1.1.1.B20: meso-2,3-butandiol dehydrogenase
This is an abbreviated version!
For detailed information about meso-2,3-butandiol dehydrogenase, go to the full flat file.
Word Map on EC 1.1.1.B20
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1.1.1.B20
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klebsiella
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diacetyl
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pneumoniae
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fed-batch
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nadh-dependent
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2s,3s-2,3-butanediol
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enterobacter
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synthesis
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serratia
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marcescens
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cloacae
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hydrolysate
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acetobacter
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biofuels
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polymyxa
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lactis
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byproduct
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acetolactate
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1,4-butanediol
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bioconversion
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aerogenes
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racemic
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refolding
- 1.1.1.B20
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klebsiella
- diacetyl
- pneumoniae
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fed-batch
-
nadh-dependent
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2s,3s-2,3-butanediol
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enterobacter
- synthesis
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serratia
- marcescens
- cloacae
- hydrolysate
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acetobacter
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biofuels
- polymyxa
- lactis
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byproduct
- acetolactate
- 1,4-butanediol
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bioconversion
- aerogenes
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racemic
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refolding
Reaction
Synonyms
(2R,3R)-2,3-butanediol dehydrogenase, 2,3-BD dehydrogenase, 2,3-butanediol dehydrogenase, 2,3-butanediol dehydrogenases, ADH-9, ARA1, BDH, BdhA, BS-BDH, BtBDH, budC, butACg, butanediol dehydrogenase, ButB, CG-BDH, Cgl2674, mbdh, meso-2,3-BD dehydrogenase, meso-2,3-BDH, meso-2,3-butanediol dehydrogenase, meso-acetoin reductase, meso-BD, meso-BDH, MF996569, More, NAD(H)-dependent meso-2,3-BDH, NAD(H)-dependent meso-2,3-butanediol dehydrogenase, PA4153, PB24_3312, PF-BDH, PF1960, PT-BDH, R,R-2,3-butanediol dehydrogenase/meso-2,3-butanediol dehydrogenase/diacetyl reductase, SmBdh
ECTree
1.1.1.B20 meso-2,3-butandiol dehydrogenaseAdvanced search results
results (
results (Engineering
Engineering on EC 1.1.1.B20 - meso-2,3-butandiol dehydrogenase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
D194G
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site-directed mutagenesis, the mutant binds the substrate but is catalytically almost inactive. The mutant is inactive with (2S,3S)-butanediol, meso-butanediol and (2R,3R)-butanediol. D194G enzyme mutant shows a similar secondary structure compared to Enterobacter aerogenes BDH. While the mutant is highly susceptible to protease digestion compared to the wild-type enzyme. Homology modeling of the mutant enzyme, with meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae, PDB ID 1GEG, as a template, reveals that Gly194 seems to lose the hydrogen bond interactions with the surrounding residues (Gly206, Gly207 and Thr209), resulting in a putative conformational changes of mutant D194G which might be responsible for the loss of activity
moe
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construction and engineering of Corynebacterium glutamicum strain DELTAaceEDELTApqoDELTAldhA(pEKEx2-als,aldB,butACg). Chromosomal inactivation of the putative BDH gene butACg (cg2958) in strain DELTAaceEDELTApqoDELTAldhA. BDH activity is nearly abolished upon inactivation of butACg indicating that Corynebacterium glutamicum expresses a single BDH under the experimental conditions examined. BDH activity increases 3fold in strain DELTAaceEDELTApqoDELTAldhA(pEKEx2-als,aldB,butACg) compared to the respective control. The inactivation of butACg gene decreases the BDH activity 75fold for the DELTAaceEDELTApqoDELTAldhADELTAbutACg(pEKEx2) strain compared to strain DELTAaceEDELTApqoDELTAldhA(pEKEx2). The major form of 2,3-butanediol is meso-2,3-butandediol, and the ratio meso-2,3-BD/optically active 2,3-BD is 95:5, the main side products are glycerol, ethanol, and acetoin
Q140I/N146F/W190H
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trace activity below 0.1 U/mg with substrate meso-butanediol, 2.9 U/mg with substrate (2S,3S)-2,3-butanediol
F212S
site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type
F212W
site-directed mutagenesis, the mutant shows reduced activity compared to wild-type
