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Jack T Pronk - One of the best experts on this subject based on the ideXlab platform.
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overproduction of threonine aldolase circumvents the biosynthetic role of Pyruvate Decarboxylase in glucose limited chemostat cultures of saccharomyces cerevisiae
Applied and Environmental Microbiology, 2003Co-Authors: Antonius J A Van Maris, Johannes P Van Dijken, Marijke A H Luttik, Aaron Adriaan Winkler, Jack T PronkAbstract:Pyruvate Decarboxylase-negative (Pdc−) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C2 requirement of Pdc− S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of l-carnitine to growth media did not restore growth of a Pdc− strain on glucose, indicating that the C2 requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc− strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of Pyruvate Decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc− GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc− strain, it failed to grow on excess glucose in batch cultures and excreted Pyruvate when transferred from glucose limitation to glucose excess.
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growth requirements of Pyruvate Decarboxylase negative saccharomyces cerevisiae
Fems Microbiology Letters, 1999Co-Authors: Marcel T Flikweert, Johannes P Van Dijken, Martin E De Swaaf, Jack T PronkAbstract:Pyruvate-Decarboxylase (Pdc)-negative Saccharomyces cerevisiae has been reported to grow in batch cultures on glucose-containing complex media, but not on defined glucose-containing media. By a combination of batch and chemostat experiments it is demonstrated that even in complex media, Pdc−S. cerevisiae does not exhibit prolonged growth on glucose. Pdc− strains do grow in carbon-limited cultures on defined media containing glucose-acetate mixtures. The acetate requirement for glucose-limited growth, estimated experimentally by continuously decreasing the acetate feed to chemostat cultures, matched the theoretical acetyl-CoA requirement for lipid and lysine synthesis, consistent with the proposed role of Pyruvate Decarboxylase in the synthesis of cytosolic acetyl-CoA.
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steady state and transient state analysis of growth and metabolite production in a saccharomyces cerevisiae strain with reduced Pyruvate Decarboxylase activity
Biotechnology and Bioengineering, 1999Co-Authors: Marcel T Flikweert, Marko Kuyper, A J A Van Maris, Peter Kotter, J P Van Dijken, Jack T PronkAbstract:Pyruvate Decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of Pyruvate Decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of Pyruvate-Decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN.PK113-7D. This caused a 3–4-fold reduction of Pyruvate-Decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h−1. Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Δ strain, formation of ethanol and acetate was reduced by 60–70%. In contrast to the wild type, the pdc2Δ strain produced substantial amounts of Pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Δ cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced Pyruvate-Decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h−1. Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h−1, growth of the pdc2Δ strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low μmax of the pdc2Δ strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for Pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial fermentations. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 42–50, 1999.
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Pyruvate Decarboxylase catalyzes decarboxylation of branched chain 2 oxo acids but is not essential for fusel alcohol production by saccharomyces cerevisiae
Applied and Environmental Microbiology, 1998Co-Authors: Eelko G Ter Schure, Marcel T Flikweert, Johannes P Van Dijken, Jack T Pronk, Theo C VerripsAbstract:The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a Decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of Pyruvate Decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether Pyruvate Decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic Pyruvate Decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via Pyruvate Decarboxylase is not an essential step in fusel alcohol production.
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metabolic responses of Pyruvate Decarboxylase negative saccharomyces cerevisiae to glucose excess
Applied and Environmental Microbiology, 1997Co-Authors: Marcel T Flikweert, J P Van Dijken, Jack T PronkAbstract:In Saccharomyces cerevisiae, oxidation of Pyruvate to acetyl coenzyme A can occur via two routes. In Pyruvate Decarboxylase-negative (Pdc-) mutants, the Pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the Pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the Pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and Pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via Pyruvate Decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that Pyruvate excretion by the mutant was not due to a decrease of the Pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the Pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, Pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.
Marcel T Flikweert - One of the best experts on this subject based on the ideXlab platform.
