Towards a Cell-Free System for the Discovery of Post-Translationally Modified Peptide Natural Products

The extraordinary chemical diversity of natural products holds the answer to many current questions in medicine and energy. Unfortunately, many valuable small molecule natural products are produced in small quantities or under unknown conditions in their native hosts. Cell-free based systems are tools that can be used to discover and probe novel biochemistries and low abundance natural products because limitations due to resource competition from endogenous pathways, concerns of cell viability, toxic products, cofactor requirements, and genetic tractability are minimized. Furthermore, cell-free extracts produced from diverse microbes with in vivo and in vitro enrichment techniques can add additional functionalities that cannot be achieved by use of a single cellular system. In brief, we demonstrate the cell-free production of a ribosomally-synthesized and post-translationally modified peptide antimicrobial lantibiotic, duramycin. Cell-free extracts enriched with tailoring enzymes, CinMXN, were generated and used to express and modify precursor peptide, DurA, in one-pot, to yield a functional peptide antimicrobial. Here, we compare the different cell-free methodologies and analysis techniques that can be employed for facile discovery and development of pathways to high-value, small molecule products.