Abstract
O-Acetyl-l-homoserine (OAH) is a versatile platform compound with extensive potential applications. It is a key precursor for the synthesis of l-methionine and S-adenosylmethionine. Currently, the microbial fermentation process for the production of OAH still faces challenges, such as low fermentation yield and long fermentation period. In this study, the supply of key precursors, including l-aspartic acid, l-homoserine, and acetyl-CoA, was firstly enhanced, which increased the OAH production from 7.25 g/l to 12.95 g/l in shaking flask fermentation. Subsequently, the non-oxidative glycolysis pathway (NOG pathway) was constructed and optimized to minimize the carbon loss and improve the carbon sources utilization, resulting in an increase in OAH production to 15.59 g/l. Finally, by accelerating cell division and enhancing the glucose transport system, OAH production was further improved to 17.23 g/l. The OAH production of the engineered strain OAH23 achieved a production level of 66.25 g/l in a 5 l bioreactor for 68 h, with the yield of 0.41 g/g glucose. The metabolic regulation strategy outlined in this study offers valuable insights for the efficient biosynthesis of OAH and other acetylated amino acids in E. coli.