We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)-water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K(m) and V(max) values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA-water medium.

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We used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (R)-(؊)-3-quinuclidinol. Microbacterium luteolum JCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated from M. luteolum JCM 9174. The bacC gene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. The qnr gene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed in Escherichia coli as proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (R)-(؊)-3-quinuclidinol. Many methods have been used for the synthesis of optically pure isomers, including enantioselective synthesis, derivatization of natural compounds, and the optical resolution of racemic compounds. Enantioselective organic synthesis is the most efficient and attractive method for producing chiral starting materials for pharmaceuticals and agrochemicals. Chiral metal catalysts such as BINAP-Ru (1) and chiral Co(II) salen complex (2, 3) have been successfully used in producing chiral alcohols or chiral diols from various ketones or epoxides. However, these catalysts are costly and leave trace metal contamination in the products, and they are sometimes difficult to handle. The enzymatic reduction of ketones to optically pure alcohols is more environmentally sustainable and thus more attractive for pharmaceutical manufacturing (4). Several excellent methods using biocatalysts have been reported for producing chiral alcohols (5-10).(R)-(Ϫ)-3-Quinuclidinol, which has a double-ring structure containing nitrogen, is a valuable intermediate for pharmaceuticals. It has been used as the chiral synthone for a cognition enhancer, a bronchodilator, and a urinary incontinence agent (11). The following enzymes have been reported to catalyze the reduction of 3-quinuclidinone to (R)-(Ϫ)-3-quinuclidinol: tropinone reductase, from the plant henbane (Solanaceae, Hyoscyamus niger) (12), and NADPH-dependent 3-quinuclidinone reductase (QNR), from the yeast Rhodotorula rubra (mucilaginosa) (13). Yamamoto et al. reported the production of (R)-(Ϫ)-3-quinuclidinol using recombinant formate dehydrogenase (FDH) and tropinone reductase (14). Uzura et al. reported a reaction system using recombinant glucose dehydrogenase and 3-quinuclidinone reductase from Rhodotorula rubra (13). The optical resolution of (Ϯ)-3-quinuclidinol esters by the hydrolysis reaction of Aspergillus melleus protease was reported by Nomoto et al. (15).In this study, we report two novel 3-quinuclidinone reductase genes, qnr and bacC, from Microbacterium luteolu...

We found two NADH-dependent reductases (QNR and bacC) in Microbacterium luteolum JCM 9174 (M. luteolum JCM 9174) that can reduce 3-quinuclidinone to optically pure (R)-(−)-3-quinuclidinol. Alcohol dehydrogenase from Leifsonia sp. (LSADH) was combined with these reductases to regenerate NAD+ to NADH in situ in the presence of 2-propanol as a hydrogen donor. The reductase and LSADH genes were efficiently expressed in E. coli cells. A number of constructed E. coli biocatalysts (intact or immobilized) were applied to the resting cell reaction and optimized. Under the optimized conditions, (R)-(−)-3-quinuclidinol was synthesized from 3-quinuclidinone (15% w/v, 939 mM) giving a conversion yield of 100% for immobilized QNR. The optical purity of the (R)-(−)-3-quinuclidinol produced by the enzymatic reactions was >99.9%. Thus, E. coli biocatalysis should be useful for the practical production of the pharmaceutically important intermediate, (R)-(−)-3-quinuclidinol.

Screening gene‐specific amplicons from metagenomes (S‐GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase ( par ) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full‐length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence‐ and activity‐based screening of these libraries identified different homologous par genes, Hpar ‐001 to ‐036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error‐prone PCR to exhibit increased activity in the presence of concentrated 2‐propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM‐based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM‐based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies.