Enzymatic Synthesis ofL-Pipecolic Acid by Δ¹-Piperideine-2-carboxylate Reductase fromPseudomonas putida

Abstract

L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine -oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Á 1 -piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of Lpipecolic acid in 99.7% e.e.Key words: Á 1 -piperideine-2-carboxylate reductase; Pseudomonas putida; L-pipecolic acid; Á 1 -piperideine-2-carboxylate L-Pipecolic acid, a nonproteinogenic -amino acid, is a key component of many bioactive molecules, such as the immunosuppressant FK506, 1) the anticancer agent VX710, 2) the antifungal antibiotic demethoxyrapamycin,3) the N-methyl-D-aspartate antagonist selfotel, 4) the antitumor antibiotic sandramycin, 5) the phytotoxic metabolite Cyl-2, 6) the anesthetic bupivacaine, 7) and the HIV protease inhibitor palinavir.8) There is an increasing demand for a convenient and efficient synthetic route to enantiomerically pure L-pipecolic acid because of its use in the synthesis of new medicaments.Current methods to obtain a pure enantiomer of L-pipecolic acid involve chemical resolution, 9) stereoselective transformation, 10) the derivatization of natural amino acids, 11) and enzymatic reactions, [12][13][14][15][16] but most of these methods fail to provide a satisfactory solution for the synthesis of chiral pipecolic acid on an industrial scale due to certain limitations, such as tedious procedures, low yields, and unavailability of starting materials. Hence, new and convenient methods for the preparation of optically active pipecolic acid are required.Recently, we identified and cloned the gene encoding17) The enzyme, which belongs to a new NAD(P)H-dependent oxidoreductase family, 18,19) catalyzes the reduction of Pip2C to L-pipecolic acid and is involved in D-lysine catabolism. 17) In this paper, we describe the production of L-pipecolic acid by an enzyme-coupled system consisting of Pip2C reductase, glucose dehydrogenase (GDH) from Bacillus subtilis, 20) and commercial L-lysine -oxidase from Trichoderma viride (Yamasa, Hiroshima, Japan) (Scheme 1).Recombinant Escherichia coli BL21(DE3) cells harboring both pDPKA, 17,21) which carries a gene for Pip2C reductase, and pSTVbsGDH, which has a GDH 20) gene between the EcoRI and PstI sites of pSTV28 (Takara Shuzo, Japan), were cultured in Luria-Bertani medium containing 100 mg/ml ampicillin and 25 mg/ml chloramphenicol at 37 C for 16 h. Three h after induction of gene expression with 1 mM isopropyl--D-thiogalactopyranoside, the cells were harvested and washed twice with 20 mM Tris-HCl (pH 7.0). The washed cells were disrupted by sonication and centrifuged. The resulting crude extract was used directly for the production of L-pipecolic acid. The standard reaction mixture for the production of L-pipecolic acid contained L-lysine (55 mM), glucose (550 mM), NADPL-lysine -oxidase (Yamasa, 3.0 U/ml), catalase from b...

Biocatalytic Imine Reduction and Reductive Amination of Ketones

“…In a very similar cascade, the Δ 1 ‐piperideine‐2‐carboxylate/Δ 1 ‐pyrroline‐2‐carboxylate reductase from Pseudomonas putida was employed in combination with L ‐lysine α‐oxidase from Trichoderma viride for the transformation of L ‐lysine into L ‐pipecolic acid (Scheme , B) 72. In contrast to the previous biotransformation using LysAT (Scheme , A), the stereogenic centre of L ‐lysine is destroyed in the oxidation step and re‐introduced in the asymmetric reduction.…”

Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle

“…RaiP activity was surveyed by determining hydrogen peroxide produced as Cheng et al reported (Cheng et al, 2018b). The reaction buffer embodied 50 mM KPB (pH 8.0), 30.0 mM L-lysine, 0.5 mM 4-aminoantipyrine (4AAP), 10 units/ml catalase and 26.5 mM phenol (Muramatsu et al, 2006). The standard reaction mixture embodied 8 μg RaiP and 180 μL reaction buffer.…”

Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle

“…RaiP activity was surveyed by determining hydrogen peroxide produced as Cheng et al reported (Cheng et al, 2018b). The reaction buffer embodied 50 mM KPB (pH 8.0), 30.0 mM L-lysine, 0.5 mM 4-aminoantipyrine (4AAP), 10 units/ml catalase and 26.5 mM phenol (Muramatsu et al, 2006). The standard reaction mixture embodied 8 μg RaiP and 180 μL reaction buffer.…”