Directed Evolution of a Gatekeeper Domain in Nonribosomal Peptide Synthesis Original Research Article

Modular natural products are biosynthesized by series of enzymes that activate, assemble, and process a nascent chain of building blocks. Adenylation domains are gatekeepers in nonribosomal peptide biosynthesis, providing the entry point for assembly of typical peptide-based natural products. We report the directed evolution of an adenylation domain based on a strategy of using a weak, promiscuous activity as a springboard for reprogramming the biosynthetic assembly line. Randomization of residues invoked in a “specificity-conferring code” and selection for a non-native substrate lead to mutant G2.1, favoring smaller amino acids with a specificity change of 105: a 170-fold improvement for l-alanine corresponds to a 103-fold decrease for its original substrate (l-phenylalanine). These results establish directed evolution as a method to change gatekeeper domain specificity and suggest that adaptation of modules in combinatorial biosynthesis is achievable with few mutations during evolution.