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

Lasso peptides are a fascinating class of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their unique threaded-ring topology. Longipeptin A, isolated from Longimycelium tulufanense, features a Trp-Trp C-N crosslink, a 5-hydroxytryptophan and a unique trivalent sulfonium S-methylmethionine. The genetic intractability of L. tulufanense hindered mechanistic studies of longipeptin biosynthesis. In this work, we heterologously expressed the biosynthetic gene cluster of longipeptins, defined its boundaries and the essential genes for its biosynthesis, and characterized the catalytic roles and timings of the two cytochromes P450 LopF/LopG and the domain of unknown function 6919 (DUF6919) protein LopH. LopF and LopG are involved in Trp-Trp C-N crosslinking and tryptophan hydroxylation, respectively. LopH acts as a novel methyltransferase for S-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.