrophic pathogen resistance in Arabidopsis hybrids

    PubMed Central

    Yang, Li; Li, Bosheng; Zheng, Xiao-yu; Li, Jigang; Yang, Mei; Dong, Xinnian; He, Guangming; An, Chengcai; Deng, Xing Wang

    2015-01-01

    Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen. PMID:26065719

  17. Localization of heme biosynthesis pathway enzymes in Plasmodium falciparum.

    PubMed

    Rao, Aditya; Yeleswarapu, Sri Jyothsna; Srinivasan, Rajgopal; Bulusu, Gopalakrishnan

    2008-12-01

    Protein trafficking in the malarial parasite Plasmodium falciparum is dictated by a complex life-cycle that involves a variety of intra-cellular and host cell destinations, such as the mitochondrion, apicoplast, rhoptries and micronemes. Of these, the apicoplast and mitochondrion are believed to account for more than 10% of this traffic. Studies have shown that mechanisms for mitochondrion and apicoplast targeting are distinct, despite their close physical proximity. The heme biosynthesis pathway spans both these organelles, making trafficking studies crucial for the spatial demarcation of the constituent interactions. This minireview highlights the challenges in identifying the possible sub-cellular destinations of the heme pathway enzymes using gleanings from literature survey as well as focussed bioinformatic analysis. PMID:19239121

  18. 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

  19. GROWTH RETARDANTS: Effects on Gibberellin Biosynthesis and Other Metabolic Pathways.

    PubMed

    Rademacher, Wilhelm

    2000-06-01

    Plant growth retardants are applied in agronomic and horticultural crops to reduce unwanted longitudinal shoot growth without lowering plant productivity. Most growth retardants act by inhibiting gibberellin (GA) biosynthesis. To date, four different types of such inhibitors are known: (a) Onium compounds, such as chlormequat chloride, mepiquat chloride, chlorphonium, and AMO-1618, which block the cyclases copalyl-diphosphate synthase and ent-kaurene synthase involved in the early steps of GA metabolism. (b) Compounds with an N-containing heterocycle, e.g. ancymidol, flurprimidol, tetcyclacis, paclobutrazol, uniconazole-P, and inabenfide. These retardants block cytochrome P450-dependent monooxygenases, thereby inhibiting oxidation of ent-kaurene into ent-kaurenoic acid. (c) Structural mimics of 2-oxoglutaric acid, which is the co-substrate of dioxygenases that catalyze late steps of GA formation. Acylcyclohexanediones, e.g. prohexadione-Ca and trinexapac-ethyl and daminozide, block particularly 3ss-hydroxylation, thereby inhibiting the formation of highly active GAs from inactive precursors, and (d) 16,17-Dihydro-GA5 and related structures act most likely by mimicking the GA precursor substrate of the same dioxygenases. Enzymes, similar to the ones involved in GA biosynthesis, are also of importance in the formation of abscisic acid, ethylene, sterols, flavonoids, and other plant constituents. Changes in the levels of these compounds found after treatment with growth retardants can mostly be explained by side activities on such enzymes. PMID:15012200

  20. Methionine salvage pathway in relation to ethylene biosynthesis

    SciTech Connect

    Miyazaki, J.H.

    1987-01-01

    The recycling of methionine during ethylene biosynthesis (the methionine cycle) was studied. During ethylene biosynthesis, the H/sub 3/CS-group of S-adenosylmethionine (SAM) is released at 5'-methylthioadenosine (MTA), which is recycled to methionine via 5'-methylthioribose (MTS). In mungbean hypocotyls and cell-free extracts of avocado fruit, (/sup 14/C)MTR was converted to labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB) as intermediates. Radioactive tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract required an amino group donor. Among several potential donors tested, L-glutamine was the most efficient. Incubation of (ribose-U-/sup 14/C)MTR with avocado extract resulted in the production of (/sup 14/C)formate, with little evolution of other /sup 14/C-labeled one-carbon compounds, indicating that the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR.

  1. Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. 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

  3. Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

    PubMed Central

    Black, Katherine A.

    2015-01-01

    ABSTRACT The 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U in Escherichia coli requires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). The E. coli MnmA adenylates and subsequently thiolates tRNA to form the s2U modification. Bacillus subtilis lacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene, yrvO, directly adjacent to mnmA. The genomic synteny of yrvO and mnmA combined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that the B. subtilis YrvO and MnmA are sufficient for s2U biosynthesis. A conditional B. subtilis knockout strain showed that s2U abundance correlates with MnmA expression, and in vivo complementation studies in E. coli IscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis. In vitro experiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between the B. subtilis proteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype of E. coli ΔiscS and ΔmnmA strains associated with s2U depletion is recovered by B. subtilis yrvO and mnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA. IMPORTANCE The 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U in Escherichia coli requires

  4. 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

  5. Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae

    PubMed Central

    Chang, Perng-Kuang; Ehrlich, Kenneth C.; Fujii, Isao

    2009-01-01

    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 what is currently known about the toxicity of CPA to animals and humans, both by itself or in combination with other mycotoxins. The review also discusses CPA biosynthesis and the genetic diversity of CPA production in A. flavus/oryzae populations. PMID:22069533

  6. Farnesol biosynthesis in Candida albicans: cellular response to sterol inhibition by zaragozic acid B.

    PubMed

    Hornby, Jacob M; Kebaara, Bessie W; Nickerson, Kenneth W

    2003-07-01

    The dimorphic fungus Candida albicans produces farnesol as a quorum-sensing molecule that regulates cellular morphology. The biosynthetic origin of farnesol has been resolved by treating these cells with zaragozic acid B, a potent inhibitor of squalene synthase in the sterol biosynthetic pathway. Treatment with zaragozic acid B leads to an eightfold increase in the amount of farnesol produced by C. albicans. Furthermore, C. albicans cell extracts contain enzymatic activity to convert [(3)H]farnesyl pyrophosphate to [(3)H]farnesol. Many common antifungal antibiotics (e.g., zaragozic acids, azoles, and allylamines) target steps in sterol biosynthesis. We suggest that the fungicidal activity of zaragozic acid derives in large part from the accumulation of farnesol that accompanies the inhibition of sterol biosynthesis. PMID:12821501

  7. Glucosinolate and Amino Acid Biosynthesis in Arabidopsis1

    PubMed Central

    Field, Ben; Cardon, Guillermo; Traka, Maria; Botterman, Johan; Vancanneyt, Guy; Mithen, Richard

    2004-01-01

    Enzymes that catalyze the condensation of acetyl coenzyme A and 2-oxo acids are likely to be important in two distinct metabolic pathways in Arabidopsis. These are the synthesis of isopropylmalate, an intermediate of Leu biosynthesis in primary metabolism, and the synthesis of methylthioalkylmalates, intermediates of Met elongation in the synthesis of aliphatic glucosinolates (GSLs), in secondary metabolism. Four Arabidopsis genes in the ecotype Columbia potentially encode proteins that could catalyze these reactions. MAM1 and MAML are adjacent genes on chromosome 5 at the Gsl-elong locus, while MAML-3 and MAML-4 are at opposite ends of chr 1. The isopropylmalate synthase activity of each member of the MAM-like gene family was investigated by heterologous expression in an isopropylmalate synthase-null Escherichia coli mutant. Only the expression of MAML-3 restored the ability of the mutant to grow in the absence of Leu. A MAML knockout line (KO) lacked long-chain aliphatic GSLs, which were restored when the KO was transformed with a functional MAML gene. Variation in expression of MAML did not alter the total levels of Met-derived GSLs, but just the ratio of chain lengths. MAML overexpression in Columbia led to an increase in long-chain GSLs, and an increase in 3C GSLs. Moreover, plants overexpressing MAML contained at least two novel amino acids. One of these was positively identified via MS/MS as homo-Leu, while the other, with identical mass and fragmentation patterns, was likely to be homo-Ile. A MAML-4 KO did not exhibit any changes in GSL profile, but had perturbed soluble amino acid content. PMID:15155874

  8. 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. PMID:25353310

  9. The novel pathway for ketodiene oxylipin biosynthesis in Jerusalem artichoke (Helianthus tuberosus) tubers.

    PubMed

    Chechetkin, Ivan R; Medvedeva, Natalia V; Grechkin, Alexander N

    2004-11-01

    The new route of the plant lipoxygenase pathway, directed specifically towards the ketodiene formation, was detected during in vitro experiments with Jerusalem artichoke (Helianthus tuberosus) tubers. Through this pathway (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD) is reduced to corresponding 13-hydroxy acid (13-HOD), which is in turn dehydrogenated into ketodiene (9Z,11E,13S)-13-oxo-9,11-octadecadienoic acid (13-KOD). Dehydrogenation of 13-HOD into 13-KOD was not dependent on the presence of either NAD or NADP, but was strongly dependent on the presence of oxygen. Under anoxic conditions, 13-HOD dehydrogenation was blocked, but addition of 2,6-dichlorophenolindophenol restored it. Sulfite addition fully suppressed the aerobic dehydrogenation of 13-HOD. Hydrogen peroxide is a by-product formed by the enzyme along with 13-KOD. These data suggest that the ketodiene biosynthesis in H. tuberosus tubers is catalyzed by flavin dehydrogenase. (9S,10E,12Z)-9-Hydroxy-10,12-octadecadienoic acid (9-HOD) is dehydrogenated by this enzyme as effectively as 13-HOD, while alpha-ketol, (9Z)-12-oxo-13-hydroxy-9-octadecenoic acid, and ricinoleic acid did not act as substrates for dehydrogenase. The enzyme was soluble and possessed a pH optimum at pH 7.0-9.0. The only 13-HOD dehydrogenase known so far was detected in rat colon. However, unlike the H. tuberosus enzyme, the rat dehydrogenase is NAD-dependent. PMID:15522817

  10. 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. PMID:27004948

  11. 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

  12. Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway.

    PubMed

    Xu, Hai; Zhang, Yuzhen; Guo, Xiaokui; Ren, Shuangxi; Staempfli, Andreas A; Chiao, Juishen; Jiang, Weihong; Zhao, Guoping

    2004-08-01

    Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded alpha-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K(m) (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed beta-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes. PMID:15292141

  13. Molecular Pathways: Inhibiting steroid biosynthesis in prostate cancer

    PubMed Central

    Ferraldeschi, Roberta; Sharifi, Nima; Auchus, Richard J.; Attard, Gerhardt

    2013-01-01

    A significant proportion of castration-resistant prostate cancers (CRPC) remain driven by ligand activation of the androgen receptor. Although the testes are the primary source of testosterone, testosterone can also be produced from peripheral conversion of adrenal sex hormone precursors dehydroepiandrosterone (DHEA) and androstenedione (AD) in the prostate and other tissues. CYP17A1 catalyzes two essential reactions in the production of DHEA and androstenedione: the hydroxylation (hydroxylase activity) and the subsequent cleavage of the C17-20 side-chain (lyase activity). Potent and selective inhibition of CYP17A1 by abiraterone depletes residual non-gonadal androgens and is an effective treatment for CRPC. Elucidation of the mechanisms that underlie resistance to abiraterone will inform on the development of novel therapeutic strategies post abiraterone. Preclinical evidence that androgen biosynthesis in prostate cancer cells does not necessarily follow a single dominant pathway and residual androgens or alternative ligands (including administered glucocorticoids) can reactivate androgen receptor signaling supports co-targeting of more than one enzyme involved in steroidogenesis and combining a CYP17A1 inhibitor with an anti-androgen. Furthermore, given the drawbacks of 17α-hydroxylase inhibition, there is considerable interest in developing new CYP17A1 inhibitors that more specifically inhibit lyase activity and are therefore less likely to require glucocorticoid co-administration. PMID:23470964

  14. Pathways of O-glycan biosynthesis in cancer cells.

    PubMed

    Brockhausen, I

    1999-12-01

    Glycoproteins with O-glycosidically linked carbohydrate chains of complex structures and functions are found in secretions and on the cell surfaces of cancer cells. The structures of O-glycans are often unusual or abnormal in cancer, and greatly contribute to the phenotype and biology of cancer cells. Some of the mechanisms of changes in O-glycosylation pathways have been determined in cancer model systems. However, O-glycan biosynthesis is a complex process that is still poorly understood. The glycosyltransferases and sulfotransferases that synthesize O-glycans appear to exist as families of related enzymes of which individual members are expressed in a tissue- and growth-specific fashion. Studies of their regulation in cancer may reveal the connection between cancerous transformation and glycosylation which may help to understand and control the abnormal biology of tumor cells. Cancer diagnosis may be based on the appearance of certain glycosylated epitopes, and therapeutic avenues have been designed to attack cancer cells via their glycans. PMID:10580130

  15. Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

    SciTech Connect

    Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

    1988-03-01

    Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added /sup 14/CO/sub 2/ was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.

  16. Auxin biosynthesis in pea: characterization of the tryptamine pathway.

    PubMed

    Quittenden, Laura J; Davies, Noel W; Smith, Jason A; Molesworth, Peter P; Tivendale, Nathan D; Ross, John J

    2009-11-01

    One pathway leading to the bioactive auxin, indole-3-acetic acid (IAA), is known as the tryptamine pathway, which is suggested to proceed in the sequence: tryptophan (Trp), tryptamine, N-hydroxytryptamine, indole-3-acetaldoxime, indole-3-acetaldehyde (IAAld), IAA. Recently, this pathway has been characterized by the YUCCA genes in Arabidopsis (Arabidopsis thaliana) and their homologs in other species. YUCCA is thought to be responsible for the conversion of tryptamine to N-hydroxytryptamine. Here we complement the genetic findings with a compound-based approach in pea (Pisum sativum), detecting potential precursors by gas chromatography/tandem-mass spectrometry. In addition, we have synthesized deuterated forms of many of the intermediates involved, and have used them to quantify the endogenous compounds, and to investigate their metabolic fates. Trp, tryptamine, IAAld, indole-3-ethanol, and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence: Trp, tryptamine, IAAld, IAA, with indole-3-ethanol as a side-branch product of IAAld. N-hydroxytryptamine was not detected, but we cannot exclude that it is an intermediate between tryptamine and IAAld, nor can we rule out the possibility of a Trp-independent pathway operating in pea roots. PMID:19710233

  17. 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

  18. 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

  19. 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.

  20. Evidence from Solanum tuberosum in support of the dual-pathway hypothesis of aromatic biosynthesis

    SciTech Connect

    Morris, P.F.; Doong, R.L.; Jensen, R.A. )

    1989-01-01

    Key branchpoint enzymes of aromatic amino acid biosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DS) and chorismate mutase (CM), have previously been shown to exist as separate compartmentalized isozymes in the chloroplasts and cytosol of tobacco, sorghum and spinach. Although additional examples of plants containing these isozyme pairs are accumulating, some studies in the literature report the presence of only the single plastidic DS or CM enzyme. Such apparent exceptions contradict the universality of pathway organization existing in higher plants that is implied by the dual-pathway hypothesis of aromatic biosynthesis. Since potato (Solanum tuberosum) exemplifies a case where only a single species of both DS and CM have been reported, we selected this system for further analysis. The DS-Mn and DS-Co isozyme pair, exhibiting all of the differential properties described in Nicotiana silvestris, have now been identified in S. tuberosum. Likwise, partial purification via DEAE-cellulose chromatography revealed two isozymes of CM in disks excised from tubers of S. tuberosum. The differential regulatory properties of these isozymes were comparable to the CM-1 and CM-2 isozymes of N. silvestris.

