谷氨酸棒杆菌L-脯氨酸外排蛋白的鉴定及功能研究

Bacterial amino acid export is evolved to keep intracellular amino acid homoeostasis, and it also plays an essential role in amino acid metabolism of bacteria. Engineering amino acid export becomes a common strategy to increase the production of industrial strains. The exporters of 14 amino acids have been identified in bacteria, however, no L-proline exporter is reported to date. Corynebacterium glutamicum, a model strain for studying amino acid metabolism, has been widely used for industrial production of various amino acids. L-proline export is also involved in osmotic regulation of bacteria including C. glutamicum. In this project, we will firstly construct a whole-genome scale repression library of membrane transport proteins in C. glutamicum by using CRISPRi technology, and the library will be screened to obtain potential L-proline export genes whose repressions cause reduced extracellular L-proline concentrations. Then, the real L-proline exporter of C. glutamicum will be identified by classic functional study of amino acid exporters. Subsequently, the efflux mechanism of L-proline such as substrate specificity, efflux rate and driving force will be determined. We will also study the effect of L-proline export on cell survival under different osmotic pressure and its response to sudden osmotic pressure changes in C. glutamicum. The outcome of this project will enrich the understanding of bacterial L-proline metabolism and osmotic pressure regulation mechanism of C. glutamicum, and will also provide a new target for increasing L-proline production of C. glutamicum.

细菌的氨基酸外排是为平衡胞内氨基酸浓度而进化形成的，是细菌氨基酸代谢的重要组成部分。强化氨基酸外排也是改造氨基酸工业菌株的重要手段，细菌中已鉴定14种氨基酸的外排蛋白，但至今没有L-脯氨酸外排蛋白被报道。谷氨酸棒杆菌是氨基酸代谢研究及工业生产的重要菌株，L-脯氨酸外排还参与谷氨酸棒杆菌等细菌的渗透压调节。本项目拟采用CRISPRi技术构建谷氨酸棒杆菌全基因组规模膜转运蛋白基因的抑制库，筛选抑制后影响L-脯氨酸外排的基因；再基于经典的功能验证鉴定L-脯氨酸外排蛋白；通过检测外排蛋白的底物特异性、外排速度及外排驱动力，阐明L-脯氨酸外排蛋白的外排机制；分析L-脯氨酸外排蛋白对谷氨酸棒杆菌适应不同渗透压环境和应对渗透压变化的作用，揭示L-脯氨酸外排蛋白的渗透压调节功能。本项目的实施将完善细菌的L-脯氨酸代谢模式，为改造L-脯氨酸工业菌株提供新靶标，也为提升谷氨酸棒杆菌的渗透压适应性提供新思路。

细菌的氨基酸外排是为平衡胞内氨基酸浓度而进化形成的，是细菌氨基酸代谢的重要组成部分。强化氨基酸外排也是改造氨基酸工业菌株的重要手段，细菌中已鉴定14种氨基酸的外排蛋白，但至今没有L-脯氨酸外排蛋白被报道。谷氨酸棒杆菌是氨基酸代谢研究及工业生产的重要菌株，L-脯氨酸外排还参与谷氨酸棒杆菌等细菌的渗透压调节。如何筛选并鉴定谷氨酸棒杆菌的L-脯氨酸外排蛋白，进而研究其L-脯氨酸外排及渗透压调节功能是本项目的重点研究内容。首先，在L-脯氨酸生产底盘基础上，采用CRISPRi技术构建了谷氨酸棒杆菌全基因组规模膜转运蛋白基因的抑制库，通过孔板筛选获得3个抑制后降低L-脯氨酸外排的基因。基因敲除验证发现只有cgl2622敲除可以降低L-脯氨酸外排，进一步测定cgl262敲除、回补及过表达对发酵过程细胞内外L-脯氨酸浓度的影响，确认Cgl2622为L-脯氨酸外排蛋白。其次，基于经典的寡肽添加实验，分析了cgl2622基因对谷氨酸棒杆菌L-脯氨酸外排速度的影响，发现基因敲除可以显著降低L-脯氨酸的外排速度。最后，通过测试cgl2622基因敲除和过表达，分析其对谷氨酸棒杆菌适应不同渗透压环境的影响，结果表明该基因对谷氨酸棒杆菌适应不同渗透压环境没有明显影响。本项目的实施，鉴定了目前唯一一个L-脯氨酸外排蛋白，完善了细菌的L-脯氨酸代谢模式，为改造L-脯氨酸工业菌株提供新靶标。