乳酸发酵是典型的产物抑制型反应，发酵过程中需添加大量中和剂，常用碳酸钙为中和剂，但乳酸提纯时会产生工业废物硫酸钙，以氢氧化钠为中和剂发酵则对环境友好，但乳酸钠对菌株的胁迫作用导致发酵产量偏低。凝结芽孢杆菌发酵生产L-乳酸培养基不需灭菌、产物光学纯度高，符合低能耗、高品质的生产要求。以前期筛选并完成基因组测序的凝结芽孢杆菌Bacillus coagulans 2-6为研究对象，根据反向代谢工程原理，先通过多次随机突变和定向筛选获得抗乳酸钠胁迫能力增强的突变株并完成基因组重测序，再利用比较基因组、生物信息学等方法鉴定3-5个有益突变基因作为靶基因，最后利用等位基因置换方法对Bacillus coagulans 2-6进行基因工程改造，构建抗乳酸钠胁迫能力强且高产L-乳酸的最小突变基因工程菌。本项目的顺利实施将为凝结芽孢杆菌在基于氢氧化钠中和的清洁乳酸发酵生产中的大规模应用奠定基础。

Lactic acid fermentation is a typical end-product inhibition process. A large amount of neutralizing agent is required during the fermentation. The commonly used neutralizer is calcium carbonate, but this process results in waste byproduct gypsum production during lactic acid purification. Sodium hydroxide as neutralizing agent for lactic acid fermentation is friendly to environment. However, the stresses of sodium lactate to strains result in reduction of fermentation yields. Fermentation of L-lactic acid with Bacillus coagulans has merits of non-sterilization and high product optical purity, which accord with low energy consumption and high quality production requirements. In this foundation, a previously screened and genome sequenced strain, Bacillus coagulans 2-6, is used to be research object. According to reverse metabolic engineering principle, first, random mutation and directional screening will be performed repeatedly to obtain a mutant strain with increased sodium lactate resistance ability. The genome of the mutant will be sequenced. Then, genome comparation and bioinformatics methods will be used to identify three to five positive mutated genes to be the targets for gene modification. Finally, Bacillus coagulans 2-6 will be genetically modified by allelic replacement to obtain a minimally mutated strain with better sodium lactate stress resistance and higher L-lactic acid production ability. The implementation of this project would lay the foundation for large scale application of Bacillus coagulans in sodium hydroxide-based clean lactic acid fermentation.