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Sample records for acid biosynthetic pathway

  1. Metabolic modeling of Rosmarinic acid biosynthetic pathway

    OpenAIRE

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

    2010-01-01

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

  2. Metabolic modeling of Rosmarinic acid biosynthetic pathway

    Science.gov (United States)

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

    2010-01-01

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

  3. Substrate specificity of the sialic acid biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

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

    2001-07-18

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

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

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    Xu Guoqiang

    2012-02-01

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

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

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    Xiao-Hui Ma

    2015-09-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  8. A Novel Antibiotic Mechanism of l-Cyclopropylalanine Blocking the Biosynthetic Pathway of Essential Amino Acid l-Leucine.

    Science.gov (United States)

    Ma, Bingji; Shen, Jinwen; Yindeeyoungyeon, Wandee; Ruan, Yuan

    2017-12-14

    The unusual amino acid l-cyclopropylalanine was isolated from the mushroom Amanita virgineoides after detection in an anti-fungal screening test. l-Cyclopropylalanine was found to exhibit broad-spectrum inhibition against fungi and bacteria. The anti-fungal activity was found to be abolished in the presence of the amino acid l-leucine, but not any other amino acids, indicating that l-cyclopropylalanine may block the biosynthesis of the essential amino acid l-leucine, thereby inhibiting fungal and bacteria growth. Further biochemical studies found l-cyclopropylalanine indeed inhibits α-isopropylmalate synthase (α-IMPS), the enzyme that catalyzes the rate-limiting step in the biosynthetic pathway of l-leucine. Inhibition of essential l-leucine synthesis in fungal and bacteria organisms, a pathway absent in host organisms such as humans, may represent a novel antibiotic mechanism to counter the ever-increasing problem of drug resistance to existing antibiotics.

  9. EPA, DHA, and Lipoic Acid Differentially Modulate the n-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

    Science.gov (United States)

    Bou, Marta; Østbye, Tone-Kari; Berge, Gerd M; Ruyter, Bente

    2017-03-01

    The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n-3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1-14C] 18:3n-3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1-14C] 18:3n-3 to 18:4n-3, 20:4n-3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n-3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n-3 by both DHA and LA. Possibly the route by 20:3n-3 and then Δ8 desaturation to 20:4n-3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health-beneficial FA in fish fed diets with low levels of EPA and/or DHA.

  10. Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin–Fatty Acid Biosynthetic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Haushalter, Robert W. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Phelan, Ryan M. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Hoh, Kristina M. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Su, Cindy [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Wang, George [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Baidoo, Edward E. K. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division; Keasling, Jay D. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Bioscience Division

    2017-03-14

    Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here in this paper we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotin and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.

  11. A biosynthetic pathway for anandamide.

    Science.gov (United States)

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

    2006-09-05

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

  12. Cloning and characterization of a putative R2R3 MYB transcriptional repressor of the rosmarinic acid biosynthetic pathway from Salvia miltiorrhiza.

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    Shuncang Zhang

    Full Text Available Salvia miltiorrhiza Bunge is one of the most renowned traditional medicinal plants in China. Phenolic acids that are derived from the rosmarinic acid pathway, such as rosmarinic acid and salvianolic acid B, are important bioactive components in S. miltiorrhiza. Accumulations of these compounds have been reported to be induced by various elicitors, while little is known about transcription factors that function in their biosynthetic pathways. We cloned a subgroup 4 R2R3 MYB transcription factor gene (SmMYB39 from S. miltiorrhiza and characterized its roles through overexpression and RNAi-mediated silencing. As the results showed, the content of 4-coumaric acid, rosmarinic acid, salvianolic acid B, salvianolic acid A and total phenolics was dramatically decreased in SmMYB39-overexpressing S. miltiorrhiza lines while being enhanced by folds in SmMYB39-RNAi lines. Quantitative real-time PCR and enzyme activities analyses showed that SmMYB39 negatively regulated transcripts and enzyme activities of 4-hydroxylase (C4H and tyrosine aminotransferase (TAT. These data suggest that SmMYB39 is involved in regulation of rosmarinic acid pathway and acts as a repressor through suppressing transcripts of key enzyme genes.

  13. Selective responses of enzymes in the two parallel pathways of rosmarinic acid biosynthetic pathway to elicitors in Salvia miltiorrhiza hairy root cultures.

    Science.gov (United States)

    Zhang, Shuncang; Yan, Yan; Wang, Bangqing; Liang, Zongsuo; Liu, Yan; Liu, Fenghua; Qi, Zhihong

    2014-05-01

    Rosmarinic acid and salvianolic acid B are two important phenolic compounds with therapeutic properties in Salvia miltiorrhiza Bunge. The biosynthesis of rosmarinic acid is initiated by two parallel pathways, namely the phenylpropanoid pathway and the tyrosine-derived pathway. Salvianolic acid B is a structural dimer of rosmarinic acid and is believed to be derived from rosmarinic acid. In the current study, methyl jasmonate (MeJA) and hyphal extracts from fungi were used as elicitors to examine the relationship between enzymes in the two parallel pathways and accumulation of phenolic compounds in S. miltiorrhiza hairy root cultures. The results showed that accumulations of rosmarinic acid, salvianolic acid B and total phenolics were enhanced by MeJA while suppressed by fugal extracts. Responses of enzymes in the tyrosine-derived pathway, at both the gene transcript and enzyme activity levels, showed a better consistency with alterations of phenolic compounds content after the two elicitors treated. Our study implied that compared with enzymes in the phenylpropanoid pathway, enzymes in the tyrosine-derived pathway are more correlated to rosmarinic acid and salvianolic acid B biosynthesis in S. miltiorrhiza hairy roots. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway.

    Science.gov (United States)

    Yan, Jinyong; Yan, Yunjun; Madzak, Catherine; Han, Bingnan

    2017-02-01

    Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.

  15. Structural and Functional Analysis of Campylobacter jejuni PseG: a Udp-sugarhydrolase from the Pseudaminic Acid Biosynthetic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    E Rangarajan; A Proteau; Q Cui; S Logan; Z Potetinova; D Whitfield; E Purisima; M Cygler; A Matte; et al.

    2011-12-31

    Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-{beta}-l-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 {angstrom} resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His{sup 17}-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His{sup 17} functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C-O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.

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

    Science.gov (United States)

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

    2014-09-01

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

  17. Enterobacter sp. I-3, a bio-herbicide inhibits gibberellins biosynthetic pathway and regulates abscisic acid and amino acids synthesis to control plant growth.

    Science.gov (United States)

    Radhakrishnan, Ramalingam; Park, Jae-Man; Lee, In-Jung

    2016-12-01

    Very few bacterial species were identified as bio-herbicides for weed control. The present research was focused to elucidate the plant growth retardant properties of Enterobacter sp. I-3 during their interaction by determining the changes in endogenous photosynthetic pigments, plant hormones and amino acids. The two bacterial isolates I-4-5 and I-3 were used to select the superior bacterium for controlling weed seeds (Echinochloa crus-galli L. and Portulaca oleracea L.) germination. The post-inoculation of I-3 (Enterobacter sp. I-3) significantly inhibited the weeds seed germination than their controls. The mechanism of bacterium induced plant growth reduction was identified in lettuce treated with I-3 bacterium and compared their effects with known chemical herbicide, trinexapac-ethyl (TE). The treatment of I-3 and TE showed a significant inhibitory effect on shoot length, leaf number, leaf length, leaf width, shoot weight, root weight and chlorophyll content in lettuce seedlings. The endogenous gibberellins (GAs) and abscisic acid (ABA) analysis showed that Enterobacter sp. I-3 treated plants had lower levels of GAs (GA 12 , GA 19 , GA 20 and GA 8 ) and GAs/ABA ratio and then, the higher level of ABA when compared to their controls. Indeed, the individual amino acids ie., aspartic acid, glutamic acid, glycine, threonine, alanine, serine, leucine, isoleucine and tyrosine were declined in TE and I-3 exposed plants. Our results suggest that the utilization of Enterobacter sp. I-3 inhibits the GAs pathway and amino acids synthesis in weeds to control their growth can be an alternative to chemical herbicides. Copyright © 2016 Elsevier GmbH. All rights reserved.

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

    Science.gov (United States)

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

    2016-03-21

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

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

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2016-03-01

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

  20. Induction of DREB2A pathway with repression of E2F, Jasmonic acid biosynthetic and photosynthesis pathways in cold acclimation specific freeze resistant wheat crown

    Science.gov (United States)

    Winter wheat lines can achieve cold acclimation (development of tolerance to freezing temperatures) and vernalization (delay in transition from vegetative to reproductive phase) in response to low non-freezing temperatures. To describe cold acclimation specific processes and pathways, we utilized co...

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

    Science.gov (United States)

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

    2013-09-10

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

  2. Bioengineering natural product biosynthetic pathways for therapeutic applications.

    Science.gov (United States)

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

    2012-12-01

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

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

    DEFF Research Database (Denmark)

    Liu, Qing; Manzano, David; Tanić, Nikola

    2014-01-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2012-08-01

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

  6. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

    Directory of Open Access Journals (Sweden)

    Shinji Kishimoto

    2016-08-01

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

  7. Evaluation of biosynthetic pathways to delta-aminolevulinic acid in Propionibacterium shermanii based on biosynthesis of vitamin B12 from D-[1-13C]glucose.

    Science.gov (United States)

    Iida, K; Kajiwara, M

    2000-04-04

    Analysis of the (13)C nuclear magnetic resonance (NMR) spectrum of (13)C-labeled vitamin B(12) biosynthesized from D-[1-(13)C]glucose by Propionibacterium shermanii provided evidence suggesting that delta-aminolevulinic acid (ALA) incorporated in the (13)C-labeled vitamin B(12) may have been synthesized via both the Shemin pathway and the C5 pathway under anaerobic conditions in the ratio of 1 < [(ratio of ALA biosynthesis from the Shemin pathway)/(that from the C5 pathway)] < 1.8. The D-ribose moiety of vitamin B(12) was labeled with (13)C at R-1, R-3, and R-5. The aminopropanol moiety of vitamin B(12) was labeled on Pr-1 and Pr-2, but not Pr-3.

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

    Science.gov (United States)

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

    2011-04-15

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

  9. A kinetic model for the penicillin biosynthetic pathway in

    DEFF Research Database (Denmark)

    Nielsen, Jens; Jørgensen, Henrik

    1996-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Michael D Barton

    2010-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Ying Xiao

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

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

    OpenAIRE

    Mikami, Koji; Hosokawa, Masashi

    2013-01-01

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

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

    Science.gov (United States)

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

    2016-10-01

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

  14. Evolution of the isoprene biosynthetic pathway in kudzu.

    Science.gov (United States)

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

    2005-02-01

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

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

    Science.gov (United States)

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

    2005-01-01

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

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

    Science.gov (United States)

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

    2014-02-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

  19. Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2008-07-01

    Full Text Available Abstract Background High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes. Results Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1 is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP increases the riboflavin accumulation significantly. Conclusion The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established.

  20. Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy.

    Science.gov (United States)

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey

    2006-08-16

    Axonal degeneration occurs in many neurodegenerative diseases and after traumatic injury and is a self-destructive program independent from programmed cell death. Previous studies demonstrated that overexpression of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) or exogenous application of nicotinamide adenine dinucleotide (NAD) can protect axons of cultured dorsal root ganglion (DRG) neurons from degeneration caused by mechanical or neurotoxic injury. In mammalian cells, NAD can be synthesized from multiple precursors, including tryptophan, nicotinic acid, nicotinamide, and nicotinamide riboside (NmR), via multiple enzymatic steps. To determine whether other components of these NAD biosynthetic pathways are capable of delaying axonal degeneration, we overexpressed each of the enzymes involved in each pathway and/or exogenously administered their respective substrates in DRG cultures and assessed their capacity to protect axons after axotomy. Among the enzymes tested, Nmnat1 had the strongest protective effects, whereas nicotinamide phosphoribosyl transferase and nicotinic acid phosphoribosyl transferase showed moderate protective activity in the presence of their substrates. Strong axonal protection was also provided by Nmnat3, which is predominantly located in mitochondria, and an Nmnat1 mutant localized to the cytoplasm, indicating that the subcellular location of NAD production is not crucial for protective activity. In addition, we showed that exogenous application of the NAD precursors that are the substrates of these enzymes, including nicotinic acid mononucleotide, nicotinamide mononucleotide, and NmR, can also delay axonal degeneration. These results indicate that stimulation of NAD biosynthetic pathways via a variety of interventions may be useful in preventing or delaying axonal degeneration.

  1. Membrane Phosphoproteomics of Yeast Early Response to Acetic Acid: Role of Hrk1 Kinase and Lipid Biosynthetic Pathways, in Particular Sphingolipids.

    Science.gov (United States)

    Guerreiro, Joana F; Mira, Nuno P; Santos, Aline X S; Riezman, Howard; Sá-Correia, Isabel

    2017-01-01

    Saccharomyces cerevisiae response and tolerance to acetic acid is critical in industrial biotechnology and in acidic food and beverages preservation. The HRK1 gene, encoding a protein kinase of unknown function belonging to the "Npr1-family" of kinases known to be involved in the regulation of plasma membrane transporters, is an important determinant of acetic acid tolerance. This study was performed to identify the alterations occurring in yeast membrane phosphoproteome profile during the adaptive early response to acetic acid stress (following 1 h of exposure to a sub-lethal inhibitory concentration; 50 mM at pH 4.0) and the effect of HRK1 expression on the phosphoproteome. Results from mass spectrometry analysis following the prefractionation and specific enrichment of phosphorylated peptides using TiO2 beads highlight the contribution of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress, with particular relevance for changes in phosphorylation of transport-related proteins, found to be highly dependent on the Hrk1 kinase. Twenty different phosphoproteins known to be involved in lipid and sterol metabolism were found to be differently phosphorylated in response to acetic acid stress, including several phosphopeptides that had not previously been described as being phosphorylated. The suggested occurrence of cellular lipid composition remodeling during the short term yeast response to acetic acid was confirmed: Hrk1 kinase-independent reduction in phytoceramide levels and a reduction in phosphatidylcholine and phosphatidylinositol levels under acetic acid stress in the more susceptible hrk1Δ strain were revealed by a lipidomic analysis.

  2. Elongating internodes of Zea mays (maize): Early steps in the GA biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Y.; Phinney, B.O. (Univ. of California, Los Angeles (USA)); Gaskin, P.; MacMillan, J. (Univ. of Bristol (England))

    1989-04-01

    The early steps in the gibberellin (GA) biosynthetic pathway have yet to be defined for tissues that show a growth response to GAs. To this end we have synthesized the ({sup 13}C,{sup 3}H)-ent-kaurenoids, ent-kaurenol, ent-kaurenal ent-kaukenoic acid. We also have double-labeled ent-kaurene and double-labeled GA{sub 12}-aldehyde. We feed 1 - 10{mu}g of each substrate, individually, to 1.0g diced internodes in the appropriate buffer plus cofactors. We have observed up to 80% metabolism. We have identified (full scan GC-MS) 7{beta}-hydroxy-ent-kaurenoic acid as the major metabolite from double-labeled ent-kaurenoic acid feeds, thus defining the step ent-kaurenoic acid to 7{beta}-hydroxy-ent-kaurenoic acid.

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

    Science.gov (United States)

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

    2014-01-01

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

  4. Ex Uno Plura: Differential Labeling of Phospholipid Biosynthetic Pathways with a Single Bioorthogonal Alcohol.

    Science.gov (United States)

    Bumpus, Timothy W; Liang, Felice J; Baskin, Jeremy M

    2018-01-16

    Imaging approaches that track biological molecules within cells are essential tools in modern biochemistry. Lipids are particularly challenging to visualize, as they are not directly genetically encoded. Phospholipids, the most abundant subgroup of lipids, are structurally diverse and accomplish many cellular functions, acting as major structural components of membranes and as signaling molecules that regulate cell growth, division, apoptosis, cytoskeletal dynamics, and numerous other physiological processes. Cells regulate the abundance, and therefore bioactivity, of phospholipids by modulating the activities of their biosynthetic enzymes. Thus, techniques that enable monitoring of flux through individual lipid biosynthetic pathways can provide key functional information. For example, the choline analogue propargylcholine (ProCho) can report on de novo biosynthesis of phosphatidylcholine by conversion to an alkynyl lipid that can be imaged following click chemistry tagging with an azido fluorophore. We report that ProCho is also a substrate of phospholipase D enzymes-which normally hydrolyze phosphatidylcholine to generate the lipid second messenger phosphatidic acid-in a transphosphatidylation reaction, generating the identical alkynyl lipid. By controlling the activities of phosphatidylcholine biosynthesis and phospholipase D enzymes, we establish labeling conditions that enable this single probe to selectively report on two different biosynthetic pathways. Just as nature exploits the economy of common metabolic intermediates to efficiently diversify biosynthesis, so can biochemists in interrogating such pathways with careful probe design. We envision that ProCho's ability to report on multiple metabolic pathways will enable studies of membrane dynamics and improve our understanding of the myriad roles that lipids play in cellular homeostasis.

  5. High night temperature strongly impacts TCA cycle, amino acid and polyamine biosynthetic pathways in rice in a sensitivity-dependent manner.

    Science.gov (United States)

    Glaubitz, Ulrike; Erban, Alexander; Kopka, Joachim; Hincha, Dirk K; Zuther, Ellen

    2015-10-01

    Global climate change combined with asymmetric warming can have detrimental effects on the yield of crop plants such as rice (Oryza sativa L.). Little is known about metabolic responses of rice to high night temperature (HNT) conditions. Twelve cultivars with different HNT sensitivity were used to investigate metabolic changes in the vegetative stage under HNT compared to control conditions. Central metabolism, especially TCA cycle and amino acid biosynthesis, were strongly affected particularly in sensitive cultivars. Levels of several metabolites were correlated with HNT sensitivity. Furthermore, pool sizes of some metabolites negatively correlated with HNT sensitivity under control conditions, indicating metabolic pre-adaptation in tolerant cultivars. The polyamines putrescine, spermidine and spermine showed increased abundance in sensitive cultivars under HNT conditions. Correlations between the content of polyamines and 75 other metabolites indicated metabolic shifts from correlations with sugar-phosphates and 1-kestose under control to correlations with sugars and amino and organic acids under HNT conditions. Increased expression levels of ADC2 and ODC1, genes encoding enzymes catalysing the first committed steps of putrescine biosynthesis, were restricted to sensitive cultivars under HNT. Additionally, transcript levels of eight polyamine biosynthesis genes were correlated with HNT sensitivity. Responses to HNT in the vegetative stage result in distinct differences between differently responding cultivars with a dysregulation of central metabolism and an increase of polyamine biosynthesis restricted to sensitive cultivars under HNT conditions and a pre-adaptation of tolerant cultivars already under control conditions with higher levels of potentially protective compatible solutes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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

    Science.gov (United States)

    Mikami, Koji; Hosokawa, Masashi

    2013-07-02

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

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

    Directory of Open Access Journals (Sweden)

    Masashi Hosokawa

    2013-07-01

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

  8. The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Marco Garavaglia

    Full Text Available Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.

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

    Science.gov (United States)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2004-04-27

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

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

    Science.gov (United States)

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

    2003-01-01

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

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

    Science.gov (United States)

    Zargar, Amin; Bailey, Constance B; Haushalter, Robert W; Eiben, Christopher B; Katz, Leonard; Keasling, Jay D

    2017-06-01

    Advances in retooling microorganisms have enabled bioproduction of 'drop-in' biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gas-turbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), 'drop-in' biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical properties (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2017-03-06

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

  14. Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga.

    Directory of Open Access Journals (Sweden)

    Michael T Guarnieri

    Full Text Available Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal-derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae. In order to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga, Chlorella vulgaris, we have established a strategy with which to bypass the necessity for genomic sequence information by using the transcriptome as a guide. Our results indicate an upregulation of both fatty acid and triacylglycerol biosynthetic machinery under oil-accumulating conditions, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of an unsequenced microalga.

  15. Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

    Energy Technology Data Exchange (ETDEWEB)

    Kyrpides, Nikos; Anderson, Iain; Rodriguez, Jason; Susanti, Dwi; Porat, Iris; Reich, Claudia; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Lykidis, Athanasios; Kim, Edwin; Thompson, Linda S.; Nolan, Matt; Land, Miriam; Copeland, Alex; Lapidus, Alla; Lucas, Susan; Detter, Chris; Zhulin, Igor B.; Olsen, Gary J.; Whitman, William; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching, hyperthermophilic member of the order Thermoproteales within the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein. Predicted highly expressed proteins do not include housekeeping genes, and instead include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins.

  16. Genome Sequence of Thermofilum pendens Reveals an Exceptional Loss of Biosynthetic Pathways without Genome Reduction

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Iain [U.S. Department of Energy, Joint Genome Institute; Rodriquez, Jason [Virginia Polytechnic Institute and State University (Virginia Tech); Susanti, Dwi [Virginia Polytechnic Institute and State University (Virginia Tech); Porat, I. [University of Georgia, Athens, GA; Reich, Claudia [University of Illinois, Urbana-Champaign; Ulrich, Luke [ORNL; Elkins, James G [ORNL; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Lykidis, A [U.S. Department of Energy, Joint Genome Institute; Kim, Edwin [U.S. Department of Energy, Joint Genome Institute; Thompson, Linda S [Los Alamos National Laboratory (LANL); Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Copeland, A [U.S. Department of Energy, Joint Genome Institute; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Detter, J C [U.S. Department of Energy, Joint Genome Institute; Zhulin, Igor B [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Olsen, Gary [ORNL; Whitman, W. B. [University of Georgia, Athens, GA; Mukhopadhyay, Biswarup [Virginia Polytechnic Institute and State University (Virginia Tech); Bristow, James [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching member of class Thermoproteales of Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first Crenarchaeote and only the second archaeon found to have transporters of the phosphotransferase system. T. pendens is known to require an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. T. pendens has fewer biosynthetic enzymes than any other free-living organism. In addition to heterotrophy, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein from a new subfamily. Predicted highly expressed proteins include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins, suggesting that defense against viruses is a high priority.

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

    Science.gov (United States)

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

    2013-01-01

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

  18. Diversity in biosynthetic pathways of galactolipids in the light of endosymbiotic origin of chloroplasts

    Directory of Open Access Journals (Sweden)

    Naoki eSato

    2016-02-01

    Full Text Available Cyanobacteria and chloroplasts perform oxygenic photosynthesis, and share a common origin. Galactolipids are present in the photosynthetic membranes of both cyanobacteria and chloroplasts, but the biosynthetic pathways of the galactolipids are significantly different in the two systems. In this minireview, we explain the history of the discovery of the cyanobacterial pathway, and present a probable scenario of the evolution of the two pathways.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

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

    Science.gov (United States)

    Trautman, Eric P.; Crawford, Jason M.

    2016-01-01

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

  1. Exploring Drug Targets in Isoprenoid Biosynthetic Pathway for Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Tabish Qidwai

    2014-01-01

    Full Text Available Emergence of rapid drug resistance to existing antimalarial drugs in Plasmodium falciparum has created the need for prediction of novel targets as well as leads derived from original molecules with improved activity against a validated drug target. The malaria parasite has a plant plastid-like apicoplast. To overcome the problem of falciparum malaria, the metabolic pathways in parasite apicoplast have been used as antimalarial drug targets. Among several pathways in apicoplast, isoprenoid biosynthesis is one of the important pathways for parasite as its multiplication in human erythrocytes requires isoprenoids. Therefore targeting this pathway and exploring leads with improved activity is a highly attractive approach. This report has explored progress towards the study of proteins and inhibitors of isoprenoid biosynthesis pathway. For more comprehensive analysis, antimalarial drug-protein interaction has been covered.

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

    Science.gov (United States)

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

    2007-01-01

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

  3. Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors.

    Science.gov (United States)

    Suzuki, Hiroyoshi; Yokokura, Junpei; Ito, Tsukasa; Arai, Ryoma; Yokoyama, Chiaki; Toshima, Hiroaki; Nagata, Shinji; Asami, Tadao; Suzuki, Yoshihito

    2014-10-01

    Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2014-10-22

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

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

    Science.gov (United States)

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

    2016-11-01

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

  6. Biosynthetic Pathway for the Epipolythiodioxopiperazine Acetylaranotin in Aspergillus terreus Revealed by Genome-based Deletion Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Chun-Jun; Yeh, Hsu-Hua; Chiang, Yi Ming; Sanchez, James F.; Chang, ShuLin; Bruno, Kenneth S.; Wang, Clay C.

    2013-04-15

    Abstract Epipolythiodioxopiperazines (ETPs) are a class of fungal secondary metabolites derived from cyclic peptides. Acetylaranotin belongs to one structural subgroup of ETPs characterized by the presence of a seven-membered dihydrooxepine ring. Defining the genes involved in acetylaranotin biosynthesis should provide a means to increase production of these compounds and facilitate the engineering of second-generation molecules. The filamentous fungus Aspergillus terreus produces acetylaranotin and related natural products. Using targeted gene deletions, we have identified a cluster of 9 genes including one nonribosomal peptide synthase gene, ataP, that is required for acetylaranotin biosynthesis. Chemical analysis of the wild type and mutant strains enabled us to isolate seventeen natural products that are either intermediates in the normal biosynthetic pathway or shunt products that are produced when the pathway is interrupted through mutation. Nine of the compounds identified in this study are novel natural products. Our data allow us to propose a complete biosynthetic pathway for acetylaranotin and related natural products.

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

    Directory of Open Access Journals (Sweden)

    Peifer Susanne

    2012-10-01

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

  8. Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Wei Sun

    Full Text Available Chalcone synthase (CHS catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1 encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

  9. Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

    Science.gov (United States)

    Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

    2015-01-01

    Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

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

    Directory of Open Access Journals (Sweden)

    Zhi-Qiang Liu

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Nicholas J. Talbot

    2006-04-01

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

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

    Science.gov (United States)

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

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

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    Juwairiah Remali

    2017-11-01

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

  14. Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera.

    Science.gov (United States)

    Pandey, Akansha; Swarnkar, Vishakha; Pandey, Tushar; Srivastava, Piush; Kanojiya, Sanjeev; Mishra, Dipak Kumar; Tripathi, Vineeta

    2016-10-05

    Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG's were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes.

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

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    Hongling Tian

    2015-01-01

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

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

    Science.gov (United States)

    Nakata, Paul A

    2011-10-20

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

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

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    Liyao Ji

    2016-12-01

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

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

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    Jaromír Cihlář

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

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

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    Sandra Fresquet-Corrales

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

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

    Science.gov (United States)

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

    2011-04-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  2. Alterations in the heme biosynthetic pathway as an index of exposure to toxins

    Energy Technology Data Exchange (ETDEWEB)

    Marks, G.S.; Zelt, D.T.; Cole, S.P.

    1982-07-01

    Under normal circumstances the heme biosynthetic pathway is carefully controlled and porphyrins are formed in only trace amounts. When control mechanisms are disturbed by xenobiotics, porphyrins may be formed and serve as a signal of the interaction between a xenobiotic and the heme biosynthetic pathway. For example, porphyrinuria was an early manifestation of a hexachlorobenzene-induced porphyria outbreak in Turkey. In humans exposed to polybrominated biphenyls and to 2,3,7,8-tetrachlorodibenzo-p-dioxin the urinary porphyrin pattern was significantly different from normal in a large number of exposed individuals. The question is raised whether measurement of urinary porphyrin profiles by improved methods will enable an estimate to be made of the extent of exposure to haloaromatic hydrocarbons in the human population. A wide variety of xenobiotics interact with the prosthetic heme of cytochrome P-450 forming novel N-alkylporphyrins. Identification of these N-alkylporphyrins in body fluids might provide a means of assessing exposure to a variety of xenobiotics in human populations.

  3. Nonconventional techniques for separation of biosynthetic amino acids.

    Science.gov (United States)

    Kloetzer, Lenuţa; Poştaru, Mădălina; Cheptea, Corina; Caşcaval, D; Galaction, Anca-Irina

    2014-01-01

    Amino acids can be obtained by biosynthesis, by protein hydrolysis or by extraction from natural sources. The most efficient methods are the first two, but the separation of amino acids from fermentation broths or protein hydrolysates is rather difficult. Amino acids dissociate in aqueous solutions, forming characteristic ionic species depending on the solution pH-value. These properties make amino acids to be hydrophilic at any pH-value. This paper presents a review of the separation studies of some amino acids by nonconventional methods, namely individual or selective reactive extraction. Separation of some amino acids from their mixture obtained either by fermentation or protein hydrolysis by reactive extraction with different extractants indicated the possibility of the amino acids selective separation as a function of the pH-value of aqueous solution correlated with the acidic or basic character of each amino acid.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xuebo Hu

    2016-07-01

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

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

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    Nori Satoh

    2013-02-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  8. Covalent immobilization of oxylipin biosynthetic enzymes on nanoporous rice husk silica for production of cis(+)-12-oxophytodienoic acid.

    Science.gov (United States)

    Le, Thu Bao; Han, Chong Soo; Cho, Kyoungwon; Han, Oksoo

    2017-09-11

    Soybean lipoxygenase, recombinant rice allene oxide synthase-1 and rice allene oxide cyclase were covalently immobilized on nanoporous rice husk silica using two types of linkers: glutardialdehyde and polyethylene glycol. The immobilization efficiency achieved using glutardialdehyde-linked rice husk silica was higher than that achieved using polyethylene glycol-linked rice husk silica (50-92% and 25-50%, respectively). Immobilization on both types of matrices significantly decreased the specific activities of the immobilized enzymes. Solid-phase reaction yields of the enzymes were determined relative to the yields observed for the solution-phase reactions. Yields of the solid-phase reactions catalyzed by immobilized soybean lipoxygenase, rice allene oxide synthase-1, and rice allene oxide cyclase ranged from 50% to 230% and were dependent on both the enzymes and linkers used. Production of cis(+)-12-oxophytodienoic acid from α-linolenic acid by consecutive reactions using all three enzymes in a co-immobilization system resulted in 83.6% and 65.1% yields on glutardialdehyde-linked and epichlorohydrin-polyethylene glycol-linked rice husk silica, respectively. Our results suggest that immobilization of biosynthetic enzymes of the octadecanoid pathway on rice husk silica may be an efficient method for the in vitro production of oxylipins. Additionally, enzyme immobilizations on rice husk silica matrices may be more broadly applicable for producing physiologically important compounds in other biosynthetic pathways.

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

    OpenAIRE

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

    2014-01-01

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

  10. Utility of the Biosynthetic Folate Pathway for Targets in Antimicrobial Discovery

    Directory of Open Access Journals (Sweden)

    Christina R. Bourne

    2014-01-01

    Full Text Available The need for new antimicrobials is great in face of a growing pool of resistant pathogenic organisms. This review will address the potential for antimicrobial therapy based on polypharmacological activities within the currently utilized bacterial biosynthetic folate pathway. The folate metabolic pathway leads to synthesis of required precursors for cellular function and contains a critical node, dihydrofolate reductase (DHFR, which is shared between prokaryotes and eukaryotes. The DHFR enzyme is currently targeted by methotrexate in anti-cancer therapies, by trimethoprim for antibacterial uses, and by pyrimethamine for anti-protozoal applications. An additional anti-folate target is dihyropteroate synthase (DHPS, which is unique to prokaryotes as they cannot acquire folate through dietary means. It has been demonstrated as a primary target for the longest standing antibiotic class, the sulfonamides, which act synergistically with DHFR inhibitors. Investigations have revealed most DHPS enzymes possess the ability to utilize sulfa drugs metabolically, producing alternate products that presumably inhibit downstream enzymes requiring the produced dihydropteroate. Recent work has established an off-target effect of sulfonamide antibiotics on a eukaryotic enzyme, sepiapterin reductase, causing alterations in neurotransmitter synthesis. Given that inhibitors of both DHFR and DHPS are designed to mimic their cognate substrate, which contain shared substructures, it is reasonable to expect such “off-target” effects. These inhibitors are also likely to interact with the enzymatic neighbors in the folate pathway that bind products of the DHFR or DHPS enzymes and/or substrates of similar substructure. Computational studies designed to assess polypharmacology reiterate these conclusions. This leads to hypotheses exploring the vast utility of multiple members of the folate pathway for modulating cellular metabolism, and includes an appealing capacity for

  11. Utility of the Biosynthetic Folate Pathway for Targets in Antimicrobial Discovery.

    Science.gov (United States)

    Bourne, Christina R

    2014-01-21

    The need for new antimicrobials is great in face of a growing pool of resistant pathogenic organisms. This review will address the potential for antimicrobial therapy based on polypharmacological activities within the currently utilized bacterial biosynthetic folate pathway. The folate metabolic pathway leads to synthesis of required precursors for cellular function and contains a critical node, dihydrofolate reductase (DHFR), which is shared between prokaryotes and eukaryotes. The DHFR enzyme is currently targeted by methotrexate in anti-cancer therapies, by trimethoprim for antibacterial uses, and by pyrimethamine for anti-protozoal applications. An additional anti-folate target is dihyropteroate synthase (DHPS), which is unique to prokaryotes as they cannot acquire folate through dietary means. It has been demonstrated as a primary target for the longest standing antibiotic class, the sulfonamides, which act synergistically with DHFR inhibitors. Investigations have revealed most DHPS enzymes possess the ability to utilize sulfa drugs metabolically, producing alternate products that presumably inhibit downstream enzymes requiring the produced dihydropteroate. Recent work has established an off-target effect of sulfonamide antibiotics on a eukaryotic enzyme, sepiapterin reductase, causing alterations in neurotransmitter synthesis. Given that inhibitors of both DHFR and DHPS are designed to mimic their cognate substrate, which contain shared substructures, it is reasonable to expect such "off-target" effects. These inhibitors are also likely to interact with the enzymatic neighbors in the folate pathway that bind products of the DHFR or DHPS enzymes and/or substrates of similar substructure. Computational studies designed to assess polypharmacology reiterate these conclusions. This leads to hypotheses exploring the vast utility of multiple members of the folate pathway for modulating cellular metabolism, and includes an appealing capacity for prokaryotic

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

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    Sacha Coesel

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

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

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    Patricia Martin

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

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

    OpenAIRE

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

    2017-01-01

    This study investigated the effects of glutamic acid on production of monacolin K and expression of the monacolin K biosynthetic gene cluster. When Monascus M1 was grown in glutamic medium instead of in the original medium, monacolin K production increased from 48.4 to 215.4?mg?l?1, monacolin K production increased by 3.5 times. Glutamic acid enhanced monacolin K production by upregulating the expression of mokB-mokI; on day 8, the expression level of mokA tended to decrease by Reverse Transc...

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

    NARCIS (Netherlands)

    Verdoes, J.C.; Sandmann, G.; Visser, H.; Diaz, M.; Mossel, van M.; Ooyen, van A.J.J.

    2003-01-01

    The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both

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

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    Bo Wu

    2009-07-01

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

  17. Biosynthetic investigation of phomopsins reveals a widespread pathway for ribosomal natural products in Ascomycetes.

    Science.gov (United States)

    Ding, Wei; Liu, Wan-Qiu; Jia, Youli; Li, Yongzhen; van der Donk, Wilfred A; Zhang, Qi

    2016-03-29

    Production of ribosomally synthesized and posttranslationally modified peptides (RiPPs) has rarely been reported in fungi, even though organisms of this kingdom have a long history as a prolific source of natural products. Here we report an investigation of the phomopsins, antimitotic mycotoxins. We show that phomopsin is a fungal RiPP and demonstrate the widespread presence of a pathway for the biosynthesis of a family of fungal cyclic RiPPs, which we term dikaritins. We characterize PhomM as an S-adenosylmethionine-dependent α-N-methyltransferase that converts phomopsin A to an N,N-dimethylated congener (phomopsin E), and show that the methyltransferases involved in dikaritin biosynthesis have evolved differently and likely have broad substrate specificities. Genome mining studies identified eight previously unknown dikaritins in different strains, highlighting the untapped capacity of RiPP biosynthesis in fungi and setting the stage for investigating the biological activities and unknown biosynthetic transformations of this family of fungal natural products.

  18. New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

    Energy Technology Data Exchange (ETDEWEB)

    Gallo, A.; Bruno, K. S.; Solfrizzo, M.; Perrone, G.; Mule, G.; Visconti, A.; Baker, S. E.

    2012-09-14

    Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been obtained in Penicillium species. In Aspergillus species only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase in OTA producing A. carbonarius ITEM 5010 has removed the ability of the fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in Aspergillus species. The absence of OTA and ochratoxin α-the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β- the dechloro analog of ochratoxin α- were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius, and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight in the biosynthetic pathway of the toxin.

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

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    Girish Deshpande

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

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

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    Wenjun Huang

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

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

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    2017-07-01

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

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

    Science.gov (United States)

    Huang, Wenjun; Lv, Haiyan; Wang, Ying

    2017-01-01

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

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

    Science.gov (United States)

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

    2014-01-10

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

  4. Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

    Energy Technology Data Exchange (ETDEWEB)

    Whicher, Jonathan R.; Florova, Galina; Sydor, Paulina K.; Singh, Renu; Alhamadsheh, Mamoun; Challis, Gregory L.; Reynolds, Kevin A.; Smith, Janet L. (Michigan); (Warwick); (Portland SU)

    2011-08-17

    Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a {beta}-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 {angstrom} resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kiyohito Yoshida

    2016-05-01

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

  7. Molecular characterization and functional analysis of chalcone synthase from Syringa oblata Lindl. in the flavonoid biosynthetic pathway.

    Science.gov (United States)

    Wang, Yu; Dou, Ying; Wang, Rui; Guan, Xuelian; Hu, Zenghui; Zheng, Jian

    2017-11-30

    The flower color of Syringa oblata Lindl., which is often modulated by the flavonoid content, varies and is an important ornamental feature. Chalcone synthase (CHS) catalyzes the first key step in the flavonoid biosynthetic pathway. However, little is known about the role of S. oblata CHS (SoCHS) in flavonoid biosynthesis in this species. Here, we isolate and analyze the cDNA (SoCHS1) that encodes CHS in S. oblata. We also sought to analyzed the molecular characteristics and function of flavonoid metabolism by SoCHS1. We successfully isolated the CHS-encoding genomic DNA (gDNA) in S. oblata (SoCHS1), and the gene structural analysis indicated it had no intron. The opening reading frame (ORF) sequence of SoCHS1 was 1170bp long and encoded a 389-amino acid polypeptide. Multiple sequence alignment revealed that both the conserved CHS active site residues and CHS signature sequence were in the deduced amino acid sequence of SoCHS1. Crystallographic analysis revealed that the protein structure of SoCHS1 is highly similar to that of FnCHS1 in Freesia hybrida. The quantitative real-time polymerase chain reaction (PCR) performed to detect the SoCHS1 transcript expression levels in flowers, and other tissues revealed the expression was significantly correlated with anthocyanin accumulation during flower development. The ectopic expression results of Nicotiana tabacum showed that SoCHS1 overexpression in transgenic tobacco changed the flower color from pale pink to pink. In conclusion, these results suggest that SoCHS1 plays an essential role in flavonoid biosynthesis in S. oblata, and could be used to modify flavonoid components in other plant species. Copyright © 2017. Published by Elsevier B.V.

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

    Science.gov (United States)

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

    2016-06-01

    Tomato (Solanum lycopersicum L.) is one of the model plant to study carotenoid biosynthesis. In the present study, the fruit carotenoid content were quantified at different developmental stages for two contrasting genotypes, viz. IIHR-249-1 and IIHR-2866 by UPLC. Lycopene content was high in IIHR-249-1 (19.45 mg/100 g fresh weight) compared to IIHR-2866 (1.88 mg/100 g fresh weight) at the ripe stage. qPCR was performed for genes that are involved in the carotenoid biosynthetic pathway to study the difference in lycopene content in fruits of both the genotypes. The expression of Phytoene synthase (PSY) increased by 36-fold and Phytoene desaturase (PDS) increased by 14-fold from immature green stage to ripe stage in IIHR-249-1. The expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta cyclase (CYC-B) decreased gradually from the initial stage to the ripe stage in IIHR-249-1. IIHR 249-1 showed 3- and 1.8-fold decrease in gene expression for Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) .The F2 hybrids derived from IIHR-249-1 and IIHR-2866 were analysed at the ripe stage for lycopene content. The gene expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) in high and low lycopene lines from F2 progenies also showed the decrease in transcript levels of both the genes in high lycopene F2 lines. We wish to suggest that the differential expression of lycopene beta-cyclases can be used in marker-assisted breeding.

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

    Directory of Open Access Journals (Sweden)

    Yong Yang

    2016-03-01

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

  10. Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways

    DEFF Research Database (Denmark)

    Vavitsas, Konstantinos; Rue, Emil Østergaard; Stefánsdóttir, Lára Kristín

    2017-01-01

    BACKGROUND: There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity......, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites. CONCLUSIONS: There is a wealth of information that can be derived by combining...... targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to 'traditional' metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies....

  11. Biosynthetic approaches to creating bioactive fungal metabolites: Pathway engineering and activation of secondary metabolism.

    Science.gov (United States)

    Motoyama, Takayuki; Osada, Hiroyuki

    2016-12-15

    The diversity of natural products is greater than that of combinatorial chemistry compounds and is similar to that of drugs. Compounds rich in sp3 carbons, such as natural products, typically exhibit high structural complexity and high specificity to molecular targets. Microorganisms can synthesize such sp3 carbon-rich compounds and can be used as excellent factories for making bioactive compounds. Here, we mainly focus on pathway engineering of two sp3 carbon-rich bioactive indole alkaloids, fumitremorgin C and terpendole E. We also demonstrate the importance of activation of secondary metabolism by focusing on tenuazonic acid, a bioactive tetramic acid compound, as an example. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Laccase‐catalysed oxidations of naturally occurring phenols: from in vivo biosynthetic pathways to green synthetic applications

    Science.gov (United States)

    Jeon, Jong‐Rok; Baldrian, Petr; Murugesan, Kumarasamy; Chang, Yoon‐Seok

    2012-01-01

    Summary Laccases are oxidases that contain several copper atoms, and catalyse single‐electron oxidations of phenolic compounds with concomitant reduction of oxygen to water. The enzymes are particularly widespread in ligninolytic basidiomycetes, but also occur in certain prokaryotes, insects and plants. Depending on the species, laccases are involved in various biosynthetic processes contributing to carbon recycling in land ecosystems and the morphogenesis of biomatrices, wherein low‐molecular‐weight naturally occurring phenols serve as key enzyme substrates. Studies of these in vivo synthetic pathways have afforded new insights into fungal laccase applicability in green synthetic chemistry. Thus, we here review fungal laccase‐catalysed oxidations of naturally occurring phenols that are particularly relevant to the synthesis of fine organic chemicals, and we discuss how the discovered synthetic strategies mimic laccase‐involved in vivo pathways, thus enhancing the green nature of such reactions. Laccase‐catalysed in vivo processes yield several types of biopolymers, including those of cuticles, lignin, polyflavonoids, humus and the melanin pigments, using natural mono‐ or poly‐phenols as building blocks. The in vivo synthetic pathways involve either phenoxyl radical‐mediated coupling or cross‐linking reactions, and can be adapted to the design of in vitro oxidative processes involving fungal laccases in organic synthesis; the laccase substrates and the synthetic mechanisms reflect in vivo processes. Notably, such in vitro synthetic pathways can also reproduce physicochemical properties (e.g. those of chromophores, and radical‐scavenging, hydration and antimicrobial activities) found in natural biomaterials. Careful study of laccase‐associated in vivo metabolic pathways has been rewarded by the discovery of novel green applications for fungal laccases. This review comprehensively summarizes the available data on laccase

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

    Science.gov (United States)

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

    2017-12-01

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

  14. Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

    Science.gov (United States)

    Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

    2012-01-01

    Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can

  15. Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

    Energy Technology Data Exchange (ETDEWEB)

    Geiger, Jim

    2013-11-30

    structure of ADP- Glucose pyrophosphorylase from potato in its inhibited conformation, and bound to both ATP and ADP-glucose. In addition, we have determined the first structure of glycogen synthase in its "closed", catalytically active conformation bound to ADP-glucose. We also determined the structure of glycogen synthase bound to malto-oligosaccharides, showing for the first time that an enzyme in the starch biosynthetic pathway recognizes glucans not just in its active site but on binding sites on the surface of the enzyme ten’s of Angstroms from the active site. In addition our structure of a glycogen branching enzyme bound to malto-oligosaccharides identified seven distinct binding sites distributed about the surface of the enzyme. We will now determine the function of these sites to get a molecular-level picture of exactly how these enzymes interact with their polymeric substrates and confer specificity leading to the complex structure of the starch granule. We will extend our studies to other isoforms of the enzymes, to understand how their structures give rise to their distinct function. Our goal is to understand what accounts for the various functional differences between SS and SBE isoforms at a molecular level.

