Expanding the repertoire of imine reductases by mining divergent biosynthetic pathways for promiscuous reactivity

Imine reductases (IREDs) are invaluable catalysts for enantioselective imine reduction and reductive amination of carbonyl compounds. Their synthetic versatility is, however, limited by their substrate scope, and new IREDs are needed. Current IREDs are closely related to the initially characterized enzymes, as their discovery has been driven by sequence homology searches. Here, we demonstrate a <i>functional</i> genomics approach based on biosynthetic promiscuity, guided by the identification of C=N reducing enzymes acting on large, complex substrates in biosynthetic pathways. These substrate-promiscuous biocatalysts share low homology to existing IREDs and fall into distinct functional enzyme families, yet they catalyze the hydrogenation of non-native imines as well as the reductive amination of simple ketones. Venturing further into sequence space without the constraints of close homology, but instead guided by functional promiscuity, has thus led us to distinct, previously unrecognized and unexplored areas of sequence space for mining IREDs for synthesis.