高产L-乳酸的最小突变凝结芽孢杆菌基因工程菌的构建

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.

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。

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，通过随机突变和定向筛选获得了1株抗乳酸钠胁迫能力最强的突变株Na-2并完成了该突变株的基因组重测序，通过比较基因组、生物信息学等方法发现一个主要协同转运蛋白（MFS）的突变可能是造成凝结芽孢杆菌抗乳酸钠胁迫能力提高的主要原因，在B. coagulans DSM1中过量表达了MFS蛋白，证实了该蛋白的过量表达能够维持发酵液pH的相对稳定，从而提高菌株耐受乳酸钠的能力。.为继续筛选凝结芽孢杆菌中抗乳酸盐胁迫相关基因和蛋白，开展了比较蛋白质组和转录组学方面的研究。利用二维电泳和质谱鉴定技术研究了凝结芽孢杆菌2-6在乳酸钠胁迫和乳酸钙胁迫条件下蛋白表达的差异，成功鉴定出四个在乳酸盐胁迫条件下高表达的蛋白。利用转录组测序技术研究了凝结芽孢杆菌2-6在乳酸盐胁迫条件下基因表达的差异。凝结芽孢杆菌对乳酸钠胁迫的应答机制包括：“氨基糖和核苷糖代谢”途径受到显著影响，“糖酵解”和“柠檬酸循环”途径受到负调节。凝结芽孢杆菌对乳酸钙胁迫的应答机制包括：“ABC转运蛋白”受到显著影响，“糖酵解”受到正调节。我们还鉴定了在乳酸盐胁迫条件下表达量上调五倍以上的基因，它们可能是基因工程改造的靶点。.总之，本研究发现了凝结芽孢杆菌抗乳酸盐胁迫的一些关键基因和蛋白，揭示了不同乳酸盐胁迫条件下该菌株的分子应答机制。这些结果对于利用代谢工程手段提高凝结芽孢杆菌抗乳酸盐胁迫能力和乳酸生产能力有着重要的用途。

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