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

    Science.gov (United States)

    Nawrath, C; Poirier, Y; Somerville, C

    1994-01-01

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

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

    Science.gov (United States)

    Nakayasu, Masaru; Ohyama, Kiyoshi; Saito, Kazuki

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Woo Tae Park

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-03-29

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-04-22

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

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

    DEFF Research Database (Denmark)

    Ono, Hajime; Rewitz, Kim; Shinoda, Tetsu

    2006-01-01

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

  3. Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Qing Liu

    Full Text Available The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS and germacrene A oxidase (GAO, which together catalyse the biosynthesis of germacra-1(10,4,11(13-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS, CYP71BL3, can catalyse the oxidation of germacra-1(10,4,11(13-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS, chicory GAO (CiGAO, and chicory COS (CiCOS in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1 FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.

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

    Science.gov (United States)

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

    2009-12-01

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

  5. Membrane Deenergization by Colicin K Affects Fluorescence of Exogenously Added but Not Biosynthetically Esterified Parinaric Acid Probes in Escherichia coli

    OpenAIRE

    Tecoma, Evelyn S.; Wu, David

    1980-01-01

    Fluorescence of the conjugated polyene fatty acid, parinaric acid (PnA), was studied in membranes of Escherichia coli during deenergization by colicin K. The free fatty acid and biosynthetically esterified forms of cis-PnA (9,11,13,15-cis,trans,trans,cis-octadecatetraenoic acid), both of which are sensitive to E. coli lipid-phase transitions, were compared. When free cis-PnA was added exogenously to respiring bacteria, dissipation of the energized state of the membrane resulted in a dramatic ...

  6. First Biosynthetic pathway of 1-hepten-3-one in Iporangaia pustulosa (Opiliones)

    Science.gov (United States)

    Rocha, Daniele F. O.; Wouters, Felipe C.; Machado, Glauco; Marsaioli, Anita J.

    2013-11-01

    Arthropods produce a great variety of natural compounds, many of which have unexplored biosynthesis. Among the armored harvestmen (Arachnida: Opiliones) of the suborder Laniatores, the defensive gland exudates contain vinyl ketones and other constituents of supposed polyketide origin. We have studied the biosynthesis of 1-hepten-3-one in the Neotropical harvestman Iporangaia pustulosa by feeding individuals with 13C-labeled precursors, demonstrating its mixed acetate/propionate origin. 13C NMR spectroscopy showed an unusual labeling pattern suggesting different propionate sources for starting and extender units. Our analysis also indicates the presence of methylmalonyl-CoA mutase, converting acetate into propionyl-CoA via succinyl-CoA, together with other C3 unit routes. This is the first biosynthetic study of alkyl vinyl ketones in arthropods. Our results shed light on the origin and diversification of chemical compounds in a major arthropod group.

  7. Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway.

    Science.gov (United States)

    Kopečná, Jana; Cabeza de Vaca, Israel; Adams, Nathan B P; Davison, Paul A; Brindley, Amanda A; Hunter, C Neil; Guallar, Victor; Sobotka, Roman

    2015-11-20

    In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Integrating nitric oxide into salicylic acid and jasmonic acid/ ethylene plant defense pathways

    Science.gov (United States)

    Mur, Luis A. J.; Prats, Elena; Pierre, Sandra; Hall, Michael A.; Hebelstrup, Kim H.

    2013-01-01

    Plant defense against pests and pathogens is known to be conferred by either salicylic acid (SA) or jasmonic acid (JA)/ethylene (ET) pathways, depending on infection or herbivore-grazing strategy. It is well attested that SA and JA/ET pathways are mutually antagonistic allowing defense responses to be tailored to particular biotic stresses. Nitric oxide (NO) has emerged as a major signal influencing resistance mediated by both signaling pathways but no attempt has been made to integrate NO into established SA/JA/ET interactions. NO has been shown to act as an inducer or suppressor of signaling along each pathway. NO will initiate SA biosynthesis and nitrosylate key cysteines on TGA-class transcription factors to aid in the initiation of SA-dependent gene expression. Against this, S-nitrosylation of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) will promote the NPR1 oligomerization within the cytoplasm to reduce TGA activation. In JA biosynthesis, NO will initiate the expression of JA biosynthetic enzymes, presumably to over-come any antagonistic effects of SA on JA-mediated transcription. NO will also initiate the expression of ET biosynthetic genes but a suppressive role is also observed in the S-nitrosylation and inhibition of S-adenosylmethionine transferases which provides methyl groups for ET production. Based on these data a model for NO action is proposed but we have also highlighted the need to understand when and how inductive and suppressive steps are used. PMID:23818890

  9. Development of fruit color in Solanaceae: a story of two biosynthetic pathways.

    Science.gov (United States)

    Dhar, Manoj K; Sharma, Rupali; Koul, Archana; Kaul, Sanjana

    2015-05-01

    This review highlights the major differences between the regulation of two important pathways namely anthocyanin and carotenoid pathways, responsible for fruit color generation in Solanaceae mediated by transcription factors (TFs). The anthocyanin pathway is regulated by a common set of TFs (MYB, MYC and WD40) belonging to specific families of DNA-binding proteins. Their regulation is aimed at controlling the type and amount of pigments produced and the physiological conditions (like pH) at which they are finally stored. In the carotenoid pathway, the color diversity depends on the quantity of pigment produced and the point where the pathway is arrested. TFs in the latter case are accordingly found to influence the sequestration and degradation of these pigments, which determines their final concentration in the tissue. TFs (phytochrome interacting factors, MADS-BOX, HB-ZIP and B-ZIP) also regulate important rate-determining steps, which decide the direction in which the pathway proceeds and the point at which it is terminated. In the absence of a clear pattern of TF-mediated regulation, it is suggested that the carotenoid pathway is more significantly influenced by other regulatory methods which need to be explored. It is expected that common factors affecting these pathways are the ones acting much before the initiation of the biosynthesis of respective pigments. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  10. Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells.

    Science.gov (United States)

    Charter, N W; Mahal, L K; Koshland, D E; Bertozzi, C R

    2000-10-01

    In this study we demonstrate that polysialyltransferases are capable of accepting unnatural substrates in terminally differentiated human neurons. Polysialyltransferases catalyze the glycosylation of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA). The unnatural sialic acid analog, N-levulinoyl sialic acid (SiaLev), was incorporated into cell surface glycoconjugates including PSA by the incubation of cultured neurons with the metabolic precursor N-levulinoylmannosamine (ManLev). The ketone group within the levulinoyl side chain of SiaLev was then used as a chemical handle for detection using a biotin probe. The incorporation of SiaLev residues into PSA was demonstrated by protection from sialidases that can cleave natural sialic acids but not those bearing unnatural N-acyl groups. The presence of SiaLev groups on the neuronal cell surface did not impede neurite outgrowth or significantly affect the distribution of PSA on neuronal compartments. Since PSA is important in neural plasticity and development, this mechanism for modulating PSA structure might be useful for functional studies.

  11. Discovery and characterization of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin S biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Jun Feng

    Full Text Available BlsE, a predicted radical S-adenosyl-L-methionine (SAM protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe-4S] cluster using UV-vis and electron paramagnetic resonance (EPR spectroscopic analysis, and investigation of the ability of flavodoxin (Fld, flavodoxin reductase (Fpr and NADPH to reduce the [4Fe-4S](2+ cluster. Mutagenesis studies demonstrated that Cys31, Cys35, Cys38 in the C×××C×MC motif and Gly73, Gly74, Glu75, Pro76 in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe-4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.

  12. Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant.

    Directory of Open Access Journals (Sweden)

    Ahmad-Faris Seman-Kamarulzaman

    Full Text Available Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate

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

    Directory of Open Access Journals (Sweden)

    Samiha Sioud

    2007-01-01

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

  14. Time Dependency of Chemodiversity and Biosynthetic Pathways: An LC-MS Metabolomic Study of Marine-Sourced Penicillium

    Directory of Open Access Journals (Sweden)

    Catherine Roullier

    2016-05-01

    Full Text Available This work aimed at studying metabolome variations of marine fungal strains along their growth to highlight the importance of the parameter “time” for new natural products discovery. An untargeted time-scale metabolomic study has been performed on two different marine-derived Penicillium strains. They were cultivated for 18 days and their crude extracts were analyzed by HPLC-DAD-HRMS (High Performance Liquid Chromatography-Diode Array Detector-High Resolution Mass Spectrometry each day. With the example of griseofulvin biosynthesis, a pathway shared by both strains, this work provides a new approach to study biosynthetic pathway regulations, which could be applied to other metabolites and more particularly new ones. Moreover, the results of this study emphasize the interest of such an approach for the discovery of new chemical entities. In particular, at every harvesting time, previously undetected features were observed in the LC-MS (Liquid Chromatography-Mass Spectrometry data. Therefore, harvesting times for metabolite extraction should be performed at different time points to access the hidden metabolome.

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

    Science.gov (United States)

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

    2014-01-01

    Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield). According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins. PMID:24722569

  16. Biosynthetic pathway for sex pheromone components produced in a Plusiinae moth, Plusia festucae

    Directory of Open Access Journals (Sweden)

    Hayaki eWatanabe

    2011-11-01

    Full Text Available While many Plusiinae species commonly secrete (Z-7-dodecenyl acetate (Z7-12:OAc as a key pheromone component, female moths of the rice looper (Plusia festucae exceptionally utilize (Z-5-dodecenyl acetate (Z5-12:OAc to communicate with their partners. GC-MS analysis of methyl esters derived from fatty acids included in the pheromone gland of Plusia festucae showed a series of esters monounsaturated at the ω7-position, i.e., (Z-5-dodecenoate, (Z-7-tetradecenoate (Z-9-hexadecenoate (Z9-16:Me, and (Z-11-octadecenoate (Z11-18:Me. By topical application of D3-labled palmitic acid (16:Acid and stearic acid (18:Acid to the pheromone glands, similar amounts of D3-Z5-12:OAc were detected. The glands treated with D13-labeled monoenoic acids (Z9-16:Acid and Z11-18:Acid, which were custom-made by utilizing an acetylene coupling reaction with D13-1-bromohexane, also produced similar amounts of D13-Z5-12:OAc. These results suggested that Z5-12:OAc was biosynthesized by ω7-desaturase with low substrate specificity, which could introduce a double bond at the 9-position of a 16:Acid derivative and the 11-position of an 18:Acid derivative. Additional experiments with the glands pretreated with an inhibitor of chain elongation supported this speculation. Furthermore, a comparative study with another Plusiinae species (Chrysodeixis eriosoma secreting Z7-12:OAc indicated that the β-oxidation systems of P. festucae and C. eriosoma were different.

  17. Reconstruction of the biosynthetic pathway for the core fungal polyketide scaffold rubrofusarin in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Naesby, Michael; Mortensen, Uffe Hasbro

    2013-01-01

    intermediate was converted into nor-rubrofusarin upon expression of the dehydratase gene aurZ from the aurofusarin gene cluster of F. graminearum. Final conversion into rubrofusarin was achieved by expression of the O-methyltransferase encoding gene aurJ, also obtained from the aurofusarin gene cluster....... CONCLUSIONS: The reconstructed pathway for rubrofusarin in S. cerevisiae allows the production of a core scaffold molecule with a branch-point role in several fungal polyketide pathways, thus paving the way for production of further natural pigments and bioactive molecules. Furthermore, the reconstruction...

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

    Science.gov (United States)

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

  19. De novo transcriptome assembly and the putative biosynthetic pathway of steroidal sapogenins of Dioscorea composita.

    Directory of Open Access Journals (Sweden)

    Xia Wang

    Full Text Available The plant Dioscorea composita has important applications in the medical and energy industries, and can be used for the extraction of steroidal sapogenins (important raw materials for the synthesis of steroidal drugs and bioethanol production. However, little is known at the genetic level about how sapogenins are biosynthesized in this plant. Using Illumina deep sequencing, 62,341 unigenes were obtained by assembling its transcriptome, and 27,720 unigenes were annotated. Of these, 8,022 unigenes were mapped to 243 specific pathways, and 531 unigenes were identified to be involved in 24 secondary metabolic pathways. 35 enzymes, which were encoded by 79 unigenes, were related to the biosynthesis of steroidal sapogenins in this transcriptome database, covering almost all the nodes in the steroidal pathway. The results of real-time PCR experiments on ten related transcripts (HMGR, MK, SQLE, FPPS, DXS, CAS, HMED, CYP51, DHCR7, and DHCR24 indicated that sapogenins were mainly biosynthesized by the mevalonate pathway. The expression of these ten transcripts in the tuber and leaves was found to be much higher than in the stem. Also, expression in the shoots was low. The nucleotide and protein sequences and conserved domains of four related genes (HMGR, CAS, SQS, and SMT1 were highly conserved between D. composita and D. zingiberensis; but expression of these four genes is greater in D. composita. However, there is no expression of these key enzymes in potato and no steroidal sapogenins are synthesized.

  20. miRNA Nomenclature : A View Incorporating Genetic Origins, Biosynthetic Pathways, and Sequence Variants

    NARCIS (Netherlands)

    Desvignes, T.; Batzel, P.; Berezikov, E.; Eilbeck, K.; Eppig, J. T.; McAndrews, M. S.; Singer, A.; Postlethwait, J. H.

    2015-01-01

    High-throughput sequencing of miRNAs has revealed the diversity and variability of mature and functional short noncoding RNAs, including their genomic origins, biogenesis pathways, sequence variability, and newly identified products such as miRNA-offset RNAs (moRs). Here we review known cases of

  1. Reconstitution of the Costunolide Biosynthetic Pathway in Yeast and Nicotiana benthamiana.

    NARCIS (Netherlands)

    Liu, Q.; Majdi, M.; Cankar, K.; Goedbloed, M.A.; Charnikhova, T.; Verstappen, F.W.A.; Vos, de R.C.H.; Beekwilder, M.J.; Krol, van der S.; Bouwmeester, H.J.

    2011-01-01

    The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene

  2. Chapter 3: Omics Advances of Biosynthetic Pathways of Isoprenoid Production in Microalgae

    Energy Technology Data Exchange (ETDEWEB)

    Paniagua-Michel, J.; Subramanian, Venkataramanan

    2017-01-01

    In this chapter, the current status of microalgal isoprenoids and the role of omics technologies, or otherwise specified, in bioproducts optimization and applications are reviewed. Emphasis is focused in the metabolic pathways of microalgae involved in the production of commercially important products, namely, hydrocarbons and biofuels, nutraceuticals, and pharmaceuticals.

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

    Science.gov (United States)

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

    1999-11-05

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

  4. Mapping and comparative proteomic analysis of the starch biosynthetic pathway in rice by 2D PAGE/MS.

    Science.gov (United States)

    Chang, Tao-Shan; Liu, Chih-Wei; Lin, Yu-Ling; Li, Chao-Yi; Wang, Arthur Z; Chien, Min-Wei; Wang, Chang-Sheng; Lai, Chien-Chen

    2017-11-01

    Our results not only provide a comprehensive overview of the starch biosynthetic pathway in the developing endosperm but also reveal some important protein markers that regulate the synthesis of starch. In human diets, rice (Oryza sativa L.) is an important source of starch, a substantial amount of which is accumulated in developing endosperm. A better understanding of the complicated pathways involved in starch biosynthesis is needed to improve the yield and quality of rice and other cereal crops through breeding. One pure line rice mutant, SA0419, was induced from a wild-type rice, TNG67, by sodium azide mutagenesis; therefore, TNG67 and SA0419 share the same genetic background. SA0419 is, however, a unique glutinous rice with a lower amylose content (8%) than that of TNG67 (20%), and the grains of SA0419 develop earlier and faster than those of TNG67. In this study, we used a comparative proteomic analysis to identify the differentially expressed proteins that may explain the differences in starch biosynthesis and the characteristics of TNG67 and SA0419. A gel-based proteomic approach was applied to profile the expressed proteome in the developing endosperm of these two rice varieties by nano-LC/MS/MS. Several over-expressed proteins were found in SA0419, such as plastidial ADP-glucose pyrophosphorylase (AGPase), phosphoglucomutase (PGM), pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFP), 6-phosphofructokinase (PFK), pyruvate phosphate dikinase (PPDK), starch branching enzymes (SBE) and starch debranching enzyme (SDBE), with those proteins mainly being involved in the pathways of starch metabolism and PPDK-mediated gluconeogenesis. Those over-expressed enzymes may contribute to the relatively early development, similar starch accumulation and rapid grain filling of SA0419 as compared with TNG67. This study provides a detailed biochemical description of starch biosynthesis and related information regarding a unique starch mutant that may assist future

  5. Ethylene and 1-MCP regulate major volatile biosynthetic pathways in apple fruit.

    Science.gov (United States)

    Yang, Xiaotang; Song, Jun; Du, Lina; Forney, Charles; Campbell-Palmer, Leslie; Fillmore, Sherry; Wismer, Paul; Zhang, Zhaoqi

    2016-03-01

    The effects of ethylene and 1-methylcyclopropene (1-MCP) on apple fruit volatile biosynthesis and gene expression were investigated. Statistical analysis identified 17 genes that changed significantly in response to ethylene and 1-MCP treatments. Genes encoding branched-chain amino acid aminotransferase (BCAT), aromatic amino acid aminotransferase (ArAT) and amino acid decarboxylases (AADC) were up-regulated during ripening and further enhanced by ethylene treatment. Genes related to fatty acid synthesis and metabolism, including acyl-carrier-proteins (ACPs), malonyl-CoA:ACP transacylase (MCAT), acyl-ACP-desaturase (ACPD), lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC2), β-oxidation, acyl-CoA synthetase (ACS), enoyl-CoA hydratase (ECHD), acyl-CoA dehydrogenase (ACAD), and alcohol acyltransferases (AATs) also increased during ripening and in response to ethylene treatment. Allene oxide synthase (AOS), alcohol dehydrogenase 1 (ADH1), 3-ketoacyl-CoA thiolase and branched-chain amino acid aminotransferase 2 (BCAT2) decreased in ethylene-treated fruit. Treatment with 1-MCP and ethylene generally produced opposite effects on related genes, which provides evidence that regulation of these genes is ethylene dependent. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  6. Hijacking the Hexosamine Biosynthetic Pathway to Promote EMT-Mediated Neoplastic Phenotypes

    Directory of Open Access Journals (Sweden)

    Kekoa eTaparra

    2016-04-01

    Full Text Available The epithelial-mesenchymal transition (EMT is a highly conserved program necessary for orchestrating distant cell migration during embryonic development. Multiple studies in cancer have demonstrated a critical role for EMT during the initial stages of tumorigenesis and later during tumor invasion. Transcription factors (TFs such as SNAIL, TWIST, and ZEB are master EMT regulators that are aberrantly overexpressed in many malignancies. Recent evidence correlates EMT-related transcriptomic alterations with metabolic reprogramming in cancer. Metabolic alterations may allow cancer to adapt to environmental stressors, supporting the irregular macromolecular demand of rapid proliferation. One potential metabolic pathway of increasing importance is the hexosamine biosynthesis pathway (HBP. The HBP utilizes glycolytic intermediates to generate the metabolite UDP-GlcNAc. This and other charged nucleotide sugars serve as the basis for biosynthesis of glycoproteins and other glycoconjugates. Recent reports in the field of glycobiology have cultivated great curiosity within the cancer research community. However, specific mechanistic relationships between the HBP and fundamental pathways of cancer, such as EMT, have yet to be elucidated. Altered protein glycosylation downstream of the HBP is well positioned to mediate many cellular changes associated with EMT including cell-cell adhesion, responsiveness to growth factors, immune system evasion, and signal transduction programs. Here we outline some of the basics of the HBP and putative roles the HBP may have in driving EMT-related cancer processes. With novel appreciation of the HBP’s connection to EMT, we hope to illuminate the potential for new therapeutic targets of cancer.

  7. Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus.

    Science.gov (United States)

    Huang, Lili; Li, Jia; Ye, Hechun; Li, Changfu; Wang, Hong; Liu, Benye; Zhang, Yansheng

    2012-11-01

    Catharanthus roseus is an important medicinal plant and the sole commercial source of monoterpenoid indole alkaloids (MIA), anticancer compounds. Recently, triterpenoids like ursolic acid and oleanolic acid have also been found in considerable amounts in C. roseus leaf cuticular wax layer. These simple pentacyclic triterpenoids exhibit various pharmacological activities such as anti-inflammatory, anti-tumor and anti-microbial properties. Using the EST collection from C. roseus leaf epidermome ( http://www.ncbi.nlm.nih.gov/dbEST ), we have successfully isolated a cDNA (CrAS) encoding 2,3-oxidosqualene cyclase (OSC) and a cDNA (CrAO) encoding amyrin C-28 oxidase from the leaves of C. roseus. The functions of CrAS and CrAO were analyzed in yeast (Saccharomyces cerevisiae) systems. CrAS was characterized as a novel multifunctional OSC producing α- and β-amyrin in a ratio of 2.5:1, whereas CrAO was a multifunctional C-28 oxidase converting α-amyrin, β-amyrin and lupeol to ursolic-, oleanolic- and betulinic acids, respectively, via a successive oxidation at the C-28 position of the substrates. In yeast co-expressing CrAO and CrAS, ursolic- and oleanolic acids were detected in the yeast cell extracts, while the yeast cells co-expressing CrAO and AtLUP1 from Arabidopsis thaliana produced betulinic acid. Both CrAS and CrAO genes show a high expression level in the leaf, which was consistent with the accumulation patterns of ursolic- and oleanolic acids in C. roseus. These results suggest that CrAS and CrAO are involved in the pentacyclic triterpene biosynthesis in C. roseus.

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

    Science.gov (United States)

    Jerga, Agoston; Rock, Charles O

    2009-06-05

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

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

    Science.gov (United States)

    Jerga, Agoston; Rock, Charles O.

    2009-01-01

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

  10. Integrated metabolome and transcriptome analysis of Magnolia champaca identifies biosynthetic pathways for floral volatile organic compounds.

    Science.gov (United States)

    Dhandapani, Savitha; Jin, Jingjing; Sridhar, Vishweshwaran; Sarojam, Rajani; Chua, Nam-Hai; Jang, In-Cheol

    2017-06-14

    Magnolia champaca, commonly known as champak is a well-known tree due to its highly fragrant flowers. Champak floral scent is attributed to a complex mix of volatile organic compounds (VOCs). These aromatic flowers are widely used in flavors and fragrances industry. Despite its commercial importance, the VOC biosynthesis pathways in these flowers are largely unknown. Here, we combine metabolite and RNA sequencing (RNA-seq) analyses of fully opened champak flowers to discover the active VOC biosynthesis pathways as well as floral scent-related genes. Volatile collection by headspace method and analysis by gas chromatography-mass spectrometry (GC-MS) identified a total of 43 VOCs from fully opened champak flowers, of which 46.9% were terpenoids, 38.9% were volatile esters and 5.2% belonged to phenylpropanoids/benzenoids. Sequencing and de novo assembly of champak flower transcriptome yielded 47,688 non-redundant unigenes. Transcriptome assembly was validated using standard polymerase chain reaction (PCR) based approach for randomly selected unigenes. The detailed profiles of VOCs led to the discovery of pathways and genes involved in floral scent biosynthesis from RNA-seq data. Analysis of expression levels of many floral-scent biosynthesis-related unigenes in flowers and leaves showed that most of them were expressed higher in flowers than in leaf tissues. Moreover, our metabolite-guided transcriptomics, in vitro and in vivo enzyme assays and transgenic studies identified (R)-linalool synthase that is essential for the production of major VOCs of champak flowers, (R)-linalool and linalool oxides. As our study is the first report on transcriptome analysis of Magnolia champaca, this transcriptome dataset that serves as an important public information for functional genomics will not only facilitate better understanding of ecological functions of champak floral VOCs, but also provide biotechnological targets for sustainable production of champak floral scent.

  11. Pseudomonas aeruginosa d-Arabinofuranose Biosynthetic Pathway and Its Role in Type IV Pilus Assembly*

    OpenAIRE

    Harvey, Hanjeong; Kus, Julianne V.; Tessier, Luc; Kelly, John; Burrows, Lori L.

    2011-01-01

    Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked d-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-d-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv...

  12. Selectively improving nikkomycin Z production by blocking the imidazolone biosynthetic pathway of nikkomycin X and uracil feeding in Streptomyces ansochromogenes

    Directory of Open Access Journals (Sweden)

    Yang Haihua

    2009-11-01

    Full Text Available Abstract Background Nikkomycins are a group of peptidyl nucleoside antibiotics and act as potent inhibitors of chitin synthases in fungi and insects. Nikkomycin X and Z are the main components produced by Streptomyces ansochromogenes. Of them, nikkomycin Z is a promising antifungal agent with clinical significance. Since highly structural similarities between nikkomycin Z and X, separation of nikkomycin Z from the culture medium of S. ansochromogenes is difficult. Thus, generating a nikkomycin Z selectively producing strain is vital to scale up the nikkomycin Z yields for clinical trials. Results A nikkomycin Z producing strain (sanPDM was constructed by blocking the imidazolone biosynthetic pathway of nikkomycin X via genetic manipulation and yielded 300 mg/L nikkomycin Z and abolished the nikkomycin X production. To further increase the yield of nikkomycin Z, the effects of different precursors on its production were investigated. Precursors of nucleoside moiety (uracil or uridine had a stimulatory effect on nikkomycin Z production while precursors of peptidyl moiety (L-lysine and L-glutamate had no effect. sanPDM produced the maximum yields of nikkomycin Z (800 mg/L in the presence of uracil at the concentration of 2 g/L and it was approximately 2.6-fold higher than that of the parent strain. Conclusion A high nikkomycin Z selectively producing was obtained by genetic manipulation combined with precursors feeding. The strategy presented here might be applicable in other bacteria to selectively produce targeted antibiotics.

  13. Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis

    Science.gov (United States)

    Aktas, Meriyem; Danne, Linna; Möller, Philip; Narberhaus, Franz

    2014-01-01

    Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylcholine (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defense response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22% of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation. PMID:24723930

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

    Science.gov (United States)

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

    2012-06-13

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

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

    Science.gov (United States)

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

    2017-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Xiaozhong Lan

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

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

    Science.gov (United States)

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

    2015-01-01

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

  18. Arctic mustard flower color polymorphism controlled by petal-specific downregulation at the threshold of the anthocyanin biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Cynthia A Dick

    2011-04-01

    Full Text Available Intra- and interspecific variation in flower color is a hallmark of angiosperm diversity. The evolutionary forces underlying the variety of flower colors can be nearly as diverse as the colors themselves. In addition to pollinator preferences, non-pollinator agents of selection can have a major influence on the evolution of flower color polymorphisms, especially when the pigments in question are also expressed in vegetative tissues. In such cases, identifying the target(s of selection starts with determining the biochemical and molecular basis for the flower color variation and examining any pleiotropic effects manifested in vegetative tissues. Herein, we describe a widespread purple-white flower color polymorphism in the mustard Parrya nudicaulis spanning Alaska. The frequency of white-flowered individuals increases with increasing growing-season temperature, consistent with the role of anthocyanin pigments in stress tolerance. White petals fail to produce the stress responsive flavonoid intermediates in the anthocyanin biosynthetic pathway (ABP, suggesting an early pathway blockage. Petal cDNA sequences did not reveal blockages in any of the eight enzyme-coding genes in white-flowered individuals, nor any color differentiating SNPs. A qRT-PCR analysis of white petals identified a 24-fold reduction in chalcone synthase (CHS at the threshold of the ABP, but no change in CHS expression in leaves and sepals. This arctic species has avoided the deleterious effects associated with the loss of flavonoid intermediates in vegetative tissues by decoupling CHS expression in petals and leaves, yet the correlation of flower color and climate suggests that the loss of flavonoids in the petals alone may affect the tolerance of white-flowered individuals to colder environments.

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

    Science.gov (United States)

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

    2013-01-01

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

  20. Progress in Understanding the Genetic Information and Biosynthetic Pathways behind Amycolatopsis Antibiotics, with Implications for the Continued Discovery of Novel Drugs.

    Science.gov (United States)

    Chen, Su; Wu, Qihao; Shen, Qingqing; Wang, Hong

    2016-01-01

    Species of Amycolatopsis, well recognized as producers of both vancomycin and rifamycin, are also known for producing other secondary metabolites, with wide usage in medicine and agriculture. The molecular genetics of natural antibiotics produced by this genus have been well studied. Since the rise of antibiotic resistance, finding new drugs to fight infection has become an urgent priority. Progress in understanding the biosynthesis of metabolites greatly helps the rational manipulation of biosynthetic pathways, and thus to achieve the goal of generating novel natural antibiotics. The efforts made in exploiting Amycolatopsis genome sequences for the discovery of novel natural products and biosynthetic pathways are summarized. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. tRNA-dependent cysteine biosynthetic pathway represents a strategy to increase cysteine contents by preventing it from thermal degradation: thermal adaptation of methanogenic archaea ancestor.

    Science.gov (United States)

    Qu, Ge; Wang, Wei; Chen, Ling-Ling; Qian, Shao-Song; Zhang, Hong-Yu

    2009-10-01

    Although cysteine (Cys) is beneficial to stabilize protein structures, it is not prevalent in thermophiles. For instance, the Cys contents in most thermophilic archaea are only around 0.7%. However, methanogenic archaea, no matter thermophilic or not, contain relatively abundant Cys, which remains elusive for a long time. Recently, Klipcan et al. correlated this intriguing property of methanogenic archaea with their unique tRNA-dependent Cys biosynthetic pathway. But, the deep reasons underlying the correlation are ambiguous. Considering the facts that free Cys is thermally labile and the tRNA-dependent Cys biosynthesis avoids the use of free Cys, we speculate that the unique Cys biosynthetic pathway represents a strategy to increase Cys contents by preventing it from thermal degradation, which may be relevant to the thermal adaptation of methanogenic archaea ancestor.

  2. Synthesis of C-Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies-Based Biosynthetic Pathway

    DEFF Research Database (Denmark)

    Andersen-Ranberg, Johan; Kongstad, Kenneth Thermann; Nafisi, Majse

    2017-01-01

    unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C-glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway...

  3. Novel acetylcholinesterase inhibitors from Zijuan tea and biosynthetic pathway of caffeoylated catechin in tea plant.

    Science.gov (United States)

    Wang, Wei; Fu, Xi-Wen; Dai, Xin-Long; Hua, Fang; Chu, Gang-Xiu; Chu, Ming-Jie; Hu, Feng-Lin; Ling, Tie-Jun; Gao, Li-Ping; Xie, Zhong-Wen; Wan, Xiao-Chun; Bao, Guan-Hu

    2017-12-15

    Zijuan tea is a special cultivar of Yunnan broad-leaf tea (Camellia sinensis var. assamica) with purple buds, leaves, and stems. Phytochemical study on this tea led to the discovery of three hydroxycinnamoylated catechins (HCCs) (1-3), seven other catechins (4-10), three proanthocyanidins (11-13), five flavones and flavone glycosides (14-18), two alkaloids (19, 20), one steroid (21), and one phenylpropanoid glycoside (22). The isolation and structural elucidation of the caffeoylated catechin (1) by means of spectroscopic techniques were described. We also provide the first evidence that 1 is synthesized via a two-step pathway in tea plant. The three HCCs (1-3) were investigated on their bioactivity through molecular modeling simulation and biochemical experiments. Our results show that they bind acetylcholinesterase (AChE) tightly and have strong AChE inhibitory activity with IC 50 value at 2.49, 11.41, 62.26μM, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.

    Science.gov (United States)

    Guo, Juan; Ma, Xiaohui; Cai, Yuan; Ma, Ying; Zhan, Zhilai; Zhou, Yongjin J; Liu, Wujun; Guan, Mengxin; Yang, Jian; Cui, Guanghong; Kang, Liping; Yang, Lei; Shen, Ye; Tang, Jinfu; Lin, Huixin; Ma, Xiaojing; Jin, Baolong; Liu, Zhenming; Peters, Reuben J; Zhao, Zongbao K; Huang, Luqi

    2016-04-01

    Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  5. Elucidation of the Vanillin Biosynthetic Pathway in Vanilla planifolia

    DEFF Research Database (Denmark)

    Gallage, Nethaji Janeshawari

    discovery and downstream processing methods due to physicochemical properties of the substrate, the product and the nature of microorganisms used. Hence, bioengineering tools have been employed to circumvent these drawbacks. The review emphasizes the major issues encountered and the solutions obtained...... peptide is transported into the vacuole for potential degradation (Chapter 3 – Manuscript in preparation). This PhD thesis also includes a review (Chapter 4), which represents the current state of biotechnology-derived vanillin synthesis based on ferulic acid, eugenol and glucose using microorganisms...... pods is a laborious and expensive process and has a stagnant supply of vanilla pods of around 2000 tons. The worldwide increasing demand for natural vanillin flavour can therefore no longer be met by the pods of vanilla orchids alone. Consequently, research in bioengineering of microorganisms...

  6. Exploring triacylglycerol biosynthetic pathway in developing seeds of Chia (Salvia hispanica L.): a transcriptomic approach.

    Science.gov (United States)

    R V, Sreedhar; Kumari, Priya; Rupwate, Sunny D; Rajasekharan, Ram; Srinivasan, Malathi

    2015-01-01

    Chia (Salvia hispanica L.), a member of the mint family (Lamiaceae), is a rediscovered crop with great importance in health and nutrition and is also the highest known terrestrial plant source of heart-healthy omega-3 fatty acid, alpha linolenic acid (ALA). At present, there is no public genomic information or database available for this crop, hindering research on its genetic improvement through genomics-assisted breeding programs. The first comprehensive analysis of the global transcriptome profile of developing Salvia hispanica L. seeds, with special reference to lipid biosynthesis is presented in this study. RNA from five different stages of seed development was extracted and sequenced separately using the Illumina GAIIx platform. De novo assembly of processed reads in the pooled transcriptome using Trinity yielded 76,014 transcripts. The total transcript length was 66,944,462 bases (66.9 Mb), with an average length of approximately 880 bases. In the molecular functions category of Gene Ontology (GO) terms, ATP binding and nucleotide binding were found to be the most abundant and in the biological processes category, the metabolic process and the regulation of transcription-DNA-dependent and oxidation-reduction process were abundant. From the EuKaryotic Orthologous Groups of proteins (KOG) classification, the major category was "Metabolism" (31.97%), of which the most prominent class was 'carbohydrate metabolism and transport' (5.81% of total KOG classifications) followed by 'secondary metabolite biosynthesis transport and catabolism' (5.34%) and 'lipid metabolism' (4.57%). A majority of the candidate genes involved in lipid biosynthesis and oil accumulation were identified. Furthermore, 5596 simple sequence repeats (SSRs) were identified. The transcriptome data was further validated through confirmative PCR and qRT-PCR for select lipid genes. Our study provides insight into the complex transcriptome and will contribute to further genome-wide research and

  7. 1. Improving the Yield of Biodiesel from Microalgae and Other Lipids. 2. Studies of the Wax Ester Biosynthetic Pathway and Potential Biotechnological Application

    OpenAIRE

    Wahlen, Bradley D.

    2012-01-01

    The production of biofuels and oleochemicals from renewable sources offers an opportunity to reduce our dependence on fossil fuels. The work contained in this dissertation has focused on developing and improving methods for the production of biodiesel from non-traditional feedstocks and understanding biosynthetic pathways that result in the production of oleochemicals and fuels. Pure vegetable oil can account for 70-80% of the total cost of biodiesel production. Many low-cost oils contain ...

  8. Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c-di-GMP production by the DgcQ protein of Escherichia coli.

    Science.gov (United States)

    Rossi, Elio; Motta, Sara; Aliverti, Alessandro; Cossu, Federica; Gourlay, Louise; Mauri, Pierluigi; Landini, Paolo

    2017-09-11

    Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  9. Exploring triacylglycerol biosynthetic pathway in developing seeds of Chia (Salvia hispanica L.: a transcriptomic approach.

    Directory of Open Access Journals (Sweden)

    Sreedhar R V

    Full Text Available Chia (Salvia hispanica L., a member of the mint family (Lamiaceae, is a rediscovered crop with great importance in health and nutrition and is also the highest known terrestrial plant source of heart-healthy omega-3 fatty acid, alpha linolenic acid (ALA. At present, there is no public genomic information or database available for this crop, hindering research on its genetic improvement through genomics-assisted breeding programs. The first comprehensive analysis of the global transcriptome profile of developing Salvia hispanica L. seeds, with special reference to lipid biosynthesis is presented in this study. RNA from five different stages of seed development was extracted and sequenced separately using the Illumina GAIIx platform. De novo assembly of processed reads in the pooled transcriptome using Trinity yielded 76,014 transcripts. The total transcript length was 66,944,462 bases (66.9 Mb, with an average length of approximately 880 bases. In the molecular functions category of Gene Ontology (GO terms, ATP binding and nucleotide binding were found to be the most abundant and in the biological processes category, the metabolic process and the regulation of transcription-DNA-dependent and oxidation-reduction process were abundant. From the EuKaryotic Orthologous Groups of proteins (KOG classification, the major category was "Metabolism" (31.97%, of which the most prominent class was 'carbohydrate metabolism and transport' (5.81% of total KOG classifications followed by 'secondary metabolite biosynthesis transport and catabolism' (5.34% and 'lipid metabolism' (4.57%. A majority of the candidate genes involved in lipid biosynthesis and oil accumulation were identified. Furthermore, 5596 simple sequence repeats (SSRs were identified. The transcriptome data was further validated through confirmative PCR and qRT-PCR for select lipid genes. Our study provides insight into the complex transcriptome and will contribute to further genome-wide research

  10. Possible Interactions between the Biosynthetic Pathways of Indole Glucosinolate and Auxin

    Directory of Open Access Journals (Sweden)

    Siva K. Malka

    2017-12-01

    Full Text Available Glucosinolates (GLS are a group of plant secondary metabolites mainly found in Cruciferous plants, share a core structure consisting of a β-thioglucose moiety and a sulfonated oxime, but differ by a variable side chain derived from one of the several amino acids. These compounds are hydrolyzed upon cell damage by thioglucosidase (myrosinase, and the resulting degradation products are toxic to many pathogens and herbivores. Human beings use these compounds as flavor compounds, anti-carcinogens, and bio-pesticides. GLS metabolism is complexly linked to auxin homeostasis. Indole GLS contributes to auxin biosynthesis via metabolic intermediates indole-3-acetaldoxime (IAOx and indole-3-acetonitrile (IAN. IAOx is proposed to be a metabolic branch point for biosynthesis of indole GLS, IAA, and camalexin. Interruption of metabolic channeling of IAOx into indole GLS leads to high-auxin production in GLS mutants. IAN is also produced as a hydrolyzed product of indole GLS and metabolized to IAA by nitrilases. In this review, we will discuss current knowledge on involvement of GLS in auxin homeostasis.

  11. p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

    Science.gov (United States)

    Petrik, Deborah L; Karlen, Steven D; Cass, Cynthia L; Padmakshan, Dharshana; Lu, Fachuang; Liu, Sarah; Le Bris, Philippe; Antelme, Sébastien; Santoro, Nicholas; Wilkerson, Curtis G; Sibout, Richard; Lapierre, Catherine; Ralph, John; Sedbrook, John C

    2014-01-01

    Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta. PMID:24372757

  12. The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways.

    Science.gov (United States)

    Dugrand-Judek, Audray; Olry, Alexandre; Hehn, Alain; Costantino, Gilles; Ollitrault, Patrick; Froelicher, Yann; Bourgaud, Frédéric

    2015-01-01

    Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the "grapefruit juice effect". Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in

  13. The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways

    Science.gov (United States)

    Dugrand-Judek, Audray; Olry, Alexandre; Hehn, Alain; Costantino, Gilles; Ollitrault, Patrick; Froelicher, Yann; Bourgaud, Frédéric

    2015-01-01

    Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in

  14. Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Nara, Takeshi, E-mail: tnara@juntendo.ac.jp [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Hashimoto, Muneaki; Hirawake, Hiroko [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Liao, Chien-Wei [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Parasitology, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan, ROC (China); Fukai, Yoshihisa; Suzuki, Shigeo; Tsubouchi, Akiko; Morales, Jorge; Takamiya, Shinzaburo [Department of Molecular and Cellular Parasitology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Division of Proteomics and Biomolecular Science, Biomedical Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Fan, Chia-Kwung [Department of Parasitology, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan, ROC (China); Inaoka, Daniel Ken [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Inoue, Masayuki [Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Tanaka, Akiko [Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045 (Japan); Harada, Shigeharu [Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Kita, Kiyoshi [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); and others

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer An Escherichia coli strain co-expressing CPSII, ATC, and DHO of Trypanosoma cruzi was constructed. Black-Right-Pointing-Pointer Molecular interactions between CPSII, ATC, and DHO of T. cruzi were demonstrated. Black-Right-Pointing-Pointer CPSII bound with both ATC and DHO. Black-Right-Pointing-Pointer ATC bound with both CPSII and DHO. Black-Right-Pointing-Pointer A functional tri-enzyme complex might precede the establishment of the fused enzyme. -- Abstract: The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.

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

    Directory of Open Access Journals (Sweden)

    Royah Vaezi

    2013-12-01

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

  16. Bioorthogonal proteomics of 15-hexadecynyloxyacetic acid chemical reporter reveals preferential targeting of fatty acid modified proteins and biosynthetic enzymes.

    Science.gov (United States)

    Yount, Jacob S; Charron, Guillaume; Hang, Howard C

    2012-01-15

    Chemical reporters are powerful tools for the detection and discovery of protein modifications following cellular labeling. The metabolism of alkyne- or azide-functionalized chemical reporters in cells can influence the efficiency and specificity of protein targeting. To evaluate the effect of degradation of chemical reporters of protein fatty acylation, we synthesized 15-hexadecynyloxyacetic acid (HDYOA), a reporter that was designed to be resistant to β-oxidation, and compared its ability to label palmitoylated proteins with an established reporter, 17-octadecynoic acid (ODYA). HDYOA was able to label known candidate S-palmitoylated proteins similarly to ODYA. Accordingly, bioorthogonal proteomic analysis demonstrated that 70% of proteins labeled with ODYA were also labeled with HDYOA. However, the proteins observed differentially in our proteomic studies suggested that a portion of ODYA protein labeling is a result of β-oxidation. In contrast, downstream enzymes involved in β-oxidation of fatty acids were not targeted by HDYOA. Since HDYOA can label S-palmitoylated proteins and is not utilized by downstream β-oxidation pathways, this fatty acid chemical reporter may be particularly useful for bioorthogonal proteomic studies in cell types metabolically skewed toward fatty acid breakdown. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Masanori Tachikawa

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  19. Cytochrome P450s from Cynara cardunculus L. CYP71AV9 and CYP71BL5, catalyze distinct hydroxylations in the sesquiterpene lactone biosynthetic pathway.

