Diversification of Tridecaptin Chemical Space via a Chimeric Biosynthetic Pathway in <i>Paenibacillus</i>

<i>Paenibacillus</i> species produce a wide array of bioactive nonribosomal peptides (NRPs). The structural diversity of NRPs is shaped by various diversification strategies that support bacterial ecological adaptation and create opportunities for new antibiotic discovery. Here, we show that chimeric biosynthesis occurs within the family of tridecaptin antibiotics. Genome mining revealed that 15 <i>Paenibacillus</i> strains harbored both a full tridecaptin BGC and a stand-alone tridecaptin-like NRPS predicted to encode a truncated decamer. The encoded NRPS domain architectures suggested the capability of these strains to produce multiple tridecaptin variants through collaborative action between this tridecaptin-like NRPS and a second NRPS homologous with TriE encoded within the complete tridecaptin BGC. Indeed, <i>Paenibacillus</i> sp. JJ-1683 produced both tridecaptin A₅ and tridecaptin B₁, while deletion of <i>triE</i> in the canonical BGC prevented the biosynthesis of all tridecaptins. This provides strong evidence for the existence of chimeric biosynthesis of lipopeptide antibiotics. Bioactivity testing revealed that the synthetic analogue of tridecaptin A₅, Oct-TriA₅, has unusual broad-spectrum activity against Gram-positive and Gram-negative ESKAPE pathogens, while Oct-TriB₁ displays moderate activity against Gram-negative strains and is not active against Gram-positive bacteria. We hypothesize that chimeric biosynthesis is a strategy that enables bacteria to produce compounds with distinct chemistry and bioactivity profiles.