Microbial Cross-Talk: Unlocking the Cytochalasin Diversity from a Termite-Associated <i>Xylaria</i>

Integrating organismal interaction studies with advanced genomic and metabolomic approaches offer great promise for discovering novel natural products and their derivatives, yet this strategy remains relatively unexplored. Here, we illustrate its potential by investigating a newly isolated <i>Xylaria</i> strain from a termite colony environment through combined genome and metabolome analyses, complemented by fungal-bacterial coculture experiments. Genome sequencing of the fungal strain allowed us to pinpoint a cytochalasin-related biosynthetic gene cluster responsible for the production of a portfolio of different bioactive epoxy-cytochalasins. Guided by the hypothesis of biosynthetic promiscuity of the underlying nonribosomal peptide synthetase (NRPS), we demonstrated for the first time that the NRPS can accept unnatural ortho- and meta-halogenated phenylalanine derivatives, leading to the isolation of multiple new chlorinated and brominated cytochalasin analogs. Second, based on the hypothesis that structural diversification can arise from interactions with commensal organisms, cocultivation with a termite-associated <i>Streptomyces</i> strain led to the discovery of a previously undescribed aspartic acid-containing cytochalasan derivative, designated xylachalasin A. Isotope labeling experiments revealed that bacterial catabolic activity is responsible for the modification of the fungal-derived cytochalasin. Isolated cytochalasins were also amiable for semisynthesis modifications, which was exemplified by the synthesis of bifunctional probes. Bioassays of a total of 26 isolated and semisynthesized derivatives demonstrated structure-dependent cytotoxicity in some cases with up to 3-fold log differences in potency and generally good plasma stability. Overall, our integrated approach underscores the vast potential of investigating fungal strains from underexplored ecological niches and their organismal interactions, offering new opportunities to discover novel natural products of potential therapeutic relevance and previously unrecognized biochemical processes.