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.     

Selection of an endogenous 2,3-butanediol pathway in Escherichia coli by fermentative redox balance

Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (k_cat/K_m≈0.4 mM⁻¹ s⁻¹) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (V_max≈6 U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3 g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme bioprospecting and metabolic evolution.     

Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases

2,3-Butanediol dehydrogenase (BDH) catalyzes the NAD-dependent redox reaction between acetoin and 2,3-butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l-BDH with a bound inhibitor at 2.0 Å. Comparison with the inhibitor binding mode of meso-BDH highlights the role of a hydrogen-bond from a conserved Trp residue¹⁹². Site-directed mutagenesis of three active site residues of meso-BDH, including Trp¹⁹⁰, which corresponds to Trp¹⁹² of l-BDH, converted its stereospecificity to that of l-BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.     

High-yield production of (R)-acetoin in Saccharomyces cerevisiae by deleting genes for NAD(P)H-dependent ketone reductases producing meso-2,3-butanediol and 2,3-dimethylglycerate

Acetoin is widely used in food and cosmetics industries as a taste and fragrance enhancer. To produce (R)-acetoin in Saccharomyces cerevisiae, acetoin biosynthetic genes encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) from Bacillus subtilis and water-forming NADH oxidase (NoxE) from Lactococcus lactis were integrated into delta-sequences in JHY605 strain, where the production of ethanol, glycerol, and (R,R)-2,3-butanediol (BDO) was largely eliminated. We further improved acetoin production by increasing acetoin tolerance by adaptive laboratory evolution, and eliminating other byproducts including meso-2,3-BDO and 2,3-dimethylglycerate, a newly identified byproduct. Ara1, Ypr1, and Ymr226c (named Ora1) were identified as (S)-alcohol-forming reductases, which can reduce (R)-acetoin to meso-2,3-BDO in vitro. However, only Ara1 and Ypr1 contributed to meso-2,3-BDO production in vivo. We elucidate that Ora1, having a substrate preference for (S)-acetoin, reduces (S)-α-acetolactate to 2,3-dimethylglycerate, thus competing with AlsD-mediated (R)-acetoin production. By deleting ARA1, YPR1, and ORA1, 101.3 g/L of (R)-acetoin was produced with a high yield (96% of the maximum theoretical yield) and high stereospecificity (98.2%).     

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.     

芽胞杆菌发酵产2,3-丁二醇的研究进展及展望

综述了近年来利用芽胞杆菌生产2,3-丁二醇(2,3-BD)的研究进展,包括生产菌株的筛选、影响芽胞杆菌发酵2,3-丁二醇的因素、芽胞杆菌2,3-丁二醇代谢途径及调控等方面,并对其研究方向进行了展望。     

Production of 2,3-butanediol from glucose byBacillus licheniformis

Bacillus licheniformis produced 2,3-butanediol from glucose with an optimum yield of 47 g/100 g glucose after 72 h of growth on a peptone/beef extract medium containing 2% (w/v) glucose at pH 6.0 and 37°C. This yield of 2,3-butanediol was higher than those previously reported forKlebsiella oxytoca (37 g/100 g glucose) andBacillus polymyxa (24 g/100 glucose).     

The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis

Bacillus subtilis produces acetoin as a major extracellular product. However, the by-products of 2,3-butanediol, lactic acid and ethanol were accompanied in the NADH-dependent pathways. In this work, metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin by manipulation the NADH levels. We first knocked out the acetoin reductase gene bdhA to block the main flux from acetoin to 2,3-butanediol. Then, among four putative candidates, we successfully screened an active water-forming NADH oxidase, YODC. Moderate-expression of YODC in the bdhA disrupted B. subtilis weakened the NADH-linked pathways to by-product pools of acetoin. Through these strategies, acetoin production was improved to 56.7 g/l with an increase of 35.3%, while the production of 2,3-butanediol, lactic acid and ethanol were decreased by 92.3%, 70.1% and 75.0%, respectively, simultaneously the fermentation duration was decreased 1.7-fold. Acetoin productivity by B. subtilis was improved to 0.639 g/(l h).     

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.     

Metabolic engineering strategies for acetoin and 2,3-butanediol production: advances and prospects

Acetoin and 2,3-butanediol (2,3-BD) have a large number of industrial applications. The production of acetoin and 2,3-BD has traditionally relied on oil supplies. Microbial production of acetoin and 2,3-BD will alleviate the dependence on oil. Acetoin and 2,3-BD are neighboring metabolites in the 2,3-BD metabolic pathway of bacteria. This review summarizes metabolic engineering strategies for improvement of microbial acetoin and 2,3-BD production. We also propose enhancements to current acetoin and 2,3-BD production strategies, by offering a metabolic engineering approach that is guided by systems biology and synthetic biology.     