F212Y
site-directed mutagenesis, the kcat of the mutant is enhanced 4-8fold compared to wild-type
N146Q
site-directed mutagenesis, the mutant shows unaltered activity compared to wild-type
Q140I
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mutation mimicking the corresponding residue in (S,S)-butanediol dehydrogenase. No activity with substrates meso-butanediol or (S,S)-butanediol
Q140I/N146F
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mutation mimicking the corresponding residues in (S,S)-butanediol dehydrogenase. Poor activity with substrates meso-butanediol or (S,S)-butanediol
Q274A
site-directed mutagenesis, the substrate binding site is occupied by a glycerol molecule in the Q247A mutant, the mutation disrupts the active site of the protein, the Q247A mutant shows a 90% loss in activity compare to wild-type
Q274A/V139Q
site-directed mutagenesis, the substrate binding site is occupied by an ethylene glycol molecule in the Q274A/V139Q mutant, the mutation disrupts the active site of the protein, the double mutant Q247A/V139Q showa 300% improvement in activity in comparison to the Q247A mutant. Although the double mutant does not completely restore the loss of Gln247 activity, significant function is regained by introducing the V139Q mutation in this protein, to about 50% activity compared to wild-type
Q274A
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site-directed mutagenesis, the substrate binding site is occupied by a glycerol molecule in the Q247A mutant, the mutation disrupts the active site of the protein, the Q247A mutant shows a 90% loss in activity compare to wild-type
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Q274A/V139Q
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site-directed mutagenesis, the substrate binding site is occupied by an ethylene glycol molecule in the Q274A/V139Q mutant, the mutation disrupts the active site of the protein, the double mutant Q247A/V139Q showa 300% improvement in activity in comparison to the Q247A mutant. Although the double mutant does not completely restore the loss of Gln247 activity, significant function is regained by introducing the V139Q mutation in this protein, to about 50% activity compared to wild-type
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additional information
additional information
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generation of a mutant strain WX-02 DELTAbudC of Bacillus licheniformis with depleted budC gene that produces high yields of the D-2,3-butanediol isomer with high optimal purity
additional information
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construction of a knockout Bacillus licheniformis mutant DELTAbudCDELTAgdh deleting two 2,3-butanediol dehydrogenases, i.e. meso-2,3-butanediol dehydrogenases BudC and GDH, through gene disruption. Escherichia coli strain S17-1 lpir is used as donor strain to allow the conjugal transfer of plasmids pKVM1-1budC and pKVM1-1gdh into Bacillus licheniformis strain MW3. Although the growth of strain MW3 (DELTAbudCDELTAgdh) is slightly lower than that of wild-type strain MW3, it can produce acetoin instead of 2,3-butanediol as its major product. Using fedbatch fermentation of Bacillus licheniformis MW3 (DELTAbudCDELTAgdh), 64.2 g/l acetoin is produced at a productivity of 2.378 g/l/h and a yield of 0.412 g/g from 156 g/l glucose in 27 h
additional information
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construction of a knockout Bacillus licheniformis mutant DELTAbudCDELTAgdh deleting two 2,3-butanediol dehydrogenases, i.e. meso-2,3-butanediol dehydrogenases BudC and GDH, through gene disruption. Escherichia coli strain S17-1 lpir is used as donor strain to allow the conjugal transfer of plasmids pKVM1-1budC and pKVM1-1gdh into Bacillus licheniformis strain MW3. Although the growth of strain MW3 (DELTAbudCDELTAgdh) is slightly lower than that of wild-type strain MW3, it can produce acetoin instead of 2,3-butanediol as its major product. Using fedbatch fermentation of Bacillus licheniformis MW3 (DELTAbudCDELTAgdh), 64.2 g/l acetoin is produced at a productivity of 2.378 g/l/h and a yield of 0.412 g/g from 156 g/l glucose in 27 h
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additional information
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generation of a mutant strain WX-02 DELTAbudC of Bacillus licheniformis with depleted budC gene that produces high yields of the D-2,3-butanediol isomer with high optimal purity
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additional information
a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
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
Bacillus subtilis BS168D
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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
a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
additional information
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a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
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additional information
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a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
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additional information
-
a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
-
additional information
-
a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
-
