Biosynthesis of Unnatural Cyclodipeptides through Genetic Code Expansion and Cyclodipeptide Synthase Evolution.

Genetic code expansion (GCE) technology has primarily been devoted to the introduction of noncanonical amino acids (ncAAs) into ribosomally synthesized proteins or peptides. Its potential for modifying nonribosomal natural products remains unexplored. In this study, we introduce a novel strategy that integrates GCE with the directed evolution of cyclodipeptide synthase (CDPS) to engineer a new class of CDPSs capable of biosynthesizing cyclodipeptides containing ncAAs. Using this approach, we achieve the efficient incorporation of 4-azido-l-phenylalanine into AlbC-derived cyclodipeptides, generating a diverse array of new-to-nature cyclodipeptides. Molecular dynamics simulations and binding free energy calculations provide insights into the potential catalytic mechanisms responsible for the enhanced recognition of ncAA-tRNAs by engineered CDPS variants. Furthermore, we expand the substrate scope to additional ncAAs and extend this approach to other CDPSs, enabling the creation of an even broader range of novel compounds. Together, these findings reveal the significant potential of GCE technology to precisely engineer biomolecules beyond proteins, unlocking new structural and functional diversity.