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.     

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.     

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).     

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.     

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。     

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%).     

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.     

Biotechnological production of 2,3-butanediol—Current state and prospects

Biotechnological production of 2,3-butanediol (hereafter referred to as 2,3-BD) from wastes and excessive biomass is a promising and attractive alternative for traditional chemical synthesis. In the face of scarcity of fossil fuel supplies the bio-based process is receiving a significant interest, since 2,3-BD may have multiple practical applications (e.g. production of synthetic rubber, plasticizers, fumigants, as an antifreeze agent, fuel additive, octane booster, and many others). Although the 2,3-BD pathway is well known, microorganisms able to ferment biomass to 2,3-BD have been isolated and described, and attempts of pilot scale production of this compound were made, still much has to be done in order to achieve desired profitability. This review summarizes hitherto gained knowledge and experience in biotechnological production of 2,3-BD, sources of biomass used, employed microorganisms both wild type and genetically improved strains, as well as operating conditions applied.     

2，3-丁二醇的发酵及盐析分离工艺

采用克雷伯氏菌（Klebsiella pneumoniae CICC 10011）发酵生产2,3-丁二醇,并对2,3-丁二醇的盐析分离工艺进行了考察。通过实验确定了以葡萄糖为底物微氧批式流加发酵的条件,发酵液中2,3-丁二醇和3-羟基丁酮的质量浓度分别为90．98g/L和12．40g/L,2,3-丁二醇的摩尔转化率为82．7％,生产强度达到2．1g/（L·h）。对发酵液中2,3-丁二醇的盐析分离研究表明,K2HPO4和K3PO4对2,3-丁二醇的盐析效果优于K2CO3。当发酵液浓缩70％后,加入质量分数为45％的K,HPO4,2,3-丁二醇的分配系数达到9．10,回收率为79．37％;上相中2,3-丁二醇的质量浓度达到420g／L;此时3-羟基丁酮的分配系数和回收率分别为11．9和83．48％。     

An energetic model for oxygen-limited metabolism

Microbial production of 2,3-butanediol by Klebsiella oxytoca occurs under conditions of an oxygen limitation. The extent to which substrate is oxidized to 2,3-butanediol and its coproducts, (acetic acid, acetoin, and ethanol) and the relative flow rates of substrate to energetic and biosynthetic pathways are controlled by the degree of oxygen limitation. Two energetic relationships which describe the response to an oxygen limitation have been derived. The first relationship describes the coupling between growth and energy production observed under oxygen-limited conditions. This allows calculation of energetic parameters and modeling of the cell mass and substrate profiles in terms of the degree of oxygen limitation only. The second relationship describes the average degree of oxidation and the rate of the end-product flow. The model has been tested with both batch and continuous culture. During these kinetic studies, two phases of growth have been observed: energy-coupled growth, which was described above; and, energy-uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal culture performance with respect to 2,3-butanediol productivity occurs during energy-coupled growth. (c) 1993 John Wiley & Sons, Inc.     

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.     

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）。     

一种简单的高产2,3-丁二醇发酵生产方法

利用一株克雷伯氏菌(Klebsiellasp.LN145)在以葡萄糖和磷酸氢二铵为主要成分的培养基中发酵生产2,3-丁二醇。在补料发酵培养过程中,通过补糖,2,3-丁二醇和3-羟基丁酮的最大产量分别达到了84.0 g/L和10.5 g/L,二醇的摩尔转化率达到理论水平的91%,转化速率达到1.8 g/(L.h)。     

Hydrolysis of Lipid-Extracted Chlorella vulgaris by Simultaneous Use of Solid and Liquid Acids

