Since its development in 1985, phage display has proven to be a powerful platform for the selection of peptide ligands. Recently, strategies for the in vitro or in vivo modification of phage-displayed peptides have been developed to allow display and maturation of monocyclic or bicyclic peptides, thereby taking advantage of the increased rigidity and improved resistance to proteolysis demonstrated by such compounds. Ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are an ideal platform for the generation of large peptide libraries containing one or more macrocycles because the primary sequence is genetically encoded and the modification enzymes responsible for macrocycle installation tolerate varied substrates. We have developed and validated a system that allows for assembly on phage of the lanthipeptide nisin, a potent antimicrobial peptide containing 5 post-translationally installed macrocycles. We first demonstrated N-terminal display of nisin. We then generated a library of more than 1 million nisin variants and enriched binders to the small molecule lipid II. We finally characterized the top hit from the selection, revealing both that the modification enzymes accept it as a substrate and that it binds lipid II. Having thus demonstrated the feasibility of N-terminal display of nisin, we expect future work to leverage this platform for display and optimization of a wide array of RiPP natural products. Support or Funding Information This work was supported by the National Institutes of Health (R37 GM 058822 to W.A.v.d.D and F31 GM113486 to K.J.H). The Bruker UltrafleXtreme MALDI TOFTOF mass spectrometer was purchased in part with a grant from the National Center for Research Resources, National Institutes of Health (S10 RR027109 A). Development of phage display of the ribosomally synthesized and post-translationally modified peptide (RiPP) nisin. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.