Structure-Guided Engineering of Carbonyl Reductase <i>Lb</i>CR to Simultaneously Enhance Catalytic Activity and Thermostability toward Bulky Ketones

(<i>S</i>)-2-Chloro-1-(2,4-dichlorophenyl)ethanol ((<i>S</i>)-TCPE) is an important building block for the synthesis of antifungal drug luliconazole. Herein, a carbonyl reductase (CR) from <i>Levilactobacillus brevis</i> (<i>Lb</i>CR) was identified for synthesis of (<i>S</i>)-TCPE. Through comprehensive Ala scanning and site-saturated mutagenesis (SSM) targeting the residues surrounding the substrate-binding pocket, the "best" variant <i>Lb</i>CR_M4 (N96V/E145A/A202L/M206A) was developed, which displays a 26.0-fold increase in catalytic activity, 83.5-fold enhancement in half-life (<i>t</i>_1/2) at 40 °C (101.4 h), excellent enantioselectivity (>99.9% <i>e.e.</i>), and broad substrate scope. Compared to the wild-type (WT) <i>Lb</i>CR, catalytic efficiency (<i>k</i>_cat/<i>K</i>_M) of <i>Lb</i>CR_M4 was increased by 28.0 folds. Furthermore, a high concentration of TCAP (400 g/L) can be transformed (99.9% conversion) within 7 h by using <i>Lb</i>CR_M4 and an isopropanol/alcohol dehydrogenase/NADPH cofactor regeneration system, giving (<i>S</i>)-TCPE in >99.9% <i>e.e</i>., which is the highest recorded space-time yield (STY, 1288.9 g/L/day) to date. Molecular dynamics (MD) simulations and dynamic cross-correlation matrix analysis elucidated the substantial catalytic performance improvement of <i>Lb</i>CR_M4. Together, the development of <i>Lb</i>CR_M4 not only overcomes the trade-offs between catalytic activity and thermostability but also affords an efficient biocatalytic approach for the synthesis of (<i>S</i>)-TCPE featuring a record STY, laying a solid foundation for industrial manufacturing of luliconazole and other active pharmaceutical intermediates.