2-Ketoisocaproate (KIC) is used as a therapeutic agent, and a KIC-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered Corynebacterium glutamicum for the production of KIC from glucose by deletion of ltbR and ilvE, encoding the transcriptional repressor LtbR and transaminase B, respectively, and additional overexpression of ilvBNCD, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. The KIC-producing strain was improved by deletion of the methylcitrate synthase genes and by decreasing citrate synthase activity by exchange of the native promoter of the citrate synthase gene. In shake-flask fermentations under l-leucine limitation, the newly constructed strain C. glutamicum VB (pJC4ilvBNCD) produced 31 +/- 2 mM (4.0 +/- 0.3 g l(-1)) KIC and showed a product yield of about 0.26 +/- 0.02 mol per mole (0.19 +/- 0.01 g per gram) of glucose. As by-product, the strain formed about 33 mM 2-ketoisovalerate, which is a precursor of KIC. KIC production was further improved by additional expression of an isopropylmalate synthase allele (leuA (EC-G462D)), encoding an enzyme resistant towards l-leucine inhibition, and by addition of acetate as additional substrate. With glucose and acetate, the newly constructed strain produced 71 +/- 3.2 mM (9.2 +/- 0.4 g l(-1)) KIC with a yield of 0.24 +/- 0.01 mol C (KIC) per mole C (in both substrates) and with nearly no 2-ketoisovalerate by-product formation (< 2 mM). Investigating the activities and regulation of the native isopropylmalate synthase and dehydratase of C. glutamicum, we observed competitive and noncompetitive inhibition, respectively, by KIC.