  1. Evidence from Solanum tuberosum in Support of the Dual-Pathway Hypothesis of Aromatic Biosynthesis.

    PubMed

    Morris, P F; Doong, R L; Jensen, R A

    1989-01-01

    Key branchpoint enzymes of aromatic amino acid biosynthesis, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DS) and chorismate mutase (CM), have previously been shown to exist as separate compartmentalized isozymes in the chloroplasts and cytosol of tobacco, sorghum and spinach. Although additional examples of plants containing these isozyme pairs are accumulating, some studies in the literature report the presence of only the single plastidic DS or CM enzyme. Such apparent exceptions contradict the universality of pathway organization existing in higher plants that is implied by the dual-pathway hypothesis of aromatic biosynthesis. Since potato (Solanum tuberosum) exemplifies a case where only a single species of both DS and CM have been reported, we selected this system for further analysis. The DS-Mn and DS-Co isozyme pair, exhibiting all of the differential properties described in Nicotiana silvestris, have now been identified in S. tuberosum. Likewise, partial purification via DEAE-cellulose chromatography revealed two isozymes of CM in disks excised from tubers of S. tuberosum. The differential regulatory properties of these isozymes were comparable to the CM-1 and CM-2 isozymes of N. silvestris. PMID:16666497

  2. Aromatic Glucosinolate Biosynthesis Pathway in Barbarea vulgaris and its Response to Plutella xylostella Infestation.

    PubMed

    Liu, Tongjin; Zhang, Xiaohui; Yang, Haohui; Agerbirk, Niels; Qiu, Yang; Wang, Haiping; Shen, Di; Song, Jiangping; Li, Xixiang

    2016-01-01

    The inducibility of the glucosinolate resistance mechanism is an energy-saving strategy for plants, but whether induction would still be triggered by glucosinolate-tolerant Plutella xylostella (diamondback moth, DBM) after a plant had evolved a new resistance mechanism (e.g., saponins in Barbara vulgaris) was unknown. In B. vulgaris, aromatic glucosinolates derived from homo-phenylalanine are the dominant glucosinolates, but their biosynthesis pathway was unclear. In this study, we used G-type (pest-resistant) and P-type (pest-susceptible) B. vulgaris to compare glucosinolate levels and the expression profiles of their biosynthesis genes before and after infestation by DBM larvae. Two different stereoisomers of hydroxylated aromatic glucosinolates are dominant in G- and P-type B. vulgaris, respectively, and are induced by DBM. The transcripts of genes in the glucosinolate biosynthesis pathway and their corresponding transcription factors were identified from an Illumina dataset of G- and P-type B. vulgaris. Many genes involved or potentially involved in glucosinolate biosynthesis were induced in both plant types. The expression patterns of six DBM induced genes were validated by quantitative PCR (qPCR), while six long-fragment genes were validated by molecular cloning. The core structure biosynthetic genes showed high sequence similarities between the two genotypes. In contrast, the sequence identity of two apparent side chain modification genes, the SHO gene in the G-type and the RHO in P-type plants, showed only 77.50% identity in coding DNA sequences and 65.48% identity in deduced amino acid sequences. The homology to GS-OH in Arabidopsis, DBM induction of the transcript and a series of qPCR and glucosinolate analyses of G-type, P-type and F1 plants indicated that these genes control the production of S and R isomers of 2-hydroxy-2-phenylethyl glucosinolate. These glucosinolates were significantly induced by P. xylostella larvae in both the susceptiple P

  3. Aromatic Glucosinolate Biosynthesis Pathway in Barbarea vulgaris and its Response to Plutella xylostella Infestation

    PubMed Central

    Liu, Tongjin; Zhang, Xiaohui; Yang, Haohui; Agerbirk, Niels; Qiu, Yang; Wang, Haiping; Shen, Di; Song, Jiangping; Li, Xixiang

    2016-01-01

    The inducibility of the glucosinolate resistance mechanism is an energy-saving strategy for plants, but whether induction would still be triggered by glucosinolate-tolerant Plutella xylostella (diamondback moth, DBM) after a plant had evolved a new resistance mechanism (e.g., saponins in Barbara vulgaris) was unknown. In B. vulgaris, aromatic glucosinolates derived from homo-phenylalanine are the dominant glucosinolates, but their biosynthesis pathway was unclear. In this study, we used G-type (pest-resistant) and P-type (pest-susceptible) B. vulgaris to compare glucosinolate levels and the expression profiles of their biosynthesis genes before and after infestation by DBM larvae. Two different stereoisomers of hydroxylated aromatic glucosinolates are dominant in G- and P-type B. vulgaris, respectively, and are induced by DBM. The transcripts of genes in the glucosinolate biosynthesis pathway and their corresponding transcription factors were identified from an Illumina dataset of G- and P-type B. vulgaris. Many genes involved or potentially involved in glucosinolate biosynthesis were induced in both plant types. The expression patterns of six DBM induced genes were validated by quantitative PCR (qPCR), while six long-fragment genes were validated by molecular cloning. The core structure biosynthetic genes showed high sequence similarities between the two genotypes. In contrast, the sequence identity of two apparent side chain modification genes, the SHO gene in the G-type and the RHO in P-type plants, showed only 77.50% identity in coding DNA sequences and 65.48% identity in deduced amino acid sequences. The homology to GS-OH in Arabidopsis, DBM induction of the transcript and a series of qPCR and glucosinolate analyses of G-type, P-type and F1 plants indicated that these genes control the production of S and R isomers of 2-hydroxy-2-phenylethyl glucosinolate. These glucosinolates were significantly induced by P. xylostella larvae in both the susceptiple P

  4. Expression of lauroyl-acyl carrier protein thioesterase in brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation

    PubMed Central

    Eccleston, VS; Ohlrogge, JB

    1998-01-01

    Expression of a California bay lauroyl-acyl carrier protein thioesterase (MCTE) in developing seeds of transgenic oilseed rape alters the fatty acid composition of the mature seed, resulting in up to 60 mol% of laurate in triacylglycerols. In this study, we examined the metabolism of lauric acid and 14C-acetate in developing seeds of oilseed rape that express high levels of MCTE. Lauroyl-CoA oxidase activity but not palmitoyl-CoA oxidase activity was increased several-fold in developing seeds expressing MCTE. In addition, isocitrate lyase and malate synthase activities were six- and 30-fold higher, respectively, in high-laurate developing seeds. Control seeds incorporated 14C-acetate almost entirely into fatty acids, whereas in seeds expressing MCTE, only 50% of the label was recovered in lipids and the remainder was in a range of water-soluble components, including sucrose and malate. Together, these results indicate that the pathways for beta-oxidation and the glyoxylate cycle have been induced in seeds expressing high levels of MCTE. Although a substantial portion of the fatty acid produced in these seeds is recycled to acetyl-CoA and sucrose through the beta-oxidation and glyoxylate cycle pathways, total seed oil is not reduced. How is oil content maintained if lauric acid is inefficiently converted to triacylglycerol? The levels of acyl carrier protein and several enzymes of fatty acid synthesis were increased two- to threefold at midstage development in high-laurate seeds. These results indicate that a coordinate induction of the fatty acid synthesis pathway occurs, presumably to compensate for the lauric acid lost through beta-oxidation or for a shortage of long-chain fatty acids. PMID:9548986

  5. Upregulation of isoprenoid pathway genes during enhanced saikosaponin biosynthesis in the hairy roots of Bupleurum falcatum.

    PubMed

    Kim, Young Soon; Cho, Jung Hyun; Ahn, Juncheul; Hwang, Baik

    2006-12-31

    In order to characterize saikosaponin biosynthesis in Bupleurum falcatum, the expression of five isoprenoid pathway genes and their relationship to saikosaponin accumulation in the hairy roots were analyzed. The hairy roots exhibited a rapid accumulation of saikosaponins when incubated in a root culture medium (3XRCM). Homology-based RT-PCR was used to isolate core fragments of five genes, HMGR, IPPI, FPS, SS, and OSC, from the hairy roots. The deduced amino acid sequences exhibited amino acid identities of more than 85% to previously reported genes. Using the fragments as probes, the expression of these five genes in the hairy roots during incubation in 3XRCM medium was examined. Expression of all five genes in the hairy roots increased soon after incubation. In particular, the SS and OSC genes were coordinately induced at 8 days of incubation, and their expression persisted throughout the incubation period. A quantitative HPLC analysis showed that the saikosaponin content of the hairy root culture also began to increase at 8 days of culture. The correlation between SS transcript level and saikosaponin content in the hairy roots suggests that transcriptional regulation plays a regulatory role in saikosaponin biosynthesis. PMID:17202854

  6. 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. PMID:21257030

  7. Disruption of cell wall fatty acid biosynthesis in Mycobacterium tuberculosis using a graph theoretic approach

    PubMed Central

    2011-01-01

    Fatty acid biosynthesis of Mycobacterium tuberculosis was analyzed using graph theory and influential (impacting) proteins were identified. The graphs (digraphs) representing this biological network provide information concerning the connectivity of each protein or metabolite in a given pathway, providing an insight into the importance of various components in the pathway, and this can be quantitatively analyzed. Using a graph theoretic algorithm, the most influential set of proteins (sets of {1, 2, 3}, etc.), which when eliminated could cause a significant impact on the biosynthetic pathway, were identified. This set of proteins could serve as drug targets. In the present study, the metabolic network of Mycobacterium tuberculosis was constructed and the fatty acid biosynthesis pathway was analyzed for potential drug targeting. The metabolic network was constructed using the KEGG LIGAND database and subjected to graph theoretical analysis. The nearness index of a protein was used to determine the influence of the said protein on other components in the network, allowing the proteins in a pathway to be ordered according to their nearness indices. A method for identifying the most strategic nodes to target for disrupting the metabolic networks is proposed, aiding the development of new drugs to combat this deadly disease. PMID:21453530

  8. Physiological insights into all-trans-retinoic acid biosynthesis

    PubMed Central

    Napoli, Joseph L.

    2011-01-01

    All-trans-retinoic acid (atRA) provides essential support to diverse biological systems and physiological processes. Epithelial differentiation and its relationship to cancer and embryogenesis have typified intense areas of interest into atRA function. Recently, however, interest in atRA action in the nervous system, the immune system, energy balance and obesity has increased considerably, especially concerning postnatal function. atRA action depends on atRA biosynthesis: defects in retinoid-dependent processes increasingly relate to defects in atRA biogenesis. Considerable evidence indicates that physiological atRA biosynthesis occurs via a regulated process, consisting of a complex interaction of retinoid binding-proteins and retinoid recognizing enzymes. An accrual of biochemical, physiological and genetic data have identified specific functional outcomes for the retinol dehydrogenases, RDH1, RDH10, and DHRS9, as physiological catalysts of the first step in atRA biosynthesis, and for the retinal dehydrogenases RALDH1, RALDH2, and RALDH3, as catalysts of the second and irreversible step. Each of these enzymes associates with explicit biological processes mediated by atRA. Redundancy occurs, but seems limited. Cumulative data supports a model of interactions among these enzymes with retinoid binding-proteins, with feedback regulation and/or control by atRA via modulating gene expression of multiple participants. The ratio apo-CRBP1/holo-CRBP1 participates by influencing retinol flux into and out of storage as retinyl esters, thereby modulating substrate to support atRA biosynthesis. atRA biosynthesis requires presence of both an RDH and an RALDH: conversely, absence of one isozyme of either step does not indicate lack of atRA biosynthesis at the site. PMID:21621639

  9. Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway

    PubMed Central

    Leon-Reyes, Antonio; Van der Does, Dieuwertje; De Lange, Elvira S.; Delker, Carolin; Wasternack, Claus; Van Wees, Saskia C. M.; Ritsema, Tita

    2010-01-01

    Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway. PMID:20839007

  10. Ribonucleic acid (RNA) biosynthesis in human cancer.

    PubMed

    Hajjawi, Omar S

    2015-01-01

    In many respects, the most remarkable chemical substances within the genome of eukaryotic cells are remarkable proteins which are the critical structural and functional units of living cells. The specifications for everything that goes in the cell are natural digital-to-digital decoding process in an archive sequence by deoxyribonucleic acid (DNA) and an articulate construction by ribonucleic acid (RNA). The products of DNA transcription are long polymers of ribonucleotides rather than deoxyribonucleotides and are termed ribonucleic acids. Certain deoxyribonucleotide sequences, or genes, give rise to transfer RNA (tRNA) and other ribosomal RNA (rRNA) when transcribed. The ribonucleotide sequences fold extensively and rRNA is associated with specific proteins to yield the essential cell components, ribosomes. Transcription of other special sequences yields messenger RNAs (mRNAs) that contain ribonucleotide sequences that will be ultimately translated into new types of amino acid sequences of functional cellular protein molecules. This switch to a different variety of cellular molecular sequences is complex, but each sequence of the three ribonucleotides specifies the insertion of one particular amino acid into the polypeptide chain under production. Whilst mRNA is considered the vehicle by which genetic information is transmitted from the genome and allocated in the appropriate cytoplasmic sites for translation into protein via cap-dependent mechanism, the actual translation depends also on the presence of other so-called household and luxury protein molecules. Recent evidence suggests RNA species are required at initiation, because treatment of cells with antibiotics or drugs that inhibit RNA synthesis cause a decrease in protein synthesis. The rRNA is necessary as a structural constituent of the ribosomes upon which translation takes place, whereas tRNA is necessary as an adaptor in amino acid activation and elongation protein chains to ribosomes. In this article

  11. Acquisition and biosynthesis of saturated and unsaturated fatty acids by trypanosomatids.

    PubMed

    Uttaro, Antonio D

    2014-08-01

    As components of phospholipids and glycosylphosphatidylinositol anchors, fatty acids are responsible for forming the core of biological membranes and the correct localization of proteins within membranes. They also contribute to anchoring proteins by direct acylation of specific amino acids. Fatty acids can be used as energy sources and serve as signaling molecules or precursors for their synthesis. All these processes highlight the important role of fatty acids in cell physiology, justifying the diverse strategies for their acquisition evolved by different organisms. This review describes several recent findings in the salvage and biosynthesis of fatty acids by parasitic protists belonging to the class Kinetoplastea. They include two biosynthetic routes, the mitochondrial one and a peculiar membrane-associated pathway, the synthesis of polyunsaturated fatty acids, and the scavenging of lysophospholipids and lipoproteins from host plasma. These different processes are also explored as putative targets for chemotherapy. PMID:24726787

