Production of 2‐ketoisocaproate with C orynebacterium glutamicum strains devoid of plasmids and heterologous genes

Summary 2‐Ketoisocaproate ( KIC ), the last intermediate in l ‐leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l ‐leucine production strains of C orynebacterium glutamicum , KIC became the major product, however, the strains were auxotrophic for l ‐isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV ‐ KICF 1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback‐resistant 2‐isopropylmalate synthase, one copy of a gene for a feedback‐resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1  mM l ‐isoleucine, MV ‐ KICF 1 accumulated 47  mM KIC (6.1 g l −1 ) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol  KIC  l −1  h −1 . Since MV ‐ KICF 1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC ‐derived compounds, such as 3‐methyl‐1‐butanol.