Enhancing ribosomal translation of backbone-altering nonproteinogenic amino acids via YebC and YeeN

Development of genetic code reprogramming methodologies has enabled the ribosomal incorporation of diverse backbone-altering nonproteinogenic amino acids (BAAs)-including d-α-amino acids, β-amino acids, and N-methyl-α-amino acids-into nascent peptides. However, their incorporation is far less efficient than that of canonical l-α-amino acids. We previously demonstrated that the ribosomal E-site-binding translation factors, EF-P and ABC-F proteins, can enhance BAA incorporation. These findings motivated us to investigate additional ribosome-binding factors that might further facilitate this process. Here, we focused on two putative A-site-binding factors, YebC and YeeN, to activate A-site BAA-tRNA and facilitate efficient incorporation. When testing 11 BAAs for consecutive incorporation, YebC and YeeN yielded average enhancements of 2.1- and 2.7-fold, respectively, with an increase of up to 6.6-fold in specific cases. Furthermore, combining YebC with EF-P and Uup (a representative ABC-F protein) produced an 8.0-fold increase in the incorporation of two consecutive N-methyl-l-leucine residues, demonstrating a clear synergistic benefit. We also found that YebC and YeeN promote ribosomal synthesis of drug-like macrocyclic peptides enriched with BAAs, such as d-serine, 1-aminocyclobutane-1-carboxylic acid, N-methyl-l-alanine, and β3-homomethionine. These results pave the way for the ribosomal synthesis of diverse macrocyclic peptide libraries and their application in mRNA display-based screening for novel bioactive compounds.