Mining and Modification of Imine Reductase for Asymmetric Reduction of Cyclic Imines

ABSTRACT A naturally sourced imine reductase (IRED) was identified from the UniProt database and thoroughly investigated, leading to the identification of an IRED ( Thermostaphylospora chromogena ) typically toward the S ‐isomer. The mutation E127P/E221A/S236A (denoted as M3) was obtained through combinatorial mutagenesis, which exhibited a wider pH range, significantly improved catalytic activity, and enhanced stability compared to the wild‐type Tc IR. M3 demonstrated an enzyme activity of 307.28 ± 3.35 U/g protein and a 7.78‐fold increase in catalytic efficiency, clearly outperforming that of Tc IR. A dual enzyme‐catalyzed asymmetric synthesis of ( S )‐1‐methyl‐1,2,3,4‐tetrahydroisoquinoline (( S )‐1‐MeTIQ) from 100 mM 1‐methyl‐3,4‐dihydroisoquinoline (1‐MeDIQ) was constructed by combining M3 with formate dehydrogenase (FDH, for coenzyme regeneration). This cascade biocatalytic system achieved remarkable results, with a high conversion (> 99%) of 1‐MeDIQ and enantioselectivity ( ee value > 98%) of ( S )‐1‐MeTIQ under optimized reaction conditions. Additionally, this dual‐enzyme system was extended to the asymmetric reduction of more cyclic imine substrates, achieving excellent conversion (> 96%) and high ee values > 96%. These findings reveal that M3 is a promising biocatalyst for the efficient, scalable, and stereoselective synthesis of chiral tetrahydroisoquinolines and other chiral N ‐heterocyclic compounds. Keywords