基于转录组-表型匹配的鼠李糖乳杆菌胁迫交叉适应机制研究

Lactobacillus rhamnosus, as an important probiotic, is one of the most widely studied starters, whose activity loss is a prominent problem in the production of fermented milk. The previous study found that the cross adaptation to stresses helped to maintain the activity of L. rhamnosus, but its specific rules and mechanisms need to be studied. This project is to begin from the cross adaptation rules of L. rhamnosus hsryfm 1301, and find the optimum pretreatment conditions. Then several cross adaptation key genes will be discovered according to the transcriptome-phenotype matching under different conditions, and the effect of the genes will be identified by using gene knockout. Subsequently, the membrane integrity, metabolome, misfolded proteins and injured DNA of the cross adaptation gene deficient strain will be measured to identify its function. The regulation of cross adaptation gene will be confirmed by EMSA which measures the binding ability of the cross adaptation gene promoter to the globle regulators (HrcA, CtsR, CcpA, LexA), constructing a regulation network of the cross-adaptation gene. Finally, this project will illustrate the molecular mechanism of cross-adaptation in L. rhamnosus, and provide new strategy and theoretical basis for increasing the viability of LAB in production.

鼠李糖乳杆菌作为重要的益生菌，是研究和应用最广的新型乳酸菌发酵剂之一，其活性损失是益生菌发酵乳生产中的一个突出问题。课题组前期研究发现，鼠李糖乳杆菌对胁迫的交叉适应有助于维持菌株活性，但其具体规律和机制有待研究。本项目拟针对鼠李糖乳杆菌建立交叉适应测试体系，确定其对发酵乳生产和贮藏过程中酸、氧、热和冷胁迫的交叉适应规律；利用转录组-表型匹配和生物信息学筛选不同胁迫下的重叠调控基因，并结合缺失突变或启动子更换验证其交叉适应作用；比较野生株与交叉适应基因缺陷型菌株的细胞膜完整性、变性蛋白比例、代谢流向和DNA损伤，确定目标基因的生理功能，利用EMSA分析目标基因启动子与胁迫耐受相关调控元件（HrcA、CtsR、CcpA、LexA）的结合能力，研究胁迫条件下目标基因的转录调控方式。本项目揭示鼠李糖乳杆菌对特定胁迫交叉适应的机制，为保持和提高菌株在生产贮藏过程中的活性提供新的策略和理论依据。

项目针对L. rhamnosus hsryfm 1301建立了交叉适应测试体系，确定了其对酸、氧、热、渗透压和冷胁迫的交叉适应规律，发现了菌株对热胁迫和氧化胁迫存在显著的交叉适应。通过转录组-表型匹配，发现热胁迫和氧化胁迫后，32个重叠调控基因与有机氮源的转运、水解和代谢有关，且全部受到下调。减少培养基中蛋白胨的含量后，菌株在热胁迫和氧化胁迫下的存活率上升了100倍。单独回补多种氨基酸均能够显著降低L. rhamnosus hsryfm 1301对热胁迫和氧化胁迫的耐受能力；莫匹罗星锂盐则能够强化菌株对热胁迫和氧化胁迫的耐受能力；缺失glnR基因后，缺陷型菌株的热胁迫-氧化胁迫交叉适应显著减弱。综上，L. rhamnosus hsryfm 1301对热胁迫-氧化胁迫交叉适应的重要机制为：菌体在受到氧化胁迫或热胁迫后，在GlnR的调控下，氮源代谢相关基因受到下调，短肽的摄入减少，肽酶减少，最终降低胞内的氨基酸浓度；较低的氨基酸浓度可能引发了紧急响应，最终提高菌株的热胁迫和氧化胁迫耐受能力。项目尝试了热胁迫-氧化胁迫交叉适应在实际生产中的应用，发现热胁迫、氧化胁迫和低氮源培养均能显著提高L. rhamnosus hsryfm 1301的喷雾干燥存活率；热预处理还能在维持活菌数的前提下抑制L. rhamnosus hsryfm 1301发酵乳的后酸化。