(1 S)− -2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) is the key chiral intermediate of antiplatelet inhibitor ticagrelor. An NADPH-dependent carbonyl reductase Km CR 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 Km CR were identified, guided by our previous research; saturation mutation and combinatorial mutation were performed to enhance the stereoselectivity of Km CR toward CFPO, generating mutant Km CR-A129W/V239N (Km CR-W 2) with strict enantioselectivity (ee p >99.7%, S). Next, a triple mutant Km CR-A129W/V239N/V268A (Km CR-W 3 , 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 Km CR coupled with the glucose dehydrogenase Es GDH from Exiguobacterium sibiricum to recycle NADPH. The "best" mutant Km CR-W 3 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 Km CR was extended. Although there is much space for improvement in the catalytic performance of Km CR-W 3 , our work enriches the knowledge of carbonyl reductase's structure and function relationships. • Strictly S -selective Km CR mutants to CFPO were constructed by combinatorial mutagenesis. • A high throughput screening method based on TLC was developed for Km CR modification. • S -selective Km CR-W 3 completely reduced 30 g/L CFPO within 2 h. [ABSTRACT FROM AUTHOR]