Optimization of 2,3-butanediol production by Klebsiella oxytoca through oxygen transfer rate control

Production of 2,3-butanediol by Klebsiella oxytoca is influenced by the degree of oxygen limitation. During batch culture studies, two phases of growth are observed: energy-coupled growth, during which cell growth and oxygen supply are coupled; and, energy-uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal 2,3-butanediol productivity occurs during the energy-coupled growth phase. In this article, a control system which maintains the batch culture at a constant level of oxygen limitation in the energy-coupled growth regime has been designed. Control, which involves feedback control on the oxygen transfer coefficient, is achieved by continually increasing the partial pressure of oxygen in the feed gas, which in turn continually increases the oxygen transfer rate. Control has resulted in a balanced state of growth, a repression of ethanol formation, and an increase in 2,3-butanediol productivity of 18%. (c) 1993 John Wiley & Sons, Inc.     

产酸克雷伯氏杆菌发酵产2，3-丁二醇的培养基优化

采用不同设计方法相结合的策略对耐高糖产酸克雷伯氏杆菌（Klebsiella oxytoca）ME—UD-3-4发酵产2,3-丁二醇的培养基进行优化。首先在单因素实验的基础上采用Plackett—Burrnan设计法对影响ME—UD-3-4发酵产2,3-丁二醇的相关因素进行研究,筛选到3种有显著效应的因素（P〈0．05）：葡萄糖、玉米浆和MgSO4·7H2O。然后利用响应曲面法（Response Surface Methodology,RSM）对这3种因素的最佳水平范围进一步探讨;对得到的回归模型进行分析,得最佳条件（g／L）：葡萄糖220、玉米浆19和MgSO4·7H2O 0．4;在最佳条件下,发酵80h,2,3-丁二醇产量从原来的57．3 g／L提高到86．1 g／L,生产强度由0．72g／（L·h）提高到1．08g/（L·h）。     

生物法生产2,3-丁二醇研究进展

2,3-丁二醇是一种重要的化工原料,可广泛应用于多个领域。二战期间由于合成橡胶需要大量1,3-丁二烯,2,3-丁二醇生产空前发展。近年来,由于聚对苯二甲酸丁烯树脂、γ-丁内酯,Spandex弹性纤维及其前体的需求增长,2,3-丁二醇的需求和产量也稳步增长。多年来,生物法生产2,3-丁二醇虽然得到了广泛的研究,但一直没有实现工业化。本文从产生2,3-丁二醇的菌种及2,3-丁二醇的生理意义、代谢途径、旋光异构体的形成机理、影响发酵的因素与产物的提纯等方面对生物法生产2,3-丁二醇进行了综述并提出了生物法生产2,3-丁二醇要解决的几个问题。     

Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2,3-butanediol

The production of biofuels by recombinant Escherichia coli is restricted by the toxicity of the products. 2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical with low toxicity to microbes, could be a promising alternative for biofuel production. However, the yield and productivity of 2,3-BD produced by recombinant E. coli strains are not sufficient for industrial scale fermentation. In this work, the production of 2,3-BD by recombinant E. coli strains was optimized by applying a systematic approach. 2,3-BD biosynthesis gene clusters were cloned from several native 2,3-BD producers, including Bacillus subtilis, Bacillus licheniformis, Klebsiella pneumoniae, Serratia marcescens, and Enterobacter cloacae, inserted into the expression vector pET28a, and compared for 2,3-BD synthesis. The recombinant strain E. coli BL21/pETP_T7-EcABC, carrying the 2,3-BD pathway gene cluster from Enterobacter cloacae, showed the best ability to synthesize 2,3-BD. Thereafter, expression of the most efficient gene cluster was optimized by using different promoters, including P_T7, P_tac, P_c, and P_abc. E. coli BL21/pET-RABC with P_abc as promoter was superior in 2,3-BD synthesis. On the basis of the results of biomass and extracellular metabolite profiling analyses, fermentation conditions, including pH, agitation speed, and aeration rate, were optimized for the efficient production of 2,3-BD. After fed-batch fermentation under the optimized conditions, 73.8 g/L of 2,3-BD was produced by using E. coli BL21/pET-RABC within 62 h. The values of both yield and productivity of 2,3-BD obtained with the optimized biological system are the highest ever achieved with an engineered E. coli strain. In addition to the 2,3-BD production, the systematic approach might also be used in the production of other important chemicals through recombinant E. coli strains.     

Optimization of sodium percarbonate pretreatment for improving 2,3-butanediol production from corncob

Sodium percarbonate (SP), a kind of alkaline strong oxidant, was applied to corncob pretreatment. The optimized pretreatment conditions were at 4% (w/v) SP concentration with solid-to-liquid (SLR) ratio of 1:10 treating for 4?hr at 60°C. This pretreatment resulted in 91.06% of cellulose and 84.08% of hemicellulose recoveries with 34.09% of lignin removal in corncob. The reducing sugar yield from SP-pretreated corncob was 0.56?g/g after 72?hr of enzymatic hydrolysis, 1.75-folds higher than that from raw corncob. 2,3-butanediol production by Enterobacer cloacae in simultaneous saccharification fermentation was 29.18?g/L using SP-pretreated corncob as a substrate, which was 11.12 times of that using raw corncob. Scanning electron microscope, X-ray diffraction, and Fourier transform infrared spectra analysis indicated that physical characteristics, crystallinity, and structure of corncob had changed obviously after SP pretreatment. This simple and novel pretreatment method was effective for delignification and carbohydrate retention in microbial production of 2,3-butanediol from lignocellulose biomass.     

