An Engineered Synthetic Pathway for Discovering Nonnatural Nonribosomal Peptides in Escherichia coli

Peptides that are synthesized independently of the ribosome in plants, fungi, and bacteria can have clinically relevant anticancer, antihemochromatosis, and antiviral activities, among many other. Despite their natural origin, discovering new natural products is challenging, and there is a need to expand the chemical diversity that is accessible. In this work, we created a novel, compressed synthetic pathway for the heterologous expression and diversification of nonribosomal peptides (NRPs) based on homologs of siderophore pathways from Escherichia coli and Vibrio cholerae. To enhance the likelihood of successful molecule production, we established a selective pressure via the iron-chelating properties of siderophores. By supplementing cells containing our synthetic pathway with different precursors that are incorporated into the pathway independently of NRP enzymes, we generated over 20 predesigned, novel, and structurally diverse NRPs. This engineering approach, where phylogenetically related genes from different organisms are integrated and supplemented with novel precursors, should enable heterologous expression and molecular diversification of NRPs.