Noncanonical Peloruside A Biosynthesis by an Uncultivated Verrucomicrobiota Symbiont.

Peloruside A, a polyketide macrolide identified in the marine sponge Mycale hentscheli, is a promising anticancer drug candidate due to its ability to stabilize tubulin at a nontaxoid binding site. Substantial efforts were made to improve the supply of this scarce substance through chemical synthesis and mariculture, however, sustainable production has yet to be achieved. Recent microbiome sequencing suggested that bacterial producers are responsible for all M. hentscheli bioactive compounds, but the identity of the peloruside producer remained unknown. Furthermore, a candidate peloruside polyketide synthase (PKS) was identified in silico, but its highly aberrant architecture prevented a confident biosynthetic prediction for the pharmacologically important exocyclic double bond. Here, we reveal an unusually complex modular PKS pathway comprising nonelongating ketosynthases (KSs), two internal thioesterases (TEs), and an acetyl coenzyme A ligase that orchestrate O-acetylation/elimination, acetyl-CoA recycling, and configurational proof-reading to facilitate selective Z-double bond incorporation. The first internal TE falls into a previously characterized O-acylating TE family but contains key amino acid substitutions that facilitate bifunctional acylation/elimination activity. Furthermore, we identified the peloruside producer as 'Candidatus Pelorusia occulta', a member of the biochemically underexplored Lentisphaeria class within the phylum Verrucomicrobiota. This work provides the biochemical and microbiological foundation for heterologous peloruside production.