Metabolic engineering of Corynebacterium glutamicum for 2-ketoisocaproate production.

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.

Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production.

2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter(-1)) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter(-1)) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.

Increased glucose utilization in Corynebacterium glutamicum by use of maltose, and its application for the improvement of L-valine productivity.

Corynebacterium glutamicum efficiently utilizes maltose as a substrate. We show here that the presence of maltose increases glucose utilization by raising the expression of ptsG, which encodes the glucose-specific EII permease of the phosphotransferase system. Consequently, the L-valine productivity of a pyruvate dehydrogenase complex-deficient C. glutamicum strain was improved by the presence of maltose.