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

Abstract

Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.The demand for enzymes in the chemical industry has expanded rapidly. Most enzymes can catalyze reactions at room temperature in a mild aqueous solution and have great substrate, stereo-, regio-, and chemoselectivity (12,15,16). Such properties are ideal for fine chemical production, particularly as chiral drug intermediates and agrochemicals.Alcohol dehydrogenase (ADH) (EC 1.1.1.1) can catalyze the asymmetric reduction of aldehyde or ketone to chiral alcohols. Phenylacetaldehyde reductase (PAR) from styrene-assimilating bacteria Rhodococcus sp. (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 (8-10).Various examples of ADH applications for asymmetric reduction have been reported that use a cofactor regeneration system with additional enzymes such as formate dehydrogenase or glucose dehydrogenase (15). 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. However, at high concentrations of 2-propanol, the efficiency of substrate conversion by PAR clearly fell. Consequently, the conversion yields with high substrate concentrations were relatively low and insufficient for industrial application.Here, we attempted to engineer PAR that can operate in relatively high concentrations of 2-propanol (Ͼ20% [vol/vol]) to achieve efficient conversion of concentrated substrates without altering the substrate specificity. Using our method with the combination of advantageous mutations, PAR was successfully improved with six amino acid replacements with significant enhancement of the substrate conversion. Although relative yield of conversion at high concentration of 2-propanol was enhanced, little change of substrate specificity was observed. MATERIALS AND METHODSMaterials. All chemicals were of reagent grade. Taq DNA poly...

Recent progress in biocatalysis for asymmetric oxidation and reduction

“…was mutated to improve the conversion efficiency in high concentrations of substrate and 2-propanol. 34 Here, 2-propanol acts as a solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates. First, the PAR library was generated by mutagenic PCR.…”

Gene‐specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases

“…During the analysis of these HPARs, we found that amino acid substitutions within them are scattered but located in quite limited sequence positions. Some of these substitutions completely coincided with those of Sar268 and Har1, which were previously engineered from PAR through application of directed evolution molecular techniques . By analyzing the properties of HPARs and comparing their amino acid sequences, we inferred the relationships between each HPAR amino acid sequence and its properties.…”

“…E. coli JM109 cells were used to host par genes fused with the pSar268‐del‐fus plasmid. This vector was derived from pSar268 , which expresses the Sar268 ( par mutant) of pUC118, by partial deletion of the Sar268 gene with Kpn I and introduction of a Not I site. To introduce fusion sites to both 5′ ends of this plasmid, PCR was performed using KOD FX DNA polymerase (Toyobo, Osaka, Japan) and the following primers: PAR‐091015 sense, 5′‐CGGGCGCGCGGTTGT‐3′ and PAR‐091015 antisense, 5′‐CCTGGCCCGGGCTC‐3′ (the underlined sequences indicate the fusion sites).…”

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

“…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.…”