Expanding the genetic code: In vivo approaches for incorporating non-proteinogenic monomers

The application of genetic code expansion has enabled the incorporation of non-canonical amino acids (ncAAs) into proteins, introducing novel functional groups and significantly broadening the scope of protein engineering. Over the past decade, this approach has extended beyond ncAAs to include non-proteinogenic monomers (npMs), such as β-amino acids and hydroxy acids. In vivo incorporation of these monomers requires maintaining orthogonality between endogenous and engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs while optimizing the use of the translational machinery. This review introduces the fundamental principles of genetic code expansion and highlights the development of orthogonal aaRS/tRNA pairs and ribosomal engineering to incorporate npMs. Despite these advancements, challenges remain in engineering aaRS/tRNA pairs to accommodate npMs, especially monomers that differ significantly from L-α-amino acids due to their incompatibility with existing translational machinery. This review also introduces recent methodologies that allow aaRSs to recognize and aminoacylate npMs without reliance on the ribosomal translation system, thereby unlocking new possibilities for synthesizing biopolymers with chemically diverse monomers.