A cell-free workflow for detecting and characterizing RiPP recognition element-precursor peptide interactions

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a promising class of new therapeutics and antimicrobials. Unfortunately, RiPP discovery efforts are hampered by low-throughput methods for characterizing RiPP recognition elements (RRE), which direct tailoring enzymes to their peptide substrates for RiPP maturation. To address this bottleneck, we report a high-throughput, cell-free workflow for parallelized expression and assaying of RREs with their associated precursor peptide substrates in a process that takes hours instead of weeks. We show the utility of our platform by rapidly scanning precursor peptide sequences for residues important for RRE binding, comprehensively mapping essential residues for RRE binding, and engineering peptides with synthetic RRE recognition sites. We also test 72 computationally predicted lasso peptide RRE and precursor peptide pairs for binding activity, enabling the discovery of a class II lasso peptide. We anticipate that our cell-free workflow will provide a tool for discovering, understanding, and engineering RiPPs.