O-Succinyl-l-homoserine overproduction with enhancement of the precursor succinyl-CoA supply by engineered Escherichia coli

Abstract

O-Succinyl-l-homoserine (OSH) is an important platform chemical in production of C₄ chemicals such as succinic acid, homoserine lactone, γ‑butyrolactone, and 1,4‑butanediol. The production of OSH through chemical method or the current engineering strain is difficult and not optimal, and thereby there remains a need to develop new engineering strategy. Here, we engineered an OSH overproducing Escherichia coli strain through deleting the degradation and competitive pathways, overexpressing thrA and metL to enhance the metabolic flux from l-asparate to l-homoserine. Additionally, increasing the precursor succinyl-CoA supply through simultaneously knocking out sucD and overexpressing sucA further increased the yield of OSH. The engineered strain OSH9/pTrc-metA11-yjeH with above strategies produced OSH at the concentration of 24.1 g/L (0.609 g/g glucose) in batch fermentation. To gain detailed insight into metabolism of the engineered strain, comparative metabolic profiling was performed between the engineered and wide-type strain. The metabolomics data deciphered that the carbon was directed toward the OSH biosynthesis resulting in less flexibility of the genetically modified strain than the wide-type strain.

Keywords: CRISPR-Cas9; Escherichia coli W3110; Metabolome analysis; O-Succinyl-l-homoserine.