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

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、thrA^C1034T)，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。

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

Abstract:

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 thrA^C1034T) 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.

Key words:Escherichia coli;O-acetyl-l-homoserine;metabolic engineering;acetic acid reuse;co-factor engineering