Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs

Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/^PyltRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/Mm^PyltRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/Mm^PyltRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/^PyltRNA_CUA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/^PyltRNA pairs that are mutually orthogonal to the MmPylRS/Mm^PyltRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/^PyltRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.