Engineering of a LysG-derived arginine-specific biosensor for high-throughput screening of arginine overproducers in Corynebacterium glutamicum

Arginine is a semi-essential amino acid and holds significant value in the pharmaceutical and nutraceutical industries. Corynebacterium glutamicum is a promising host for arginine production, yet its industrial titers remain limited. Biosensor-based high-throughput screening enables rapid isolation of overproducers. However, the native LysG-based biosensor in C. glutamicum lacks arginine specificity, limiting its efficiency for screening high arginine-producing strains. Here, we developed an arginine-specific biosensor pLysGF222I in C. glutamicum, by combining semi-rational design and directed evolution of LysG, an endogenous regulator responsive to basic amino acids. Structural and interaction analyses revealed that the F222I substitution in LysG retained high affinity for arginine while significantly decreasing its responsiveness to histidine and lysine. Subsequently, this arginine-specific biosensor was successfully applied to screen a library of N-acetylglutamate kinase (NAGK) mutants, identifying 18 novel NAGK mutants with enhanced arginine production compared with previously reported variants. The strain integrated with the optimal NAGK mutant was further subjected to mutagenesis and screening, yielding an engineered strain that achieved an arginine titer of 56.07 g/L in a 5-L bioreactor, a 42% increase relative to the original starting strain. Overall, this study provides a powerful tool for accelerating the metabolic engineering of C. glutamicum for high-level arginine production.