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G198D/S199 V/P201E/Y218A
mutant displays no detectable activity with NADPH as cofactor, but uses NADH with kcat/Km value of 12 per s and mM
S199A
increase in catalytic efficiencies
S199C
decrease in catalytic efficiencies, with no significant changes in substrate preference
S199G
decrease in catalytic efficiencies, with no significant changes in substrate preference
S199R
increase in catalytic efficiencies
S199W
decrease in catalytic efficiencies, with no significant changes in substrate preference
E221S the
mutation produces a 170fold decrease in the Vm/Km with NADH because of a simultaneous 16fold increase in the Km value and an 11fold decrease in the Vm value, the mutation provides a positive effect on NADPH coenzyme specificity
E221S/I222R
mutant with preference for NADPH as coenzyme
E221S/I222R/A223S
mutant with preference for NADPH as coenzyme
D194G
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the mutant enzyme has almost lost its entire enzymatic activity
D194G
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the mutant enzyme has almost lost its entire enzymatic activity
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additional information
the biocatalytic reduction of 5-methyl-2,3-hexanedione to mainly 5-methyl-3-hydroxy-2-hexanone with only small amounts of 5-methyl-2-hydroxy-3-hexanone within an enzyme membrane reactor is demonstrated
additional information
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the biocatalytic reduction of 5-methyl-2,3-hexanedione to mainly 5-methyl-3-hydroxy-2-hexanone with only small amounts of 5-methyl-2-hydroxy-3-hexanone within an enzyme membrane reactor is demonstrated
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expression of 4 genes encoding alpha-acetolactate synthase from Bacillus subtilis, alpha-acetolactate decarboxylase and 2,3-butanediol dehydrogenase from Bacillus amyloliquefaciens, and NADH oxidase from Lactococcus lactis in Saccharomyces cerevisiae strain YPH499 is modulated using a cocktail delta-integration strategy. The resultant strain, YPH499/dPdAdG/BD6-10, is used in a fed-batch cultivation for the production of 2,3-butanediol. The concentration, production rate, and yield obtained are 80.0 g/l, 4.00 g/l/h, and 41.7%, respectively. The cocktail delta-integration strategy leads to the upregulation of BsAlsS and BaAlsD genes in the modified strain, which results in higher production rate of 2,3-butanediol. The conversion rate of pyruvate to alpha-acetolactate, catalyzed by BsAlsS, and of alpha-acetolactate into acetoin (catalyzed by BaAlsD) must be slower compared to that of acetoin being converted to 2,3-butanediol catalyzed by BaBdhA in the control strain. The conversion of alpha-acetolactate into acetoin catalyzed by alpha-acetolactate decarboxylase might be the rate limiting step in 2,3-butanediol production in recombinant Saccharomyces cerevisiae. Method development and evaluation, overview
additional information
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expression of 4 genes encoding alpha-acetolactate synthase from Bacillus subtilis, alpha-acetolactate decarboxylase and 2,3-butanediol dehydrogenase from Bacillus amyloliquefaciens, and NADH oxidase from Lactococcus lactis in Saccharomyces cerevisiae strain YPH499 is modulated using a cocktail delta-integration strategy. The resultant strain, YPH499/dPdAdG/BD6-10, is used in a fed-batch cultivation for the production of 2,3-butanediol. The concentration, production rate, and yield obtained are 80.0 g/l, 4.00 g/l/h, and 41.7%, respectively. The cocktail delta-integration strategy leads to the upregulation of BsAlsS and BaAlsD genes in the modified strain, which results in higher production rate of 2,3-butanediol. The conversion rate of pyruvate to alpha-acetolactate, catalyzed by BsAlsS, and of alpha-acetolactate into acetoin (catalyzed by BaAlsD) must be slower compared to that of acetoin being converted to 2,3-butanediol catalyzed by BaBdhA in the control strain. The conversion of alpha-acetolactate into acetoin catalyzed by alpha-acetolactate decarboxylase might be the rate limiting step in 2,3-butanediol production in recombinant Saccharomyces cerevisiae. Method development and evaluation, overview
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additional information
