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biofuel production
ethanol production by the hyperthermophilic archaeon Pyrococcus furiosus by expression of bacterial bifunctional alcohol dehydrogenase from Thermoanaerobacter sp. X514. Ethanol and acetate are the only major carbon end-products from glucose under these conditions. The amount of ethanol produced per estimated glucose consumed is increased from the background level 0.7 respectively. Although ethanol production from acetyl-CoA is demonstrated in Pyrococcus furiosus, the highest ethanol yield (from strain Te-AdhEA) is still lower than that of the AAA pathway in Pyrococcus furiosus, which functions via the native enzymes acetyl-CoA synthetase (ACS) and aldehyde oxidoreductase (AOR) along with heterologously expressed alcohol dehydrogenase (AdhA)
biofuel production
C7IV28
expression in Pyrococcus furiosus from which the native aldehyde oxidoreductase (AOR) gene is deleted supports ethanol production. The highest amount of ethanol (estimated 61% theoretical yield) is produced when adhE and adhA from Thermoanaerobacter are co-expressed. A strain containing the Thermoanaerobacter ethanolicus AdhE in a synthetic operon with AdhA is constructed. The AdhA gene is amplified from Thermoanaerobacter sp. X514. The amino acid sequence of AdhA from Thermoanaerobacter sp. X514 is identical to that of AdhA from Thermoanaerobacter ethanolicus. Of the bacterial strains expressing the various heterologous AdhE genes, only those containing AdhE and AdhA from Thermoanaerobacter sp. produced ethanol above background. The Thermoanaerobacter ethanolicus AdhEA strain containing both AdhE and AdhA produces the most ethanol (4.2 mM), followed by Thermoanaerobacter ethanolicus AdhE strain (2.6 mM), Thermoanaerobacter ethanolicus AdhA strain (1.8 mM) and Thermoanaerobacter sp. X514 AdhE strain (1.5 mM). Ethanol and acetate are the only major carbon end-products from glucose under these conditions. For these four strains, the amount of ethanol produced per estimated glucose consumed is increased from the background level to 1.2, 1.0, 0.8 and 0.7 respectively. Although ethanol production from acetyl-CoA is demonstrated in Pyrococcus furiosus, the highest ethanol yield (from strain Thermoanaerobacter ethanolicus AdhEA) is still lower than that of the previously reported AAA pathway in Pyrococcus furiosus, which functions via native enzymes acetyl-CoA synthetase (ACS) and aldehyde oxidoreductase (AOR) along with heterologously expressed alcohol dehydrogenase (AdhA)
biofuel production
proteome analysis as well as enzyme assays performed in cell-free extracts demonstrates that glycerol is degraded via glyceraldehyde-3-phosphate, which is further metabolized through the lower part of glycolysis leading to formation of mainly ethanol and hydrogen. Fermentation of glycerol to ethanol and hydrogen by this bacterium represents a remarkable option to add value to the biodiesel industries by utilization of surplus glycerol
biofuel production
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expression in Pyrococcus furiosus from which the native aldehyde oxidoreductase (AOR) gene is deleted supports ethanol production. The highest amount of ethanol (estimated 61% theoretical yield) is produced when adhE and adhA from Thermoanaerobacter are co-expressed. A strain containing the Thermoanaerobacter ethanolicus AdhE in a synthetic operon with AdhA is constructed. The AdhA gene is amplified from Thermoanaerobacter sp. X514. The amino acid sequence of AdhA from Thermoanaerobacter sp. X514 is identical to that of AdhA from Thermoanaerobacter ethanolicus. Of the bacterial strains expressing the various heterologous AdhE genes, only those containing AdhE and AdhA from Thermoanaerobacter sp. produced ethanol above background. The Thermoanaerobacter ethanolicus AdhEA strain containing both AdhE and AdhA produces the most ethanol (4.2 mM), followed by Thermoanaerobacter ethanolicus AdhE strain (2.6 mM), Thermoanaerobacter ethanolicus AdhA strain (1.8 mM) and Thermoanaerobacter sp. X514 AdhE