    Science.gov (United States)

    Eljounaidi, Kaouthar; Cankar, Katarina; Comino, Cinzia; Moglia, Andrea; Hehn, Alain; Bourgaud, Frédéric; Bouwmeester, Harro; Menin, Barbara; Lanteri, Sergio; Beekwilder, Jules

    2014-06-01

    Cynara cardunculus (Asteraceae) is a cross pollinated perennial crop which includes the two cultivated taxa globe artichoke and cultivated cardoon. The leaves of these plants contain high concentrations of sesquiterpene lactones (STLs) among which cynaropicrin is the most represented, and has recently attracted attention because of its therapeutic potential as anti-tumor and anti-photoaging agent. Costunolide is considered the common precursor of the STLs and three enzymes are involved in its biosynthetic pathway: i.e. the germacrene A synthase (GAS), the germacrene A oxidase (GAO) and the costunolide synthase (COS). Here we report on the isolation of two P450 genes, (i.e. CYP71AV9 and CYP71BL5), in a set of ∼19,000 C. cardunculus unigenes, and their functional characterization in yeast and in planta. The metabolite analyses revealed that the co-expression of CYP71AV9 together with GAS resulted in the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid in yeast. The co-expression of CYP71BL5 and CYP71AV9 with GAS led to biosynthesis of the free costunolide in yeast and costunolide conjugates in Nicotiana benthamiana, demonstrating their involvement in STL biosynthesis as GAO and COS enzymes. The substrate specificity of CYP71AV9 was investigated by testing its ability to convert amorpha-4,11-diene, (+)-germacrene D and cascarilladiene to their oxidized products when co-expressed in yeast with the corresponding terpene synthases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  20. The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae.

    Science.gov (United States)

    Rouhbakhsh, D; Lai, C Y; von Dohlen, C D; Clark, M A; Baumann, L; Baumann, P; Moran, N A; Voegtlin, D J

    1996-04-01

    The bacterial endosymbionts (Buchnera) from the aphids Rhopalosiphum padi, R. maidis, Schizaphis graminum, and Acyrthosiphon pisum contain the genes for anthranilate synthase (trpEG) on plasmids made up of one or more 3.6-kb units. Anthranilate synthase is the first as well as the rate-limiting enzyme in the tryptophan biosynthetic pathway. The amplification of trpEG on plasmids may result in an increase of enzyme protein and overproduction of this essential amino acid, which is required by the aphid host. The nucleotide sequence of trpEG from endosymbionts of different species of aphids is highly conserved, as is an approximately 500-bp upstream DNA segment which has the characteristics of an origin of replication. Phylogenetic analyses were performed using trpE and trpG from the endosymbionts of these four aphids as well as from the endosymbiont of Schlechtendalia chinensis, in which trpEG occurs on the chromosome. The resulting phylogeny was congruent with trees derived from sequences of two chromosome-located bacterial genes (part of trpB and 16S ribosomal DNA). In turn, trees obtained from plasmid-borne and bacterial chromosome-borne sequences were congruent with the tree resulting from phylogenetic analysis of three aphid mitochondrial regions (portions of the small and large ribosomal DNA subunits, as well as cytochrome oxidase II). Congruence of trees based on genes from host mitochondria and from bacteria adds to previous support for exclusively vertical transmission of the endosymbionts within aphid lineages. Congruence with trees based on plasmid-borne genes supports the origin of the plasmid-borne trpEG from the chromosomal genes of the same lineage and the absence of subsequent plasmid exchange among endosymbionts of different species of aphids.

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  2. Biosynthetic production of universally (13)C-labelled polyunsaturated fatty acids as reference materials for natural health product research.

    Science.gov (United States)

    Le, Phuong Mai; Fraser, Catherine; Gardner, Graeme; Liang, Wei-Wan; Kralovec, Jaroslav A; Cunnane, Stephen C; Windust, Anthony J

    2007-09-01

    Long-chain polyunsaturated fatty acids (LCPUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have become important natural health products with numerous proven benefits related to brain function and cardiovascular health. Not only are omega-3 fatty acids available in a plethora of dietary supplements, but they are also increasingly being incorporated as triglycerides into conventional foods, including bread, milk, yoghurt and confectionaries. Recently, transgenic oil seed crops and livestock have been developed that enhance omega-3 fatty acid content. This diverse array of matrices presents a difficult analytical challenge and is compounded further by samples generated through clinical research. Stable isotope (13)C-labelled LCPUFA standards offer many advantages as research tools because they may be distinguished from their naturally abundant counterparts by mass spectrometry and directly incorporated as internal standards into analytical procedures. Further, (13)C-labelled LCPUFAs are safe to use as metabolic tracers to study uptake and metabolism in humans. Currently, (13)C-labelled LCPUFAs are expensive, available in limited supply and not in triglyceride form. To resolve these issues, marine heterotrophic microorganisms are being isolated and screened for LCPUFA production with a view to the efficient biosynthetic production of U-(13)C-labelled fatty acids using U-(13)C glucose as a carbon source. Of 37 isolates obtained, most were thraustochytrids, and either DHA or omega-6 docosapentaenoic acid (22:5n-6) were produced as the major LCPUFA. The marine protist Hyalochlorella marina was identified as a novel source of EPA and omega-3 docosapentaenoic acid (22:5n-3). As proof of principle, gram-level production of (13)C-labelled DHA has been achieved with high chemical purity ( >99%) and high (13)C incorporation levels (>90%), as confirmed by NMR and MS analyses. Finally, U-(13)C-DHA was enzymatically re-esterified to

  3. Amino Acid Biosynthesis Pathways in Diatoms

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    Mariusz A. Bromke

    2013-04-01

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

  4. Structure of ThiM from Vitamin B1 biosynthetic pathway of Staphylococcus aureus - Insights into a novel pro-drug approach addressing MRSA infections

    Science.gov (United States)

    Drebes, Julia; Künz, Madeleine; Windshügel, Björn; Kikhney, Alexey G.; Müller, Ingrid B.; Eberle, Raphael J.; Oberthür, Dominik; Cang, Huaixing; Svergun, Dmitri I.; Perbandt, Markus; Betzel, Christian; Wrenger, Carsten

    2016-03-01

    Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin). Thiamin in its activated form, thiamin pyrophosphate, is an essential co-factor for all organisms. Therefore, thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional co-factors by the bacterium can function as pro-drugs which thus block various co-factor dependent pathways. We characterized one of the key enzymes within the S. aureus vitamin B1 biosynthetic pathway, 5-(hydroxyethyl)-4-methylthiazole kinase (SaThiM; EC 2.7.1.50), a potential target for pro-drug compounds and analyzed the native structure of SaThiM and complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues.

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

    Directory of Open Access Journals (Sweden)

    Clark Shawn M

    2013-01-01

    Full Text Available Abstract 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

  6. Salicylic acid-independent plant defence pathways

    NARCIS (Netherlands)

    Pieterse, C.M.J.; Loon, L.C. van

    1999-01-01

    Salicylic acid is an important signalling molecule involved in both locally and systemically induced disease resistance responses. Recent advances in our understanding of plant defence signalling have revealed that plants employ a network of signal transduction pathways, some of which are

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

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    2016-07-01

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

  8. Isolation and identification of dihydroartemisinic acid hydroperoxide from Artemisia annua : A novel biosynthetic precursor of artemisinin

    NARCIS (Netherlands)

    Wallaart, TE; Pras, N; Quax, WJ

    Dihydroartemisinic acid hydroperoxide (2) was isolated for the first time as a natural product from the plant Artemisia annua in a 29% yield. Its structure was identified by H-1 and C-13 NMR spectroscopy. Compound 2 is known as an intermediate of the photochemical oxidation of dihydroartemisinic

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

    Science.gov (United States)

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

    2015-07-01

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

  10. Influence of the dissolved oxygen concentration on the penicillin biosynthetic pathway in steady-state cultures of Penicillium chrysogenum

    DEFF Research Database (Denmark)

    Henriksen, Claus Maxel; Nielsen, Jens Bredal; Villadsen, John

    1997-01-01

    The influence the of dissolved oxygen concentration on penicillin biosynthesis was studied in steady-state continuous cultures of a high-yielding strain of Penicillium chrysogenum operated at a dilution rate of 0.05 h-l. The dissolved oxygen concentration was varied between 0.019 and 0.344 m...... recovered to its maximum value when the dissolved oxygen concentration was reset to a value above 0.08 mM. The specific formation rates of a number of typical byproducts of the penicillin production, i.e., delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, isopenicillin N, 6-aminopenicillanic acid, 8......-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and glutathione is suggested. The intracellular pools of the pathway intermediates delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N increased respectively 2- and 3-fold when the dissolved oxygen concentration was lowered from 0.344 to 0.042 mM, whereas the intracellular pools of glutathione...

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

    Science.gov (United States)

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

    2013-01-01

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

  12. Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae down-regulates the sphingolipid biosynthetic pathway leading to growth arrest.

    Science.gov (United States)

    Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N; Barny, Marie-Anne

    2014-06-27

    Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of Wolinella succinogenes

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Timothy H. [Cornell University, Ithaca, New York 14853-1301 (United States); Krishnamoorthy, Kalyanaraman; Begley, Tadhg P., E-mail: begley@tamu.edu [Texas A& M University, College Station, TX 77842 (United States); Ealick, Steven E., E-mail: begley@tamu.edu [Cornell University, Ithaca, New York 14853-1301 (United States)

    2011-10-01

    MetY is the first reported structure of an O-acetylhomoserine sulfhydrylase that utilizes a protein thiocarboxylate intermediate as the sulfur source in a novel methionine-biosynthetic pathway instead of catalyzing a direct sulfhydrylation reaction. O-Acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5′-phosphate (PLP) dependent sulfide-utilizing enzyme in the l-cysteine and l-methionine biosynthetic pathways of various enteric bacteria and fungi. OAHS catalyzes the conversion of O-acetylhomoserine to homocysteine using sulfide in a process known as direct sulfhydrylation. However, the source of the sulfur has not been identified and no structures of OAHS have been reported in the literature. Here, the crystal structure of Wolinella succinogenes OAHS (MetY) determined at 2.2 Å resolution is reported. MetY crystallized in space group C2 with two monomers in the asymmetric unit. Size-exclusion chromatography, dynamic light scattering and crystal packing indicate that the biological unit is a tetramer in solution. This is further supported by the crystal structure, in which a tetramer is formed using a combination of noncrystallographic and crystallographic twofold axes. A search for structurally homologous proteins revealed that MetY has the same fold as cystathionine γ-lyase and methionine γ-lyase. The active sites of these enzymes, which are also PLP-dependent, share a high degree of structural similarity, suggesting that MetY belongs to the γ-elimination subclass of the Cys/Met metabolism PLP-dependent family of enzymes. The structure of MetY, together with biochemical data, provides insight into the mechanism of sulfur transfer to a small molecule via a protein thiocarboxylate intermediate.

  14. De Novo Biosynthesis of Glutarate via α-Keto Acid Carbon Chain Extension and Decarboxylation Pathway in Escherichia coli.

    Science.gov (United States)

    Wang, Jian; Wu, Yifei; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

    2017-10-20

    Microbial based bioplastics are promising alternatives to petroleum based synthetic plastics due to their renewability and economic feasibility. Glutarate is one of the most potential building blocks for bioplastics. The recent biosynthetic routes for glutarate were mostly based on the l-lysine degradation pathway from Pseudomonas putida that required lysine either by feeding or lysine overproduction via genetic manipulations. Herein, we established a novel glutarate biosynthetic pathway by incorporation of a "+1" carbon chain extension pathway from α-ketoglutarate (α-KG) in combination with α-keto acid decarboxylation pathway in Escherichia coli. Introduction of homocitrate synthase (HCS), homoaconitase (HA) and homoisocitrate dehydrogenase (HICDH) from Saccharomyces cerevisiae into E. coli enabled "+1" carbon extension from α-KG to α-ketoadipate (α-KA), which was subsequently converted into glutarate by a promiscuous α-keto acid decarboxylase (KivD) and a succinate semialdehyde dehydrogenase (GabD). The recombinant E. coli coexpressing all five genes produced 0.3 g/L glutarate from glucose. To further improve the titers, α-KG was rechanneled into carbon chain extension pathway via the clustered regularly interspersed palindromic repeats system mediated interference (CRISPRi) of essential genes sucA and sucB in tricarboxylic acid (TCA) cycle. The final strain could produce 0.42 g/L glutarate, which was increased by 40% compared with the parental strain.

  15. Lactic Acid Bacterial Starter Culture with Antioxidant and γ-Aminobutyric Acid Biosynthetic Activities Isolated from Flatfish-Sikhae Fermentation.

    Science.gov (United States)

    Won, Yeong Geol; Yu, Hyun-Hee; Chang, Young-Hyo; Hwang, Han-Joon

    2015-12-01

    The aim of this study is to select a lactic acid bacterial strain as a starter culture for flatfish-Sikhae fermentation and to evaluate its suitability for application in a food system. Four strains of lactic acid bacteria isolated from commercial flatfish-Sikhae were identified and selected as starter culture candidates through investigation of growth rates, salt tolerance, food safety, and functional properties such as antioxidative and antimicrobial activities. The fermentation properties of the starter candidates were also examined in food systems prepared with these strains (candidate batch) in comparison with a spontaneous fermentation process without starter culture (control batch) at 15°C. The results showed that the candidate YG331 batch had better fermentation properties such as viable cell count, pH, and acidity than the other experimental batches, including the control batch. The results are expressed according to selection criteria based on a preliminary sensory evaluation and physiochemical investigation. Also, only a small amount of histamine was detected with the candidate YG331 batch. The radical scavenging activity of the candidate batches was better compared with the control batch, and especially candidate YG331 batch showed the best radical scavenging activity. Also, we isolated another starter candidate (identified as Lactobacillus brevis PM03) with γ-aminobutyric acid (GABA)-producing activity from commercial flatfish-Sikhae products. The sensory scores of the candidate YG331 batch were better than those of the other experimental batches in terms of flavor, color, and overall acceptance. In this study, we established selection criteria for the lactic acid bacterial starter for the flatfish-Sikhae production and finally selected candidate YG331 as the most suitable starter.

  16. Genetic, metabolomic and transcriptomic analyses of the de novo L-cysteine biosynthetic pathway in the enteric protozoan parasite Entamoeba histolytica.

    Science.gov (United States)

    Jeelani, Ghulam; Sato, Dan; Soga, Tomoyoshi; Nozaki, Tomoyoshi

    2017-11-15

    The de novo L-cysteine biosynthetic pathway is critical for the growth, antioxidative stress defenses, and pathogenesis of bacterial and protozoan pathogens, such as Salmonella typhimurium and Entamoeba histolytica. This pathway involves two key enzymes, serine acetyltransferase (SAT) and cysteine synthase (CS), which are absent in mammals and therefore represent rational drug targets. The human parasite E. histolytica possesses three SAT and CS isozymes; however, the specific roles of individual isoforms and significance of such apparent redundancy remains unclear. In the present study, we generated E. histolytica cell lines in which CS and SAT expression was knocked down by transcriptional gene silencing. The strain in which CS1, 2 and 3 were simultaneously silenced and the SAT3 gene-silenced strain showed impaired growth when cultured in a cysteine lacking BI-S-33 medium, whereas silencing of SAT1 and SAT2 had no effects on growth. Combined transcriptomic and metabolomic analyses revealed that, CS and SAT3 are involved in S-methylcysteine/cysteine synthesis. Furthermore, silencing of the CS1-3 or SAT3 caused upregulation of various iron-sulfur flavoprotein genes. Taken together, these results provide the first direct evidence of the biological importance of SAT3 and CS isoforms in E. histolytica and justify the exploitation of these enzymes as potential drug targets.

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

    Science.gov (United States)

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

  18. A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

    Directory of Open Access Journals (Sweden)

    Belshoff Alex C

    2011-05-01

    Full Text Available Abstract Background Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes. Results We utilized a stable isotope resolved metabolomics (SIRM approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits

  19. Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

    Science.gov (United States)

    Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

    2009-03-04

    The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

  20. Genome of Diaporthe sp. provides insights into the potential inter-phylum transfer of a fungal sesquiterpenoid biosynthetic pathway

    Science.gov (United States)

    de Sena Filho, Jose Guedes; Quin, Maureen B.; Spakowicz, Daniel J; Shaw, Jeffrey J.; Kucera, Kaury; Dunican, Brian; Strobel, Scott A.; Schmidt-Dannert, Claudia

    2016-01-01

    Fungi have highly active secondary metabolic pathways which enable them to produce a wealth of sesquiterpenoids that are bioactive. One example is Δ6-protoilludene, the precursor to the cytotoxic illudins, which are pharmaceutically relevant as anticancer therapeutics. To date, this valuable sesquiterpene has only been identified in members of the fungal division Basidiomycota. To explore the untapped potential of fungi belonging to the division Ascomycota in producing Δ6-protoilludene, we isolated the a fungal endophyte Diaporthe sp. BR109 and show that it produces a diversity of terpenoids including Δ6-protoilludene. Using a genome sequencing and mining approach 17 putative novel sesquiterpene synthases were identified in Diaporthe sp. BR109. A phylogenetic approach was used to predict which gene encodes Δ6-protoilludene synthase, which was then confirmed experimentally. These analyses reveal that the sesquiterpene synthase and its putative sesquiterpene scaffold modifying cytochrome P450(s) may have been acquired by inter-phylum horizontal gene transfer from Basidiomycota to Ascomycota. Bioinformatic analyses indicate that inter-phylum transfer of these minimal sequiterpenoid secondary metabolic pathways may have occurred in other fungi. This work provides insights into the evolution of fungal sesquiterpenoid secondary metabolic pathways in the production of pharmaceutically relevant bioactive natural products. PMID:27521636

  1. Fungal peroxisomes as biosynthetic organelles.

    Science.gov (United States)

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

    2014-12-01

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

  2. Omega-3 phospholipids from fish suppress hepatic steatosis by integrated inhibition of biosynthetic pathways in dietary obese mice

    NARCIS (Netherlands)

    Rossmeisl, M.; Medrikova, D.; Schothorst, van E.M.; Pavlisova, J.; Kuda, O.; Hensler, M.; Bardova, K.; Flachs, P.; Stankova, B.; Vecka, M.; Tvrizicka, E.; Zak, A.; Keijer, J.; Kopecky, J.

    2014-01-01

    Non-alcoholic fatty liver disease (NAFLD) accompanies obesity and insulin resistance. Recent meta-analysis suggested omega-3 polyunsaturated fatty acids DHA and EPA to decrease liver fat in NAFLD patients. Anti-inflammatory, hypolipidemic, and insulin-sensitizing effects of DHA/EPA depend on their

  3. Modification of the mannitol biosynthetic pathway in Aureobasidium pullulans to alter the structure of the polyol lipid liamocin

    Science.gov (United States)

    Aureobasidium pullulans is an important industrial organism that is utilized for the production of numerous bioproducts, such as pullulan, ß-glucan, aureobasidin, and polymalic acid. Our laboratory is also interested in production of the extracellular polyol lipids, called liamocins, produced by cer...

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

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere

    Filamentous fungi produce a wide range of bioactive compounds, classified as secondary metabolites,which have the potential to be used as pharmaceuticals, insecticides, fungicides and food additives.Secondary metabolites also include mycotoxins, which are produced by fungi that contaminate food...... as a model organism for a range of research disciplines and manygenetic engineering tools are available for working in this organism. This PhD study therefore employed A.nidulans as a model system to address the following on two aspects: 1) Developing A. nidulans as aplatform for pathway discovery...

  5. Patulin transformation products and last intermediates in its biosynthetic pathway, E- and Z-ascladiol, are not toxic to human cells.

    Science.gov (United States)

    Tannous, Joanna; Snini, Selma P; El Khoury, Rhoda; Canlet, Cécile; Pinton, Philippe; Lippi, Yannick; Alassane-Kpembi, Imourana; Gauthier, Thierry; El Khoury, André; Atoui, Ali; Zhou, Ting; Lteif, Roger; Oswald, Isabelle P; Puel, Olivier

    2017-06-01

    Patulin is the main mycotoxin contaminating apples. During the brewing of alcoholic beverages, this mycotoxin is degraded to ascladiol, which is also the last precursor of patulin. The present study aims (1) to characterize the last step of the patulin biosynthetic pathway and (2) to describe the toxicity of ascladiol. A patE deletion mutant was generated in Penicillium expansum. In contrast to the wild strain, this mutant does not produce patulin but accumulates high levels of E-ascladiol with few traces of Z-ascladiol. This confirms that patE encodes the patulin synthase involved in the conversion of E-ascladiol to patulin. After purification, cytotoxicities of patulin and E- and Z-ascladiol were investigated on human cell lines from liver, kidney, intestine, and immune system. Patulin was cytotoxic for these four cell lines in a dose-dependent manner. By contrast, both E- and Z-ascladiol were devoid of cytotoxicity. Microarray analyses on human intestinal cells treated with patulin and E-ascladiol showed that the latter, unlike patulin, did not alter the whole human transcription. These results demonstrate that E- and Z-ascladiol are not toxic and therefore patulin detoxification strategies leading to the accumulation of ascladiol are good approaches to limit the patulin risk.

  6. Inhibition of green tea and the catechins against 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    Science.gov (United States)

    Hui, Xian; Liu, Hui; Tian, Fang-Lin; Li, Fei-Fei; Li, Heng; Gao, Wen-Yun

    2016-09-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the MEP terpenoid biosynthetic pathway and also a validated antimicrobial target. Green tea which is rich in polyphenolic components such as the catechins, possesses a plenty of pharmacological activities, in particular an antibacterial effect. To uncover the antibacterial mechanism of green tea and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of green tea and its main antimicrobial catechins were investigated in this study. The results show that the raw extract of green tea and its ethyl acetate fraction are able to suppress DXR activity explicitly. Further determination of the DXR inhibitory capacity of eight catechin compounds demonstrates that the most active compound is gallocatechin gallate that is able to inhibit around 50% activity of DXR at 25μM. Based on these data, the primary structure-activity relationship of the catechins against DXR is discussed. This study would be very helpful to elucidate the antimicrobial mechanism of green tea and the catechins and also would be very useful to direct the rational utilization of them as food additives. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Antimicrobial mechanism of theaflavins: They target 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    Science.gov (United States)

    Hui, Xian; Yue, Qiao; Zhang, Dan-Dan; Li, Heng; Yang, Shao-Qing; Gao, Wen-Yun

    2016-12-12

    1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the 2-methyl-D-erythritol 4-phosphate (MEP) terpenoid biosynthetic pathway and is also a validated antimicrobial target. Theaflavins, which are polyphenolic compounds isolated from fermented tea, possess a wide range of pharmacological activities, especially an antibacterial effect, but little has been reported on their modes of antimicrobial action. To uncover the antibacterial mechanism of theaflavins and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of theaflavins were investigated in this study. The results show that all four theaflavin compounds could specifically suppress the activity of DXR, with theaflavin displaying the lowest effect against DXR (IC50 162.1 μM) and theaflavin-3,3'-digallate exhibiting the highest (IC50 14.9 μM). Moreover, determination of inhibition kinetics of the theaflavins demonstrates that they are non-competitive inhibitors of DXR against 1-deoxy-D-xylulose 5-phosphate (DXP) and un-competitive inhibitors with respect to NADPH. The possible interactions between DXR and the theaflavins were simulated via docking experiments.

  8. Structure and Functional Analysis of ClbQ, an Unusual Intermediate-Releasing Thioesterase from the Colibactin Biosynthetic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Guntaka, Naga Sandhya; Healy, Alan R.; Crawford, Jason M.; Herzon, Seth B.; Bruner, Steven D. (Yale); (Florida); (Yale-MED)

    2017-09-08

    Colibactin is a genotoxic hybrid nonribosomal peptide/polyketide secondary metabolite produced by various pathogenic and probiotic bacteria residing in the human gut. The presence of colibactin metabolites has been correlated to colorectal cancer formation in several studies. The specific function of many gene products in the colibactin gene cluster can be predicted. However, the role of ClbQ, a type II editing thioesterase, has not been established. The importance of ClbQ has been demonstrated by genetic deletions that abolish colibactin cytotoxic activity, and recent studies suggest an atypical role in releasing pathway intermediates from the assembly line. Here we report the 2.0 Å crystal structure and biochemical characterization of ClbQ. Our data reveal that ClbQ exhibits greater catalytic efficiency toward acyl-thioester substrates as compared to precolibactin intermediates and does not discriminate among carrier proteins. Cyclized pyridone-containing colibactins, which are off-pathway derivatives, are not viable substrates for ClbQ, while linear precursors are, supporting a role of ClbQ in facilitating the promiscuous off-loading of premature precolibactin metabolites and novel insights into colibactin biosynthesis.

  9. A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici.

    Science.gov (United States)

    Zhang, Peng; Wang, Xiuna; Fan, Aili; Zheng, Yanjing; Liu, Xingzhong; Wang, Shihua; Zou, Huixi; Oakley, Berl R; Keller, Nancy P; Yin, Wen-Bing

    2017-08-01

    Spore pigmentation is very common in the fungal kingdom. The best studied pigment in fungi is melanin which coats the surface of single cell spores. What and how pigments function in a fungal species with multiple cell conidia is poorly understood. Here, we identified and deleted a polyketide synthase (PKS) gene PfmaE and showed that it is essential for multicellular conidial pigmentation and development in a plant endophytic fungus, Pestalotiopsis fici. To further characterize the melanin pathway, we utilized an advanced Aspergillus nidulans heterologous system for the expression of the PKS PfmaE and the Pfma gene cluster. By structural elucidation of the pathway metabolite scytalone in A. nidulans, we provided chemical evidence that the Pfma cluster synthesizes DHN melanin. Combining genetic deletion and combinatorial gene expression of Pfma cluster genes, we determined that the putative reductase PfmaG and the PKS are sufficient for the synthesis of scytalone. Feeding scytalone back to the P. fici ΔPfmaE mutant restored pigmentation and multicellular adherence of the conidia. These results cement a growing understanding that pigments are essential not simply for protection of spores from biotic and abiotic stresses but also for spore structural development. © 2017 John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2015-05-01

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

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

    Science.gov (United States)

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

    2015-09-01

    miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  12. Insights into the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in Leishmania parasites and characterization of a choline kinase from Leishmania infantum.

    Science.gov (United States)

    Pulido, Sergio A; Nguyen, Victoria H; Alzate, Juan F; Cedeño, David L; Makurath, Monika A; Ríos-Vásquez, Amalia; Duque-Benítez, Sandra M; Jones, Marjorie A; Robledo, Sara M; Friesen, Jon A

    2017-11-01

    The protozoan parasite Leishmania infantum is a causative agent of the disease visceral leishmaniasis, which can be fatal if not properly treated. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthesis pathways are attractive targets for new antileishmanial compounds since these Leishmania cell membrane phospholipids are important for parasite morphology and physiology. In this work we observed Leishmania synthesize PC and PE from extracellular choline and ethanolamine, respectively, suggesting the presence of CDP-choline and CDP-ethanolamine pathways. In addition, Leishmania converted PE to PC, indicating the parasite possesses phosphatidylethanolamine N-methyltransferase (PEMT) activity. The first step in the biosynthesis of PC or PE requires the phosphorylation of choline or ethanolamine by a kinase. We cloned the gene encoding a putative choline/ethanolamine kinase from Leishmania infantum and expressed and purified the encoded recombinant protein. The enzyme possesses choline kinase activity with a Vmax of 3.52μmol/min/mg and an apparent Km value of 0.089mM with respect to choline. The enzyme can also phosphorylate ethanolamine in vitro, but the apparent Km for ethanolamine is 850-fold greater than for choline. In an effort to probe requirements for small molecule inhibition of Leishmania choline kinase, the recombinant enzyme was evaluated for the ability to be inhibited by novel quaternary ammonium salts. The most effective inhibitor was N-iodomethyl-N,N,-dimethyl-N-(6,6-diphenyl hex-5-en-1-yle) ammonium iodide, denoted compound C6. In the presence of 4mM compound C6, the Vmax/Km decreased to approximately 1% of the wild-type catalytic efficiency. In addition, in Leishmania cells treated with compound C6 choline transport was inhibited. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    KAUST Repository

    Chen, Hao

    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. © 2010 Blackwell Publishing Ltd.

  14. Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: strategies and applications.

    Science.gov (United States)

    Ludwig-Müller, Jutta; Jahn, Linda; Lippert, Annemarie; Püschel, Joachim; Walter, Antje

    2014-11-01

    A plethora of bioactive plant metabolites has been explored for pharmaceutical, food chemistry and agricultural applications. The chemical synthesis of these structures is often difficult, so plants are favorably used as producers. While whole plants can serve as a source for secondary metabolites and can be also improved by metabolic engineering, more often cell or organ cultures of relevant plant species are of interest. It should be noted that only in few cases the production for commercial application in such cultures has been achieved. Their genetic manipulation is sometimes faster and the production of a specific metabolite is more reliable, because of less environmental influences. In addition, upscaling in bioreactors is nowadays possible for many of these cultures, so some are already used in industry. There are approaches to alter the profile of metabolites not only by using plant genes, but also by using bacterial genes encoding modifying enzymes. Also, strategies to cope with unwanted or even toxic compounds are available. The need for metabolic engineering of plant secondary metabolite pathways is increasing with the rising demand for (novel) compounds with new bioactive properties. Here, we give some examples of recent developments for the metabolic engineering of plants and organ cultures, which can be used in the production of metabolites with interesting properties. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. The effect of methyl jasmonate and light irradiation treatments on the stilbenoid biosynthetic pathway in Vitis vinifera cell suspension cultures.

    Science.gov (United States)

    Andi, Seyed Ali; Gholami, Mansour; Ford, Christopher M

    2017-08-29

    Grape stilbenes are a well-known family of plant polyphenolics that have been confirmed to have many biological activities in relation to health benefits. In the present study, we investigated the effect of methyl jasmonate (MeJA) elicitor at four different concentrations (25, 50, 100 and 200 μM) in combination or not with high-level light irradiation (10,000 LUX) on a cell line obtained from the pulp of Vitis vinifera cv. Shahani. Our results showed that the stilbene synthesis pathway is inhibited by high-light conditions. A concentration of 50 μM MeJA was optimum for efficient production and high accumulation of total phenolics and total flavonoids as well as total stilbenoids. Furthermore, we showed that there is a significant negative correlation between the production of these metabolites and cell growth. These data provide valuable information for the future scale-up of cell cultures for the production of these very high value compounds in bioreactor system.

  16. Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway.

    Science.gov (United States)

    Ong, Seong Siang; Wickneswari, Ratnam

    2011-11-30

    Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem. In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in epigenetic silencing

  17. Violet/blue chrysanthemums--metabolic engineering of the anthocyanin biosynthetic pathway results in novel petal colors.

    Science.gov (United States)

    Brugliera, Filippa; Tao, Guo-Qing; Tems, Ursula; Kalc, Gianna; Mouradova, Ekaterina; Price, Kym; Stevenson, Kim; Nakamura, Noriko; Stacey, Iolanda; Katsumoto, Yukihisa; Tanaka, Yoshikazu; Mason, John G

    2013-10-01

    Chrysanthemums (Chrysanthemum×morifolium Ramat.) are an important cut-flower and potted plant crop in the horticultural industry world wide. Chrysanthemums express the flavonoid 3'-hydroxylase (F3'H) gene and thus accumulate anthocyanins derived from cyanidin in their inflorescences which appear pink/red. Delphinidin-based anthocyanins are lacking due to the deficiency of a flavonoid 3', 5'-hydroxylase (F3'5'H), and so violet/blue chrysanthemum flower colors are not found. In this study, together with optimization of transgene expression and selection of the host cultivars and gene source, F3'5'H genes have been successfully utilized to produce transgenic bluish chrysanthemums that accumulate delphinidin-based anthocyanins. HPLC analysis and feeding experiments with a delphinidin precursor identified 16 cultivars of chrysanthemums out of 75 that were predicted to turn bluish upon delphinidin accumulation. A selection of eight cultivars were successfully transformed with F3'5'H genes under the control of different promoters. A pansy F3'5'H gene under the control of a chalcone synthase promoter fragment from rose resulted in the effective diversion of the anthocyanin pathway to produce delphinidin in transgenic chrysanthemum flower petals. The resultant petal color was bluish, with 40% of total anthocyanidins attributed to delphinidin. Increased delphinidin levels (up to 80%) were further achieved by hairpin RNA interference-mediated silencing of the endogenous F3'H gene. The resulting petal colors were novel bluish hues, not possible by hybridization breeding. This is the first report of the production of anthocyanins derived from delphinidin in chrysanthemum petals leading to novel flower color.

  18. Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes.

    Directory of Open Access Journals (Sweden)

    Fei Xia

    Full Text Available Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17 and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19 are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high

  19. Amino Acids Attenuate Insulin Action on Gluconeogenesis and Promote Fatty Acid Biosynthesis via mTORC1 Signaling Pathway in trout Hepatocytes

    Directory of Open Access Journals (Sweden)

    Weiwei Dai

    2015-06-01

    Full Text Available Background/Aims: Carnivores exhibit poor utilization of dietary carbohydrates and glucose intolerant phenotypes, yet it remains unclear what are the causal factors and underlying mechanisms. We aimed to evaluate excessive amino acids (AAs-induced effects on insulin signaling, fatty acid biosynthesis and glucose metabolism in rainbow trout and determine the potential involvement of mTORC1 and p38 MAPK pathway. Methods: We stimulated trout primary hepatocytes with different AA levels and employed acute administration of rapamycin to inhibit mTORC1 activation. Results: Increased AA levels enhanced the phosphorylation of ribosomal protein S6 kinase (S6K1, S6, and insulin receptor substrate 1 (IRS-1 on Ser302 but suppressed Akt and p38 phosphorylation; up-regulated the expression of genes related to gluconeogenesis and fatty acid biosynthesis. mTORC1 inhibition not only inhibited the phosphorylation of mTORC1 downstream targets, but also blunted IRS-1 Ser302 phosphorylation and restored excessive AAs-suppressed Akt phosphorylation. Rapamycin also inhibited fatty acid biosynthetic and gluconeogenic gene expression. Conclusion: High levels of AAs up-regulate hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner, while attenuate insulin-mediated repression of gluconeogenesis through elevating IRS-1 Ser302 phosphorylation, which in turn impairs Akt activation and thereby weakening insulin action. We propose that p38 MAPK probably also involves in these AAs-induced metabolic changes.

  20. Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl., a Non-Model Plant with Potent Laxative Properties.

    Directory of Open Access Journals (Sweden)

    Nagaraja Reddy Rama Reddy

    Full Text Available Senna (Cassia angustifolia Vahl. is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG, Cluster of Orthologous Gene (COG and Gene Ontology (GO. Out of the total transcripts, 40138 (95.0% and 36349 (97.7% from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf and 32077 (mature leaf transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7% CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in

  1. Formal modeling and analysis of the hexosamine biosynthetic pathway: role of O-linked N-acetylglucosamine transferase in oncogenesis and cancer progression

    Directory of Open Access Journals (Sweden)

    Muhammad Tariq Saeed

    2016-09-01

    Full Text Available The alteration of glucose metabolism, through increased uptake of glucose and glutamine addiction, is essential to cancer cell growth and invasion. Increased flux of glucose through the Hexosamine Biosynthetic Pathway (HBP drives increased cellular O-GlcNAcylation (hyper-O-GlcNAcylation and contributes to cancer progression by regulating key oncogenes. However, the association between hyper-O-GlcNAcylation and activation of these oncogenes remains poorly characterized. Here, we implement a qualitative modeling framework to analyze the role of the Biological Regulatory Network in HBP activation and its potential effects on key oncogenes. Experimental observations are encoded in a temporal language format and model checking is applied to infer the model parameters and qualitative model construction. Using this model, we discover step-wise genetic alterations that promote cancer development and invasion due to an increase in glycolytic flux, and reveal critical trajectories involved in cancer progression. We compute delay constraints to reveal important associations between the production and degradation rates of proteins. O-linked N-acetylglucosamine transferase (OGT, an enzyme used for addition of O-GlcNAc during O-GlcNAcylation, is identified as a key regulator to promote oncogenesis in a feedback mechanism through the stabilization of c-Myc. Silencing of the OGT and c-Myc loop decreases glycolytic flux and leads to programmed cell death. Results of network analyses also identify a significant cycle that highlights the role of p53-Mdm2 circuit oscillations in cancer recovery and homeostasis. Together, our findings suggest that the OGT and c-Myc feedback loop is critical in tumor progression, and targeting these mediators may provide a mechanism-based therapeutic approach to regulate hyper-O-GlcNAcylation in human cancer.

  2. Formal modeling and analysis of the hexosamine biosynthetic pathway: role of O-linked N-acetylglucosamine transferase in oncogenesis and cancer progression.

    Science.gov (United States)

    Saeed, Muhammad Tariq; Ahmad, Jamil; Kanwal, Shahzina; Holowatyj, Andreana N; Sheikh, Iftikhar A; Zafar Paracha, Rehan; Shafi, Aamir; Siddiqa, Amnah; Bibi, Zurah; Khan, Mukaram; Ali, Amjad

    2016-01-01

    The alteration of glucose metabolism, through increased uptake of glucose and glutamine addiction, is essential to cancer cell growth and invasion. Increased flux of glucose through the Hexosamine Biosynthetic Pathway (HBP) drives increased cellular O-GlcNAcylation (hyper-O-GlcNAcylation) and contributes to cancer progression by regulating key oncogenes. However, the association between hyper-O-GlcNAcylation and activation of these oncogenes remains poorly characterized. Here, we implement a qualitative modeling framework to analyze the role of the Biological Regulatory Network in HBP activation and its potential effects on key oncogenes. Experimental observations are encoded in a temporal language format and model checking is applied to infer the model parameters and qualitative model construction. Using this model, we discover step-wise genetic alterations that promote cancer development and invasion due to an increase in glycolytic flux, and reveal critical trajectories involved in cancer progression. We compute delay constraints to reveal important associations between the production and degradation rates of proteins. O-linked N-acetylglucosamine transferase (OGT), an enzyme used for addition of O-GlcNAc during O-GlcNAcylation, is identified as a key regulator to promote oncogenesis in a feedback mechanism through the stabilization of c-Myc. Silencing of the OGT and c-Myc loop decreases glycolytic flux and leads to programmed cell death. Results of network analyses also identify a significant cycle that highlights the role of p53-Mdm2 circuit oscillations in cancer recovery and homeostasis. Together, our findings suggest that the OGT and c-Myc feedback loop is critical in tumor progression, and targeting these mediators may provide a mechanism-based therapeutic approach to regulate hyper-O-GlcNAcylation in human cancer.

  3. Discovery and Engineering of Pathways for Production of α-Branched Organic Acids.

    Science.gov (United States)

    Blaisse, Michael R; Dong, Hongjun; Fu, Beverly; Chang, Michelle C Y

    2017-10-18

    Cell-based synthesis offers many opportunities for preparing small molecules from simple renewable carbon sources by telescoping multiple reactions into a single fermentation step. One challenge in this area is the development of enzymatic carbon-carbon bond forming cycles that enable a modular disconnection of a target structure into cellular building blocks. In this regard, synthetic pathways based on thiolase enzymes to catalyze the initial carbon-carbon bond forming step between acyl coenzyme A (CoA) substrates offer a versatile route for biological synthesis, but the substrate diversity of such pathways is currently limited. In this report, we describe the identification and biochemical characterization of a thiolase-ketoreductase pair involved in production of branched acids in the roundworm, Ascaris suum, that demonstrates selectivity for forming products with an α-methyl branch using a propionyl-CoA extender unit. Engineering synthetic pathways for production of α-methyl acids in Escherichia coli using these enzymes allows the construction of microbial strains that produce either chiral 2-methyl-3-hydroxy acids (1.1 ± 0.2 g L(-1)) or branched enoic acids (1.12 ± 0.06 g L(-1)) in the presence of a dehydratase at 44% and 87% yield of fed propionate, respectively. In vitro characterization along with in vivo analysis indicates that the ketoreductase is the key driver for selectivity, forming predominantly α-branched products even when paired with a thiolase that highly prefers unbranched linear products. Our results expand the utility of thiolase-based pathways and provide biosynthetic access to α-branched compounds as precursors for polymers and other chemicals.

  4. Biosynthetic studies on terpenoids produced by Streptomyces.

    Science.gov (United States)

    Kuzuyama, Tomohisa

    2017-07-01

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

  5. Genetic and metabolomic dissection of the ergothioneine and selenoneine biosynthetic pathway in the fission yeast, S. pombe, and construction of an overproduction system.

    Directory of Open Access Journals (Sweden)

    Tomáš Pluskal

    Full Text Available Ergothioneine is a small, sulfur-containing metabolite (229 Da synthesized by various species of bacteria and fungi, which can accumulate to millimolar levels in tissues or cells (e.g. erythrocytes of higher eukaryotes. It is commonly marketed as a dietary supplement due to its proposed protective and antioxidative functions. In this study we report the genes forming the two-step ergothioneine biosynthetic pathway in the fission yeast, Schizosaccharomyces pombe. We identified the first gene, egt1+ (SPBC1604.01, by sequence homology to previously published genes from Neurospora crassa and Mycobacterium smegmatis. We showed, using metabolomic analysis, that the Δegt1 deletion mutant completely lacked ergothioneine and its precursors (trimethyl histidine/hercynine and hercynylcysteine sulfoxide. Since the second step of ergothioneine biosynthesis has not been characterized in eukaryotes, we examined four putative homologs (Nfs1/SPBC21D10.11c, SPAC11D3.10, SPCC777.03c, and SPBC660.12c of the corresponding mycobacterial enzyme EgtE. Among deletion mutants of these genes, only one (ΔSPBC660.12c, designated Δegt2 showed a substantial decrease in ergothioneine, accompanied by accumulation of its immediate precursor, hercynylcysteine sulfoxide. Ergothioneine-deficient strains exhibited no phenotypic defects during vegetative growth or quiescence. To effectively study the role of ergothioneine, we constructed an egt1+ overexpression system by replacing its native promoter with the nmt1+ promoter, which is inducible in the absence of thiamine. We employed three versions of the nmt1 promoter with increasing strength of expression and confirmed corresponding accumulations of ergothioneine. We quantified the intracellular concentration of ergothioneine in S. pombe (0.3, 157.4, 41.6, and up to 1606.3 µM in vegetative, nitrogen-starved, glucose-starved, and egt1+-overexpressing cells, respectively and described its gradual accumulation under long

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  7. A biosynthetic pathway for anandamide

    OpenAIRE

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

    2006-01-01

    The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released “on demand” by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE–PLD). Here we document a biosynt...

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

    Directory of Open Access Journals (Sweden)

    Javier Fernández

    2014-05-01

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

  9. Biosynthetic infochemical communication.

    Science.gov (United States)

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

    2015-07-09

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

  10. Minimum Information about a Biosynthetic Gene cluster

    NARCIS (Netherlands)

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

    2015-01-01

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

  11. Two alternative pathways for docosahexaenoic acid (DHA, 22:6n-3) biosynthesis are widespread among teleost fish.

    Science.gov (United States)

    Oboh, Angela; Kabeya, Naoki; Carmona-Antoñanzas, Greta; Castro, L Filipe C; Dick, James R; Tocher, Douglas R; Monroig, Oscar

    2017-06-20

    Docosahexaenoic acid (DHA) plays important physiological roles in vertebrates. Studies in rats and rainbow trout confirmed that DHA biosynthesis proceeds through the so-called "Sprecher pathway", a biosynthetic process requiring a Δ6 desaturation of 24:5n-3 to 24:6n-3. Alternatively, some teleosts possess fatty acyl desaturases 2 (Fads2) that enable them to biosynthesis DHA through a more direct route termed the "Δ4 pathway". In order to elucidate the prevalence of both pathways among teleosts, we investigated the Δ6 ability towards C24 substrates of Fads2 from fish with different evolutionary and ecological backgrounds. Subsequently, we retrieved public databases to identify Fads2 containing the YXXN domain responsible for the Δ4 desaturase function, and consequently enabling these species to operate the Δ4 pathway. We demonstrated that, with the exception of Δ4 desaturases, fish Fads2 have the ability to operate as Δ6 desaturases towards C24 PUFA enabling them to synthesise DHA through the Sprecher pathway. Nevertheless, the Δ4 pathway represents an alternative route in some teleosts and we identified the presence of putative Δ4 Fads2 in a further 11 species and confirmed the function as Δ4 desaturases of Fads2 from medaka and Nile tilapia. Our results demonstrated that two alternative pathways for DHA biosynthesis exist in teleosts.