  12. Indole-3-acetic acid biosynthetic pathways in the basidiomycetous yeast Rhodosporidium paludigenum.

    PubMed

    Nutaratat, Pumin; Srisuk, Nantana; Arunrattiyakorn, Panarat; Limtong, Savitree

    2016-07-01

    Microorganisms produce plant growth regulators, such as auxins, cytokinins and gibberellins, to promote plant growth. Auxins are a group of compounds with an indole ring that have a positive effect on plant growth. Indole-3-acetic acid (IAA) is a plant growth hormone classified as an indole derivative of the auxin family. IAA biosynthesis pathways have been reported and widely studied in several groups of bacteria. Only a few studies on IAA biosynthesis pathways have been conducted in yeast. This study aimed to investigate IAA biosynthesis pathways in a basidiomycetous yeast (Rhodosporidium paludigenum DMKU-RP301). Investigations were performed both with and without a tryptophan supplement. Indole compound intermediates were detected by gas chromatography-mass spectrometry. Indole-3-lactic acid and indole-3-ethanol were found as a result of the enzymatic reduction of indole-3-pyruvic acid and indole-3-acetaldehyde, in IAA biosynthesis via an indole-3-pyruvic acid pathway. In addition, we also found indole-3-pyruvic acid in culture supernatants determined by high-performance liquid chromatography. Identification of tryptophan aminotransferase activity supports indole-3-pyruvic acid-routed IAA biosynthesis in R. paludigenum DMKU-RP301. We hence concluded that R. paludigenum DMKU-RP301 produces IAA through an indole-3-pyruvic acid pathway. PMID:26899734

  13. Starch biosynthesis in cassava: a genome-based pathway reconstruction and its exploitation in data integration

    PubMed Central

    2013-01-01

    Background Cassava is a well-known starchy root crop utilized for food, feed and biofuel production. However, the comprehension underlying the process of starch production in cassava is not yet available. Results In this work, we exploited the recently released genome information and utilized the post-genomic approaches to reconstruct the metabolic pathway of starch biosynthesis in cassava using multiple plant templates. The quality of pathway reconstruction was assured by the employed parsimonious reconstruction framework and the collective validation steps. Our reconstructed pathway is presented in the form of an informative map, which describes all important information of the pathway, and an interactive map, which facilitates the integration of omics data into the metabolic pathway. Additionally, to demonstrate the advantage of the reconstructed pathways beyond just the schematic presentation, the pathway could be used for incorporating the gene expression data obtained from various developmental stages of cassava roots. Our results exhibited the distinct activities of the starch biosynthesis pathway in different stages of root development at the transcriptional level whereby the activity of the pathway is higher toward the development of mature storage roots. Conclusions To expand its applications, the interactive map of the reconstructed starch biosynthesis pathway is available for download at the SBI group’s website (http://sbi.pdti.kmutt.ac.th/?page_id=33). This work is considered a big step in the quantitative modeling pipeline aiming to investigate the dynamic regulation of starch biosynthesis in cassava roots. PMID:23938102

  14. Biosynthesis of amino acids in Clostridium pasteurianum

    PubMed Central

    Dainty, R. H.; Peel, J. L.

    1970-01-01

    1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) 14C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus 14C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of 14C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate → threonine → glycine ⇌ serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine. PMID:5419750

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

    PubMed Central

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

    2011-01-01

    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 β-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. PMID:22332000

  16. The catalytic machinery of a key enzyme in amino Acid biosynthesis.

    PubMed

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

    2011-01-01

    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 β-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. PMID:22332000

  17. 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.

  18. Possible evolution of alliarinoside biosynthesis from the glucosinolate pathway in Alliaria petiolata.

    PubMed

    Frisch, Tina; Møller, Birger L

    2012-05-01

    Nitrile formation in plants involves the activity of cytochrome P450s. Hydroxynitrile glucosides are widespread among plants but generally do not occur in glucosinolate producing species. Alliaria petiolata (garlic mustard, Brassicaceae) is the only species known to produce glucosinolates as well as a γ-hydroxynitrile glucoside. Furthermore, A. petiolata has been described to release diffusible cyanide, which indicates the presence of unidentified cyanogenic glucoside(s). Our research on A. petiolata addresses the molecular evolution of P450s. By integrating current knowledge about glucosinolate and hydroxynitrile glucoside biosynthesis in other species and new visions on recurrent evolution of hydroxynitrile glucoside biosynthesis, we propose a pathway for biosynthesis of the γ-hydroxynitrile glucoside, alliarinoside. Homomethionine and the corresponding oxime are suggested as shared intermediates in the biosynthesis of alliarinoside and 2-propenyl glucosinolate. The first committed step in the alliarinoside pathway is envisioned to be catalysed by a P450, which has been recruited to metabolize the oxime. Furthermore, alliarinoside biosynthesis is suggested to involve enzyme activities common to secondary modification of glucosinolates. Thus, we argue that biosynthesis of alliarinoside may be the first known case of a hydroxynitrile glucoside pathway having evolved from the glucosinolate pathway. An intriguing question is whether the proposed hydroxynitrile intermediate may also be converted to novel homomethionine-derived cyanogenic glucoside(s), which could release cyanide. Elucidation of the pathway for biosynthesis of alliarinoside and other putative hydroxynitrile glucosides in A. petiolata is envisioned to offer significant new knowledge on the emerging picture of P450 functional dynamics as a basis for recurrent evolution of pathways for bioactive natural product biosynthesis. PMID:22212644

  19. Biosynthesis and Elongation of Short- and Medium-Chain-Length Fatty Acids

    PubMed Central

    van der Hoeven, Rutger S.; Steffens, John C.

    2000-01-01

    Short- and medium-chain-length fatty acids (FAs) are important constituents of a wide array of natural products. Branched and straight short-chain-length FAs originate from branched chain amino acid metabolism, and serve as primers for elongation in FA synthase-like reactions. However, a recent model proposes that the one-carbon extension reactions that utilize 2-oxo-3-methylbutyric acid in leucine biosynthesis also catalyze a repetitive one-carbon elongation of short-chain primers to medium-chain-length FAs. The existence of such a mechanism would require a novel form of regulation to control carbon flux between amino acid and FA biosynthesis. A critical re-analysis of the data used to support this pathway fails to support the hypothesis for FA elongation by one-carbon extension cycles of α-ketoacids. Therefore, we tested the hypothesis experimentally using criteria that distinguish between one- and two-carbon elongation mechanisms: (a) isotopomer patterns in terminal carbon atom pairs of branched and straight FAs resulting from differential labeling with [13C]acetate; (b) [13C]threonine labeling patterns in odd- and even chain length FAs; and (c) differential sensitivity of elongation reactions to inhibition by cerulenin. All three criteria indicated that biosynthesis of medium-chain length FAs is mediated primarily by FA synthase-like reactions. PMID:10631271

  20. Branched Chain Amino Acid Metabolism in the Biosynthesis of Lycopersicon pennellii Glucose Esters 1

    PubMed Central

    Walters, Donald S.; Steffens, John C.

    1990-01-01

    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 C4 and C5 acids, and branched and straight chain C10, C11, and C12 acids. The structure of the branched acyl constituents suggests that the branched chain amino acid biosynthetic pathway participates in their biosynthesis. [14C]Valine and deuterated branched chain amino acids (and their oxo-acid derivatives) were incorporated into branched C4 and C5 acid groups of glucose esters by a process of transamination, oxidative decarboxylation and subsequent acylation. C4 and C5 branched acids were elongated by two carbon units to produce the branched C10-C12 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. PMID:16667654

  1. The Biosynthesis of δ-Aminolevulinic Acid in Higher Plants

    PubMed Central

    Beale, Samuel I.; Castelfranco, Paul A.

    1974-01-01

    δ-Aminolevulinic acid dehydrase activity in cucumber (Cucumis sativus L. var. Alpha green) cotyledons did not change as the tissue was allowed to green for 24 hours. δ-Aminolevulinic acid accumulated in greening cucumber cotyledons, and barley (Hordeum sativum L. var. Numar) and bean (Phaseolus vulgaris L. var. Red Kidney) leaves incubated in the presence of levulinic acid, a specific competitive inhibitor of δ-aminolevulinic acid dehydrase. The rate of δ-aminolevulinic acid accumulation in levulinic acid-treated cucumber cotyledons paralleled the rate of chlorophyll accumulation in the controls, and the quantity of δ-aminolevulinic acid accumulated compensated for the decrease in chlorophyll accumulation. When levulinic acid-treated cucumber cotyledons were returned to darkness, δ-aminolevulinic acid accumulation ceased. δ-Aminolevulinic acid accumulation showed an absolute requirement for oxygen and was inhibited drastically by cyanide and azide, and to a lesser extent by arsenite and malonate. 2,4-Dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, sodium fluoroacetate, and hydroxylamine hydrochloride showed no effect under the conditions tested. Freezing and thawing of the tissue completely prevented the accumulation of δ-aminolevulinic acid. The findings of this investigation are consistent with the hypothesis that δ-aminolevulinic acid is a chlorophyll precursor in higher plants, and that chlorophyll biosynthesis is regulated at the level of the formation of δ-aminolevulinic acid. PMID:16658693

  2. Deciphering Carbamoylpolyoxamic Acid Biosynthesis Reveals Unusual Acetylation Cycle Associated with Tandem Reduction and Sequential Hydroxylation.

    PubMed

    Qi, Jianzhao; Wan, Dan; Ma, Hongmin; Liu, Yuanzhen; Gong, Rong; Qu, Xudong; Sun, Yuhui; Deng, Zixin; Chen, Wenqing

    2016-08-18

    Polyoxin, produced by Streptomcyes cacaoi var. asoensis and Streptomyces aureochromogenes, contains two non-proteinogenic amino acids, carbamoylpolyoxamic acid (CPOAA) and polyoximic acid. Although the CPOAA moiety is highly unusual, its biosynthetic logic has remained enigmatic for decades. Here, we address CPOAA biosynthesis by reconstitution of its pathway. We demonstrated that its biosynthesis is initiated by a versatile N-acetyltransferase, PolN, catalyzing L-glutamate (1) to N-acetyl glutamate (2). Remarkably, we verified that PolM, a previously annotated dehydrogenase, catalyzes an unprecedented tandem reduction of acyl-phosphate to aldehyde, and subsequently to alcohol. We also unveiled a distinctive acetylation cycle catalyzed by PolN to synthesize α-amino-δ-hydroxyvaleric acid (6). Finally, we report that PolL is capable of converting a rare sequential hydroxylation of α-amino-δ-carbamoylhydroxyvaleric acid (7) to CPOAA. PolL represents an intriguing family of Fe(II)-dependent α-ketoglutarate dioxygenase with a cupin fold. These data illustrate several novel enzymatic reactions, and also set a foundation for rational pathway engineering for polyoxin production. PMID:27541195

  3. Metabolic engineering of Kluyveromyces lactis for L-ascorbic acid (vitamin C) biosynthesis

    PubMed Central

    2013-01-01

    Background L-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this “L” enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana. Results Plasmids were constructed and modified such that the cloned plant genes were targeted to the K. lactis LAC4 Locus by homologous recombination and that the expression was associated to the growth on D-galactose or lactose. Upon K. lactis transformation, GME was under the control of the native LAC4 promoter whereas VTC2 and VTC4 were expressed from the S. cerevisiae promoters GPD1 and ADH1 respectively. The expression in K. lactis, of the L-galactose biosynthesis genes was determined by Reverse Transcriptase-PCR and western blotting. The recombinant yeasts were capable to produce about 30 mg.L-1 of L-ascorbic acid in 48 hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We also evaluated the L-AA production culturing recombinant recombinant strains in cheese whey, a waste product during cheese production, as an alternative source of lactose. Conclusions This work is the first attempt to engineer K. lactis cells for L-ascorbic acid biosynthesis by a fermentation process without any trace of “L” isomers precursors in the culture medium. We have engineered K. lactis

  4. 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

  5. Biosynthesis of the Halogenated Auxin, 4-Chloroindole-3-Acetic Acid1[W][OA

    PubMed Central

    Tivendale, Nathan D.; Davidson, Sandra E.; Davies, Noel W.; Smith, Jason A.; Dalmais, Marion; Bendahmane, Abdelhafid I.; Quittenden, Laura J.; Sutton, Lily; Bala, Raj K.; Le Signor, Christine; Thompson, Richard; Horne, James; Reid, James B.; Ross, John J.

    2012-01-01

    Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea. PMID:22573801

  6. 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.

  7. Pathways of Nucleotide Biosynthesis in Mycoplasma mycoides subsp. mycoides

    PubMed Central

    Mitchell, Alana; Finch, Lloyd R.

    1977-01-01

    By measuring the specific activity of nucleotides isolated from ribonucleic acid after the incorporation of 14C-labeled precursors under various conditions of growth, we have defined the major pathways of ribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. M. mycoides did not possess pathways for the de novo synthesis of nucleotides but was capable of interconversion of nucleotides. Thus, uracil provided the requirement for both pyrimidine ribonucleotides. Thymine is also required, suggesting that the methylation step is unavailable. No use was made of cytosine. Uridine was rapidly degraded to uracil. Cytidine competed effectively with uracil to provide most of the cytidine nucleotide and also provided an appreciable proportion of uridine nucleotide. In keeping with these results, there was a slow deamination of cytidine to uridine with further degradation to uracil in cultures of M. mycoides. Guanine was capable of meeting the full requirement of the organism for purine nucleotide, presumably by conversion of guanosine 5′-monophosphate to adenosine 5′-monophosphate via the intermediate inosine 5′-monophosphate. When available with guanine, adenine effectively gave a complete provision of adenine nucleotide, whereas hypoxanthine gave a partial provision. Neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleotide. Exogenous guanosine, inosine, and adenosine underwent rapid cleavage to the corresponding bases and so show a pattern of utilization similar to that of the latter. PMID:324972

  8. Farnesylation mediates brassinosteroid biosynthesis to regulate abscisic acid responses.

    PubMed

    Northey, Julian G B; Liang, Siyu; Jamshed, Muhammad; Deb, Srijani; Foo, Eloise; Reid, James B; McCourt, Peter; Samuel, Marcus A

    2016-01-01

    Protein farnesylation is a post-translational modification involving the addition of a 15-carbon farnesyl isoprenoid to the carboxy terminus of select proteins(1-3). Although the roles of this lipid modification are clear in both fungal and animal signalling, many of the mechanistic functions of farnesylation in plant signalling are still unknown. Here, we show that CYP85A2, the cytochrome P450 enzyme that performs the last step in brassinosteroid biosynthesis (conversion of castasterone to brassinolide)(4), must be farnesylated to function in Arabidopsis. Loss of either CYP85A2 or CYP85A2 farnesylation results in reduced brassinolide accumulation and increased plant responsiveness to the hormone abscisic acid (ABA) and overall drought tolerance, explaining previous observations(5). This result not only directly links farnesylation to brassinosteroid biosynthesis but also suggests new strategies to maintain crop yield under challenging climatic conditions. PMID:27455172

