Semirational Engineering of Aldo–Keto Reductases for the Asymmetric Reduction of Ethyl 3,3,3‐Trifluoro‐2‐Oxopropanoate

Ethyl 3,3,3‐trifluoro‐2‐hydroxypropanoate (TFLAEt) is an important multifunctional intermediate, and the asymmetric reduction of its corresponding fluorinated keto ester, ethyl 3,3,3‐trifluoro‐2‐oxopropanoate (TFPyEt), to generate chiral TFLAEt represents a valuable yet challenging task in biocatalysis. In this study, several aldo–keto reductases (AKRs) from Saccharomyces cerevisiae were cloned and heterologously expressed in Escherichia coli . Their reductase activities toward TFPyEt were evaluated, and YJR096w and YDL124w exhibited moderate R ‐selectivity (72.6% ee) and S ‐selectivity (82.8% ee), respectively. Homology modeling and molecular docking with TFPyEt and NADPH were conducted to elucidate the molecular basis of their enantioselectivity. Through semi‐rational engineering, two mutants—YJR096w‐L49Y/D275H and YDL124w‐T26G—were obtained, exhibiting excellent enantioselectivity with enantiomeric excess (ee) values of 99.9% ( R ) and 97.6% ( S ), respectively, for the reduction of TFPyEt. An NADPH regeneration system was constructed using glucose dehydrogenase ( Ba GDH) from Bacillus amyloliquefaciens , enabling the efficient production of chiral TFLAEt from TFPyEt under optimized biocatalytic conditions using the two mutants. This work presents an effective strategy for enhancing the enantioselectivity of native biocatalysts and provides valuable insights into the development of novel green catalysts for asymmetric transformations.