Engineering Imine Reductase for Efficient Biosynthesis of 1-Aryl-Tetrahydro-β-Carbolines and Their N-Methylation Products

Chiral 1-aryl-tetrahydro-β-carboline (THβC) is an important substructure in natural products and pharmaceuticals. The application of imine reductase (IRED) for their enantioselective synthesis is attractive yet has not been realized, owing to the bulkiness and rigidness of the framework. In this study, through in silico and mutational analysis of the steric hindrance-tolerant IRED IR45, we identified a critical binding mode for 1-phenyl dihydro-β-carbolines (DHβCs). Engineering the key residues at L228′, M250′, and E251′ in the subunit B significantly expanded the substrate tolerance of the enzyme, enabling efficient, stereoselective synthesis of a series of ortho-, meta-, para-, and multi-substituted (S)-1-phenyl-THβCs. By combining enzymatic imine reduction and whole-cell N-methylation in one pot, we further developed a cost-effective strategy to directly synthesize (S)-N-methyl 1-phenyl-THβCs from DHβC substrates. Our results not only provide an effective route to the biosynthesis of the important 1-aryl THβC frameworks but also significantly expand the substrate specificity of IRED, which will be useful for the broad application of IREDs in the synthesis of other sterically hindered amines.