谷氨酸棒杆菌双组份系统HrrSA、ChrSA分子调控机制及其对血红素合成调控机制研究

血红素（heme）在细胞内发挥着重要的生理功能，其胞内浓度水平受到严格调控。探索heme的调控机制不仅为模式菌株的代谢调控奠定基础，同时也为近源致病菌的侵染机理提供有价值的线索。本研究从产5-氨基乙酰丙酸（ALA）谷氨酸棒杆菌工程菌出发，发现并探索双组份调控系统HrrSA、ChrSA调控heme合成途径的分子机制，利用分子生物学手段DNase I足迹法、5′-RACE法等确定调控蛋白与DNA的结合位点及方式；解析其生物学功能及交叉调控关系；通过代谢产物检测、qPCR、13C代谢流分析手段揭示双组份对heme及ALA合成的影响，进一步揭示heme合成调控机制；最终利用双组份的调控模式动态调控heme生物合成来探索ALA合成途径的优化机制。本项目开展的对双组份调控系统深入分子调控机制研究及对heme、ALA合成的调控机制研究，为代谢工程中高效生物合成途径提供新的理论基础。

Heme plays an important physiological function in the cell. Its intracellular concentration levels are tightly regulated. Exploring the regulation mechanism of heme not only lays the foundation for the metabolic regulation of model strain, but also provide valuable clues to the infection mechanism of pathogenic relative important pathogens. This study, based on the engineered 5-aminolevulinic acid (ALA) producing Corynebacterium glutamicum, is to find and explore the molecular mechanisms of regulation of the two-component regulatory systems HrrSA, ChrSA in heme biosynthesis, by using the new technologies of molecular biology like DNase I footprinting, 5'-RACE method to determine the binding sites and the manner of the regulatory protein acting on DNA. This study also analyzing its biological function and cross-regulation of HrrSA and ChrSA; revealing the impact of the two-component on heme and ALA synthesis by metabolites detected, qPCR and 13C metabolic flux analysis, thus further uncovering heme synthesis regulation mechanism. Then using the two-component regulation model dynamically regulates heme biosynthesis and explores the optimization mechanisms ALA synthesis pathway. This project conducted the in-depth research of the molecular mechanisms of the two-component regulatory system and the regulation mechanism of heme and ALA synthesis, providing a new theoretical foundation for high efficient biosynthetic pathway in metabolic engineering.