Orthogonal (O) ribosome-mediated translation of O-mRNAs enables the incorporation of up to three distinct non-canonical amino acids (ncAAs) into proteins in Escherichia coli (E. coli). However, the general and efficient incorporation of multiple distinct ncAAs by O-ribosomes requires scalable strategies for both creating efficiently and specifically translated O-mRNAs, and the compact expression of multiple O-aminoacyl-tRNA synthetase (O-aaRS)/O-tRNA pairs. We automate the discovery of O-mRNAs that lead to up to 40 times more protein, and are up to 50-fold more orthogonal, than previous O-mRNAs; protein yields from our O-mRNAs match or exceed those from wild-type mRNAs. These advances enable a 33-fold increase in yield for incorporating three distinct ncAAs. We automate the creation of operons for O-tRNA genes, and develop operons for O-aaRS genes. Combining our advances creates a 68-codon, 24-amino-acid genetic code to efficiently incorporate four distinct ncAAs into a single protein in response to four distinct quadruplet codons.
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The code for the O-mRNA design method and the tRNA operon designer are available at https://www2.mrc-lmb.cam.ac.uk/research/technology-transfer/chinlab.
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This work was supported by the UK Medical Research Council (MRC; MC_U105181009 and MC_UP_A024_1008) and an ERC Advanced Grant SGCR (all to J.W.C.). D.L.D. and S.B.O. were supported by the Boehringer Ingelheim Fonds. We thank M. Skehel at the MRC-LMB mass spectrometry facility for performing mass spectrometry.
The authors declare no competing interests.
Peer review information Nature Chemistry thanks the anonymous reviewers for their contribution to the peer review of this work.
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Dunkelmann, D.L., Oehm, S.B., Beattie, A.T. et al. A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. Nat. Chem. 13, 1110–1117 (2021). https://doi.org/10.1038/s41557-021-00764-5