Protein engineering of carbonyl reductase for asymmetric synthesis of ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol

(1S)− -2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) is the key chiral intermediate of antiplatelet inhibitor ticagrelor. An NADPH-dependent carbonyl reductase KmCR from Kluyveromyces marxianus ZJB14056 displayed enantioselective reduction activity toward 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) with moderate stereoselectivity (eep =80.3%, S). Key residues controlling the stereoselectivity of KmCR were identified, guided by our previous research; saturation mutation and combinatorial mutation were performed to enhance the stereoselectivity of KmCR toward CFPO, generating mutant KmCR-A129W/V239N (KmCR-W2) with strict enantioselectivity (eep >99.7%, S). Next, a triple mutant KmCR-A129W/V239N/V268A (KmCR-W3, eep >99.7%, S) with 83 % increase in catalytic activity was obtained by regional error-prone PCR (epPCR). The enantioselective reduction of (S)-CFPL was achieved by using KmCR coupled with the glucose dehydrogenase EsGDH from Exiguobacterium sibiricum to recycle NADPH. The "best" mutant KmCR-W3 was able to transform 30 g/L CFPO completely with strict enantioselectivity (eep >99.7%, S, yield=95.5%). Moreover, the substrate scope of KmCR was extended. Although there is much space for improvement in the catalytic performance of KmCR-W3, our work enriches the knowledge of carbonyl reductase's structure and function relationships.