Directed Evolution of an Adenylation Domain Alters\nSubstrate Specificity and Generates a New Catechol Siderophore in Escherichia coli

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