EF-P Inhibits Ribosomal α-Hydroxy Acid Incorporation: Strategic tRNA Body Selection for Co-incorporating α-Hydroxy Acids and Nonproteinogenic Amino Acids into Depsipeptides

Genetic code reprogramming has enabled the ribosomal incorporation of diverse nonproteinogenic amino acids (npAAs) into nascent peptide chains. While backbone-altering npAAs—such as β-amino, γ-amino, D-amino, and N-methylamino acids—are typically poor substrates, the development of engineered tRNAs, notably tRNA Pro1E2 , has facilitated their efficient incorporation. tRNA Pro1E2 possesses a distinct D-arm motif recognized by the translation factor EF-P, which enhances peptide bond formation with these npAAs. α-Hydroxy acids can also be ribosomally incorporated to form ester bonds, serving as valuable building blocks for novel bioactive depsipeptides. In this study, α-hydroxy acid incorporation was unexpectedly found to be significantly inhibited by EF-P when charged onto EF-P-responsive tRNAs, such as tRNA Pro1 and tRNA Pro1E2 . This inhibition was effectively disrupted by a single mutation in the D-arm motif. To enable the efficient co-incorporation of α-hydroxy acids and other backbone-altering npAAs (e.g., β- and D-amino acids) in a single translation, a dual-tRNA strategy was employed: α-hydroxy acids were charged onto an EF-P-insensitive tRNA (tRNA AsnE2 ), while β-/D-amino acids were charged onto tRNA Pro1E2 . Using this approach, a model depsipeptide containing two α-hydroxy acids, one β-amino acid, and one D-amino acid was successfully synthesized in an EF-P-dependent manner.