Characterization of a (2R,3R)-2,3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060W gene product. Disruption and induction of the gene

The completion of the Saccharomyces cerevisiae genome project in 1996 showed that almost 60% of the potential open reading frames of the genome had no experimentally determined function. Using a conserved sequence motif present in the zinc-containing medium-chain alcohol dehydrogenases, we found several potential alcohol dehydrogenase genes with no defined function. One of these, YAL060W, was overexpressed using a multicopy inducible vector, and its protein product was purified to homogeneity. The enzyme was found to be a homodimer that, in the presence of NAD(+), but not of NADP, could catalyze the stereospecific oxidation of (2R,3R)-2, 3-butanediol (K(m) = 14 mm, k(cat) = 78,000 min(-)(1)) and meso-butanediol (K(m) = 65 mm, k(cat) = 46,000 min(-)(1)) to (3R)-acetoin and (3S)-acetoin, respectively. It was unable, however, to further oxidize these acetoins to diacetyl. In the presence of NADH, it could catalyze the stereospecific reduction of racemic acetoin ((3R/3S)- acetoin; K(m) = 4.5 mm, k(cat) = 98,000 min(-)(1)) to (2R,3R)-2,3-butanediol and meso-butanediol, respectively. The substrate stereospecificity was determined by analysis of products by gas-liquid chromatography. The YAL060W gene product can therefore be classified as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase (BDH). S. cerevisiae could grow on 2,3-butanediol as the sole carbon and energy source. Under these conditions, a 3. 5-fold increase in (2R,3R)-2,3-butanediol dehydrogenase activity was observed in the total cell extracts. The isoelectric focusing pattern of the induced enzyme coincided with that of the pure BDH (pI 6.9). The disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions. The disrupted strain could also grow on 2,3-butanediol, although attaining a lesser cell density than the wild-type strain. Taking into consideration the substrate specificity of the YAL060W gene product, we propose the name of BDH for this gene. The corresponding enzyme is the first eukaryotic (2R, 3R)-2,3-butanediol dehydrogenase characterized of the medium-chain dehydrogenase/reductase family.     

Cloning, expression and characterization of meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae

The budC gene encoding the meso-2,3-BDH from Klebsiella pneumoniae XJ-Li was expressed in E. coli BL21 (DE3) pLys. Hypothetical amino acid sequence alignments revealed that the enzyme belongs to the short chain dehydrogenase/reductase family. After purification and refolding, the recombinant enzyme had activities of 218 U/mg for reduction of acetoin and 66 U/mg for oxidation of meso-2,3-butanediol. Highest activities were at pH 8.0 and 9.0 respectively. These are higher than other meso-2,3-butanediol dehydrogenases from K. pneumoniae. The low K (m) value (0.65 mM) for acetoin indicated that the enzyme can easily reduce acetoin to meso-2,3-butanediol. There were no significant activities towards 2R,3R-2,3-butanediol, 1,4-butanediol and 2S,3S-2,3-butanediol, suggesting that the enzyme has a high stereospecificity for the meso-dihydric alcohol.     

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K(i) of 34 microM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.     

Assay of physiological levels of 2,3-butanediol diastereomers in blood and urine by gas chromatography-mass spectrometry

We present an assay for 2,3-butanediol by gas chromatography-mass spectrometry of its trimethylsilyl ethers. 2R,3R- and/or 2S,3S-2,3-butanediol and meso-2,3-butanediol are quantitated with corresponding internal standards of [2,3-2H2]butanediol. Limits of detection are 1 and 0.1 microM for split and splitless injections, respectively. Blood concentrations of 2,3-butanediol in nonalcoholics are 0.5 +/- 0.3 (SD) microM for 2R,3R- and/or 2S,3S-2,3-butanediol and 0.8 +/- 0.4 microM for meso-2,3-butanediol (n = 9). Two hours after alcohol ingestion, blood levels had risen in eight of nine subjects to 1.2 +/- 0.7 microM for 2R,3R-/2S,3S-2,3-butanediol and to 1.2 +/- 0.6 microM for meso-2,3-butanediol. Baseline urinary excretion of 2,3-butanediol is 0.4 +/- 0.2 mumol/mmol creatinine for 2R,3R-/2S,3S-2,3-butanediol and 0.9 +/- 0.5 mumol/mmol creatinine for meso-2,3-butanediol.     

