Chiral alcohol production by NADH‐dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH

Abstract

Phenylacetaldehyde reductase (PAR) produced by styreneassimilating Corynebacterium strain ST-10 was used to synthesize chiral alcohols. This enzyme with a broad substrate range reduced various prochiral aromatic ketones and b-ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.). The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R)-2-chloro-1-(3-chlorophenyl)ethanol (28 mgAEmL )1 ) from m-chlorophenacyl chloride, ethyl (R)-4-chloro-3-hydroxy butanoate) (28 mgAEmL )1 ) from ethyl 4-chloro-3-oxobutanoate and (S)-N-tert-butoxycarbonyl(Boc)-3-pyrrolidinol from N-Boc-3-pyrrolidinone (51 mgAEmL )1 ), with more than 86% yields. The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3-7% (v/v) 2-propanol in the reaction mixture. This biocatalytic process provided one of the best asymmetric reductions ever reported.

Gene Cloning and Expression ofLeifsoniaAlcohol Dehydrogenase (LSADH) Involved in Asymmetric Hydrogen-Transfer Bioreduction to Produce (R)-Form Chiral Alcohols

“…strain ST-10 is a unique NADH-dependent alcohol dehydrogenase (ADH) with a broad substrate range and high enantioselectivity to give (S)-form alcohols without an additional coenzyme regeneration system, because the enzyme itself is able to regenerate NADH in the presence of 2-propanol. [12][13][14] Therefore, a recombinant PAR or its mutated enzyme (Sar268) system 15) and chemotolerant ADH reported in the R. ruber DSM44541 strain, 16) are regarded as superior asymmetric hydrogentransfer biocatalysts with which to produce (S)-alcohols.…”

“…1). 14,15,17) In this report, we describe the cloning, sequence analysis, and expression in Escherichia coli of the gene encoding LSADH from Leifsonia sp. S749, and the purification of the recombinant enzyme.…”

Purification and Characterization of a Yeast Carbonyl Reductase for Synthesis of Optically Active (R)-Styrene Oxide Derivatives

“…Asymmetric reductions of phenacyl halide by Geotrichum 5,6) baker's yeast 7,8) and phenylacetaldehyde reductase 9) from Corynebacterium sp. have been reported.…”

Engineering of Phenylacetaldehyde Reductase for Efficient Substrate Conversion in Concentrated 2-Propanol

“…(formerly identified as Corynebacterium sp.) ST-10 belongs to the family of zinc-containing medium-chain ADHs (8). PAR has broad substrate specificity and catalyzes asymmetric reduction at high enantioselectivity in an NADH-dependent manner (8)(9)(10).…”

“…Recent reports have used the advantage of ADHs for NADH self regeneration with secondary alcohols as hydrogen donors (13,21). We previously reported the ability of PAR to reduce various carbonyl compounds coupled with NADH self regeneration in the presence of 2-propanol as a proton donor (8). 2-Propanol can also profitably act as a solvent for the PAR substrates that cannot dissolve in aqueous media.…”