Wei Liang, Wang Qian, Xu Ning, Cheng Jian, Zhou Wei, Han Guoqiang, Jiang Huifeng, Liu Jun, Ma Yanhe
Tianjin Institute of Industrial Biotechnology , Chinese Academy of Sciences , Tianjin 300308 , China.
Key Laboratory of Systems Microbial Biotechnology , Chinese Academy of Sciences , Tianjin 300308 , China.
ACS Synth Biol. 2019 May 17;8(5):1153-1167. doi: 10.1021/acssynbio.9b00042. Epub 2019 Apr 22.
O-acetylhomoserine (OAH) is a promising platform chemical for the production of l-methionine and other valuable compounds. However, the relative low titer and yield of OAH greatly limit its industrial production and cost-effective application. In this study, we successfully constructed an efficient OAH-producing strain with high titer and yield by combining protein and metabolic engineering strategies in E. coli. Initially, an OAH-producing strain was created by reconstruction of biosynthetic pathway and deletion of degradation and competitive pathways, which accumulated 1.68 g/L of OAH. Subsequently, several metabolic engineering strategies were implemented to improve the production of OAH. The pathway flux of OAH was enhanced by eliminating byproduct accumulation, increasing oxaloacetate supply and promoting the biosynthesis of precursor homoserine, resulting in a 1.79-fold increase in OAH production. Moreover, protein engineering was applied to improve the properties of the rate-limiting enzyme homoserine acetyltransferase (MetXlm) based on evolutionary conservation analysis and structure-guided engineering. The resulting triple F147L-M182I-M240A mutant of MetXlm exhibited a 12.15-fold increase in specific activity, and the optimized expression of the MetXlm mutant led to a 57.14% improvement in OAH production. Furthermore, the precursor acetyl-CoA supply and NADPH generation were also enhanced to facilitate the biosynthesis of OAH by promoting CoA biosynthesis, overexpressing heterogeneous acetyl-CoA synthetase (ACS), and introducing NADP-dependent pyruvate dehydrogenase (PDH). Finally, the engineered strain OAH-7 produced 62.7 g/L of OAH with yield and productivity values of 0.45 g/g glucose and 1.08 g/L/h, respectively, in a 7.5 L fed-batch fermenter, which was the highest OAH production ever reported.
O-乙酰高丝氨酸（OAH）是用于生产L-甲硫氨酸和其他有价值化合物的一种很有前景的平台化学品。然而，OAH相对较低的滴度和产量极大地限制了其工业化生产和经济高效的应用。在本研究中，我们通过在大肠杆菌中结合蛋白质工程和代谢工程策略，成功构建了一株具有高滴度和高产量的高效OAH生产菌株。最初，通过重建生物合成途径以及删除降解和竞争途径创建了一株OAH生产菌株，该菌株积累了1.68 g/L的OAH。随后，实施了几种代谢工程策略来提高OAH的产量。通过消除副产物积累、增加草酰乙酸供应以及促进前体高丝氨酸的生物合成，增强了OAH的途径通量，导致OAH产量提高了1.79倍。此外，基于进化保守性分析和结构导向工程，应用蛋白质工程来改善限速酶高丝氨酸乙酰转移酶（MetXlm）的特性。所得的MetXlm三重F147L-M182I-M240A突变体的比活性提高了12.15倍，MetXlm突变体的优化表达使OAH产量提高了57.14%。此外，还通过促进辅酶A生物合成、过表达异源乙酰辅酶A合成酶（ACS）以及引入NADP依赖性丙酮酸脱氢酶（PDH）来增强前体乙酰辅酶A的供应和NADPH的生成，以促进OAH的生物合成。最后，工程菌株OAH-7在7.5 L补料分批发酵罐中产生了62.7 g/L的OAH，产量和生产率分别为0.45 g/g葡萄糖和1.08 g/L/h，这是迄今为止报道的最高OAH产量。
