Genetic encoding of multiple distinct noncanonical amino acids

The site-specific incorporation of noncanonical amino acids (ncAAs) has revolutionized protein research. However, synthesizing multifunctional biopolymers demands moving beyond single-type ncAA incorporation to simultaneously encoding multiple distinct ncAAs. This review summarizes transformative progress in the genetic encoding of multiple distinct ncAAs, including the development of mutually orthogonal translation systems and the expansion of codon repertoires. We highlight recent breakthroughs that have achieved the incorporation of multiple distinct ncAAs. Notably, integrating rare codon recoding with stop codon suppression in mammalian systems has recently enabled the genetic encoding of five distinct ncAAs. These innovations establish a robust platform for advanced biosensors, next-generation therapeutics, and synthetic biology with expanded chemical repertoires.