  9. Alternative route for the biosynthesis of polyunsaturated fatty acids in K562 cells.

    PubMed Central

    Naval, J; Martínez-Lorenzo, M J; Marzo, I; Desportes, P; Piñeiro, A

    1993-01-01

    K562 human leukaemia cells lack a significant delta 6-desaturase activity. However, they synthesize long-chain polyunsaturated fatty acids (PUFA) from linoleic (C18:2(9,12)) and linolenic (C18:3(9,12,15)) acids, by reactions involving a C2 chain elongation followed by a delta 5-desaturation step and, to some extent, a further elongation. The main products formed were separated by argentation t.l.c. and identified by g.l.c. as the uncommon fatty acids C20:3(5,11,14) and C20:4(5,11,14,17) respectively. These acids were also produced when cells were supplemented with C20:2(11,14) or C20:3(11,14,17) respectively. The presence of a delta 5-desaturase was further confirmed by using its corresponding normal substrates, C20:3(8,11,14) and C20:4(8,11,14,17), which led to C20:4(5,8,11,14) and C20:5(5,8,11,14,17) respectively. On the other hand, a high delta 9-desaturase activity, but no significant delta 4-desaturase activity, were detected in K562 cells. These results indicate the existence of an alternative pathway, involving delta 5-desaturase, which is the only route for PUFA biosynthesis in K562 cells. This pathway may be relevant for the biosynthesis of PUFA in cells lacking delta 6-desaturase activity. PMID:8489510

  10. Cloning and optimization of a nisin biosynthesis pathway for bacteriocin harvest.

    PubMed

    Kong, Wentao; Lu, Ting

    2014-07-18

    Nisin is an important antimicrobial peptide that has enormous applications in biotechnology. Despite many encouraging efforts, its overproduction has been a long-standing challenge due to the complexity of the underlying pathway and the difficulty in genetic modification of lactic acid bacteria. Here, we cloned an entire nisin biosynthesis pathway from a nisin-producing strain (Lactococcus lactis K29) into a plasmid and transplanted the plasmid into a nisin deficient strain Lactococcus lactis MG1363, resulting in successful heterologous expression of bioactive recombinant nisin. To increase nisin harvest, we also overexpressed nisA, a gene responsible for nisin precursor production, with a set of constitutive promoters. To further optimize nisin yield, we minimized the metabolic cost of the engineered strains by integrating nisA overexpression cassettes and the recombinant pathway into a single circuit. With our rational construction and optimization, our engineered optimized strain is able to produce bioactive nisin with a yield of 1098 IU/mL, which is more than six times higher than that of the original strain. PMID:24847677

  11. 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

  12. Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis

    PubMed Central

    2014-01-01

    Background Purine nucleotides are essential metabolites for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and biosynthesis of several amino acids and riboflavin. Owing to the pivotal roles of purines in cell physiology, the pool of intracellular purine nucleotides must be maintained under strict control, and hence the de novo purine biosynthetic pathway is tightly regulated by transcription repression and inhibition mechanism. Deregulation of purine pathway is essential for this pathway engineering in Bacillus subtilis. Results Deregulation of purine pathway was attempted to improve purine nucleotides supply, based on a riboflavin producer B. subtilis strain with modification of its rib operon. To eliminate transcription repression, the pur operon repressor PurR and the 5’-UTR of pur operon containing a guanine-sensing riboswitch were disrupted. Quantitative RT-PCR analysis revealed that the relative transcription levels of purine genes were up-regulated about 380 times. Furthermore, site-directed mutagenesis was successfully introduced into PRPP amidotransferase (encoded by purF) to remove feedback inhibition by homologous alignment and analysis. Overexpression of the novel mutant PurF (D293V, K316Q and S400W) significantly increased PRPP amidotransferase activity and triggered a strong refractory effect on purine nucleotides mediated inhibition. Intracellular metabolite target analysis indicated that the purine nucleotides supply in engineered strains was facilitated by a stepwise gene-targeted deregulation. With these genetic manipulations, we managed to enhance the metabolic flow through purine pathway and consequently increased riboflavin production 3-fold (826.52 mg/L) in the purF-VQW mutant strain. Conclusions A sequential optimization strategy was applied to deregulate the rib operon and purine pathway of B. subtilis to create genetic diversities and to improve riboflavin production

  13. Genetic control of abscisic acid biosynthesis in maize.

    PubMed

    Tan, B C; Schwartz, S H; Zeevaart, J A; McCarty, D R

    1997-10-28

    Abscisic acid (ABA), an apocarotenoid synthesized from cleavage of carotenoids, regulates seed maturation and stress responses in plants. The viviparous seed mutants of maize identify genes involved in synthesis and perception of ABA. Two alleles of a new mutant, viviparous14 (vp14), were identified by transposon mutagenesis. Mutant embryos had normal sensitivity to ABA, and detached leaves of mutant seedlings showed markedly higher rates of water loss than those of wild type. The ABA content of developing mutant embryos was 70% lower than that of wild type, indicating a defect in ABA biosynthesis. vp14 embryos were not deficient in epoxy-carotenoids, and extracts of vp14 embryos efficiently converted the carotenoid cleavage product, xanthoxin, to ABA, suggesting a lesion in the cleavage reaction. vp14 was cloned by transposon tagging. The VP14 protein sequence is similar to bacterial lignostilbene dioxygenases (LSD). LSD catalyzes a double-bond cleavage reaction that is closely analogous to the carotenoid cleavage reaction of ABA biosynthesis. Southern blots indicated a family of four to six related genes in maize. The Vp14 mRNA is expressed in embryos and roots and is strongly induced in leaves by water stress. A family of Vp14-related genes evidently controls the first committed step of ABA biosynthesis. These genes are likely to play a key role in the developmental and environmental control of ABA synthesis in plants. PMID:9342392

  14. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

    SciTech Connect

    Lu, JN; Brigham, CJ; Plassmeier, JK; Sinskey, AJ

    2014-08-01

    2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC50 = 1.2 mM), l-isoleucine (IC50 = 2.3 mM), and l-leucine (IC50 = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (K-M = 10.5 mu M) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2

  15. 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. PMID:26873116

  16. 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. PMID:27242809

  17. 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

  18. 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

  19. Auxin biosynthesis.

    PubMed

    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

  20. 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

  1. Fatty acid biosynthesis as a target for novel antibacterials

    PubMed Central

    Rock, Charles O

    2006-01-01

    The bacterial fatty acid synthesis pathway has significant potential as a target for the development of novel antibacterials. The pathway has been extensively studied in Escherichia coli, the crystal structures of the compounds involved are known and homologous genes are readily identified in the genomes of important pathogens. The, currently used drugs triclosan and isoniazid are known to target one step in the pathway. Other experimental compounds such as thiolactomycin and cerulenin effectively inhibit other steps. These known pathway inhibitors are reviewed and the areas for potential future developments are explored. PMID:15043388

  2. The astaxanthin dideoxyglycoside biosynthesis pathway in Sphingomonas sp. PB304.

    PubMed

    Kim, Se Hyeuk; Kim, Jin Ho; Lee, Bun Yeol; Lee, Pyung Cheon

    2014-12-01

    A major carotenoid in Sphingomonas sp. PB304, originally isolated from a river in Daejon City, South Korea, was identified as astaxanthin dideoxyglycoside. Gene clusters encoding the astaxanthin dideoxyglycoside biosynthetic enzymes were identified by screening Sphingomonas sp. PB304 fosmid libraries using degenerate probes that harbor highly conserved sequences from the Sphigomonas elodea-derived crtI and Nostoc sp. PCC 7120-dervied crtW genes. Selected positive gene clusters were fully sequenced and annotated, revealing genes encoding six putative carotenogenic enzymes: phytoene synthase (CrtB), phytoene desaturase (CrtI), lycopene cyclase (CrtY), carotene hydroxylase (CrtZ), carotene ketolase (CrtW), and glycosyltransferase (CrtX). All of the carotenogenic enzymes, except for CrtX, were functional in the recombinant host Escherichia coli expressing synthetic carotenogenic modules from Pantoea agglomerans. CrtX did not take up UDP-glucose or GDP-fucose as sugar substrates during the in vitro reaction. Although no direct experimental evidence was obtained for the function of Sphingomonas sp. PB304 CrtX, it can be categorized as a putative deoxyglycosyltransferase based on the presence of astaxanthin dideoxyglycoside in Sphingomonas sp. PB304, a putative corresponding gene in the carotenoid biosynthetic gene cluster, and high amino acid sequence homology to the existing glycosyltransferases. Therefore, we propose that astaxanthin dideoxyglycoside can be synthesized in Sphingomonas sp. PB304 via sequential reactions of six pathway enzymes, including CrtX on the phytoene intermediate. PMID:25193422

  3. Exploring cyanobacterial genomes for natural product biosynthesis pathways.

    PubMed

    Micallef, Melinda L; D'Agostino, Paul M; Al-Sinawi, Bakir; Neilan, Brett A; Moffitt, Michelle C

    2015-06-01

    Cyanobacteria produce a vast array of natural products, some of which are toxic to human health, while others possess potential pharmaceutical activities. Genome mining enables the identification and characterisation of natural product gene clusters; however, the current number of cyanobacterial genomes remains low compared to other phyla. There has been a recent effort to rectify this issue by increasing the number of sequenced cyanobacterial genomes. This has enabled the identification of biosynthetic gene clusters for structurally diverse metabolites, including non-ribosomal peptides, polyketides, ribosomal peptides, UV-absorbing compounds, alkaloids, terpenes and fatty acids. While some of the identified biosynthetic gene clusters correlate with known metabolites, genome mining also highlights the number and diversity of clusters for which the product is unknown (referred to as orphan gene clusters). A number of bioinformatic tools have recently been developed in order to predict the products of orphan gene clusters; however, in some cases the complexity of the cyanobacterial pathways makes the prediction problematic. This can be overcome by the use of mass spectrometry-guided natural product genome mining, or heterologous expression. Application of these techniques to cyanobacterial natural product gene clusters will be explored. PMID:25482899

  4. Genomic Analysis of Genes Involved in the Biosynthesis of Very Long Chain Polyunsaturated Fatty Acids in Thraustochytrium sp. 26185.

    PubMed

    Zhao, Xianming; Dauenpen, Meesapyodsuk; Qu, Cunmin; Qiu, Xiao

    2016-09-01

    Thraustochytrium sp. 26185 is a marine protist that can produce a large amount of docosahexaenoic acid (DHA, 22:6n-3), an ω3 very long chain polyunsaturated fatty acid (VLCPUFA) of nutritional importance. However, the mechanism of how this fatty acid is synthesized and assembled into the storage lipid triacylglycerol is unclear. Here we report sequencing of the whole genome and genomic analysis of genes involved in the biosynthesis and assembly of the fatty acids in this species. Genome sequencing produced a total of 2,418,734,139 bp clean sequences with about 62 fold genome coverage. Annotation of the genome sequences revealed 10,797 coding genes. Among them, 10,216 genes could be assigned into 25 KOG classes where 451 genes were specifically assigned to the group of lipid transport and metabolism. Detailed analysis of these genes revealed co-existence of both aerobic pathway and anaerobic pathways for the biosynthesis of DHA in this species. However, in the aerobic pathway, a key gene encoding stearate Δ9 desaturase introducing the first double bond to long chain saturated fatty acid 18:0 was missing from the genome. Genomic survey of genes involved in the acyl trafficking among glycerolipids showed that, unlike plants, this protist did not possess phosphatidylcholine:diacylglycerol cholinephosphotransferase, an important enzyme in bridging two types of glycerolipids, diacylglycerols (DAG) and phosphatidylcholines (PtdCho). These results shed new insight on the biosynthesis and assembly of VLCPUFA in the Thraustochytrium. PMID:27514858

  5. Terminal Olefin (1-Alkene) Biosynthesis by a Novel P450 Fatty Acid Decarboxylase from Jeotgalicoccus Species ▿ †

    PubMed Central

    Rude, Mathew A.; Baron, Tarah S.; Brubaker, Shane; Alibhai, Murtaza; Del Cardayre, Stephen B.; Schirmer, Andreas

    2011-01-01

    Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleTJE), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleTJE is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in α- and/or β-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family. PMID:21216900

  6. 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

  7. A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway.

    PubMed

    Lange, B M; Wildung, M R; McCaskill, D; Croteau, R

    1998-03-01

    Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants. PMID:9482845

  8. 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. PMID:27035483

  9. Vitamin and co-factor biosynthesis pathways in Plasmodium and other apicomplexan parasites

    PubMed Central

    Müller, Sylke; Kappes, Barbara

    2007-01-01

    Vitamins are essential components of the human diet. By contrast, the malaria parasite Plasmodium falciparum and related apicomplexan parasites synthesise certain vitamins, de novo, either completely or in parts. The occurrence of the various biosynthesis pathways is specific to different apicomplexan parasites, emphasising their distinct requirements for nutrients and growth factors. The absence of vitamin biosynthesis from the human host implies that inhibition of the parasite pathways may be a way to interfere specifically with parasite development. However, the precise role of biosynthesis and potential uptake of vitamins for the overall regulation of vitamin homeostasis in the parasites needs to be established first. In this review Sylke Müller and Barbara Kappes focus mainly on the procurement of vitamin B1, B5 and B6 by Plasmodium and other apicomplexan parasites. PMID:17276140

  10. An Alternative Pathway for Formononetin Biosynthesis in Pueraria lobata.

    PubMed

    Li, Jia; Li, Changfu; Gou, Junbo; Wang, Xin; Fan, Rongyan; Zhang, Yansheng

    2016-01-01

    The O-methylation is an important tailing process in Pueraria lobata isoflavone metabolism, but the molecular mechanism governing it remains not elucidated. This manuscript describes the mining of key O-methyltransferases (OMTs) involved in the process. Using our previously constructed P. lobata transcriptome, the OMT candidates were searched, extensively analyzed, and their functions were investigated by expression in yeast, Escherichia coli, or Glycine max hairy roots. Here, we report the identification of the key OMT gene responsible for formononetin production in P. lobata (designated as PlOMT9). PlOMT9 primarily functions as an isoflavone-specific 4'-O-methyltransferase, although it shows high sequence identities with isoflavone 7-O-methyltransferases. Moreover, unlike the previously reported OMTs that catalyze the 4'-O-methylation for formononetin biosynthesis at the isoflavanone stage, PlOMT9 performs this modifying step at the isoflavone level, using daidzein rather than 2,7,4'-trihydroxy-isoflavanone as the substrate. Gene expression analyses and metabolite profiling supported its proposed roles in P. lobata. Using the system of transgenic G. max hairy roots, the role of PlOMT9 in the biosynthesis of formononetin was further demonstrated in vivo. PMID:27379141

  11. An Alternative Pathway for Formononetin Biosynthesis in Pueraria lobata

    PubMed Central

    Li, Jia; Li, Changfu; Gou, Junbo; Wang, Xin; Fan, Rongyan; Zhang, Yansheng

    2016-01-01

    The O-methylation is an important tailing process in Pueraria lobata isoflavone metabolism, but the molecular mechanism governing it remains not elucidated. This manuscript describes the mining of key O-methyltransferases (OMTs) involved in the process. Using our previously constructed P. lobata transcriptome, the OMT candidates were searched, extensively analyzed, and their functions were investigated by expression in yeast, Escherichia coli, or Glycine max hairy roots. Here, we report the identification of the key OMT gene responsible for formononetin production in P. lobata (designated as PlOMT9). PlOMT9 primarily functions as an isoflavone-specific 4′-O-methyltransferase, although it shows high sequence identities with isoflavone 7-O-methyltransferases. Moreover, unlike the previously reported OMTs that catalyze the 4′-O-methylation for formononetin biosynthesis at the isoflavanone stage, PlOMT9 performs this modifying step at the isoflavone level, using daidzein rather than 2,7,4′-trihydroxy-isoflavanone as the substrate. Gene expression analyses and metabolite profiling supported its proposed roles in P. lobata. Using the system of transgenic G. max hairy roots, the role of PlOMT9 in the biosynthesis of formononetin was further demonstrated in vivo. PMID:27379141

  12. 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. PMID:24594312

  13. New prospects for deducing the evolutionary history of metabolic pathways in prokaryotes: Aromatic biosynthesis as a case-in-point

    NASA Astrophysics Data System (ADS)

    Ahmad, Suhail; Jensen, Roy A.

