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

Abstract Cyclic peptides are powerful scaffolds for modulating protein–protein interactions; however, methods 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 reactions with cysteine to generate macrocyclic peptides directly on the bacterial outer membrane. Using high-resolution mass spectrometry, we show that peptide cyclization proceeds efficiently and selectively with no detectable off-target reactions. Further, we demonstrate that this platform supports simultaneous incorporation of two distinct uAAs, enabling the surface display of chemically diverse, drug-like macrocycles. To demonstrate the utility of this platform, we constructed and screened a naïve macrocyclic peptide library against a model target, yielding de novo ligands with conserved sequence motifs and binding affinities similar to those previously identified by phage display. Together, these results establish a robust approach for generating diverse cyclic peptide libraries, thereby expanding the scope of cell-surface display for research and drug discovery.