In this study, we present a significant advancement in the field of enzymatic asymmetric reductive amination (ARA) of ketones, a pivotal reaction for chiral amine synthesis. Through a combination of semirational enzyme design and bioprocess development, we achieve the dual activation and stabilization of amine dehydrogenase (AmDH) to meet industrial demands. The engineered AmDH exhibits remarkable catalytic efficiency (turnover number, TON >1,000,000) and exceptional stability (half-life >7 days at 50 °C), with a broadened substrate scope including various aryl alkyl ketones and fatty ketones. Leveraging biobased oleic acid as an activator and stabilizer, we achieved kilogram-scale synthesis of chiral amines. Furthermore, the integration of AmDH with chemical catalysts in chemoenzymatic cascades has enabled the synthesis of a wide array of pharmaceutically relevant amines from diverse substrates, demonstrating the enzyme’s versatility and potential to transform synthetic chemistry.

Chemoenzymatic cascade catalysis has emerged as a revolutionary tool for streamlining traditional retrosynthetic disconnections, creating new possibilities for the asymmetric synthesis of valuable chiral compounds. Here we construct a one-pot concurrent chemoenzymatic cascade by integrating organobismuth-catalyzed aldol condensation with ene-reductase (ER)-catalyzed enantioselective reduction, enabling the formal asymmetric α-benzylation of cyclic ketones. To achieve this, we develop a pair of enantiocomplementary ERs capable of reducing α-arylidene cyclic ketones, lactams, and lactones. Our engineered mutants exhibit significantly higher activity, up to 37-fold, and broader substrate specificity compared to the parent enzyme. The key to success is due to the well-tuned hydride attack distance/angle and, more importantly, to the synergistic proton-delivery triade of Tyr28-Tyr69-Tyr169. Molecular docking and density functional theory (DFT) studies provide important insights into the bioreduction mechanisms. Furthermore, we demonstrate the synthetic utility of the best mutants in the asymmetric synthesis of several key chiral synthons.