代谢工程改造大肠杆菌高效合成L-瓜氨酸  

Metabolic engineering of Escherichia coli for efficient biosynthesis of L-citrulline

作　　者：胥琳峰 于文文 朱学文 张权威 武耀康 李江华 堵国成 吕雪芹 陈坚 刘龙 XU Linfeng;YU Wenwen;ZHU Xuewen;ZHANG Quanwei;WU Yaokang;LI Jianghua;DU Guocheng;LV Xueqin;CHEN Jian;LIU Long(Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,Jiangsu,China;Science Center for Future Foods,Jiangnan University,Wuxi 214122,Jiangsu,China;Yixing Institute of Food and Biotechnology,Yixing 214200,Jiangsu,China;Food Laboratory of Zhongyuan,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学糖化学与生物技术教育部重点实验室,江苏无锡214122 [2]江南大学未来食品科学中心,江苏无锡214122 [3]宜兴食品与生物技术研究院,江苏宜兴214200 [4]江南大学中原食品实验室,江苏无锡214122

出　　处：《生物工程学报》2025年第1期242-255,共14页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2023YFA0914500)。

摘　　要：L-瓜氨酸是一种非蛋白质氨基酸,在人体健康方面发挥着重要作用,具有很大的市场需求。尽管微生物细胞工厂已被广泛用于生物合成,但在L-瓜氨酸生物合成方面仍存在着遗传不稳定和效率低等挑战。本研究以大肠杆菌BL21(DE3)为出发菌株构建了一株高效、无质粒、无需诱导的L-瓜氨酸生产菌株。首先阻断L-瓜氨酸降解并解除反馈抑制,构建出L-瓜氨酸合成底盘菌株,其产量达到0.43 g/L。随后采用推拉抑制策略增强了L-瓜氨酸生物合成,使产量提高到6.0 g/L。接着,强化了NADPH合成和L-瓜氨酸转运系统以提高合成效率,最终L-瓜氨酸产量达到11.6 g/L。最后,在3 L发酵罐中进行分批补料发酵,L-瓜氨酸产量达到44.9 g/L。本研究为L-瓜氨酸的工业化生产奠定了基础,为其他氨基酸代谢网络的改造提供了思路。L-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand.Although microbial cell factories have been widely used for biosynthesis,there are still challenges such as genetic instability and low efficiency in the biosynthesis of L-citrulline.In this study,an efficient,plasmid-free,non-inducible L-citrulline-producing strain of Escherichia coli BL21(DE3)was engineered by combined strategies.Firstly,a chassis strain capable of synthesizing L-citrulline was constructed by block of L-citrulline degradation and removal of feedback inhibition,with the L-citrulline titer of 0.43 g/L.Secondly,a push-pull-restrain strategy was employed to enhance the L-citrulline biosynthesis,which realized the L-citrulline titer of 6.0 g/L.Thirdly,the NADPH synthesis and L-citrulline transport were strengthened to promote the synthesis efficiency,which achieved the L-citrulline titer of 11.6 g/L.Finally,fed-batch fermentation was performed with the engineered strain in a 3 L fermenter,in which the L-citrulline titer reached 44.9 g/L.This study lays the foundation for the industrial production of L-citrulline and provides insights for the modification of other amino acid metabolic networks.

关 键 词：L-瓜氨酸 大肠杆菌 代谢工程 L-脯氨酸 反馈抑制 

分 类 号：TQ922[轻工技术与工程—发酵工程]