additional information
-
a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
-
additional information
-
a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
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additional information
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expression of gene bdh1 from Saccharomyces cervisiae in Escherichia coi strain YYC202(DE3) ldhA-/- ilvC-/- expressing ilvBN from Escherichia coli and aldB from Lactobacillus lactis, encoding acetolactate synthase and acetolactate decarboxylase activities, respectively. Disruption of the lactate biosynthesis pathway in the strain increases pyruvate precursor availability to this strain, increased availability of NADH for acetoin reduction to meso-2,3-butanediol is the most important consequence of ldhA deletion. Optimization of 2,3-butanediol production in Escherichia coli, overview
additional information
Enterobacter aerogenes is metabolically engineered for acetoin production. To remove the pathway enzymes that catalyze the formation of by-products, the three genes encoding a lactate dehydrogenase (ldhA) and two 2,3-butanediol dehydrogenases (budC, and dhaD), respectively, are deleted from the genome. The acetoin production is higher under highly aerobic conditions. An extracellular glucose oxidative pathway in Enterobacter aerogenes is activated under the aerobic conditions, resulting in the accumulation of 2-ketogluconate. To decrease the accumulation of this by-product, the gene encoding a glucose dehydrogenase (gcd) is also deleted. The resulting strain does not produce 2-ketogluconate but produces significant amounts of acetoin, with concentration reaching 71.7 g/l with 2.87 g/l/h productivity in fed-batch fermentation. The resulting strains are EMY-01 (DELTAldhA), EJW-0 (DELTAldhA-DELTAbudC), EJW-02 (DELTAldhA-DELTAbudC-DELTAdhaD), and EJW-03 (DELTAldhA-DELTAbudC-DELTAdhaD-DELTAgcd), evaluation for acetoin production, overview
additional information
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Enterobacter aerogenes is metabolically engineered for acetoin production. To remove the pathway enzymes that catalyze the formation of by-products, the three genes encoding a lactate dehydrogenase (ldhA) and two 2,3-butanediol dehydrogenases (budC, and dhaD), respectively, are deleted from the genome. The acetoin production is higher under highly aerobic conditions. An extracellular glucose oxidative pathway in Enterobacter aerogenes is activated under the aerobic conditions, resulting in the accumulation of 2-ketogluconate. To decrease the accumulation of this by-product, the gene encoding a glucose dehydrogenase (gcd) is also deleted. The resulting strain does not produce 2-ketogluconate but produces significant amounts of acetoin, with concentration reaching 71.7 g/l with 2.87 g/l/h productivity in fed-batch fermentation. The resulting strains are EMY-01 (DELTAldhA), EJW-0 (DELTAldhA-DELTAbudC), EJW-02 (DELTAldhA-DELTAbudC-DELTAdhaD), and EJW-03 (DELTAldhA-DELTAbudC-DELTAdhaD-DELTAgcd), evaluation for acetoin production, overview
additional information
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Enterobacter aerogenes is metabolically engineered for acetoin production. To remove the pathway enzymes that catalyze the formation of by-products, the three genes encoding a lactate dehydrogenase (ldhA) and two 2,3-butanediol dehydrogenases (budC, and dhaD), respectively, are deleted from the genome. The acetoin production is higher under highly aerobic conditions. An extracellular glucose oxidative pathway in Enterobacter aerogenes is activated under the aerobic conditions, resulting in the accumulation of 2-ketogluconate. To decrease the accumulation of this by-product, the gene encoding a glucose dehydrogenase (gcd) is also deleted. The resulting strain does not produce 2-ketogluconate but produces significant amounts of acetoin, with concentration reaching 71.7 g/l with 2.87 g/l/h productivity in fed-batch fermentation. The resulting strains are EMY-01 (DELTAldhA), EJW-0 (DELTAldhA-DELTAbudC), EJW-02 (DELTAldhA-DELTAbudC-DELTAdhaD), and EJW-03 (DELTAldhA-DELTAbudC-DELTAdhaD-DELTAgcd), evaluation for acetoin production, overview
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additional information
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Bacillus subtilis is engineered to produce chiral pure meso-2,3-BD. D-2,3-butanediol production is abolished by deleting D-2,3-butanediol dehydrogenase (EC 1.1.1.4) coding gene bdhA, and acoA gene is knocked out to prevent the degradation of acetoin, the immediate precursor of 2,3-butanediol. Next, both pta and ldh gene are deleted to decrease the accumulation of the byproducts, acetate and L-lactate. The meso-2,3-butanediol dehydrogenase coding gene from Klebsiella pneumoniae CICC10011 is introduced, as well as alsSD overexpressed in the tetra mutant (DELTAacoADELTAbdhADELTAptaDELTAldh) to achieve the efficient production of chiral meso-2,3-butanediol. Finally, the pool of NADH availability is further increased to facilitate the conversion of meso-2,3-butanediol from acetoin by overexpressing the udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-butanediol with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct acetoin is no more than 1.1 g/L. Method optimization. The titer of meso-2,3-butanediol is almost unchanged at 37°C, 42°C, and 46°C, while the meso-2,3-butanediol productivity increases when the cultivation temperature is increased from 37°C to 46°C. The titer and productivity at 50°C decreases by 28.6% and 36.3% compared to those at 37°C
