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.     

Formation of a chiral acetoinic compound from diacetyl by Escherichia coli expressing meso-2,3-butanediol dehydrogenase

L-Acetoin (L-AC) was produced from diacetyl (DA) by Escherichia coli JM109/pBUD119 containing the meso-2,3-butanediol dehydrogenase (D-AC forming) gene. However, when the strain was cultured in the presence of isopropyl-beta-D-thiogalacto-pyranoside, the enzyme formed catalysed not only D-AC but also L-AC. L-AC was further reduced to L-2,3-butanediol (BD). The yield of L-AC or L-BD from DA (3 gl-1) was about 70% (w/w).     

Purification and Characterization of L-2,3-Butanediol Dehydrogenase of Brevibacterium saccharolyticum C-1012 Expressed in Escherichia coli

The L-2,3-butanediol dehydrogenase produced in E. coli JM109/pLBD2-CTC was purified by 5 steps. The molecular mass of this enzyme was estimated at 110 kDa and the subunit was mesured to be 30 kDa. The L-BDH had some differences from the BDHs from other sources in substrate specificity, pI value, pH stability, effects of divalent cations, and organic acids.     

Purification and characterization of L-2,3-butanediol dehydrogenase of Brevibacterium saccharolyticum C-1012 expressed in Escherichia coli

The L-2,3-butanediol dehydrogenase produced in E. coli JM109/pLBD2-CTC was purified by 5 steps. The molecular mass of this enzyme was estimated at 110 kDa and the subunit was measured to be 30 kDa. The L-BDH had some differences from the BDHs from other sources in substrate specificity, pI value, pH stability, effects of divalent cations, and organic acids.     

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 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 and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli.

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an M(r) of 142,000 +/- 2,000 and had same the catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase.     

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.     

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.     

Reduction of acetoin to 2,3-butanediol in Klebsiella pneumoniae: a new model

Fermentation of xylose by Klebsiella pneumoniae (ATCC 8724, formerly known as Aerobacter aerogenes) carried out in our laboratory yields 2,3-butanediol as the major product. Experimental data obtained in this work cannot be explained by the model presently in the literature for the formation of 2,3-butanediol isomers from acetoin isomers. A new model is proposed with the existence of two acetoin reductases and an acetoin racemase. The two reductases were separated and their stereospecificity determined. Extension of the model of other microorganisms is discussed.     

Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum"

A lambda recombinant phage expressing beta-mannanase activity in Escherichia coli has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." The gene was cloned into pBR322 on a 5-kb BamHI fragment, and its location was obtained by deletion analysis. The sequence of a 2.1-kb fragment containing the mannanase gene has been determined. One open reading frame was found which could code for a protein of Mr 38,904. The mannanase gene (manA) was overexpressed in E. coli by cloning the gene downstream from the lacZ promoter of pUC18. The enzyme was most active at pH 6 and 80 degrees C and degraded locust bean gum, guar gum, Pinus radiata glucomannan, and konjak glucomannan. The noncoding region downstream from the mannanase gene showed strong homology to celB, a gene coding for a cellulase from the same organism, suggesting that the manA gene might have been inserted into its present position on the "C. saccharolyticum" genome by homologous recombination.     

Characterization of a stereospecific acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 and its application for the synthesis of (2S,3S)-2,3-butanediol

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K _m values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.     

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.     

Characterization of acetoin production in a budC gene disrupted mutant of Serratia marcescens G12

The 2,3-butanediol (2,3-BD) dehydrogenase gene budC of Serratia marcescens G12 was disrupted to construct the acetoin (AC) producing strain G12M. In shake-flask cultures, AC production was enhanced by increased concentrations of glucose or sodium acetate in G12M. In fed-batch fermentation, G12M produced 47.5 g/L AC along with 9.8 g/L 2,3-BD. The expression of the key enzymes for AC synthesis was further investigated. Alpha-acetolactate synthase gene budB decreased its expression significantly in G12M compared with G12. This probably explained the moderate AC production in G12M cultures. Additionally, overexpression of budB gene and α-acetolactate decarboxylase gene budA was conducted in G12M and no significant increase of AC was observed. The results suggested that intracellular AC accumulation might inhibit the expression of budB and budA gene and induce budC gene expression in G12M. Our analyses offered the bases for further genetic manipulations in improving AC production in microbial fermentations.     

Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway

Abstract The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), acoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA , acoB , and acoC for E1α ( M _r 34639), E1β ( M _r 37268), and E2 ( M _r 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M _r 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.     

D-2,3-butanediol production due to heterologous expression of an acetoin reductase in Clostridium acetobutylicum

Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both d- and l-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM d-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both d-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum.     

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.     

Separation of Racemic frommeso-2,3-Butanediol

2,3-Butanediol containing less than 3% of themesoform has been obtained from samples containing up to 50% of themesoform. The diacetate was obtained by esterification with acetic anhydride in the presence of traces of sulfuric acid as a catalyst and was then purified. When the diacetate was held at 4°C, crystals of racemic 2,3-butanediol diacetate formed, and these were separated by filtration. The diacetate was then transformed back to 2,3-butanediol by transesterification with methanol in the presence of sodium methylate as a catalyst. The resulting 2,3-butanediol contained less than 3% of themesoform. For an original batch of 2,3-butanediol containing 50%dland 50%meso,this method can isolate up to 70% of the racemate content. If the original 2,3-butanediol contains too muchmesoform, racemic 2,3-butanediol diacetate does not crystallize, but 2,3-butanediol containing up to 60% of themesoform can be enriched up to 70% racemate by distillation.     

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.     

Cryoprotection of red blood cells by a 2,3-butanediol containing mainly the levo and dextro isomers.

A 2,3-butanediol containing 96.7% (w/w) racemic mixture of the levo and dextro isomers and only 3.1% (w/w) of the meso isomer (called 2,3-butanediol 97% dl) has been used for the cryoprotection of red blood cells. The erythrocytes were cooled to -196 degrees C at rates between 2 and 3500 degrees C/min, followed by slow or rapid warming. Up to 20% (w/w) of this polyalcohol, only the classical peak of survival is observed, as with up to 20% (w/w) 1,2-propanediol or 1,3-butanediol. Twenty percent 2,3-butanediol 97% dl can protect red blood cells very efficiently. The maximum survival, of 90%, as with 20% glycerol, is a little lower than with 20% 1,2-propanediol and higher than with 20% 1,3-butanediol. Fifteen percent 2,3-butanediol protects fewer red blood cells than 15% glycerol or 1,2-propanediol, with a maximum survival of about 80%. The best cryoprotection by 30% 2,3-butanediol 97% dl is obtained at the slowest cooling and warming rates, where survival approaches 90%. After a minimum, an increase of survival is observed at the fastest cooling rates, which would correspond to complete vitrification. These rates are lower than with 30%, 1,2-propanediol or 1,3-butanediol, in agreement with the higher glass-forming tendency of 2,3-butanediol 97% dl solutions. In agreement with the remarkable physical properties of its aqueous solutions, the present experiments also suggest that 2,3-butanediol containing mainly the levo and dextro isomers could be a very useful cryoprotectant for organ cryopreservation. However, it would perhaps be better to use it in combination with other cryoprotectants, since it is a little more toxic than glycerol or 1,2-propanediol at high concentrations.