Directed Evolution of an Adenylation Domain Alters Substrate Specificity and Generates a New Catechol Siderophore in <i>Escherichia coli</i>

Nonribosomal peptide synthetases (NRPS) biosynthesize numerous natural products with therapeutic, agricultural, and industrial significance. Reliably altering substrate selection in these enzymes has been a longstanding goal, as this would enable the production of tailor-made peptides with desired activities. In this study, the NRPS EntF and the associated biosynthesis of the siderophore enterobactin (ENT) were used as a model system to interrogate substrate selection by an adenylation (A) domain. We employed a directed evolution pipeline that harnesses an <i>in vivo</i> genetic selection for siderophore production to alter A domain substrate selection. Surprisingly, this led to the formation of a new, physiologically active catechol siderophore in <i>Escherichia coli</i>. We characterized the enzyme variants <i>in vitro</i> and demonstrated transferability of our findings to the well-studied TycC and GrsB NRPSs. This work identifies critical binding pocket residues that allow for altered substrate selection in our model system and expands upon our understanding of iron acquisition in <i>E. coli</i>.