Bacillus subtilis芽孢合成期乙偶姻合成代谢流重排研究

Acetoin, also named as 3-hydroxy-2-butanone, plays a key role in avoiding acidification, participating in the regulation of NADH/ NAD+ ratio and storing carbon in microorganisms. It is a wide-used flavor in the food industry, and it also serves as a precursor in the synthesis of many important compounds. Bacillus subtilis JNA 3-10 was isolated as an acetoin high-producing strain by this lab when fermented with glucose as substrate. However, from the stationary stage of fermentation, B. subtilis begins to form spores, accompanied by cell lysis and the intercellular metabolic flux redistribution, which led to the decrease of biomass and acetoin productivity. Using metabolic engineering strategies, we first intend to inactivate the sporulation related feed-forward loops by disruption of the genes of corresponding Sigma factors in B. subtilis. Then the spontaneously inducible promoters in the middle or late phase of acetoin fermentation will be isolated by 2-DE technology to improve the expression level of ALsR, the positive regulator of acetoin synthesis pathway, and regulate the cofactor ratio in the middle and late phase of fermentation. For further improve acetoin productivity, the metabolic synthesis pathway of the major by-products will also be disrupted or weakened. This work provides reference for construction of acetoin high-producing metabolic engineered B. subtilis.

乙偶姻（3-羟基-2-丁酮），可降低微生物细胞内酸性物质对自身的毒害、起到维持胞内NADH/NAD+平衡、贮存碳源等作用。作为食用香精，乙偶姻广泛应用食品工业，也可用于合成其它重要化合物。课题组前期筛选到一株可利用葡萄糖高效生产乙偶姻的环境安全型枯草芽孢杆菌JNA 3-10，该菌在发酵中后期会发生形成芽孢、细胞溶解等现象，引起细胞内代谢物流向较大的改变和碳源损耗，导致发酵体系中生物量和乙偶姻产率下降。为解决上述问题，本课题拟利用代谢工程手段，首先阻断芽孢形成信号反馈通路关键Sigma因子的编码基因获得芽孢缺陷型枯草芽孢杆菌；其次利用蛋白质二维电泳技术筛选枯草芽孢杆菌发酵中后期自发诱导型启动子用于乙偶姻合成途径正向调控蛋白(ALsR)的加强表达和辅因子水平的调控，并阻断或弱化乙偶姻竞争性副产物合成代谢流，进一步提高乙偶姻合成效率。本项目将为利用代谢工程改造枯草芽孢杆菌生产乙偶姻提供有益借鉴。

项目针对枯草芽孢杆菌发酵过程中芽孢形成与乙偶姻积累在代谢流量分配方面存在的矛盾，通过代谢工程手段从微生物代谢水平对枯草芽孢杆菌芽孢形成途径进行阻断，在此基础上对乙偶姻合成代谢途径进行优化并探索发酵过程理性调控策略。首先利用Cre/lox敲除体系分别敲除芽孢合成关键应答调节蛋白基因spo0A和芽孢特异性σ因子编码基因sigF、sigE，得到重组菌B. subtilis△spo0A和BSD1。spo0A基因的敲除成功的在芽孢合成起始阶段阻断了芽孢的产生；而sigF和sigE基因的共敲除成功的在芽孢合成不对称分裂阶段阻断了芽孢的产生，同时细胞形态在芽孢形成阶段发生了变化，在细胞的两端各形成了一个类似前芽孢的细胞室。spo0A基因的缺失在一定程度上抑制了B. subtilis△spo0A的生长和乙偶姻产量；而sigF和sigE基因的缺失对重组菌生长及产物合成均无显著的影响。通过进一步敲除乙偶姻还原酶基因bdhA、乳酸脱氢酶基因ldh和乙酸激酶基因ackA，分别阻断副产物2,3-丁二醇、乳酸和乙酸的合成。乙偶姻产量达到32.87 g·L-1，乳酸产量降低了79.4%，然而乙酸和琥珀酸的产量分别提高了52.7%、12.3%。比较了几个自诱导启动子在发酵中后期的表达能力，研究发现启动子PsrfA在发酵前期活性较低，在36 h后活性迅速上升，显著的提高了ALDC和ALS的酶活。通过摇瓶发酵，重组菌BSD4/pMA5-PsrfA-alsSD的乙偶姻产量达到39.08 g·L-1，摩尔得率达到0.79 mol·mol-1。乙偶姻合成途径代谢流的加强，使副产物乙酸和琥珀酸的产量分别下降了25.0%、24.3%，而2,3-丁二醇和乳酸的产量基本无变化。对重组菌BSD4/ pMA5-PsrfA-alsSD进行分批补料发酵试验，乙偶姻产量提高至46.57 g·L-1，生产效率达到0.49 g·L-1·h-1，摩尔得率为0.74 mol·mol-1，实现了乙偶姻的高效合成。