转录调节因子CpxR与OmpR协同调控Xenocoumacin1生物合成的分子机制

新农药创制是我国实现化学农药减施增效战略的途径。Xcn1是嗜线虫致病杆菌产生的一种优秀抑菌物质，具有开发为新农药的潜质。转录调节因子CpxR和OmpR以相同的方式调控Xcn1的合成，且CpxR调控ompR的转录，但调控机制不明确。生物信息学分析发现ompR启动子区存在CpxR的结合位点，xcn基因簇各操纵子启动子区存在OmpR的结合位点，两者通过级联方式调控Xcn1的合成。在此基础上，本项目拟采用DNA affinity捕获结合在ompR与xcn各操纵子启动子区的调控蛋白，初步验证生物信息学分析结果；进一步通过分子遗传学方法明确CpxR和OmpR分别是直接调控ompR与xcn的调控蛋白；同时分析其各结合位点的功能及其对Xcn1产量的影响，阐明CpxR与OmpR协同调控Xcn1合成的分子机制，为Xcn1高产菌株的改造提供理论依据，为Xcn1新农药的研发提供技术支撑。

The development of new pesticides is the way to realize the strategy of reducing and increasing the efficiency of chemical pesticides in China. Xenocoumacin 1 (Xcn1), a major antimicrobial compound produced by Xenorhabdus nematophila, has great potential for use in agricultural productions. CpxR, a global response regulator associated with the mutualism and pathogenesis of X. nematophila negatively regulates the production of xenocoumacin1. OmpR also negatively regulates the production of xenocoumacin1. Moreover, CpxR positivity regulates the expression of ompR. But the regulatory mechanism is not clear. Bioinformatics analysis shows that there are some CpxR-binding sites located on the promoter region of ompR, and some OmpR-binding sites located on the promoter region of six major xcn transcripts. These means that CpxR may directly regulate the expression of ompR, and OmpR may directly regulate the xcn genes. In order to test the hypothesis, the method of DNA affinity will be used to capture the potential regulators, which specifically bind to the promoter region of ompR and six major xcn transcripts. Then, electrophoretic mobility shift assays (EMSAs), DNase I footprinting in combination with site-directed DNA mutagenesis will be used to determine the precise CpxR binding sites upstream of ompR and OmpR binding sites upstream of six major xcn transcripts. Furthermore, the function of each CpxR-binding site and OmpR-binding site will be analyzed by xylE reporter gene, and the effects of each binding site on the production of Xcn1 will be determined by constructing the mutant strains of each binding site. The discovery of the project will not only improve our understanding the molecular mechanism underlying the effect of CpxR on Xcn1 production, but also provide insishts into the characteristics and network of metabolic regulation in X. nematophila, the rational manipulation of other industrially or medically important Xenorhabdus via metabolic engineering and synthetic biology.