The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
Charles K Singleton - One of the best experts on this subject based on the ideXlab platform.
-
sensitivity to thiamine deficiency in cultured human cells is dependent on cell type and is enhanced in cells from thiamine responsive megaloblastic anemia patients
Journal of Nutritional Biochemistry, 1998Co-Authors: Stevan R Pekovich, Peter R Martin, Vincenzo Poggi, Charles K SingletonAbstract:Abstract To address tissue-specific variation in sensitivity to thiamine deficiency, three human cell types were grown in medium with various thiamine concentrations. The activity of a cytosolic and a mitochondrial thiamine diphosphate-dependent enzyme was examined. Each cell type displayed a unique response to thiamine depletion with respect to α-ketoglutarate dehydrogenase and Transketolase activity and to inhibition of cell growth. Loss of α-ketoglutarate dehydrogenase activity was similar in lymphoblasts and fibroblasts, whereas loss of activity in neuroblastoma cells was significantly more resistant to thiamine depletion. Transketolase activity in neuroblastoma cells was only moderately resistant to thiamine depletion, with the activity in fibroblasts being the most and in lymphoblasts the least resistant. Total Transketolase activity was 33% higher in fibroblasts than in lymphoblasts and neuroblastoma cells, indicating a differential requirement for production and maintenance of Transketolase activity in this cell type. Compared with normal lymphoblasts, those derived from patients with thiamine-responsive megaloblastic anemia were 100 to 1000 times more sensitive to thiamine depletion. Although fibroblasts from these patients also demonstrated a 1000-fold increase in sensitivity with respect to Transketolase activity, α-ketoglutarate dehydrogenase activity demonstrated no enhanced sensitivity. The results indicate a complex, cell-type dependent regulation of intracellular pools of thiamine and its phosphorylated derivatives in response to fluctuating extracellular thiamine levels.
-
a Transketolase assembly defect in a wernicke korsakoff syndrome patient
Alcoholism: Clinical and Experimental Research, 1997Co-Authors: James J L Wang, Peter R Martin, Charles K SingletonAbstract:Thiamine deficiency, a frequent complication of alcoholism, contributes significantly to the development of damage in various organ systems, including the brain. The molecular mechanisms that underlie the differential vulnerabilities to thiamine deficiency of tissue and cell types and among individuals are not understood. Investigations into these mechanisms have examined potential variations in thiamine utilizing enzymes. Transketolase is a homodimeric enzyme containing two molecules of noncovalently bound thiamine pyrophosphate. In the present study, we examined a his-tagged human Transketolase that was produced in and purified from fscherichia coli cells. Previous findings demonstrated that purified his-Transketolase had a K, app for cofactor and a thiamine pyrophosphatedependent lag period for attaining steady-state kinetics that was similar to Transketolase purified from human tissues. Interestingly, the time of the lag period, which is normally independent of enzyme concentration, was found herein to be dependent on the concentration of the recombinant protein. This atypical behavior was due to production in E. coli. Generation of the normal, enzyme concentration-independent state required a cytosolic factor(s) derived from human cells. Importantly, the required factor(@ was found to be defective in a Wernicke-Korsakoff patient whose cells in culture show
-
a Transketolase assembly defect in a wernicke korsakoff syndrome patient
Alcoholism: Clinical and Experimental Research, 1997Co-Authors: James H C Wang, Peter R Martin, Charles K SingletonAbstract:Thiamine deficiency, a frequent complication of alcoholism, contributes significantly to the development of damage in various organ systems, including the brain. The molecular mechanisms that underlie the differential vulnerabilities to thiamine deficiency of tissue and cell types and among individuals are not understood. Investigations into these mechanisms have examined potential variations in thiamine utilizing enzymes. Transketolase is a homodimeric enzyme containing two molecules of noncovalently bound thiamine pyrophosphate. In the present study, we examined a his-tagged human Transketolase that was produced in and purified from Escherichia coli cells. Previous findings demonstrated that purified his-Transketolase had a Km app for cofactor and a thiamine pyrophosphate-dependent lag period for attaining steady-state kinetics that was similar to Transketolase purified from human tissues. Interestingly, the time of the lag period, which is normally independent of enzyme concentration, was found herein to be dependent on the concentration of the recombinant protein. This atypical behavior was due to production in E. coli. Generation of the normal, enzyme concentration-independent state required a cytosolic factor(s) derived from human cells. Importantly, the required factor(s) was found to be defective in a Wernicke-Korsakoff patient whose cells in culture show an enhanced sensitivity to thiamine deficiency.
