Abstract
In this study, we demonstrate the feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl–tRNA synthetase and tRNA (TrpRS–tRNATrp) pair. This was made possible by first functionally replacing this endogenous pair with an E. coli–optimized counterpart from Saccharomyces cerevisiae, and then reintroducing the liberated E. coli TrpRS–tRNATrp pair into the resulting strain as a nonsense suppressor, which was then followed by its directed evolution to genetically encode several new unnatural amino acids (UAAs). These engineered TrpRS–tRNATrp variants were also able to drive efficient UAA mutagenesis in mammalian cells. Since bacteria-derived aminoacyl–tRNA synthetase (aaRS)–tRNA pairs are typically orthogonal in eukaryotes, our work provides a general strategy to develop additional aaRS–tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes.
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Acknowledgements
The EcNR1 strain was a kind gift from G.M. Church (Harvard). A.C. is thankful for support from the Richard and Susan Smith Family Foundation, Newton, MA.
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J.S.I. designed the experiments, performed strain development/characterization, cloning/mutagenesis, directed evolution, and protein expression, and wrote the manuscript; P.S.A. synthesized 5AzW and 5PrW; C.J.J.W. performed cloning/mutagenesis, directed evolution, and protein expression; L.A.C. performed the digestion/MS analysis of reporter proteins; M.J.L. assisted with the design of the recombination experiments; Y.Z. assisted with the protein expression in mammalian cells; A.C. conceived the idea, designed the experiments, and wrote the manuscript.
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Supplementary information
Supplementary Text and Figures
Supplementary Results, Supplementary Figures 1–13 and Supplementary Tables 1–3. (PDF 2035 kb)
Supplementary Note 1
Synthesis of 5PrW and associated characterization data. (PDF 727 kb)
Supplementary Note 2
Sequences for plasmids and other gene cassettes. (PDF 1690 kb)
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Italia, J., Addy, P., Wrobel, C. et al. An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes. Nat Chem Biol 13, 446–450 (2017). https://doi.org/10.1038/nchembio.2312
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DOI: https://doi.org/10.1038/nchembio.2312
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