Microalgal biomass was hydrolyzed using a solid acid catalyst with the aid of liquid acid. The use of solid acid as the main catalyst instead of liquid acid was to omit subsequent neutralization and/or desalination steps, which are commonly required in using the resulting hydrolysates for microbial fermentation. The hydrolysis of 10 g/L of lipid-extracted Chlorella vulgaris containing 12.2% carbohydrates using 7.6 g/L Amberlyst 36 and 0.0075 N nitric acid at 150°C resulted in 1.08 g/L of mono-sugars with a yield of 88.5%. For hydrolysis of higher concentrations of the biomass over 10 g/L, the amount of Amberlyst 36 needed to be increased in proportion to the biomass concentration to maintain similar levels of hydrolysis performance. Increasing the solid acid concentration protected the surface of the solid acid from being severely covered by cell debris during the reaction. A hydrolysate of lipid-extracted C. vulgaris 50 g/L was used, with no post-treatment of desalination, for the cultivation of Klebsiella oxytoca producing 2,3-butanediol. Cell growth in the hydrolysate was found to be almost the same as in the conventional medium with the same monosaccharide composition, confirming its fermentation compatibility. It was noticeable that the yield of 2,3-butanediol with the hydrolysate was observed to be 2.6 times higher than that with the conventional medium. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2729, 2019     

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.     

Breakage of Chromosomal DNA with Aromatic Reductones

Strand breakages of mammalian cellular chromosomal DNA with aromatic reductones were ascertained by use of a cultured cell strain of the rat fetal lung (RFL). The mode of the breakages was investigated by ultracentrifugal analyses. The reductones induced the breakages of the cellular DNA in two different fashions; one is single strand breaks and another double strand breaks. Although the single strand breaks were rapidly repaired, double strand breaks were only partially repaired. Both breaks were not cytocidal. Some physiological alterations were observed to follow the strand breaks.     

Combinatorial optimization of cyanobacterial 2,3-butanediol production

A vital goal of renewable technology is the capture and re-energizing of exhausted CO₂ into usable carbon products. Cyanobacteria fix CO₂ more efficiently than plants, and can be engineered to produce carbon feedstocks useful for making plastics, solvents, and medicines. However, fitness of this technology in the economy is threatened by low yields in engineered strains. Robust engineering of photosynthetic microorganisms is lagging behind model microorganisms that rely on energetic carbon, such as Escherichia coli, due in part to slower growth rates and increased metabolic complexity. In this work we show that protein expression from characterized parts is unpredictable in Synechococcus elongatus sp. strain PCC 7942, and may contribute to slow development. To overcome this, we apply a combinatorial approach and show that modulation of the 5'-untranslated region (UTR) can produce a range of protein expression sufficient to optimize chemical feedstock production from CO₂.     

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。     

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.     

Use of respiratory quotient as a control parameter for optimum oxygen supply and scale-up of 2,3-butanediol production under microaerobic conditions

The respiratory quotient (RQ) was found to be a suitable control parameter for optimum oxygen supply for the production of 2,3-butanediol + acetoin under microaerobic conditions. In laboratory scale continuous cultures optimum production of 2,3-butanediol + acetoin was obtained at an RQ value between 4.0 to 4.5. This agreed well with optimum RQ value (4.0) stoichiometrically derived from the bioreactions involved. In fed-batch cultures product concentrations as high as 102.9 g/L (96.0 g/L butanediol + 6.9 g/L acetoin) can be achieved within 32 h cultivation with an RQ control algorithm for oxygen supply. Under similar conditions only 85.7 g/L product (77.6 g/L butanediol + 8.1 g/L acetoin) was obtained with control of constant oxygen supply rate throughout the cultivation.In pilot scale batch cultures under identical oxygen supply rate the achievable RQ value was found to be strongly influenced by the reactor type and scale. The initial oxygen supply rate influenced the achievable RQ as well. However, in all the reactors studied the specific product formation rate of cells in the exponential growth phase was only a function of RQ. The same optimum RQ value as found in continuous cultures was obtained. It was thus concluded that RQ can be used as a control parameter for optimum production of 2,3-butanediol + acetoin in both laboratory and pilot plant scale reactors. (c) 1994 John Wiley & Sons, Inc.