construction of an engineered Bacillus subtilis strain 168 in which the bdhA gene is knocked out by the cre/lox system using the lox71-zeo-lox66 resistance marker cassette. The effects of bdhA gene deletion on production of acetoin and 2,3-butanediol are evaluated. By increasing the glucose concentration, the acetoin yield is improved from 6.61 g/l to 24.6 g/l. Deletion of the gene bdhA efficiently blocks the transformation of acetoin and 2,3-butanediol during the fermentation of strain BS168D, overview
additional information
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construction of an engineered Bacillus subtilis strain 168 in which the bdhA gene is knocked out by the cre/lox system using the lox71-zeo-lox66 resistance marker cassette. The effects of bdhA gene deletion on production of acetoin and 2,3-butanediol are evaluated. By increasing the glucose concentration, the acetoin yield is improved from 6.61 g/l to 24.6 g/l. Deletion of the gene bdhA efficiently blocks the transformation of acetoin and 2,3-butanediol during the fermentation of strain BS168D, overview
additional information
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construction of an engineered Bacillus subtilis strain 168 in which the bdhA gene is knocked out by the cre/lox system using the lox71-zeo-lox66 resistance marker cassette. The effects of bdhA gene deletion on production of acetoin and 2,3-butanediol are evaluated. By increasing the glucose concentration, the acetoin yield is improved from 6.61 g/l to 24.6 g/l. Deletion of the gene bdhA efficiently blocks the transformation of acetoin and 2,3-butanediol during the fermentation of strain BS168D, overview
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additional information
coexpression of 2,3-BD biosynthesis pathway genes alsS, alsD, and bdhA in synthetically engineered strains Corynebacterium crenatumDELTAbutADELTAldh SD and Corynebacterium crenatumDELTAldh SDA for selective acetoin (AC) and 2,3-butanediol (2,3-BD) production. Production of AC and 2,3-BD using resting cell bioconversion with glucose as substrate, method evaluation, overview
additional information
in vitro bioreduction of 2-hydroxyacetophenone (2-HAP) is catalyzed by BDHA coupled with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration. The two coexpressed enantiocomplementary carbonyl reductases, BDHA and GoSCR (polyol dehydrogenase from Gluconobacter oxydans) are used for asymmetric reduction of 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) or (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity, method optimization, overview. Products (R)-PED and (S)-PED are obtained with 99% yield, over 99% enantiomeric excess and 18.0 g/l/h volumetric productivity. The reaction is carried out in 5 ml sodium phosphate buffer (pH 7.0, 100 mM) at 30°C, containing 10 U/ml BDHA (cell free extract of Escherichia coli (BDHA)), 15 U/ml GoSCR (cell free extract of Escherichia coli (GoSCR)), 10 U/ml GDH (cell free extract of Escherichia coli (GDH)), 50-200 mM 2-HAP (with 10% DMSO as co-solvent), 60-250 mM D-glucose. Strong tolerance of BDHA and GoSCR against high substrate concentration
additional information
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in vitro bioreduction of 2-hydroxyacetophenone (2-HAP) is catalyzed by BDHA coupled with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration. The two coexpressed enantiocomplementary carbonyl reductases, BDHA and GoSCR (polyol dehydrogenase from Gluconobacter oxydans) are used for asymmetric reduction of 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) or (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity, method optimization, overview. Products (R)-PED and (S)-PED are obtained with 99% yield, over 99% enantiomeric excess and 18.0 g/l/h volumetric productivity. The reaction is carried out in 5 ml sodium phosphate buffer (pH 7.0, 100 mM) at 30°C, containing 10 U/ml BDHA (cell free extract of Escherichia coli (BDHA)), 15 U/ml GoSCR (cell free extract of Escherichia coli (GoSCR)), 10 U/ml GDH (cell free extract of Escherichia coli (GDH)), 50-200 mM 2-HAP (with 10% DMSO as co-solvent), 60-250 mM D-glucose. Strong tolerance of BDHA and GoSCR against high substrate concentration
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additional information
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coexpression of 2,3-BD biosynthesis pathway genes alsS, alsD, and bdhA in synthetically engineered strains Corynebacterium crenatumDELTAbutADELTAldh SD and Corynebacterium crenatumDELTAldh SDA for selective acetoin (AC) and 2,3-butanediol (2,3-BD) production. Production of AC and 2,3-BD using resting cell bioconversion with glucose as substrate, method evaluation, overview