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growth requirements of Pyruvate Decarboxylase negative saccharomyces cerevisiae
Fems Microbiology Letters, 1999Co-Authors: Marcel T Flikweert, Johannes P Van Dijken, Martin E De Swaaf, Jack T PronkAbstract:Pyruvate-Decarboxylase (Pdc)-negative Saccharomyces cerevisiae has been reported to grow in batch cultures on glucose-containing complex media, but not on defined glucose-containing media. By a combination of batch and chemostat experiments it is demonstrated that even in complex media, Pdc−S. cerevisiae does not exhibit prolonged growth on glucose. Pdc− strains do grow in carbon-limited cultures on defined media containing glucose-acetate mixtures. The acetate requirement for glucose-limited growth, estimated experimentally by continuously decreasing the acetate feed to chemostat cultures, matched the theoretical acetyl-CoA requirement for lipid and lysine synthesis, consistent with the proposed role of Pyruvate Decarboxylase in the synthesis of cytosolic acetyl-CoA.
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steady state and transient state analysis of growth and metabolite production in a saccharomyces cerevisiae strain with reduced Pyruvate Decarboxylase activity
Biotechnology and Bioengineering, 1999Co-Authors: Marcel T Flikweert, Marko Kuyper, A J A Van Maris, Peter Kotter, J P Van Dijken, Jack T PronkAbstract:Pyruvate Decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of Pyruvate Decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of Pyruvate-Decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN.PK113-7D. This caused a 3–4-fold reduction of Pyruvate-Decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h−1. Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Δ strain, formation of ethanol and acetate was reduced by 60–70%. In contrast to the wild type, the pdc2Δ strain produced substantial amounts of Pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Δ cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced Pyruvate-Decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h−1. Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h−1, growth of the pdc2Δ strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low μmax of the pdc2Δ strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for Pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial fermentations. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 42–50, 1999.
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Pyruvate Decarboxylase catalyzes decarboxylation of branched chain 2 oxo acids but is not essential for fusel alcohol production by saccharomyces cerevisiae
Applied and Environmental Microbiology, 1998Co-Authors: Eelko G Ter Schure, Marcel T Flikweert, Johannes P Van Dijken, Jack T Pronk, Theo C VerripsAbstract:The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a Decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of Pyruvate Decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether Pyruvate Decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic Pyruvate Decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via Pyruvate Decarboxylase is not an essential step in fusel alcohol production.
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metabolic responses of Pyruvate Decarboxylase negative saccharomyces cerevisiae to glucose excess
Applied and Environmental Microbiology, 1997Co-Authors: Marcel T Flikweert, J P Van Dijken, Jack T PronkAbstract:In Saccharomyces cerevisiae, oxidation of Pyruvate to acetyl coenzyme A can occur via two routes. In Pyruvate Decarboxylase-negative (Pdc-) mutants, the Pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the Pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the Pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and Pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via Pyruvate Decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that Pyruvate excretion by the mutant was not due to a decrease of the Pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the Pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, Pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.
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Pyruvate Decarboxylase an indispensable enzyme for growth of saccharomyces cerevisiae on glucose
Yeast, 1996Co-Authors: Marcel T Flikweert, Johannes P Van Dijken, Linda Van Der Zanden, Wouter Th M M Janssen, Yde H Steensma, Jack T PronkAbstract:In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active Pyruvate Decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild-type strain, using the dominant marker genes APT1 and Tn5ble. A Pyruvate-Decarboxylase-negative (Pdc-) mutant lacking all three PDC genes exhibited a three-fold lower growth rate in complex medium with glucose than the isogenic wild-type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc- strains. Furthermore, in ethanol-limited chemostat cultures, the biomass yield of Pdc- and wild-type S. cerevisiae were identical. However, Pdc- S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol-limited chemostat cultures (D = 0 center dot 10 h-1) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed-substrate cultures were switched to a feed containing glucose as the sole carbon source, wash-out occurred. It is concluded that the mitochondrial Pyruvate dehydrogenase complex cannot function as the sole source of acetyl-CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose-limited chemostat cultures.
