Enhancing the Imine Reductase Activity of a Promiscuous Glucose Dehydrogenase for Scalable Manufacturing of a Chiral Neprilysin Inhibitor Precursor

Imine reductases (IREDs) have been identified as an important class of biocatalysts to synthesize chiral amines with substantial promise for industrial application. Here, we report the promiscuous imine reductase activity of a glucose dehydrogenase (GDH). Starting from enzyme GDH Rd1bb, a commercial glucose dehydrogenase variant typically used for NAD(P)H regeneration, eight rounds of directed evolution were used to convert this enzyme into a highly active IRED for the chemo- and stereoselective manufacture of a chiral neprilysin inhibitor precursor, improved NADH cofactor specificity, and superior thermal stability. The evolved variant GDH Rd6bb achieved high productivity, with 99% conversion over 3 h at 50 g/L keto substrate concentration and 10% enzyme loading with respect to the keto substrate. Early process development studies at multigram scale provided the product in 94% yield with >99% purity as a single stereoisomer with an er of >99.9:0.1 and a dr of >99.9:0.1.