凝结芽孢杆菌产高光学纯L-乳酸的机理研究

L-Lactic acid is a building block widely used in food, pharmaceutical, and chemical industries. Use of L-lactic acid in the synthesis of polylactic acid has grown over the years, and high optical purity is an inevitable prerequisite for lactic acid polymerization. Currently, microbial fermentation accounts for approximately 90% of the total lactic acid production worldwide. However, the optical purity of L-lactic acid produced by industrial strains cannot meet the standards of high quality polylactic acid. A heat-resistant strain (Bacillus coagulans 2-6), isolated by our team, is an efficient polymer-grade L-lactic acid producer with the optical purity of 99%. Recently, the whole genome sequence of Bacillus coagulans 2-6 is reported by our team. NAD-dependent L-lactate dehydrogenase, NAD-dependent D-lactate dehydrogenase and glycolate oxidase are identified as the key enzymes during polymer-grade L-lactic acid production. Based on the preliminary studies, key enzymes, involved in lactic acid metabolism, are going to be purified from B. coagulans 2-6, Lactobacillus bulgaricus ATCC 11842 (D-lactic acid producing bacteria) and L. plantarum ATCC 14917(DL-lactic acid producing bacteria) in this study, and the biochemical and kinetic characterization of those enzymes will be comparatively studied. The aim of research is to determine the mechanism of the high optical purity of L-lactic acid produced by B. coagulans 2-6 through the combination of gene knockout in vivo and enzymatic studies in vitro. And the genes encoding key enzymes will be the targets for solving the problem involved in the industrialized production. The research will provide the fundamental information for further Bacillus coagulans improvement, and will also provide a theoretical basis for industrial application.

光学纯L-乳酸是近年来迅速发展起来的生物塑料─聚乳酸的主要前体。微生物发酵法是乳酸生产的主要方法。然而现有工业菌株生产的L-乳酸光学纯度难以满足高质量聚乳酸生产要求。本实验室已筛选获得一株产高光学纯L-乳酸（光学纯度99%）的耐热凝结芽孢杆菌2-6。前期完成了该菌的全基因组测序，注释结果显示在该菌中只存在三个与乳酸代谢相关的酶：NAD依赖型L-乳酸脱氢酶，NAD依赖型D-乳酸脱氢酶和乙醇酸氧化酶，并对乳酸代谢关键酶展开了研究。本申请拟在前期工作的基础上，对比分析凝结芽孢杆菌2-6，保加利亚乳杆菌ATCC 11842（D-乳酸生产菌）和植物乳杆菌ATCC 14917（DL-乳酸生产菌）中乳酸代谢关键酶酶学特性；结合体内基因敲除和体外酶学方法探讨凝结芽孢杆菌2-6产高光学纯L-乳酸机理；并以此为指导，解决光学纯L-乳酸工业化生产中的问题，为其进一步分子改造和在工业化的应用提供理论依据。

乳酸是一种重要的多用途有机酸，光学纯L-乳酸是近年来迅速发展起来的生物塑料─聚乳酸的主要前体。以本课题组以已获得的一株产高光学纯L-乳酸，且完成全基因组测序的耐热芽孢杆菌为研究对象，通过蛋白异源表达与纯化，RT-PCR技术，Western blot技术等，对比分析不同构型乳酸生产菌株乳酸代谢关键酶的体内与体外酶学性质。研究结果表明，凝结芽孢杆菌中L-乳酸脱氢酶在蛋白水平和转录水平的高效表达是该菌产高光学纯L-乳酸的关键因素，此外，其它乳酸代谢酶协同作用，减少了产物乳酸对菌株的毒害作用。该研究揭示了凝结芽孢杆菌产高光学纯L-乳酸的机制。通过基因敲除与回补技术，分析关键基因敲除菌株乳酸代谢的变化，确定了不同乳酸代谢关键酶的作用机制，为凝结芽孢杆菌细胞工厂的构建提供理论基础。