Improved l -Cysteine Production in Corynebacterium glutamicum through Metabolic Engineering and High-Throughput Screening

l -Cysteine as a high-value amino acid has irreplaceable application value in food processing, pharmaceutical synthesis, cosmetic preparation, and animal feed and other fields. Corynebacterium glutamicum ( C. glutamicum ) is commonly used as a chassis strain for cysteine synthesis due to its characteristics of high safety, controllable metabolic regulation, and strong stress resistance. However, the efficient synthesis capability of this strain remains challenged. In C. glutamicum, the major limitations now reside in product-associated metabolic toxicity and the complex regulatory network. The accumulation of l -cysteine perturbs cellular redox homeostasis and restricts further increase in metabolic flux. To address this bottleneck, metabolic engineering was first employed to optimize the synthesis pathway, and a glycine-auxotrophic strategy was applied to decouple one-carbon metabolism from product synthesis. Building upon this, a key aspect of this study was the construction of a high-throughput screening platform that integrates a high-performance biosensor, atmospheric and room-temperature plasma mutagenesis, and fluorescence-activated cell sorting. This platform directly tackles the “high-production-high-toxicity” conflict, enabling efficient screening of mutants with enhanced tolerance and biosynthetic capacity for l -cysteine accumulation from a large-scale random mutagenesis library. An l -cysteine titer of 665 mg/L was achieved by the final engineered strain, representing a 4.04 fold increase compared with that of the strain before mutagenesis. demonstrating the platform’s powerful capability for mining and optimizing complex physiological regulatory traits. This work provides an integrated solution from rational design to nonrational evolutionary screening to overcome metabolic toxicity bottlenecks in microbial production.