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.

Enhancement of Sulfur Conversion Rate in the Production of l-Cysteine by Engineered Escherichia coli.

Cysteine is a commercially important sulfur-containing amino acid widely used as a supplement in the agricultural and food industries. It is extremely desirable to achieve a high sulfur conversion rate in the fermentation-based cysteine production. Here, the metabolic engineering of Escherichia coli was performed to enhance the sulfur conversion rate in cysteine biosynthesis. Accordingly, the reduction of sulfur loss by the regulator decR and its yhaOM operons were deleted. serACB was integrated into chromosome with constitutive promoter to coordinately increase sulfur utilization. The sulfur assimilation pathways and sulfur transcriptional regulator cysB were overexpressed to regulate sulfur metabolism and enhance sulfur conversion significantly. After the process optimization in fed-batch fermentation, LH16 [SLH02 ΔyhaM Ptrc1-serACB-cysM-nrdH-(pLH03, pTrc99a-cysB)] produced 7.5 g/L of cysteine with a sulfur conversion rate of 90.11%. These results indicate that cysteine production by LH16 is a valuable process in the agricultural and food industries.