-
thiamine pyrophosphate requiring enzymes are altered during pyrithiamine induced thiamine deficiency in cultured human lymphoblasts
Journal of Nutrition, 1996Co-Authors: Stevan R Pekovich, Peter R Martin, Charles K SingletonAbstract:The most common of the severe compli cations of thiamine deficiency are beriberi and Wer- nicke-Korsakoff syndrome. To help clarify the bio chemical basis for these disorders, a cell culture sys tem has been established in which pyrithiamine, a potent thiamine transport inhibitor, was used to mimic different degrees of thiamine deficiency within human lymphoblasts. Activities of both Transketolase and a- ketoglutarate dehydrogenase (a-KGDH) decreased at the same rate and to roughly the same levels in re sponse to thiamine deficiency within a given cell line. However, variation in sensitivity to thiamine deficiency, as judged by the relative percentage of loss of enzy matic activities, was found when different cell lines were compared. When exogenous thiamine pyrophos- phate was added to the activity assays, differences between Transketolase and a-KGDH became readily apparent. Only 25% of the lost Transketolase activity was present as apo-enzyme, whereas 70% of the lost a-KGDH activity was present in the apo-enzyme form. For Transketolase, the non-recoverable activity was due mainly to a decrease in the synthesis rate of the protein during thiamine deficiency, suggesting that thi amine has a direct effect on the expression of the Transketolase gene and/or protein. J. Nutr. 126: 1791-1798, 1996.
-
cloning of human Transketolase cdnas and comparison of the nucleotide sequence of the coding region in wernicke korsakoff and non wernicke korsakoff individuals
Journal of Biological Chemistry, 1993Co-Authors: Brian A Mccool, Peter R Martin, S G Plonk, Charles K SingletonAbstract:Variants of the enzyme Transketolase which possess reduced affinity for its cofactor thiamine pyrophosphate (high apparent Km) have been described in chronic alcoholic patients with Wernicke-Korsakoff syndrome. Since the syndrome has been shown to be directly related to thiamine deficiency, it has been hypothesized that such Transketolase variants may represent a genetic predisposition to the development of this syndrome. To test this hypothesis, human Transketolase cDNA clones were isolated, and their nucleotide and predicted amino acid sequence were determined. Transketolase was found to be a single copy gene which produces a single mRNA of approximately 2100 nucleotides. Additionally, the nucleotide sequence of the Transketolase coding region in fibroblasts derived from two Wernicke-Korsakoff (WK) patients was compared to that of two nonalcoholic controls. Although nucleotide and predicted amino acid differences were detected between fibroblast cultures and the original cDNAs and among the cultures themselves, no specific nucleotide variations, which would encode a variant amino acid sequence, were associated exclusively with the coding region from WK patients. Thus, allelic variants of the Transketolase gene cannot account for the biochemically distinct forms of the enzyme found in these patients nor be considered as a mechanism for genetic predisposition to the development of Wernicke-Korsakoff syndrome. Instead, the underlying mechanism must be extragenic and may be a result of differences in post-translational processing/modification of the Transketolase polypeptide.
Andriy Sibirny - One of the best experts on this subject based on the ideXlab platform.
-
Peroxisomes and peroxisomal Transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha.
Biotechnology for biofuels, 2018Co-Authors: Olena O. Kurylenko, Kostyantyn V. Dmytruk, Justyna Ruchala, Roksolana V. Vasylyshyn, Oleh V. Stasyk, Olena V. Dmytruk, Andriy SibirnyAbstract:Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve high-temperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol. Involvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal Transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal Transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic Transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30–40 times more ethanol than is produced by the wild-type strain. Our results indicate the importance of the peroxisomal enzymes, Transketolase (dihydroxyacetone synthase, Das1), and transaldolase (Tal2), in the xylose alcoholic fermentation of O. polymorpha.
-
Peroxisomes and peroxisomal Transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha
BMC, 2018Co-Authors: Olena O. Kurylenko, Kostyantyn V. Dmytruk, Justyna Ruchala, Roksolana V. Vasylyshyn, Oleh V. Stasyk, Olena V. Dmytruk, Andriy SibirnyAbstract:Abstract Background Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve high-temperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol. Results Involvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal Transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal Transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic Transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30–40 times more ethanol than is produced by the wild-type strain. Conclusions Our results indicate the importance of the peroxisomal enzymes, Transketolase (dihydroxyacetone synthase, Das1), and transaldolase (Tal2), in the xylose alcoholic fermentation of O. polymorpha
Peter R Martin - One of the best experts on this subject based on the ideXlab platform.
