title: Reductive amination by recombinant Escherichia coli: whole cell biotransformation of 2-keto-3-methylvalerate to L-isoleucine.
abstract: A whole cell biotransformation system for reductive amination has been studied in recombinant Escherichia coli cells. Reductive amination of 2-keto-3-methylvalerate to L-isoleucine by a two-enzyme-cascade was achieved by overproduction of endogenous L-alanine dependent transaminase AvtA and heterologous L-alanine dehydrogenase from Bacillus subtilis in recombinant E. coli. Up to 100 mM L-isoleucine were produced from 100 mM 2-keto-3-methylvalerate and 100 mM ammonium sulfate. Regeneration of NADH as cofactor in the whole cell system was driven by glucose catabolism. The effects of defined gene deletions in the central carbon metabolism on biotransformation were tested. Strains lacking the NuoG subunit of NADH:ubiquinone oxidoreductase (complex I) or aceA encoding the glyoxylate cycle enzyme isocitrate lyase exhibited increased biotransformation rates.
authors.affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, D-33615 Bielefeld, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Lorenz
authors.forename: Stephanie
authors.initials: S
authors.lastname: Klatte
authors.forename: Volker F
authors.initials: VF
authors.lastname: Wendisch
mesh_terms: D050788:Alanine Dehydrogenase / Q000378:metabolism*; D000586:Amination; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005947:Glucose / Q000378:metabolism; D007522:Isocitrate Lyase / Q000235:genetics; D007532:Isoleucine / Q000378:metabolism*; D007651:Keto Acids / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D000637:Transaminases / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007651:Keto Acids; D011994:Recombinant Proteins; D007532:Isoleucine; D009243:NAD; C016211:alpha-keto-beta-methylvaleric acid; D010088:Oxidoreductases; D050788:Alanine Dehydrogenase; D000637:Transaminases; D007522:Isocitrate Lyase; D005947:Glucose
keywords: 2-keto-3-methylvalerate; Reductive amination; Transaminase; Whole cell biotransformation; l-Alanine dehydrogenase