Structural and enzymatic characterization of Bacillus subtilis R,R-2,3-butanediol dehydrogenase

2,3-butanediol dehydrogenase (BDH, EC 1.1.1.76) also known as acetoin reductase (AR, EC 1.1.1.4) is the key enzyme converting acetoin (AC) into 2,3-butanediol (BD) and undertaking the irreversible conversion of diacetyl to acetoin in various microorganisms. The existence of three BDHs (R,R-, meso-, and S,S-BDH) product different BD isomers. Catalyzing mechanisms of meso- and S,S-BDH have been understood with the assistance of their X-ray crystal structures. However, the lack of structural data for R,R-BDH restricts the integral understanding of the catalytic mechanism of BDHs. In this study, we successfully crystallized and solved the X-ray crystal structure of Bacillus subtilis R,R-BDH. A zinc ion was found locating in the catalytic center and coordinated by Cys37, His70 and Glu152, helping to stabilize the chiral substrates observed in the predicted molecular docking model. The interaction patterns of different chiral substrates in the molecular docking model explained the react priority measured by the enzyme activity assay of R,R-BDH. Site-directed mutation experiments determined that the amino acids Cys37, Thr244, Ile268 and Lys340 are important in the catalytically active center. The structural information of R,R-BDH presented in this study accomplished the understanding of BDHs catalytic mechanism and more importantly provides useful guidance for the directional engineering of R,R-BDH to obtain high-purity monochiral BD and AC.     

Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase

Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversibly R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2R,3R)-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms—purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)—to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of the delitto perfetto methodology to place BDH1 under the control of the GAL1 promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol, and (3R,4R)-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.     

D194G突变对meso-2,3-丁二醇脱氢酶催化特性的影响

目的:比较来源于Enterobacter aerogenes CICC10293和Bacillus subtilis的meso-2,3-丁二醇脱氢酶(E. a-BDH和D194G B. s-BDH)活性和动力学参数,分析D194氨基酸对BDH催化特性的影响。方法:利用E. coli BL21(DE3)原核表达E. a-BDH和D194G B. s-BDH,经HiTrap Q FF阴离子交换柱和Superdex 75凝胶柱纯化后,用MALDI-TOF MS确定其分子质量;检测NADH/NAD⁺氧化还原的吸光度变化确定BDH活性、辅酶和底物的特异性、最适pH、温度及动力学参数。结果:重组表达E. a-BDH和D194G B. s-BDH是同源四聚体蛋白,基因序列有两处碱基不同(g.27A/T和g.581A/G),其中g.581A/G导致BDH的一处氨基酸发生改变(p.D194G)。D194G B. s-BDH的活性约为E. a-BDH的2.3%,并且丧失了氧化meso-2,3-丁二醇的能力。二者均以乙偶姻/NADH为最适底物,但D194G B. s-BDH的K_m是E. a-BDH的5.63倍。结论:D194G氨基酸突变降低了BDH的活性。     

Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.Acetoin and 2,3-butanediol are minor products generated by Saccharomyces cerevisiae during alcohol fermentation. Their sensory impacts on wine are poorly documented. Acetoin may affect the wine bouquet, although its perception threshold in wine is relatively high, around 150 mg/liter (21, 31). On the other hand, 2,3-butanediol is odorless (33) and cannot be expected to appreciably affect the sensory quality of wine. However, the compound may contribute to the wine body (28).Acetaldehyde, pyruvate, and α-acetolactate are the main precursors of acetoin in S. cerevisiae. Acetoin can be formed from acetaldehyde and/or pyruvate through an anomalous reaction of pyruvate decarboxylase. Thus, although its main activity is to irreversibly decarboxylate pyruvate to acetaldehyde, it can also catalyze carbon-carbon bond formation, yielding acetoin from pyruvate and/or acetaldehyde (2, 4). In addition, α-acetolactate would produce acetoin through its nonenzymatic decarboxylation to diacetyl and subsequent reduction to acetoin through the action of several NADH- and NADPH-dependent oxidoreductases (12). However, the situation is more complex in wine fermentation, where other yeasts and bacteria display supplementary enzymatic activities capable of producing both acetoin and 2,3-butanediol (1, 27).We have previously characterized a butanediol dehydrogenase (Bdh1p) as a medium-chain dehydrogenase/reductase (MDR) that can reversibly transform R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively, in a NAD(H)-dependent reaction (10). BDH2 is a gene adjacent to BDH1 whose uncharacterized protein product (Bdh2p) shares 51% sequence identity with Bdh1p. To evaluate the in vivo roles of Bdh1p and Bdh2p, we compared the levels of several extracellular metabolites in cultures of wild-type and deficient strains. The results show that, although Bdh1p is the main enzyme in 2,3-butanediol production [essentially the (2R,3R)-2,3-butanediol stereoisomer], some meso-2,3-butanediol is still produced by the bdh1Δ strains. We have characterized Ara1p as an oxidoreductase that can reduce racemic acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol in the presence of NADPH.Furthermore, we have overexpressed Bdh2p with a histidine tag at its carboxyl terminus and have shown it to be inactive toward acetoin and 2,3-butanediol. A microarray study indicated that BDH1 and BDH2 are reciprocally regulated under the conditions studied.     

