Heterologous expression and in vivo characterization of the longipeptin biosynthetic gene cluster

-methylation to afford trivalent sulfonium. All the three secondary modifications occur post-macrolactamization, with the LopF-catalyzed crosslinking serving as an essential prerequisite for LopH-mediated methylation. Our results establish a framework for investigating P450-catalyzed lasso peptide crosslinking, represent the first functional characterization of a DUF6919 family protein and reveal distinct strategies for sulfonium installation in RiPPs.