    1988-03-01

    Metabolic pathways of prokaryotes are more biochemically diverse than is generally recognized. Distinctive biochemical features are shared by phylogenetic clusters. The hierarchical levels of characterstate clustering depends upon evolutionary events which fortuitously became fixed in the genome of a common ancestor. Prokaryotes can now be ordered on a phylogenetic tree. This allows the evolutionary steps that underlie the construction and regulation of appropriately complex biochemical pathways to be traced in an evolutionary progression of prokaryote types that house these pathways. Essentially the approach is to deduce ancestral character states at ever deeper phylogenetic levels, utilizing logical principles of maximum parsimony. The current perspective on the evolution of the biochemical pathway for biosynthesis of aromatic amino acids is developed as a case-in-point model for analyses that should be feasible with many major metabolic systems. Phenylalanine biosynthesis probably arose prior to the addition of branches leading to tyrosine and tryptophan. An evolutionary scenario is developed that begins with non-enzymatic reactions which may have operated in primitive systems, followed by the evolution of an enzymatic system that pre-dated the divergence of major lineages of modern eubacteria (Gram-positive bacteria, Gram-negative purple bacteria, and cyanobacteria).

  14. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.

    PubMed

    Jomaa, H; Wiesner, J; Sanderbrand, S; Altincicek, B; Weidemeyer, C; Hintz, M; Türbachova, I; Eberl, M; Zeidler, J; Lichtenthaler, H K; Soldati, D; Beck, E

    1999-09-01

    A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured. PMID:10477522

  15. Restoration of nucleic acid biosynthesis after clinical death and factors stimulating the process in vivo.

    PubMed

    Konikova, A S; Petukhova, L M; Pogossova, A V; Vinarskaya, A A; Nikulin, V I

    1975-01-01

    The biosynthesis of RNA and DNA falls almost to zero in 60 min after the death of rabbits from anoxia, in all the organs of the body. Rapid artificial cooling of the rabbits to 20 degrees C undertaken within 10 min after death preserved nucleic acid biosynthesis and permitted restoration of life 3-4 h after death, with recovery beginning in 60 min. During the reanimation the addition of ATP to the blood stimulated the restoration of RNA biosynthesis in the spinal cord to a considerable extent; the addition of cocarboxylase to the blood promoted cardiac RNA biosynthesis as well as cardiac and pancreatic DNA biosynthesis during recovery. PMID:1197938

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in Ralstonia eutropha on short chain fatty acids.

    PubMed

    Yu, Jian; Si, Yingtao

    2004-01-01

    Short chain fatty acids such as acetic, propionic, and butyric acids can be synthesized into polyhydroxyalkanoates (PHAs) by Ralstonia eutropha. Metabolic carbon fluxes of the acids in living cells have significant effect on the yield, composition, and thermomechanical properties of PHA bioplastics. Based on the general knowledge of central metabolism pathways and the unusual metabolic pathways in R. eutropha, a metabolic network of 41 bioreactions is constructed to analyze the carbon fluxes on utilization of the short chain fatty acids. In fed-batch cultures with constant feeding of acid media, carbon metabolism and distribution in R. eutropha were measured involving CO2, PHA biopolymers, and residual cell mass. As the cells underwent unsteady state metabolism and PHA biosynthesis under nitrogen-limited conditions, accumulative carbon balance was applied for pseudo-steady-state analysis of the metabolic carbon fluxes. Cofactor NADP/NADPH balanced between PHA synthesis and the C3/C4 pathway provided an independent constraint for solution of the underdetermined metabolic network. A major portion of propionyl-CoA was directed to pyruvate via the 2-methylcitrate cycle and further decarboxylated to acetyl-CoA. Only a small amount of propionate carbon (<15% carbon) was directly condensed with acetyl-CoA for 3-hydroxyvalerate. The ratio of glyoxylate shunt to TCA cycle varies from 0 to 0.25, depending on the intracellular acetyl-CoA level and acetic acid in the medium. Malate is the node of the C3/C4 pathway and TCA cycle and its decarboxylation to dehydrogenation ranges from 0.33 to 1.28 in response to the demands on NADPH and oxaloacetate for short chain fatty acids utilization. PMID:15296425

  18. Proteomic Dissection of Endosperm Starch Granule Associated Proteins Reveals a Network Coordinating Starch Biosynthesis and Amino Acid Metabolism and Glycolysis in Rice Endosperms.

    PubMed

    Yu, Huatao; Wang, Tai

    2016-01-01

    Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms. PMID:27252723

  19. Proteomic Dissection of Endosperm Starch Granule Associated Proteins Reveals a Network Coordinating Starch Biosynthesis and Amino Acid Metabolism and Glycolysis in Rice Endosperms

    PubMed Central

    Yu, Huatao; Wang, Tai

    2016-01-01

    Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms. PMID:27252723

  20. A comprehensive survey on isoleucine biosynthesis pathways in seven epidemic Leptospira interrogans reference strains of China.

    PubMed

    Zou, Ying; Guo, Xiaokui; Picardeau, Mathieu; Xu, Hai; Zhao, Guoping

    2007-04-01

    Previous studies have indicated that different species of Leptospira synthesize isoleucine via either pyruvate and/or threonine pathways. Seven epidemic Leptospira interrogans reference strains from China belonging to different serovars, together with three saprophytic strains of Leptospira biflexa and Leptospira meyeri, were analysed. The isoleucine biosynthesis properties were studied firstly by measuring the key enzymes of the two pathways, citramalate synthase (CimA, CE4.1.3.-) and threonine deaminase (IlvA, CE4.2.1.16), from cell extracts of the bacteria. Meanwhile, alpha-isopropylmalate synthase (LeuA, CE4.2.1.12), the key enzyme of leucine biosynthesis, was also measured as a control. It was found that all L. interrogans strains synthesized isoleucine via the pyruvate pathway exclusively, but L. biflexa and L. meyeri used both pathways. Dot-Blot and PCR amplification of both cimA and ilvA genes in the corresponding strains provided additional evidence consistent with the data of enzymatic assays. Although it is evident that leptospires' isoleucine biosynthesis may preferentially adapt either to the pyruvate pathway exclusively for pathogens or to the combination of both pyruvate and threonine pathways for saprophytes, broader sampling with careful genomospecies identification is needed for a solid conclusion. PMID:17227461

  1. 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

  2. A natural protecting group strategy to carry an amino acid starter unit in the biosynthesis of macrolactam polyketide antibiotics.

    PubMed

    Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2011-11-16

    Macrolactam antibiotics are an important class of macrocyclic polyketides that contain a unique nitrogen-containing starter unit. In the present study, a set of starter biosynthetic enzymes in the macrolactam antibiotic vicenistatin was characterized. We found that the protection-deprotection strategy of the aminoacyl-ACP intermediate was critical in this system. On the basis of bioinformatics, the described pathway is also proposed as a common method for carrying amino acids in the biosynthesis of other macrolactam antibiotics. PMID:22010945

  3. Genetic effects in the leukotriene biosynthesis pathway and association with atherosclerosis

    PubMed Central

    Crosslin, David R.; Shah, Svati H.; Nelson, Sarah C.; Haynes, Carol S.; Connelly, Jessica J.; Gadson, Shera; Goldschmidt-Clermont, Pascal J.; Vance, Jeffery M.; Rose, Jason; Granger, Chris B.; Seo, David; Gregory, Simon G.; Kraus, William E.

    2009-01-01

    Leukotrienes are arachidonic acid derivatives long known for their inflammatory properties and their involvement with a number of human diseases, most particularly asthma. Recently, leukotriene-based inflammation has also been shown to play an important role in atherosclerosis: ALOX5AP and LTA4H, both genes in the leukotriene biosynthesis pathway, have individually been shown to be associated with various cardiovascular disease (CVD) phenotypes. To assess the role of the leukotriene pathway in CVD pathogenesis, we performed genetic association studies of ALOX5AP and LTA4H in a family based study of early onset coronary artery disease (EOCAD) (GENECARD, 1,101 families) and in a non-familial dataset of EOCAD (CATHGEN, 656 cases and 405 controls). We found weak to moderate association between single nucleotide polymorphisms (SNPs) in ALOX5AP and LTA4H with EOCAD. The previously reported four-SNP haplotype (HapA) in ALOX5AP showed association with EOCAD in CATHGEN (P = 0.02), while controlling for age, race and CVD risk factors. HapK, the previously reported ten-SNP haplotype in LTA4H was associated with EOCAD in CATHGEN (P = 0.04). Another previously reported four-SNP haplotype in ALOX5AP (HapB) was not significant in our sample (P = 0.39). The overall lack of (or weak) association of single SNPs as compared with the haplotype results demonstrates the need for analyzing multiple SNPs within each gene in such studies. Interestingly, we detected an association of SNPs in ALOX5 (P < 0.05), the target of ALOX5AP, with CVD. Using a pathway-based approach, we also detected statistical evidence for interactions among ALOX5, ALOX5AP and LTA4H using RNA expression data from a collection of freshly harvested human aortas with varying degrees of atherosclerosis. The GENECARD families did not demonstrate evidence for linkage or association with ALOX5, ALOX5AP or LTA4H. Our results support a modest role for the leukotriene pathway in atherosclerosis pathogenesis, reveal important

  4. Fatty acid biosynthesis during the life cycle of Debaryomyces etchellsii.

    PubMed

    Arous, Fatma; Mechichi, Tahar; Nasri, Moncef; Aggelis, George

    2016-07-01

    Fatty acid biosynthesis during the life cycle of the ascomycetous yeast Debaryomyces etchellsii cultivated on a non-fermentable substrate, i.e. glycerol, in nitrogen rich media (NRM) and nitrogen limited media (NLM) has been studied. Although considerable activities of key lipogenic enzymes, such as ATP citrate lyase (ACL) and malic enzyme (ME), were detected in vegetative cells during asexual proliferation (which occurred in the first growth stages in both NRM and NLM), lipid accumulation was restricted due to the high activities of NAD+-isocitrate dehydrogenase (NAD+-ICDH). A similar enzymatic profile has been found in ascii and free ascospores produced in NRM; thus lipid accumulation was low. On the contrary, very high activities of both ACL and ME and low activities of NAD+-ICDH were detected in ascii and free ascospores produced in NLM resulting in lipid accumulation. Neutral lipids (NL) were the predominant fraction of cellular lipids produced in vegetative cells and ascospores in both NRM and NLM. On the other hand, phospholipids (P) were the major polar lipids while glycolipids (G) were synthesized in low proportions. During transition from asexual to sexual phase, the percentage of NL increased with a significant decrease of P and, to a lesser extent, of G. High quantities of linoleic acid were found esterified in polar lipids, especially in P, during the vegetative stage of growth, while, with a few exceptions, during transition from asexual to sexual stage, linoleic acid concentration decreased markedly, mainly in P, while oleic acid concentration increased. PMID:27129978

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

    PubMed

    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

  6. 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

  7. TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci.

    PubMed

    Lee, Sang Ho; Wang, Hao; Labroli, Marc; Koseoglu, Sandra; Zuck, Paul; Mayhood, Todd; Gill, Charles; Mann, Paul; Sher, Xinwei; Ha, Sookhee; Yang, Shu-Wei; Mandal, Mihir; Yang, Christine; Liang, Lianzhu; Tan, Zheng; Tawa, Paul; Hou, Yan; Kuvelkar, Reshma; DeVito, Kristine; Wen, Xiujuan; Xiao, Jing; Batchlett, Michelle; Balibar, Carl J; Liu, Jenny; Xiao, Jianying; Murgolo, Nicholas; Garlisi, Charles G; Sheth, Payal R; Flattery, Amy; Su, Jing; Tan, Christopher; Roemer, Terry

    2016-03-01

    The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci. PMID:26962156

  8. 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

  9. Backdoor pathway for dihydrotestosterone biosynthesis: implications for normal and abnormal human sex development.

    PubMed

    Fukami, Maki; Homma, Keiko; Hasegawa, Tomonobu; Ogata, Tsutomu

    2013-04-01

    We review the current knowledge about the "backdoor" pathway for the biosynthesis of dihydrotestosterone (DHT). While DHT is produced from cholesterol through the conventional "frontdoor" pathway via testosterone, recent studies have provided compelling evidence for the presence of an alternative "backdoor" pathway to DHT without testosterone intermediacy. This backdoor pathway is known to exist in the tammar wallaby pouch young testis and the immature mouse testis, and has been suggested to be present in the human as well. Indeed, molecular analysis has identified pathologic mutations of genes involved in the backdoor pathway in genetic male patients with undermasculinized external genitalia, and urine steroid profile analysis has argued for the relevance of the activated backdoor pathway to abnormal virilization in genetic females with cytochrome P450 oxidoreductase deficiency and 21-hydroxylase deficiency. It is likely that the backdoor pathway is primarily operating in the fetal testis in a physiological condition to produce a sufficient amount of DHT for male sex development, and that the backdoor pathway is driven with a possible interaction between fetal and permanent adrenals in pathologic conditions with increased 17-hydroxyprogesterone levels. These findings provide novel insights into androgen biosynthesis in both physiological and pathological conditions. PMID:23073980

  10. 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

  11. The putrescine biosynthesis pathway in Lactococcus lactis is transcriptionally regulated by carbon catabolic repression, mediated by CcpA.

    PubMed

    Linares, Daniel M; del Río, Beatriz; Ladero, Victor; Redruello, Begoña; Martín, María Cruz; Fernández, María; Alvarez, Miguel A

    2013-07-01

    Lactococcus lactis is the lactic acid bacterium most widely used by the dairy industry as a starter for the manufacture of fermented products such as cheese and buttermilk. However, some strains produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The proteins involved in this pathway, including those necessary for agmatine uptake and conversion into putrescine, are encoded by the aguB, aguD, aguA and aguC genes, which together form an operon. This paper reports the mechanism of regulation of putrescine biosynthesis in L. lactis. It is shown that the aguBDAC operon, which contains a cre site at the promoter of aguB (the first gene of the operon), is transcriptionally regulated by carbon catabolic repression (CCR) mediated by the catabolite control protein CcpA. PMID:23688550

  12. 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

  13. Transcriptome Analysis of Manganese-deficient Chlamydomonas reinhardtii Provides Insight on the Chlorophyll Biosynthesis Pathway

    SciTech Connect

    Lockhart, Ainsley; Zvenigorodsky, Natasha; Pedraza, Mary Ann; Lindquist, Erika

    2011-08-11

    The biosynthesis of chlorophyll and other tetrapyrroles is a vital but poorly understood process. Recent genomic advances with the unicellular green algae Chlamydomonas reinhardtii have created opportunity to more closely examine the mechanisms of the chlorophyll biosynthesis pathway via transcriptome analysis. Manganese is a nutrient of interest for complex reactions because of its multiple stable oxidation states and role in molecular oxygen coordination. C. reinhardtii was cultured in Manganese-deplete Tris-acetate-phosphate (TAP) media for 24 hours and used to create cDNA libraries for sequencing using Illumina TruSeq technology. Transcriptome analysis provided intriguing insight on possible regulatory mechanisms in the pathway. Evidence supports similarities of GTR (Glutamyl-tRNA synthase) to its Chlorella vulgaris homolog in terms of Mn requirements. Data was also suggestive of Mn-related compensatory up-regulation for pathway proteins CHLH1 (Manganese Chelatase), GUN4 (Magnesium chelatase activating protein), and POR1 (Light-dependent protochlorophyllide reductase). Intriguingly, data suggests possible reciprocal expression of oxygen dependent CPX1 (coproporphyrinogen III oxidase) and oxygen independent CPX2. Further analysis using RT-PCR could provide compelling evidence for several novel regulatory mechanisms in the chlorophyll biosynthesis pathway.

