Adenylation Domain Engineering Enables Biosynthesis of an Improved Polymyxin Antibiotic

ABSTRACT Polymyxins are potent nonribosomal peptide antibiotics, yet their clinical use is limited by a narrow therapeutic window and nephro‐/neurotoxicity. Conventional efforts to improve these agents rely on the incorporation of nonnatural building blocks that cannot be accessed biosynthetically and therefore must be introduced by total synthesis. Here we report a biosynthetic route to a next‐generation polymyxin that circumvents this limitation. By engineering the substrate‐binding pocket of a nonribosomal adenylation (A‐)domain, we created a mutationally expanded pocket that selectively activates the fatty amino acid l ‐2‐aminodecanoic acid ( l ‐Ada). Incorporation of this engineered A‐domain into the native polymyxin biosynthesis enabled production of Ada‐polymyxin with high potential for lowered toxicity directly in Paenibacillus polymyxa fermentation. This work demonstrates that rational redesign of nonribosomal A‐domains can redirect natural product biosynthesis toward chemically diverse, therapeutically superior antibiotics, opening new avenues for combating drug‐resistant bacteria.