  12. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods

    Science.gov (United States)

    Sofia, Heidi J.; Chen, Guang; Hetzler, Beth G.; Reyes-Spindola, Jorge F.; Miller, Nancy E.

    2001-01-01

    A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships. PMID:11222759

  13. Integrating nitric oxide into salicylic acid and jasmonic acid/ethylene plant defense pathways

    DEFF Research Database (Denmark)

    Mur, Luis A J; Prats, Elena; Pierre, Sandra

    2013-01-01

    Plant defence against pests and pathogens is known to be conferred by either salicylic acid (SA) or jasmonic acid (JA)/ethylene (ET) pathways, depending on infection or herbivore-grazing strategy. It is well attested that SA and JA/ET pathways are mutually antagonistic allowing defence responses...

  14. Regulation of L-ascorbic acid content in strawberry fruits

    National Research Council Canada - National Science Library

    Eduardo Cruz-Rus; Iraida Amaya; José F. Sánchez-Sevilla; Miguel A. Botella; Victoriano Valpuesta

    2011-01-01

    Plants have several L-ascorbic acid (AsA) biosynthetic pathways, but the contribution of each one to the synthesis of AsA varyies between different species, organs, and developmental stages. Strawberry (Fragaria×ananassa...

  15. Early lignin pathway enzymes and routes to chlorogenic acid in switchgrass (Panicum virgatum L.).

    Science.gov (United States)

    Escamilla-Treviño, Luis L; Shen, Hui; Hernandez, Timothy; Yin, Yanbin; Xu, Ying; Dixon, Richard A

    2014-03-01

    Studying lignin biosynthesis in Panicum virgatum (switchgrass) has provided a basis for generating plants with reduced lignin content and increased saccharification efficiency. Chlorogenic acid (CGA, caffeoyl quinate) is the major soluble phenolic compound in switchgrass, and the lignin and CGA biosynthetic pathways potentially share intermediates and enzymes. The enzyme hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) is responsible for CGA biosynthesis in tobacco, tomato and globe artichoke, but there are no close orthologs of HQT in switchgrass or in other monocotyledonous plants with complete genome sequences. We examined available transcriptomic databases for genes encoding enzymes potentially involved in CGA biosynthesis in switchgrass. The protein products of two hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) genes (PvHCT1a and PvHCT2a), closely related to lignin pathway HCTs from other species, were characterized biochemically and exhibited the expected HCT activity, preferring shikimic acid as acyl acceptor. We also characterized two switchgrass coumaroyl shikimate 3'-hydroxylase (C3'H) enzymes (PvC3'H1 and PvC3'H2); both of these cytochrome P450s had the capacity to hydroxylate 4-coumaroyl shikimate or 4-coumaroyl quinate to generate caffeoyl shikimate or CGA. Another switchgrass hydroxycinnamoyl transferase, PvHCT-Like1, is phylogenetically distant from HCTs or HQTs, but exhibits HQT activity, preferring quinic acid as acyl acceptor, and could therefore function in CGA biosynthesis. The biochemical features of the recombinant enzymes, the presence of the corresponding activities in plant protein extracts, and the expression patterns of the corresponding genes, suggest preferred routes to CGA in switchgrass.

  16. Hydrogen isotope analysis of amino acids and whole cells reflects biosynthetic processing of nutrient- and water-derived hydrogen

    Science.gov (United States)

    Griffin, P.; Newsome, S.; Steele, A.; Fogel, M. L.

    2011-12-01

    Hydrogen (H) isotopes serve as sensitive tracers of biochemical processes that can be exploited to answer critical questions in biogeochemistry, ecology, and microbiology. Despite this apparent utility, relatively little is known about the specific mechanisms of H isotope fractionation involved in biosynthesis. In order to understand how organisms incorporate hydrogen from their chemical milieu into biomass, we have cultured the model bacterium E. coli MG1655 in a variety of media composed of deuterium-labeled nutrients and waters. Isotopic analysis of bulk cell mass reveals that the H fractionation between media water and cell material varies as a function of the nutrient source, with commonly used organic food sources (glucose and tryptone) leading to far smaller fractionation signals than non-standard ones (such as formamide, adenine, and urea). In addition, we have completed compound specific isotope analysis of amino acids using combined GC-IRMS. Amino acids harvested from E. coli cultured on glucose in water of varied D/H composition posses an extraordinary range of isotopic compositions (400-600 %). Furthermore, these amino acids follow a systematic distribution of D/H where proline is always heaviest and glycine is always lightest. However, when the short-chain peptide tryptone is used in place of glucose, only the non-essential amino acids reflect media water D/H values, suggesting the direct incorporation of some media-borne amino acids into cellular protein. These observations provide a foundation for understanding the cellular routing of hydrogen obtained from food and water sources and indicate that D/H analysis can serve as a powerful probe of biological function.

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

    Directory of Open Access Journals (Sweden)

    Ni-Hao Jiang

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

  18. Deciphering ascorbic acid regulatory pathways in ripening tomato fruit using a weighted gene correlation network analysis approach.

    Science.gov (United States)

    Gao, Chao; Ju, Zheng; Li, Shan; Zuo, Jinhua; Fu, Daqi; Tian, Huiqin; Luo, Yunbo; Zhu, Benzhong

    2013-11-01

    Genotype is generally determined by the co-expression of diverse genes and multiple regulatory pathways in plants. Gene co-expression analysis combining with physiological trait data provides very important information about the gene function and regulatory mechanism. L-Ascorbic acid (AsA), which is an essential nutrient component for human health and plant metabolism, plays key roles in diverse biological processes such as cell cycle, cell expansion, stress resistance, hormone synthesis, and signaling. Here, we applied a weighted gene correlation network analysis approach based on gene expression values and AsA content data in ripening tomato (Solanum lycopersicum L.) fruit with different AsA content levels, which leads to identification of AsA relevant modules and vital genes in AsA regulatory pathways. Twenty-four modules were compartmentalized according to gene expression profiling. Among these modules, one negatively related module containing genes involved in redox processes and one positively related module enriched with genes involved in AsA biosynthetic and recycling pathways were further analyzed. The present work herein indicates that redox pathways as well as hormone-signal pathways are closely correlated with AsA accumulation in ripening tomato fruit, and allowed us to prioritize candidate genes for follow-up studies to dissect this interplay at the biochemical and molecular level. © 2013 Institute of Botany, Chinese Academy of Sciences.

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

    Science.gov (United States)

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

    2003-11-01

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

  20. Anthocyanin biosynthetic genes in Brassica rapa.

    Science.gov (United States)

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

    2014-06-04

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

  1. Biosynthesis of the C(7)-cyclitol moiety of acarbose in Actinoplanes species SE50/110. 7-O-phosphorylation of the initial cyclitol precursor leads to proposal of a new biosynthetic pathway.

    Science.gov (United States)

    Zhang, Chang-Sheng; Stratmann, Ansgar; Block, Oliver; Brückner, Ralph; Podeschwa, Michael; Altenbach, Hans-Josef; Wehmeier, Udo F; Piepersberg, Wolfgang

    2002-06-21

    characterized by mass spectrometric methods. The product of the AcbM-catalyzed reaction, 2-epi-5-epi-valiolone 7-phosphate, was purified on a preparative scale and identified by NMR spectroscopy. A biosynthetic pathway for the pseudodisaccharidic acarviosyl moiety of acarbose is proposed on the basis of these data.

  2. Isolation and dynamic expression of four genes involving in shikimic acid pathway in Camellia sinensis 'Baicha 1' during periodic albinism.

    Science.gov (United States)

    Zhu, Xu-Jun; Zhao, Zhen; Xin, Hua-Hong; Wang, Ming-Le; Wang, Wei-Dong; Chen, Xuan; Li, Xing-Hui

    2016-10-01

    Flavonoids are the main flavor components and functional ingredients in tea, and the shikimic acid pathway is considered as one of the most important pathways in flavonoid biosynthesis, but little was known about the function of regulatory genes in the metabolism phenolic compounds in tea plant (Camellia sinensis), especially related genes in shikimic acid pathway. The dynamic changes of catechin (predominant flavonoid) contents were analyzed in this study, and four genes (CsPPT, CsDAHPS, CsSDH and CsCS) involving in shikimic acid pathway in C. sinensis albino cultivar 'Baicha 1' were cloned and characterized. The full-length cDNA sequences of these genes were obtained using reverse transcription-PCR and rapid amplification of cDNA ends. At the albinistic stage, the amounts of all catechins decreased to the lowest levels, when epigallocatechin gallate was the highest, whereas gallocatechin-3-O-gallate the lowest. Gene expression patterns analyzed by qRT-PCR showed that CsPPT and CsDAHPS were highly expressed in flowers and buds, while CsSDH and CsCS showed high expression levels in buds and leaves. It was also found that the transcript abundance of shikimic acid biosynthetic genes followed a tightly regulated biphasic pattern, and was affected by albinism. The transcript levels of CsPPT and CsDAHPS were decreased at albinistic stage followed elevated expression, whereas CsSDH and CsCS were increased only at re-greening stage. Taken together, these findings suggested that these four genes in C. sinensis may play different roles in shikimic acid biosynthesis and these genes may have divergent functions.

  3. Amino acids as regulators and components of nonproteinogenic pathways

    NARCIS (Netherlands)

    Meijer, Alfred J.

    2003-01-01

    Amino acids are not only important precursors for the synthesis of proteins and other N-containing compounds, but also participate in the regulation of major metabolic pathways. Glutamate and aspartate, for example, are components of the malate/aspartate shuttle and their concentrations control the

  4. Ring cleavage and degradative pathway of cyanuric acid in bacteria

    OpenAIRE

    Cook, Alasdair M.; Beilstein, Paul; Grossenbacher, Hugo; Hütter, Ralf

    1985-01-01

    The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was...

  5. Expanding metabolic pathway for de novo biosynthesis of the chiral pharmaceutical intermediate L-pipecolic acid in Escherichia coli.

    Science.gov (United States)

    Ying, Hanxiao; Tao, Sha; Wang, Jing; Ma, Weichao; Chen, Kequan; Wang, Xin; Ouyang, Pingkai

    2017-03-27

    The six-carbon circular non-proteinogenic compound L-pipecolic acid is an important chiral drug intermediate with many applications in the pharmaceutical industry. In the present study, we developed a metabolically engineered strain of Escherichia coli for the overproduction of L-pipecolic acid from glucose. The metabolic pathway from L-lysine to L-pipecolic acid was constructed initially by introducing lysine cyclodeaminase (LCD). Next, L-lysine metabolic flux from glucose was amplified by the plasmid-based overexpression of dapA, lysC, and lysA under the control of the strong trc promoter to increase the biosynthetic pool of the precursor L-lysine. Additionally, since the catalytic efficiency of the key enzyme LCD is limited by the cofactor NAD(+), the intracellular pyridine nucleotide concentration was rebalanced by expressing the pntAB gene encoding the transhydrogenase, which elevated the proportion of LCD with bound NAD(+) and enhanced L-pipecolic acid production significantly. Further, optimization of Fe(2+) and surfactant in the fermentation process resulted in 5.33 g/L L-pipecolic acid, with a yield of 0.13 g/g of glucose via fed-batch cultivation. We expanded the metabolic pathway for the synthesis of the chiral pharmaceutical intermediate L-pipecolic acid in E. coli. Using the engineered E. coli, a fast and efficient fermentative production of L-pipecolic acid was achieved. This strategy could be applied to the biosynthesis of other commercially and industrially important chiral compounds containing piperidine rings.

  6. AMPK-independent pathways regulate skeletal muscle fatty acid oxidation

    DEFF Research Database (Denmark)

    Dzamko, Nicolas; Schertzer, Jonathan D.; Ryall, James G.

    2008-01-01

    The activation of AMP-activated protein kinase (AMPK) and phosphorylation/inhibition of acetyl-CoA carboxylase 2 (ACC2) is believed to be the principal pathway regulating fatty acid oxidation. However, during exercise AMPK activity and ACC Ser-221 phosphorylation does not always correlate...... with rates of fatty acid oxidation. To address this issue we have investigated the requirement for skeletal muscle AMPK in controlling aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside (AICAR) and contraction-stimulated fatty acid oxidation utilizing transgenic mice expressing a muscle-specific kinase...... dead (KD) AMPK alpha2. In wild-type (WT) mice, AICAR and contraction increased AMPK alpha2 and alpha1 activities, the phosphorylation of ACC2 and rates of fatty acid oxidation while tending to reduce malonyl-CoA levels. Despite no activation of AMPK in KD mice, ACC2 phosphorylation was maintained...

  7. Expression, Crystallization and Preliminary X-ray Diffraction Analyses of Med-ORF10 in the Biosynthetic Pathway of an Antitumor Antibiotic Medermycin.

    Science.gov (United States)

    Liu, Yanli; Liu, Shasha; Yang, Tingting; Guo, Xiaoxia; Jiang, Yali; Zahid, Kashif Rafiq; Liu, Ke; Liu, Jinlin; Yang, Jihong; Zhao, Haobin; Yang, Yi; Li, Aiying; Qi, Chao

    2015-12-01

    Medermycin, as a prominent member of benzoisochromanequinones, possesses strong antitumor activity and is biosynthesized under the control of a 29-ORF-containing biosynthetic gene cluster. Most of ORFs in this gene cluster have not been characterized, including a small protein encoding gene med-ORF10, proposed to play a regulatory role in biosynthesis of medermycin in an unknown mode. In this study, we reported the expression, protein preparation, crystallization and preliminary X-ray diffraction analyses of Med-ORF10 of the wild type Streptomyces strain. Firstly, we cloned and overexpressed med-ORF10 in Escherichia coli and purified the protein with 98% purity and 3 mg/L yield. Then, we crystallized the protein at concentration of 20 mg/mL in condition 22% PEG 3350, 0.2 M magnesium formate and collected the data at 1.78 Å resolution. Finally, we detected the expression of Med-ORF10 in Streptomyces by western blotting. In conclusion, this study confirmed the expression of Med-ORF10 protein in the wild-type strain of Streptomyces AM-7161 and collected the X-ray diffraction data of Med-ORF10 crystal at 1.78 Å resolution. These studies provide evidences for the functional Med-ORF10 protein in Streptomyces strains and facilitate our further investigation.

  8. Tissue-specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding.

    Science.gov (United States)

    Drew, Janice E; Farquharson, Andrew J; Horgan, Graham W; Williams, Lynda M

    2016-11-01

    The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Constrained Multistate Sequence Design for Nucleic Acid Reaction Pathway Engineering.

    Science.gov (United States)

    Wolfe, Brian R; Porubsky, Nicholas J; Zadeh, Joseph N; Dirks, Robert M; Pierce, Niles A

    2017-03-01

    We describe a framework for designing the sequences of multiple nucleic acid strands intended to hybridize in solution via a prescribed reaction pathway. Sequence design is formulated as a multistate optimization problem using a set of target test tubes to represent reactant, intermediate, and product states of the system, as well as to model crosstalk between components. Each target test tube contains a set of desired "on-target" complexes, each with a target secondary structure and target concentration, and a set of undesired "off-target" complexes, each with vanishing target concentration. Optimization of the equilibrium ensemble properties of the target test tubes implements both a positive design paradigm, explicitly designing for on-pathway elementary steps, and a negative design paradigm, explicitly designing against off-pathway crosstalk. Sequence design is performed subject to diverse user-specified sequence constraints including composition constraints, complementarity constraints, pattern prevention constraints, and biological constraints. Constrained multistate sequence design facilitates nucleic acid reaction pathway engineering for diverse applications in molecular programming and synthetic biology. Design jobs can be run online via the NUPACK web application.

  10. Flavonolignans inhibit the arachidonic acid pathway in blood platelets.

    Science.gov (United States)

    Bijak, Michal; Saluk-Bijak, Joanna

    2017-08-10

    Arachidonic acid metabolism by cyclooxygenase (COX) is a major pathway for blood platelets' activation, which is associated with pro-thrombotic platelet activity and the production of pro-inflammatory mediators. Inhibition of COX activity is one of the major means of anti-platelet pharmacotherapy preventing arterial thrombosis and reducing the incidence of cardiovascular events. Recent studies have presented that a silymarin (standardized extract of Milk thistle (Silybum marianum)) can inhibit the COX pathway. Accordingly, the aim of our study was to determine the effects of three major flavonolignans (silybin, silychristin and silydianin) on COX pathway activity in blood platelets. We determined the effect of flavonolignans on arachidonic acid induced blood platelet aggregation, COX pathway metabolites formation, as well as COX activity in platelets. Additionally, we analysed the potential mechanism of this interaction using the bioinformatic ligand docking method. We observed that tested compounds decrease the platelet aggregation level, both thromboxane A2 and malondialdehyde formation, as well as inhibit the COX activity. The strongest effect was observed for silychristin and silybin. In our in silico study we showed that silychristin and silybin have conformations which interact with the active COX site as competitive inhibitors, blocking the possibility of substrate binding. The results obtained from this study clearly present the potential of flavonolignans as novel antiplatelet and anti-inflammatory agents.

  11. Conservation and Expression Patterns Divergence of Ascorbic Acid d-mannose/l-galactose Pathway Genes in Brassica rapa.

    Science.gov (United States)

    Duan, Weike; Ren, Jun; Li, Yan; Liu, Tongkun; Song, Xiaoming; Chen, Zhongwen; Huang, Zhinan; Hou, Xilin; Li, Ying

    2016-01-01

    Ascorbic acid (AsA) participates in diverse biological processes, is regulated by multiple factors and is a potent antioxidant and cellular reductant. The D-Mannose/L-Galactose pathway is a major plant AsA biosynthetic pathway that is highly connected within biosynthetic networks, and generally conserved across plants. Previous work has shown that, although most genes of this pathway are expressed under standard growth conditions in Brassica rapa, some paralogs of these genes are not. We hypothesize that regulatory evolution in duplicate AsA pathway genes has occurred as an adaptation to environmental stressors, and that gene retention has been influenced by polyploidation events in Brassicas. To test these hypotheses, we explored the conservation of these genes in Brassicas and their expression patterns divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A genome), B. oleracea (C genome) and B. napus (AC genome). However, the number of genes that encode the same type of enzymes varied among the three plant species. With the exception of GMP, which has nine genes, there were one to four genes that encoded the other enzymes. Moreover, we found that expression patterns divergence widely exists among these genes. (i) VTC2 and VTC5 are paralogous genes, but only VTC5 is influenced by FLC. (ii) Under light treatment, PMI1 co-regulates the AsA pool size with other D-Man/L-Gal pathway genes, whereas PMI2 is regulated only by darkness. (iii) Under NaCl, Cu(2+), MeJA and wounding stresses, most of the paralogs exhibit different expression patterns. Additionally, GME and GPP are the key regulatory enzymes that limit AsA biosynthesis in response to these treatments. In conclusion, our data support that the conservative and divergent expression patterns of D-Man/L-Gal pathway genes not only avoid AsA biosynthesis network instability but also allow B. rapa to better adapt to complex environments.

  12. Conservation and expression patterns divergence of ascorbic acid D-mannose/L-galactose pathway genes in Brassica rapa

    Directory of Open Access Journals (Sweden)

    Weike eDuan

    2016-06-01

    Full Text Available Ascorbic acid (AsA participates in diverse biological processes, is regulated by multiple factors and is a potent antioxidant and cellular reductant. The D-mannose/L-galactose pathway is a major plant AsA biosynthetic pathway that is highly connected within biosynthetic networks, and generally conserved across plants. Previous work has shown that, although most genes of this pathway are expressed under standard growth conditions in Brassica rapa, some paralogs of these genes are not. We hypothesize that regulatory evolution in duplicate AsA pathway genes has occurred as an adaptation to environmental stressors, and that gene retention has been influenced by polyploidation events in Brassicas. To test these hypotheses, we explored the conservation of these genes in Brassicas and their expression patterns divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A genome, Brassica oleracea (C genome and Brassica napus (AC genome. However, the number of genes that encode the same type of enzymes varied among the three plant species. With the exception of GMP, which has nine genes, there were one to four genes that encoded the other enzymes. Moreover, we found that expression patterns divergence widely exists among these genes. i VTC2 and VTC5 are paralogous genes, but only VTC5 is influenced by FLC. ii Under light treatment, PMI1 co-regulates the AsA pool size with other D-Man/L-Gal pathway genes, whereas PMI2 is regulated only by darkness. iii Under NaCl, Cu2+, MeJA and wounding stresses, most of the paralogs exhibit different expression patterns. Additionally, GME and GPP are the key regulatory enzymes that limit AsA biosynthesis in response to these treatments. In conclusion, our data support that the conservative and divergent expression patterns of D-Man/L-Gal pathway genes not only avoid AsA biosynthesis network instability but also allow B. rapa to better adapt to complex environments.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  14. Niacin Sensitivity and the Arachidonic Acid Pathway in Schizophrenia

    Science.gov (United States)

    Messamore, Erik; Hoffman, William F.; Yao, Jeffrey K.

    2010-01-01

    Objective Schizophrenia is associated with a blunted flush response to niacin. Since niacin-induced skin flushing is mediated by vasodilators derived from arachidonic acid (AA), we tested whether the blunted flush response to niacin is a marker of AA deficiency. Methods Eight concentrations of methylnicotinate were applied to the forearms of 20 adults with schizophrenia and 20 controls. Laser Doppler measurement of blood flow responses was used to derive values for niacin sensitivity (defined as the concentration eliciting half-maximal response, i.e., EC50 value) and efficacy (defined as the maximal evoked blood flow response). RBC membrane fatty acids were analyzed by gas chromatography. Results Niacin sensitivity and efficacy were reduced in schizophrenia. In the control group, there was significant correlation between AA levels and niacin sensitivity as well as a trend toward correlation between AA levels and niacin efficacy. In contrast, neither sensitivity nor efficacy of niacin correlated with AA levels in schizophrenia. An expected correlation between the levels of AA and its elongation product (adrenic acid) was absent in schizophrenia. Adrenic acid levels correlated with niacin efficacy in schizophrenia. Conclusions The schizophrenia-associated niacin response abnormality involves both diminished sensitivity and reduced efficacy. The lack of expected correlation between levels of AA and adrenic acid suggests homeostatic imbalance within the n-6 polyunsaturated fatty acid (PUFA) pathway in schizophrenia. Though AA levels were unrelated to measures of niacin response in schizophrenia, the correlation between adrenic acid and niacin efficacy in schizophrenia suggests relevance of the n-6 PUFA pathway to the blunted niacin response. PMID:20417059

  15. γ-Aminobutyric Acid Pathway and Modified Tricarboxylic Acid Cycle Activity During Growth and Sporulation of Bacillus thuringiensis

    Science.gov (United States)

    Aronson, John N.; Borris, David P.; Doerner, Jeffrey F.; Akers, Evelyn

    1975-01-01

    Enzymatic analyses of Bacillus thuringiensis extracts suggested that a modified Krebs tricarboxylic acid cycle (without α-ketoglutarate dehydrogenase) can operate during sporulation in conjunction with the glyoxylic acid cycle and the γ-aminobutyric acid pathway. PMID:1180554

  16. Structure of the Bifunctional Acyltransferase/Decarboxylase LnmK from the Leinamycin Biosynthetic Pathway Revealing Novel Activity for a Double-Hot-Dog Fold

    Energy Technology Data Exchange (ETDEWEB)

    Lohman, Jeremy R. [Scripps Research Inst., Jupiter, FL (United States); Bingman, Craig A. [Univ. of Wisconsin, Madison, WI (United States); Phillips Jr., George N. [Univ. of Wisconsin, Madison, WI (United States); Rice Univ., Houston, TX (United States); Shen, Ben [Scripps Research Inst., Jupiter, FL (United States)

    2013-01-15

    The β-branched C3 unit in leinamycin biosynthesis is installed by a set of four proteins, LnmFKLM. In vitro biochemical investigation confirmed that LnmK is a bifunctional acyltransferase/decarboxylase (AT/DC) that catalyzes first self-acylation using methylmalonyl-CoA as a substrate and subsequently transacylation of the methylmalonyl group to the phosphopantetheinyl group of the LnmL acyl carrier protein [Liu, T., Huang, Y., and Shen, B. (2009) J. Am. Chem. Soc. 131, 6900–6901]. LnmK shows no sequence homology to proteins of known function, representing a new family of AT/DC enzymes. Here we report the X-ray structure of LnmK. LnmK is homodimer with each of the monomers adopting a double-hot-dog fold. Cocrystallization of LnmK with methylmalonyl-CoA revealed an active site tunnel terminated by residues from the dimer interface. But, to canonical AT and ketosynthase enzymes that employ Ser or Cys as an active site residue, none of these residues are found in the vicinity of the LnmK active site. Instead, three tyrosines were identified, one of which, Tyr62, was established, by site-directed mutagenesis, to be the most likely active site residue for the AT activity of LnmK. Moreover, LnmK represents the first AT enzyme that employs a Tyr as an active site residue and the first member of the family of double-hot-dog fold enzymes that displays an AT activity known to date. The LnmK structure sets the stage for probing of the DC activity of LnmK through site-directed mutagenesis. These findings highlight natural product biosynthetic machinery as a rich source of novel enzyme activities, mechanisms, and structures.

  17. Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis.

    Science.gov (United States)

    Wang, Yaya; Deng, Zixin; Qu, Xudong

    2014-01-01

    Fluorination has been widely used in chemical synthesis, but is rare in nature. The only known biological fluorination scope is represented by the fl pathway from Streptomyces cattleya that produces fluoroacetate (FAc) and 4-fluorothreonine (4-FT). Here we report the identification of a novel pathway for FAc and 4-FT biosynthesis from the actinomycetoma-causing pathogen Nocardia brasiliensis ATCC 700358. The new pathway shares overall conservation with the fl pathway in S. cattleya. Biochemical characterization of the conserved domains revealed a novel fluorinase NobA that can biosynthesize 5'-fluoro-5'-deoxyadenosine (5'-FDA) from inorganic fluoride and S-adenosyl-l-methionine (SAM). The NobA shows similar halide specificity and characteristics to the fluorination enzyme FlA of the fl pathway. Kinetic parameters for fluoride ( K m 4153 μM, k cat 0.073 min (-1)) and SAM ( K m 416 μM, k cat 0.139 min (-1)) have been determined, revealing that NobA is slightly (2.3 fold) slower than FlA. Upon sequence comparison, we finally identified a distinct loop region in the fluorinases that probably accounts for the disparity of fluorination activity.

  18. Ring cleavage and degradative pathway of cyanuric acid in bacteria.

    Science.gov (United States)

    Cook, A M; Beilstein, P; Grossenbacher, H; Hütter, R

    1985-10-01

    The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was conclusively identified. Biuret (1 mol) was converted stoichiometrically into 1.1 mol of NH4+, about 1 mol of CO2 and 1.0 mol of urea, which was conclusively identified. Urea (1 mol) was converted into 1.9 mol of NH4+ and 1.0 mol of CO2. The reactions proceeded under aerobic or anoxic conditions and were presumed to be hydrolytic. Data indicate that the same pathway occurred in another pseudomonad and a strain of Klebsiella pneumoniae.

  19. New Biosynthetic Step in the Melanin Pathway of Wangiella (Exophiala) dermatitidis: Evidence for 2-Acetyl-1,3,6,8-Tetrahydroxynaphthalene as a Novel Precursor

    Science.gov (United States)

    The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced as a pentaketide directly by an iter...

  20. Identification of Two Suppressors of CSG2 Calcium Sensitivity, SCS7 and SUR2, as Genes Encoding Hydroxylases of the Sphingolipid Biosynthetic Pathway of Saccharomyces cerevisiae

    National Research Council Canada - National Science Library

    Haak, Dale A

    1997-01-01

    .... The biochemical significance of much of this structural variability is not well understood. The sphingolipids of the yeast Saccharomyces cerevisiae undergo the a-hydroxylation of the very long chain fatty acid (VLCFA...

  1. Biosynthetic inorganic chemistry.

    Science.gov (United States)

    Lu, Yi

    2006-08-25

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

  2. Metabolic pathways leading from amino acids to vitamin B12 in Propionibacterium shermanii, and the sources of the seven methyl carbons.

    Science.gov (United States)

    Iida, Katsumi; Kajiwara, Masahiro

    2007-10-01

    The metabolic pathways leading from l-[2-13C]aspartic acid, [2-13C]glycine and l-[methyl-13C]methionine to vitamin B12 were investigated, focusing on the biosynthetic pathways leading to the aminopropanol moiety of vitamin B12 and on the role of the Shemin pathway leading to delta-aminolevulinic acid (a biosynthetic intermediate of tetrapyrrole), by means of feeding experiments with Propionibacterium shermanii in combination with 13C-NMR spectroscopy. The 13C-methylene carbons of l-[2-(13)C]aspartic acid, which is transformed to [2-13C]glycine via l-[2-13C]threonine, and [2-13C]glycine added to the culture medium served mainly to enrich the seven methyl carbons of the corrin ring through C-methylation by S-adenosyl-l-[methyl-13C]methionine derived from catabolically generated l-[methyl-13C]methionine in the presence of tetrahydrofolic acid. The results indicate that the catabolism of these amino acids predominates over pathways leading to (2R)-1-amino-2-propanol or delta-aminolevulinic acid in P. shermanii. Feeding of l-[methyl-13C]methionine efficiently enriched all seven methyl carbons. In the cases of [2-13C]glycine and l-[methyl-13C]methionine, the 13C-enrichment ratio of the methyl carbon at C-25 (the site of the first C-methylation) was less than those of the other six methyl carbons, probably due to the influence of endogenous d-glucose in P. shermanii. The almost identical 13C-enrichment ratios of the other six methyl carbons indicated that these C-methylations during vitamin B12 biosynthesis were completed before the amino acids were completely consumed. However, in the case of l-[2-13C]aspartic acid, the 13C-enrichment ratios of five methyl carbons were low and similar, whereas the last two sites of C-methylation (C-53 and C-35) were not labeled, presumably because of complete consumption of the smaller amount of added label. The ratios of 13C-incorporation into the seven methyl carbons are influenced by the conditions of amino acid feeding experiments in

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-05-20

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

  4. Intracellular hydrogen peroxide and superoxide poison 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, the first committed enzyme in the aromatic biosynthetic pathway of Escherichia coli.

    Science.gov (United States)

    Sobota, Jason M; Gu, Mianzhi; Imlay, James A

    2014-06-01

    In Escherichia coli, aromatic compound biosynthesis is the process that has shown the greatest sensitivity to hydrogen peroxide stress. This pathway has long been recognized to be sensitive to superoxide as well, but the molecular target was unknown. Feeding experiments indicated that the bottleneck lies early in the pathway, and the suppressive effects of fur mutations and manganese supplementation suggested the involvement of a metalloprotein. The 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHP synthase) activity catalyzes the first step in the pathway, and it is provided by three isozymes known to rely upon a divalent metal. This activity progressively declined when cells were stressed with either oxidant. The purified enzyme was activated more strongly by ferrous iron than by other metals, and only this metalloform could be inactivated by hydrogen peroxide or superoxide. We infer that iron is the prosthetic metal in vivo. Both oxidants displace the iron atom from the enzyme. In peroxide-stressed cells, the enzyme accumulated as an apoprotein, potentially with an oxidized cysteine residue. In superoxide-stressed cells, the enzyme acquired a nonactivating zinc ion in its active site, an apparent consequence of the repeated ejection of iron. Manganese supplementation protected the activity in both cases, which matches the ability of manganese to metallate the enzyme and to provide substantial oxidant-resistant activity. DAHP synthase thus belongs to a family of mononuclear iron-containing enzymes that are disabled by oxidative stress. To date, all the intracellular injuries caused by physiological doses of these reactive oxygen species have arisen from the oxidation of reduced iron centers.

  5. Proteolytic pathways induced by herbicides that inhibit amino acid biosynthesis.

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    Amaia Zulet

    Full Text Available 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.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.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.

  6. Minimum Information about a Biosynthetic Gene cluster : commentary

    NARCIS (Netherlands)

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

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

  7. Antimicrobial mechanism of epigallocatechin gallate and gallocatechin gallate: They target 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    Science.gov (United States)

    Hui, Xian; Hua, Shui-Hong; Wu, Qian-Qian; Li, Heng; Gao, Wen-Yun

    2017-05-15

    The catechins EGCG and GCG show a variety of pharmacological activities, especially an antibacterial capacity, but their modes of antimicrobial action have not been fully elucidated. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), the first key enzyme in the MEP pathway for terpenoid biosynthesis, is a recently validated antimicrobial target. In order to disclose the antibacterial mechanism of EGCG and GCG, the DXR inhibitory activity of them was investigated in this study. The data show that EGCG and GCG both could specifically suppress the activity of DXR, with EGCG exhibiting relatively low effect against DXR (IC 50 about 210 μM) and GCG displaying strong activity (IC 50 27.5 μM). In addition, studies on inhibition kinetics of the catechins against DXR demonstrate that they are competitive inhibitors of DXR against DXP and uncompetitive inhibitors with respect to NADPH. Meanwhile, the possible interactions between DXR and the catechine, esyth onlols were simulated via docking experiments. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway.

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    Kamran Jawed

    Full Text Available Short-chain fatty acids (SCFAs, such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.

  9. The floral transcriptome of ylang ylang (Cananga odorata var. fruticosa) uncovers biosynthetic pathways for volatile organic compounds and a multifunctional and novel sesquiterpene synthase.

    Science.gov (United States)

    Jin, Jingjing; Kim, Mi Jung; Dhandapani, Savitha; Tjhang, Jessica Gambino; Yin, Jun-Lin; Wong, Limsoon; Sarojam, Rajani; Chua, Nam-Hai; Jang, In-Cheol

    2015-07-01

    The pleasant fragrance of ylang ylang varieties (Cananga odorata) is mainly due to volatile organic compounds (VOCs) produced by the flowers. Floral scents are a key factor in plant-insect interactions and are vital for successful pollination. C. odorata var. fruticosa, or dwarf ylang ylang, is a variety of ylang ylang that is popularly grown in Southeast Asia as a small shrub with aromatic flowers. Here, we describe the combined use of bioinformatics and chemical analysis to discover genes for the VOC biosynthesis pathways and related genes. The scented flowers of C. odorata var. fruticosa were analysed by gas chromatography/mass spectrometry and a total of 49 VOCs were identified at four different stages of flower development. The bulk of these VOCs were terpenes, mainly sesquiterpenes. To identify the various terpene synthases (TPSs) involved in the production of these essential oils, we performed RNA sequencing on mature flowers. From the RNA sequencing data, four full-length TPSs were functionally characterized. In vitro assays showed that two of these TPSs were mono-TPSs. CoTPS1 synthesized four products corresponding to β-thujene, sabinene, β-pinene, and α-terpinene from geranyl pyrophosphate and CoTPS4 produced geraniol from geranyl pyrophosphate. The other two TPSs were identified as sesqui-TPSs. CoTPS3 catalysed the conversion of farnesyl pyrophosphate to α-bergamotene, whereas CoTPS2 was found to be a multifunctional and novel TPS that could catalyse the synthesis of three sesquiterpenes, β-ylangene, β-copaene, and β-cubebene. Additionally, the activities of the two sesqui-TPSs were confirmed in planta by transient expression of these TPS genes in Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  10. Volatile profiles of members of the USDA Geneva Malus Core Collection: utility in evaluation of a hypothesized biosynthetic pathway for esters derived from 2-methylbutanoate and 2-methylbutan-1-ol.

    Science.gov (United States)

    Sugimoto, Nobuko; Forsline, Philip; Beaudry, Randolph

    2015-02-25

    The volatile ester and alcohol profiles of ripening apple fruit from 184 germplasm lines in the USDA Malus Germplasm Repository at the New York Agricultural Experiment Station in Geneva, NY, USA, were evaluated. Cluster analysis suggested biochemical relationships exist between several ester classes. A strong linkage was revealed between 2-methylbutanoate, propanoate, and butanoate esters, suggesting the influence of the recently proposed "citramalic acid pathway" in apple fruit. Those lines with a high content of esters formed from 2-methylbutan-1-ol and 2-methylbutanoate (2MB) relative to straight-chain (SC) esters (high 2MB/SC ratio) exhibited a marked increase in isoleucine and citramalic acid during ripening, but those lines with a low content did not. Thus, the data were consistent with the existence of the hypothesized citramalic acid pathway and suggest that the Geneva Malus Germplasm Repository, appropriately used, could be helpful in expanding our understanding of mechanisms for fruit volatile synthesis and other aspects of secondary metabolism.

  11. The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds.

    Science.gov (United States)

    Bates, Philip D; Browse, John

    2011-11-01

    Engineering of oilseed plants to accumulate unusual fatty acids (FAs) in seed triacylglycerol (TAG) requires not only the biosynthetic enzymes for unusual FAs but also efficient utilization of the unusual FAs by the host-plant TAG biosynthetic pathways. Competing pathways of diacylglycerol (DAG) and subsequent TAG synthesis ultimately affect TAG FA composition. The membrane lipid phosphatidylcholine (PC) is the substrate for many FA-modifying enzymes (desaturases, hydroxylases, etc.) and DAG can be derived from PC for TAG synthesis. The relative proportion of PC-derived DAG versus de novo synthesized DAG utilized for TAG synthesis, and the ability of each pathway to utilize unusual FA substrates, are unknown for most oilseed plants, including Arabidopsis thaliana. Through metabolic labeling experiments we demonstrate that the relative flux of de novo DAG into the PC-derived DAG pathway versus direct conversion to TAG is ∼14/1 in wild-type Arabidopsis. Expression of the Ricinus communis FA hydroxylase reduced the flux of de novo DAG into PC by ∼70%. Synthesis of TAG directly from de novo DAG did not increase, resulting in lower total synthesis of labeled lipids. Hydroxy-FA containing de novo DAG was rapidly synthesized, but it was not efficiently accumulated or converted to PC and TAG, and appeared to be in a futile cycle of synthesis and degradation. However, FA hydroxylation on PC and conversion to DAG allowed some hydroxy-FA to accumulate in sn-2 TAG. Therefore, the flux of DAG through PC represents a major bottleneck for the accumulation of unusual FAs in TAG of transgenic Arabidopsis seeds. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

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

    Science.gov (United States)

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

    2017-04-01

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

  13. Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis

    Science.gov (United States)

    Zhao, Lishan; Chang, Wei-chen; Xiao, Youli; Liu, Hung-wen; Liu, Pinghua

    2016-01-01

    Isoprenoids are a class of natural products with more than 50,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway (the methylerythritol phosphate (MEP) pathway) and a modified mevalonate (MVA) pathway. In this review, mechanistic insights on the MEP pathway enzymes are summarized. Since many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both the MVA and MEP pathway-based synthetic biology efforts are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations. PMID:23746261

  14. Study of Arachidonic Acid Pathway in Human Bladder Tumor

    Directory of Open Access Journals (Sweden)

    Masahide Matsuyama

    2009-12-01

    Full Text Available Recent epidemiological studies and animal experiments have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs reduce the incidence of colorectal carcinoma. Cyclooxygenase (COX is the principal target of NSAIDs. COX is the first oxidase in the process of prostaglandin production from arachidonic acid. COX enzyme may be involved in the initiation and/or the promotion of tumorigenesis due to NSAIDs inhibition of COX. Lipoxygenase (LOX is also an initial enzyme in the pathway for producing leukotrienes from arachidonic acid. Similar to COX, LOX enzyme may also be involved in the initiation and/or promotion of tumorigenesis. Peroxisome proliferator activator-receptor (PPAR-γ is a ligand-activated transcriptional factor belonging to the steroid receptor superfamily. PPAR-γ plays a role in both adipocyte differentiation and tumorigenesis. PPAR-γ is one target for cell growth modulation of NSAIDs. In this review, we report the expression of COX-2, LOX and PPAR-γ in human bladder tumor tissues as well as the effects of COX-2 and LOX inhibitors and PPAR-γ ligand.

  15. Study of Arachidonic Acid Pathway in Human Bladder Tumor

    Directory of Open Access Journals (Sweden)

    Masahide Matsuyama

    2009-01-01

    Full Text Available Recent epidemiological studies and animal experiments have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs reduce the incidence of colorectal carcinoma. Cyclooxygenase (COX is the principal target of NSAIDs. COX is the first oxidase in the process of prostaglandin production from arachidonic acid. COX enzyme may be involved in the initiation and/or the promotion of tumorigenesis due to NSAIDs inhibition of COX. Lipoxygenase (LOX is also an initial enzyme in the pathway for producing leukotrienes from arachidonic acid. Similar to COX, LOX enzyme may also be involved in the initiation and/or promotion of tumorigenesis. Peroxisome proliferator activator-receptor (PPAR-γ is a ligand-activated transcriptional factor belonging to the steroid receptor superfamily. PPAR-γ plays a role in both adipocyte differentiation and tumorigenesis. PPAR-γ is one target for cell growth modulation of NSAIDs. In this review, we report the expression of COX-2, LOX and PPAR-γ in human bladder tumor tissues as well as the effects of COX-2 and LOX inhibitors and PPAR-γ ligand.

  16. Chlorination of Amino Acids: Reaction Pathways and Reaction Rates.

    Science.gov (United States)

    How, Zuo Tong; Linge, Kathryn L; Busetti, Francesco; Joll, Cynthia A

    2017-05-02

    Chlorination of amino acids can result in the formation of organic monochloramines or organic dichloramines, depending on the chlorine to amino acid ratio (Cl:AA). After formation, organic chloramines degrade into aldehydes, nitriles and N-chloraldimines. In this paper, the formation of organic chloramines from chlorination of lysine, tyrosine and valine were investigated. Chlorination of tyrosine and lysine demonstrated that the presence of a reactive secondary group can increase the Cl:AA ratio required for the formation of N,N-dichloramines, and potentially alter the reaction pathways between chlorine and amino acids, resulting in the formation of unexpected byproducts. In a detailed investigation, we report rate constants for all reactions in the chlorination of valine, for the first time, using experimental results and modeling. At Cl:AA = 2.8, the chlorine was found to first react quickly with valine (5.4 × 10 4 M -1 s -1 ) to form N-monochlorovaline, with a slower subsequent reaction with N-monochlorovaline to form N,N-dichlorovaline (4.9 × 10 2 M -1 s -1 ), although some N-monochlorovaline degraded into isobutyraldehyde (1.0 × 10 -4 s -1 ). The N,N-dichlorovaline then competitively degraded into isobutyronitrile (1.3 × 10 -4 s -1 ) and N-chloroisobutyraldimine (1.2 × 10 -4 s -1 ). In conventional drinking water disinfection, N-chloroisobutyraldimine can potentially be formed in concentrations higher than its odor threshold concentration, resulting in aesthetic challenges and an unknown health risk.