Stereochemical applications of the expression of the L-2,3-butanediol dehydrogenase gene in Escherichia coli

The L-2,3-butanediol dehydrogenase (L-BDH) gene of Brevibacterium saccharolyticum was strongly expressed in Escherichia coli using the tac promoter. However, the stereospecificity of the resulting L-BDH was reduced. The region upstream of the meso-BDH gene of Klebsiella pneumoniae was also involved in the expression of the B. saccharolyticum gene. However, in this case, the resulting L-BDH exhibited more stable stereospecificity. A stereospecificity recognition region was located within the rear sequence (Hpa I site, carboxy terminal) of the BDH open reading frame. Using a transformed strain of E. coli, the conversion of L-acetoin (L-AC), in the commercially available racemic mixture of AC, to L-2,3-butanediol (L-BD) was attempted. As a result, 0.37% L-BD was formed from 1% AC added to the culture.     

Production of 2,3-butanediol by Klebsiella pneumoniae BLh-1 and Pantoea agglomerans BL1 cultivated in acid and enzymatic hydrolysates of soybean hull

We investigated the production of 2,3-butanediol by two enterobacteria isolated from an environmental consortium, Klebsiella pneumoniae BLh-1 and Pantoea agglomerans BL1, in a bioprocess using acid and enzymatic hydrolysates of soybean hull as substrates. Cultivations were carried out in orbital shaker under microaerophilic conditions, at 30°C and 37°C, for both bacteria. Both hydrolysates presented high osmotic pressures, around 2,000 mOsm/kg, with varying concentrations of glucose, xylose, and arabinose. Both bacteria were able to grow in the hydrolysates, at both temperatures, and they efficiently converted sugars into 2,3-butanediol, showing yields varying from 0.25 to 0.51 g/g of sugars and maximum 2,3-butanediol concentrations varying from 6.4 to 21.9 g/L. Other metabolic products were also obtained in lower amounts, notably ethanol, which peaked at 3.6 g/L in cultures using the enzymatic hydrolysate at 30°C. These results suggest the potential use of these recently isolated bacteria to convert lignocellulosic biomass hydrolysates into value-added products.     

Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing

2,3-Butanediol is a promising valuable chemical that can be used in various areas as a liquid fuel and a platform chemical. Here, 2,3-butanediol production in Saccharomyces cerevisiae was improved stepwise by eliminating byproduct formation and redox rebalancing. By introducing heterologous 2,3-butanediol biosynthetic pathway and deleting competing pathways producing ethanol and glycerol, metabolic flux was successfully redirected to 2,3-butanediol. In addition, the resulting redox cofactor imbalance was restored by overexpressing water-forming NADH oxidase (NoxE) from Lactococcus lactis. In a flask fed-batch fermentation with optimized conditions, the engineered adh1Δadh2Δadh3Δadh4Δadh5Δgpd1Δgpd2Δ strain overexpressing Bacillus subtilis α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD), S. cerevisiae 2,3-butanediol dehydrogenase (Bdh1), and L. lactis NoxE from a single multigene-expression vector produced 72.9 g/L 2,3-butanediol with the highest yield (0.41 g/g glucose) and productivity (1.43 g/(L·h)) ever reported in S. cerevisiae.     

Effects of corn steep liquor on production of 2,3-butanediol and acetoin by Bacillus subtilis

The initial concentration of corn steep liquor (CSL) have remarkable effects on not only 2,3-butanediol (2,3-BD) and acetoin (metabolic precursor) production, but also on the ratio of 2,3-BD to acetoin. When a high concentration of CSL was supplemented, cell growth was improved, acetoin reductase (ACR) was stimulated, the concentration of 2,3-BD increased by 78.6%, acetoin decreased by 61.9%, and the ratio of 2,3-BD to acetoin increased by 3.69-fold. The acr gene, encoding ACR, was over-expressed in Bacillus subtilis. Compared to the control (parent strain), low levels of CSL in the engineered strain increased 2,3-BD concentration and the ratio 2,3-BD to acetoin by 13.9% and 39.5%, respectively, and decreased acetoin titer by 18.3%. Acetoin became a major product under low levels of CSL. Also, a knockout strain carrying an acr::cat insertion mutation was constructed. As expected, the loss of ACR activity led to an accumulation of acetoin in the supernatants of acr:: cat mutant cultures. Additionally, the productivity of acetoin was improved by high concentration of CSL. The results above demonstrate the feasibility of using B. subtilis for the production of not only 2,3-BD but also acetoin as a major product.