代谢工程改造大肠杆菌生产O-乙酰-L-高丝氨酸  

Metabolic engineering of Escherichia coli for the biosynthesis of O-acetyl-L-homoserine

作　　者：黄良刚 高峰 许诺然 周俊平 牛坤 张博 柳志强 郑裕国 HUANG Lianggang;GAO Feng;XU Nuoran;ZHOU Junping;NIU Kun;ZHANG Bo;LIU Zhiqiang;ZHENG Yuguo(College of Biotechnology and Bioengineering,Zhejiang University of Technology,Hangzhou 310014,Zhejiang,China)

机构地区：[1]浙江工业大学生物工程学院,浙江杭州310014

出　　处：《生物工程学报》2025年第1期256-270,共15页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2018YFA0901400)。

摘　　要：O-乙酰-L-高丝氨酸(O-acetyl-L-homoserine,OAH)是一种平台化合物,可用于生产L-蛋氨酸和其他有价值的化合物,但产量低和转化率低等问题限制了其工业化生产和应用。为了解决这一问题,本研究以前期构建的L-高丝氨酸宿主大肠杆菌HS33为底盘,采用系统代谢工程策略构建了一株高产OAH的菌株。首先,强化磷酸烯醇式丙酮酸(phosphoenolpyruvate,PEP)积累、丙酮酸利用以及OAH合成途径(过表达aspB、aspA、thrAC1034T),获得积累13.37 g/L OAH的初始菌株;随后,整合筛选的辅因子供应基因解决还原力和能量供应问题,将产量提升至15.79 g/L;之后,进一步强化乙酸回用途径,改善乙酰辅酶A供应,结合多源乙酰基转移酶MetX表达使得改造获得的工程菌株OAH28的OAH产量提升至17.49 g/L。最终,在5 L发酵罐中进行生产性能测试,工程菌株OAH产量达到47.12 g/L,葡萄糖转化率为32%,生产强度为0.59 g/(L·h)。上述研究结果为OAH的代谢工程改造实现产量提升提供了一定的理论基础,也为工业化生产提供了有效的借鉴和参考。O-acetyl-L-homoserine(OAH)is a promising platform compound for the production of L-methionine and other valuable compounds,while its low yield and low conversion rate limit the industrial application.To solve these problems,we constructed a strain for high OAH production with the previously constructed L-homoserine producer Escherichia coli HS33 as the chassis by systematic metabolic engineering.Firstly,PEP accumulation,pyruvate utilization,and OAH synthesis pathway(overexpressing aspB,aspA,and thrAC1034T)were enhanced to obtain an initial strain accumulating 13.37 g/L OAH.Subsequently,the co-factor synthesis genes were integrated to supply reducing power and energy,which increased the yield to 15.79 g/L.The OAH yield of the engineered strain OAH28 was further increased to 17.49 g/L by strengthening the acetic acid reuse pathway,improving the supply of acetyl-CoA,and regulating the expression of MetX from different sources.Finally,in a 5 L fermenter,OAH28 achieved an OAH titer of 47.12 g/L,with a glucose conversion rate of 32%and productivity of 0.59 g/(L·h).The results lay a foundation for increasing the OAH production by metabolic engineering and give insights into the industrial production of OAH.

关 键 词：大肠杆菌 O-乙酰-L-高丝氨酸 代谢工程 乙酸回用 辅因子工程 

分 类 号：TQ922[轻工技术与工程—发酵工程]