Identification Of Novel Stereospecific Aldo-keto Reductases From Candida Parapsilosis For Highly Enantioselective Reduction Of Carbonyl Compounds

Biocatalytic reduction of prochiral ketones offers significant potential in synthesis ofoptically active alcohols, due to its highly chemo-, regio-and stereoselectivities. However, sofar the application of aldo-keto reductases (AKRs) in asymmetric reduction has beenhampered due to limited availability of AKRs with high enantioselectivity and catalyticefficiency. The disclosure of microbial genome sequence allows scientists to search for novelenzymes with potential applications. Based on the genome sequence of Candida parapsilosis,a versatile bioresource for asymmetric reduction, eight open reading frames encoding putativeAKRs were discovered and expressed, and the resulted enzymes (CPARs), comprising anAKR toolbox, were evaluated towards various carbonyl substrates. Meanwhile, eightaldo-keto reductases were evaluated towards N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) and built a cell-free system for asymmetric reduction of DKTP to(S)-N,N-dimethyl-3-hydroxy-(2-thienyl)-1-propanamine (DHTP), a key intermediate of thechiral drug Duloxetine.(1) Genomic mining based on sequence similarity was carried out against the genomesequence of C. Parapsilosis (http://www.sanger.ac.uk/sequencing/Candida/parapsilosis/) andeight homologous ORFs, coding for putative AKRs, were revealed. From the amino acidsequence and secondary structure prediction, the putative enzymes exhibit a classic (α/β)8structure containing the NADP-binding motif, the catalytic tetrad (DxxxxY, K, and H) andtwo AKR family signature sequences. The genes encoding CPARs were ligated into pET21cexpression vector and transformed into the E.coli BL21(DE3) competent cells. Theexpressions of target recombinant proteins were initiated with the optimization ofthe external expression conditions.(2) The capability of CPARs for catalyzing reduction of carbonyl compounds wasinvestigated towards different kinds of substrates including aryl ketones, aliphatic ketones andketoesters. The CPARs were active to the selected substrates, obviously higher activities toaryl ketones and ketoesters, especially of2-hydroxyacetophenone and ethyl4-chloro-3-oxobutyrate. Additionally, most of them were obviously enantioselective to the substratesand gave alcohol products with optical purity up to99%e.e. Of the enzymes, CPAR4wasoutstanding with excellent enantioselectivity and broad substrate spectrum. All these positivefeatures demonstrate that genomic mining is powerful in searching for novel and efficientbiocatalysts of desired reactions for pharmaceuticals and fine chemicals synthesis.(3) After assessing of the discovered AKR toolbox for the asymmetric reduction ofDKTP,(S)-DHTP (>99.9%e.e.) was obtained. Of the enzymes, CPAR4performed highercatalytic ability for asymmetric reduction. Then the biomediated reaction conditions using thecell-free system involving the expressed CPAR4were optimized and (S)-DHTP (>99.9%e.e.)was produced with the yield of94.5%at the initial substrate concentration of3g·L-1. Andeven when the substrate concentration was increased to5g·L-1.the enantiopure product canalso be obtained with the yield over70%. Therefore, the newly identified enzyme and itscell-free system would be promising for highly stereospecific bioreduction of prochiral pharmaceutical intermediates.