植物乳杆菌AR195共轭亚油酸生物合成的转录调控机制研究

共轭亚油酸（CLA）是一种新型功能性油脂，乳酸菌因能高效合成c9,t11-CLA和t10,c12-CLA活性异构体成为当前研究热点。改变外界环境和CLA相关基因表达都可调控CLA合成，但具体调控机制尚不明确。申请人前期发现LttR转录调控CLA生物合成，且ArgR2是CLA合成潜在调控因子。基于此，本项目以一株高产c9,t11-CLA的植物乳杆菌AR195为研究对象，通过DNA亲和层析和LC-MS/MS进一步挖掘影响CLA生物合成的其他调控因子；构建各调控因子突变菌株，从代谢和转录水平分析调控因子对靶基因的调控方式，利用DNA足迹法鉴定特异结合位点，通过RNA-seq和分子互作数据分析构建CLA代谢转录调控因子的协作调控网络，从分子水平解析植物乳杆菌CLA生物合成的转录调控机制。通过本项目的实施，深化对乳酸菌CLA生物合成的认识，为调控乳酸菌CLA生物合成、提升CLA产量奠定理论基础。

Conjugated linoleic acid（CLA）is a new type of functional oil. Lactic acid bacteria have become the research hotspot because of their ability to specifically synthesize the active isomers of cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers with stronger probiotic functions. Changing the external environment and the expression of CLA genes can regulate CLA synthesis, but the regulation mechanism is not clear. The applicant previously found that LttR transcriptionally regulates CLA biosynthesis, and ArgR2 is a potential regulator in CLA synthesis. Based on this, this project takes Lactobacillus plantarum AR195 as the research object, further explores other regulators affecting CLA biosynthesis by DNA affinity chromatography and LC-MS/MS. Then constructs the mutant strains, and analyzes the regulation of regulators on the target gene from the metabolic and transcriptional levels. The specific binding sites will be located by DNase I footprinting method. A cooperative regulatory network of CLA metabolic transcriptional regulators will be constructed by RNA-seq and molecular interaction data analysis. The regulatory mechanism of CLA biosynthesis will be studied at the molecular level. Through the implementation of this project, it will deepen the understanding of CLA biosynthesis of lactic acid bacteria and lay a theoretical foundation for the design of metabolic engineering of lactic acid bacteria to increase the yield of CLA.