  17. Effects of EPSPS Copy Number Variation (CNV and Glyphosate Application on the Aromatic and Branched Chain Amino Acid Synthesis Pathways in Amaranthus palmeri

    Directory of Open Access Journals (Sweden)

    Manuel Fernández-Escalada

    2017-11-01

    Full Text Available A key enzyme of the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19, is the known target of the widely used herbicide glyphosate. Glyphosate resistance in Amaranthus palmeri, one of the most troublesome weeds in agriculture, has evolved through increased EPSPS gene copy number. The aim of this work was to study the pleiotropic effects of (i EPSPS increased transcript abundance due to gene copy number variation (CNV and of (ii glyphosate application on the aromatic amino acid (AAA and branched chain amino acid (BCAA synthesis pathways. Hydroponically grown glyphosate sensitive (GS and glyphosate resistant (GR plants were treated with glyphosate 3 days after treatment. In absence of glyphosate treatment, high EPSPS gene copy number had only a subtle effect on transcriptional regulation of AAA and BCAA pathway genes. In contrast, glyphosate treatment provoked a general accumulation of the transcripts corresponding to genes of the AAA pathway leading to synthesis of chorismate in both GS and GR. After chorismate, anthranilate synthase transcript abundance was higher while chorismate mutase transcription showed a small decrease in GR and remained stable in GS, suggesting a regulatory branch point in the pathway that favors synthesis toward tryptophan over phenylalanine and tyrosine after glyphosate treatment. This was confirmed by studying enzyme activities in vitro and amino acid analysis. Importantly, this upregulation was glyphosate dose dependent and was observed similarly in both GS and GR populations. Glyphosate treatment also had a slight effect on the expression of BCAA genes but no general effect on the pathway could be observed. Taken together, our observations suggest that the high CNV of EPSPS in A. palmeri GR populations has no major pleiotropic effect on the expression of AAA biosynthetic genes, even in response to glyphosate treatment. This finding supports the idea that the fitness cost associated

  18. HPLC-MS/MS analyses show that the near-Starchless aps1 and pgm leaves accumulate wild type levels of ADPglucose: further evidence for the occurrence of important ADPglucose biosynthetic pathway(s alternative to the pPGI-pPGM-AGP pathway.

    Directory of Open Access Journals (Sweden)

    Abdellatif Bahaji

    Full Text Available In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a involves plastidic phosphoglucomutase (pPGM, ADPglucose (ADPG pyrophosphorylase (AGP and starch synthase (SS, and (b is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI. This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b pPGM and AGP are not major determinants of intracellular ADPG content, and (c the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.

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

    Science.gov (United States)

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

    2015-07-03

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

  20. Integrated Interactive Chart as a Tool for Teaching Metabolic Pathways

    Science.gov (United States)

    Kalogiannis, Stavros; Pagkalos, Ioannis; Koufoudakis, Panagiotis; Dashi, Ino; Pontikeri, Kyriaki; Christodoulou, Constantina

    2014-01-01

    An interactive chart of energy metabolism with didactic function, complementary to the already existing metabolic maps, located at the URL www.metpath.teithe.gr is being presented. The chart illustrates the major catabolic and biosynthetic pathways of glucose, fatty acids, and aminoacids, individually as well as in an integrated view. For every…

  1. AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes.

    Science.gov (United States)

    Ma, Ruifang; Xiao, Ying; Lv, Zongyou; Tan, Hexin; Chen, Ruibing; Li, Qing; Chen, Junfeng; Wang, Yun; Yin, Jun; Zhang, Lei; Chen, Wansheng

    2017-01-01

    Lignans, such as lariciresinol and its derivatives, have been identified as effective antiviral ingredients in Isatis indigotica. Evidence suggests that the APETALA2/ethylene response factor (AP2/ERF) family might be related to the biosynthesis of lignans in I. indigotica. However, the special role played by the AP2/ERF family in the metabolism and its underlying putative mechanism still need to be elucidated. One novel AP2/ERF gene, named Ii049, was isolated and characterized from I. indigotica in this study. The quantitative real-time PCR analysis revealed that Ii049 was expressed highest in the root and responded to methyl jasmonate, salicylic acid (SA) and abscisic acid treatments to various degrees. Subcellular localization analysis indicated that Ii049 protein was localized in the nucleus. Knocking-down the expression of Ii049 caused a remarkable reduction of lignan/lignin contents and transcript levels of genes involved in the lignan/lignin biosynthetic pathway. Ii049 bound to the coupled element 1, RAV1AAT and CRTAREHVCBF2 motifs of genes IiPAL and IiCCR, the key structural genes in the lignan/lignin pathway. Furthermore, Ii049 was also essential for SA biosynthesis, and SA induced lignan accumulation in I. indigotica. Notably, the transgenic I. indigotica hairy roots overexpressing Ii049 showed high expression levels of lignan/lignin biosynthetic genes and SA content, resulting in significant accumulation of lignan/lignin. The best-engineered line (OVX049-10) produced 425.60 μg·g(-1) lariciresinol, an 8.3-fold increase compared with the wild type production. This study revealed the function of Ii049 in regulating lignan/lignin biosynthesis, which had the potential to increase the content of valuable lignan/lignin in economically significant medicinal plants.

  2. AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes

    Directory of Open Access Journals (Sweden)

    Ruifang Ma

    2017-08-01

    Full Text Available Lignans, such as lariciresinol and its derivatives, have been identified as effective antiviral ingredients in Isatis indigotica. Evidence suggests that the APETALA2/ethylene response factor (AP2/ERF family might be related to the biosynthesis of lignans in I. indigotica. However, the special role played by the AP2/ERF family in the metabolism and its underlying putative mechanism still need to be elucidated. One novel AP2/ERF gene, named Ii049, was isolated and characterized from I. indigotica in this study. The quantitative real-time PCR analysis revealed that Ii049 was expressed highest in the root and responded to methyl jasmonate, salicylic acid (SA and abscisic acid treatments to various degrees. Subcellular localization analysis indicated that Ii049 protein was localized in the nucleus. Knocking-down the expression of Ii049 caused a remarkable reduction of lignan/lignin contents and transcript levels of genes involved in the lignan/lignin biosynthetic pathway. Ii049 bound to the coupled element 1, RAV1AAT and CRTAREHVCBF2 motifs of genes IiPAL and IiCCR, the key structural genes in the lignan/lignin pathway. Furthermore, Ii049 was also essential for SA biosynthesis, and SA induced lignan accumulation in I. indigotica. Notably, the transgenic I. indigotica hairy roots overexpressing Ii049 showed high expression levels of lignan/lignin biosynthetic genes and SA content, resulting in significant accumulation of lignan/lignin. The best-engineered line (OVX049-10 produced 425.60 μg·g−1 lariciresinol, an 8.3-fold increase compared with the wild type production. This study revealed the function of Ii049 in regulating lignan/lignin biosynthesis, which had the potential to increase the content of valuable lignan/lignin in economically significant medicinal plants.

  3. Retinoic acid activates two pathways required for meiosis in mice.

    Directory of Open Access Journals (Sweden)

    Jana Koubova

    2014-08-01

    Full Text Available In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA, the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function.

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

    Science.gov (United States)

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

    2017-08-01

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

  5. Elucidation of Enzymatic Mechanism of Phenazine Biosynthetic Protein PhzF Using QM/MM and MD Simulations.

    Directory of Open Access Journals (Sweden)

    Fei Liu

    Full Text Available The phenazine biosynthetic pathway is of considerable importance for the pharmaceutical industry. The pathway produces two products: phenazine-1,6-dicarboxylic acid and phenazine-1-carboxylic acid. PhzF is an isomerase that catalyzes trans-2,3-dihydro-3-hydroxyanthranilic acid isomerization and plays an essential role in the phenazine biosynthetic pathway. Although the PhzF crystal structure has been determined recently, an understanding of the detailed catalytic mechanism and the roles of key catalytic residues are still lacking. In this study, a computational strategy using a combination of molecular modeling, molecular dynamics simulations, and quantum mechanics/molecular mechanics simulations was used to elucidate these important issues. The Apo enzyme, enzyme-substrate complexes with negatively charged Glu45, enzyme-transition state analog inhibitor complexes with neutral Glu45, and enzyme-product complexes with negatively charged Glu45 structures were optimized and modeled using a 200 ns molecular dynamics simulation. Residues such as Gly73, His74, Asp208, Gly212, Ser213, and water, which play important roles in ligand binding and the isomerization reaction, were comprehensively investigated. Our results suggest that the Glu45 residue at the active site of PhzF acts as a general base/acid catalyst during proton transfer. This study provides new insights into the detailed catalytic mechanism of PhzF and the results have important implications for PhzF modification.

  6. In vitro reconstitution of an abscisic acid signalling pathway

    KAUST Repository

    Fujii, Hiroaki

    2009-11-18

    The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants; it has critical functions in stress resistance and in growth and development. Several proteins have been reported to function as ABA receptors, and many more are known to be involved in ABA signalling. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear. Here we show that by combining the recently identified ABA receptor PYR1 with the type 2C protein phosphatase (PP2C) ABI1, the serine/threonine protein kinase SnRK2.6/OST1 and the transcription factor ABF2/AREB1, we can reconstitute ABA-triggered phosphorylation of the transcription factor in vitro. Introduction of these four components into plant protoplasts results in ABA-responsive gene expression. Protoplast and test-tube reconstitution assays were used to test the function of various members of the receptor, protein phosphatase and kinase families. Our results suggest that the default state of the SnRK2 kinases is an autophosphorylated, active state and that the SnRK2 kinases are kept inactive by the PP2Cs through physical interaction and dephosphorylation. We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway. © 2009 Macmillan Publishers Limited. All rights reserved.

  7. Biochemical characterization of dTDP-D-Qui4N and dTDP-D-Qui4NAc biosynthetic pathways in Shigella dysenteriae type 7 and Escherichia coli O7.

    Science.gov (United States)

    Wang, Ying; Xu, Yanli; Perepelov, Andrei V; Qi, Yuanyuan; Knirel, Yuriy A; Wang, Lei; Feng, Lu

    2007-12-01

    O-antigen variation due to the presence of different types of sugars and sugar linkages is important for the survival of bacteria threatened by host immune systems. The O antigens of Shigella dysenteriae type 7 and Escherichia coli O7 contain 4-(N-acetylglycyl)amino-4,6-dideoxy-d-glucose (d-Qui4NGlyAc) and 4-acetamido-4,6-dideoxy-d-glucose (d-Qui4NAc), respectively, which are sugars not often found in studied polysaccharides. In this study, we characterized the biosynthetic pathways for dTDP-d-Qui4N and dTDP-d-Qui4NAc (the nucleotide-activated precursors of d-Qui4NGlyAc and d-Qui4NAc in O antigens). Predicted genes involved in the synthesis of the two sugars were cloned, and the gene products were overexpressed and purified as His-tagged fusion proteins. In vitro enzymatic reactions were carried out using the purified proteins, and the reaction products were analyzed by capillary electrophoresis, electrospray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. It is shown that in S. dysenteriae type 7 and E. coli O7, dTDP-d-Qui4N is synthesized from alpha-d-glucose-1-phosphate in three reaction steps catalyzed by glucose-1-phosphate thymidyltransferase (RmlA), dTDP-d-glucose 4,6-dehydratase (RmlB), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (VioA). An additional acetyltransferase (VioB) catalyzes the conversion of dTDP-d-Qui4N into dTDP-d-Qui4NAc in E. coli O7. Kinetic parameters and some other properties of VioA and VioB are described and differences between VioA proteins from S. dysenteriae type 7 (VioA(D7)) and E. coli O7 (VioA(O7)) discussed. To our knowledge, this is the first time that functions of VioA and VioB have been biochemically characterized. This study provides valuable enzyme sources for the production of dTDP-d-Qui4N and dTDP-d-Qui4NAc, which are potentially useful in the pharmaceutical industry for drug development.

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

    Indian Academy of Sciences (India)

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

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

    Science.gov (United States)

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

    2012-01-01

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

  10. Regulation of L-ascorbic acid content in strawberry fruits

    OpenAIRE

    Cruz-Rus, Eduardo; Amaya, Iraida; José F Sánchez-Sevilla; Botella, Miguel A; Valpuesta, Victoriano

    2011-01-01

    Plants have several L-ascorbic acid (AsA) biosynthetic pathways, but the contribution of each one to the synthesis of AsA varyies between different species, organs, and developmental stages. Strawberry (Fragaria×ananassa) fruits are rich in AsA. The pathway that uses D-galacturonate as the initial substrate is functional in ripe fruits, but the contribution of other pathways to AsA biosynthesis has not been studied. The transcription of genes encoding biosynthetic enzymes such as D-galacturon...

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

    Directory of Open Access Journals (Sweden)

    Valerio Mori

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

  12. Caffeic Acid Induces Apoptosis in Human Cervical Cancer Cells Through the Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Wei-Chun Chang

    2010-12-01

    Conclusion: Caffeic acid induces apoptosis by inhibiting Bcl-2 activity, leading to release of cytochrome c and subsequent activation of caspase-3, indicating that caffeic acid induces apoptosis via the mitochondrial apoptotic pathway. This also suggests that caffeic acid has a strong anti-tumor effect and may be a promising chemopreventive or chemotherapeutic agent.

  13. Kinetics and reaction pathways of total acid number reduction of cyclopentane carboxylic acid using subcritical methanol

    Directory of Open Access Journals (Sweden)

    Mandal Pradip C.

    2016-09-01

    Full Text Available Cyclopentane carboxylic acid (CPCA is a model compound of Naphthenic acids (NAs. This objective of this paper is to discover total acid number (TAN reduction kinetics and pathways of the reaction between CAPA and subcritical methanol (SubC-MeOH. The experiments were carried out in an autoclave reactor at temperatures of 180-220°C, a methanol partial pressure (MPP of 3 MPa, reaction times of 0-30 min and CPCA initial gas phase concentrations of 0.016-0.04 g/mL. TAN content of the samples were analyzed using ASTM D 974 techniques. The reaction products were identified and quantified with the help of GC/MS and GC-FID respectively. Experimental results reveal that TAN removal kinetics followed first order kinetics with an activation energy of 13.97 kcal/mol and a pre-exponential factor of 174.21 s-1. Subcritical methanol is able to reduce TAN of CPCA decomposing CPCA into new compounds such as cyclopentane, formaldehyde, methyl acetate and 3-pentanol.

  14. Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

    DEFF Research Database (Denmark)

    Liu, Chengwei; Tagami, Koichi; Minami, Atsushi

    2015-01-01

    KULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products....

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

    Science.gov (United States)

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

    2015-09-10

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

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

    Science.gov (United States)

    2011-01-01

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

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

    Science.gov (United States)

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

    1998-01-01

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

  18. Retrobiosynthetic study of salicylic acid in Catharanthus roseus cell suspension cultures

    NARCIS (Netherlands)

    Mustafa, Natali Rianika

    2007-01-01

    Salicylic acid (SA) is an important signal compound in systemic acquired resistance in plants. The level of this C6C1 compound in plants increases after a pathogenic attack. There are two biosynthetic pathways of SA, the phenylalanine pathway, which is thought to occur in plants, and the

  19. A plant thiolase involved in benzoic acid biosynthesis and volatile benzenoid production

    NARCIS (Netherlands)

    Van Moerkercke, A.; Schauvinhold, I.; Pichersky, E.; Haring, M.A.; Schuurink, R.C.

    2009-01-01

    The exact biosynthetic pathways leading to benzoic acid (BA) formation in plants are not known, but labeling experiments indicate the contribution of both β-oxidative and non-β-oxidative pathways. In Petunia hybrida BA is a key precursor for the production of volatile benzenoids by its flowers.

  20. Metabolic engineering of the fungal D-galacturonate pathway for L-ascorbic acid production.

    Science.gov (United States)

    Kuivanen, Joosu; Penttilä, Merja; Richard, Peter

    2015-01-08

    Synthetic L-ascorbic acid (vitamin C) is widely used as a preservative and nutrient in food and pharmaceutical industries. In the current production method, D-glucose is converted to L-ascorbic acid via several biochemical and chemical steps. The main source of L-ascorbic acid in human nutrition is plants. Several alternative metabolic pathways for L-ascorbic acid biosynthesis are known in plants. In one of them, D-galacturonic acid is the precursor. D-Galacturonic acid is also the main monomer in pectin, a plant cell wall polysaccharide. Pectin is abundant in biomass and is readily available from several waste streams from fruit and sugar processing industries. In the present work, we engineered the filamentous fungus Aspergillus niger for the conversion of D-galacturonic acid to L-ascorbic acid. In the generated pathway, the native D-galacturonate reductase activity was utilized while the gene coding for the second enzyme in the fungal D-galacturonic acid pathway, an L-galactonate consuming dehydratase, was deleted. Two heterologous genes coding for enzymes from the plant L-ascorbic acid pathway--L-galactono-1,4-lactone lactonase from Euglena gracilis (EgALase) and L-galactono-1,4-lactone dehydrogenase from Malpighia glabra (MgGALDH)--were introduced into the A. niger strain. Alternatively, an unspecific L-gulono-1,4-lactone lactonase (smp30) from the animal L-ascorbic acid pathway was introduced in the fungal strain instead of the plant L-galactono-1,4-lactone lactonase. In addition, a strain with the production pathway inducible with D-galacturonic acid was generated by using a bidirectional and D-galacturonic acid inducible promoter from the fungus. Even though, the lactonase enzyme activity was not observed in the resulting strains, they were capable of producing L-ascorbic acid from pure D-galacturonic acid or pectin-rich biomass in a consolidated bioprocess. Product titers up to 170 mg/l were achieved. In the current study, an L-ascorbic acid pathway using

  1. Sorbic acid stress activates the Candida glabrata high osmolarity glycerol MAP kinase pathway

    Directory of Open Access Journals (Sweden)

    Zeljkica eJandric

    2013-11-01

    Full Text Available Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in Candida glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologues CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species, and required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.

  2. Sorbic acid stress activates the Candida glabrata high osmolarity glycerol MAP kinase pathway.

    Science.gov (United States)

    Jandric, Zeljkica; Gregori, Christa; Klopf, Eva; Radolf, Martin; Schüller, Christoph

    2013-01-01

    Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in C. glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologs CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG) pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species and it required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.

  3. Dual responsive physical networks from asymmetric biosynthetic triblock copolymers

    NARCIS (Netherlands)

    Pham, T.H.T.

    2013-01-01

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

  4. De novo transcriptome assembly and characterization of nine tissues of Lonicera japonica to identify potential candidate genes involved in chlorogenic acid, luteolosides, and secoiridoid biosynthesis pathways.

    Science.gov (United States)

    Rai, Amit; Kamochi, Hidetaka; Suzuki, Hideyuki; Nakamura, Michimi; Takahashi, Hiroki; Hatada, Tomoki; Saito, Kazuki; Yamazaki, Mami

    2017-01-01

    Lonicera japonica is one of the most important medicinal plants with applications in traditional Chinese and Japanese medicine for thousands of years. Extensive studies on the constituents of L. japonica extracts have revealed an accumulation of pharmaceutically active metabolite classes, such as chlorogenic acid, luteolin and other flavonoids, and secoiridoids, which impart characteristic medicinal properties. Despite being a rich source of pharmaceutically active metabolites, little is known about the biosynthetic enzymes involved, and their expression profile across different tissues of L. japonica. In this study, we performed de novo transcriptome assembly for L. japonica, representing transcripts from nine different tissues. A total of 22 Gbps clean RNA-seq reads from nine tissues of L. japonica were used, resulting in 243,185 unigenes, with 99,938 unigenes annotated based on a homology search using blastx against the NCBI-nr protein database. Unsupervised principal component analysis and correlation studies using transcript expression data from all nine tissues of L. japonica showed relationships between tissues, explaining their association at different developmental stages. Homologs for all genes associated with chlorogenic acid, luteolin, and secoiridoid biosynthesis pathways were identified in the L. japonica transcriptome assembly. Expression of unigenes associated with chlorogenic acid was enriched in stems and leaf-2, unigenes from luteolin were enriched in stems and flowers, while unigenes from secoiridoid metabolic pathways were enriched in leaf-1 and shoot apex. Our results showed that different tissues of L. japonica are enriched with sets of unigenes associated with specific pharmaceutically important metabolic pathways and, therefore, possess unique medicinal properties. The present study will serve as a resource for future attempts for functional characterization of enzyme coding genes within key metabolic processes.

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

    Science.gov (United States)

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

    2011-01-21

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

  6. Precipitation pathways for ferrihydrite formation in acidic solutions

    DEFF Research Database (Denmark)

    Zhu, Mengqiang; Frandsen, Cathrine; Wallace, Adam F.

    2016-01-01

    Iron oxides and oxyhydroxides form via Fe3+ hydrolysis and polymerization in many aqueous environments, but the pathway from Fe3+ monomers to oligomers and then to solid phase nuclei is unknown. In this work, using combined X-ray, UV-vis, and Mossbauer spectroscopic approaches, we were able to id...

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

    Science.gov (United States)

    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...

  8. Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation.

    Science.gov (United States)

    Kandasamy, Vijayalakshmi; Vaidyanathan, Hema; Djurdjevic, Ivana; Jayamani, Elamparithi; Ramachandran, K B; Buckel, Wolfgang; Jayaraman, Guhan; Ramalingam, Subramanian

    2013-02-01

    Fermentation-derived products are in greater demand to meet the increasing global market as well as to overcome environmental problems. In this work, Escherichia coli has been metabolically engineered with acrylate pathway genes from Clostridium propionicum for the conversion of D-lactic acid to propionic acid. The introduced synthetic pathway consisted of seven genes encoding the enzymes propionate CoA-transferase (Pct), lactoyl-CoA dehydratase (Lcd) and acryloyl-CoA reductase (Acr). The engineered strain synthesised propionic acid at a concentration of 3.7 ± 0.2 mM upon fermentation on glucose. This low production level could be attributed to the low activity of the recombinant enzymes in particular the rate-limiting enzyme, Acr. Interestingly, the recombinant pathway caused an increased lactate production in E. coli with a yield of 1.9 mol/mol of glucose consumed along with a decrease in other by-products. Down-regulation of the pfl (pyruvate formate lyase) genes and a possible inhibition of Pfl activity by the acrylate pathway intermediate, acryloyl-CoA, could have reduced carbon flow to the Pfl pathway with a concomitant increase in lactate production. This study reports a novel way of synthesising propionic acid by employing a non-native, user-friendly organism through metabolic engineering.

  9. Linking fungal secondary metabolites and pathways to their genes in Aspergillus

    DEFF Research Database (Denmark)

    Petersen, Lene Maj

    . oryzae metabolites, however, revealed the chemical link between the two species. In two parallel projects, involving A. niger and A. aculeatus respectively, the polyketide 6-methyl salicylic acid (6-MSA), and corresponding biosynthetic pathways, were investigated. In A. niger, 6-MSA was converted...

  10. Decreased Biosynthesis of Jasmonic Acid via Lipoxygenase Pathway Compromised Caffeine-Induced Resistance to Colletotrichum gloeosporioides Under Elevated CO2 in Tea Seedlings.

    Science.gov (United States)

    Li, Xin; Ahammed, Golam Jalal; Li, Zhixin; Tang, Meijun; Yan, Peng; Han, Wenyan

    2016-11-01

    Caffeine, the major purine alkaloid in tea has long been known for its role in plant defense. However, its effect on Colletotrichum gloeosporioides that causes brown blight disease in tea is largely unknown especially under elevated CO2. Here we show that elevated CO2 reduced endogenous caffeine content in tea leaves, but sharply increased susceptibility of tea to C. gloeosporioides. The expression of C. gloeosporioides actin gene was gradually increased during the postinoculation period. In contrast, foliar application of caffeine decreased the C. gloeosporioides-induced necrotic lesions and the expression of C. gloeosporioides actin. Analysis of endogenous jasmonic acid (JA) content revealed that exogenous caffeine could induce JA content under both CO2 conditions in absence of fungal infection; however, in presence of fungal infection, caffeine increased JA content only under elevated CO2. Furthermore, exogenous caffeine enhanced lipoxygenase (LOX) activity and its biosynthetic gene expression under both CO2 conditions, indicating that increased JA biosynthesis via LOX pathway by caffeine might strengthen plant defense only under elevated CO2, while caffeine-induced defense under ambient CO2 might be associated with JA-independent LOX pathway in tea. These results provide novel insights into caffeine-induced plant defense mechanisms that might help to develop an eco-friendly approach for disease control.

  11. Nucleic acid sensing and innate immunity: signaling pathways controlling viral pathogenesis and autoimmunity

    Science.gov (United States)

    Ahlers, Laura R. H.; Goodman, Alan G.

    2016-01-01

    Innate immunity refers to the body’s initial response to curb infection upon exposure to invading organisms. While the detection of pathogen-associated molecules is an ancient form of host defense, if dysfunctional, autoimmune disease may result. The innate immune response during pathogenic infection is initiated through the activation of receptors recognizing conserved molecular patterns, such as nucleic acids from a virus’ genome or replicative cycle. Additionally, the host’s own nucleic acids are capable of activating an immune response. Therefore, it follows that the nucleic acid-sensing pathways must be tightly controlled to avoid an autoimmune response from recognition of self, yet still be unimpeded to respond to viral infections. In this review, we will describe the nucleic acid sensing pathways and how they respond to virus infection. Moreover, we will discuss autoimmune diseases that develop when these pathways fail to signal properly and identify knowledge gaps that are prime for interrogation. PMID:27857881

  12. Muconic Acid Production via Alternative Pathways and a Synthetic "Metabolic Funnel".

    Science.gov (United States)

    Thompson, Brian; Pugh, Shawn; Machas, Michael; Nielsen, David R

    2017-11-02

    Muconic acid is a promising platform biochemical and precursor to adipic acid, which can be used to synthesize various plastics and polymers. In this study, the systematic construction and comparative evaluation of a modular network of non-natural pathways for muconic acid biosynthesis was investigated in Escherichia coli, including via three distinct and novel pathways proceeding via phenol as a common intermediate. However, poor recombinant activity and high promiscuity of phenol hydroxylase ultimately limited "phenol-dependent" muconic acid production. A fourth pathway proceeding via p-hydroxybenzoate, protocatechuate, and catechol was accordingly developed, though with muconic acid titers by this route reaching just 819 mg/L, its performance lagged behind that of the established, "3-dehydroshikimiate-derived" route. Finally, these two most promising pathways were coexpressed in parallel to create a synthetic "metabolic funnel" that, by enabling maximal net precursor assimilation and flux while preserving native chorismate biosynthesis, nearly doubled muconic acid production to up to >3.1 g/L at a glucose yield of 158 mg/g while introducing only a single auxotrophy. This generalizable, "funneling" strategy is expected to have broad applications in metabolic engineering for further enhancing production of muconic acid, as well as other important bioproducts of interest.

  13. Analysis of Chlorogenic Acid Oxidation Pathway in Simulated ...

    African Journals Online (AJOL)

    properties used in traditional Chinese herbal medicine. Because it possesses a variety of biological activities, honeysuckle is widely used in pharmaceuticals, beverages, health foods, and other products. One of the major bioactive components of honeysuckle is chlorogenic acid. (CA). Phenolic compounds in plant-based ...

  14. Humic acids of vermicompost as an ecological pathway to increase ...

    African Journals Online (AJOL)

    This paper discussed the potential role of humic acids (HA) in preventing oxidative stress in rice plants submitted to water stress. The rice seedlings (Oryza sativa L. cv. IACUB-30) was grown in nutrient solution and HA were extracted from vermicompost and analysed using Fourier-transform infrared (FTIR) spectroscopic ...

  15. Parallel proton transfer pathways in aqueous acid-base reactions

    NARCIS (Netherlands)

    Cox, M.J.; Bakker, H.J.

    2008-01-01

    We study the mechanism of proton transfer (PT) between the photoacid 8-hydroxy-1,3, 6-pyrenetrisulfonic acid (HPTS) and the base chloroacetate in aqueous solution. We investigate both proton and deuteron transfer reactions in solutions with base concentrations ranging from 0.25M to 4M. Using

  16. Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae.

    Science.gov (United States)

    Weber, Heike E; Gottardi, Manuela; Brückner, Christine; Oreb, Mislav; Boles, Eckhard; Tripp, Joanna

    2017-05-15

    Biotechnological production of cis , cis -muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis , cis -muconic acid has already been established in the host organism Saccharomyces cerevisiae , the generation of industrially relevant amounts of cis , cis -muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit C iso (AroY-C iso ), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-C iso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1 , which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-C iso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-C iso , although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-C iso , suggesting that mitochondrial localization has no major impact on cofactor biosynthesis. IMPORTANCE In Saccharomyces cerevisiae , the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis , cis -muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate

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

    Science.gov (United States)

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

    2009-08-11

    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 approximately 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.

  18. Carnosic acid biosynthesis elucidated by a synthetic biology platform

    Science.gov (United States)

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

    2016-01-01

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

  19. In response to 'Can sugars be produced from fatty acids? A test case for pathway analysis tools'

    National Research Council Canada - National Science Library

    Faust, Karoline; Croes, Didier; van Helden, Jacques

    2009-01-01

    Motivation: In their article entitled 'Can sugars be produced from fatty acids? A test case for pathway analysis tools' de Figueiredo and co-authors assess the performance of three pathway prediction tools...

  20. Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics.

    Science.gov (United States)

    Price, Morgan N; Zane, Grant M; Kuehl, Jennifer V; Melnyk, Ryan A; Wall, Judy D; Deutschbauer, Adam M; Arkin, Adam P

    2018-01-01

    For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

  1. Engineering the fatty acid metabolic pathway in Saccharomyces cerevisiae for advanced biofuel production

    Directory of Open Access Journals (Sweden)

    Xiaoling Tang

    2015-12-01

    Full Text Available Fatty acid-derived fuels and chemicals have attracted a great deal of attention in recent decades, due to their following properties of high compatibility to gasoline-based fuels and existing infrastructure for their direct utilization, storage and distribution. The yeast Saccharomyces cerevisiae is the ideal biofuel producing candidate, based on the wealth of available genetic information and versatile tools designed to manipulate its metabolic pathways. Engineering the fatty acid metabolic pathways in S. cerevisiae is an effective strategy to increase its fatty acid biosynthesis and provide more pathway precursors for production of targeted products. This review summarizes the recent progress in metabolic engineering of yeast cells for fatty acids and fatty acid derivatives production, including the regulation of acetyl-CoA biosynthesis, NADPH production, fatty acid elongation, and the accumulation of activated precursors of fatty acids for converting enzymes. By introducing specific enzymes in the engineered strains, a powerful platform with a scalable, controllable and economic route for advanced biofuel production has been established.

  2. Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis.

    Science.gov (United States)

    Yoon, Mee-Sup; Chen, Jie

    2013-12-01

    Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner. Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.

  3. Reduction of nitrobenzene with alkaline ascorbic acid: Kinetics and pathways

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Chenju, E-mail: cliang@nchu.edu.tw [Department of Environmental Engineering, National Chung Hsing University 250, Kuo-kuang Road, Taichung 402, Taiwan (China); Lin, Ya-Ting [Department of Environmental Engineering, Chung Yuan Christian University, 200 Chung Pei Road, Chung Li District, Taoyuan City 320, Taiwan (China); Shiu, Jia-Wei [Department of Environmental Engineering, National Chung Hsing University 250, Kuo-kuang Road, Taichung 402, Taiwan (China)

    2016-01-25

    Highlights: • Alkaline ascorbic acid (a.k.a. vitamin C) is capable of reductively degrading NB. • The pH above the pK{sub a2} of ascorbic acid increases reductive electron transfer to NB. • The rate equation for the reactions between NB and AA is determined. • NSB, AZOXY, and AZO are identified as intermediates and aniline as a final product. • Alkaline pH is essential for AA remediation of NB contaminated soils. - Abstract: Alkaline ascorbic acid (AA) exhibits the potential to reductively degrade nitrobenzene (NB), which is the simplest of the nitroaromatic compounds. The nitro group (NO{sub 2}{sup −}) of NB has a +III oxidation state of the N atom and tends to gain electrons. The effect of alkaline pH ranging from 9 to 13 was initially assessed and the results demonstrated that the solution pH, when approaching or above the pK{sub a2} of AA (11.79), would increase reductive electron transfer to NB. The rate equation for the reactions between NB and AA at pH 12 can be described as r = ((0.89 ± 0.11) × 10{sup −4} mM{sup 1−(a} {sup +} {sup b)} h{sup −1}) × [NB]{sup a} {sup =} {sup 1.35} {sup ±} {sup 0.10}[AA]{sup b} {sup =} {sup 0.89} {sup ±} {sup 0.01}. The GC/MS analytical method identified nitrosobenzene, azoxybenzene, and azobenzene as NB reduction intermediates, and aniline (AN) as a final product. These experimental results indicate that the alkaline AA reduction of NB to AN mainly proceeds via the direct route, consisting of a series of two-electron or four-electron transfers, and the condensation reaction plays a minor route. Preliminary evaluation of the remediation of spiked NB contaminated soils revealed that maintenance of alkaline pH and a higher water to soil ratio are essential for a successful alkaline AA application.

  4. Lysophosphatidic acid targets vascular and oncogenic pathways via RAGE signaling

    Science.gov (United States)

    Touré, Fatouma; Chitayat, Seth; Pei, Renjun; Song, Fei; Li, Qing; Zhang, Jinghua; Rosario, Rosa; Ramasamy, Ravichandran; Chazin, Walter J.

    2012-01-01

    The endogenous phospholipid lysophosphatidic acid (LPA) regulates fundamental cellular processes such as proliferation, survival, motility, and invasion implicated in homeostatic and pathological conditions. Hence, delineation of the full range of molecular mechanisms by which LPA exerts its broad effects is essential. We report avid binding of LPA to the receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily, and mapping of the LPA binding site on this receptor. In vitro, RAGE was required for LPA-mediated signal transduction in vascular smooth muscle cells and C6 glioma cells, as well as proliferation and migration. In vivo, the administration of soluble RAGE or genetic deletion of RAGE mitigated LPA-stimulated vascular Akt signaling, autotaxin/LPA-driven phosphorylation of Akt and cyclin D1 in the mammary tissue of transgenic mice vulnerable to carcinogenesis, and ovarian tumor implantation and development. These findings identify novel roles for RAGE as a conduit for LPA signaling and suggest targeting LPA–RAGE interaction as a therapeutic strategy to modify the pathological actions of LPA. PMID:23209312

  5. Reduction of nitrobenzene with alkaline ascorbic acid: Kinetics and pathways.

    Science.gov (United States)

    Liang, Chenju; Lin, Ya-Ting; Shiu, Jia-Wei

    2016-01-25

    Alkaline ascorbic acid (AA) exhibits the potential to reductively degrade nitrobenzene (NB), which is the simplest of the nitroaromatic compounds. The nitro group (NO2(-)) of NB has a +III oxidation state of the N atom and tends to gain electrons. The effect of alkaline pH ranging from 9 to 13 was initially assessed and the results demonstrated that the solution pH, when approaching or above the pKa2 of AA (11.79), would increase reductive electron transfer to NB. The rate equation for the reactions between NB and AA at pH 12 can be described as r=((0.89±0.11)×10(-4) mM(1-(a+b))h(-1))×[NB](a=1.35±0.10)[AA](b=0.89±0.01). The GC/MS analytical method identified nitrosobenzene, azoxybenzene, and azobenzene as NB reduction intermediates, and aniline (AN) as a final product. These experimental results indicate that the alkaline AA reduction of NB to AN mainly proceeds via the direct route, consisting of a series of two-electron or four-electron transfers, and the condensation reaction plays a minor route. Preliminary evaluation of the remediation of spiked NB contaminated soils revealed that maintenance of alkaline pH and a higher water to soil ratio are essential for a successful alkaline AA application. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Methylseleninic acid suppresses pancreatic cancer growth involving multiple pathways.

    Science.gov (United States)

    Wang, Lei; Hu, Hongbo; Wang, Zhe; Xiong, Hua; Cheng, Yan; Liao, Joshua Dezhong; Deng, Yibin; Lü, Junxuan

    2014-01-01

    As a potential novel agent for treating pancreatic cancer, methylseleninic acid (MSeA) was evaluated in cell culture and xenograft models. Results showed that MSeA induced G1 cell cycle arrest and apoptosis in a majority of human and mouse pancreatic cancer cell lines, but G2 arrest in human PANC-1 and PANC-28 cell lines. In contrast to our previous finding in human prostate cancer LNCaP cells having a lack of P53 activation by MSeA, induction of G2 arrest in PANC-1 cells was accompanied by increased mutant P53 Ser15 phosphorylation, upregulation of P53-targets P21Cip1 and GADD45 and G2 checkpoint kinase (Chk2) activation, suggestive of DNA damage responses. A rapid inhibition of AKT phosphorylation was followed by reduced mTOR signaling and increased autophagy in PANC-1 cells attenuating caspase-mediated apoptosis execution. Furthermore, daily oral treatment with MSeA (3 mg Se/kg body weight) significantly suppressed growth of subcutaneously inoculated PANC-1 xenograft in SCID mice. Immunohistochemical analyses detected increased p-Ser15 P53, P21Cip1, pS139-H2AX (DNA damage responses), and caspase-3 cleavage and decreased pSer473AKT and Ki67 proliferative index and reduced intratumor vascular density in MSeA-treated xenograft. These results provide impetus for further research of MSeA in the therapy and/or chemoprevention of pancreatic cancer.

  7. Modulation of nonessential amino acid biosynthetic pathways in virulent Hessian fly larvae (Mayetiola destructor), feeding on susceptible host wheat (Triticum aestivum)

    Science.gov (United States)

    Hessian fly (Mayetiola destructor), an obligate plant-parasitic gall midge, is an important dipteran pest of wheat (Triticum aestivum). The insect employs an effector-based feeding strategy to reprogram the host plant to be nutritionally beneficial for the developing larva by inducing formation of p...

  8. Metabolic engineering of the mixed-acid fermentation pathway of Escherichia coli for anaerobic production of glutamate and itaconate

    National Research Council Canada - National Science Library

    Vuoristo, Kiira S; Mars, Astrid E; Sangra, Jose Vidal; Springer, Jan; Eggink, Gerrit; Sanders, Johan P.M; Weusthuis, Ruud A

    2015-01-01

    .... The purpose of this study was to establish proof of principle for an anaerobic fermentation process for the production of itaconic acid by modification of the mixed acid fermentation pathway of E. coli. E. coli BW25113 (DE3...

  9. Pimelic acid, the first precursor of the Bacillus subtilis biotin synthesis pathway, exists as the free acid and is assembled by fatty acid synthesis.

    Science.gov (United States)

    Manandhar, Miglena; Cronan, John E

    2017-05-01

    Biotin synthetic pathways are readily separated into two stages, synthesis of the seven carbon α, ω-dicarboxylic acid pimelate moiety and assembly of the fused heterocyclic rings. The biotin pathway genes responsible for pimelate moiety synthesis vary widely among bacteria whereas the ring synthesis genes are highly conserved. Bacillus subtilis seems to have redundant genes, bioI and bioW, for generation of the pimelate intermediate. Largely consistent with previous genetic studies it was found that deletion of bioW caused a biotin auxotrophic phenotype whereas deletion of bioI did not. BioW is a pimeloyl-CoA synthetase that converts pimelic acid to pimeloyl-CoA. The essentiality of BioW for biotin synthesis indicates that the free form of pimelic acid is an intermediate in biotin synthesis although this is not the case in E. coli. Since the origin of pimelic acid in Bacillus subtilis is unknown, 13 C-NMR studies were carried out to decipher the pathway for its generation. The data provided evidence for the role of free pimelate in biotin synthesis and the involvement of fatty acid synthesis in pimelate production. Cerulenin, an inhibitor of the key fatty acid elongation enzyme, FabF, markedly decreased biotin production by B. subtilis resting cells whereas a strain having a cerulenin-resistant FabF mutant produced more biotin. In addition, supplementation with pimelic acid fully restored biotin production in cerulenin-treated cells. These results indicate that pimelic acid originating from fatty acid synthesis pathway is a bona fide precursor of biotin in B. subtilis. © 2017 John Wiley & Sons Ltd.

  10. Ketol-acid reductoisomerase enzymes and methods of use

    Science.gov (United States)

    Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O'Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

    2015-10-27

    Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

  11. Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation.

    Science.gov (United States)

    Zhang, Yuan; Yan, Ya-Ping; Wu, Yu-Cui; Hua, Wen-Ping; Chen, Chen; Ge, Qian; Wang, Zhe-Zhi

    2014-01-01

    To produce beneficial phenolic acids for medical and commercial purposes, researchers are interested in improving the normally low levels of salvianolic acid B (Sal B) produced by Salvia miltiorrhiza. Here, we present a strategy of combinational genetic manipulation to enrich the precursors available for Sal B biosynthesis. This approach, involving the lignin pathway, requires simultaneous, ectopic expression of an Arabidopsis Production of Anthocyanin Pigment 1 transcription factor (AtPAP1) plus co-suppression of two endogenous, key enzyme genes: cinnamoyl-CoA reductase (SmCCR) and caffeic acid O-methyltransferase (SmCOMT). Compared with the untransformed control, we achieved a greater accumulation of Sal B (up to 3-fold higher) along with a reduced lignin concentration. This high-Sal B phenotype was stable in roots during vegetative growth and was closely correlated with increased antioxidant capacity for the corresponding plant extracts. Although no outward change in phenotype was apparent, we characterized the molecular phenotype through integrated analysis of transcriptome and metabolome profiling. Our results demonstrated the far-reaching consequences of phenolic pathway perturbations on carbohydrate metabolism, respiration, photo-respiration, and stress responses. This report is the first to describe the production of valuable end products through combinational genetic manipulation in S. miltiorrhiza plants. Our strategy will be effective in efforts to metabolically engineer multi-branch pathway(s), such as the phenylpropanoid pathway, in economically significant medicinal plants. © 2013 International Metabolic Engineering Society Published by International Metabolic Engineering Society All rights reserved.

  12. Kinetic Footprinting of an RNA-Folding Pathway Using Peroxynitrous Acid.

    Science.gov (United States)

    Chaulk; MacMillan

    2000-02-01

    Following footprints to discover a path is easier with peroxynitrous acid. The folding of the Tetrahymena ribozyme was studied using this readily available reagent to generate hydroxyl radicals for kinetic footprinting studies. The different domains of the ribozyme appear to assemble at different rates following an ordered, hierarchical pathway (see scheme).

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

    Science.gov (United States)

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

    2017-01-01

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

  14. Stromal uptake and transmission of acid is a pathway for venting cancer cell-generated acid.

    Science.gov (United States)

    Hulikova, Alzbeta; Black, Nicholas; Hsia, Lin-Ting; Wilding, Jennifer; Bodmer, Walter F; Swietach, Pawel

    2016-09-06

    Proliferation and invasion of cancer cells require favorable pH, yet potentially toxic quantities of acid are produced metabolically. Membrane-bound transporters extrude acid from cancer cells, but little is known about the mechanisms that handle acid once it is released into the poorly perfused extracellular space. Here, we studied acid handling by myofibroblasts (colon cancer-derived Hs675.T, intestinal InMyoFib, embryonic colon-derived CCD-112-CoN), skin fibroblasts (NHDF-Ad), and colorectal cancer (CRC) cells (HCT116, HT29) grown in monoculture or coculture. Expression of the acid-loading transporter anion exchanger 2 (AE2) (SLC4A2 product) was detected in myofibroblasts and fibroblasts, but not in CRC cells. Compared with CRC cells, Hs675.T and InMyoFib myofibroblasts had very high capacity to absorb extracellular acid. Acid uptake into CCD-112-CoN and NHDF-Ad cells was slower and comparable to levels in CRC cells, but increased alongside SLC4A2 expression under stimulation with transforming growth factor β1 (TGFβ1), a cytokine involved in cancer-stroma interplay. Myofibroblasts and fibroblasts are connected by gap junctions formed by proteins such as connexin-43, which allows the absorbed acid load to be transmitted across the stromal syncytium. To match the stimulatory effect on acid uptake, cell-to-cell coupling in NHDF-Ad and CCD-112-CoN cells was strengthened with TGFβ1. In contrast, acid transmission was absent between CRC cells, even after treatment with TGFβ1. Thus, stromal cells have the necessary molecular apparatus for assembling an acid-venting route that can improve the flow of metabolic acid through tumors. Importantly, the activities of stromal AE2 and connexin-43 do not place an energetic burden on cancer cells, allowing resources to be diverted for other activities.

