谷氨酸棒状杆菌mepA基因影响外源蛋白表达的机制研究

The mepA gene encoding a metallopeptidase A of Corynebacterium glutamicum (C. glutamicum), plays a predominated role in regulating cell growth and cell wall metabolism of bacteria. In previous works, it was found that dissolved oxygen concentration (DO), the signal transduction system of MtrAB and MprAB two-component could regulate the expression level of the mepA gene in C. glutamicum, and further could affect the expression levels of foreign proteins, which strongly suggest that it could be a promising gene editing site for the construction of C. glutamicum chassis cell. Therefore, in this proposal, the effect of the regulatory network of the mepA gene on cellular carbon center metabolism and cell’s responses to environment will be studied systematically from three aspects of gene regulation, metabolic network and protein expression, using the techniques, i.e. chemostat culture, transcriptome and metabonomics analysis, interactions of protein to protein / DNA, insertion or inactivation or overexpression of target genes, bioinformatic and physiological and biochemical analysis. It is expected that the mechanism of the mepA gene of Corynebacterium glutamicum involved in the process of recombinant protein expression, mainly about the processes of protein synthesis and protein secretion, will be understood. The knowledges obtained from the present study will provide valuable information for the optimization of C. glutamicum chassis aiming at enhancing expression level of recombinant protein.

谷氨酸棒状杆菌（Corynebacterium glutamicum, C. glutamicum）中金属内肽酶编码基因mepA在细菌的生长和细胞壁代谢调控中发挥重要作用。前期研究发现MepA表达受溶氧状态和MtrAB、MprAB双组份系统调控，且显著影响外源蛋白的表达，是C. glutamicum底盘细胞优化的重要基因编辑位点。据此本项目将借助恒化培养、组学、蛋白与蛋白/基因互作、靶基因插入和失活、生物信息学、生理生化分析等研究方案，从基因调控、代谢网络、蛋白表达三方面系统地研究mepA基因调控网络对细胞碳中心代谢和细胞应答外界环境能力的影响，进而从外源蛋白合成所需底物能量和分泌运输两个角度解析mepA影响外源蛋白表达的调控机制。研究结果将为优化C. glutamicum底盘细胞，提高其表达外源蛋白的能力提供新途径和理论支撑，具有重要的理论及实际意义。

谷氨酸棒状杆菌（Corynebacterium glutamicum, C. glutamicum）中金属内肽酶编码基因mepA在细菌的生长和细胞壁代谢调控中发挥重要作用。前期研究发现MepA表达受溶氧状态和MtrAB、MprAB双组份系统调控，且显著影响外源蛋白的表达，是C. glutamicum底盘细胞优化的重要基因编辑位点。本项目通过荧光定量PCR技术和转录组学数据解析了mepA与MtrAB、MprAB、PhoRP双组份系统以及sigma因子之间的相互作用关系，进而解析了mepA基因调控网络，揭示mepA参与细胞转运、细胞分裂以及双组分系统的调控。研究MepA不同菌株细胞壁细胞膜变化，证明MepA的过表达导致肽聚糖降解失调，进而导致细胞分裂缺陷，导致细胞伸长；研究MepA不同菌株生存能力和细胞通透性的变化，证明MepA过表达增加了细胞的通透性和细胞对异烟肼的敏感性。通过研究mepA与外源蛋白表达关系，发现虽然mepA敲除能够提高外源蛋白表达水平，且引起胞内ATP水平变化，但mepA与外源蛋白分泌途径没有直接相关性。因此，我们得出结论mepA通过影响双组份系统、细胞分裂、细胞转运，ATP水平，间接影响外源蛋白的表达。本项目为了提升C. glutamicum分泌表达能力，还开发了表达元件、宿主细胞以及与之高度匹配的分泌表达系统。研究结果为优化C. glutamicum底盘细胞，提高其表达外源蛋白的能力提供新途径和理论支撑，具有重要的理论及实际意义。