title: Heterologous production of L-lysine ε-oxidase by directed evolution using a fusion reporter method.
abstract: For the heterologous production of l-lysine ε-oxidase (LodA), we constructed a new plasmid carrying LodA gene fused in-frame with an antibiotic (phleomycine) resistant gene. The new plasmid was randomly mutated and the mutated plasmids were transformed into Escherichia coli BL21 (DE3) harboring lodB, which encodes a protein (LodB) acting in posttranslational modification of LodA, and active mutants were selected by phleomycin resistance and oxidase activities. One soluble LodA variant isolated by this method contained six silent mutations and one missense mutation. At these mutation points, the codon adaptations at Lys92, Ala550, and Thr646, and the amino acid substitution at His286 to Arg contributed to the production of its functional form. The active form of LodA variant was induced by post-modification of LodB in the heterologous coexpression, and the activity increased with additional NaCl and heat treatment. This is the first report of heterologous production of LodA by random mutagenesis.
authors.affiliation: a Biotechnology Research Center and Department of Biotechnology , Toyama Prefectural University , Imizu , Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Matsui
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003062:Codon; D004151:Dipeptides / Q000096:biosynthesis*; D019020:Directed Molecular Evolution; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D045563:Indolequinones / Q000096:biosynthesis*; D008239:Lysine / Q000378:metabolism; D053544:Marinomonas / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011499:Protein Processing, Post-Translational / Q000235:genetics
chemical_list: D003062:Codon; D004151:Dipeptides; D045563:Indolequinones; C447619:cysteine tryptophylquinone; D000594:Amino Acid Oxidoreductases; C041123:L-lysine oxidase; D008239:Lysine
keywords: LodB; cysteine tryptophylquinone cofactor; directed mutagenesis; fusion reporter method; l-lysine ε-oxidase (LodA)