Fitness of Chassis Cells and Metabolic Pathways for l-Cysteine Overproduction in Escherichia coli

l-Cysteine is a ubiquitous and unique sulfur-containing amino acid with numerous applications in agricultural and food industries. The efficient production of l-cysteine via microbial fermentation has received a great deal of attention. In this study, the fitness of different Escherichia coli K-12 strains harboring plasmid pLH03 was investigated. The enhancement of the precursor synthetic pathway and thiosulfate assimilation pathway resulted in the good performance of the E. coli BW25113 strain. The expression levels of synthetic pathway genes were optimized by two constitutive promoters to assess their effects on cysteine production. In conjunction, the main degradation pathway genes were also deleted for more efficient production of cysteine. l-Cysteine production was further increased through the manipulation of the sulfur transcription regulator cysB and sulfur supplementation. After process optimization in a 1.5 L bioreactor, LH2A1M0BΔYTS-pLH03 [BW25113 Ptrc2-serA Ptrc1-cysMPtrc-cysBΔyhaMΔtnaAΔsdaA-(pLH03)] accumulated 8.34 g/L cysteine, laying a foundation for application in the cysteine fermentation industry.