LAUSR

Significance Fatty-acylated lipopeptides are metabolites of high medical and biotechnological importance, exemplified by the clinically used daptomycin, polymyxin, and echinocandin anti-infectives. The known representatives are produced by giant nonribosomal enzymes that usually generate heterogeneously acylated peptides requiring elaborate procedures to obtain defined compounds. While a few interesting acylated, ribosomally synthesized peptides have been described, none of them use fatty acids in their biosynthesis. Computational and synthetic biology methods identified a cryptic family of ribosomal lipopeptides present in diverse bacteria. A multifaceted structure elucidation approach revealed specific and distinct fatty acylations of amino acid side chains for three pathways. The results bring the production of tailored, gene-encoded lipopeptides using minimalistic gene sets into reach and provide guidelines for synthetic biology–based discovery. Lipopeptides represent a large group of microbial natural products that include important antibacterial and antifungal drugs and some of the most-powerful known biosurfactants. The vast majority of lipopeptides comprise cyclic peptide backbones N-terminally equipped with various fatty acyl moieties. The known compounds of this type are biosynthesized by nonribosomal peptide synthetases, giant enzyme complexes that assemble their products in a non–gene-encoded manner. Here, we report the genome-guided discovery of ribosomally derived, fatty-acylated lipopeptides, termed selidamides. Heterologous reconstitution of three pathways, two from cyanobacteria and one from an arctic, ocean-derived alphaproteobacterium, allowed structural characterization of the probable natural products and suggest that selidamides are widespread over various bacterial phyla. The identified representatives feature cyclic peptide moieties and fatty acyl units attached to (hydroxy)ornithine or lysine side chains by maturases of the GCN5-related N-acetyltransferase superfamily. In contrast to nonribosomal lipopeptides that are usually produced as congener mixtures, the three selidamides are selectively fatty acylated with C10, C12, or C16 fatty acids, respectively. These results highlight the ability of ribosomal pathways to emulate products with diverse, nonribosomal-like features and add to the biocatalytic toolbox for peptide drug improvement and targeted discovery.