Combinatorial RiPP biosynthesis for the generation of gene-encoded new-to-nature peptide antimicrobials

The excessive and inappropriate use of antibiotics has escalated antimicrobial resistance, driving the urgent need for novel antibiotics with innovative structures and modes of action. Non-ribosomal peptides (NRPs), such as the last-resort antibiotics daptomycin and polymyxin, exhibit potent antibacterial activity, but their significant side effects limit their application. Various endeavours have been made to create improved NRP analogues, but with limited success. In contrast, ribosomally synthesized and post-translationally modified peptides (RiPPs) are highly amenable to engineering. As RiPPs encompass much of the chemical diversity found in NRPs, they offer a new avenue for creating structural and functional NRP mimics via ribosomal pathways. In this thesis, we developed multiple novel RiPP combinatorial biosynthesis pathways that combine modifications from diverse RiPP enzymes into a single peptide, yielding such NRP mimics. We devised a chimeric leader that facilitates recognition for modification by three different RiPP modification enzymes, the epimerase OspD, the arginase OspR, and the lanthionine macrocyclase SyncM, resulting in the in vivo production of macrocyclic peptides containing D-amino acids and ornithines. Advancing further, sequences of a wide variety of NRP antibiotics were closely mimicked, successfully generating various NRP mimics with antibacterial activity against both Gram-negative and Gram-positive ESKAPE-pathogens. In addition, we established another hybrid pathway, introducing a β-amino acid and macrocycle in a single product by the ketoamide-installing splicease PlpYX, and SyncM, creating a structural mimic of the anti-cancer NRP aplidin. Overall, this work demonstrates the feasibility of generating gene-encoded NRP-emulating antimicrobial peptides through RiPP biosynthesis, offering promise in addressing antibiotic resistance.