理性代谢工程改造促进谷氨酸棒杆菌高效合成L-谷氨酰胺  

Efficient Synthesis of L-Glutamine by Corynebacterium glutamicum Promoted via Rational Metabolic Engineering

作　　者：乔郅钠 吕青兰 李翔飞 王晴 徐美娟[1] 饶志明[1] QIAO Zhina;LYU Qinglan;LI Xiangfei;WANG Qing;XU Meijuan;RAO Zhiming(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2022年第8期65-74,共10页Journal of Food Science and Biotechnology

基　　金：国家重点研发计划项目(2021YFC2100900)。

摘　　要：L-谷氨酰胺(L-glutamine,L-Gln)是人体液中含量最丰富的一种半必需氨基酸,具有多种营养和药理功能,被广泛应用于医药、食品添加剂、营养保健品、饲料等领域。目前,微生物发酵法是生产L-Gln的主要方法,发酵产酸效率低、副产物多、菌种性能欠缺等问题严重限制了其工业化应用。为解决这一问题,作者以实验室前期构建的L-Gln生产菌株CGQ03为出发菌株,通过强化谷氨酸脱氢酶表达及辅因子NADPH供应促进前体L-谷氨酸合成、增强辅因子ATP胞内含量、过表达溶氧相关蛋白VHb提高细胞携氧能力、增强关键酶谷氨酰胺合成酶催化活性及发酵工艺优化等策略提高了L-Gln产量。最终的基因工程菌CGQ08/pDXW10-glnASc-gdhCg在5 L发酵罐上补料分批发酵66 h,L-Gln的产量最高达(94.5±1.8)g/L,糖酸转化率为34.8%,生产强度为1.43 g/(L·h)。本研究为L-Gln及其相关化合物的工业化生产提供了借鉴。L-glutamine is the most abundant semi-essential amino acid in human fluids.It has a variety of nutritional and pharmacological functions and is widely used in medicine,food additives,nutritional health products,feed and other fields.At present,microbial fermentation is the main method to produce L-Gln,but its industrial application is seriously limited by the problems of low fermentation acid production efficiency,many by-products,and lack of bacterial performance.In order to solve those problems,the L-Gln production strain CGQ03 constructed early in our laboratory was used as the starting strain in this study.The synthesis of precursor L-glutamate was promoted by enhancing the expression of glutamate dehydrogenase and the supply of cofactor NADPH.The yield of L-Gln production was increased by enhancing the intracellular content of cofactor ATP,overexpressing the dissolved oxygen-related protein VHb to improve the oxygen carrying capacity of cells,enhancing the catalytic activity of key enzyme glutamine synthetase,and optimizing the fermentation process.The final genetically engineered strain CGQ08/pDXW10-glnASc-gdhCg was fed-batch fermented in a 5 L fermenter for 66 h,and the yield of L-Gln was up to(94.5±1.8)g/L,with the sugar-acid conversion rate of 34.8%and the production intensity of 1.43 g/(L·h).This study could provide an important reference for the industrial production of L-Gln and its related compounds.

关 键 词：L-谷氨酰胺 谷氨酸棒杆菌 谷氨酰胺合酶 NADPH供应 ATP供应 发酵工艺优化 

分 类 号：Q815[生物学—生物工程]