Metabolic engineering of Bacillus subtilis for enhanced production of acetoin

Acetoin is widely used in food and other industries. A bdhA and acoA double-knockout strain of Bacillus subtilis produced acetoin at 0.72?mol/mol, a 16.4?% increased compared to the wild type. Subsequent overexpression of the alsSD operon enhanced the acetolactate synthase activity by 52 and 66?% in growth and stationary phases, respectively. However, deletion of pta gene caused little increase of acetoin production. For acetoin production by the final engineered strain, BSUW06, acetoin productivity was improved from 0.087?g/l?h, using M9 medium plus 30?g glucose/l under micro-aerobic conditions, to 0.273?g/h?l using LB medium plus 50?g glucose/l under aerobic conditions. In fermentor culture, BSUW06 produced acetoin up to 20?g/l.     

Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway.

A recent study indicated that Bacillus subtilis catabolizes acetoin by enzymes encoded by the acu gene cluster (F. J. Grundy, D. A. Waters, T. Y. Takova, and T. M. Henkin, Mol. Microbiol. 10:259-271, 1993) that are completely different from those in the multicomponent acetoin dehydrogenase enzyme system (AoDH ES) encoded by aco gene clusters found before in all other bacteria capable of utilizing acetoin as the sole carbon source for growth. By hybridization with a DNA probe covering acoA and acoB of the AoDH ES from Clostridium magnum, genomic fragments from B. subtilis harboring acoA, acoB, acoC, acoL, and acoR homologous genes were identified, and some of them were functionally expressed in E. coli. Furthermore, acoA was inactivated in B. subtilis by disruptive mutagenesis; these mutants were impaired to express PPi-dependent AoDH E1 activity to remove acetoin from the medium and to grow with acetoin as the carbon source. Therefore, acetoin is catabolized in B. subtilis by the same mechanism as all other bacteria investigated so far, leaving the function of the previously described acu genes obscure.     

Mechanism of uridine production by Bacillus subtilis mutants

Summary Pyrimidine analogue-resistant mutants of Bacillus subtilis were found to produce a large amount of uridine. One of them accumulated 55 mg/ml of uridine in culture medium. The changes in enzymes involved in the metabolism of uridine 5-monophosphate (UMP) were examined with this mutant. All six enzymes of de novo UMP biosynthesis were completely free from regulation by uridine compounds, and the activities of these enzymes were 16- to 30-fold higher than those of the enzymes of the parental strain. In the mutant strain, the level of uridine phosphorylase, responsible for converting uridine to uracil, was extremely low, compared with that of the parental strain. No apparent change was observed between the strains in the activity of UMP dephosphorylation or uracil phosphoribosyltransferase. The implication of these findings is discussed in relation to the overproduction of uridine by the mutant.Microbial production of uridine. Part III     

Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories

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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.     

Metabolic engineering of Bacillus subtilis fueled by systems biology: Recent advances and future directions

By combining advanced omics technology and computational modeling, systems biologists have identified and inferred thousands of regulatory events and system-wide interactions of the bacterium Bacillus subtilis, which is commonly used both in the laboratory and in industry. This dissection of the multiple layers of regulatory networks and their interactions has provided invaluable information for unraveling regulatory mechanisms and guiding metabolic engineering. In this review, we discuss recent advances in the systems biology and metabolic engineering of B. subtilis and highlight current gaps in our understanding of global metabolism and global pathway engineering in this organism. We also propose future perspectives in the systems biology of B. subtilis and suggest ways that this approach can be used to guide metabolic engineering. Specifically, although hundreds of regulatory events have been identified or inferred via systems biology approaches, systematic investigation of the functionality of these events in vivo has lagged, thereby preventing the elucidation of regulatory mechanisms and further rational pathway engineering. In metabolic engineering, ignoring the engineering of multilayer regulation hinders metabolic flux redistribution. Post-translational engineering, allosteric engineering, and dynamic pathway analyses and control will also contribute to the modulation and control of the metabolism of engineered B. subtilis, ultimately producing the desired cellular traits. We hope this review will aid metabolic engineers in making full use of available systems biology datasets and approaches for the design and perfection of microbial cell factories through global metabolism optimization.     

枯草芽孢杆菌代谢工程改造的策略与工具

作为一种食品安全级的典型工业模式微生物,枯草芽孢杆菌Bacillus subtilis由于具有非致病性、胞外分泌蛋白能力强以及无明显的密码子偏爱性等特点,现已被广泛应用于代谢工程领域。近年来,随着分子生物学和基因工程技术等的迅速发展,多种研究策略和工具被用于构建枯草芽孢杆菌底盘细胞进行生物制品的高效合成。文中从启动子工程、基因编辑、基因回路、辅因子工程以及途径酶组装等方面介绍枯草芽孢杆菌在代谢工程领域的研究历程,并总结其在生物制品生产中的相关应用,最后对其未来的研究方向进行展望。     

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.     

枯草芽孢杆菌氧化还原感应全局调控因子Rex对乙偶姻合成的影响

【背景】枯草芽孢杆菌体内含有一种可响应胞内氧化还原水平的因子,称之为氧化还原感应全局调控因子Rex (由基因ydiH编码)。Rex可通过感知辅酶NADH/NAD+水平的变化来调节胞内氧化还原平衡。【目的】研究Rex对枯草芽孢杆菌乙偶姻合成和辅因子代谢的相关性。【方法】利用比较转录组挖掘乙偶姻和2,3-丁二醇可逆转化过程中显著差异的基因,并通过Cre/lox基因敲除技术敲除ydiH、acuA (乙酰AcsA)和acoC (二氢脂酰胺乙酰转移酶)。随后,利用实时荧光定量PCR (RT-qPCR)技术分析敲除菌株中乙偶姻相关基因的转录水平。【结果】通过发酵实验发现,敲除ydiH会在一定程度上抑制菌体的生长速率,但发酵前期乙偶姻单位细胞产量和底物转化率都得到了显著提高;敲除acuA和acoC后,对乙偶姻合成、菌体生长和糖耗速率均影响不大;敲除ydiH后,与乙偶姻合成相关基因alsR (alsSD的正转录调控因子)、alsS (α-乙酰乳酸合成酶)、alsD (α-乙酰乳酸脱羧酶)和bdhA (2,3-丁二醇脱氢酶)的转录水平显著上调。【结论】枯草芽孢杆菌氧化还原感应全局调控因子Rex通过抑制与...     

Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 microg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p(R) promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.     

A simulation model for the continuous production of acetoin and butanediol using Bacillus subtilis with integrated pervaporation separation

The potential for producing acetoin and butanediol with a Bacillus subtilis strain was investigated with continuous culture using molasses as carbon substrate. The steady-state results were influenced by both oxygen and undetermined limiting compounds. Employing the known metabolic pathways, four overall stoichiometry relations were used with an energetic assumption on the energy requirements for biomass formation to establish a linear relations were used with an energetic assumption on the energy requirements for biomass formation to establish a linear relation between the overall rates, whose parameters were determined by linear regression. This provided a relationship for the product formation rate. The chemostat culture data were described with a growth kinetics model, which included limitation by molasses and oxygen as well as diauxic effects and product inhibition. The biokinetics model was combined with an experimentally verified model for the membrane Pervaporation. From this combined model were determined the influence of the membrane characteristics (enrichment factors and membrane area) and the dilution rate on the performance of the integrated process. Simulations revealed that an increase of the enrichment factor, possible by membrane improvement, would have counteracting influences, owing to decreased product inhibition but with lower biomass concentration. (c) 1993 Wiley & Sons, Inc.