  15. Metabolic engineering of the mixed-acid fermentation pathway of Escherichia coli for anaerobic production of glutamate and itaconate

    NARCIS (Netherlands)

    Vuoristo, Kiira S.; Mars, Astrid E.; Sangra, Jose Vidal; Springer, Jan; Eggink, Gerrit; Sanders, Johan P.M.; Weusthuis, Ruud A.

    2015-01-01

    Itaconic acid, an unsaturated C5-dicarboxylic acid, is a biobased building block for the polymer industry. The purpose of this study was to establish proof of principle for an anaerobic fermentation process for the production of itaconic acid by modification of the mixed acid fermentation pathway

  16. Effects of serum uric acid levels on the arginase pathway in women with metabolic syndrome.

    Science.gov (United States)

    Uslu, S; Ozcelik, E; Kebapci, N; Temel, H E; Demirci, F; Ergun, B; Demirustu, C

    2016-02-01

    Elevated serum uric acid levels and increased arginase activity are risk factors for cardiovascular diseases (CVD). The aim of the present study was to investigate effects of serum uric acid levels on the arginase pathway in women with metabolic syndrome (MetS). Serum arginase activity, and nitrite and uric acid levels were measured in 48 women with MetS and in 20 healthy controls. The correlation of these parameters with components of MetS was also evaluated. Our data show statistically higher arginase activity and uric acid levels but lower nitrite levels in women with MetS compared to controls. Serum uric acid levels were negatively correlated with HDL cholesterol, nitrite levels and positively with Body Mass Index, waist to hip ratio, triglyceride and total cholesterol levels, systolic blood pressure, Homeostasis Model Assessment-Insulin Resistance-Index, serum arginase activity, and LDL-cholesterol levels in women with MetS. Results of the present study suggest that serum uric acid levels may contribute to the pathogenesis of MetS through a process mediated by arginase pathway, and serum arginase activity and nitrite and uric acid levels can be used as indicators of CVD in women with MetS.

  17. Microbial effectors target multiple steps in the salicylic acid production and signaling pathway

    Directory of Open Access Journals (Sweden)

    Shigeyuki eTanaka

    2015-05-01

    Full Text Available Microbes attempting to colonize plants are recognized through the plant immune surveillance system. This leads to a complex array of global as well as specific defense responses, which are often associated with plant cell death and subsequent arrest of the invader. The responses also entail complex changes in phytohormone signaling pathways. Among these, salicylic acid signaling is an important pathway because of its ability to trigger plant cell death. As biotrophic and hemibiotrophic pathogens need to invade living plant tissue to cause disease, they have evolved efficient strategies to downregulate salicylic acid signaling by virulence effectors, which can be proteins or secondary metabolites. Here we review the strategies prokaryotic pathogens have developed to target salicylic acid biosynthesis and signaling, and contrast this with recent insights into how plant pathogenic eukaryotic fungi and oomycetes accomplish the same goal.

  18. Metabolic solutions to the biosynthesis of some diaminomonocarboxylic acids in nature: Formation in cyanobacteria of the neurotoxins 3-N-methyl-2,3-diaminopropanoic acid (BMAA) and 2,4-diaminobutanoic acid (2,4-DAB).

    Science.gov (United States)

    Nunn, Peter B; Codd, Geoffrey A

    2017-12-01

    The non-encoded diaminomonocarboxylic acids, 3-N-methyl-2,3-diaminopropanoic acid (syn: α-amino-β-methylaminopropionic acid, MeDAP; β-N-methylaminoalanine, BMAA) and 2,4-diaminobutanoic acid (2,4-DAB), are distributed widely in cyanobacterial species in free and bound forms. Both amino acids are neurotoxic in whole animal and cell-based bioassays. The biosynthetic pathway to 2,4-DAB is well documented in bacteria and in one higher plant species, but has not been confirmed in cyanobacteria. The biosynthetic pathway to BMAA is unknown. This review considers possible metabolic routes, by analogy with reactions used in other species, by which these amino acids might be biosynthesised by cyanobacteria, which are a widespread potential environmental source of these neurotoxins. Where possible, the gene expression that might be implicated in these biosyntheses is discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Characterization of the Tetrahymena ribozyme folding pathway using the kinetic footprinting reagent peroxynitrous acid.

    Science.gov (United States)

    Chaulk, S G; MacMillan, A M

    2000-01-11

    Large RNAs fold into complex structures which determine their biological activities. A full understanding of both RNA structure and dynamics will include the description of the pathways by which these structures are formed. Kinetic footprinting [Sclavi, B., et al. (1997) J. Mol. Biol. 266, 144-159] has been shown to be a powerful method for the study of dynamic processes involving RNA. Here we describe the use of a readily available reagent, peroxynitrous acid, as a kinetic footprinting tool for the study of RNA folding. Hydroxyl radicals generated from this reagent were used to footprint the Tetrahymena ribozyme during its magnesium-dependent folding-in agreement with synchroton X-ray footprinting [Sclavi, B., et al. (1998) Science 279, 1940-1943] and oligonucleotide/hybridization cleavage experiments [Zarrinkar, P. P., and Williamson, J. R. (1994) Science 265, 918-924], this work suggests an ordered, hierarchical folding pathway for the ribozyme. Several slow steps in the folding pathway were observed in the peroxynitrous acid footprinting, but none of these corresponded to the rate-determining step of folding. This suggests that the formation of the global, protected structure is followed by one or more slow local rearrangements to yield the final active structure. These studies illustrate the utility of peroxynitrous acid as a reagent for the elucidation of RNA folding pathways and the study of RNA dynamics.

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

    Science.gov (United States)

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

    2014-09-22

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

  1. Flux balance analysis of mycolic acid pathway: targets for anti-tubercular drugs.

    Directory of Open Access Journals (Sweden)

    Karthik Raman

    2005-10-01

    Full Text Available Mycobacterium tuberculosis is the focus of several investigations for design of newer drugs, as tuberculosis remains a major epidemic despite the availability of several drugs and a vaccine. Mycobacteria owe many of their unique qualities to mycolic acids, which are known to be important for their growth, survival, and pathogenicity. Mycolic acid biosynthesis has therefore been the focus of a number of biochemical and genetic studies. It also turns out to be the pathway inhibited by front-line anti-tubercular drugs such as isoniazid and ethionamide. Recent years have seen the emergence of systems-based methodologies that can be used to study microbial metabolism. Here, we seek to apply insights from flux balance analyses of the mycolic acid pathway (MAP for the identification of anti-tubercular drug targets. We present a comprehensive model of mycolic acid synthesis in the pathogen M. tuberculosis involving 197 metabolites participating in 219 reactions catalysed by 28 proteins. Flux balance analysis (FBA has been performed on the MAP model, which has provided insights into the metabolic capabilities of the pathway. In silico systematic gene deletions and inhibition of InhA by isoniazid, studied here, provide clues about proteins essential for the pathway and hence lead to a rational identification of possible drug targets. Feasibility studies using sequence analysis of the M. tuberculosis H37Rv and human proteomes indicate that, apart from the known InhA, potential targets for anti-tubercular drug design are AccD3, Fas, FabH, Pks13, DesA1/2, and DesA3. Proteins identified as essential by FBA correlate well with those previously identified experimentally through transposon site hybridisation mutagenesis. This study demonstrates the application of FBA for rational identification of potential anti-tubercular drug targets, which can indeed be a general strategy in drug design. The targets, chosen based on the critical points in the pathway, form a

  2. Flux balance analysis of mycolic Acid pathway: targets for anti-tubercular drugs.

    Directory of Open Access Journals (Sweden)

    2005-10-01

    Full Text Available Mycobacterium tuberculosis is the focus of several investigations for design of newer drugs, as tuberculosis remains a major epidemic despite the availability of several drugs and a vaccine. Mycobacteria owe many of their unique qualities to mycolic acids, which are known to be important for their growth, survival, and pathogenicity. Mycolic acid biosynthesis has therefore been the focus of a number of biochemical and genetic studies. It also turns out to be the pathway inhibited by front-line anti-tubercular drugs such as isoniazid and ethionamide. Recent years have seen the emergence of systems-based methodologies that can be used to study microbial metabolism. Here, we seek to apply insights from flux balance analyses of the mycolic acid pathway (MAP for the identification of anti-tubercular drug targets. We present a comprehensive model of mycolic acid synthesis in the pathogen M. tuberculosis involving 197 metabolites participating in 219 reactions catalysed by 28 proteins. Flux balance analysis (FBA has been performed on the MAP model, which has provided insights into the metabolic capabilities of the pathway. In silico systematic gene deletions and inhibition of InhA by isoniazid, studied here, provide clues about proteins essential for the pathway and hence lead to a rational identification of possible drug targets. Feasibility studies using sequence analysis of the M. tuberculosis H37Rv and human proteomes indicate that, apart from the known InhA, potential targets for anti-tubercular drug design are AccD3, Fas, FabH, Pks13, DesA1/2, and DesA3. Proteins identified as essential by FBA correlate well with those previously identified experimentally through transposon site hybridisation mutagenesis. This study demonstrates the application of FBA for rational identification of potential anti-tubercular drug targets, which can indeed be a general strategy in drug design. The targets, chosen based on the critical points in the pathway, form a

  3. Wnt/β-catenin signalling pathway mediated aberrant hippocampal neurogenesis in kainic acid-induced epilepsy.

    Science.gov (United States)

    Qu, Zhengyi; Su, Fang; Qi, Xueting; Sun, Jianbo; Wang, Hongcai; Qiao, Zhenkui; Zhao, Hong; Zhu, Yulan

    2017-10-01

    Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis with massive neuronal loss and severe gliosis. Aberrant neurogenesis has been shown in the epileptogenesis process of temporal lobe epilepsy. However, the molecular mechanisms underlying aberrant neurogenesis remain unclear. The roles of Wnt signalling cascade have been well established in neurogenesis during multiple aspects. Here, we used kainic acid-induced rat epilepsy model to investigate whether Wnt/β-catenin signalling pathway is involved in the aberrant neurogenesis in temporal lobe epilepsy. Immunostaining and western blotting results showed that the expression levels of β-catenin, Wnt3a, and cyclin D1, the key regulators in Wnt signalling pathway, were up-regulated during acute epilepsy induced by the injection of kainic acids, indicating that Wnt signalling pathway was activated in kainic acid-induced temporal lobe epilepsy. Moreover, BrdU labelling results showed that blockade of the Wnt signalling by knocking down β-catenin attenuated aberrant neurogenesis induced by kainic acids injection. Altogether, Wnt/β-catenin signalling pathway mediated hippocampal neurogenesis during epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy. Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis. Aberrant neurogenesis has been shown to involve in the epileptogenesis process of temporal lobe epilepsy. In the present study, we discovered that Wnt3a/β-catenin signalling pathway serves as a link between aberrant neurogenesis and underlying remodelling in the hippocampus, leading to temporal lobe epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy. Copyright © 2017 John Wiley & Sons, Ltd.

  4. Fatty acid omega‐oxidation as a rescue pathway for fatty acid oxidation disorders in humans

    National Research Council Canada - National Science Library

    Wanders, Ronald J. A; Komen, Jasper; Kemp, Stephan

    2011-01-01

    Fatty acids (FAs) can be degraded via different mechanisms including α‐, β‐ and ω‐oxidation. In humans, a range of different genetic diseases has been identified in which either mitochondrial FA...

  5. Molecular cloning of a novel glucuronokinase/putative pyrophosphorylase from zebrafish acting in an UDP-glucuronic acid salvage pathway.

    Directory of Open Access Journals (Sweden)

    Roman Gangl

    Full Text Available In animals, the main precursor for glycosaminoglycan and furthermore proteoglycan biosynthesis, like hyaluronic acid, is UDP-glucuronic acid, which is synthesized via the nucleotide sugar oxidation pathway. Mutations in this pathway cause severe developmental defects (deficiency in the initiation of heart valve formation. In plants, UDP-glucuronic acid is synthesized via two independent pathways. Beside the nucleotide sugar oxidation pathway, a second minor route to UDP-glucuronic acid exist termed the myo-inositol oxygenation pathway. Within this myo-inositol is ring cleaved into glucuronic acid, which is subsequently converted to UDP-glucuronic acid by glucuronokinase and UDP-sugar pyrophosphorylase. Here we report on a similar, but bifunctional enzyme from zebrafish (Danio rerio which has glucuronokinase/putative pyrophosphorylase activity. The enzyme can convert glucuronic acid into UDP-glucuronic acid, required for completion of the alternative pathway to UDP-glucuronic acid via myo-inositol and thus establishes a so far unknown second route to UDP-glucuronic acid in animals. Glucuronokinase from zebrafish is a member of the GHMP-kinase superfamily having unique substrate specificity for glucuronic acid with a Km of 31 ± 8 µM and accepting ATP as the only phosphate donor (Km: 59 ± 9 µM. UDP-glucuronic acid pyrophosphorylase from zebrafish has homology to bacterial nucleotidyltransferases and requires UTP as nucleosid diphosphate donor. Genes for bifunctional glucuronokinase and putative UDP-glucuronic acid pyrophosphorylase are conserved among some groups of lower animals, including fishes, frogs, tunicates, and polychaeta, but are absent from mammals. The existence of a second pathway for UDP-glucuronic acid biosynthesis in zebrafish likely explains some previous contradictory finding in jekyll/ugdh zebrafish developmental mutants, which showed residual glycosaminoglycans and proteoglycans in knockout mutants of UDP

  6. Effect of uric acid on inflammatory COX-2 and ROS pathways in vascular smooth muscle cells.

    Science.gov (United States)

    Oğuz, Nurgül; Kırça, Mustafa; Çetin, Arzu; Yeşilkaya, Akın

    2017-10-01

    Hyperuricemia is thought to play a role in cardiovascular diseases (CVD), including hypertension, coronary artery disease and atherosclerosis. However, exactly how uric acid contributes to these pathologies is unknown. An underlying mechanism of inflammatory diseases, such as atherosclerosis, includes enhanced production of cyclooxygenase-2 (COX-2) and superoxide anion. Here, we aimed to examine the effect of uric acid on inflammatory COX-2 and superoxide anion production and to determine the role of losartan. Primarily cultured vascular smooth muscle cells (VSMCs) were time and dose-dependently induced by uric acid and COX-2 and superoxide anion levels were measured. COX-2 levels were determined by ELISA, and superoxide anion was measured by the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c method. Uric acid elevated COX-2 levels in a time-dependent manner. Angiotensin-II receptor blocker, losartan, diminished uric-acid-induced COX-2 elevation. Uric acid also increased superoxide anion level in VSMCs. Uric acid plays an important role in CVD pathogenesis by inducing inflammatory COX-2 and ROS pathways. This is the first study demonstrating losartan's ability to reduce uric-acid-induced COX-2 elevation.

  7. [Metabolic pathway and metabolites of total diterpene acid isolated from Pseudolarix kaempferi].

    Science.gov (United States)

    Liu, Peng; Guo, Hong-Zhu; Sun, Jiang-Hao; Xu, Man; Guo, Hui; Sun, Shi-Feng; Guo, De-An

    2014-08-01

    The preliminary metabolic profile of total diterpene acid (TDA) isolated from Pseudolarix kaempferi was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the predominant metabolite in plasma, urine, bile and feces after both oral and intravenous administrations to rats using HPLC-UV and HPLC-ESI/MS(n), and demethoxydeacetoxypseudolaric acid B (DDPB), a metabolite proposed to be the glucoside of PC2 (PC2G), as well as pseudolaric acid C (PC), pseudolaric acid A (PA), pseudolaric acid A O-beta-D glucopyranoside (PAG), pseudolaric acid B O-beta-D glucopyranoside (PBG) and deacetylpseudolaric acid A (DPA) originated from TDA could also be detected. It was demonstrated by tests that the metabolism of TDA is independent of intestinal microflora, and neither of pepsin and trypsin is in charge of metabolism of TDA, TDA is also stable in both pH environments of gastric tract and intestinal tract. The metabolites of TDA in whole blood in vitro incubation were found to be PC2, DDPB and PC2G, which demonstrated that the metabolic reaction of TDA in vivo is mainly occurred in blood and contributed to be the hydrolysis of plasma esterase to ester bond, as well as the glucosylation reaction. These results clarified the metabolic pathway of TDA for the first time, which is of great significance to the in vivo active form and acting mechanism research of P. kaempferi.

  8. Uric acid priming in human monocytes is driven by the AKT–PRAS40 autophagy pathway

    Science.gov (United States)

    Crişan, Tania O.; Cleophas, Maartje C. P.; Novakovic, Boris; Erler, Kathrin; van de Veerdonk, Frank L.; Stunnenberg, Hendrik G.; Netea, Mihai G.; Dinarello, Charles A.; Joosten, Leo A. B.

    2017-01-01

    Metabolic triggers are important inducers of the inflammatory processes in gout. Whereas the high serum urate levels observed in patients with gout predispose them to the formation of monosodium urate (MSU) crystals, soluble urate also primes for inflammatory signals in cells responding to gout-related stimuli, but also in other common metabolic diseases. In this study, we investigated the mechanisms through which uric acid selectively lowers human blood monocyte production of the natural inhibitor IL-1 receptor antagonist (IL-1Ra) and shifts production toward the highly inflammatory IL-1β. Monocytes from healthy volunteers were first primed with uric acid for 24 h and then subjected to stimulation with lipopolysaccharide (LPS) in the presence or absence of MSU. Transcriptomic analysis revealed broad inflammatory pathways associated with uric acid priming, with NF-κB and mammalian target of rapamycin (mTOR) signaling strongly increased. Functional validation did not identify NF-κB or AMP-activated protein kinase phosphorylation, but uric acid priming induced phosphorylation of AKT and proline-rich AKT substrate 40 kDa (PRAS 40), which in turn activated mTOR. Subsequently, Western blot for the autophagic structure LC3-I and LC3-II (microtubule-associated protein 1A/1B-light chain 3) fractions, as well as fluorescence microscopy of LC3-GFP–overexpressing HeLa cells, revealed lower autophagic activity in cells exposed to uric acid compared with control conditions. Interestingly, reactive oxygen species production was diminished by uric acid priming. Thus, the Akt–PRAS40 pathway is activated by uric acid, which inhibits autophagy and recapitulates the uric acid-induced proinflammatory cytokine phenotype. PMID:28484006

  9. Uric acid priming in human monocytes is driven by the AKT-PRAS40 autophagy pathway.

    Science.gov (United States)

    Crişan, Tania O; Cleophas, Maartje C P; Novakovic, Boris; Erler, Kathrin; van de Veerdonk, Frank L; Stunnenberg, Hendrik G; Netea, Mihai G; Dinarello, Charles A; Joosten, Leo A B

    2017-05-23

    Metabolic triggers are important inducers of the inflammatory processes in gout. Whereas the high serum urate levels observed in patients with gout predispose them to the formation of monosodium urate (MSU) crystals, soluble urate also primes for inflammatory signals in cells responding to gout-related stimuli, but also in other common metabolic diseases. In this study, we investigated the mechanisms through which uric acid selectively lowers human blood monocyte production of the natural inhibitor IL-1 receptor antagonist (IL-1Ra) and shifts production toward the highly inflammatory IL-1β. Monocytes from healthy volunteers were first primed with uric acid for 24 h and then subjected to stimulation with lipopolysaccharide (LPS) in the presence or absence of MSU. Transcriptomic analysis revealed broad inflammatory pathways associated with uric acid priming, with NF-κB and mammalian target of rapamycin (mTOR) signaling strongly increased. Functional validation did not identify NF-κB or AMP-activated protein kinase phosphorylation, but uric acid priming induced phosphorylation of AKT and proline-rich AKT substrate 40 kDa (PRAS 40), which in turn activated mTOR. Subsequently, Western blot for the autophagic structure LC3-I and LC3-II (microtubule-associated protein 1A/1B-light chain 3) fractions, as well as fluorescence microscopy of LC3-GFP-overexpressing HeLa cells, revealed lower autophagic activity in cells exposed to uric acid compared with control conditions. Interestingly, reactive oxygen species production was diminished by uric acid priming. Thus, the Akt-PRAS40 pathway is activated by uric acid, which inhibits autophagy and recapitulates the uric acid-induced proinflammatory cytokine phenotype.

  10. Branched-Chain Amino Acids.

    Science.gov (United States)

    Yamamoto, Keisuke; Tsuchisaka, Atsunari; Yukawa, Hideaki

    Branched-chain amino acids (BCAAs), viz., L-isoleucine, L-leucine, and L-valine, are essential amino acids that cannot be synthesized in higher organisms and are important nutrition for humans as well as livestock. They are also valued as synthetic intermediates for pharmaceuticals. Therefore, the demand for BCAAs in the feed and pharmaceutical industries is increasing continuously. Traditional industrial fermentative production of BCAAs was performed using microorganisms isolated by random mutagenesis. A collection of these classical strains was also scientifically useful to clarify the details of the BCAA biosynthetic pathways, which are tightly regulated by feedback inhibition and transcriptional attenuation. Based on this understanding of the metabolism of BCAAs, it is now possible for us to pursue strains with higher BCAA productivity using rational design and advanced molecular biology techniques. Additionally, systems biology approaches using augmented omics information help us to optimize carbon flux toward BCAA production. Here, we describe the biosynthetic pathways of BCAAs and their regulation and then overview the microorganisms developed for BCAA production. Other chemicals, including isobutanol, i.e., a second-generation biofuel, can be synthesized by branching the BCAA biosynthetic pathways, which are also outlined.

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

    Science.gov (United States)

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

    2017-11-17

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

  12. Biochemical and Structural Characterization of a Ureidoglycine Aminotransferase in the Klebsiella pneumoniae Uric Acid Catabolic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    French, Jarrod B.; Ealick, Steven E. (Cornell)

    2010-09-03

    Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an {alpha}-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

  13. Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis [v1; ref status: indexed, http://f1000r.es/2tz

    Directory of Open Access Journals (Sweden)

    Yaya Wang

    2014-02-01

    Full Text Available Fluorination has been widely used in chemical synthesis, but is rare in nature. The only known biological fluorination scope is represented by the fl pathway from Streptomyces cattleya that produces fluoroacetate (FAc and 4-fluorothreonine (4-FT. Here we report the identification of a novel pathway for FAc and 4-FT biosynthesis from the actinomycetoma-causing pathogen Nocardia brasiliensis ATCC 700358. The new pathway shares overall conservation with the fl pathway in S. cattleya. Biochemical characterization of the conserved domains revealed a novel fluorinase NobA that can biosynthesize 5’-fluoro-5’-deoxyadenosine (5’-FDA from inorganic fluoride and S-adenosyl-l-methionine (SAM. The NobA shows similar halide specificity and characteristics to the fluorination enzyme FlA of the fl pathway. Kinetic parameters for fluoride (Km 4153 μM, kcat 0.073 min-1 and SAM (Km 416 μM, kcat 0.139 min-1 have been determined, revealing that NobA is slightly (2.3 fold slower than FlA. Upon sequence comparison, we finally identified a distinct loop region in the fluorinases that probably accounts for the disparity of fluorination activity.

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

    Science.gov (United States)

    García-Estrada, Carlos; Martín, Juan-Francisco

    2016-10-01

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

  15. Fatty acid omega-oxidation as a rescue pathway for fatty acid oxidation disorders in humans

    NARCIS (Netherlands)

    Wanders, Ronald J. A.; Komen, Jasper; Kemp, Stephan

    2011-01-01

    Fatty acids (FAs) can be degraded via different mechanisms including alpha-, beta- and omega-oxidation. In humans, a range of different genetic diseases has been identified in which either mitochondrial FA beta-oxidation, peroxisomal FA beta-oxidation or FA alpha-oxidation is impaired. Treatment

  16. Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants

    Energy Technology Data Exchange (ETDEWEB)

    Schenck, Craig A.; Holland, Cynthia K.; Schneider, Matthew R.; Men, Yusen; Lee, Soon Goo; Jez, Joseph M.; Maeda , Hiroshi A. (UW); (WU)

    2017-06-26

    L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

  17. Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.

    Science.gov (United States)

    Schenck, Craig A; Holland, Cynthia K; Schneider, Matthew R; Men, Yusen; Lee, Soon Goo; Jez, Joseph M; Maeda, Hiroshi A

    2017-09-01

    L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

  18. A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes.

    Science.gov (United States)

    Andersen, Gorm; Björnberg, Olof; Polakova, Silvia; Pynyaha, Yuriy; Rasmussen, Anna; Møller, Kasper; Hofer, Anders; Moritz, Thomas; Sandrini, Michael Paolo Bastner; Merico, Anna-Maria; Compagno, Concetta; Akerlund, Hans-Erik; Gojković, Zoran; Piskur, Jure

    2008-07-18

    Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil. Six loci, named URC1-6 (for uracil catabolism), are involved in the novel catabolic pathway. Four of them, URC3,5, URC6, and URC2 encode urea amidolyase, uracil phosphoribosyltransferase, and a putative transcription factor, respectively. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). Urc1p and Urc4p are therefore likely the core components of this novel biochemical pathway. A combination of genetic and analytical chemistry methods demonstrates that uridine monophosphate and urea are intermediates, and 3-hydroxypropionic acid, ammonia and carbon dioxide the final products of degradation. The URC pathway does not require the presence of an active respiratory chain and is therefore different from the oxidative and rut pathways described in prokaryotes, although the latter also gives 3-hydroxypropionic acid as the end product. The genes of the URC pathway are not homologous to any of the eukaryotic or prokaryotic genes involved in pyrimidine degradation described to date.

  19. Bacillus amyloliquefaciens FZB42 repressed plant miR846 to induce systemic resistance via jasmonic acid-dependent signaling pathway.

    Science.gov (United States)

    Xie, Shanshan; Jiang, Haiyang; Ding, Ting; Xu, Qianqian; Chai, Wenbo; Cheng, Beijiu

    2017-11-01

    Bacillus amyloliquefaciens FZB42 is a kind of plant-growth-promoting rhizobacteria (PGPR) that activates induced systemic resistance (ISR) in Arabidopsis. Blocking in cyclic lipopeptides and 2,3-butanediol synthesis of FZB42, which were proved to be involved in priming ISR, resulted in abolishment of plant defense responses. To further clarify the ISR activated by PGPR at miRNA level, sRNA libraries from Arabidopsis leaves after root-irrigated with FZB42, FZB42ΔsfpΔalsS and control were constructed and sequenced. After fold-change selection, promoter analysis and target prediction, miR846-5p and miR846-3p from the same precursor were selected as candidate ISR-associated miRNA. miR846, belongs to non-conserved miRNAs, specifically exists in Arabidopsis and its function in plant defense response remains unclear. The disease severities of transgenic Arabidopsis overexpression miR846 (OEmiR846) and knockdown miR846 (STTM846) against Pseudomonas syringae DC3000 suggested that the miR846 expression level in Arabidopsis was negatively correlated with disease resistance. Moreover, miR846 in Arabidopsis Col-0 was repressed after methyl jasmonate treatment. In addition, jasmonic acid (JA) signaling related genes were up-regulated in STTM846 and stomatal apertures of STTM846 were also less than Arabidopsis Col-0 after methyl jasmonate treatment. Furthermore, the disease resistance of STTM846 transgenic Arabidopsis against pstDC3000 was blocked by adding JA biosynthetic inhibitor diethyldiethiocarbamic acid (DIECA). Taken together, our results suggested that Bacillus amyloliquefaciens FZB42 inoculation suppressed miR846 expression to induce Arabidopsis systemic resistance via JA-dependent signaling pathway. This article is protected by copyright. All rights reserved. © 2017 BSPP and John Wiley & Sons Ltd.

  20. [Optimization of synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid].

    Science.gov (United States)

    Wang, Dong; Wang, Bei-Bei; Liu, Yi; Shi, Ming-Yu; Xiao, Dong-Guang; Huang, Lu-Qi; Dai, Zhu-Bo; Zhang, Xue-Li

    2014-07-01

    To optimize the synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid. Using the DNA assembler method, one copy of Glycyrrhiza glabra beta-amyrin synthase (GgbAS), Medicago truncatula oleanolic acid synthase (MtOAS) and Arabidopsis thaliana cytochrome P450 reductase 1 (AtCPR1) genes were introduced into Saccharomyces cerevisiae strain BY-OA, resulting in strain BY-20A. YPD medium with different glucose concentration were then used to cultivate strain BY-2OA. Increasing gene copies of GgbAS, MtOAS and AtCPR1 resulted in increased beta-amyrin and oleanolic acid production. The strain BY-2OA produced 136.5 mg x L(-1) beta-amyrin and 92.5 mg x L(-1) oleanolic acid, which were 54% and 30% higher than the parent strain BY-OA. Finally, the titer of oleanolic acid increased to 165.7 mg x L(-1) when cultivated in YPD medium with 40 mg x L(-1) glucose. Production of oleanoic acid increased significantly in the yeast strain BY-2OA, which can provide the basis for creating an alternative way for production of oleanoic acid in place of extraction from plant sources.

  1. Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine

    DEFF Research Database (Denmark)

    Borodina, Irina; Kildegaard, Kanchana Rueksomtawin; Jensen, Niels Bjerg

    2015-01-01

    . With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for highlevel production of 3HP, we identified the ß-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of ß......-alanine and its subsequent conversion into 3HP using a novel ß-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3 g・L-1 with a 0.14±0.0 C-mol・C-mol-1 yield on glucose in 80 hours in controlled fed-batch fermentation in mineral medium at pH 5...

  2. Autotaxin-Lysophosphatidic Acid Pathway in Intraocular Pressure Regulation and Glaucoma Subtypes.

    Science.gov (United States)

    Honjo, Megumi; Igarashi, Nozomi; Kurano, Makoto; Yatomi, Yutaka; Igarashi, Koji; Kano, Kuniyuki; Aoki, Junken; Weinreb, Robert N; Aihara, Makoto

    2018-02-01

    To compare the levels of autotaxin (ATX), lysophosphatidic acid (LPA), and lysophosphatidylcholine (LPC) in the aqueous humor (AH) of healthy control subjects with those of patients with different subtypes of glaucoma, and also to investigate the relationship of the ATX-LPA pathway with IOP and subtype of glaucoma. This study included 164 eyes of 164 consecutive cases of cataract and glaucoma surgery (37 healthy, 31 normal tension glaucoma, 49 primary open angle glaucoma, 28 secondary open angle glaucoma, and 19 exfoliation glaucoma). Aqueous levels of LPA, LPC, and ATX were quantified using liquid chromatography-tandem mass spectrometry and a two-site immunoenzymetric assay. The association between aqueous levels of ATX/LPA/LPC and IOP elevation in different glaucoma subtypes was investigated. The diagnostic values of indices of the ATX-LPA pathway were compared using receiver operating characteristic curve analysis. Notable increases in ATX/LPA/LPC levels in glaucoma patients were observed. The ATX-LPA pathway was significantly related to IOP elevation and the subtype of glaucoma, especially in SOAG and XFG patients, and the area under the curve was significant for discriminating glaucoma eyes from healthy eyes. Bioactive ATX/LPA/LPC concentrations were present in aqueous humor, and higher ATX and LPA concentrations were significantly correlated with IOP in all study subjects. Furthermore, the ATX-LPA pathway was significantly related to glaucoma subtype. These results reveal the potentially important role of the ATX-LPA pathway for IOP regulation in healthy subjects and glaucoma patients.

  3. Caffeic Acid Inhibits NFkappaB Activation of Osteoclastogenesis Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Ferry Sandra

    2011-12-01

    Full Text Available BACKGROUND: Caffeic acid (3,4-dihydroxycinnamic acids is involved in various green plants. Based on our previous report, a major component of sweet potato extracts, possibly caffeic acid, was shown as a promising inhibitor of osteoclastogenesis. However, the effect of caffeic acid in inhibiting osteoclastogenesis needs to be confirmed. The underlying mechanism needs to be disclosed as well. METHODS: Caffeic acid in various concentrations was added to in vitro osteoclastogenesis of receptor activator nuclear factor kB ligand (RANKL-tumor necrosis factor alpha (TNF-α-macrophage colony stimulating factor (M-CSF-induced bone marrow-derived monocyte/macrophage precursor cells (BMMs and RANKL-TNF-α-induced RAW264 cells D-Clone (RAW-D cells. Tartrate resistant acid phosphatase (TRAP staining was performed and TRAP-positive polynucleated cells (PNCs were counted. For apoptosis analysis, caffeic acid-treated BMMs, RAW-D cells and osteoclast-like PNCs were subjected to Sub-G1 Apoptosis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assays. To measure NFkB activity, RAW-D cells were transfected with pNFkB-TA-Luc and subjected to Dual Luciferase Reporter Assay System. RESULTS: Caffeic acid inhibited osteoclastogenesis of RANKL-TNF-α-M-CSF-induced BMMs as well as RANKL-TNF-α-induced RAW-D cells in a dose dependent manner. Caffeic acid did not induce apoptosis in BMMs, RAW-D cells and osteoclast-like PNCs. RANKL-TNF-α-induced NFkB activity in RAW-D was diminished by caffeic acid in a dose dependent manner. Significant NFkB activity inhibtion was observed starting from 1µg/mL caffeic acid. CONCLUSIONS: Caffeic acid could be a potent osteoclastogenesis inhibitor through inhibition of NFkB activity. Our present study should be further followed up to disclose caffeic acid's possible overlying signaling pathways in inhibiting osteoclastogenesis. KEYWORDS: caffeic acid, osteoclastogenesis, NFkB, RANKL, TNF-α.

  4. Impairment of PPARαand the Fatty Acid Oxidation Pathway Aggravates Renal Fibrosis during Aging.

    Science.gov (United States)

    Chung, Ki Wung; Lee, Eun Kyeong; Lee, Mi Kyung; Oh, Goo Taeg; Yu, Byung Pal; Chung, Hae Young

    2018-02-12

    Defects in the renal fatty acid oxidation (FAO) pathway have been implicated in the development of renal fibrosis. Although, compared with young kidneys, aged kidneys show significantly increased fibrosis with impaired kidney function, the mechanisms underlying the effects of aging on renal fibrosis have not been investigated. In this study, we investigated peroxisome proliferator-activated receptor α (PPAR α ) and the FAO pathway as regulators of age-associated renal fibrosis. The expression of PPAR α and the FAO pathway-associated proteins significantly decreased with the accumulation of lipids in the renal tubular epithelial region during aging in rats. In particular, decreased PPAR α protein expression associated with increased expression of PPAR α -targeting microRNAs. Among the microRNAs with increased expression during aging, miR-21 efficiently decreased PPAR α expression and impaired FAO when ectopically expressed in renal epithelial cells. In cells pretreated with oleic acid to induce lipid stress, miR-21 treatment further enhanced lipid accumulation. Furthermore, treatment with miR-21 significantly exacerbated the TGF- β -induced fibroblast phenotype of epithelial cells. We verified the physiologic importance of our findings in a calorie restriction model. Calorie restriction rescued the impaired FAO pathway during aging and slowed fibrosis development. Finally, compared with kidneys of aged littermate controls, kidneys of aged PPAR α -/- mice showed exaggerated lipid accumulation, with decreased activity of the FAO pathway and a severe fibrosis phenotype. Our results suggest that impaired renal PPAR α signaling during aging aggravates renal fibrosis development, and targeting PPAR α is useful for preventing age-associated CKD. Copyright © 2018 by the American Society of Nephrology.

  5. Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Wang, Zheng; Chen, Yun

    2015-01-01

    biomass into the products of interest. We engineered Saccharomyces cerevisiae for production of 3-hydroxypropionic acid (3HP), a potential building block for acrylates, from glucose and xylose. We introduced the 3HP biosynthetic pathways via malonyl-CoA or β-alanine intermediates into a xylose...

  6. Decomposition Pathways of Peroxynitrous Acid: Gas-Phase and Solution Energetics

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, David A.(BATTELLE (PACIFIC NW LAB)); Feller, David F.(BATTELLE (PACIFIC NW LAB)); Zhan, Chang-Guo N.; Francisco, Joseph S.(6408)

    2002-03-08

    It was recently suggested that HOONO, which forms after protonation of the ONOO- anion under biological conditions, decomposes into HNO and (1Dg)O2. Subsequent workers argued that the mechanism for HOONO decomposition proceeds via homolytic bond fission, producing the radical pair, OH and NO2 and another recent study argued that a cyclic form of the peroxynitrous acid results in the products H++ O2(1Dg)+ NO-. Calculations on the reaction pathway for the process showed that it required a high activation energy, and is thus implausible. High level ab initio molecular orbital theory including extrapolation to the complete basis set limit has been used to calculate the heats of formation of reactants and products for the homolytic bond fission pathways for decomposition of HOONO and the molecular pathway that yields HNO and 1O2 as well as the transition state for the latter process. These data are used to evaluate the probability of whether the decomposition of peroxynitrous acid can produce HNO and 1O2. Isomerization of HOONO to HONO2 is also discussed.

  7. Putrescine production via the ornithine decarboxylation pathway improves the acid stress survival of Lactobacillus brevis and is part of a horizontally transferred acid resistance locus.

    Science.gov (United States)

    Romano, Andrea; Ladero, Victor; Alvarez, Miguel A; Lucas, Patrick M

    2014-04-03

    Decarboxylation pathways are widespread among lactic acid bacteria; their physiological role is related to acid resistance through the regulation of the intracellular pH and to the production of metabolic energy via the generation of a proton motive force and its conversion into ATP. These pathways include, among others, biogenic amine (BA) production pathways. BA accumulation in foodstuffs is a health risk; thus, the study of the factors involved in their production is of major concern. The analysis of several lactic acid bacterial strains isolated from different environments, including fermented foods and beverages, revealed that the genes encoding these pathways are clustered on the chromosome, which suggests that these genes are part of a genetic hotspot related to acid stress resistance. Further attention was devoted to the ornithine decarboxylase pathway, which affords putrescine from ornithine. Studies were performed on three lactic acid bacteria belonging to different species. The ODC pathway was always shown to be involved in cytosolic pH alkalinisation and acid shock survival, which were observed to occur with a concomitant increase in putrescine production. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Santosh Kumar Upadhyay

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

  10. Omega-3 polyunsaturated fatty acids antagonize macrophage inflammation via activation of AMPK/SIRT1 pathway.

    Directory of Open Access Journals (Sweden)

    Bingzhong Xue

    Full Text Available Macrophages play a key role in obesity-induced inflammation. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA exert anti-inflammatory functions in both humans and animal models, but the exact cellular signals mediating the beneficial effects are not completely understood. We previously found that two nutrient sensors AMP-activated protein kinase (AMPK and SIRT1 interact to regulate macrophage inflammation. Here we aim to determine whether ω-3 PUFAs antagonize macrophage inflammation via activation of AMPK/SIRT1 pathway. Treatment of ω-3 PUFAs suppresses lipopolysaccharide (LPS-induced cytokine expression in macrophages. Luciferase reporter assays, electrophoretic mobility shift assays (EMSA and Chromatin immunoprecipitation (ChIP assays show that treatment of macrophages with ω-3 PUFAs significantly inhibits LPS-induced NF-κB signaling. Interestingly, DHA also increases expression, phosphorylation and activity of the major isoform α1AMPK, which further leads to SIRT1 over-expression. More importantly, DHA mimics the effect of SIRT1 on deacetylation of the NF-κB subunit p65, and the ability of DHA to deacetylate p65 and inhibit its signaling and downstream cytokine expression require SIRT1. In conclusion, ω-3 PUFAs negatively regulate macrophage inflammation by deacetylating NF-κB, which acts through activation of AMPK/SIRT1 pathway. Our study defines AMPK/SIRT1 as a novel cellular mediator for the anti-inflammatory effects of ω-3 PUFAs.

  11. An effective method and pathways of acrylonitrile degradation to acrylic acid through an alkaline hydrothermal system.

    Science.gov (United States)

    Dong, Wenjie; Peng, Boyu; Wang, Ke; Miao, Jia; Zhang, Wei; Zhang, Yalei; Shen, Zheng

    2017-07-01

    Degradation of pollution for specific chemicals represents an optimal approach to high-strength wastewater treatment. One-pot selective conversion of acrylonitrile to acrylic acid in a hydrothermal system with NaOH as a catalyst was carried out. The influence factors were evaluated, including initial acrylonitrile concentration, reaction temperature, reaction time and amount of alkali. Experimental results showed that the highest yield of acrylic acid (55%) was obtained at the initial acrylonitrile concentration of 3 × 10(3) mg/L, 300°C for 90 s with 1.0 M NaOH. To determine the reaction path, intermediates analysis and calculation of carbon and nitrogen balance were carried out by means of HPLC, GC and TOC/TN methods. Two probable reaction pathways were proposed as follows: (1) Acrylonitrile was hydrolyzed into acrylamide, and acrylic acid was obtained via further hydrolysis. (2) Acrylonitrile was converted into 3-hydroxy-propionitrile via additive reaction, and this product was readily converted to 3-hydroxy-propionic acid through two steps of hydrolysis, followed by dehydration reaction to produce acrylic acid. This study offered not only an efficient method to transfer highly toxic pollutants into valuable chemical, but also a better understanding of hydrothermal alkali catalytic reaction.

  12. Rewiring a secondary metabolite pathway towards itaconic acid production in Aspergillus niger.

    Science.gov (United States)

    Hossain, Abeer H; Li, An; Brickwedde, Anja; Wilms, Lars; Caspers, Martien; Overkamp, Karin; Punt, Peter J

    2016-07-28

    The industrially relevant filamentous fungus Aspergillus niger is widely used in industry for its secretion capabilities of enzymes and organic acids. Biotechnologically produced organic acids promise to be an attractive alternative for the chemical industry to replace petrochemicals. Itaconic acid (IA) has been identified as one of the top twelve building block chemicals which have high potential to be produced by biotechnological means. The IA biosynthesis cluster (cadA, mttA and mfsA) has been elucidated in its natural producer Aspergillus terreus and transferred to A. niger to enable IA production. Here we report the rewiring of a secondary metabolite pathway towards further improved IA production through the overexpression of a putative cytosolic citrate synthase citB in a A. niger strain carrying the IA biosynthesis cluster. We have previously shown that expression of cadA from A. terreus results in itaconic acid production in A. niger AB1.13, albeit at low levels. This low-level production is boosted fivefold by the overexpression of mttA and mfsA in itaconic acid producing AB1.13 CAD background strains. Controlled batch cultivations with AB1.13 CAD + MFS + MTT strains showed increased production of itaconic acid compared with AB1.13 CAD strain. Moreover, preliminary RNA-Seq analysis of an itaconic acid producing AB1.13 CAD strain has led to the identification of the putative cytosolic citrate synthase citB which was induced in an IA producing strain. We have overexpressed citB in a AB1.13 CAD + MFS + MTT strain and by doing so hypothesize to have targeted itaconic acid production to the cytosolic compartment. By overexpressing citB in AB1.13 CAD + MFS + MTT strains in controlled batch cultivations we have achieved highly increased titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h while no CA was produced. Expression of the IA biosynthesis cluster in Aspergillus niger AB1.13 strain enables IA production. Moreover, in the AB1.13 CAD

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

  15. Origin of saxitoxin biosynthetic genes in cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Ahmed Moustafa

    2009-06-01

    Full Text Available Paralytic shellfish poisoning (PSP is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX. STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway.We generated a draft genome assembly of the saxitoxin-producing (STX+ cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxin-genes (named sxtA to sxtZ that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non-toxic (STX- sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria.Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX- strains among Anabaena circinalis strains.