  14. An integrated approach to demonstrating the ANR pathway of proanthocyanidin biosynthesis in plants.

    PubMed

    Peng, Qing-Zhong; Zhu, Yue; Liu, Zhong; Du, Ci; Li, Ke-Gang; Xie, De-Yu

    2012-09-01

    Proanthocyanidins (PAs) are oligomers or polymers of plant flavan-3-ols and are important to plant adaptation in extreme environmental conditions. The characterization of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) has demonstrated the different biogenesis of four stereo-configurations of flavan-3-ols. It is important to understand whether ANR and the ANR pathway widely occur in the plant kingdom. Here, we report an integrated approach to demonstrate the ANR pathway in plants. This includes different methods to extract native ANR from different tissues of eight angiosperm plants (Lotus corniculatus, Desmodium uncinatum, Medicago sativa, Hordeum vulgare, Vitis vinifera, Vitis bellula, Parthenocissus heterophylla, and Cerasus serrulata) and one fern plant (Dryopteris pycnopteroides), a general enzymatic analysis approach to demonstrate the ANR activity, high-performance liquid chromatography-based fingerprinting to demonstrate (-)-epicatechin and other flavan-3-ol molecules, and phytochemical analysis of PAs. Results demonstrate that in addition to leaves of M. sativa, tissues of other eight plants contain an active ANR pathway. Particularly, the leaves, flowers and pods of D. uncinatum, which is a model plant to study LAR and the LAR pathways, are demonstrated to express an active ANR pathway. This finding suggests that the ANR pathway involves PA biosynthesis in D. uncinatum. In addition, a sequence BLAST analysis reveals that ANR homologs have been sequenced in plants from both gymnosperms and angiosperms. These data show that the ANR pathway to PA biosynthesis occurs in both seed and seedless vascular plants. PMID:22678031

  15. Biosynthesis of Unusual Moth Pheromone Components Involves Two Different Pathways in the Navel Orangeworm, Amyelois transitella

    PubMed Central

    Wang, Hong-Lei; Zhao, Cheng-Hua; Millar, Jocelyn G.; Cardé, Ring T.

    2010-01-01

    The sex pheromone of the navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), consists of two different types of components, one type including (11Z,13Z)-11,13-hexadecadienal (11Z,13Z-16:Ald) with a terminal functional group containing oxygen, similar to the majority of moth pheromones reported, and another type including the unusual long-chain pentaenes, (3Z,6Z,9Z,12Z,15Z)-3,6,9,12,15-tricosapentaene (3Z,6Z,9Z,12Z,15Z-23:H) and (3Z,6Z,9Z,12Z,15Z)- 3,6,9,12,15-pentacosapentaene (3Z,6Z,9Z,12Z,15Z-25:H). After decapitation of females, the titer of 11Z,13Z-16:Ald in the pheromone gland decreased significantly, whereas the titer of the pentaenes remained unchanged. Injection of a pheromone biosynthesis activating peptide (PBAN) into the abdomens of decapitated females restored the titer of 11Z,13Z-16:Ald and even increased it above that in intact females, whereas the titer of the pentaenes in the pheromone gland was not affected by PBAN injection. In addition to common fatty acids, two likely precursors of 11Z,13Z-16:Ald, i.e., (Z)-11-hexadecenoic and (11Z,13Z)-11,13-hexadecadienoic acid, as well as traces of (Z)-6-hexadecenoic acid, were found in gland extracts. In addition, pheromone gland lipids contained (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-icosapentaenoic acid, which also was found in extracts of the rest of the abdomen. Deuterium-labeled fatty acids, (16,16,16-D3)-hexadecanoic acid and (Z)-[13,13,14,14,15,15,16,16,16-D9]-11-hexadecenoic acid, were incorporated into 11Z,13Z-16:Ald after topical application to the sex pheromone gland coupled with abdominal injection of PBAN. Deuterium label was incorporated into the C23 and C25 pentaenes after injection of (9Z,12Z,15Z)- [17,17,18,18,18-D5]-9,12,15-octadecatrienoic acid into 1–2 d old female pupae. These labeling results, in conjunction with the composition of fatty acid intermediates found in pheromone gland extracts, support different pathways leading to the two pheromone components. 11Z,13Z-16

  16. Retinoic acid biosynthesis is impaired in human and murine endometriosis.

    PubMed

    Pierzchalski, Keely; Taylor, Robert N; Nezhat, Ceana; Jones, Jace W; Napoli, Joseph L; Yang, Guixiang; Kane, Maureen A; Sidell, Neil

    2014-10-01

    Endometriosis is characterized by the presence of endometrial glands and stroma in extrauterine sites. Our objective was to determine whether endometriotic lesions (ELs) from women with endometriosis have altered retinoid levels compared with their eutopic endometrium, and to test the hypothesis that defects in all-trans retinoic acid (ATRA) biosynthesis in EL is related to reduced expression of cellular retinol-binding protein type 1 (RBP1). Retinoids were evaluated by liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography in eutopic endometrial biopsies (EBs) and ELs from 42 patients with pathologically confirmed endometriosis. The ATRA levels were reduced, whereas the retinol and retinyl ester concentrations were elevated in EL compared with EB tissue. Similar results were found in a mouse model of endometriosis that used green fluorescent protein-positive endometrial tissue injected into the peritoneum of syngeneic hosts to mimic retrograde menses. The ATRA biosynthesis in vitro in retinol-treated primary human endometrial stromal cell (ESC) cultures derived from ELs was reduced compared with that of ESCs derived from patient-matched EBs. Correspondingly, RBP1 expression was reduced in tissue and ESCs derived from EL versus EB. Rbp1(-/-) mice showed reduced endometrial ATRA concentrations compared with wild type, associated with loss of tissue organization and hypercellularity. These findings provide the first quantitative measurements of ATRA in human endometrium and endometriosis, demonstrating reduced ATRA in ectopic tissue and corresponding ESC cultures. Quantitation of retinoids in murine endometriosis and in Rbp1(-/-) mice supports the contention that impaired ATRA synthesis caused by reduced RBP1 promotes an "endometriosis phenotype" that enables cells to implant and grow at ectopic sites. PMID:25143356

  17. Characterization of a C3 Deoxygenation Pathway Reveals a Key Branch Point in Aminoglycoside Biosynthesis.

    PubMed

    Lv, Meinan; Ji, Xinjian; Zhao, Junfeng; Li, Yongzhen; Zhang, Chen; Su, Li; Ding, Wei; Deng, Zixin; Yu, Yi; Zhang, Qi

    2016-05-25

    Apramycin is a clinically interesting aminoglycoside antibiotic (AGA) containing a highly unique bicyclic octose moiety, and this octose is deoxygenated at the C3 position. Although the biosynthetic pathways for most 2-deoxystreptamine-containing AGAs have been well characterized, the pathway for apramycin biosynthesis, including the C3 deoxygenation process, has long remained unknown. Here we report detailed investigation of apramycin biosynthesis by a series of genetic, biochemical and bioinformatical studies. We show that AprD4 is a novel radical S-adenosyl-l-methionine (SAM) enzyme, which uses a noncanonical CX3CX3C motif for binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis. We also show that AprD3 is an NADPH-dependent reductase that catalyzes the reduction of the dehydrated product from AprD4-catalyzed reaction to generate lividamine, a C3' deoxygenated product of paromamine. AprD4 and AprD3 do not form a tight catalytic complex, as shown by protein complex immunoprecipitation and other assays. The AprD4/AprD3 enzyme system acts on different pseudodisaccharide substrates but does not catalyze the deoxygenation of oxyapramycin, an apramycin analogue containing a C3 hydroxyl group on the octose moiety, suggesting that oxyapramycin and apramycin are partitioned into two parallel pathways at an early biosynthetic stage. Functional dissection of the C6 dehydrogenase AprQ shows the crosstalk between different AGA biosynthetic gene clusters from the apramycin producer Streptomyces tenebrarius, and reveals the remarkable catalytic versatility of AprQ. Our study highlights the intriguing chemistry in apramycin biosynthesis and nature's ingenuity in combinatorial biosynthesis of natural products. PMID:27120352

  18. Multicellular compartmentation of catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate

    PubMed Central

    St-Pierre, B; Vazquez-Flota, FA; De Luca V

    1999-01-01

    In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed. PMID:10330473

  19. Biosynthesis of Jasmonic Acid by Several Plant Species 1

    PubMed Central

    Vick, Brady A.; Zimmerman, Don C.

    1984-01-01

    Six plant species metabolized 18O-labeled 12-oxo-cis,cis-10,15-phytodienoic acid (12-oxo-PDA) to short chain cyclic fatty acids. The plant species were corn (Zea mays L.), eggplant (Solanum melongena L.), flax (Linum usitatissimum L.), oat (Avena sativa L.), sunflower (Helianthus annuus L.), and wheat (Triticum aestivum L.). Among the products was jasmonic acid, a natural plant constituent with growth-regulating properties. The pathway is the same as the one recently reported by us for jasmonic acid synthesis in Vicia faba L. pericarp. First, the ring double bond of 12-oxo-PDA is saturated; then β-oxidation enzymes remove six carbons from the carboxyl side chain of the ring. Substrate specificity studies indicated that neither the stereochemistry of the side chain at carbon 13 of 12-oxo-PDA nor the presence of the double bond at carbon 15 was crucial for either enzyme step. The presence of enzymes which convert 12-oxo-PDA to jasmonic acid in several plant species indicates that this may be a general metabolic pathway in plants. PMID:16663643

  20. Reconstruction of diaminopimelic acid biosynthesis allows characterisation of Mycobacterium tuberculosis N-succinyl-L,L-diaminopimelic acid desuccinylase

    PubMed Central

    Usha, Veeraraghavan; Lloyd, Adrian J.; Roper, David I.; Dowson, Christopher G.; Kozlov, Guennadi; Gehring, Kalle; Chauhan, Smita; Imam, Hasan T.; Blindauer, Claudia A.; Besra, Gurdyal S.

    2016-01-01

    With the increased incidence of tuberculosis (TB) caused by Mycobacterium tuberculosis there is an urgent need for new and better anti-tubercular drugs. N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a key enzyme in the succinylase pathway for the biosynthesis of meso-diaminopimelic acid (meso-DAP) and L-lysine. DapE is a zinc containing metallohydrolase which hydrolyses N-succinyl L,L diaminopimelic acid (L,L-NSDAP) to L,L-diaminopimelic acid (L,L-DAP) and succinate. M. tuberculosis DapE (MtDapE) was cloned, over-expressed and purified as an N-terminal hexahistidine ((His)6) tagged fusion containing one zinc ion per DapE monomer. We redesigned the DAP synthetic pathway to generate L,L-NSDAP and other L,L-NSDAP derivatives and have characterised MtDapE with these substrates. In contrast to its other Gram negative homologues, the MtDapE was insensitive to inhibition by L-captopril which we show is consistent with novel mycobacterial alterations in the binding site of this drug. PMID:26976706

  1. Identification of an itaconic acid degrading pathway in itaconic acid producing Aspergillus terreus.

    PubMed

    Chen, Mei; Huang, Xuenian; Zhong, Chengwei; Li, Jianjun; Lu, Xuefeng

    2016-09-01

    Itaconic acid, one of the most promising and flexible bio-based chemicals, is mainly produced by Aspergillus terreus. Previous studies to improve itaconic acid production in A. terreus through metabolic engineering were mainly focused on its biosynthesis pathway, while the itaconic acid-degrading pathway has largely been ignored. In this study, we used transcriptomic, proteomic, bioinformatic, and in vitro enzymatic analyses to identify three key enzymes, itaconyl-CoA transferase (IctA), itaconyl-CoA hydratase (IchA), and citramalyl-CoA lyase (CclA), that are involved in the catabolic pathway of itaconic acid in A. terreus. In the itaconic acid catabolic pathway in A. terreus, itaconic acid is first converted by IctA into itaconyl-CoA with succinyl-CoA as the CoA donor, and then itaconyl-CoA is hydrated into citramalyl-CoA by IchA. Finally, citramalyl-CoA is cleaved into acetyl-CoA and pyruvate by CclA. Moreover, IctA can also catalyze the reaction between citramalyl-CoA and succinate to generate succinyl-CoA and citramalate. These results, for the first time, identify the three key enzymes, IctA, IchA, and CclA, involved in the itaconic acid degrading pathway in itaconic acid producing A. terreus. The results will facilitate the improvement of itaconic acid production by metabolically engineering the catabolic pathway of itaconic acid in A. terreus. PMID:27102125

  2. The Methymycin/Pikromycin Biosynthetic Pathway: A Model for Metabolic Diversity in Natural Product Biosynthesis

    PubMed Central

    Kittendorf, Jeffrey D.; Sherman, David H.

    2010-01-01

    The methymycin/pikromycin (Pik) macrolide pathway represents a robust metabolic system for analysis of modular polyketide biosynthesis. The enzymes that comprise this biosynthetic pathway display unprecedented substrate flexibility, combining to produce six structurally diverse macrolide antibiotics in Streptomyces venezuelae. Thus, it is appealing to consider that the pikromycin biosynthetic enzymes could be leveraged for high throughput production of novel macrolide antibiotics. Accordingly, efforts over the past decade have focused on the detailed investigation of the six-module polyketide synthase, desosamine sugar assembly and glycosyl transfer, and the cytochrome P450 monooxygenase that is responsible for hydroxylation. This review summarizes the advances in understanding of pikromycin biosynthesis that have been gained during the course of these investigations. PMID:19027305

  3. Tailoring pathway modularity in the biosynthesis of erythromycin analogs heterologously engineered in E. coli

    PubMed Central

    Zhang, Guojian; Li, Yi; Fang, Lei; Pfeifer, Blaine A.

