辅因子NADPH调控L-赖氨酸高效合成的机制研究

Cofactor NADPH is one of the important redox cofactor, which remarkably influenced the production efficiency of a target metabolite. Previous studies have showed that the L-lysine biosynthesis is closely correlated with the availability of NADPH. However, these studies mainly focus on reconstructing the pathway of NADPH regeneration to improve the production of L-lysine, relatively little is known about the mechanism of high-efficient L-lysine production regulated by NADPH from cell physiology, protein expression and metabolic flux distribution. This project takes L-lysine producer C. glutamicum lysCfbr as research model, and studies the effects of NADPH on the physiological and metabolic functions of cells from three aspects: intracellular micro-environment, protein expression and metabolic flux using genetic engineering, physiological and biochemical determination, omics technology, etc. The purpose of this project is to research the mechanism of high-efficient L-lysine production regulated by NADPH from the perspective of relationship among NADPH metabolism, intracellular micro-environment, transcriptional regulatory protein, metabolic flux distribution and high-efficient L-lysine production. And the experimental results obtained in this study will provide theoretical foundation for breeding L-lysine high-producing strains by cofactor engineering and for optimizing the fermentation process of L-lysine.

NADPH是细胞内重要的氧化还原辅因子，显著影响目标代谢产物的生产效率。前期研究发现，辅因子NADPH水平与L-赖氨酸高效合成密切相关。然而，已有研究主要针对NADPH再生途径的改造，关于NADPH是如何通过细胞生理过程、蛋白质表达和代谢通量分布调控L-赖氨酸高效合成的机制缺乏全局性和机理性的认识。本项目以遗传背景清晰的L-赖氨酸产生菌Corynebacterium glutamicum lysCfbr为研究模型，借助靶基因敲除或过表达、生理生化分析和组学技术等研究策略，从胞内微环境、蛋白质表达和碳代谢流三个方面系统地研究在L-赖氨酸合成时辅因子NADPH对细胞生理代谢功能的影响，从NADPH代谢-胞内微环境-转录调控蛋白-代谢通量分布-赖氨酸高效合成的关联角度解析NADPH调控L-赖氨酸高效合成的机制。研究结果可望为采用辅因子工程选育L-赖氨酸高产菌和优化L-赖氨酸发酵工艺提供理论依据。

本项目针对基于NADPH供给的调控策略无法实现精细化调控L-赖氨酸的碳代谢途径这一问题，以L-赖氨酸产生菌为研究模型，重构辅因子NADPH再生途径来获得不同NADPH水平的菌株，并分析改造后细胞生理调节作用、碳代谢途径中功能模块基因的表达量和碳物质流分配及其对L-赖氨酸合成的影响。研究结果可为采用辅因子工程精细化调控L-赖氨酸合成途径，选育L-赖氨酸高产菌株和优化L-赖氨酸发酵工艺提供理论依据。研究结果如下：.1).构建了不同NADPH水平的工程菌株，并初步解析了胞内NADPH对胞内微环境的调节作用. 胞内高水平NADPH浓度有效调节胞内ATP和NADH水平，从而调节胞内pHi，提高细胞对酸胁迫的适应力。另外，胞内高水平NADPH可以降低胞内ROS浓度，提高细胞对氧胁迫的适应力。.2).利用转录组学分析了受NADPH调控的转录调控蛋白，并明确了转录调节因子Cgl2680对NADPH再生具有负调控作用. 胞内NADPH水平的改变，显著改变众多基因表达，这些基因参与分子功能、细胞成分和生物过程，且与NADPH的代谢密切相关。此外，AraC家族转录调节因子Cgl2680负调控NADPH再生。.3).理性改变C. glutamicum中二氢吡啶二羧酸还原酶(DHDPR)辅因子亲和性，确定了DHDPR在改变胞内微环境和L-赖氨酸合成的作用. CgDHDPR对NADH的偏好性比EcDHDPR低，且受NADH的抑制作用。此外，CgDHDPR突变子K11A和R13A以及EcDHDPR突变子R16A和R39A对NADPH的亲和性远低于野生型，其中R39A表现最好。将R39A插入到L-赖氨酸产生菌基因组中更有利于改变胞内NADPH和NADH水平，改善重组菌株的L-赖氨酸生产性能。.4).组合表达不同辅因子偏好性酶，优化NADPH与碳代谢途径中功能模块的适配，实现了高效发酵生产L-赖氨酸。. 组合表达NADP+依赖型甘油醛-3-磷酸脱氢酶和NAD+依赖型异柠檬酸脱氢酶，可平衡胞内NAD(H/+)和NADP(H/+)水平，促进胞内糖代谢速率，使目的菌株L-赖氨酸产量由85.6 g/L提升到121.4 g/L，葡萄糖转化率由33%提高到46%。