(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 (ee_p =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 (ee_p >99.7%, S). Next, a triple mutant KmCR-A129W/V239N/V268A (KmCR-W₃, ee_p >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 (ee_p >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.

(1 S )- -2-chloro-1-(3,4-difluorophenyl)ethanol (( S )-CFPL) 是抗血小板抑制剂替格瑞洛的关键手性中间体。来自马克斯克鲁维酵母 ZJB14056的 NADPH 依赖性羰基还原酶Km CR显示出对 2-氯-1-(3,4-二氟苯基)乙酮 (CFPO) 的对映选择性还原活性，具有中等立体选择性 ( ee _p =80.3%, S )。在我们之前的研究指导下，确定了控制Km CR立体选择性的关键残基；进行饱和突变和组合突变以增强Km CR 对 CFPO 的立体选择性，产生突变体KmCR-A129W/V239N ( Km CR-W ₂ ) 具有严格的对映选择性 ( ee _p >99.7%, S )。接下来，通过区域易错PCR (epPCR)获得催化活性增加83%的三重突变体Km CR-A129W/V239N/V268A ( Km CR-W ₃ , ee _p >99.7%, S )。( S )-CFPL的对映选择性还原是通过使用Km CR与来自Exiguobacterium sibiricum的葡萄糖脱氢酶Es GDH结合来回收NADPH来实现的。“最佳”突变体Km CR-W ₃能够以严格的对映选择性完全转化 30 g/L CFPO（ee _p >99.7%，S，产率=95.5%）。此外，扩大了Km CR的底物范围。虽然Km CR-W ₃的催化性能还有很大的提升空间，但我们的工作丰富了羰基还原酶的结构和功能关系的知识。