additional information
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Bacillus subtilis is engineered to produce chiral pure meso-2,3-BD. D-2,3-butanediol production is abolished by deleting D-2,3-butanediol dehydrogenase (EC 1.1.1.4) coding gene bdhA, and acoA gene is knocked out to prevent the degradation of acetoin, the immediate precursor of 2,3-butanediol. Next, both pta and ldh gene are deleted to decrease the accumulation of the byproducts, acetate and L-lactate. The meso-2,3-butanediol dehydrogenase coding gene from Klebsiella pneumoniae CICC10011 is introduced, as well as alsSD overexpressed in the tetra mutant (DELTAacoADELTAbdhADELTAptaDELTAldh) to achieve the efficient production of chiral meso-2,3-butanediol. Finally, the pool of NADH availability is further increased to facilitate the conversion of meso-2,3-butanediol from acetoin by overexpressing the udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-butanediol with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct acetoin is no more than 1.1 g/L. Method optimization. The titer of meso-2,3-butanediol is almost unchanged at 37°C, 42°C, and 46°C, while the meso-2,3-butanediol productivity increases when the cultivation temperature is increased from 37°C to 46°C. The titer and productivity at 50°C decreases by 28.6% and 36.3% compared to those at 37°C
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additional information
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a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
additional information
-
a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
-
additional information
a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
additional information
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a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
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additional information
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a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
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additional information
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a two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase (CT-XR) from Candida tenuis is constructed to co-produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidate organisms (Bacillus subtilis, Corynebacterium glutamicum, Parageobacillus thermoglucosidans, and Pyrococcus furiosus), as well as xylose reductase from Candida tenuis are purified and analyzed The best BDH is then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, and the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g/l D-(-)-acetoin with an optical purity of 95.2% is produced in 6 h. The yield and productivity of acetoin is 0.97 g/g and 4.75 g/l/h. The titer of co-product xylitol is 40.29 g/l, and the yield and productivity of xylitol reaches 0.98 g/g and 6.72 g/l/h. Method development, evaluation, and optimization for production of optically pure D-(-)-acetoin, overview. Enzyme CT-XR acts most effectively with BDH from Corynebacterium glutamicum (CG-BDH)
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enzyme SmBdh is superior to other Bdhs for expression in Zymomonas mobilis for 2,3-BDO production. Structurally guided changes of recombinantly SmBdh expressed in Zymomonas mobilis can explain its superiority over lower activity Bdh enzymes from the same family of proteins. Development of two mutants of SmBdh: (1) Q247A where the Gln247 side chain is removed, leaving alanine at position 247 and (2) the double mutant Q247A/V139Q, where the missing glutamine side chain is reinstated at the position 139 that is present in KpBdh. Whereas Q247A will disrupt the active site of the protein, Q247A/V139Q is expected to restore the active site via a compensatory mechanism resulting in the active site being established by a single protein chain without contribution from a symmetry-related molecule (i.e. from the C-terminus of the opposite molecule in the tetramer)
additional information
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enzyme SmBdh is superior to other Bdhs for expression in Zymomonas mobilis for 2,3-BDO production. Structurally guided changes of recombinantly SmBdh expressed in Zymomonas mobilis can explain its superiority over lower activity Bdh enzymes from the same family of proteins. Development of two mutants of SmBdh: (1) Q247A where the Gln247 side chain is removed, leaving alanine at position 247 and (2) the double mutant Q247A/V139Q, where the missing glutamine side chain is reinstated at the position 139 that is present in KpBdh. Whereas Q247A will disrupt the active site of the protein, Q247A/V139Q is expected to restore the active site via a compensatory mechanism resulting in the active site being established by a single protein chain without contribution from a symmetry-related molecule (i.e. from the C-terminus of the opposite molecule in the tetramer)
