嗜盐四联球菌胁迫应答分子机制及其在酱油酿造中的应用研究

酱油发酵是盐度、温度、pH和渗透压等胁迫压力不断变化的动态过程，嗜盐四联球菌作为促成酱油风味形成的主要乳酸菌，主要通过其分子伴侣及蛋白水解酶系统应对发酵过程所产生的胁迫压力。本课题将从DNA、RNA、蛋白质等水平上深入研究嗜盐四联球菌分子伴侣及蛋白水解酶系统的胁迫应答机制，并据此遗传改造新菌种，探讨其在酱油酿造中的应用。采用基因敲除、荧光定量PCR、蛋白酶活性测定等方法，研究HrcA/CIRCE对dnaK、groESL、shsp操纵子的负调控作用；借助尺寸排阻色谱法、免疫印迹、蛋白复性等技术，揭示ClpB/ClpP/DnaK的相互作用机制；通过胁迫表达分析hrcA、dnaK、butA mRNA和蛋白酶活性，以及敲除hrcA对butA的表达影响，阐明甜菜碱运输系统与分子伴侣系统的相互关系；结合基因工程筛选突变菌种，应用于酱油酿造，为提高酿造酱油的品质和安全性奠定坚实基础。

Soy sauce fermentation is a dynamic process of stresses, including changing salinity, temperature, pH and osmotic pressure. As the major lactic acid bacteria (LAB) contributing to the formation of soy sauce flavor, Tetragenococcus halophilus adjusts to various stresses produced by fermentation mainly through the molecular chaperone and protease system. This project focuses on the stress response mechanisms of molecular chaperones and proteases from T. halophilus at the DNA, RNA, and protein levels, respectively; accordingly generates new strains with genetic manipulation methods to explore their applications in soy sauce brewing. To investigate the negative regulation of the dnaK, groESL, and shsp operons, exerted by HrcA/CIRCE, gene knockout, quantitative PCR, protease activity analysis are employed; with size exclusion chromatography, Western blotting, protein renaturation technology, the interaction mechanisms of ClpB/ClpP/DnaK are deliberated; the relationship between the molecular chaperone system and the glycine betaine transport system are clarified by means of expression analysis of hrcA, dnaK and butA mRNA and protease activity under stresses, as well as the effect of knockout of hrcA on the expression of butA; apply mutation strains to soy sauce brewing with genetic engineering approach, which lays a solid foundation to improve the quality and safety of soy sauce.