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 Bryopsis sp. and its molluscan predator Elysia rufescens. Recent studies have pinpointed a bacterial endosymbiont of the green alga, Candidatus Endobryopsis kahalalidefaciens, as the true producer of kahalalide F. In the present work, we characterize a closely related kahalalide F analog, kahalalide Z5, as well as several related compounds from a marine cyanobacterium Limnoraphis sp. collected in the Las Perlas Islands, Panama. To uncover and prioritize novel metabolites 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 Limnoraphis sp. as an independent source of kahalalide F-like molecules. This work not only broadens the phylogenetic spectrum of organisms capable of producing these bioactive compounds but also illustrates the potential of AI-enhanced metabolomic and genomic analyses to streamline the discovery and characterization of complex biomedically-relevant natural products.