Systems metabolic engineering overcomes bottlenecks for high-level L-cysteine production

The sustainable biomanufacturing of L-cysteine, a sulfur-containing amino acid with extensive industrial applications, is hindered by interconnected metabolic bottlenecks including stringent regulation mechanisms, product cytotoxicity, and severe carbon loss. Here, we systematically addressed these challenges to develop an efficient Escherichia coli cell factory for L-cysteine production. First, we employed a synergistic approach combining multicopy integration and carbon flux redirection to achieve deregulation and alleviate metabolic burden. Subsequently, the stepwise identification and resolution of toxic 2-hydroxyglutarate accumulation overcame the L-serine supply bottleneck. Furthermore, we achieved customized expression of exporters by integrating growth phase-dependent dynamic regulation with promoter engineering, thereby simultaneously reduced cytotoxicity and carbon loss. Scale-up evaluation in a 5-L bioreactor demonstrated the superiority of glycerol over glucose, achieving a titer of 38.50 g/L with a yield of 0.17 g/g glycerol, which are the highest titer and yield reported to date. This work presents a systematic framework for resolving interconnected metabolic bottlenecks and paves the way for industrial production of L-cysteine.