组合调控解淀粉芽孢杆菌胞内辅因子NADH/NAD+强化2,3-丁二醇生物合成的分子机制

Interest in this bioprocess has increased remarkably because 2,3-butanediol (2,3-BD) has a large number of industrial applications, and microbial production will alleviate the dependence on oil supply for the production of platform chemicals. Additionally, 2,3-BD has potential applications in the manufacture of printing inks, perfumes, fumigants, moistening and softening agents, explosives, plasticizers, foods, and pharmaceuticals. So far, absolutely unbeatable in efficient production of 2,3-BD are Klebsiella and Serratia marcescens. It is important to note that mainly class 2 (pathogenic) microorganisms are employed in the 2,3-BD fermentation. Industrial-scale fermentation requires obeying safety regulations, which implies that class 2 microorganisms are unwanted in such applications. In our previous study, a class 1 (safe) strain of Bacillus amyloliquefaciens producing 2,3-BD designated as B10-127 was isolated in our lab. However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production. Low levels of NADH may limit the BDH reaction, since this coenzyme is preferentially used in 2,3-BD synthesis. Based on this perspective, we designed a combinatorial regulation of cofactor engineering for effective improvement of 2,3-BD production while inhibiting the accumulation of unwanted by-products.

2,3-丁二醇是一种重要的四碳平台化合物，被广泛应用于化工、食品、医药、燃料及航空航天等多个领域。微生物法生产2,3-丁二醇已成为国内研究的热点之一。2,3-丁二醇高产菌株为克雷伯氏菌属和沙雷氏菌属，但这些菌具有潜在的致病性, 难以符合生物技术产业化安全生产的要求。本项目以安全菌株解淀粉芽孢杆菌为生产2,3-丁二醇的出发菌，但该菌株在发酵过程中会有一定量的2,3-丁二醇前体物质乙偶姻等副产物的积累。而影响乙偶姻向2,3-丁二醇转化的重要因素是辅因子NADH水平和NADH/NAD+再生效率。因此，针对前体物质乙偶姻过量积累的现象，解析解淀粉芽孢杆菌产物积累和辅因子代谢的相互关系。在此基础上，通过理性设计、定点改造、基因敲除等手段进行与辅因子再生相关的关键酶的分子改造，提出通过组合调控解淀粉芽孢杆菌胞内辅因子NADH/NAD+强化2,3-丁二醇合成和抑制前体物质乙偶姻积累的策略，并构建高产和单

生物能源作为可再生能源，有望减少能源供给对石油的依赖程度。2,3-丁二醇作为一种重要的生物基四碳平台化合物，广泛应用于化工、能源、燃料等多个领域。课题针对解淀粉芽孢杆菌发酵过程中2,3-丁二醇前体物质乙偶姻过量积累的现象，通过组合调控胞内辅因子NADH/NAD+的策略，就如何减少副产物乙偶姻的积累和提高2,3-丁二醇产量的分子机制进行深入研究。具体结论如下：.（1）通过添加不同氧化还原态态底物、引入甲酸/甲酸脱氢酶NADH再生体系和敲除NADH氧化酶基因，可以调控芽孢杆菌胞内辅因子的种类和水平，从而可使芽孢杆菌选择性的积累乙偶姻或2,3-丁二醇。.（2）通过过量共表达芽孢杆菌的NAD+依赖型的3-磷酸甘油醛脱氢酶和NADH依赖型2,3-丁二醇脱氢酶，强化了辅酶NAD+/NADH循环再生效率，成功的提高了发酵液中2,3-丁二醇的产量和生产强度，显著降低了乙偶姻等副产物积累。.（3）通过适度表达2,3-丁二醇合成支路中关键酶α-乙酰乳酸合成酶和α-乙酰乳酸合成酶调控因子ALsR，增强了从丙酮酸到2,3-丁二醇合成途径的代谢流通量；阻断磷酸戊糖途径（PPP）中的关键酶葡糖糖-6-磷酸脱氢酶，使代谢更多地流向EMP途径，同时增强胞内的NADH的水平，进一步提高2,3-BD的产量。.本研究通过组合调控芽孢杆菌胞内辅因子NADH/NAD+调控策略，成功解析了强化2,3-丁二醇生物合成的分子机制，为芽孢杆菌胞内辅因子调控策略提供借鉴。在项目资助期间，在Critical Reviews in Biotechnology, Biotechnology Biofuels, Microbial Cell Factories等期刊发表与本项目相关的论文6篇，其中SCI论文5篇（IF>5的2篇，IF>3的4篇）；申请相关专利10项，其中申请国际专利1项，授权专利3项。通过发酵工艺控制优化，2,3-丁二醇产量处于国际先进水平。