Biosynthesis of Collinodin Unveils Iterative Oxidative and Prenylation Modifications

Lasso peptides are a unique family belonging to ribosomally synthesized and post-translationally modified peptides (RiPPs), characterized by unique isopeptide bonds and knotted structures. In recent years, genome mining for RiPPs has significantly expanded the diversity of post-translational modification chemistries. However, lasso peptides with multiple modifications are rarely reported. In this study, we used genome mining tools to identify a lasso peptide biosynthetic gene cluster that encodes unusual tailoring enzymes, including an α-KG-dependent dioxygenase and a prenyltransferase. Through heterologous expression, we successfully produced a lasso peptide with prenylation modification and elucidated its biosynthetic pathway in detail through in vivo and in vitro experiments. We identified a rare α-KG-dependent dioxygenase that acts twice during the maturation of the lasso peptide. Additionally, we verified the function of the prenyltransferase and its substrate broad specificity through in vitro experiments.