ABCT、AACT、SecG基因共调控提高谷氨酸棒杆菌底盘细胞的外源蛋白表达及机理解析

C. glutamicum is a promising chassis cell for expressing recombiant protein, but the disadvantage of its low protein expressing efficiency restricts its application. Therefore nowadays it is a quite dynamatic area of optimizing the C. glutamicum expression system by genetically modifying the energy metabolism of the chassis cells and enhancing their protein synthesis ability. In our previous works, ABCT（encoding ABC transporter ATPase, AACT（encoding acetyl-CoA acetyltransferases） and SecG （encoding Sec secretory system SecG protein）have been identified that are closely related to cellular energy utilization and efficiency of recombinant protein expression. Therefore we propose to investigate the regulatory roles of ABCT, AACT and SecG on the transport capacity of substrates for protein synthesis, the efficiency of energy utilization, and the capability of protein secretion. The mechanism of the genes for co-regulation of exogenous proteins expression in the chassis cells will be unveiled as well, on the basis of technology of CRISPR/Cas9 gene editing system, DNA/protein-protein interaction technology, metabolomics and physiological and biochemical analysis, etc. As a result, a high-version C. glutamicum chassis cell with high efficiency of energy utilization and enhanced ability of recombinant protein expression will be constructed. The outcomes of the proposed study will pave a new way for developing C. glutamicum to be an excellent host for recombinant protein production.

谷氨酸棒杆菌（C. glutamicum）是一种新型外源蛋白表达生产用的底盘细胞，但存在蛋白表达效率较低的问题。理性改造底盘细胞能量代谢，增强其蛋白合成能力是优化C. glutamicum表达系统的有效研究方向。前期研究发现ABCT（编码ABC转运体ATP酶）、AACT（编码乙酰辅酶A酰基转移酶）、SecG（编码Sec分泌系统SecG蛋白）三个基因均与细胞能量代谢和外源蛋白表达密切相关。据此本项目采用CRISPR/Cas9基因编辑系统、蛋白质与基因/蛋白互作、代谢组学、生理生化分析等研究策略，通过研究ABCT、AACT、SecG在蛋白合成底物运输、能量利用、蛋白分泌三方面的作用，探讨它们对底盘细胞表达外源蛋白的共调控机制，并据此构建高效利用能量、外源蛋白表达能力提高的高版本C. glutamicum底盘细胞。研究结果为进一步优化C. glutamicum蛋白表达系统提供新途径和理论支撑。

谷氨酸棒杆菌（C. glutamicum）是一种新型外源蛋白表达生产用的底盘细胞，但存在蛋白表达效率较低的问题。理性改造底盘细胞能量代谢，增强其蛋白合成能力是优化C. glutamicum表达系统的有效研究方向。前期研究发现ABCT（编码ABC转运体ATP酶）、AACT（编码乙酰辅酶A酰基转移酶）、SecG（编码Sec分泌系统SecG蛋白）三个基因均与细胞能量代谢和外源蛋白表达密切相关。项目完成了C. glutamicum SecG（编码基因NCgl1522）、ABCT（编码基因NCgl0909）、AACT（编码基因NCgl2632）功能研究，并解析了它们对外源蛋白表达的影响机制以及它们对细胞代谢特别是能量代谢过程的作用。从多基因共同改造的角度对C. glutamicum宿主菌进行了综合改造，获得了外源蛋白表达量提升的宿主即C.g 15647ΔNCgl2632ΔNCgl0909，并对此菌株生产蛋白的培养条件进行了优化。同时项目开发了新的宿主细胞和相应3A（three antibiotic）表达系统和高效双顺反子表达系统。研究结果不仅丰富了谷氨酸棒杆菌基因调控的分子机理研究，也为生产重组蛋白开发了新型高效的细胞工厂，具有重要的理论价值和应用价值。