Recoding multiple rare codons enables the simultaneous incorporation of up to five distinct noncanonical amino acids.

Expanding the genetic code has revolutionized our ability to study and manipulate biological systems through site-specific incorporation of noncanonical amino acids (ncAAs). However, current methods are primarily limited to single-type ncAA incorporation in mammalian cells owing to translation inefficiency. Here we introduce a multi-type rare codon recoding strategy that addresses this limitation. By systematically evaluating and repurposing rare codons, alongside engineering mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs, we achieve the expression of proteins containing two or three distinct ncAAs at site-specific positions with recoding rates of up to 90% at wild-type protein expression levels in mammalian cells. This approach facilitates a broad range of applications, including dual bioorthogonal labelling and sequential protein activation. We further demonstrate the utility of this strategy by incorporating up to five distinct ncAAs into a single protein, revealing a redefinable nature of the genetic code and opening unprecedented avenues for future applications in biomedicine and synthetic biology.