Polymyxins remain a last-resort defence against Gram-negative bacterial infections, yet their clinical utility is constrained by acute nephrotoxicity and spreading resistance fuelled by agricultural overuse. Analogues carrying L-2-aminodecanoic acid, L-Ada, in place of the fatty amino acid side chain have shown activity against resistant clinical isolates of Acinetobacter baumannii and good tolerability in mouse thigh models, but until now they have only been accessible through total synthesis. Biosynthetic production from the native nonribosomal peptide synthetase, NRPS, machinery of Paenibacillus polymyxa was blocked because the adenylation domain of PmxA1 selects for L-leucine: the bulky L-Ada side chain sterically clashes with the wild-type binding pocket and cannot be adenylated.
Researchers in the Kries Lab at the University of Stuttgart, published in Angewandte Chemie International Edition, reasoned that the obstacle was geometric: a homology model built with the Chai-1 pipeline showed that Trp454 caps the base of the substrate-binding pocket, forcing the L-Ada side chain into a distorted conformation that, as the authors note, only suggests rather than proves an explanation for the lack of adenylation activity. Precedent in homologous A-domains indicated that replacing the bulky tryptophan with alanine could open the pocket to larger substrates. Integrating W454A into P. polymyxa DSM 365 and supplementing cultures with exogenous L-Ada enabled direct fermentative production of Ada-polymyxin at 6.3 ± 0.3 μM, quantified assuming identical LC-MS/MS responses for Ada-polymyxin and a polymyxin B1 standard, though the engineered strain displayed cell lysis and declining viable counts after 50 h, indicating self-intoxication at that concentration.
The authors describe this result as a solid foundation for further strain improvement, and they identify endogenous L-Ada biosynthesis, enhanced producer resistance to self-intoxication, and optimisation of downstream domains as the next engineering priorities. More broadly, the work indicates a strategy of replacing bulky pocket residues to admit larger noncanonical building blocks, an approach that, the authors suggest, may extend to other NRPS systems. The full kinetic characterisation, strain engineering details, and antimicrobial data are available in the original publication.
