Artificial Multidomain Ribosomally Synthesized and Post-translationally Modified Peptide Enzymes for Farnesylated Peptide Library Generation

Cyanobactin biosynthetic pathways are used in synthetic biology approaches to create large, peptide-based chemical libraries with drug-like features such as N-C macrocyclization and prenylation. It remains challenging to express enzymes from multiple RiPP pathways to rationally produce the desired products. Here, we designed a simple yet robust method aimed to produce and assess multiple enzymes, fusing biosynthetic genes together in a well-expressed, soluble construct that enables production of macrocyclic peptides and selectively appends C₅, C₁₀, or C₁₅ isoprenoids to tyrosine side chains. A library was developed and assayed, defining the sequence features necessary for prenylation and providing an overall >40% success rate of using a library with an estimated maximum size of 2.6 million peptide derivatives. This flexible and robust system enables the generation of novel compounds and libraries of such compounds with minimal side products in living organisms.