Layered modification for enhancing O-Succinyl-L-homoserine biosynthesis with a non-auxotrophic probiotic Escherichia coli Nissle 1917

O-Succinyl-L-homoserine (OSH) is a promising platform compound with extensive application prospects in medicine, feed, agriculture, and food. Many scholars have dedicated significant efforts to the rational engineering of microbial cell factories, particularly in Escherichia coli, aiming to achieve efficient production of OSH. In this study, modular genetic engineering was performed to achieve layered modification of the OSH biosynthesis pathway. Firstly, an OSH producing chassis strain was constructed based on the wild-type probiotic E. coli Nissle 1917 (EcN) via knocking out the negative genes, dynamically regulating the synthesis of by-product, relieving the feedback inhibition of key enzymes, and weakening the degradation pathway. Furthermore, the supply of the key precursors L-homoserine, L-aspartate and oxaloacetate was strengthened to drive the carbon flux into the OSH biosynthesis. In addition, the regeneration of cofactor NADPH further increased the OSH titer to 13.44 ± 0.77 g/L in the shake flask. Fed-batch fermentation in the 5-L bioreactor showed strain OSHY33 produced 80.79 ± 2.10 g/L of OSH within 44 h, representing the highest OSH productivity of 1.84 g/L/h to date. This research has laid a solid foundation for OSH production through microbial fermentation.