title: Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.
abstract: 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 Corynebacterium 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-KICF1, 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-KICF1 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-KICF1 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.
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425, Jülich, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Vogt
authors.forename: Sabine
authors.initials: S
authors.lastname: Haas
authors.forename: Tino
authors.initials: T
authors.lastname: Polen
authors.forename: Jan
authors.initials: J
authors.lastname: van Ooyen
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
mesh_terms: D053898:Biosynthetic Pathways; D018387:Codon, Initiator; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007651:Keto Acids / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D020125:Mutation, Missense
chemical_list: D018387:Codon, Initiator; D003470:Culture Media; D007651:Keto Acids; C013082:alpha-ketoisocaproic acid