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

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。

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

Abstract:

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 W3110 ΔIJAHFEBC/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.

Key words:biosynthesis pathway;coordinated gene expression;L-methionine;metabolic engineering;fermentation engineering