-
sensitivity to thiamine deficiency in cultured human cells is dependent on cell type and is enhanced in cells from thiamine responsive megaloblastic anemia patients
Journal of Nutritional Biochemistry, 1998Co-Authors: Stevan R Pekovich, Peter R Martin, Vincenzo Poggi, Charles K SingletonAbstract:Abstract To address tissue-specific variation in sensitivity to thiamine deficiency, three human cell types were grown in medium with various thiamine concentrations. The activity of a cytosolic and a mitochondrial thiamine diphosphate-dependent enzyme was examined. Each cell type displayed a unique response to thiamine depletion with respect to α-ketoglutarate dehydrogenase and Transketolase activity and to inhibition of cell growth. Loss of α-ketoglutarate dehydrogenase activity was similar in lymphoblasts and fibroblasts, whereas loss of activity in neuroblastoma cells was significantly more resistant to thiamine depletion. Transketolase activity in neuroblastoma cells was only moderately resistant to thiamine depletion, with the activity in fibroblasts being the most and in lymphoblasts the least resistant. Total Transketolase activity was 33% higher in fibroblasts than in lymphoblasts and neuroblastoma cells, indicating a differential requirement for production and maintenance of Transketolase activity in this cell type. Compared with normal lymphoblasts, those derived from patients with thiamine-responsive megaloblastic anemia were 100 to 1000 times more sensitive to thiamine depletion. Although fibroblasts from these patients also demonstrated a 1000-fold increase in sensitivity with respect to Transketolase activity, α-ketoglutarate dehydrogenase activity demonstrated no enhanced sensitivity. The results indicate a complex, cell-type dependent regulation of intracellular pools of thiamine and its phosphorylated derivatives in response to fluctuating extracellular thiamine levels.
-
a Transketolase assembly defect in a wernicke korsakoff syndrome patient
Alcoholism: Clinical and Experimental Research, 1997Co-Authors: James J L Wang, Peter R Martin, Charles K SingletonAbstract:Thiamine deficiency, a frequent complication of alcoholism, contributes significantly to the development of damage in various organ systems, including the brain. The molecular mechanisms that underlie the differential vulnerabilities to thiamine deficiency of tissue and cell types and among individuals are not understood. Investigations into these mechanisms have examined potential variations in thiamine utilizing enzymes. Transketolase is a homodimeric enzyme containing two molecules of noncovalently bound thiamine pyrophosphate. In the present study, we examined a his-tagged human Transketolase that was produced in and purified from fscherichia coli cells. Previous findings demonstrated that purified his-Transketolase had a K, app for cofactor and a thiamine pyrophosphatedependent lag period for attaining steady-state kinetics that was similar to Transketolase purified from human tissues. Interestingly, the time of the lag period, which is normally independent of enzyme concentration, was found herein to be dependent on the concentration of the recombinant protein. This atypical behavior was due to production in E. coli. Generation of the normal, enzyme concentration-independent state required a cytosolic factor(s) derived from human cells. Importantly, the required factor(@ was found to be defective in a Wernicke-Korsakoff patient whose cells in culture show
-
a Transketolase assembly defect in a wernicke korsakoff syndrome patient
Alcoholism: Clinical and Experimental Research, 1997Co-Authors: James H C Wang, Peter R Martin, Charles K SingletonAbstract:Thiamine deficiency, a frequent complication of alcoholism, contributes significantly to the development of damage in various organ systems, including the brain. The molecular mechanisms that underlie the differential vulnerabilities to thiamine deficiency of tissue and cell types and among individuals are not understood. Investigations into these mechanisms have examined potential variations in thiamine utilizing enzymes. Transketolase is a homodimeric enzyme containing two molecules of noncovalently bound thiamine pyrophosphate. In the present study, we examined a his-tagged human Transketolase that was produced in and purified from Escherichia coli cells. Previous findings demonstrated that purified his-Transketolase had a Km app for cofactor and a thiamine pyrophosphate-dependent lag period for attaining steady-state kinetics that was similar to Transketolase purified from human tissues. Interestingly, the time of the lag period, which is normally independent of enzyme concentration, was found herein to be dependent on the concentration of the recombinant protein. This atypical behavior was due to production in E. coli. Generation of the normal, enzyme concentration-independent state required a cytosolic factor(s) derived from human cells. Importantly, the required factor(s) was found to be defective in a Wernicke-Korsakoff patient whose cells in culture show an enhanced sensitivity to thiamine deficiency.
