Fermentative production of α-ketoisovalerate and α-ketoisocaproate by metabolically engineered Corynebacterium glutamicum.

Alpha-ketoisovalerate (KIV) and α-ketoisocaproate (KIC) are widely used as food additives and in the synthesis of pharmaceuticals and higher alcohols. Current chemical synthesis methods are environmentally harmful, and whole-cell catalysis processes are costly due to expensive substrates. Direct fermentative production of KIV and KIC from glucose is a promising alternative, although research in this area remains limited. In this study, we engineered an L-valine-overproducing Corynebacterium glutamicum strain for KIV and KIC production. We inactivated leucine dehydrogenase and isopropylmalate synthase to block the formation of L-valine and KIC, resulting in the production of 53.5 g/L KIV with a yield of 0.16 g/g glucose and a productivity of 0.70 g/L·h⁻¹ in a 5-L fermentor. Next, we overexpressed genes in the L-leucine biosynthesis pathway (leuA, leuCD, and leuB) by introducing a feedback-resistant leuA (leuA) in a plasmid-based system, deleting the transcriptional repressor gene ltbR, and increasing the gene copy numbers of leuCD and leuB under a strong promoter, creating a high-KIC-producing strain. Acetate supplementation enhanced acetyl-CoA supply, increasing KIC production while reducing KIV accumulation. The final strain produced 79.8 g/L KIC with a yield of 0.29 g/g glucose and a productivity of 1.05 g/L·h⁻¹ in a 5-L fermentor, surpassing previous fermentation results and most whole-cell catalysis processes, highlighting its industrial application potential.