Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Continuous directed evolution of aminoacyl-tRNA synthetases

Nature Chemical Biology volume 13, pages 1253–1260 (2017)Cite this article

Subjects

An Erratum to this article was published on 16 January 2018

This article has been updated

Abstract

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing noncanonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of PACE positive selections for the continuous evolution of AARS activity and the noncanonical amino acids used in this study.
Figure 2: Evolution of AARS activity during mock PACE.
Figure 3: Continuous evolution and characterization of chimeric pyrrolysyl-tRNA synthetase (chPylRS) variants with enhanced aminoacylation activity.
Figure 4: Evolution of AARS variants from dual positive- and negative-selection PACE with greatly improved amino acid specificity.

Accession codes

Accessions

Protein Data Bank

Change history

  • 01 December 2017

    In the version of this article initially published, the label colors for p-NF and p-IF in the key for Figure 4c were transposed. The cyan bars should correspond to p-NF and the fuchsia bars to p-IF. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Liu, C.C. & Schultz, P.G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413–444 (2010).

    CAS  PubMed  Google Scholar 

  2. Wan, W., Tharp, J.M. & Liu, W.R. Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim. Biophys. Acta 1844, 1059–1070 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Chin, J.W. Expanding and reprogramming the genetic code of cells and animals. Annu. Rev. Biochem. 83, 379–408 (2014).

    CAS  PubMed  Google Scholar 

  4. Umehara, T. et al. N-acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo. FEBS Lett. 586, 729–733 (2012).

    CAS  PubMed  Google Scholar 

  5. O'Donoghue, P., Ling, J., Wang, Y.-S. & Söll, D. Upgrading protein synthesis for synthetic biology. Nat. Chem. Biol. 9, 594–598 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Esvelt, K.M., Carlson, J.C. & Liu, D.R. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Leconte, A.M. et al. A population-based experimental model for protein evolution: effects of mutation rate and selection stringency on evolutionary outcomes. Biochemistry 52, 1490–1499 (2013).

    CAS  PubMed  Google Scholar 

  8. Carlson, J.C., Badran, A.H., Guggiana-Nilo, D.A. & Liu, D.R. Negative selection and stringency modulation in phage-assisted continuous evolution. Nat. Chem. Biol. 10, 216–222 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Dickinson, B.C., Packer, M.S., Badran, A.H. & Liu, D.R. A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations. Nat. Commun. 5, 5352 (2014).

    CAS  PubMed  Google Scholar 

  10. Hubbard, B.P. et al. Continuous directed evolution of DNA-binding proteins to improve TALEN specificity. Nat. Methods 12, 939–942 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Badran, A.H. et al. Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance. Nature 533, 58–63 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Riechmann, L. & Holliger, P. The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli. Cell 90, 351–360 (1997).

    CAS  PubMed  Google Scholar 

  13. Yin, Y.W. & Steitz, T.A. Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science 298, 1387–1395 (2002).

    CAS  PubMed  Google Scholar 

  14. