Cyanobacteria Join the Kahalalide Conversation: Genome and Metabolite Evidence for Structurally Related Peptides

Kahalalide F is a cyclic depsipeptide with notable anticancer properties, initially discovered from the green alga <i>Bryopsis</i> sp. and its molluscan predator <i>Elysia rufescens</i>. Recent studies have pinpointed a bacterial endosymbiont of the green alga, <i>Candidatus</i> Endobryopsis kahalalidefaciens, as the true producer of kahalalide F. In the present work, we characterize a closely related kahalalide F analog, kahalalide Z₅, from the marine cyanobacterium <i>Limnoraphis</i> sp. collected in the Las Perlas Islands, Panama, and propose the structures of several related compounds by detailed MS analysis. To uncover novel metabolites and prioritize them for targeted isolation from this organism, we employed a robust metabolomics strategy combining LC-MS/MS with SMART NMR and DeepSAT, artificial intelligence platforms trained to infer chemical structures from ¹H-¹³C HSQC NMR data. This integrated approach annotated a compound with structural similarities to kahalalide F, which we subsequently characterized using a suite of spectroscopic techniques and chemical degradation studies. Whole-genome sequencing of the producing strain further revealed a NRPS biosynthetic gene cluster that aligns with the structural features of kahalalide Z₅. This study identifies the marine cyanobacterium <i>Limnoraphis</i> sp. as an independent source of kahalalide F-like molecules. This work broadens the phylogenetic spectrum of organisms capable of producing these bioactive compounds, reveals marine cyanobacteria as producers of an increased repertoire of unique natural products, and illustrates the potential of AI-enhanced metabolomic and genomic analyses to streamline the discovery and characterization of complex biomedically relevant natural products.