    2015-01-01

    Type I modular polyketide synthases are responsible for potent therapeutic compounds that include avermectin (antihelinthic), rapamycin (immunosuppressant), pikromycin (antibiotic), and erythromycin (antibiotic). However, compound access and biosynthetic manipulation are often complicated by properties of native production organisms, prompting an approach (termed heterologous biosynthesis) illustrated in this study through the reconstitution of the erythromycin pathway through Escherichia coli. Using this heterologous system, 16 tailoring pathways were introduced, systematically producing eight chiral pairs of deoxysugar substrates. Successful analog formation for each new pathway emphasizes the remarkable flexibility of downstream enzymes to accommodate molecular variation. Furthermore, analogs resulting from three of the pathways demonstrated bioactivity against an erythromycin-resistant Bacillus subtilis strain. The approach and results support a platform for continued molecular diversification of the tailoring components of this and other complex natural product pathways in a manner that mirrors the modular nature of the upstream megasynthases responsible for aglycone polyketide formation. PMID:26601183

  4. RNA-Seq analysis for indigo biosynthesis pathway genes in Indigofera tinctoria and Polygonum tinctorium.

    PubMed

    Sarangi, Bijaya K; Minami, Yoshiko; Thul, Sanjog T

    2015-12-01

    Natural indigo is the most important blue dye for textile dyeing and valuable secondary metabolite biosynthesized in Indigofera tinctoria and Polygonum tinctorium plants. Present investigation is made to generation of gene resource for pathway enrichment and to understand possible gene expression involved in indigo biosynthesis. The data about raw reads and the transcriptome assembly project has been deposited at GenBank under the accessions SRA180766 and SRX692542 for I. tinctoria and P. tinctorium, respectively. PMID:26697377

  5. Putative pathway of sex pheromone biosynthesis and degradation by expression patterns of genes identified from female pheromone gland and adult antenna of Sesamia inferens (Walker).

    PubMed

    Zhang, Ya-Nan; Xia, Yi-Han; Zhu, Jia-Yao; Li, Sheng-Yun; Dong, Shuang-Lin

    2014-05-01

    The general pathway of biosynthesis and degradation for Type-I sex pheromones in moths is well established, but some genes involved in this pathway remain to be characterized. The purple stem borer, Sesamia inferens, employs a pheromone blend containing components with three different terminal functional groups (Z11-16:OAc, Z11-16:OH, and Z11-16:Ald) of Type-I sex pheromones. Thus, it provides a good model to study the diversity of genes involved in pheromone biosynthesis and degradation pathways. By analyzing previously obtained transcriptomic data of the sex pheromone glands and antennae, we identified 73 novel genes that are possibly related to pheromone biosynthesis (46 genes) or degradation (27 genes). Gene expression patterns and phylogenetic analysis revealed that one desaturase (SinfDes4), one fatty acid reductase (SinfFAR2), and one fatty acid xtransport protein (SinfFATP1) genes were predominantly expressed in pheromone glands, and clustered with genes involved in pheromone synthesis in other moth species. Ten genes including five carboxylesterases (SinfCXE10, 13, 14, 18, and 20), three aldehyde oxidases (SinfAOX1, 2 and 3), and two alcohol dehydrogenases (SinfAD1 and 3) were expressed specifically or predominantly in antennae, and could be candidate genes involved in pheromone degradation. SinfAD1 and 3 are the first reported alcohol dehydrogenase genes with antennae-biased expression. Based on these results we propose a pathway involving these potential enzyme-encoding gene candidates in sex pheromone biosynthesis and degradation in S. inferens. This study provides robust background information for further elucidation of the genetic basis of sex pheromone biosynthesis and degradation, and ultimately provides potential targets to disrupt sexual communication in S. inferens for control purposes. PMID:24817326

  6. Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

    PubMed

    Dey, Sanghamitra; Lane, James M; Lee, Richard E; Rubin, Eric J; Sacchettini, James C

    2010-08-10

    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 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue 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 A, 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

  7. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway.

    PubMed

    Cárdenas, Pablo D; Sonawane, Prashant D; Pollier, Jacob; Vanden Bossche, Robin; Dewangan, Veena; Weithorn, Efrat; Tal, Lior; Meir, Sagit; Rogachev, Ilana; Malitsky, Sergey; Giri, Ashok P; Goossens, Alain; Burdman, Saul; Aharoni, Asaph

    2016-01-01

    Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops. PMID:26876023

  8. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway

    PubMed Central

    Cárdenas, Pablo D.; Sonawane, Prashant D.; Pollier, Jacob; Vanden Bossche, Robin; Dewangan, Veena; Weithorn, Efrat; Tal, Lior; Meir, Sagit; Rogachev, Ilana; Malitsky, Sergey; Giri, Ashok P.; Goossens, Alain; Burdman, Saul; Aharoni, Asaph

    2016-01-01

    Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops. PMID:26876023

  9. 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.

  10. Local and Systemic Biosynthesis of Salicylic Acid in Infected Cucumber Plants.

    PubMed Central

    Meuwly, P.; Molders, W.; Buchala, A.; Metraux, J. P.

    1995-01-01

    Radiolabeling studies showed that salicylic acid (SA), an essential component in the signal transduction pathway leading to systemic acquired resistance, is synthesized from phenylalanine (Phe) and benzoic acid in cucumber (Cucumis sativus L.) plants inoculated with pathogens. Leaf discs from plants inoculated with either tobacco necrosis virus or Pseudomonas lachrymans incorporated more [14C]Phe into [14C]SA than mock-inoculated controls. The identity of SA was confirmed by gas chromatography-mass spectrometry. No reduction in specific activity of [14C]SA was observed for either free or bound SA between control and infected plants after feeding [14C]Phe. A specific inhibitor of Phe ammonia-lyase, 2-aminoindan-2-phosphonic acid, completely inhibited the incorporation of [14C]Phe into [14C]SA, although plants treated with 2-aminoindan-2-phosphonic acid could still produce [14C]SA from [14C]benzoic acid. Biosynthesis of SA in tissue inoculated with tobacco necrosis virus followed a transient pattern with the highest induction occurring 72 h postinoculation. Uninfected tissues from an infected plant synthesized de novo more SA than did controls. This suggests the involvement of a systemic signal triggering SA synthesis in tissue distant from the site of infection that display systemic acquired resistance. PMID:12228656

  11. 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.

  12. Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

    PubMed Central

    Lambou, Karine; Pennati, Andrea; Valsecchi, Isabel; Tada, Rui; Sherman, Stephen; Sato, Hajime; Beau, Remi

    2013-01-01

    The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD+ to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom. PMID:23563483

  13. LTP3 contributes to disease susceptibility in Arabidopsis by enhancing abscisic acid (ABA) biosynthesis.

    PubMed

    Gao, Shan; Guo, Wenya; Feng, Wen; Liu, Liang; Song, Xiaorui; Chen, Jian; Hou, Wei; Zhu, Hongxia; Tang, Saijun; Hu, Jian

    2016-04-01

    Several plant lipid transfer proteins (LTPs) act positively in plant disease resistance. Here, we show that LTP3 (At5g59320), a pathogen and abscisic acid (ABA)-induced gene, negatively regulates plant immunity in Arabidopsis. The overexpression of LTP3 (LTP3-OX) led to an enhanced susceptibility to virulent bacteria and compromised resistance to avirulent bacteria. On infection of LTP3-OX plants with Pseudomonas syringae pv. tomato, genes involved in ABA biosynthesis, NCED3 and AAO3, were highly induced, whereas salicylic acid (SA)-related genes, ICS1 and PR1, were down-regulated. Accordingly, in LTP3-OX plants, we observed increased ABA levels and decreased SA levels relative to the wild-type. We also showed that the LTP3 overexpression-mediated enhanced susceptibility was partially dependent on AAO3. Interestingly, loss of function of LTP3 (ltp3-1) did not affect ABA pathways, but resulted in PR1 gene induction and elevated SA levels, suggesting that LTP3 can negatively regulate SA in an ABA-independent manner. However, a double mutant consisting of ltp3-1 and silent LTP4 (ltp3/ltp4) showed reduced susceptibility to Pseudomonas and down-regulation of ABA biosynthesis genes, suggesting that LTP3 acts in a redundant manner with its closest homologue LTP4 by modulating the ABA pathway. Taken together, our data show that LTP3 is a novel negative regulator of plant immunity which acts through the manipulation of the ABA-SA balance. PMID:26123657

  14. 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

  15. Systematic profiling of indole-3-acetic acid biosynthesis in bacteria using LC-MS/MS.

    PubMed

    Lin, Guang-Huey; Chang, Chung-Yu; Lin, Huei-Ru

    2015-04-15

    Indole-3-acetic acid (IAA) is produced from tryptophan through five synthesis pathways. A comprehensive method for the quantification of IAA and biosynthesis-related intermediates in a culture medium was developed. Sample preparation was simple with protein precipitation. The analytes were separated on a superficially porous C18 silica column and detected by electrospray ionization-tandem mass spectrometry in the positive ion multiple reaction monitoring mode. The limit of detection was 0.05 μM, and the lower limits of quantification ranged from 0.05 to 2 μM. The intra-day and inter-day precision and accuracy were less than 13.96%. Ion suppression was observed, and the deuterated internal standards were used to compensate for the matrix effect. The method was applied to analyze changes in tryptophan catabolism in a culture medium of Pseudomonas putida. The proposed method is robust and suitable for the systematic profiling of IAA biosynthesis in culture supernatant. PMID:25746752

  16. Lactate Racemization as a Rescue Pathway for Supplying d-Lactate to the Cell Wall Biosynthesis Machinery in Lactobacillus plantarum

    PubMed Central

    Goffin, Philippe; Deghorain, Marie; Mainardi, Jean-Luc; Tytgat, Isabelle; Champomier-Vergès, Marie-Christine; Kleerebezem, Michiel; Hols, Pascal

    2005-01-01

    Lactobacillus plantarum is a lactic acid bacterium that produces d- and l-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for d-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production of d-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for d-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires l-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by l-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of l-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is d-lactate dependent. We also show that the growth defect can be partially restored by expression of the d-alanyl-d-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various d-2-hydroxy acids but not d-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in d-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in d-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for d-lactate production upon

  17. Abscisic acid biosynthesis in isolated embryos of Zea mays L

    SciTech Connect

    Gage, D.A.; Fong, F.; Zeevaart, J.A.D. Texas A M Univ., College Station )

    1989-04-01

    Previous labeling experiments with {sup 18}O{sub 2} have supported the hypothesis that stress-induced abscisic acid (ABA) is synthesized through an indirect pathway involving an oxygenated carotenoid (xanthophyll) as a precursor. To investigate ABA formation under nonstress conditions, an {sup 18}O{sub 2} labeling experiment was conducted with isolated embryos from in vitro grown maize (Zea mays L.) kernels. Of the ABA produced during the incubation in {sup 18}O{sub 2}, three-fourths contained a single {sup 18}O atom located in the carboxyl group. Approximately one-fourth of the ABA synthesized during the experiment contained two {sup 18}O atoms. These results suggest that ABA synthesized in maize embryos under nonstress conditions also proceeds via the indirect pathway, requiring a xanthophyll precursor. It was also found that the newly synthesized ABA was preferentially released into the surrounding medium.

  18. 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. PMID:25366131

  19. 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. PMID:27178211

  20. Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

    SciTech Connect

    Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen

    2012-04-15

    Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

  1. 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

  2. Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii.

    PubMed

    Chi, Xiaoyuan; Zhang, Xiaowen; Guan, Xiangyu; Ding, Ling; Li, Youxun; Wang, Mingqing; Lin, Hanzhi; Qin, Song

    2008-04-01

    Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal delta 12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae). PMID:18545969

  3. Rhamnose biosynthesis pathway supplies precursors for primary and secondary metabolism in Saccharopolyspora spinosa.

    PubMed

    Madduri, K; Waldron, C; Merlo, D J

    2001-10-01

    Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in Saccharopolyspora spinosa are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of spinosyn fermentation products. PMID:11544225

  4. Dissecting Abscisic Acid Signaling Pathways Involved in Cuticle Formation.

    PubMed

    Cui, Fuqiang; Brosché, Mikael; Lehtonen, Mikko T; Amiryousefi, Ali; Xu, Enjun; Punkkinen, Matleena; Valkonen, Jari P T; Fujii, Hiroaki; Overmyer, Kirk

    2016-06-01

    The cuticle is the outer physical barrier of aerial plant surfaces and an important interaction point between plants and the environment. Many environmental stresses affect cuticle formation, yet the regulatory pathways involved remain undefined. We used a genetics and gene expression analysis in Arabidopsis thaliana to define an abscisic acid (ABA) signaling loop that positively regulates cuticle formation via the core ABA signaling pathway, including the PYR/PYL receptors, PP2C phosphatase, and SNF1-Related Protein Kinase (SnRK) 2.2/SnRK2.3/SnRK2.6. Downstream of the SnRK2 kinases, cuticle formation was not regulated by the ABA-responsive element-binding transcription factors but rather by DEWAX, MYB16, MYB94, and MYB96. Additionally, low air humidity increased cuticle formation independent of the core ABA pathway and cell death/reactive oxygen species signaling attenuated expression of cuticle-biosynthesis genes. In Physcomitrella patens, exogenous ABA suppressed expression of cuticle-related genes, whose Arabidopsis orthologs were ABA-induced. Hence, the mechanisms regulating cuticle formation are conserved but sophisticated in land plants. Signaling specifically related to cuticle deficiency was identified to play a major role in the adaptation of ABA signaling pathway mutants to increased humidity and in modulating their immunity to Botrytis cinerea in Arabidopsis. These results define a cuticle-specific downstream branch in the ABA signaling pathway that regulates responses to the external environment. PMID:27060495

  5. 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

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

    PubMed

    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

  7. Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid

    PubMed Central

    Zheng, Xiao-yu; Zhou, Mian; Yoo, Heejin; Pruneda-Paz, Jose L.; Spivey, Natalie Weaver; Kay, Steve A.; Dong, Xinnian

    2015-01-01

    The plant hormone salicylic acid (SA) is essential for local defense and systemic acquired resistance (SAR). When plants, such as Arabidopsis, are challenged by different pathogens, an increase in SA biosynthesis generally occurs through transcriptional induction of the key synthetic enzyme isochorismate synthase 1 (ICS1). However, the regulatory mechanism for this induction is poorly understood. Using a yeast one-hybrid screen, we identified two transcription factors (TFs), NTM1-LIKE 9 (NTL9) and CCA1 HIKING EXPEDITION (CHE), as activators of ICS1 during specific immune responses. NTL9 is essential for inducing ICS1 and two other SA synthesis-related genes, PHYTOALEXIN-DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), in guard cells that form stomata. Stomata can quickly close upon challenge to block pathogen entry. This stomatal immunity requires ICS1 and the SA signaling pathway. In the ntl9 mutant, this response is defective and can be rescued by exogenous application of SA, indicating that NTL9-mediated SA synthesis is essential for stomatal immunity. CHE, the second identified TF, is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for the pathogen-induced SA synthesis in systemic tissues during SAR. CHE may also regulate ICS1 through the known transcription activators CALMODULIN BINDING PROTEIN 60g (CBP60g) and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) because induction of these TF genes is compromised in the che-2 mutant. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner and therefore fills a gap in the signal transduction pathway between pathogen recognition and SA production. PMID:26139525

  8. Arabidopsis Chlorophyll Biosynthesis: An Essential Balance between the Methylerythritol Phosphate and Tetrapyrrole Pathways[C][W

    PubMed Central

    Kim, Se; Schlicke, Hagen; Van Ree, Kalie; Karvonen, Kristine; Subramaniam, Anant; Richter, Andreas; Grimm, Bernhard; Braam, Janet

    2013-01-01

    Chlorophyll, essential for photosynthesis, is composed of a chlorin ring and a geranylgeranyl diphosphate (GGPP)–derived isoprenoid, which are generated by the tetrapyrrole and methylerythritol phosphate (MEP) biosynthesis pathways, respectively. Although a functional MEP pathway is essential for plant viability, the underlying basis of the requirement has been unclear. We hypothesized that MEP pathway inhibition is lethal because a reduction in GGPP availability results in a stoichiometric imbalance in tetrapyrrolic chlorophyll precursors, which can cause deadly photooxidative stress. Consistent with this hypothesis, lethality of MEP pathway inhibition in Arabidopsis thaliana by fosmidomycin (FSM) is light dependent, and toxicity of MEP pathway inhibition is reduced by genetic and chemical impairment of the tetrapyrrole pathway. In addition, FSM treatment causes a transient accumulation of chlorophyllide and transcripts associated with singlet oxygen-induced stress. Furthermore, exogenous provision of the phytol molecule reduces FSM toxicity when the phytol can be modified for chlorophyll incorporation. These data provide an explanation for FSM toxicity and thereby provide enhanced understanding of the mechanisms of FSM resistance. This insight into MEP pathway inhibition consequences underlines the risk plants undertake to synthesize chlorophyll and suggests the existence of regulation, possibly involving chloroplast-to-nucleus retrograde signaling, that may monitor and maintain balance of chlorophyll precursor synthesis. PMID:24363312

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

    PubMed Central

    2013-01-01

    Background 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. Results 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. Conclusions 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

  10. Cross-functionalities of Bacillus deacetylases involved in bacillithiol biosynthesis and bacillithiol-S-conjugate detoxification pathways.

