Bacterial Display of Genetically Encoded Macrocyclic Peptide Libraries using Cysteine-Reactive Unnatural Amino Acids

Cyclic peptides are powerful scaffolds for modulating protein–protein interactions, yet strategies for constructing cyclic peptide libraries for cell-surface display applications remain limited. Here we present a bacterial surface display platform that enables site-specific incorporation of diverse unnatural amino acids (uAAs) into peptides displayed on the surface of Escherichia coli. These uAAs include several cysteine-reactive residues that undergo spontaneous intramolecular reaction with cysteine to generate macrocyclic peptides directly on the bacterial outer membrane. We show, using high-resolution mass spectrometry, that peptide cyclization proceeds efficiently and selectively without detectable side products. Further, we demonstrate that this platform supports simultaneous incorporation of two distinct uAAs, enabling surface display of chemically diverse, drug-like macrocycles. To demonstrate the utility of this platform, we constructed and screened a naïve macrocyclic peptide library, yielding de novo ligands that contain conserved sequence motifs previously identified by phage display. This work establishes a robust approach for generating diverse cyclic peptide libraries, thereby expanding the scope of cell-surface display for ligand discovery.