Mutagenesis and Analysis for Enhancing (2R,3R)-2,3-Butanediol Tolerance and Production in Corynebacterium glutamicum

2,3-Butanediol (2,3-BD) is a versatile bulk chemical, serving as a high-boiling green solvent, a key monomer for polyesters and polyurethanes, and a crucial intermediate in the production of fine chemicals and cosmetics. Additionally, it is a key intermediate for pharmaceuticals and agrochemicals, supporting green chemistry. Microbial production of 2,3-BD offers a sustainable bioprocess but faces the challenge of high-concentration product inhibition. In an effort to improve 2,3-BD tolerance and production in Corynebacterium glutamicum, the engineered strain CGK4 was subjected to atmospheric and room temperature plasma (ARTP) mutagenesis, from which three superior mutant strains were isolated. Genomic and reverse engineering analyses revealed two mutations beneficial for tolerance and seven mutations beneficial for production. Among these, mutations atpG^L250P, cobT^V192V, and ctaE^L79P increased 2,3-BD titer by 18.0%, 14.5%, and 15.0%, respectively. Furthermore, we found that while certain mutations markedly enhanced stress tolerance, the highest-producing strains achieved a balanced metabolic state rather than maximal tolerance, indicating a non-linear relationship between the two traits. Finally, through mutation combination optimization, the optimal strain K4-cobT-ctaE was constructed. In shake-flask fed-batch fermentation, this strain produced 84.35 ± 3.04 g/L (2R,3R)-2,3-BD, which was 38.3% higher than that of the parental strain. Our findings provide a promising approach to addressing 2,3-BD tolerance challenges and promote the development of microbial production platforms for industrial application.