Erica Oduaran - One of the best experts on this subject based on the ideXlab platform.
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the bifunctional Pyruvate Decarboxylase Pyruvate ferredoxin oxidoreductase from thermococcus guaymasensis
Archaea, 2014Co-Authors: Mohammad S. Eram, Erica OduaranAbstract:The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from Pyruvate was purified and characterized from T. guaymasensis under strictly anaerobic conditions. The enzyme had both Pyruvate Decarboxylase (PDC) and Pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of 3.8 ± 0.22 U mg(-1) and 20.2 ± 1.8 U mg(-1), with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilic β -keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding β -keto acids.
Frank Jordan - One of the best experts on this subject based on the ideXlab platform.
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novel synthesis of 2 oxo 4 phenyl 3 butynoic acid a new inhibitor and alternate substrate of Pyruvate Decarboxylase
Journal of Organic Chemistry, 1994Co-Authors: Chingfan C Chiu, Frank JordanAbstract:An improved method is reported for the synthesis of 2-oxo acids and is applied to the synthesis of 2-oxo-4-phenyl-3-butynoic acid. The compound is synthesized by reacting the N-methoxy-N-methylamide of monoethyloxalic acid with lithium phenylacetylide yielding ethyl 2-oxo-4-phenyl3-butynoate (78% yield), followed by strictly pH-controlled hydrolysis to the free acid in nearly quantitative yield. The compound in shown to be a potent irreversible inhibitor of brewers'yeast Pyruvate Decarboxylase, in addition to producing both cis- and trans-cinnamic acids as products of turnover. The formation of these isomeric cinnamic acids can be rationalized if the thiamin diphosphate-bound α-carbanion/enamine intermediate resulting from decarboxylation is protonated at the side chain γ carbon to form two diastereomeric allenols, whose tautomerization and hydrolysis lead to the two products
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a thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase Pyruvate oxidase and Pyruvate Decarboxylase
Structure, 1993Co-Authors: Yves A Muller, William Furey, Frank Jordan, Gunter Schneider, Ylva Lindqvist, Georg E SchulzAbstract:Abstract Background: The crystal structures of three thiamin diphosphate-dependent enzymes that catalyze distinct reactions in basic metabolic pathways are known. These enzymes — transketolase, Pyruvate oxidase and Pyruvate Decarboxylase — also require metal ions such as Ca 2+ and Mg 2+ as cofactors and have little overall sequence similarity. Here, the crystal structures of these three enzymes are compared. Results: The three enzymes share a similar pattern of binding of thiamin diphosphate and the metal ion cofactors. The enzymes function as multisubunit proteins, with each polypeptide chain folded into three αβ domains. Two of these domains are involved in binding of the thiamin diphosphate and the metal ion. These domains have the same topology of six parallel β-strands and surrounding α-helices. The thiamin diphosphate is bound in a cleft, formed by two domains from two different subunits. Only a few residues are conserved in all three enzymes and these are responsible for proper binding of the cofactors. Conclusions: Despite considerable differences in quaternary structure and lack of overall sequence homology, thiamin diphosphate binds to the three enzymes in a very similar fashion, and a general thiamin-binding fold can be revealed.
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catalytic centers in the thiamin diphosphate dependent enzyme Pyruvate Decarboxylase at 2 4 a resolution
Biochemistry, 1993Co-Authors: Fred Dyda, William Furey, Subramanyam Swaminathan, Martin Sax, Bruce Farrenkopf, Frank JordanAbstract:The crystal structure of brewers' yeast Pyruvate Decarboxylase, a thiamin diphosphate dependent alpha-keto acid Decarboxylase, has been determined to 2.4-A resolution. The homotetrameric assembly contains two dimers, exhibiting strong intermonomer interactions within each dimer but more limited ones between dimers. Each monomeric subunit is partitioned into three structural domains, all folding according to a mixed alpha/beta motif. Two of these domains are associated with cofactor binding, while the other is associated with substrate activation. The catalytic centers containing both thiamin diphosphate and Mg(II) are located deep in the intermonomer interface within each dimer. Amino acids important in cofactor binding and likely to participate in catalysis and substrate activation are identified.
