Rational Engineering of Mesorhizobium Imine Reductase for Improved Synthesis of N-Benzyl Cyclo-tertiary Amines

The effective synthesis ofN-benzyl cyclo-tertiary amines using imine reductase, key components in natural products and pharmaceutical synthesis, is a green approach. Traditional methods faced challenges with enzyme activity and selectivity. This study focused on enhancingMesorhizobiumimine reductase (MesIRED) for betterN-benzyl cyclo-tertiary amine production. Through alanine scanning and consensus mutation, 12 single-siteMesIRED mutants were identified from 23 candidates, showing improved conversion ofN-benzylpyrrolidine andN-benzylpiperidine. Notably, mutants from I177, V212, I213, and A241 significantly boosted conversions. The best-performing mutant forN-benzylpyrrolidine,MesIREDV212A/I213V(M1), increased conversion from 23.7% to 74.3%. ForN-benzylpiperidine,MesIREDV212A/I177A/A241I(M2) enhanced conversion from 22.8% to 66.8%. Tunnel analysis revealed M1 and M2 have more efficient tunnels for larger product movement compared to wild-typeMesIRED. Using recombinantE. colicoexpressingMesIRED and glucose dehydrogenase (GDH), high conversions were achieved: 75.1% forN-benzylpyrrolidine (M1) and 88.8% forN-benzylpiperidine (M2). A preparative experiment resulted in 86.2% conversion and 60.2% yield forN-benzylpiperidine. This research offers an efficient method for engineering IRED, significantly improving conversion and selectivity forN-benzyl cyclo-tertiary amines, aiding drug synthesis and providing insights into rational design of other enzymes.Keywords:imine reductase;N-benzyl cyclo-tertiary amines;alanine scanning;consensus mutation;whole-cell biocatalysis;coexpression