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

    Directory of Open Access Journals (Sweden)

    Dragana Božić

    Full Text Available 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  19. Defining and Modeling Known Adverse Outcome Pathways: Domoic Acid and Neuronal Signaling as a Case Study

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Karen H.; Andersen, Melvin E.; Basu, Nil; Carvan, Michael J.; Crofton, Kevin M.; King, Kerensa A.; Sunol, Cristina; Tiffany-Castiglioni, Evelyn; Schultz, Irvin R.

    2011-01-01

    An adverse outcome pathway (AOP) is a sequence of key events from a molecular-level initiating event and an ensuing cascade of steps to an adverse outcome with population level significance. To implement a predictive strategy for ecotoxicology, the multiscale nature of an AOP requires computational models to link salient processes (e.g., in chemical uptake, toxicokinetics, toxicodynamics, and population dynamics). A case study with domoic acid was used to demonstrate strategies and enable generic recommendations for developing computational models in an effort to move toward a toxicity testing paradigm focused on toxicity pathway perturbations applicable to ecological risk assessment. Domoic acid, an algal toxin with adverse effects on both wildlife and humans, is a potent agonist for kainate receptors (ionotropic glutamate receptors whose activation leads to the influx of Na+ and Ca2+). Increased Ca2+ concentrations result in neuronal excitotoxicity and cell death primarily in the hippocampus, which produces seizures, impairs learning and memory, and alters behavior in some species. Altered neuronal Ca2+ is a key process in domoic acid toxicity which can be evaluated in vitro. Further, results of these assays would be amenable to mechanistic modeling for identifying domoic acid concentrations and Ca2+ perturbations that are normal, adaptive, or clearly toxic. In vitro assays with outputs amenable to measurement in exposed populations can link in vitro to in vivo conditions, and toxicokinetic information will aid in linking in vitro results to the individual organism. Development of an AOP required an iterative process with three important outcomes: (1) a critically reviewed, stressor-specific AOP; (2) identification of key processes suitable for evaluation with in vitro assays; and (3) strategies for model development.

  20. A comprehensive proteome map of glandular trichomes of hop (Humulus lupulus L.) female cones: Identification of biosynthetic pathways of the major terpenoid-related compounds and possible transport proteins.

    Science.gov (United States)

    Champagne, Antoine; Boutry, Marc

    2017-04-01

    Female flowers of hop (Humulus lupulus) are an essential source of terpenoid-related compounds, which are mainly used as flavoring in the beer brewing process. The compounds involved are bitter acids, terpenophenolics, as well as mono- and sesquiterpenoids. In this work, we analyzed the proteome of purified glandular trichomes (lupulin glands) from female flowers, which produce and accumulate these compounds. An extensive 2D-LC-MS/MS analysis identified 1015 proteins. Of these, most correspond to housekeeping and primary metabolism-related proteins, albeit predominantly including amino acid and lipid metabolism, which feeds the specialized (secondary) metabolism. Indeed, 75 proteins belong to the specialized metabolism. No less than 40 enzymes are involved in the synthesis of terpenoid-derived compounds and 21 are predicted transporters, some of which might be involved in the transport of specialized metabolites. We discuss the possible routes involved in the intra- and intercellular translocation of terpenoids and their precursors. This comprehensive proteomic map of the glandular trichomes of hop female flowers represents a valuable resource to improve our knowledge on the function of glandular trichomes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways

    Directory of Open Access Journals (Sweden)

    V. Barquissau

    2016-05-01

    Conclusions: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.

  2. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

    KAUST Repository

    Ross, Avena C.

    2013-01-23

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

  3. Chitosan oligosaccharide and salicylic acid up-regulate gene expression differently in relation to the biosynthesis of artemisinin in Artemisia annua L

    DEFF Research Database (Denmark)

    Yin, Heng; Kjær, Anders; Fretté, Xavier

    2012-01-01

    oligosaccharide (COS) and salicylic acid (SA) on both artemisinin production and gene expression related to the biosynthetic pathway of artemisinin. COS up-regulated the transcriptional levels of the genes ADS and TTG1 2.5 fold and 1.8 fold after 48 h individually, whereas SA only up-regulated ADS 2.0 fold after...

  4. Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment

    Energy Technology Data Exchange (ETDEWEB)

    Ichijo, Yuta; Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555 (Japan); Satou, Kazuhiro; Negishi, Jun [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Itabashi-Ku, Tokyo 173-8605 (Japan); Oshima, Natsuki [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Mogi, Chihiro; Sato, Koichi [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555 (Japan); Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

    2016-01-01

    Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the G{sub s}-protein/cAMP/CRE, G{sub 12/13}-protein/Rho/SRE, and G{sub q}-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174{sup th} position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A. - Highlights: • Zebrafish two G2A homologs are proton

  5. Effect of okadaic acid on cultured clam heart cells: involvement of MAPkinase pathways

    Directory of Open Access Journals (Sweden)

    Houda Hanana

    2012-09-01

    Okadaic acid (OA is one of the main diarrhetic shellfish poisoning toxins and a potent inhibitor of protein phosphatases 1 and 2A. The downstream signal transduction pathways following the protein phosphatase inhibition are still unknown and the results of most of the previous studies are often conflicting. The aim of the present study was to evaluate the effects of OA on heart clam cells and to analyse its possible mechanisms of action by investigating the signal transduction pathways involved in OA cytotoxicity. We showed that OA at 1 µM after 24 h of treatment induces disorganization of the actin cytoskeleton, rounding and detachment of fibroblastic cells. Moreover, treatment of heart cells revealed a sequential activation of MAPK proteins depending on the OA concentration. We suggest that the duration of p38 and JNK activation is a critical factor in determining cell apoptosis in clam cardiomyocytes. In the opposite, ERK activation could be involved in cell survival. The cell death induced by OA is a MAPK modulated pathway, mediated by caspase 3-dependent mechanism. OA was found to induce no significant effect on spontaneous beating rate or inward L-type calcium current in clam cardiomyocytes, suggesting that PP1 was not inhibited even by the highest dose of OA.

  6. Effect of okadaic acid on cultured clam heart cells: involvement of MAPkinase pathways.

    Science.gov (United States)

    Hanana, Houda; Talarmin, Hélène; Pennec, Jean-Pierre; Droguet, Mickael; Morel, Julie; Dorange, Germaine

    2012-12-15

    Okadaic acid (OA) is one of the main diarrhetic shellfish poisoning toxins and a potent inhibitor of protein phosphatases 1 and 2A. The downstream signal transduction pathways following the protein phosphatase inhibition are still unknown and the results of most of the previous studies are often conflicting. The aim of the present study was to evaluate the effects of OA on heart clam cells and to analyse its possible mechanisms of action by investigating the signal transduction pathways involved in OA cytotoxicity. We showed that OA at 1 µM after 24 h of treatment induces disorganization of the actin cytoskeleton, rounding and detachment of fibroblastic cells. Moreover, treatment of heart cells revealed a sequential activation of MAPK proteins depending on the OA concentration. We suggest that the duration of p38 and JNK activation is a critical factor in determining cell apoptosis in clam cardiomyocytes. In the opposite, ERK activation could be involved in cell survival. The cell death induced by OA is a MAPK modulated pathway, mediated by caspase 3-dependent mechanism. OA was found to induce no significant effect on spontaneous beating rate or inward L-type calcium current in clam cardiomyocytes, suggesting that PP1 was not inhibited even by the highest dose of OA.

  7. Mitochondrial acetyl-CoA utilization pathway for terpenoid productions.

    Science.gov (United States)

    Yuan, Jifeng; Ching, Chi-Bun

    2016-11-01

    Acetyl-CoA is a central molecule in the metabolism of the cell, which is also a precursor molecule to a variety of value-added products such as terpenoids and fatty acid derived molecules. Considering subcellular compartmentalization of metabolic pathways allows higher concentrations of enzymes, substrates and intermediates, and bypasses competing pathways, mitochondrion-compartmentalized acetyl-CoA utilization pathways might offer better pathway activities with improved product yields. As a proof-of-concept, we sought to explore a mitochondrial farnesyl pyrophosphate (FPP) biosynthetic pathway for the biosynthesis of amorpha-4,11-diene in budding yeast. In the present study, the eight-gene FPP biosynthetic pathway was successfully expressed inside yeast mitochondria to enable high-level amorpha-4,11-diene production. In addition, we also found the mitochondrial compartment serves as a partial barrier for the translocation of FPP from mitochondria into the cytosol, which would potentially allow minimized loss of FPP to cytosolic competing pathways. To our best knowledge, this is the first report to harness yeast mitochondria for terpenoid productions from the mitochondrial acetyl-CoA pool. We envision subcellular metabolic engineering might also be employed for an efficient production of other bio-products from the mitochondrial acetyl-CoA in other eukaryotic organisms. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    2011-01-01

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

  9. Enzyme analyses demonstrate that beta-methylbutyric acid is converted to beta-hydroxy-beta-methylbutyric acid via the leucine catabolic pathway by galactomyces reessii

    Science.gov (United States)

    Lee; Rosazza

    1998-03-01

    Galactomyces reessii accomplishes the enzymatic transformation of beta-methylbutyric acid (isovaleric acid) to beta-hydroxy-beta-methylbutyric acid. The enzymatic basis for this bioconversion was evaluated by analyzing cell-free extracts of G. reessii for enzyme activities commonly associated with leucine catabolism. G. reessii extracts contained activities for acyl-CoA synthetase, acyl-CoA dehydrogenase, and enoyl-CoA hydratase, whereas beta-methylbutyric acid hydroxylase, alpha-ketoisocaproate oxygenase, and acyl-CoA oxidase (with isovaleryl-CoA as substrate) were not observed. Furthermore, beta-methylbutyric acid is initially activated to isovaleryl-CoA by acyl-CoA synthetase, dehydrogenated to methylcrotonyl-CoA by acyl-CoA dehydrogenase, hydrated to beta-hydroxy-beta-methylbutyric acid-CoA by enoyl-CoA hydratase, and hydrolyzed to beta-hydroxy-beta-methylbutyric acid in G. reessii extracts. Cell-free extracts converted both isovaleryl-CoA and methylcrotonyl-CoA into beta-hydroxy-beta-methylbutyric acid, thus demonstrating that beta-methylbutyric acid is part of the leucine catabolic pathway. The rate of beta-methylbutyric acid conversion to beta-hydroxy-beta-methylbutyric acid with cell-free extract was 0. 013 &mgr;mol beta-hydroxy-beta-methylbutyric acid (mg protein)-1 h-1, while the conversion rate of leucine was fivefold lower. With whole cells, the highest production rate [0.042 &mgr;mol beta-hydroxy-beta-methylbutyric acid (g cells)-1 h-1] was also observed with beta-methylbutyric acid. The results indicate that beta-methylbutyric acid is transformed to beta-hydroxy-beta-methylbutyric acid through the leucine catabolic pathway.

  10. Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berries

    Directory of Open Access Journals (Sweden)

    Soole Kathleen L

    2009-12-01

    Full Text Available Abstract Background Fresh fruits are well accepted as a good source of the dietary antioxidant ascorbic acid (Asc, Vitamin C. However, fruits such as grapes do not accumulate exceptionally high quantities of Asc. Grapes, unlike most other cultivated fruits do however use Asc as a precursor for the synthesis of both oxalic (OA and tartaric acids (TA. TA is a commercially important product in the wine industry and due to its acidifying effect on crushed juice it can influence the organoleptic properties of the wine. Despite the interest in Asc accumulation in fruits, little is known about the mechanisms whereby Asc concentration is regulated. The purpose of this study was to gain insights into Asc metabolism in wine grapes (Vitis vinifera c.v. Shiraz. and thus ascertain whether the developmental demand for TA and OA synthesis influences Asc accumulation in the berry. Results We provide evidence for developmentally differentiated up-regulation of Asc biosynthetic pathways and subsequent fluctuations in Asc, TA and OA accumulation. Rapid accumulation of Asc and a low Asc to dehydroascorbate (DHA ratio in young berries was co-ordinated with up-regulation of three of the primary Asc biosynthetic (Smirnoff-Wheeler pathway genes. Immature berries synthesised Asc in-situ from the primary pathway precursors D-mannose and L-galactose. Immature berries also accumulated TA in early berry development in co-ordination with up-regulation of a TA biosynthetic gene. In contrast, ripe berries have up-regulated expression of the alternative Asc biosynthetic pathway gene D-galacturonic acid reductase with only residual expression of Smirnoff-Wheeler Asc biosynthetic pathway genes and of the TA biosynthetic gene. The ripening phase was further associated with up-regulation of Asc recycling genes, a secondary phase of increased accumulation of Asc and an increase in the Asc to DHA ratio. Conclusion We demonstrate strong developmental regulation of Asc biosynthetic

  11. Pathway engineering of Propionibacterium jensenii for improved production of propionic acid.

    Science.gov (United States)

    Liu, Long; Guan, Ningzi; Zhu, Gexin; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian

    2016-01-27

    Propionic acid (PA) is an important chemical building block widely used in the food, pharmaceutical, and chemical industries. In our previous study, a shuttle vector was developed as a useful tool for engineering Propionibacterium jensenii, and two key enzymes-glycerol dehydrogenase and malate dehydrogenase-were overexpressed to improve PA titer. Here, we aimed to improve PA production further via the pathway engineering of P. jensenii. First, the phosphoenolpyruvate carboxylase gene (ppc) from Klebsiella pneumoniae was overexpressed to access the one-step synthesis of oxaloacetate directly from phosphoenolpyruvate without pyruvate as intermediate. Next, genes encoding lactate dehydrogenase (ldh) and pyruvate oxidase (poxB) were deleted to block the synthesis of the by-products lactic acid and acetic acid, respectively. Overexpression of ppc and deleting ldh improved PA titer from 26.95 ± 1.21 g·L(-1) to 33.21 ± 1.92 g·L(-1) and 30.50 ± 1.63 g·L(-1), whereas poxB deletion decreased it. The influence of this pathway engineering on gene transcription, enzyme expression, NADH/NAD(+) ratio, and metabolite concentration was also investigated. Finally, PA production in P. jensenii with ppc overexpression as well as ldh deletion was investigated, which resulted in further increases in PA titer to 34.93 ± 2.99 g·L(-1) in a fed-batch culture.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  13. Drosophila fatty acid taste signals through the PLC pathway in sugar-sensing neurons.

    Science.gov (United States)

    Masek, Pavel; Keene, Alex C

    2013-01-01

    Taste is the primary sensory system for detecting food quality and palatability. Drosophila detects five distinct taste modalities that include sweet, bitter, salt, water, and the taste of carbonation. Of these, sweet-sensing neurons appear to have utility for the detection of nutritionally rich food while bitter-sensing neurons signal toxicity and confer repulsion. Growing evidence in mammals suggests that taste for fatty acids (FAs) signals the presence of dietary lipids and promotes feeding. While flies appear to be attracted to fatty acids, the neural basis for fatty acid detection and attraction are unclear. Here, we demonstrate that a range of FAs are detected by the fly gustatory system and elicit a robust feeding response. Flies lacking olfactory organs respond robustly to FAs, confirming that FA attraction is mediated through the gustatory system. Furthermore, flies detect FAs independent of pH, suggesting the molecular basis for FA taste is not due to acidity. We show that low and medium concentrations of FAs serve as an appetitive signal and they are detected exclusively through the same subset of neurons that sense appetitive sweet substances, including most sugars. In mammals, taste perception of sweet and bitter substances is dependent on phospholipase C (PLC) signaling in specialized taste buds. We find that flies mutant for norpA, a Drosophila ortholog of PLC, fail to respond to FAs. Intriguingly, norpA mutants respond normally to other tastants, including sucrose and yeast. The defect of norpA mutants can be rescued by selectively restoring norpA expression in sweet-sensing neurons, corroborating that FAs signal through sweet-sensing neurons, and suggesting PLC signaling in the gustatory system is specifically involved in FA taste. Taken together, these findings reveal that PLC function in Drosophila sweet-sensing neurons is a conserved molecular signaling pathway that confers attraction to fatty acids.

  14. Drosophila fatty acid taste signals through the PLC pathway in sugar-sensing neurons.

    Directory of Open Access Journals (Sweden)

    Pavel Masek

    Full Text Available Taste is the primary sensory system for detecting food quality and palatability. Drosophila detects five distinct taste modalities that include sweet, bitter, salt, water, and the taste of carbonation. Of these, sweet-sensing neurons appear to have utility for the detection of nutritionally rich food while bitter-sensing neurons signal toxicity and confer repulsion. Growing evidence in mammals suggests that taste for fatty acids (FAs signals the presence of dietary lipids and promotes feeding. While flies appear to be attracted to fatty acids, the neural basis for fatty acid detection and attraction are unclear. Here, we demonstrate that a range of FAs are detected by the fly gustatory system and elicit a robust feeding response. Flies lacking olfactory organs respond robustly to FAs, confirming that FA attraction is mediated through the gustatory system. Furthermore, flies detect FAs independent of pH, suggesting the molecular basis for FA taste is not due to acidity. We show that low and medium concentrations of FAs serve as an appetitive signal and they are detected exclusively through the same subset of neurons that sense appetitive sweet substances, including most sugars. In mammals, taste perception of sweet and bitter substances is dependent on phospholipase C (PLC signaling in specialized taste buds. We find that flies mutant for norpA, a Drosophila ortholog of PLC, fail to respond to FAs. Intriguingly, norpA mutants respond normally to other tastants, including sucrose and yeast. The defect of norpA mutants can be rescued by selectively restoring norpA expression in sweet-sensing neurons, corroborating that FAs signal through sweet-sensing neurons, and suggesting PLC signaling in the gustatory system is specifically involved in FA taste. Taken together, these findings reveal that PLC function in Drosophila sweet-sensing neurons is a conserved molecular signaling pathway that confers attraction to fatty acids.

  15. Phenotypic, genetic and molecular characterization of a maize low phytic acid mutant (lpa241)

    DEFF Research Database (Denmark)

    Pilu, R.; Panzeri, D.; Gavazzi, G.

    2003-01-01

    90% reduction of phytic acid and about a tenfold increase in seed-free phosphate content. Although germination rate was decreased by about 30% compared to wild-type, developement of mutant plants was apparentely unaffected. The results of the genetic, biochemical and molecular characterization...... experiments carried out by SSR mapping, MDD-HPLC and RT-PCR are consistent with a mutation affecting the MIPS1S gene, coding for the first enzyme of the phytic acid biosynthetic pathway....

  16. Rosmarinic acid stimulates liver regeneration through the mTOR pathway.

    Science.gov (United States)

    Lou, Kaihan; Yang, Min; Duan, Erdan; Zhao, Jiahui; Yu, Cong; Zhang, Rongping; Zhang, Lanchun; Zhang, Ming; Xiao, Zhicheng; Hu, Weiyan; He, Zhiyong

    2016-12-01

    Rosemary (Rosmarinus offcinsalis L) has a liver protection function under various conditions of liver damage. Rosmarinic acid, one of the pharmacological constituents of rosemary, exhibited protective effects against organ injury, including acute liver injury. We hypothesize that RA stimulates liver regeneration. In the present study, we investigated the effects and mechanism of RA administration on liver regeneration using partial hepatectomy (PH), a well-validated liver regeneration model in mice. We use a 2/3 partial hepatectomy (PH) model to induce liver regeneration. RA was administered prior to and simultaneously with PH. The regeneration process was estimated by the index of the liver to body weight (ILBW) and the expression of proliferating cell nuclear antigen (PCNA) and liver transaminases. The administration of rosmarinic acid stimulated hepatocyte proliferation based on activation of the mTOR/S6K pathway. Rosmarinic acid treatment also rescued impaired liver function due to PH. These data demonstrate that RA is potentially useful to promote liver regeneration. Copyright © 2016 Elsevier GmbH. All rights reserved.

  17. The Arabidopsis DELAYED DEHISCENCE1 Gene Encodes an Enzyme in the Jasmonic Acid Synthesis Pathway

    Science.gov (United States)

    Sanders, Paul M.; Lee, Pei Yun; Biesgen, Christian; Boone, James D.; Beals, Thomas P.; Weiler, Elmar W.; Goldberg, Robert B.

    2000-01-01

    delayed dehiscence1 is an Arabidopsis T-DNA mutant in which anthers release pollen grains too late for pollination to occur. The delayed dehiscence1 defect is caused by a delay in the stomium degeneration program. The gene disrupted in delayed dehiscence1 encodes 12-oxophytodienoate reductase, an enzyme in the jasmonic acid biosynthesis pathway. We rescued the mutant phenotype by exogenous application of jasmonic acid and obtained seed set from previously male-sterile plants. In situ hybridization studies showed that during the early stages of floral development, DELAYED DEHISCENCE1 mRNA accumulated within all floral organs. Later, DELAYED DEHISCENCE1 mRNA accumulated specifically within the pistil, petals, and stamen filaments. DELAYED DEHISCENCE1 mRNA was not detected in the stomium and septum cells of the anther that are involved in pollen release. The T-DNA insertion in delayed dehiscence1 eliminated both DELAYED DEHISCENCE1 mRNA accumulation and 12-oxophytodienoate reductase activity. These experiments suggest that jasmonic acid signaling plays a role in controlling the time of anther dehiscence within the flower. PMID:10899973

  18. Nitrate reduction pathway in an anaerobic acidification reactor and its effect on acid fermentation.

    Science.gov (United States)

    Xie, Li; Ji, Chi; Wang, Rui; Zhou, Qi

    2015-01-01

    This study investigated the performance of a reactor in which denitrification was integrated into the anaerobic acidogenic process. Industrial wastewater cassava stillage was used as the carbon source, and the nitrate reduction pathway and its effects on acid fermentation were examined. Results from batch and semi-continuous tests showed that the presence of nitrate did not inhibit anaerobic acidification but altered the distribution of volatile fatty acid (VFA) species. Nitrate reduction was attributable to denitrification and to dissimilatory nitrate reduction to ammonia (DNRA). The ratio of DNRA to denitrification was proportional to the ratio of [Formula: see text] . After 130 days of semi-continuous operation, denitrification removal efficiency accounted for about 60% at a [Formula: see text] of 50. The proportional distribution of VFAs was acetate, followed by propionate and then butyrate. The polymerase chain reaction-denaturing gradient gel electrophoresis results confirmed the contributions of denitrification and DNRA in the nitrate-amended reactor and showed that the addition of nitrate enriched the structure of the bacterial community, but did not suppress the activity of acid-producing bacteria. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Ketogenic essential amino acids modulate lipid synthetic pathways and prevent hepatic steatosis in mice.

    Directory of Open Access Journals (Sweden)

    Yasushi Noguchi

    2010-08-01

    Full Text Available Although dietary ketogenic essential amino acid (KAA content modifies accumulation of hepatic lipids, the molecular interactions between KAAs and lipid metabolism are yet to be fully elucidated.We designed a diet with a high ratio (E/N of essential amino acids (EAAs to non-EAAs by partially replacing dietary protein with 5 major free KAAs (Leu, Ile, Val, Lys and Thr without altering carbohydrate and fat content. This high-KAA diet was assessed for its preventive effects on diet-induced hepatic steatosis and whole-animal insulin resistance. C57B6 mice were fed with a high-fat diet, and hyperinsulinemic ob/ob mice were fed with a high-fat or high-sucrose diet. The high-KAA diet improved hepatic steatosis with decreased de novo lipogenesis (DNL fluxes as well as reduced expressions of lipogenic genes. In C57B6 mice, the high-KAA diet lowered postprandial insulin secretion and improved glucose tolerance, in association with restored expression of muscle insulin signaling proteins repressed by the high-fat diet. Lipotoxic metabolites and their synthetic fluxes were also evaluated with reference to insulin resistance. The high-KAA diet lowered muscle and liver ceramides, both by reducing dietary lipid incorporation into muscular ceramides and preventing incorporation of DNL-derived fatty acids into hepatic ceramides.Our results indicate that dietary KAA intake improves hepatic steatosis and insulin resistance by modulating lipid synthetic pathways.

  20. Aquaglyceroporins Are the Entry Pathway of Boric Acid in Trypanosoma brucei.

    Science.gov (United States)

    Marsiccobetre, Sabrina; Rodríguez-Acosta, Alexis; Lang, Florian; Figarella, Katherine; Uzcátegui, Néstor L

    2017-05-01

    The boron element possesses a range of different effects on living beings. It is essential to beneficial at low concentrations, but toxic at excessive concentrations. Recently, some boron-based compounds have been identified as promising molecules against Trypanosoma brucei, the causative agent of sleeping sickness. However, until now, the boron metabolism and its access route into the parasite remained elusive. The present study addressed the permeability of T. brucei aquaglyceroporins (TbAQPs) for boric acid, the main natural boron species. To this end, the three TbAQPs were expressed in Saccharomyces cerevisiae and Xenopus laevis oocytes. Our findings in both expression systems showed that all three TbAQPs are permeable for boric acid. Especially TbAQP2 is highly permeable for this compound, displaying one of the highest conductances reported for a solute in these channels. Additionally, T. brucei aquaglyceroporin activities were sensitive to pH. Taken together, these results establish that TbAQPs are channels for boric acid and are highly efficient entry pathways for boron into the parasite. Our findings stress the importance of studying the physiological functions of boron and their derivatives in T. brucei, as well as the pharmacological implications of their uptake by trypanosome aquaglyceroporins. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Salicylic-Acid-Induced Chilling- and Oxidative-Stress Tolerance in Relation to Gibberellin Homeostasis, C-Repeat/Dehydration-Responsive Element Binding Factor Pathway, and Antioxidant Enzyme Systems in Cold-Stored Tomato Fruit.

    Science.gov (United States)

    Ding, Yang; Zhao, Jinhong; Nie, Ying; Fan, Bei; Wu, Shujuan; Zhang, Yu; Sheng, Jiping; Shen, Lin; Zhao, Ruirui; Tang, Xuanming

    2016-11-02

    Effects of salicylic acid (SA) on gibberellin (GA) homeostasis, C-repeat/dehydration-responsive element binding factor (CBF) pathway, and antioxidant enzyme systems linked to chilling- and oxidative-stress tolerance in tomato fruit were investigated. Mature green tomatoes (Solanum lycopersicum L. cv. Moneymaker) were treated with 0, 0.5, and 1 mM SA solution for 15 min before storage at 4 °C for 28 days. In comparison to 0 or 0.5 mM SA, 1 mM SA significantly decreased the chilling injury (CI) index in tomato fruit. In the SA-treated fruit, the upregulation of GA biosynthetic gene (GA3ox1) expression was followed by gibberellic acid (GA3) surge and DELLA protein degradation. CBF1 participated in the SA-modulated tolerance and stimulated the expression of GA catabolic gene (GA2ox1). Furthermore, 1 mM SA enhanced activities of antioxidant enzymes and, thus, reduced reactive oxygen species accumulation. Our findings suggest that SA might protect tomato fruit from CI and oxidative damage through regulating GA metabolism, CBF1 gene expression, and antioxidant enzyme activities.

  2. Palmitic acid interferes with energy metabolism balance by adversely switching the SIRT1-CD36-fatty acid pathway to the PKC zeta-GLUT4-glucose pathway in cardiomyoblasts.

    Science.gov (United States)

    Chen, Yeh-Peng; Tsai, Chia-Wen; Shen, Chia-Yao; Day, Cecilia-Hsuan; Yeh, Yu-Lan; Chen, Ray-Jade; Ho, Tsung-Jung; Padma, V Vijaya; Kuo, Wei-Wen; Huang, Chih-Yang

    2016-05-01

    Metabolic regulation is inextricably linked with cardiac function. Fatty acid metabolism is a significant mechanism for creating energy for the heart. However, cardiomyocytes are able to switch the fatty acids or glucose, depending on different situations, such as ischemia or anoxia. Lipotoxicity in obesity causes impairments in energy metabolism and apoptosis in cardiomyocytes. We utilized the treatment of H9c2 cardiomyoblast cells palmitic acid (PA) as a model for hyperlipidemia to investigate the signaling mechanisms involved in these processes. Our results show PA induces time- and dose-dependent lipotoxicity in H9c2 cells. Moreover, PA enhances cluster of differentiation 36 (CD36) and reduces glucose transporter type 4 (GLUT4) pathway protein levels following a short period of treatment, but cells switch from CD36 back to the GLUT4 pathway after during long-term exposure to PA. As sirtuin 1 (SIRT1) and protein kinase Cζ (PKCζ) play important roles in CD36 and GLUT4 translocation, we used the SIRT1 activator resveratrol and si-PKCζ to identify the switches in metabolism. Although PA reduced CD36 and increased GLUT4 metabolic pathway proteins, when we pretreated cells with resveratrol to activate SIRT1 or transfected si-PKCζ, both were able to significantly increase CD36 metabolic pathway proteins and reduce GLUT4 pathway proteins. High-fat diets affect energy metabolism pathways in both normal and aging rats and involve switching the energy source from the CD36 pathway to GLUT4. In conclusion, PA and high-fat diets cause lipotoxicity in vivo and in vitro and adversely switch the energy source from the CD36 pathway to the GLUT4 pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress.

    Science.gov (United States)

    Roychoudhury, Aryadeep; Paul, Saikat; Basu, Supratim

    2013-07-01

    Salinity, drought and low temperature are the common forms of abiotic stress encountered by land plants. To cope with these adverse environmental factors, plants execute several physiological and metabolic responses. Both osmotic stress (elicited by water deficit or high salt) and cold stress increase the endogenous level of the phytohormone abscisic acid (ABA). ABA-dependent stomatal closure to reduce water loss is associated with small signaling molecules like nitric oxide, reactive oxygen species and cytosolic free calcium, and mediated by rapidly altering ion fluxes in guard cells. ABA also triggers the expression of osmotic stress-responsive (OR) genes, which usually contain single/multiple copies of cis-acting sequence called abscisic acid-responsive element (ABRE) in their upstream regions, mostly recognized by the basic leucine zipper-transcription factors (TFs), namely, ABA-responsive element-binding protein/ABA-binding factor. Another conserved sequence called the dehydration-responsive element (DRE)/C-repeat, responding to cold or osmotic stress, but not to ABA, occurs in some OR promoters, to which the DRE-binding protein/C-repeat-binding factor binds. In contrast, there are genes or TFs containing both DRE/CRT and ABRE, which can integrate input stimuli from salinity, drought, cold and ABA signaling pathways, thereby enabling cross-tolerance to multiple stresses. A strong candidate that mediates such cross-talk is calcium, which serves as a common second messenger for abiotic stress conditions and ABA. The present review highlights the involvement of both ABA-dependent and ABA-independent signaling components and their interaction or convergence in activating the stress genes. We restrict our discussion to salinity, drought and cold stress.

  4. The Expression Analysis of Genes in Fatty Acid Biosynthesis Pathway during the Seed Development of Tung Tree

    Directory of Open Access Journals (Sweden)

    Guixiong Jiang

    2013-07-01

    Full Text Available Tung tree (Vernicia fordii is one of the important oil plants in China. The current researches on tung tree are mainly focused on the cultivation and breeding while the molecular mechanisms hidden in the back of tung tree's phenotypes are still uncovered. This research compared the transcriptome of three different stages during tung tree's seed development using RNA-Seq and obtained a lot of differentially expressed Unigenes. Through GO classification and pathway enrichment analysis, all of these differentially expressed Unigenes were classified into 128 signaling pathways including fatty acid biosynthesis. Fourteen homologous proteins were obtained when the sequences of 54 Unigenes within fatty acid biosynthesis pathway were aligned against KEGG database and the expression profiles of the genes encoding these proteins during seed development were analyzed. This research provides necessary data platform to elucidate the molecular mechanism underlies fatty acid biosynthesis of tung tree's seeds and theoretical guidance of tung tree varieties' improvement to increase output of tung oil.

  5. Studies on the Biochemical Formation Pathway of the Amino Acid l-Theanine in Tea (Camellia sinensis) and Other Plants.

    Science.gov (United States)

    Cheng, Sihua; Fu, Xiumin; Wang, Xiaoqin; Liao, Yinyin; Zeng, Lanting; Dong, Fang; Yang, Ziyin

    2017-08-23

    Tea (Camellia sinensis) is the most widely consumed beverage aside from water. The flavor of tea is conferred by certain metabolites, especially l-theanine, in C. sinensis. To determine why more l-theanine accumulates in C. sinensis than in other plants, we compare l-theanine contents between C. sinensis and other plant species (Camellia nitidissima, Camellia japonica, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum) and use a stable isotope labeling approach to elucidate its biosynthetic route. We quantify relevant intermediates and metabolites by mass spectrometry. l-Glutamic acid, a precursor of l-theanine, is present in most plants, while ethylamine, another precursor of l-theanine, specifically accumulates in Camellia species, especially C. sinensis. Most plants contain the enzyme/gene catalyzing the conversion of ethylamine and l-glutamic acid to l-theanine. After supplementation with [ 2 H 5 ]ethylamine, all the plants produce [ 2 H 5 ]l-theanine, which suggests that ethylamine availability is the reason for the difference in l-theanine accumulation between C. sinensis and other plants.

  6. Maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase.

    Science.gov (United States)

    Liu, Jun; Wang, Xue; Chen, Yu-Peng; Mao, Li-Fei; Shang, Jing; Sun, Hong-Bin; Zhang, Lu-Yong

    2014-04-01

    To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid (MA) exerts anti-diabetic effects. HepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a (GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β (IRβ), protein kinase B (also known as Akt), and glycogen synthase kinase-3β (GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet (HFD). The results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. The results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  7. The synthetic xylulose-1 phosphate pathway increases production of glycolic acid from xylose-rich sugar mixtures

    OpenAIRE

    Trichez, Débora; Cam, Yvan; Spina, Lucie; François, Jean-Marie

    2016-01-01

    Background: Glycolic acid (GA) is a two-carbon hydroxyacid with applications in the cosmetic, textile, and medical industry. Microbial GA production from all sugars can be achieved by engineering the natural glyoxylate shunt. The synthetic (D)-xylulose-1 phosphate (X1P) pathway provides a complementary route to produce GA from (D)-xylose. The simultaneous operation of the X1P and glyoxylate pathways increases the theoretical GA yield from xylose by 20 %, which may strongly improve GA producti...

  8. Abscisic acid interacts antagonistically with classical defense pathways in rice-migratory nematode interaction.

    Science.gov (United States)

    Nahar, Kamrun; Kyndt, Tina; Nzogela, Yasinta Beda; Gheysen, Godelieve

    2012-11-01

    Studies involving plant-nematode interactions provide an opportunity to unravel plant defense signaling in root tissues. In this study, we have characterized the roles of salicylate (SA), jasmonate (JA), ethylene (ET) and abscisic acid (ABA) in plant defense against the migratory nematode Hirschmanniella oryzae in the monocot model plant rice (Oryza sativa). Experiments with exogenous hormone applications, biosynthesis inhibition and mutant/transgenic lines were executed to test the effect on H. oryzae parasitism in rice roots. Our results demonstrate that an intact ET, JA and SA biosynthesis pathway is a prerequisite for defense against H. oryzae. By contrast, exogenous ABA treatment drastically compromised the rice defense towards this nematode. Gene expression analyses using quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrate that the disease-inducing effect of ABA is likely to be the result of an antagonistic interaction between this hormone and the SA/JA/ET-dependent basal defense system. Collectively, in rice defense against H. oryzae, at least three pathways, namely SA, JA and ET, are important, while ABA plays a negative role in defense. Our results suggest that the balance of ABA and SA/JA/ET signaling is an important determinant for the outcome of the rice-H. oryzae interaction. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

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

    Science.gov (United States)

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

    2014-06-01

    Photosynthesis in "green" seeds, such as rapeseed, soybean, and Arabidopsis, plays a substantial role in the improved efficiency of oil accumulation. However, the molecular mechanism underpinning the coordinated expression of fatty acid (FA) biosynthesis- and photosynthesis-related genes in such developing seeds remains to be elucidated. Here, we found that seed-specific overexpression of BnWRI1, a WRI1 homolog from rapeseed (Brassica napus cv. ZGY2), results in enhanced chlorophyll content in developing seeds and increased oil content and seed mass in matured seeds. BnWRI1 was co-expressed with BnBCCP and BnCAB, two marker genes of FA biosynthesis and photosynthesis during seed development, respectively. Overexpression of BnWRI1 increased expression of both marker genes. Further, the nuclear-localized BnWRI1 protein was found to act as a transcription activator. It could bind to the GT1-element and/or GCC-box, which are widespread in the upstream regions of genes involved in FA biosynthesis and photosynthesis pathways. Accordingly, BnWRI1 could interact with promoters of BCCP2 and LHB1B2 in vivo. These results suggested that BnWRI1 may coordinate FA biosynthesis and photosynthesis pathways in developing seeds via directly stimulating expression of GT1-element and/or GCC-box containing genes. © 2014 Institute of Botany, Chinese Academy of Sciences.

  10. Transformation of halobenzoquinones with the presence of amino acids in water: Products, pathways and toxicity.

    Science.gov (United States)

    Du, Penghui; Zhao, He; Cao, Hongbin; Huang, Ching-Hua; Liu, Wen; Li, Yao

    2017-10-01

    The transformation and detoxification of halobenzoquinones (HBQs), a class of emerging disinfection byproducts (DBPs), was studied in the presence of amino acids (AAs). The reaction activity of three HBQs with AAs generally ranked as 2-chlorobenzoquinone (CBQ) density functional theory (DFT) calculations, AAs can easily covalently incorporate into HBQs via nucleophilic addition (CBQ and DCBQ) or substitution (TCBQ) through CNC or CSC linkages. Hydroxylation, nucleophilic reaction and decarboxylation were proposed to be the three major reaction pathways for HBQs transformation with AAs. HBQs firstly underwent the spontaneous hydrolysis, resulting in OH-HBQs formation. Then, nucleophilic addition/substitution of AAs occurred on HBQs and OH-HBQs to produce AA-HBQs/AA-HBQs-OH adducts. These adducts were subsequently oxidized into their corresponding decarboxylated forms. Based on the results of Luminous bacterium Q67 acute toxicity test, the toxicity of HBQs solution greatly decreased with AAs presented. The toxicity change was well explained by the lowest unoccupied molecular orbital energy (ELUMO) of formed products. Notably, the step that AAs nucleophilic bonded with HBQs led to the highest rise of ELUMO, which should be the most effective pathway for HBQs detoxification. This study shows that binding with amino nitrogen compounds should be an important process for HBQs transformation and detoxification, which helps to better understand the fate of this typical DBP in surface water. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Acidic Fibroblast Growth Factor Promotes Endothelial Progenitor Cells Function via Akt/FOXO3a Pathway.

    Directory of Open Access Journals (Sweden)

    Liya Huang

    Full Text Available Acidic fibroblast growth factor (FGF1 has been suggested to enhance the functional activities of endothelial progenitor cells (EPCs. The Forkhead homeobox type O transcription factors (FOXOs, a key substrate of the survival kinase Akt, play important roles in regulation of various cellular processes. We previously have shown that FOXO3a is the main subtype of FOXOs expressed in EPCs. Here, we aim to determine whether FGF1 promotes EPC function through Akt/FOXO3a pathway. Human peripheral blood derived EPCs were transduced with adenoviral vectors either expressing a non-phosphorylable, constitutively active triple mutant of FOXO3a (Ad-TM-FOXO3a or a GFP control (Ad-GFP. FGF1 treatment improved functional activities of Ad-GFP transduced EPCs, including cell viability, proliferation, antiapoptosis, migration and tube formation, whereas these beneficial effects disappeared by Akt inhibitor pretreatment. Moreover, EPC function was declined by Ad-TM-FOXO3a transduction and failed to be attenuated even with FGF1 treatment. FGF1 upregulated phosphorylation levels of Akt and FOXO3a in Ad-GFP transduced EPCs, which were repressed by Akt inhibitor pretreatment. However, FGF1 failed to recover Ad-TM-FOXO3a transduced EPCs from dysfunction. These data indicate that FGF1 promoting EPC function is at least in part mediated through Akt/FOXO3a pathway. Our study may provide novel ideas for enhancing EPC angiogenic ability and optimizing EPC transplantation therapy in the future.

  12. Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.

    Science.gov (United States)

    Kildegaard, Kanchana R; Jensen, Niels B; Schneider, Konstantin; Czarnotta, Eik; Özdemir, Emre; Klein, Tobias; Maury, Jérôme; Ebert, Birgitta E; Christensen, Hanne B; Chen, Yun; Kim, Il-Kwon; Herrgård, Markus J; Blank, Lars M; Forster, Jochen; Nielsen, Jens; Borodina, Irina

    2016-03-15

    In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacs (L641P). Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L(-1) 3HP with a yield of 13% C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by (13)C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents

  13. The bile acid chenodeoxycholic acid directly modulates metabolic pathways in white adipose tissue in vitro: insight into how bile acids decrease obesity.

    Science.gov (United States)

    Teodoro, João Soeiro; Rolo, Anabela Pinto; Jarak, Ivana; Palmeira, Carlos Marques; Carvalho, Rui Albuquerque

    2016-10-01

    Obesity is a worldwide epidemic, and associated pathologies, including type 2 diabetes and cardiovascular alterations, are increasingly escalating morbidity and mortality. Despite intensive study, no effective simple treatment for these conditions exists. As such, the need for go-to drugs is serious. Bile acids (BAs) present the possibility of reversing these problems, as various in vivo studies and clinical trials have shown significant effects with regard to weight and obesity reduction, insulin sensitivity restoration and cardiovascular improvements. However, the mechanism of action of BA-induced metabolic improvement has yet to be fully established. The currently most accepted model involves non-shivering thermogenesis for energy waste, but this is disputed. As such, we propose to determine whether the BA chenodeoxycholic acid (CDCA) can exert anti-obesogenic effects in vitro, independent of thermogenic brown adipose tissue activation. By exposing differentiated 3 T3-L1 adipocytes to high glucose and CDCA, we demonstrate that this BA has anti-obesity effects in vitro. Nuclear magnetic resonance spectroscopic analysis of metabolic pathways clearly indicates an improvement in metabolic status, as these cells become more oxidative rather than glycolytic, which may be associated with an increase in fatty acid oxidation. Our work demonstrates that CDCA-induced metabolic alterations occur in white and brown adipocytes and are not totally dependent on endocrine/nervous system signaling, as thought until now. Furthermore, future exploration of the mechanisms behind these effects will undoubtedly reveal interesting targets for clinical modulation. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Terpenoid pathway discovery in Tripterygium wilfordii

    DEFF Research Database (Denmark)

    Hansen, Nikolaj Lervad

    . To achieve this feat requires knowledge of the underlying biosynthetic pathways that give rise to the metabolites of interest in the native plants. This thesis presents an investigation of the biosynthetic pathways of medicinal compounds in Tripterygium wilfordii (Celastraceae), a widely used plant...

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Ginkgolic acid suppresses the development of pancreatic cancer by inhibiting pathways driving lipogenesis.

    Science.gov (United States)

    Ma, Jiguang; Duan, Wanxing; Han, Suxia; Lei, Jianjun; Xu, Qinhong; Chen, Xin; Jiang, Zhengdong; Nan, Ligang; Li, Jiahui; Chen, Ke; Han, Liang; Wang, Zheng; Li, Xuqi; Wu, Erxi; Huo, Xiongwei

    2015-08-28

    Ginkgolic acid (GA) is a botanical drug extracted from the seed coat of Ginkgo biloba L. with a wide range of bioactive properties, including anti-tumor effect. However, whether GA has antitumor effect on pancreatic cancer cells and the underlying mechanisms have yet to be investigated. In this study, we show that GA suppressed the viability of cancer cells but has little toxicity on normal cells, e.g, HUVEC cells. Furthermore, treatment of GA resulted in impaired colony formation, migration, and invasion ability and increased apoptosis of cancer cells. In addition, GA inhibited the de novo lipogenesis of cancer cells through inducing activation of AMP-activated protein kinase (AMPK) signaling and downregulated the expression of key enzymes (e.g. acetyl-CoA carboxylase [ACC], fatty acid synthase [FASN]) involved in lipogenesis. Moreover, the in vivo experiment showed that GA reduced the expression of the key enzymes involved in lipogenesis and restrained the tumor growth. Taken together, our results suggest that GA may serve as a new candidate against tumor growth of pancreatic cancer partially through targeting pathway driving lipogenesis.

