代谢工程改造大肠杆菌一碳模块高效合成L-甲硫氨酸  

Efficient synthesis of L-methionine by engineering the one carbon module of Escherichia coli

作　　者：张博[1,2] 王莹 牛坤[1,2] 柳志强 郑裕国[1,2] ZHANG Bo;WANG Ying;NIU Kun;LIU Zhiqiang;ZHENG Yuguo(The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals,Zhejiang University of Technology,Hangzhou 310014,Zhejiang,China;Key Laboratory of Bioorganic Synthesis of Zhejiang Province,College of Biotechnology and Bioengineering,Zhejiang University of Technology,Hangzhou 310014,Zhejiang,China)

机构地区：[1]浙江工业大学手性生物制造国家地方联合工程研究中心,浙江杭州310014 [2]浙江工业大学生物工程学院浙江省生物有机合成技术研究重点实验室,浙江杭州310014

出　　处：《生物工程学报》2023年第8期3302-3317,共16页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2018YFA0901400);国家自然科学基金(32070099,31971342)。

摘　　要：L-甲硫氨酸又名L-蛋氨酸,是人体必需8种氨基酸之一,在饲料、医药、食品领域具有重要应用。以实验室前期构建的M2(Escherichia coli W3110?IJAHFEBC/PAM)为出发菌株,以模块化代谢工程策略构建了一株L-甲硫氨酸高产菌株。首先通过过表达亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MetF)和筛选不同来源的丝氨酸羟甲基转移酶(hydroxymethyltransferase,GlyA),增强了一碳模块甲基供体的生成,优化了一碳模块。随后针对一碳模块的前体供应,过表达了胱醚裂解酶(cysteamine lyase,MalY)和半胱氨酸内运基因(fliY),有效地提高了L-高半胱氨酸和L-半胱氨酸的供应。最终摇瓶发酵L-甲硫氨酸的产量由2.8 g/L提高至4.05 g/L,5 L发酵罐中达到18.26 g/L。研究结果表明,一碳模块对L-甲硫氨酸的生物合成具有十分重要的影响,在细胞内通过优化一碳模块,可以实现L-甲硫氨酸的高效生物合成。本研究为进一步提高微生物发酵生产L-甲硫氨酸的水平奠定了基础。L-methionine,also known as L-aminomethane,is one of the eight essential amino acids required by the human body and has important applications in the fields of feed,medicine,and food.In this study,an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain(Escherichia coli W3110ΔIJAHFEBC/PAM)previously constructed in our laboratory.Firstly,the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase(methylenetetrahydrofolate reductase,MetF)and screening of hydroxymethyltransferase(GlyA)from different sources,optimizing the one-carbon module.Subsequently,cysteamine lyase(hydroxymethyltransferase,MalY)and cysteine internal transporter gene(fliY)were overexpressed to improve the supply of L-homocysteine and L-cysteine,two precursors of the one-carbon module.The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L,and up to 18.26 g/L in a 5 L fermenter.The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine,and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module.This study may facilitate further improvement of microbial fermentation production of L-methionine.

关 键 词：生物合成 基因协同表达 L-甲硫氨酸 代谢工程 发酵工程 

分 类 号：TQ922[轻工技术与工程—发酵工程]