Engineering of Corynebacterium glutamicum for the enhanced production of optically pure (meso)-2,3-butanediol.

2,3-Butanediol (2,3-BDO) is a versatile platform chemical with diverse applications in cosmetics, pharmaceuticals, agricultural and food manufacturing. Among its stereoisomers, optically pure (meso)-2,3-BDO is particularly valuable; however, achieving high titers with stereoselectivity remains challenging in conventional hosts due to byproduct formation, low tolerance, and plasmid instability. In this study, we established Corynebacterium glutamicum as an efficient and robust chassis for the industrial-level production of optically pure (meso)-2,3-BDO. A structure-guided engineering approach was applied to 2,3-butanediol dehydrogenase (KpBDH), where α6-helix truncation enhanced catalytic efficiency and enabled near-complete conversion of acetoin to the target isomer. To further improve productivity, competing byproduct pathways were deleted, and cofactor homeostasis was reinforced by integrating UdhA for NADH regeneration and DrPPK for ATP regeneration. Finally, all biosynthetic modules were stably integrated into the chromosome, generating the plasmid-free strain ES11. In 5L fed-batch fermentation, ES11 produced 100.4 ± 0.4 g/L (meso)-2,3-BDO with > 99% optical purity, a yield of 0.33 ± 0.04 g/g glucose, and productivity of 0.82 ± 0.06 g/L/h. This work represents the first demonstration of > 100 g/L optically pure (meso)-2,3-BDO using C. glutamicum and establishes an integrated strategy of enzyme engineering, pathway optimization, and process design.