  18. Investigating sources and pathways of perfluoroalkyl acids (PFAAs) in aquifers in Tokyo using multiple tracers

    Energy Technology Data Exchange (ETDEWEB)

    Kuroda, Keisuke, E-mail: keisukekr@gmail.com [Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656 (Japan); Murakami, Michio [Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8505 (Japan); Oguma, Kumiko [Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656 (Japan); Takada, Hideshige [Laboratory of Organic Geochemistry (LOG), Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509 (Japan); Takizawa, Satoshi [Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656 (Japan)

    2014-08-01

    We employed a multi-tracer approach to investigate sources and pathways of perfluoroalkyl acids (PFAAs) in urban groundwater, based on 53 groundwater samples taken from confined aquifers and unconfined aquifers in Tokyo. While the median concentrations of groundwater PFAAs were several ng/L, the maximum concentrations of perfluorooctane sulfonate (PFOS, 990 ng/L), perfluorooctanoate (PFOA, 1800 ng/L) and perfluorononanoate (PFNA, 620 ng/L) in groundwater were several times higher than those of wastewater and street runoff reported in the literature. PFAAs were more frequently detected than sewage tracers (carbamazepine and crotamiton), presumably owing to the higher persistence of PFAAs, the multiple sources of PFAAs beyond sewage (e.g., surface runoff, point sources) and the formation of PFAAs from their precursors. Use of multiple methods of source apportionment including principal component analysis–multiple linear regression (PCA–MLR) and perfluoroalkyl carboxylic acid ratio analysis highlighted sewage and point sources as the primary sources of PFAAs in the most severely polluted groundwater samples, with street runoff being a minor source (44.6% sewage, 45.7% point sources and 9.7% street runoff, by PCA–MLR). Tritium analysis indicated that, while young groundwater (recharged during or after the 1970s, when PFAAs were already in commercial use) in shallow aquifers (< 50 m depth) was naturally highly vulnerable to PFAA pollution, PFAAs were also found in old groundwater (recharged before the 1950s, when PFAAs were not in use) in deep aquifers (50–500 m depth). This study demonstrated the utility of multiple uses of tracers (pharmaceuticals and personal care products; PPCPs, tritium) and source apportionment methods in investigating sources and pathways of PFAAs in multiple aquifer systems. - Highlights: • Aquifers in Tokyo had high levels of perfluoroalkyl acids (up to 1800 ng/L). • PFAAs were more frequently detected than sewage

  19. Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Mononucleotide 5′-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside*

    OpenAIRE

    Bogan, Katrina L.; Evans, Charles; Belenky, Peter; Song, Peng; Burant, Charles F.; Kennedy, Robert; Brenner, Charles

    2009-01-01

    Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD+, which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast...

  20. Identification and analysis of the paulomycin biosynthetic gene cluster and titer improvement of the paulomycins in Streptomyces paulus NRRL 8115.

    Directory of Open Access Journals (Sweden)

    Jine Li

    Full Text Available The paulomycins are a group of glycosylated compounds featuring a unique paulic acid moiety. To locate their biosynthetic gene clusters, the genomes of two paulomycin producers, Streptomyces paulus NRRL 8115 and Streptomyces sp. YN86, were sequenced. The paulomycin biosynthetic gene clusters were defined by comparative analyses of the two genomes together with the genome of the third paulomycin producer Streptomyces albus J1074. Subsequently, the identity of the paulomycin biosynthetic gene cluster was confirmed by inactivation of two genes involved in biosynthesis of the paulomycose branched chain (pau11 and the ring A moiety (pau18 in Streptomyces paulus NRRL 8115. After determining the gene cluster boundaries, a convergent biosynthetic model was proposed for paulomycin based on the deduced functions of the pau genes. Finally, a paulomycin high-producing strain was constructed by expressing an activator-encoding gene (pau13 in S. paulus, setting the stage for future investigations.

  1. Fusarium Mycotoxins: Biosynthetic Pathways and Role in Virulence

    Science.gov (United States)

    Fusarium head blight (FHB) of wheat and barley is a devastating disease that has reached global proportions. Not only does this disease result in lower yields, but the mycotoxins produced by the fungus affect the quality of the grain. Fusarium sp. can produce a number of mycotoxins, including tric...

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

    Indian Academy of Sciences (India)

    2016-04-08

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

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

    OpenAIRE

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

    2016-01-01

    Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes ...

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

    DEFF Research Database (Denmark)

    Weber, Tilmann

    2014-01-01

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

  5. Behavior of Free Aromatic Amino Acid Pools in Rosmarinic Acid-Producing Cell Cultures of Anchusa officinalis L. 1

    Science.gov (United States)

    De-Eknamkul, Wanchai; Ellis, Brian E.

    1989-01-01

    The pool sizes of free l-phenylalanine and l-tyrosine, the precursors of rosmarinic acid in Anchusa officinalis L. cell suspension cultures, fluctuated during the culture cycle. The major increase in pool sizes was preceded by a peak of prephenate aminotransferase activity, while the subsequent decrease coincided with the presence of high activities of phenylalanine ammonia-lyase and tyrosine aminotransferase, the two entrypoint enzymes of the rosmarinic acid biosynthesis pathway. Timecourse feeding studies with linear growth stage cells revealed that the tyrosine pool turned over rapidly, consistent with direct participation in rosmarinic acid synthesis. Since externally applied l-tyrosine was rapidly incorporated into rosmarinic acid with little evidence of radioactively labeled intermediates, it is suggested that there exists a close coupling between the l-tyrosine pool and the rosmarinic acid biosynthetic pathway, which may involve the channelling of intermediates both into and within the pathway. PMID:16666560

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

    Directory of Open Access Journals (Sweden)

    Oksana Bilyk

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

  7. Up-regulation of abscisic acid signaling pathway facilitates aphid xylem absorption and osmoregulation under drought stress

    Science.gov (United States)

    Guo, Huijuan; Sun, Yucheng; Peng, Xinhong; Wang, Qinyang; Harris, Marvin; Ge, Feng

    2016-01-01

    The activation of the abscisic acid (ABA) signaling pathway reduces water loss from plants challenged by drought stress. The effect of drought-induced ABA signaling on the defense and nutrition allocation of plants is largely unknown. We postulated that these changes can affect herbivorous insects. We studied the effects of drought on different feeding stages of pea aphids in the wild-type A17 of Medicago truncatula and ABA signaling pathway mutant sta-1. We examined the impact of drought on plant water status, induced plant defense signaling via the abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) pathways, and on the host nutritional quality in terms of leaf free amino acid content. During the penetration phase of aphid feeding, drought decreased epidermis/mesophyll resistance but increased mesophyll/phloem resistance of A17 but not sta-1 plants. Quantification of transcripts associated with ABA, JA and SA signaling indicated that the drought-induced up-regulation of ABA signaling decreased the SA-dependent defense but increased the JA-dependent defense in A17 plants. During the phloem-feeding phase, drought had little effect on the amino acid concentrations and the associated aphid phloem-feeding parameters in both plant genotypes. In the xylem absorption stage, drought decreased xylem absorption time of aphids in both genotypes because of decreased water potential. Nevertheless, the activation of the ABA signaling pathway increased water-use efficiency of A17 plants by decreasing the stomatal aperture and transpiration rate. In contrast, the water potential of sta-1 plants (unable to close stomata) was too low to support xylem absorption activity of aphids; the aphids on sta-1 plants had the highest hemolymph osmolarity and lowest abundance under drought conditions. Taken together this study illustrates the significance of cross-talk between biotic-abiotic signaling pathways in plant-aphid interaction, and reveals the mechanisms leading to alter

  8. Rhabdastrellic acid-A induced autophagy-associated cell death through blocking Akt pathway in human cancer cells.

    Directory of Open Access Journals (Sweden)

    Dan-Dan Li

    Full Text Available BACKGROUND: Autophagy is an evolutionarily conserved protein degradation pathway. A defect in autophagy may contribute to tumorigenesis. Autophagy inducers could have a potential function in tumor prevention and treatment. METHODOLOGY/PRINCIPAL FINDINGS: Our results showed that Rhabdastrellic acid-A, an isomalabaricane triterpenoid isolated from the sponge Rhabdastrella globostellata, inhibited proliferation of human cancer cell lines Hep3B and A549 and induced caspase-independent cell death in both the cell lines. Further investigation showed that Rhabdastrellic acid-A induced autophagy of cancer cells determined by YFP-LC3 punctation and increased LC3-II. The pretreatment with autophagy inhibitor 3-MA inhibited Rhabdastrellic acid-A-induced cell death. Knockdown of autophagy-related gene Atg5 inhibited Rhabdastrellic acid-A-induced cell death in A549 cells. Also, phospho-Akt and its downstream targets significantly decreased after treatment with Rhabdastrellic acid-A in both cancer cell lines. Transfection of constitutive active Akt plasmid abrogated autophagy and cell death induced by Rhabdastrellic acid-A. CONCLUSIONS/SIGNIFICANCE: These results suggest that Rhabdastrellic acid-A could induce autophagy-associated cell death through blocking Akt pathway in cancer cells. It also provides the evidence that Rhabdastrellic acid-A deserves further investigation as a potential anticancer or cancer preventive agent.

  9. Activation of glycerol metabolic pathway by evolutionary engineering of Rhizopus oryzae to strengthen the fumaric acid biosynthesis from crude glycerol.

    Science.gov (United States)

    Huang, Di; Wang, Ru; Du, Wenjie; Wang, Guanyi; Xia, Menglei

    2015-11-01

    Rhizopus oryzae is strictly inhibited by biodiesel-based by-product crude glycerol, which results in low fumaric acid production. In this study, evolutionary engineering was employed to activate the glycerol utilization pathway for fumaric acid production. An evolved strain G80 was selected, which could tolerate and utilize high concentrations of crude glycerol to produce 14.9g/L fumaric acid with a yield of 0.248g/g glycerol. Key enzymes activity analysis revealed that the evolved strain displayed a significant upregulation in glycerol dissimilation, pyruvate consumption and reductive tricarboxylic acid pathways, compared with the parent strain. Subsequently, intracellular metabolic profiling analysis showed that amino acid biosynthesis, tricarboxylic acid cycle, fatty acid and stress response metabolites accounted for metabolic difference between two strains. Moreover, a glycerol fed-batch strategy was optimized to obtain the highest fumaric acid production of 25.5g/L, significantly increased by 20.9-fold than that of the parent strain of 1.2g/L. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Jungsuwadee Paiboon

    2011-02-01

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

  11. The influence of alternative pathways of respiration that utilize branched-chain amino acids following water shortage in Arabidopsis.

    Science.gov (United States)

    Pires, Marcel V; Pereira Júnior, Adilson A; Medeiros, David B; Daloso, Danilo M; Pham, Phuong Anh; Barros, Kallyne A; Engqvist, Martin K M; Florian, Alexandra; Krahnert, Ina; Maurino, Veronica G; Araújo, Wagner L; Fernie, Alisdair R

    2016-06-01

    During dark-induced senescence isovaleryl-CoA dehydrogenase (IVDH) and D-2-hydroxyglutarate dehydrogenase (D-2HGDH) act as alternate electron donors to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) pathway. However, the role of this pathway in response to other stresses still remains unclear. Here, we demonstrated that this alternative pathway is associated with tolerance to drought in Arabidopsis. In comparison with wild type (WT) and lines overexpressing D-2GHDH, loss-of-function etfqo-1, d2hgdh-2 and ivdh-1 mutants displayed compromised respiration rates and were more sensitive to drought. Our results demonstrated that an operational ETF/ETFQO pathway is associated with plants' ability to withstand drought and to recover growth once water becomes replete. Drought-induced metabolic reprogramming resulted in an increase in tricarboxylic acid (TCA) cycle intermediates and total amino acid levels, as well as decreases in protein, starch and nitrate contents. The enhanced levels of the branched-chain amino acids in loss-of-function mutants appear to be related to their increased utilization as substrates for the TCA cycle under water stress. Our results thus show that mitochondrial metabolism is highly active during drought stress responses and provide support for a role of alternative respiratory pathways within this response. © 2015 John Wiley & Sons Ltd.

  12. Excretion pathways and ruminal disappearance of glyphosate and its degradation product aminomethylphosphonic acid in dairy cows.

    Science.gov (United States)

    von Soosten, D; Meyer, U; Hüther, L; Dänicke, S; Lahrssen-Wiederholt, M; Schafft, H; Spolders, M; Breves, G

    2016-07-01

    From 6 balance experiments with total collection of feces and urine, samples were obtained to investigate the excretion pathways of glyphosate (GLY) in lactating dairy cows. Each experiment lasted for 26d. The first 21d served for adaptation to the diet, and during the remaining 5d collection of total feces and urine was conducted. Dry matter intake and milk yield were recorded daily and milk and feed samples were taken during the sampling periods. In 2 of the 6 experiments, at the sampling period for feces and urine, duodenal contents were collected for 5d. Cows were equipped with cannulas at the dorsal sac of the rumen and the proximal duodenum. Duodenal contents were collected every 2h over 5 consecutive days. The daily duodenal dry matter flow was measured by using chromium oxide as a volume marker. All samples (feed, feces, urine, milk and duodenal contents were analyzed for GLY and aminomethylphosphonic acid (AMPA). Overall, across the 6 experiments (n=32) the range of GLY intake was 0.08 to 6.67mg/d. The main proportion (61±11%; ±SD) of consumed GLY was excreted with feces; whereas excretion by urine was 8±3% of GLY intake. Elimination via milk was negligible. The GLY concentrations above the limit of quantification were not detected in any of the milk samples. A potential ruminal degradation of GLY to AMPA was derived from daily duodenal GLY flow. The apparent ruminal disappearance of GLY intake was 36 and 6%. In conclusion, the results of the present study indicate that the gastrointestinal absorption of GLY is of minor importance and fecal excretion represents the major excretion pathway. A degradation of GLY to AMPA by rumen microbes or a possible retention in the body has to be taken into account. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  13. Folic Acid Alters Methylation Profile of JAK-STAT and Long-Term Depression Signaling Pathways in Alzheimer's Disease Models.

    Science.gov (United States)

    Li, Wen; Liu, Huan; Yu, Min; Zhang, Xumei; Zhang, Yan; Liu, Hongbo; Wilson, John X; Huang, Guowei

    2016-11-01

    Dementia has emerged as a major societal issue because of the worldwide aging population and the absence of any effective treatment. DNA methylation is an epigenetic mechanism that evidently plays a role in Alzheimer's disease (AD). Folate acts through one-carbon metabolism to support the methylation of multiple substrates including DNA. We aimed to test the hypothesis that folic acid supplementation alters DNA methylation profiles in AD models. Mouse Neuro-2a cells expressing human APP695 (N2a-APP cells) were incubated with folic acid (2.8-20 μmol/L). AD transgenic mice were fed either folate-deficient or control diets and gavaged daily with water or folic acid (600 μg/kg). Gene methylation profiles were determined by methylated DNA immunoprecipitation-DNA microarray (MeDIP-chip). Differentially methylated regions (DMRs) were determined by Quantitative Differentially Methylated Regions analysis, and differentially methylated genes (DMGs) carrying at least three DMRs were selected for pathway analysis. Folic acid up-regulated DNA methylation levels in N2a-APP cells and AD transgenic mouse brains. Functional network analysis of folic acid-induced DMGs in these AD models revealed subnetworks composed of 24 focus genes in the janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway and 12 focus genes in the long-term depression (LTD) signaling pathway. In conclusion, these results revealed a role for folic acid in the JAK-STAT and LTD signaling pathways which may be relevant to AD pathogenesis. This novel finding may stimulate reinvestigation of folic acid supplementation as a prophylactic or therapeutic treatment for AD.

  14. Bardoxolone methyl (BARD) ameliorates aristolochic acid (AA)-induced acute kidney injury through Nrf2 pathway.

    Science.gov (United States)

    Wu, Juan; Liu, Xinhui; Fan, Jinjin; Chen, Wenfang; Wang, Juan; Zeng, Youjia; Feng, Xiaorang; Yu, Xueqing; Yang, Xiao

    2014-04-06

    Bardoxolone methyl (BARD) is an antioxidant modulator that acts through induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. This study aimed to investigate the role of BARD in protecting kidneys from aristolochic acid (AA)-induced acute kidney injury (AKI). Male C57BL/6 mice received intraperitoneal (i.p.) injections of aristolochic acid I (AAI) (5mg/kg/day) for 5 days to produce acute AA nephropathy (AAN) model. BARD (10mg/kg/day, i.p.) was applied for 7 consecutive days, starting 2 days prior to AAI administration. The mice in the AA group showed AKI as evidenced by worsening kidney function evaluated by blood urea nitrogen (BUN) and serum creatinine (SCr) levels, and severe tubulointerstitial injury marked by massive tubule necrosis in kidney tissues. BARD significantly reduced BUN and SCr levels which were elevated by AAI. Additionally, AAI-induced histopathological renal damage was ameliorated by BARD. Furthermore, the expression of Nrf2 was reduced, and its repressor Kelch-like ECH-associated protein 1 (Keap1) was increased significantly, whereas heme oxygenase-1 (HO-1) was upregulated and NAD(P)H quinone oxidoreductase-1 (NQO1) was barely increased in the cytoplasm of tubules in kidneys after treatment with AAI. BARD significantly upregulated renal Nrf2, NQO1 and HO-1 expression and downregulated Keap1 expression compared with those in the AA group. Moreover, it was found that Nrf2 was expressed both in the cytoplasm and nuclear of glomeruli and tubules, whereas NQO1 and HO-1 were localized in the cytoplasm of tubules only. In conclusion, AA-induced acute renal injury was associated with impaired Nrf2 activation and expression of its downstream target genes in renal tissues. BARD prevented renal damage induced by AAI, and this renoprotective effect may be exerted by activating the Nrf2 signaling pathway and increasing expression of the downstream target genes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  15. Engineering the Saccharomyces cerevisiae β-oxidation pathway to increase medium chain fatty acid production as potential biofuel.

    Science.gov (United States)

    Chen, Liwei; Zhang, Jianhua; Chen, Wei Ning

    2014-01-01

    Fatty acid-derived biofuels and biochemicals can be produced in microbes using β-oxidation pathway engineering. In this study, the β-oxidation pathway of Saccharomyces cerevisiae was engineered to accumulate a higher ratio of medium chain fatty acids (MCFAs) when cells were grown on fatty acid-rich feedstock. For this purpose, the haploid deletion strain Δpox1 was obtained, in which the sole acyl-CoA oxidase encoded by POX1 was deleted. Next, the POX2 gene from Yarrowia lipolytica, which encodes an acyl-CoA oxidase with a preference for long chain acyl-CoAs, was expressed in the Δpox1 strain. The resulting Δpox1 [pox2+] strain exhibited a growth defect because the β-oxidation pathway was blocked in peroxisomes. To unblock the β-oxidation pathway, the gene CROT, which encodes carnitine O-octanoyltransferase, was expressed in the Δpox1 [pox2+] strain to transport the accumulated medium chain acyl-coAs out of the peroxisomes. The obtained Δpox1 [pox2+, crot+] strain grew at a normal rate. The effect of these genetic modifications on fatty acid accumulation and profile was investigated when the strains were grown on oleic acids-containing medium. It was determined that the engineered strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] had increased fatty acid accumulation and an increased ratio of MCFAs. Compared to the wild-type (WT) strain, the total fatty acid production of the strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] were increased 29.5% and 15.6%, respectively. The intracellular level of MCFAs in Δpox1 [pox2+] and Δpox1 [pox2+, crot+] increased 2.26- and 1.87-fold compared to the WT strain, respectively. In addition, MCFAs in the culture medium increased 3.29-fold and 3.34-fold compared to the WT strain. These results suggested that fatty acids with an increased MCFAs ratio accumulate in the engineered strains with a modified β-oxidation pathway. Our approach exhibits great potential for transforming low value fatty acid-rich feedstock into high

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

    Science.gov (United States)

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

    2017-11-16

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

  17. Salvianolic Acid B Ameliorates Hyperglycemia and Dyslipidemia in db/db Mice through the AMPK Pathway

    Directory of Open Access Journals (Sweden)

    Ming-Qing Huang

    2016-12-01

    Full Text Available Background/Aims: Salvianolic acid B (Sal B, a major polyphenolic compound of Salvia miltiorrhiza Bunge, has been shown to possess potential antidiabetic activities. However, the action mechanism of SalB in type 2 diabetes has not been investigated extensively. The present study was designed to investigate the effects of Sal B on diabetes-related metabolic changes in a spontaneous model of type 2 diabetes, as well as its potential molecular mechanism. Methods: Male C57BL/KsJ-db/db mice were orally treated with Sal B (50 and 100 mg/kg or metformin (positive drug, 300 mg/kg for 6 weeks. Results: Both doses of Sal B significantly decreased fasting blood glucose, serum insulin, triglyceride and free fatty acid levels, reduced hepatic gluconeogenic gene expression and improved insulin intolerance in db/db mice. High dose Sal B also significantly improved glucose intolerance, increased hepatic glycolytic gene expression and muscle glycogen content, and ameliorated histopathological alterations of pancreas, similar to metformin. Sal B treatment resulted in increased phosphorylated AMP-activated protein kinase (p-AMPK protein expression in skeletal muscle and liver, increased glucose transporter 4 (GLUT4 and glycogen synthase protein expressions in skeletal muscle, and increased peroxisome proliferator-activated receptor alpha (PPARα and phosphorylated acetyl CoA carboxylase (p-ACC protein expressions in liver. Conclusion: Our data suggest that Sal B displays beneficial effects in the prevention and treatment of type 2 diabetes at least in part via modulation of the AMPK pathway.

  18. E-2-hexenal promotes susceptibility to Pseudomonas syringae by activating jasmonic acid pathways in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Alessandra eScala

    2013-04-01

    Full Text Available Green Leaf Volatiles (GLVs are C6-molecules - alcohols, aldehydes and esters - produced by plants upon herbivory or during pathogen infection. Exposure to this blend of volatiles induces defence-related responses in neighboring undamaged plants, thus assigning a role to GLVs in regulating plant defences. Here we compared Arabidopsis thaliana ecotype Ler with a hydroperoxide lyase line, hpl1, unable to synthesize GLVs, for susceptibility to Pseudomonas syringae pv. tomato (DC3000. We found that the growth of DC3000 was significantly reduced in the hpl1 mutant. This phenomenon correlated with lower jasmonic acid (JA levels and higher salicylic acid (SA levels in the hpl1 mutant. Furthermore, upon infection, the JA-responsive genes VSP2 and LEC were only slightly or not induced, respectively, in hpl1. This suggests that the reduced growth of DC3000 in hpl1 plants is due to the constraint of JA-dependent responses. Treatment of hpl1 plants with E-2-hexenal, one of the more reactive GLVs, prior to infection with DC3000, resulted in increased growth of DC3000 in hpl1, thus complementing this mutant. Interestingly, the growth of DC3000 also increased in Ler plants treated with E-2-hexenal. This stronger growth was not dependent on the JA-signaling component MYC2, but on ORA59, an integrator of JA and ethylene signaling pathways, and on the production of coronatine by DC3000. GLVs may have multiple effects on plant-pathogen interactions, in this case reducing resistance to P. syringae via JA and ORA59.

  19. The arachidonic acid-LTB4-BLT2 pathway enhances human B-CLL aggressiveness.

    Science.gov (United States)

    Guriec, Nathalie; Le Jossic-Corcos, Catherine; Simon, Brigitte; Ianotto, Jean-Christophe; Tempescul, Adrian; Dréano, Yvonne; Salaün, Jean-Pierre; Berthou, Christian; Corcos, Laurent

    2014-11-01

    Deregulation of the oxidative cascade of poly-unsaturated fatty acids (PUFAs) has been associated with several cancers, including chronic lymphocytic leukemia (B-CLL). Leukotriene B4 (LTB4), a metabolite of arachidonic acid (AA), is produced by B-CLL and contributes to their survival. The aim of the present study was to analyze the activity of the oxidative cascade of PUFAs in B-CLL. Purified B cells from patients and normal B CD5 positive cells were subjected to flow cytometry, Western-blot and RT-qPCR analyses. LTB4 plasma and intracellular concentrations were determined by ELISA. Our results showed that aggressive B-CLL tumor cells, i.e. cells with an annual proliferation index above 2, over-expressed calcium-dependent and calcium-independent phospholipases A2 (cPLA2-alpha and iPLA2-beta, respectively), 5-lipoxygenase (5LOX) and leukotriene A4 hydroxylase (LTA4H). Intracellular LTB4 levels were lower in the most aggressive cells than in cells with a smaller proliferation index, despite equivalent plasma levels, and lower expression of cytochrome P450 4F3A (CYP4F3A), one major enzyme involved in LTB4 inactivation. Since BLT2, a LTB4 membrane receptor was also more often expressed on aggressive tumor cells, and since a BLT2 inhibitor significantly impaired B-CLL viability in vitro, we propose that LTB4 was efficiently trapped onto BLT2 present on aggressive tumors, thereby eliciting an autocrine response. Taken together our results demonstrate a major deregulation of the pathway leading to LTB4 synthesis and degradation in B-CLL cells, and provide a framework for understanding how these modifications promote cell survival and proliferation, especially in the most aggressive BCLL. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Bile acids evoke placental inflammation by activating Gpbar1/NF-κB pathway in intrahepatic cholestasis of pregnancy.

    Science.gov (United States)

    Zhang, YouHua; Pan, YouDong; Lin, ChangDong; Zheng, YaJuan; Sun, Hao; Zhang, HaiLong; Wang, JunLei; Yuan, MengYa; Duan, Tao; Du, QiaoLing; Chen, JianFeng

    2016-12-01

    Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic disorder with potentially deleterious consequences for fetuses. Although a clear correlation between the elevated levels of maternal serum bile acids and deficient fetal outcome has been established in clinical practice, the underlying mechanisms remain elusive. Herein, we report that bile acids induce NF-κB pathway activation via G protein-coupled bile acid receptor 1 (Gpbar1), with consequent upregulation of inflammatory genes in trophoblasts, leading to aberrant leukocyte infiltration and inflammation in placenta. Ursodeoxycholic acid (UDCA), a drug used clinically to treat ICP, competes with other bile acids for binding with Gpbar1 and thus inhibits bile acid-induced inflammatory response in trophoblasts and improves fetal survival in pregnant rats with obstructive cholestasis. Notably, inhibition of NF-κB by andrographolide is more effective than UDCA in benefiting placentas and fetuses. Thus, anti-inflammation therapy targeting Gpbar1/NF-κB pathway could be effective in suppressing bile acid-induced inflammation and alleviating ICP-associated fetal disorders. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  1. Effect of Saturated Stearic Acid on MAP Kinase and ER Stress Signaling Pathways during Apoptosis Induction in Human Pancreatic β-Cells Is Inhibited by Unsaturated Oleic Acid

    Directory of Open Access Journals (Sweden)

    Jan Šrámek

    2017-11-01

    Full Text Available It has been shown that saturated fatty acids (FAs have a detrimental effect on pancreatic β-cells function and survival, leading to apoptosis, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction and regulation by FAs in β-cells remain unclear; however, mitogen-activated protein (MAP kinase and endoplasmic reticulum (ER stress signaling pathways may be involved. In this study, we tested how unsaturated oleic acid (OA affects the effect of saturated stearic acid (SA on the p38 mitogen-activated protein kinase (MAPK and extracellular signal-regulated kinase (ERK pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic β-cells NES2Y. We demonstrated that OA is able to inhibit all effects of SA. OA alone has only minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.

  2. Nucleotide Metabolism and its Control in Lactic Acid Bacteria

    DEFF Research Database (Denmark)

    Kilstrup, Mogens; Hammer, Karin; Jensen, Peter Ruhdal

    2005-01-01

    Most metabolic reactions are connected through either their utilization of nucleotides or their utilization of nucleotides or their regulation by these metabolites. In this review the biosynthetic pathways for pyrimidine and purine metabolism in lactic acid bacteria are described including...... the interconversion pathways, the formation of deoxyribonucleotides and the salvage pathways for use of exogenous precursors. The data for the enzymatic and the genetic regulation of these pathways are reviewed, as well as the gene organizations in different lactic acid bacteria. Mutant phenotypes and methods...... for manipulation of nucleotide pools are also discussed. Our aim is to provide an overview of the physiology and genetics of nucleotide metabolism and its regulation that will facilitate the interpretation of data arising from genetics, metabolomics, proteomics, and transcriptomics in lactic acid bacteria....

  3. Priming by Hexanoic acid induce activation of mevalonic and linolenic pathways and promotes the emission of plant volatiles.

    Directory of Open Access Journals (Sweden)

    Eugenio eLlorens

    2016-04-01

    Full Text Available Hexanoic acid is a short natural monocarboxylic acid present in some fruits and plants. Previous studies reported that soil drench application of this acid induces effective resistance in tomato plants against Botrytis cinerea and Pseudomonas syringae and in citrus against Alternaria alternata and Xanthomonas citri. In this work, we performed an in deep study of the metabolic changes produced in citrus by the application of hexanoic acid in response to the challenge pathogen Alternaria alternata, focusing on the response of the plant. Moreover, we used 13C labeled hexanoic to analyze its behavior inside the plants. Finally, we studied the volatile emission of the treated plants after the challenge inoculation. Drench application of 13C labeled hexanoic demonstrated that this molecule stays in the roots and is not mobilized to the leaves, suggesting long distance induction of resistance. Moreover, the study of the metabolic profile showed an alteration of more than two hundred molecules differentially induced by the application of the compound and the inoculation with the fungus. Bioinformatics analysis of data showed that most of these altered molecules could be related with the mevalonic and linolenic pathways suggesting the implication of these pathways in the induced resistance mediated by hexanoic acid. Finally, the application of this compound showed an enhancement of the emission of 17 volatile metabolites. Taken together, this study indicates that after the application of hexanoic acid this compound remains in the roots, provoking molecular changes that may trigger the defensive response in the rest of the plant mediated by changes in the mevalonic and linolenic pathways and enhancing the emission of volatile compounds, suggesting for the first time the implication of mevalonic pathway in response to hexanoic application.

  4. Carnosic acid induces apoptosis of hepatocellular carcinoma cells via ROS-mediated mitochondrial pathway.

    Science.gov (United States)

    Zhang, Xinrui; Chen, Yiling; Cai, Guangsheng; Li, Xin; Wang, Di

    2017-11-01

    Carnosic acid (CA), an important bioactive phenolic diterpene mainly found in labiate plants, exerts various biological functions, including antioxidant, anti-inflammatory, antitumor, and neuroprotective activities. In the present study, we proved the deleterious effects of CA against hepatocellular carcinoma (HCC) in both in vitro and in vivo models. In vitro, CA significantly decreased cell viability, inhibited cell proliferation and migration, enhanced apoptosis, and increased caspase-3, -8, and -9 activities in HepG2 and SMMC-7721 cells. Specifically, CA led to a decreased mitochondrial membrane potential (MMP) and increases in intracellular reactive oxygen species (ROS) levels and apoptosis-related protein expression. Pre-incubation of HCC cells with N-Acetyl-l-cysteine (NAC), a ROS inhibitor, strongly suppressed CA-induced apoptotic phenomena, including reduced cell viability, excessive ROS levels, MMP decreases, and abnormal protein expression, suggesting an association of CA-induced apoptosis with oxidative stress-mediated mitochondrial pathways. In HepG2-and SMMC-7721-xenograft tumor mouse models, treatment with CA inhibited tumor growth and modulated apoptosis-related protein expression, confirming the anti-HCC effects of this chemical. Moreover, the CA-mediated anti-HCC effects associated with oxidative stress provide experimental evidence to support the potential use of CA as a drug therapy for HCC. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. From ether to acid: A plausible degradation pathway of glycerol dialkyl glycerol tetraethers

    Science.gov (United States)

    Liu, Xiao-Lei; Birgel, Daniel; Elling, Felix J.; Sutton, Paul A.; Lipp, Julius S.; Zhu, Rong; Zhang, Chuanlun; Könneke, Martin; Peckmann, Jörn; Rowland, Steven J.; Summons, Roger E.; Hinrichs, Kai-Uwe

    2016-06-01

    Glycerol dialkyl glycerol tetraethers (GDGTs) are ubiquitous microbial lipids with extensive demonstrated and potential roles as paleoenvironmental proxies. Despite the great attention they receive, comparatively little is known regarding their diagenetic fate. Putative degradation products of GDGTs, identified as hydroxyl and carboxyl derivatives, were detected in lipid extracts of marine sediment, seep carbonate, hot spring sediment and cells of the marine thaumarchaeon Nitrosopumilus maritimus. The distribution of GDGT degradation products in environmental samples suggests that both biotic and abiotic processes act as sinks for GDGTs. More than a hundred newly recognized degradation products afford a view of the stepwise degradation of GDGT via (1) ether bond hydrolysis yielding hydroxyl isoprenoids, namely, GDGTol (glycerol dialkyl glycerol triether alcohol), GMGD (glycerol monobiphytanyl glycerol diether), GDD (glycerol dibiphytanol diether), GMM (glycerol monobiphytanol monoether) and bpdiol (biphytanic diol); (2) oxidation of isoprenoidal alcohols into corresponding carboxyl derivatives and (3) chain shortening to yield C39 and smaller isoprenoids. This plausible GDGT degradation pathway from glycerol ethers to isoprenoidal fatty acids provides the link to commonly detected head-to-head linked long chain isoprenoidal hydrocarbons in petroleum and sediment samples. The problematic C80 to C82 tetraacids that cause naphthenate deposits in some oil production facilities can be generated from H-shaped glycerol monoalkyl glycerol tetraethers (GMGTs) following the same process, as indicated by the distribution of related derivatives in hydrothermally influenced sediments.

  6. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues.

    Science.gov (United States)

    Myers, Jennifer S; von Lersner, Ariana K; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  7. Linoleic acid and stearic acid elicit opposite effects on AgRP expression and secretion via TLR4-dependent signaling pathways in immortalized hypothalamic N38 cells.

    Science.gov (United States)

    Wang, Songbo; Xiang, Nana; Yang, Liusong; Zhu, Canjun; Zhu, Xiaotong; Wang, Lina; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

    2016-03-18

    The regulation of food intake is a promising way to combat obesity. It has been implicated that various fatty acids exert different effects on food intake and body weight. However, the underlying mechanism remains poorly understood. The aim of the present study was to investigate the effects of linoleic acid (LA) and stearic acid (SA) on agouti-related protein (AgRP) expression and secretion in immortalized mouse hypothalamic N38 cells and to explore the likely underlying mechanisms. Our results demonstrated that LA inhibited, while SA stimulated AgRP expression and secretion of N38 cells in a dose-dependent manner. In addition, LA suppressed the protein expression of toll-like receptor 4 (TLR4), phosphorylation levels of JNK and IKKα/β, suggesting the inhibition of TLR4-dependent inflammation pathway. However, the above mentioned inhibitory effects of LA were eliminated by TLR4 agonist lipopolysaccharide (LPS). In contrast, SA promoted TLR4 protein expression and activated TLR4-dependent inflammation pathway, with elevated ratio of p-JNK/JNK. While TLR4 siRNA reversed the stimulatory effects of SA on AgRP expression and TLR4-dependent inflammation. Moreover, we found that TLR4 was also involved in LA-enhanced and SA-impaired leptin/insulin signal pathways in N38 cells. In conclusion, our findings indicated that LA elicited inhibitory while SA exerted stimulatory effects on AgRP expression and secretion via TLR4-dependent inflammation and leptin/insulin pathways in N38 cells. These data provided a better understanding of the mechanism underlying fatty acids-regulated food intake and suggested the potential role of long-chain unsaturated fatty acids such as LA in reducing food intake and treating obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Rewiring the reductive tricarboxylic acid pathway and L-malate transport pathway of Aspergillus oryzae for overproduction of L-malate.

    Science.gov (United States)

    Liu, Jingjing; Xie, Zhipeng; Shin, Hyun-Dong; Li, Jianghua; Du, Guocheng; Chen, Jian; Liu, Long

    2017-07-10

    Aspergillus oryzae finds wide application in the food, feed, and wine industries, and is an excellent cell factory platform for production of organic acids. In this work, we achieved the overproduction of L-malate by rewiring the reductive tricarboxylic acid (rTCA) pathway and L-malate transport pathway of A. oryzae NRRL 3488. First, overexpression of native pyruvate carboxylase and malate dehydrogenase in the rTCA pathway improved the L-malate titer from 26.1gL-1 to 42.3gL-1 in shake flask culture. Then, the oxaloacetate anaplerotic reaction was constructed by heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase from Escherichia coli, increasing the L-malate titer to 58.5gL-1. Next, the export of L-malate from the cytoplasm to the external medium was strengthened by overexpression of a C4-dicarboxylate transporter gene from A. oryzae and an L-malate permease gene from Schizosaccharomyces pombe, improving the L-malate titer from 58.5gL-1 to 89.5gL-1. Lastly, guided by transcription analysis of the expression profile of key genes related to L-malate synthesis, the 6-phosphofructokinase encoded by the pfk gene was identified as a potential limiting step for L-malate synthesis. Overexpression of pfk with the strong sodM promoter increased the L-malate titer to 93.2gL-1. The final engineered A. oryzae strain produced 165gL-1 L-malate with a productivity of 1.38gL-1h-1 in 3-L fed-batch culture. Overall, we constructed an efficient L-malate producer by rewiring the rTCA pathway and L-malate transport pathway of A. oryzae NRRL 3488, and the engineering strategy adopted here may be useful for the construction of A. oryzae cell factories to produce other organic acids. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Dian Anggraini Suroto

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

  10. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    Energy Technology Data Exchange (ETDEWEB)

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G., E-mail: deborah.murdock@vanderbilt.edu

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  11. Analysis of hydroxycinnamic acid degradation in Agrobacterium fabrum reveals a coenzyme A-dependent, beta-oxidative deacetylation pathway.

    Science.gov (United States)

    Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Nesme, Xavier; Lavire, Céline; Hommais, Florence

    2014-06-01

    The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl-CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)-CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.

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

    DEFF Research Database (Denmark)

    Weber, Tilmann; Blin, Kai; Duddela, Srikanth

    2015-01-01

    Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we...

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

    NARCIS (Netherlands)

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

    Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of Gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway

  14. Biosynthetic controls on the 13C-contents of organic components in the photoautotrophic bacterium Chloroflexus aurantiacus

    NARCIS (Netherlands)

    Sinninghe Damsté, J.S.; Meer, M.T.J. van der; Schouten, S.; Dongen, B.E. van; Rijpstra, W.I.C.; Fuchs, G.; Leeuw, J.W. de; Ward, D.M.

    2001-01-01

    To assess the effects related to known and proposed biosynthetic pathways on the 13C content of lipids and storage products of the photoautotrophic bacterium Chloroflexus aurantiacus, the isotopic compositions of bulk cell material, alkyl and isoprenoid lipids, and storage products such as glycogen

  15. Construction of energy-conserving sucrose utilization pathways for improving poly-γ-glutamic acid production in Bacillus amyloliquefaciens.

    Science.gov (United States)

    Feng, Jun; Gu, Yanyan; Quan, Yufen; Gao, Weixia; Dang, Yulei; Cao, Mingfeng; Lu, Xiaoyun; Wang, Yi; Song, Cunjiang; Wang, Shufang

    2017-06-06

    Sucrose is an naturally abundant and easily fermentable feedstock for various biochemical production processes. By now, several sucrose utilization pathways have been identified and characterized. Among them, the pathway consists of sucrose permease and sucrose phosphorylase is an energy-conserving sucrose utilization pathway because it consumes less ATP when comparing to other known pathways. Bacillus amyloliquefaciens NK-1 strain can use sucrose as the feedstock to produce poly-γ-glutamic acid (γ-PGA), a highly valuable biopolymer. The native sucrose utilization pathway in NK-1 strain consists of phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-P hydrolase and consumes more ATP than the energy-conserving sucrose utilization pathway. In this study, the native sucrose utilization pathway in NK-1 was firstly deleted and generated the B. amyloliquefaciens 3Δ strain. Then four combination of heterologous energy-conserving sucrose utilization pathways were constructed and introduced into the 3Δ strain. Results demonstrated that the combination of cscB (encodes sucrose permease) from Escherichia coli and sucP (encodes sucrose phosphorylase) from Bifidobacterium adolescentis showed the highest sucrose metabolic efficiency. The corresponding mutant consumed 49.4% more sucrose and produced 38.5% more γ-PGA than the NK-1 strain under the same fermentation conditions. To our best knowledge, this is the first report concerning the enhancement of the target product production by introducing the heterologous energy-conserving sucrose utilization pathways. Such a strategy can be easily extended to other microorganism hosts for reinforced biochemical production using sucrose as substrate.

  16. Genomes of rumen bacteria encode atypical pathways for fermenting hexoses to short-chain fatty acids

    KAUST Repository

    Hackmann, Timothy J.

    2017-09-11

    Bacteria have been thought to follow only a few well-recognized biochemical pathways when fermenting glucose or other hexoses. These pathways have been chiseled in the stone of textbooks for decades, with most sources rendering them as they appear in the classic 1986 text by Gottschalk. Still, it is unclear how broadly these pathways apply, given that they were established and delineated biochemically with only a few model organisms. Here we show that well-recognized pathways often cannot explain fermentation products formed by bacteria. In the most extensive analysis of its kind, we reconstructed pathways for glucose fermentation from genomes of 48 species and subspecies of bacteria from one environment (the rumen). In total, 44% of these bacteria had atypical pathways, including several that are completely unprecedented for bacteria or any organism. In detail, 8% of bacteria had an atypical pathway for acetate formation; 21% for propionate or succinate formation; 6% for butyrate formation; and 33% had an atypical or incomplete Embden-Meyerhof-Parnas pathway. This study shows that reconstruction of metabolic pathways-a common goal of omics studies-could be incorrect if well-recognized pathways are used for reference. Further, it calls for renewed efforts to delineate fermentation pathways biochemically. This article is protected by copyright. All rights reserved.

  17. Allicin Alleviates Inflammation of Trinitrobenzenesulfonic Acid-Induced Rats and Suppresses P38 and JNK Pathways in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Chen Li

    2015-01-01

    Full Text Available Background. Allicin has anti-inflammatory, antioxidative and proapoptotic properties. Aims. To evaluate the effects and investigate the mechanism of allicin on trinitrobenzenesulfonic acid-induced colitis, specifically with mesalazine or sulfasalazine. Methods. 80 rats were divided equally into 8 groups: control; trinitrobenzenesulfonic acid; allicin prevention; allicin; mesalazine; sulfasalazine; allicin + sulfasalazine, and mesalazine + allicin. Systemic and colonic inflammation parameters were analysed. In addition, protein and culture medium of Caco-2 cells treated with various concentrations of IL-1β or allicin were collected for investigation of IL-8, NF-κB p65 P38, ERK, and JNK. One-way ANOVA and Kruskal-Wallis H test were used for parametric and nonparametric tests, respectively. Results. Allicin reduced the body weight loss of trinitrobenzenesulfonic acid-induced rats, histological score, serum TNF-α and IL-1β levels, and colon IL-1β mRNA level and induced serum IL-4 level, particularly in combination with mesalazine. In addition, 1 ng/mL IL-1β stimulated the P38, ERK, and JNK pathways, whereas