Advances in the Biosynthesis of Noncanonical Amino Acids for Genetic Code Expansion.

Genetic code expansion (GCE) enables the in situ site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. This technology has accelerated the development of next-generation protein therapeutics and expanded the utility of enzymes in advanced biocatalysis. Despite its transformative potential, current GCE technology relies on the cellular uptake of chemically synthesized, exogenously supplied ncAAs. The need for high ncAA concentrations significantly limits the technology's practicality, particularly for in vivo applications, where rapid systemic clearance, low bioavailability, metabolic instability, and poor tissue distribution prevent ncAAs from reaching effective intracellular levels. In this review, we summarize recent advances in fully autonomous ncAA biosynthesis for GCE technology across microbial, zebrafish, and mammalian systems. We discuss how metabolic engineering can be integrated with orthogonal translation machinery to establish self-sufficient GCE platforms, outline key pathway design principles, and examine remaining technical and translational bottlenecks. Finally, we highlight emerging applications of these biosynthetic strategies in biological research and therapeutic development.