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

Area of Science:

• Microbial Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• l-Cysteine is a valuable amino acid with broad applications in food, pharmaceuticals, cosmetics, and animal feed.
• Corynebacterium glutamicum (C. glutamicum) is a preferred microbial chassis for l-cysteine synthesis due to its safety and metabolic control, but faces challenges in efficient production.
• Key limitations in C. glutamicum for l-cysteine production include product-associated metabolic toxicity and intricate regulatory networks, which perturb cellular redox homeostasis and limit metabolic flux.

Purpose of the Study:

• To overcome metabolic toxicity and enhance l-cysteine biosynthesis in C. glutamicum.
• To develop a high-throughput screening platform for identifying mutants with improved l-cysteine tolerance and production capabilities.
• To integrate rational metabolic engineering with evolutionary screening for microbial production optimization.

Main Methods:

• Metabolic engineering strategies were employed to optimize the l-cysteine synthesis pathway.
• A glycine-auxotrophic strategy was implemented to decouple one-carbon metabolism from product synthesis.
• A high-throughput screening platform was constructed, combining a biosensor, atmospheric and room-temperature plasma mutagenesis, and fluorescence-activated cell sorting.

Main Results:

• The developed screening platform effectively addressed the conflict between high production and high toxicity.
• Engineered strains exhibited enhanced tolerance and biosynthetic capacity for l-cysteine accumulation.
• The final engineered strain achieved an l-cysteine titer of 665 mg/L, a 4.04-fold increase compared to the pre-mutagenesis strain.

Conclusions:

• The integrated approach of rational design and evolutionary screening successfully overcame metabolic toxicity bottlenecks in microbial l-cysteine production.
• The high-throughput screening platform demonstrates powerful capability for mining and optimizing complex physiological regulatory traits.
• This study provides a robust framework for enhancing microbial production of high-value chemicals like l-cysteine.