Lipid-donor-anchored genome mining uncovers dioxanopeptins, antibacterial lipopeptides with a 1,3-dioxane...

Non-ribosomal lipopeptide antibiotics derive much of their antibacterial performance from the structure of their lipid tail, yet genome mining remains largely peptide-centric and rarely prioritizes lipid donor formation as an entry point. Herein, we establish a lipid-donor-anchored discovery strategy using pfa-type polyunsaturated fatty acid synthase (PUFAS) core genes as the genomic anchor for long polyunsaturated acyl donors, followed by filtering for co-localization of non-ribosomal peptide synthetase (NRPS) assembly lines. This approach delineated 60 curated PUFAS-NRPS biosynthetic gene clusters, including a previously unassigned dxp family, which is distributed across Proteobacteria. Cultivation of Chitinimonas koreensis DSM 17726 enabled the isolation of dioxanopeptins, a new set of non-ribosomal lipopeptides featuring a long polyunsaturated lipid tail further functionalized by a pyruvyl-derived 1,3-dioxane ketal motif. Recombinant DxpH catalyzed the in vitro conversion of dedioxanopeptin to dioxanopeptin using phosphoenolpyruvate as the pyruvyl donor, establishing pyruvylation as a post-assembly tailoring step. Dioxanopeptins displayed selective activity against methicillin-resistant Staphylococcus aureus, and their antibacterial phenotype correlated with disruption of proton motive force homeostasis and ATP production. Removal of the pyruvyl-derived ketal markedly diminished potency, highlighting its contribution to antibacterial activity. Together, these results expand the chemical space of lipopeptides and illustrate how lipid-donor-anchored genome mining can complement NRPS-focused strategies to access functionally differentiated antibiotic scaffolds.