strain (1.5 mM). Ethanol and acetate are the only major carbon end-products from glucose under these conditions. For these four strains, the amount of ethanol produced per estimated glucose consumed is increased from the background level to 1.2, 1.0, 0.8 and 0.7 respectively. Although ethanol production from acetyl-CoA is demonstrated in Pyrococcus furiosus, the highest ethanol yield (from strain Thermoanaerobacter ethanolicus AdhEA) is still lower than that of the previously reported AAA pathway in Pyrococcus furiosus, which functions via native enzymes acetyl-CoA synthetase (ACS) and aldehyde oxidoreductase (AOR) along with heterologously expressed alcohol dehydrogenase (AdhA)
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synthesis
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50 microg of alcohol dehydrogenase AdhA, EC 1.1.1.2, and 50 microg actaldehyde dehydrogenase AldH, EC 1.2.1.10, in buffer solution (pH 8.0) containing NADPH, NADH and acetyl-CoA at 60°C, produce 1.6 mM ethanol from 3 mM acetyl-CoA after 90 min
synthesis
construction of a bypassed pyruvate decarboxylation pathway, through which pyruvate can be converted to acetyl-CoA, by using a coupled enzyme system consisting of pyruvate decarboxylase from Acetobacter pasteurianus and the CoA-acylating aldehyde dehydrogenase from Thermus thermophilus. A cofactor-balanced and CoA-recycling synthetic pathway for N-acetylglutamate production is designed by coupling the bypassed pathway with the glutamate dehydrogenase from Thermus thermophilus and N-acetylglutamate synthase from Thermotoga maritima. N-Acetylglutamate can be produced from an equimolar mixture of pyruvate and alpha-ketoglutarate with a molar yield of 55% through the synthetic pathway consisting of a mixture of four recombinant Escherichia coli strains having either one of the thermostable enzymes. The overall recycling number of CoA is 27
synthesis
synthetic pathway for n-butanol production from acetyl coenzyme at 70°C, using beta-ketothiolase Thl, 3-hydroxybutyryl-CoA dehydrogenase Hbd, and 3-hydroxybutyryl-CoA dehydratase Crt from Caldanaerobacter subterraneus subsp. tengcongensis, trans-2-enoyl-CoA reductase Ter from Spirochaeta thermophila and bifunctional aldehyde dehydrogenase AdhE and and butanol dehydrogenase in vitro. n-Butanol is produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, is used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol is observed in vitro, but with a 60% decrease in the predicted pathway flux
synthesis
synthetic pathway for n-butanol production from acetyl-CoA at 70°C, using beta-ketothiolase Thl, 3-hydroxybutyryl-CoA dehydrogenase Hbd, and 3-hydroxybutyryl-CoA dehydratase Crt from Caldanaerobacter subterraneus subsp. tengcongensis, trans-2-enoyl-CoA reductase Ter from Spirochaeta thermophila and bifunctional aldehyde dehydrogenase AdhE and and butanol dehydrogenase in vitro. n-Butanol is produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, is used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol is observed in vitro, but with a 60% decrease in the predicted pathway flux
synthesis
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construction of a bypassed pyruvate decarboxylation pathway, through which pyruvate can be converted to acetyl-CoA, by using a coupled enzyme system consisting of pyruvate decarboxylase from Acetobacter pasteurianus and the CoA-acylating aldehyde dehydrogenase from Thermus thermophilus. A cofactor-balanced and CoA-recycling synthetic pathway for N-acetylglutamate production is designed by coupling the bypassed pathway with the glutamate dehydrogenase from Thermus thermophilus and N-acetylglutamate synthase from Thermotoga maritima. N-Acetylglutamate can be produced from an equimolar mixture of pyruvate and alpha-ketoglutarate with a molar yield of 55% through the synthetic pathway consisting of a mixture of four recombinant Escherichia coli strains having either one of the thermostable enzymes. The overall recycling number of CoA is 27
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