Combinatorial Metabolic Engineering of Escherichia coli for Enhanced L-Cysteine Production: Insights into Crucial Regulatory Modes and Optimization of Carbon-Sulfur Metabolism and Cofactor Availability

Microbial production of valuable compounds can be enhanced by various metabolic strategies. This study proposed combinatorial metabolic engineering to develop an effective Escherichia coli cell factory dedicated to L-cysteine production. First, the crucial regulatory modes that control L-cysteine levels were investigated to guide metabolic modifications. A two-stage fermentation was achieved by employing multi-copy gene expression, improving the balance between production and growth. Subsequently, carbon flux distribution was further optimized by modifying the C1 unit metabolism and the glycolytic pathway. The modifications of sulfur assimilation demonstrated superior performance of thiosulfate utilization pathways in enhancing L-cysteine titer. Furthermore, the studies focusing on cofactor availability and preference emphasized the vital role of synergistic enhancement of sulfur-carbon metabolism in L-cysteine overproduction. In a 5 L bioreactor, the strain BW15-3/pED accumulated 12.6 g/L of L-cysteine. This work presented an effective metabolic engineering strategy for the development of L-cysteine-producing strains.

Automated summary

This study proposed a combinatorial metabolic engineering strategy to develop an efficient Escherichia coli cell factory for L-cysteine production. The researchers investigated the crucial regulatory modes and employed multi-copy gene expression to achieve a two-stage fermentation for improved balance between production and growth. They further optimized carbon flux distribution by modifying the C1 unit metabolism and the glycolytic pathway, and demonstrated the superior performance of thiosulfate utilization pathways in enhancing L-cysteine production. The work also highlighted the vital role of synergistic enhancement of sulfur-carbon metabolism in L-cysteine overproduction. In a 5 L bioreactor, the engineered strain accumulated 12.6 g/L of L-cysteine.