-
thiamine pyrophosphate requiring enzymes are altered during pyrithiamine induced thiamine deficiency in cultured human lymphoblasts
Journal of Nutrition, 1996Co-Authors: Stevan R Pekovich, Peter R Martin, Charles K SingletonAbstract:The most common of the severe compli cations of thiamine deficiency are beriberi and Wer- nicke-Korsakoff syndrome. To help clarify the bio chemical basis for these disorders, a cell culture sys tem has been established in which pyrithiamine, a potent thiamine transport inhibitor, was used to mimic different degrees of thiamine deficiency within human lymphoblasts. Activities of both Transketolase and a- ketoglutarate dehydrogenase (a-KGDH) decreased at the same rate and to roughly the same levels in re sponse to thiamine deficiency within a given cell line. However, variation in sensitivity to thiamine deficiency, as judged by the relative percentage of loss of enzy matic activities, was found when different cell lines were compared. When exogenous thiamine pyrophos- phate was added to the activity assays, differences between Transketolase and a-KGDH became readily apparent. Only 25% of the lost Transketolase activity was present as apo-enzyme, whereas 70% of the lost a-KGDH activity was present in the apo-enzyme form. For Transketolase, the non-recoverable activity was due mainly to a decrease in the synthesis rate of the protein during thiamine deficiency, suggesting that thi amine has a direct effect on the expression of the Transketolase gene and/or protein. J. Nutr. 126: 1791-1798, 1996.
-
cloning of human Transketolase cdnas and comparison of the nucleotide sequence of the coding region in wernicke korsakoff and non wernicke korsakoff individuals
Journal of Biological Chemistry, 1993Co-Authors: Brian A Mccool, Peter R Martin, S G Plonk, Charles K SingletonAbstract:Variants of the enzyme Transketolase which possess reduced affinity for its cofactor thiamine pyrophosphate (high apparent Km) have been described in chronic alcoholic patients with Wernicke-Korsakoff syndrome. Since the syndrome has been shown to be directly related to thiamine deficiency, it has been hypothesized that such Transketolase variants may represent a genetic predisposition to the development of this syndrome. To test this hypothesis, human Transketolase cDNA clones were isolated, and their nucleotide and predicted amino acid sequence were determined. Transketolase was found to be a single copy gene which produces a single mRNA of approximately 2100 nucleotides. Additionally, the nucleotide sequence of the Transketolase coding region in fibroblasts derived from two Wernicke-Korsakoff (WK) patients was compared to that of two nonalcoholic controls. Although nucleotide and predicted amino acid differences were detected between fibroblast cultures and the original cDNAs and among the cultures themselves, no specific nucleotide variations, which would encode a variant amino acid sequence, were associated exclusively with the coding region from WK patients. Thus, allelic variants of the Transketolase gene cannot account for the biochemically distinct forms of the enzyme found in these patients nor be considered as a mechanism for genetic predisposition to the development of Wernicke-Korsakoff syndrome. Instead, the underlying mechanism must be extragenic and may be a result of differences in post-translational processing/modification of the Transketolase polypeptide.
Olena O. Kurylenko - One of the best experts on this subject based on the ideXlab platform.
-
Peroxisomes and peroxisomal Transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha.
Biotechnology for biofuels, 2018Co-Authors: Olena O. Kurylenko, Kostyantyn V. Dmytruk, Justyna Ruchala, Roksolana V. Vasylyshyn, Oleh V. Stasyk, Olena V. Dmytruk, Andriy SibirnyAbstract:Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve high-temperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol. Involvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal Transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal Transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic Transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30–40 times more ethanol than is produced by the wild-type strain. Our results indicate the importance of the peroxisomal enzymes, Transketolase (dihydroxyacetone synthase, Das1), and transaldolase (Tal2), in the xylose alcoholic fermentation of O. polymorpha.
