Characterization of the Biosynthetic Gene Cluster for the Ribosomally Synthesized Cyclic Peptide Epichloëcyclins in <i>Epichloë festucae</i>

<p dir="ltr">The various grass-induced epichloëcyclins of the <i>Epichloë </i>spp. are ribosomally synthesized and post-translationally modified peptides (RiPPs), produced as small, secreted cyclopeptides from a single gene, <i>gigA</i>. Here, four clustered and coregulated genes (<i>gigA</i>, <i>gigB</i>, <i>gigC</i>, and <i>kexB</i>) 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 <i>gigA</i>, <i>gigC</i>, or <i>kexB </i>mutants, although Δ<i>gigB</i> infection correlated with increased host tiller height and biomass, while only Δ<i>kexB</i> exhibited an effect on endophyte morphology. Disrupting epichloëcyclin biosynthesis showed negligible influence on the biosynthesis of <i>E. festucae</i>-associated alkaloids. Epichloëcyclins may perform other secondary metabolism functions in <i>Epichloë </i>and other fungi.</p>