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 alpha-oxidase and a dagger(1)-piperideine-2-carboxylae (Pip2C) reductase. In this study, we focused on an l-lysine alpha-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 A degrees C and 7.4, respectively. The enzyme was stable below 60 A degrees C and at a pH range of 5.5-7.5 but was markedly inhibited by Co2+. 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).