[Abstract]

Codon expansion has become a powerful tool for overcoming the limitations of the standard genetic code system, which restricts the building block of proteins to canonical amino acids. The incorporation of non-canonical amino acids (ncAAs) into proteins presents a significant opportunity to expand their functional diversity. The precise incorporation of ncAAs in vivo requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair and a blank codon to assign them. Studies have focused on the biosynthesis of proteins with novel chemical properties alongside biotechnological advancements in codon expansion research. The three principal strategies for codon expansion are: stop codon utilization, quadruplet codon generation, and sense codon compression. Although using stop codons as blank codons remains an effective approach, the need for additional blank codons has expanded research into quadruplet codons and sense-codon compression. This review presents an overview of each strategy by integrating recent advances in research. We discuss the advantages and limitations of these strategies, as well as the challenges encountered. Subsequently, we propose potential approaches to enhance the efficiency and fidelity of ncAA incorporation. The insights presented in this review provide perspectives for future research and facilitate the advancement of codon expansion and its applications in biotechnology.

DOI: 10.1016/j.biortech.2025.132691

LINK: https://www.sciencedirect.com/science/article/pii/S0960852425006571