Mutation breeding of acetoin high producing Bacillus subtilis blocked in 2,3-butanediol dehydrogenase

Bacillus subtilis mutants were obtained after the wild strain JNA 3-10 was mutagenized by UV irradiation coupled with diethyl sulfate. A visual filter assay was employed for the qualitative identification of 2,3-butanediol dehydrogenase (BDH) blocked B. subtilis. Selected mutants were tested for the activities of acetoin reductase (AR) and BDH. According to further batch fermentation, one mutant named JNA-UD-6 that produced 24.3 % more acetoin than JNA 3-10 with the corresponding byproducts of 2,3-butanediol decreased by 39.8 % was isolated. A nonsense mutation (p.Tyr118X) that precluded the synthesis of a full-length functional AR/BDH within the bdhA gene of JNA-UD-6 was detected. Acetoin production of JNA-UD-6 was further improved to about 53.9 g/L in a 5-L fermentor with 150 g/L glucose consumed. However,a small amount of 2,3-butanediol was found in late phase of JNA-UD-6 fermentation, and it was due to the existence of a putative gene that encoding a minor AR. This work proved a strategy to efficiently breeding an acetoin high producing strain by traditional mutation methods.     

Effect of deletion of 2,3-butanediol dehydrogenase gene (bdhA) on acetoin production of Bacillus subtilis

The present work aims to block 2,3-butanediol synthesis in acetoin fermentation of Bacillus subtilis. First, we constructed a recombinant strain BS168D by deleting the 2,3-butanediol dehydrogenase gene bdhA of the B. subtilis168, and there was almost no 2,3-butanediol production in 20?g/L of glucose media. The acetoin yield of BS168D reached 6.61?g/L, which was about 1.5 times higher than that of the control B. subtilis168 (4.47?g/L). Then, when the glucose concentration was increased to 100?g/L, the acetoin yield reached 24.6?g/L, but 2.4?g/L of 2,3-butanediol was detected at the end of fermentation. The analysis of 2,3-butanediol chiral structure indicated that the main 2,3-butanediol production of BS168D was meso-2,3-butanediol, and the bdhA gene was only responsible for (2R,3R)-2,3-butanediol synthesis. Therefore, we speculated that there may exit another pathway relating to the meso-2,3-butanediol synthesis in the B. subtilis. In addition, the results of low oxygen condition fermentation showed that deletion of bdhA gene successfully blocked the reversible transformation between acetoin and 2,3-butanediol and eliminated the effect of dissolved oxygen on the transformation.     

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K_i of 34 μM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.     

Mechanism for the formation of 2,3-butanediol stereoisomers in Bacillus polymyxa

The mechanism of the formation of 2,3-butanediol isomers in Bacillus polymyxa was studied. We proposed a new model with NADPH-linked diacetyl reductase (S-acetoin forming) and R(−)-2,3-butanediol dehydrogenase. The two enzymes were separated by Blue Sepharose CL-6B and their stereospecificities were identified using all of the pure isomers of 2,3-butanediol (R(−), S(+)m, and meso), acetoin (R(−) and S(+)) and the separation and measurement of these isomers. The presence of acetoin or butanediol racemase was not confirmed in our experiments.     

Identification and characterization of a mycobacterial (2R,3R)-2,3-butanediol dehydrogenase

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD(+)-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.     

Characterization of a zinc-containing alcohol dehydrogenase with stereoselectivity from the hyperthermophilic archaeon Thermococcus guaymasensis

An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l-threonine dehydrogenases with binding motifs of catalytic zinc and NADP(+). Metal analyses revealed that this NADP(+)-dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K(m) values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP(+), respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.     

