title: Functional expression of L-lysine α-oxidase from Scomber japonicus in Escherichia coli for one-pot synthesis of L-pipecolic acid from DL-lysine.
abstract: L-Pipecolic acid is a key component of biologically active molecules and a pharmaceutically important chiral building block. It can be stereoselectively produced from L-lysine by a two-step bioconversion involving L-lysine α-oxidase and ∆(1)-piperideine-2-carboxylae (Pip2C) reductase. In this study, we focused on an L-lysine α-oxidase from Scomber japonicus that was originally identified as an apoptosis-inducing protein (AIP) and applied the enzyme to one-pot fermentation of L-pipecolic acid in Escherichia coli. A synthetic gene coding for an AIP was expressed in E. coli, and the recombinant enzyme was purified and characterized. The purified enzyme was determined to be a homodimer with a molecular mass of 133.9 kDa. The enzyme essentially exhibited the same substrate specificity as the native enzyme. Optimal temperature and pH for the enzymatic reaction were 70 °C and 7.4, respectively. The enzyme was stable below 60 °C and at a pH range of 5.5-7.5 but was markedly inhibited by Co(2+). To establish a one-pot fermentation system for the synthesis of optically pure L-pipecolic acid from DL-lysine, an E. coli strain carrying a plasmid encoding AIP, Pip2C reductase from Pseudomonas putida, lysine racemase from P. putida, and glucose dehydrogenase from Bacillus subtilis was constructed. The one-pot process produced 45.1 g/L of L-pipecolic acid (87.4 % yield from DL-lysine) after a 46-h reaction with high optical purity (>99.9 % enantiomeric excess).
authors.affiliation: Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Tani
authors.forename: Ryoma
authors.initials: R
authors.lastname: Miyake
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Yukami
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Dekishima
authors.forename: Hideyasu
authors.initials: H
authors.lastname: China
authors.forename: Shigeki
authors.initials: S
authors.lastname: Saito
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawabata
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Mihara
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005399:Fishes / Q000235:genetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D060847:Metabolic Engineering; D010875:Pipecolic Acids / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D029941:Fish Proteins; D010875:Pipecolic Acids; D000594:Amino Acid Oxidoreductases; C041123:L-lysine oxidase; C031345:pipecolic acid; D008239:Lysine