-
Peroxisomes and peroxisomal Transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha
BMC, 2018Co-Authors: Olena O. Kurylenko, Kostyantyn V. Dmytruk, Justyna Ruchala, Roksolana V. Vasylyshyn, Oleh V. Stasyk, Olena V. Dmytruk, Andriy SibirnyAbstract:Abstract Background Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve high-temperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol. Results Involvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal Transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal Transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic Transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30–40 times more ethanol than is produced by the wild-type strain. Conclusions Our results indicate the importance of the peroxisomal enzymes, Transketolase (dihydroxyacetone synthase, Das1), and transaldolase (Tal2), in the xylose alcoholic fermentation of O. polymorpha
Laurence Hecquet - One of the best experts on this subject based on the ideXlab platform.
-
One-pot, two-step cascade synthesis of naturally rare L-erythro (3S,4S) ketoses by coupling thermostable transaminase and Transketolase.
Green Chemistry, 2017Co-Authors: Marion Lorillière, Franck Charmantray, Wolfdieter Fessner, Thangavelu Saravanan, Maxime De Sousa, Felipe Bruna, Egon Heuson, Thierry Gefflaut, Véronique De Berardinis, Laurence HecquetAbstract:An efficient simultaneous cascade of two enzymatic steps catalyzed by a thermostable transaminase and Transketolase was performed at elevated temperatures allowing the synthesis of naturally rare L-erythro (3S,4S) ketoses. L-ribulose, 5-deoxy-L-ribulose, D-tagatose and L-psicose, which are highly valuable chiral building blocks and display prominent biological properties, were obtained on a preparative scale with excellent stereoselectivities and good yields. A thermostable Transketolase from Geobacillus stearothermophilus catalyzed at high temperatures the stereospecific synthesis of L-erythro (3S,4S)-configured ketoses from (2S)-hydroxylated aldehydes and β-hydroxypyruvate in which the latter is generated in an unprecedented manner in situ from natural L-serine and pyruvate using a novel thermostable L-α-transaminase from the thermophilic bacterium Thermosinus carboxydivorans. Overall, this cascade synthesis prevents the thermal decomposition of the labile β-hydroxypyruvate and offers an efficient and environmentally friendly procedure.
-
a thermostable Transketolase evolved for aliphatic aldehyde acceptors
Chemical Communications, 2015Co-Authors: Dong Yi, Franck Charmantray, Laurence Hecquet, Thangavelu Saravanan, Titu Devamani, Wolfdieter FessnerAbstract:Directed evolution of the thermostable Transketolase from Geobacillus stearothermophilus based on a pH-based colorimetric screening of smart libraries yielded several mutants with up to 16-fold higher activity for aliphatic aldehydes and high enantioselectivity (>95% ee) in the asymmetric carboligation step.
-
fructose 1 6 bisphosphate aldolase and Transketolase complementary tools for the de novo syntheses of monosaccharides and analogues
Journal of Molecular Catalysis B-enzymatic, 1998Co-Authors: Corinne Andre, Laurence Hecquet, Colette Demuynck, Thierry Gefflaut, Christine Guerard, Marielle Lemaire, Jean BolteAbstract:Abstract This paper reports a new synthesis of bromoacetol phosphate and dihydroxyacetone phosphate for use in fructose-1,6-bisphosphate aldolase (FB-aldolase) catalyzed syntheses. Then the activities of FB-aldolase and Transketolase towards polyhydroxybutanal analogues of erythrose and erythrose-4-phosphate were studied. These activities were high enough to allow the syntheses of rare heptulose-1-phosphates of the d and l series.
-
chemoenzymatic synthesis of 6 deoxy d fructose and 6 deoxy l sorbose using Transketolase
Tetrahedron, 1994Co-Authors: Laurence Hecquet, Jean Bolte, Colette DemuynckAbstract:Abstract A chemoenzymatic route to 6-deoxy (D-fructose 5b and L-sorbose 5a) is described. This method is based on conversion of racemic 2,3-dihydroxybutyraldehyde to 5a and 5b catalyzed by spinach leaves Transketolase. Only the (R,R) and (R,S) isomers of 2,3-dihydroxybutyraldehyde react, yielding 6-deoxy-D-fructose and 6-deoxy-L-sorbose.
-
enzyme catalyzed synthesis of carbohydrates synthetic potential of Transketolase
ChemInform, 1992Co-Authors: C Demuynck, Laurence Hecquet, Jean Bolte, Valerie DalmasAbstract:Abstract The synthetic potential of yeast or spinach Transketolases was studied, using hydroxypyruvate as a donor substrate and 31 aldehydes as acceptors. All of them react, including aromatic, heteroaromatic and α,β unsaturated aldehydes.