The Bacillus subtilis ydjL (bdhA) Gene Encodes Acetoin Reductase/2,3-Butanediol Dehydrogenase

Bacillus subtilis is capable of producing 2,3-butanediol from acetoin by fermentation, but to date, the gene encoding the enzyme responsible, acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), has remained unknown. A search of the B. subtilis genome database with the amino acid sequences of functional AR/BDHs from Saccharomyces cerevisiae and Bacillus cereus resulted in the identification of a highly similar protein encoded by the B. subtilis ydjL gene. A knockout strain carrying a ydjL::cat insertion mutation was constructed, which (i) abolished 2,3-butanediol production in early stationary phase, (ii) produced no detectable AR or BDH activity in vitro, and (iii) accumulated the precursor acetoin in early stationary phase. The ydjL::cat mutation also affected the kinetics of lactate but not acetate production during stationary-phase cultivation with glucose under oxygen limitation. A very small amount of 2,3-butanediol was detected in very-late-stationary-phase (96-hour) cultures of the ydjL::cat mutant, suggesting the existence of a second gene encoding a minor AR activity. From the data, it is proposed that the major AR/BDH-encoding gene ydjL be renamed bdhA.     

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.     

Production of L-2,3-butanediol by a new pathway constructed in Escherichia coli

AIMS: A metabolic pathway for L-2,3-butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce L-BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and L-2,3-butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD-comb. METHODS AND RESULTS: The meso-BDH of Klebsiella pneumoniae was used for its DAR activity to convert DA to L-acetoin (AC) and the L-BDH of Brevibacterium saccharolyticum was used to reduce L-AC to L-BD. The respective gene coding each enzyme was connected in tandem to the MCS of pFLAG-CTC (pBUD-comb). The divided addition of DA as a source, addition of 2% glucose, and the combination of static and shaking culture was effective for the production. CONCLUSIONS: L-BD (2200 mg l(-1)) was generated from 3000 mg l(-1) added of DA, which corresponded to a 73% conversion rate. Meso-BD as a by-product was mixed by 2% at most. SIGNIFICANCE AND IMPACT OF THE STUDY: An enzyme system for converting DA to L-BD was constructed with a view to using DA-producing bacteria in the future.     

Reducing diacetyl production of wine by overexpressing <Emphasis Type="Italic">BDH1</Emphasis> and <Emphasis Type="Italic">BDH2</Emphasis> in <Emphasis Type="Italic">Saccharomyces uvarum</Emphasis>

As a byproduct of yeast valine metabolism during fermentation, diacetyl can produce a buttery aroma in wine. However, high diacetyl concentrations generate an aromatic off-flavor and poor quality in wine. 2,3-Butanediol dehydrogenase encoded by BDH1 can catalyze the two reactions of acetoin from diacetyl and 2,3-butanediol from acetoin. BDH2 is a gene adjacent to BDH1, and these genes are regulated reciprocally. In this study, BDH1 and BDH2 were overexpressed in Saccharomyces uvarum to reduce the diacetyl production of wine either individually or in combination. Compared with those in the host strain WY1, the diacetyl concentrations in the recombinant strains WY1-1 with overexpressed BDH1, WY1-2 with overexpressed BDH2 alone, and WY1-12 with co-overexpressed BDH1 and BDH2 were decreased by 39.87, 33.42, and 46.71%, respectively. BDH2 was only responsible for converting diacetyl into acetoin, but not for the metabolic pathway of acetoin to 2,3-butanediol in S. uvarum. This study provided valuable insights into diacetyl reduction in wine.     

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with K_m values of the order of 10^?4m. A K_m value of 60 mm was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(?)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(?)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.     

Production of (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis

Production of highly pure (2S,3S)-2,3-butanediol ((2S,3S)-2,3-BD) and (3S)-acetoin ((3S)-AC) in high concentrations is desirable but difficult to achieve. In the present study, glucose was first transformed to a mixture of (2S,3S)-2,3-BD and meso-2,3-BD by resting cells of Klebsiella pneumoniae CICC 10011, followed by biocatalytic resolution of the mixture by resting cells of Bacillus subtilis 168. meso-2,3-BD was transformed to (3S)-AC, leaving (2S,3S)-2,3-BD in the reaction medium. Using this approach, 12.5 g l(-1) (2S,3S)-2,3-BD and 56.7 g l(-1) (3S)-AC were produced. Stereoisomeric purity of (2S,3S)-2,3-BD and enantiomeric excess of (3S)-AC was 96.9 and 96.2%, respectively.