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additional information
variation of residue S199 does not yield any NADH-dependent enzymes
additional information
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mutagenesis of wild-type strain ME-303 enzyme induced by UV-light coupled with diethyl sulfate and a modified proton suicide method leading to 7.8% increased butane-2,3-diol production, and a decrease in the corresponding byproducts of lactic and acetic acid by 88% and 92%, respectively, overview
additional information
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mutagenesis of wild-type strain ME-303 enzyme induced by UV-light coupled with diethyl sulfate and a modified proton suicide method leading to 7.8% increased butane-2,3-diol production, and a decrease in the corresponding byproducts of lactic and acetic acid by 88% and 92%, respectively, overview
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additional information
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Paenibacillus polymyxa is used for the production of R-,R-2,3-butanediol in exceptionally high enantiomeric purity. Rational metabolic engineering efforts to increase productivity and product titers have been restricted due to limited genetic accessibility of the organism. By use of CRISPR-Cas9 mediated genome editing, six metabolic mutant variants are generated and compared in batch fermentations. Downstream processing is facilitated by completely eliminating exopolysaccharide formation through the combined knockout of the sacB gene and the clu1 region, encoding for the underlying enzymatic machinery of levan and paenan synthesis. Knockout constructs are generated to eliminate undesirable side-products of 2,3-BDL fermentations. Spore formation is inhibited by deletion of spoIIE, thereby disrupting the sporulation cascade of Paenibacillus polymyxa. Optimization of the carbon flux towards 2,3-butanediol is achieved by deletion of the lactate dehydrogenase ldh1 and decoupling of the butanediol dehydrogenase from its natural regulation via constitutive episomal expression. The improved strain shows 45% increased productivity, reaching a final concentration of 43.8 g/l butanediol. A yield of 0.43 g/g glucose is achieved, accounting for 86% of the theoretical maximum. Paenibacillus polymyxa is transformed by conjugation using Escherichia coli strain S17-1 harboring the various plasmids. Method optimization and evaluation, overview
additional information
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Paenibacillus polymyxa is used for the production of R-,R-2,3-butanediol in exceptionally high enantiomeric purity. Rational metabolic engineering efforts to increase productivity and product titers have been restricted due to limited genetic accessibility of the organism. By use of CRISPR-Cas9 mediated genome editing, six metabolic mutant variants are generated and compared in batch fermentations. Downstream processing is facilitated by completely eliminating exopolysaccharide formation through the combined knockout of the sacB gene and the clu1 region, encoding for the underlying enzymatic machinery of levan and paenan synthesis. Knockout constructs are generated to eliminate undesirable side-products of 2,3-BDL fermentations. Spore formation is inhibited by deletion of spoIIE, thereby disrupting the sporulation cascade of Paenibacillus polymyxa. Optimization of the carbon flux towards 2,3-butanediol is achieved by deletion of the lactate dehydrogenase ldh1 and decoupling of the butanediol dehydrogenase from its natural regulation via constitutive episomal expression. The improved strain shows 45% increased productivity, reaching a final concentration of 43.8 g/l butanediol. A yield of 0.43 g/g glucose is achieved, accounting for 86% of the theoretical maximum. Paenibacillus polymyxa is transformed by conjugation using Escherichia coli strain S17-1 harboring the various plasmids. Method optimization and evaluation, overview
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additional information
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construction of a an BDH1 deletion strain, WY1DELTA1, which shows 1.37fold increased diacetyl production
additional information
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construction of a an BDH1 deletion strain, WY1DELTA1, which shows 1.37fold increased diacetyl production
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