EnvZ/OmpR双组分系统中转录因子OmpR调控粘质沙雷氏菌合成灵菌红素的机制

Prodiginines are a group of secondary metabolites produced by Serratia marcescens and other microbes and attracting more and more attentions because of their excellent bioactivities. Biosynthesis and regulation mechanisms of prodiginines are complex and still uncovered clearly now, which becomes the main limiting factor for the production and applications of prodiginines. Though our group previously found that prodigiosin synthesis of Serratia marcescens FZSF02 are strictly regulated by OmpR the transcriptional regulatory protein of EnvZ/OmpR two-component system, and several genes including the pigA-O gene cluster genes were found transcriptionally regulated by OmpR by comparing the RNAseq data of the wild type strain and OmpR mutant strain. But the molecular mechanisms of OmpR that regulates the prodigiosin synthesis in this strain are still unknown. In this project, the wild strain of FZSF02 and its OmpR mutant strain were used as the research materials, and the question is focused on if the OmpR regulates prodigiosin synthesis by activating the promoter of the pigA-O gene cluster directly or through regulating the downstream genes to activate the cluster indirectly. With the help of electrophoretic mobility shift assay method, LacZ-promoter fusion method, gene knockout and over expression method, mechanisms that how OmpR regulates prodigiosin synthesis would be researched.This study would enhance the understanding of the prodigionsin synthesis process and provide new knowledge to enhance the engineering production of prodigiosin.

灵菌红素是粘质沙雷氏菌等产生的一类重要次生代谢产物，由于其良好的生物活性而受到越来越多关注。灵菌红素的合成过程及其调控机制复杂，尚未得到最终阐明，这成为限制其生产和应用的的重要因素。本课题组前期发现粘质沙雷氏菌FZSF02合成灵菌红素严格依赖EnvZ/OmpR双组分系统转录因子OmpR的调控，并从OmpR突变体和野生型菌株RNAseq数据比较中发现包括pigA-O基因簇在内的多个表达变化的基因，但是其调控机制尚不清楚。本项目拟以FZSF02及其OmpR突变菌株为研究材料，围绕OmpR是通过直接激活pigA-O基因簇还是通过调控其下游基因间接激活pigA-O基因簇而调控灵菌红素合成这个关键问题，系统采用凝胶迁移、LacZ报告基因启动子活性检测、基因的敲除和过表达等实验技术，深入研究OmpR调控灵菌红素合成的机制。研究结果将增进对灵菌红素合成过程的认识，为其生产工程化提供新的理论知识。

课题组前期研究发现双组份调控系统EnvZ/OmpR调控粘质沙雷氏菌FZSF02菌株灵菌红素合成，但是其调控机制未知。本研究首先构建了ompR突变体的互补菌株和ompR敲除菌株。通过对比野生型菌株，OmpR敲除菌株和基因互补菌株进一步验证了OmpR特异性调控该菌株灵菌红素合成，此外对菌株的生物被膜形成能力有明显影响，但是对菌株生长，泳动性和胁迫响应能力无明显影响。LacZ报告基因实验和凝胶迁移实验证明OmpR可通过与灵菌红素合成基因簇启动子直接结合而正向调控该菌灵菌红素合成。另外，OmpR还可通过直接与自身基因启动子序列结合而激活自身基因的表达。基于上述结果，推测双组份系统EnvZ/OmpR调控灵菌红素合成的机制如下：首先某些未知因子诱导envZ和ompR表达，OmpR被EnvZ磷酸化。磷酸化的OmpR进一步与envZ/ompR启动子结合促进OmpR大量表达。当OmpR浓度达到一定程度将与灵菌红素合成基因簇的启动子牢固结合而诱导该基因簇上相关基因在转录水平大量表达。