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.
作者
张博
王莹
牛坤
柳志强
郑裕国
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)
出处
《生物工程学报》
CAS
CSCD
北大核心
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)。
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
biosynthesis pathway
coordinated gene expression
L-methionine
metabolic engineering
fermentation engineering