Michele M Bianchi - One of the best experts on this subject based on the ideXlab platform.
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Autoregulation of the Kluyveromyces lactis Pyruvate Decarboxylase gene KlPDC1 involves the regulatory gene RAG3.
Microbiology, 2014Co-Authors: Daniela Ottaviano, Chiara Micolonghi, Lorenza Tizzani, Marc Lemaire, Micheline Wésolowski-louvel, Maria Egle De Stefano, Danilo Ranieri, Michele M BianchiAbstract:In the yeast Kluyveromyces lactis, the Pyruvate Decarboxylase gene KlPDC1 is strongly regulated at the transcription level by different environmental factors. Sugars and hypoxia act as inducers of transcription, while ethanol acts as a repressor. Their effects are mediated by gene products, some of which have been characterized. KlPDC1 transcription is also strongly repressed by its product--KlPdc1--through a mechanism called autoregulation. We performed a genetic screen that allowed us to select and identify the regulatory gene RAG3 as a major factor in the transcriptional activity of the KlPDC1 promoter in the absence of the KlPdc1 protein, i.e. in the autoregulatory mechanism. We also showed that the two proteins Rag3 and KlPdc1 interact, co-localize in the cell and that KlPdc1 may control Rag3 nuclear localization.
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optimization of recombinant fungal laccase production with strains of the yeast kluyveromyces lactis from the Pyruvate Decarboxylase promoter
Fems Yeast Research, 2009Co-Authors: Danilo Ranieri, Maria Chiara Colao, Maurizio Ruzzi, Gabriele Romagnoli, Michele M BianchiAbstract:Laccases are multicopper oxidases of wide specificity that catalyze the oxidation of phenolic and related compounds using molecular oxygen as the electron acceptor. Here, we report the production of the Lcc1 laccase of the fungus Trametes trogii in strains of the yeast Kluyveromyces lactis, using the Pyruvate Decarboxylase promoter (KlPDC1) as an expression system. We assayed laccase production in various strains, with replicative and integrative transformants and with different cultivation parameters. A comparison with Lcc1 enzymes from other yeasts and from the original organism was also performed. The best production conditions were obtained with integrative transformants of an individual strain, whereas cultivation conditions were less stringent than the use of the regulated KlPDC1 promoter could anticipate. The secreted recombinant laccase showed better enzyme properties than the native enzyme or recombinant enzyme from other yeasts. We conclude that selected K. lactis strains, with opportune physiological properties and transcription regulation of the heterologous gene, could be optimal hosts for laccase isoenzyme production.
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the petite negative yeast kluyveromyces lactis has a single gene expressing Pyruvate Decarboxylase activity
Molecular Microbiology, 1996Co-Authors: Michele M Bianchi, Lorenza Tizzani, Monika Destruelle, Laura Frontali, Micheline WesolowskilouvelAbstract:Summary We cloned and sequenced the Pyruvate Decarboxylase (PDC; EC 4.1.1.1) structural gene KIPDCA in the yeast Kluyveromyces lactis and found it to be allelic to the previously isolated rag6 mutation. The putative amino acid sequence of the KIPdcAp appeared to be highly homologous to those of the yeast Pdc proteins identified so far. The disruption of KIPDCA indicated that it is the only PDC structural gene in K. lactis, as evidenced by the lack of PDC activity and ethanol production in the pdcAΔ strains and by the absence of growth on glucose in the presence of respiratory inhibitors. It was observed that expression of the KIPDCA gene is induced by glucose at the transcriptional level. Transcription of the gene was reduced in the rag1, rag2, rag5 and rag8 mutants, which are defective for the low-affinity glucose permease, phosphoglucose isomerase, hexokinase, and a positive regulator of RAG1 expression, respectively.