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the enzyme from Serratia marcescens is overexpressed in Lactobacillus diolivorans to produce meso-2,3-butanediol (meso-2,3-BTD). A two-step cultivation process with Serratia marcescens is developed for production of 2-butanol in Lactobacillus diolivorans via vitamin B12-dependent diol dehydratase (PduCDE) and alcohol dehydrogenase (pduQ). In the first step of the process, Serratia marcescens is used to produce stereospecifically meso-2,3-BTD from glucose followed by heat inactivation of Serratia marcescens. The accumulated meso-2,3-BTD is then converted during anaerobic fermentation with glucose into 2-butanol by Lactobacillus diolivorans. The process yields a butanol concentration of 10 g/l relying on wild-type bacterial strains. A further improvement of the maximum butanol titer is achieved using an engineered Lactobacillus diolivorans strain overexpressing the endogenous alcohol dehydrogenase pduQ. The two-step cultivation process based on this engineered strain leads to a maximum 2-butanol titer of 13.4 g/l, which means an increase of 34%
additional information
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enzyme SmBdh is superior to other Bdhs for expression in Zymomonas mobilis for 2,3-BDO production. Structurally guided changes of recombinantly SmBdh expressed in Zymomonas mobilis can explain its superiority over lower activity Bdh enzymes from the same family of proteins. Development of two mutants of SmBdh: (1) Q247A where the Gln247 side chain is removed, leaving alanine at position 247 and (2) the double mutant Q247A/V139Q, where the missing glutamine side chain is reinstated at the position 139 that is present in KpBdh. Whereas Q247A will disrupt the active site of the protein, Q247A/V139Q is expected to restore the active site via a compensatory mechanism resulting in the active site being established by a single protein chain without contribution from a symmetry-related molecule (i.e. from the C-terminus of the opposite molecule in the tetramer)
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additional information
Serratia marcescens DSMZ 14187
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the enzyme from Serratia marcescens is overexpressed in Lactobacillus diolivorans to produce meso-2,3-butanediol (meso-2,3-BTD). A two-step cultivation process with Serratia marcescens is developed for production of 2-butanol in Lactobacillus diolivorans via vitamin B12-dependent diol dehydratase (PduCDE) and alcohol dehydrogenase (pduQ). In the first step of the process, Serratia marcescens is used to produce stereospecifically meso-2,3-BTD from glucose followed by heat inactivation of Serratia marcescens. The accumulated meso-2,3-BTD is then converted during anaerobic fermentation with glucose into 2-butanol by Lactobacillus diolivorans. The process yields a butanol concentration of 10 g/l relying on wild-type bacterial strains. A further improvement of the maximum butanol titer is achieved using an engineered Lactobacillus diolivorans strain overexpressing the endogenous alcohol dehydrogenase pduQ. The two-step cultivation process based on this engineered strain leads to a maximum 2-butanol titer of 13.4 g/l, which means an increase of 34%
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additional information
AEF50077
efficient (3R)-acetoin production from meso-2,3-butanediol using a whole-cell biocatalyst with coexpression of Serratia sp. meso-2,3-butanediol dehydrogenase, Lactobacillus brevis NADH oxidase and Vitreoscilla sp. hemoglobin
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chiral (3R)-AC production from meso-2,3-butanediol (meso-2,3-BD) is obtained using recombinant Escherichia coli cells co-expressing meso-2,3-butanediol dehydrogenase (meso-2,3-BDH), NADH oxidase (NOX), and hemoglobin protein (VHB) from Serratia sp. T241, Lactobacillus brevis, and Vitreoscilla, respectively. The biocatalysis system of Escherichia coli/pET-mbdh-nox-vgb is developed and the bioconversion conditions are optimized. Under the optimal conditions, 86.74 g/l of (3R)-acetoin with the productivity of 3.61 g/l/h and the stereoisomeric purity of 97.89% is achieved from 93.73 g/l meso-2,3-BD using the whole-cell biocatalysis system, pH 7.0 at 30°C for 12 h. The results show the industrial potential for (3R)-acetoin production via whole-cell biocatalysis. Escherichia coli/pET-mbdh cannot produce acetoin from (2R,3R)-2,3-BD as substrate. To obtain high (3R)-acetoin productivity, a cofactor regeneration system involved in co-expression of meso-2,3-BDH and NOX enzymes from Serratia sp. T241 Lactobacillus brevis is developed in Escherichia coli. The NOX enzyme efficiently oxidizes NADH, which is formed by meso-2,3-BDH, and regenerate NAD+ for the biocatalytic process. The feasibility of (3R)-AC production from the substrate of meso-2,3-BD by whole-cell biocatalysis is conducted, method optimization, overview. A small amount of (3S)-acetoin (1.86 g/l) can also be produced from (2S,3S)-2,3-BD in the substrate 2,3-BD (2.23% of (2S,3S)-2,3-BD) by the biocatalyst
additional information
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the regeneration of oxidised nicotinamide adenine dinucleotide is a key point in preparative application of dehydrogenases for the oxidative route. An electrochemical regeneration system is successfully combined with the BDH catalysed reaction. Up to 48 mM (R)-acetoin is produced in the reaction system while productivities up to 2 mM/h are reached. Possibility to apply an electrochemical system in a semi-preparative synthesis. Lyophilised recombinant ADH-9 from Escherichia coli BL21(DE3) cells is immobilized onto Amberlite FPA54 to 0.01 U per mg carrier leading to increased productivity compared to the immobilised form, method optimization
