The <i>Alteromonas macleodii</i> ribosome enables consecutive incorporation of bulky D-amino acids into peptides

Genetic code reprogramming allows for ribosomal incorporation of exotic amino acids, such as d-α- and d-β-amino acids, into peptides. However, their incorporation efficiency remains much lower than that of canonical l-amino acids, making their multiple/consecutive incorporations difficult. The side chain of d-α-amino acids clashes with U2506 of 23S rRNA, hindering the incorporation of d-α-amino acids with bulky side chains; therefore, consecutive incorporation has been limited to small d-α-amino acids. To overcome this limitation, we screened ribosomes from phylogenetically diverse bacterial species to identify variants that enable consecutive incorporation of bulky d-amino acids. The Alteromonas macleodii (AM) ribosome is capable of incorporating d-α-, d-β-, and α,α-disubstituted amino acids with efficiencies superior to the Escherichia coli (EC) ribosome. It elongates six consecutive d-Ser and two consecutive 1-aminocyclobutane-1-carboxylic acid with yields 6.1- and 7.3-fold higher than the EC ribosome. Moreover, consecutive incorporation of nine types of bulky d-α-amino acids (d-Asn, d-Asp, d-Gln, d-Met, d-Phe, d-Thr, d-Trp, d-Tyr, d-Val) was achieved for the first time. A model macrocyclic peptide containing four d-amino acids and one α,α-disubstituted amino acid was also synthesized by the AM ribosome. These results expand the potential of ribosomal synthesis for peptide libraries containing structurally diverse, bulky d-amino acids.