De novo design of ribosomally synthesized and post-translationally modified peptides

In nature, peptides are enzymatically modified to constrain their structure and introduce functional moieties. De novo peptide structures could be built by combining enzymes from different pathways, but determining the rules of their use is difficult. We present a biophysical model to combine enzymes sourced from bacterial ribosomally synthesized and post-translationally modified peptide (RiPP) gene clusters. Using a pipeline to evaluate more than 1,000 peptides, the model was parameterized under uniform conditions in Escherichia coli for enzymes from different classes (graspetide, spliceotide, pantocin, cyanobactin, glycocin, lasso peptide and lanthipeptide). Synthetic leader peptides with recognition sequences for up to three enzymes were designed to modify core sequences sharing no identity to natural RiPPs. Empirically, RiPPs with the desired modifications constituted 7–67% of the total peptides produced, and 6 of our 8 peptide designs were successfully modified. This work is an example of the design of enzyme-modified peptides and libraries, using a framework that can be expanded to include new enzymes and chemical moieties.