    PubMed

    Fang, Zhong; Roberts, Alexandra A; Weidman, Karissa; Sharma, Sunil V; Claiborne, Al; Hamilton, Christopher J; Dos Santos, Patricia C

    2013-09-01

    BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the acetyl group from N-acetylglucosamine malate to generate glucosamine malate. In Bacillus anthracis, BA1557 has been identified as the N-acetylglucosamine malate deacetylase (BshB); however, a high content of bacillithiol (~70%) was still observed in the B. anthracis ∆BA1557 strain. Genomic analysis led to the proposal that another deacetylase could exhibit cross-functionality in bacillithiol biosynthesis. In the present study, BA1557, its paralogue BA3888 and orthologous Bacillus cereus enzymes BC1534 and BC3461 have been characterized for their deacetylase activity towards N-acetylglucosamine malate, thus providing biochemical evidence for this proposal. In addition, the involvement of deacetylase enzymes is also expected in bacillithiol-detoxifying pathways through formation of S-mercapturic adducts. The kinetic analysis of bacillithiol-S-bimane conjugate favours the involvement of BA3888 as the B. anthracis bacillithiol-S-conjugate amidase (Bca). The high degree of specificity of this group of enzymes for its physiological substrate, along with their similar pH-activity profile and Zn²⁺-dependent catalytic acid-base reaction provides further evidence for their cross-functionalities. PMID:23758290

  11. The De-Etiolated 1 Homolog of Arabidopsis Modulates the ABA Signaling Pathway and ABA Biosynthesis in Rice.

    PubMed

    Zang, Guangchao; Zou, Hanyan; Zhang, Yuchan; Xiang, Zheng; Huang, Junli; Luo, Li; Wang, Chunping; Lei, Kairong; Li, Xianyong; Song, Deming; Din, Ahmad Ud; Wang, Guixue

    2016-06-01

    DEETIOLATED1 (DET1) plays a critical role in developmental and environmental responses in many plants. To date, the functions of OsDET1 in rice (Oryza sativa) have been largely unknown. OsDET1 is an ortholog of Arabidopsis (Arabidopsis thaliana) DET1 Here, we found that OsDET1 is essential for maintaining normal rice development. The repression of OsDET1 had detrimental effects on plant development, and leaded to contradictory phenotypes related to abscisic acid (ABA) in OsDET1 interference (RNAi) plants. We found that OsDET1 is involved in modulating ABA signaling in rice. OsDET1 RNAi plants exhibited an ABA hypersensitivity phenotype. Using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays, we determined that OsDET1 interacts physically with DAMAGED-SPECIFIC DNA-BINDING PROTEIN1 (OsDDB1) and CONSTITUTIVE PHOTOMORPHOGENIC10 (COP10); DET1- and DDB1-ASSOCIATED1 binds to the ABA receptors OsPYL5 and OsDDB1. We found that the degradation of OsPYL5 was delayed in OsDET1 RNAi plants. These findings suggest that OsDET1 deficiency disturbs the COP10-DET1-DDB1 complex, which is responsible for ABA receptor (OsPYL) degradation, eventually leading to ABA sensitivity in rice. Additionally, OsDET1 also modulated ABA biosynthesis, as ABA biosynthesis was inhibited in OsDET1 RNAi plants and promoted in OsDET1-overexpressing transgenic plants. In conclusion, our data suggest that OsDET1 plays an important role in maintaining normal development in rice and mediates the cross talk between ABA biosynthesis and ABA signaling pathways in rice. PMID:27208292

  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. PMID:27214242

  13. 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.

  14. 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

  15. para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae*

    PubMed Central

    Marbois, Beth; Xie, Letian X.; Choi, Samuel; Hirano, Kathleen; Hyman, Kyle; Clarke, Catherine F.

    2010-01-01

    Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable 13C6-isotope of pABA (p- amino[aromatic-13C6]benzoic acid ([13C6]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[13C6]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [13C6]pABA or [13C6]4HB generate both 13C6-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product 13C6-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ6. This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ6 quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates. PMID:20592037

  16. MRE: a web tool to suggest foreign enzymes for the biosynthesis pathway design with competing endogenous reactions in mind

    PubMed Central

    Kuwahara, Hiroyuki; Alazmi, Meshari; Cui, Xuefeng; Gao, Xin

    2016-01-01

    To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. We developed a novel web server, called MRE, which, for a given pair of starting and desired compounds in a given chassis organism, ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. These unique, chassis-centered features distinguish MRE from existing pathway design tools and allow synthetic biologists to evaluate the design of their biosynthesis systems from a different angle. By using biosynthesis of a range of high-value natural products as a case study, we show that MRE is an effective tool to guide the design and optimization of heterologous biosynthesis pathways. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/. PMID:27131375

  17. MRE: a web tool to suggest foreign enzymes for the biosynthesis pathway design with competing endogenous reactions in mind.

    PubMed

    Kuwahara, Hiroyuki; Alazmi, Meshari; Cui, Xuefeng; Gao, Xin

    2016-07-01

    To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. We developed a novel web server, called MRE, which, for a given pair of starting and desired compounds in a given chassis organism, ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. These unique, chassis-centered features distinguish MRE from existing pathway design tools and allow synthetic biologists to evaluate the design of their biosynthesis systems from a different angle. By using biosynthesis of a range of high-value natural products as a case study, we show that MRE is an effective tool to guide the design and optimization of heterologous biosynthesis pathways. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/. PMID:27131375

  18. A propionate-inducible expression system based on the Corynebacterium glutamicum prpD2 promoter and PrpR activator and its application for the redirection of amino acid biosynthesis pathways.

    PubMed

    Plassmeier, Jens K; Busche, Tobias; Molck, Stella; Persicke, Marcus; Pühler, Alfred; Rückert, Christian; Kalinowski, Jörn

    2013-01-20

    A novel expression system for Corynebacterium glutamicum, based on the transcriptional activator of propionate metabolism genes PrpR and its target promoter/operator sequence, was developed and tested. The activator PrpR is co-activated by propionate added to the growth medium. In a minimal medium a propionate concentration of only 1 mg l⁻¹ was found to be sufficient for full induction (up to 120-fold) of its native target, the propionate metabolism operon prpDBC2. Then, an artificial transcription and translation reporter system, using the cat gene encoding chloramphenicol acetyl transferase was constructed and tested. The induction was found to be as fast and as high as in the natural system, reaching its maximal transcriptional induction rate within 2 min and a significant accumulation of Cat protein at around 30 min. The duration of the induced transcription was found to be controllable by the propionate concentration applied. The prpD2 promoter and PrpR activator based expression system revealed very similar characteristics in minimal and complex media, making it ideal for applications in large scale industrial fermentations. As a proof-of-principle, the expression system was employed for the propionate-inducible redirection of metabolites in a lysine-production C. glutamicum strain at the homoserine dehydrogenase (hom) branching point, which resulted in an up to 2.5-fold increase of the concentrations of the three amino acids (threonine, homoserine and isoleucine) in the supernatant. PMID:22982516

  19. Characterization of a Citrus R2R3-MYB Transcription Factor that Regulates the Flavonol and Hydroxycinnamic Acid Biosynthesis.

    PubMed

    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

  20. 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

  1. 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. PMID:25921651

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

    PubMed

    Bonaventure, Gustavo; Baldwin, Ian T

    2010-03-01

    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

  3. Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid.

    PubMed

    Haeuptle, Micha A; Welti, Michael; Troxler, Heinz; Hülsmeier, Andreas J; Imbach, Timo; Hennet, Thierry

    2011-02-25

    The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells. PMID:21183681

  4. Improvement of Dolichol-linked Oligosaccharide Biosynthesis by the Squalene Synthase Inhibitor Zaragozic Acid*

    PubMed Central

    Haeuptle, Micha A.; Welti, Michael; Troxler, Heinz; Hülsmeier, Andreas J.; Imbach, Timo; Hennet, Thierry

    2011-01-01

    The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc2Man5 in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells. PMID:21183681

  5. A biosynthetic pathway for a prominent class of microbiota-derived bile acids

    PubMed Central

    Devlin, A. Sloan; Fischbach, Michael A.

    2015-01-01

    The gut bile acid pool is millimolar in concentration, varies widely in composition among individuals, and is linked to metabolic disease and cancer. Although these molecules derive almost exclusively from the microbiota, remarkably little is known about which bacterial species and genes are responsible for their biosynthesis. Here, we report a biosynthetic pathway for the second most abundant class in the gut, iso (3β-hydroxy) bile acids, whose levels exceed 300 µM in some humans and are absent in others. We show, for the first time, that iso bile acids are produced by Ruminococcus gnavus, a far more abundant commensal than previously known producers; and that the iso bile acid pathway detoxifies deoxycholic acid, favoring the growth of the keystone genus Bacteroides. By revealing the biosynthetic genes for an abundant class of bile acids, our work sets the stage for predicting and rationally altering the composition of the bile acid pool. PMID:26192599

  6. Artemether Exhibits Amoebicidal Activity against Acanthamoeba castellanii through Inhibition of the Serine Biosynthesis Pathway

    PubMed Central

    Deng, Yihong; Ran, Wei; Man, Suqin; Li, Xueping; Gao, Hongjian; Tang, Wei

    2015-01-01

    Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner and induced ultrastructural modification and cell apoptosis. The iTRAQ quantitative proteomic analysis identified 707 proteins that were differentially expressed after artemether treatment. We focused on phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthesis pathway because of their importance to the growth and proliferation of protozoan and cancer cells. The expression of these proteins in Acanthamoeba was validated using quantitative real-time PCR and Western blotting after artemether treatment. The changes in the expression levels of phosphoserine aminotransferase were consistent with those of phosphoglycerate dehydrogenase. Therefore, the downregulation of phosphoserine aminotransferase may be due to the downregulation of phosphoglycerate dehydrogenase. Furthermore, exogenous serine might antagonize the activity of artemether against Acanthamoeba trophozoites. These results indicate that the serine biosynthesis pathway is important to amoeba survival and that targeting these enzymes would improve the treatment of Acanthamoeba infections. Artemether may be used as a phosphoglycerate dehydrogenase inhibitor to control or block Acanthamoeba infections. PMID:26014935

  7. 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.

  8. 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

  9. Long Chain (C20 and C22) Fatty Acid Biosynthesis in Developing Seeds of Tropaeolum majus

    PubMed Central

    Pollard, Michael R.; Stumpf, Paul K.

    1980-01-01

    The storage triacylglycerols of nasturtium (Tropaeolum majus) seeds are composed principally of cis-11-eicosenoate and cis-13-docosenoate. To investigate the biosynthesis of these C20 and C22 fatty acids, developing seed tissue was incubated with various 14C-labeled precursors. Incubation with [1-14C]acetate produced primarily cis-11-[1-14C]eicosenoate and cis-13-[1,3-14C]docosenoate in the triacylglycerol fraction, the odd-carbon [U-14C]oleate also formed from [14C] acetate was in the polar lipid fraction. Kinetic data showed that this oleate was not channeled into cis-11-eicosenoate nor cis-13-docosenoate over a 24-hour period. Under suitable conditions, nasturtium seed could also produce [14C]stearate, [14C]eicosenoate, and [14C]docosenoate from [1-14C]acetate. The results are discussed in terms of the number of pathways producing fatty acids. From pool size and other considerations, the results can be rationalized only in terms of different de novo systems for oleate biosythesis, one supplying oleate for incorporation into phospholipids and the other supplying oleate for chain elongation and subsequent esterification into triacylglycerols. Because of the probable heterogeneous nature of the seed tissue, it is not known if these two systems are operating in different cell types, in the same cell type at different stages of development, or in the same cell type concurrently. PMID:16661495

  10. Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae.

    PubMed

    Franken, Jaco; Burger, Anita; Swiegers, Jan H; Bauer, Florian F

    2015-08-01

    Industrial synthesis of L-carnitine is currently performed by whole-cell biotransformation of industrial waste products, mostly D-carnitine and cronobetaine, through specific bacterial species. No comparable system has been established using eukaryotic microorganisms, even though there is a significant and growing international demand for either the pure compound or carnitine-enriched consumables. In eukaryotes, including the fungus Neurospora crassa, L-carnitine is biosynthesized through a four-step metabolic conversion of trimethyllysine to L-carnitine. In contrast, the industrial yeast, Saccharomyces cerevisiae lacks the enzymes of the eukaryotic biosynthesis pathway and is unable to synthesize carnitine. This study describes the cloning of all four of the N. crassa carnitine biosynthesis genes and the reconstruction of the entire pathway in S. cerevisiae. The engineered yeast strains were able to catalyze the synthesis of L-carnitine, which was quantified using hydrophilic interaction liquid chromatography electrospray ionization mass spectrometry (HILIC-ESI-MS) analyses, from trimethyllysine. Furthermore, the yeast threonine aldolase Gly1p was shown to effectively catalyze the second step of the pathway, fulfilling the role of a serine hydroxymethyltransferase. The analyses also identified yeast enzymes that interact with the introduced pathway, including Can1p, which was identified as the yeast transporter for trimethyllysine, and the two yeast serine hydroxymethyltransferases, Shm1p and Shm2p. Together, this study opens the possibility of using an engineered, carnitine-producing yeast in various industrial applications while providing insight into possible future strategies aimed at tailoring the production capacity of such strains. PMID:25851717