Adenylation Domain Engineering Enables Biosynthesis of an Improved Polymyxin Antibiotic

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.