Expression and in Vitro Reconstitution of Novel Lasso Peptide Gene Clusters

Lasso peptides are members of a class of small molecules called ribosomally synthesized post-translationally modified peptides (or RiPPs) characterized by a unique three-dimensional structure. Lasso peptides are defined by a single isopeptide bond formed between the N-terminus and an acidic side chain, forming a ring. The remaining C-terminus of the peptide is threaded through this ring, forming the characteristic lariat shape. Recent research has shown that potential lasso peptide clusters are widespread in a range of genomes, but the set of isolated lasso peptides is more limited. A set of gene clusters were found through genome mining with peptides predicted as more hydrophobic than known examples and with previously unobserved N-terminal residues. These clusters were heterologously expressed and the properties of the products were investigated. In addition, the associated maturation proteins for each cluster were expressed and purified. Using purified components, in vitro experiments were performed and mature peptides were identified by LC-MS, confirming the activity of the proteins. The in vitro reconstituted system also allowed quantification of the activity of the enzymes in a manner that was not possible through heterologous expression of clusters and enables future studies to further understand the details of lasso peptide production.