Characterization of the Biosynthetic Gene Cluster for the Ribosomally Synthesized Cyclic Peptide Epichloëcyclins in Epichloë festucae .

The various grass-induced epichloëcyclins of the Epichloë spp. are ribosomally synthesized and post-translationally modified peptides (RiPPs), produced as small, secreted cyclopeptides from a single gene, gigA. Here, four clustered and coregulated genes ( gigA , gigB , gigC , and kexB ) with predicted roles in epichloëcyclin production in Epichloë festucae were evaluated through gene disruption. Subsequent chemical analysis indicates that GigB is a DUF3328 domain-containing protein associated with cyclization of epichloëcyclins; GigC is a methyltransferase enzyme responsible for N-methylation of desmethylepichloëcyclins; and KexB is a subtilisin-like enzyme, partly responsible for the propeptide cleavage of epichloëcyclin intermediates. Symbiotic effects on the host phenotype were not observed for gigA , gigC , or kexB mutants, although Δ gigB infection correlated with increased host tiller height and biomass, while only Δ kexB exhibited an effect on endophyte morphology. Disrupting epichloëcyclin biosynthesis showed negligible influence on the biosynthesis of E. festucae -associated alkaloids. Epichloëcyclins may perform other secondary metabolism functions in Epichloë and other fungi.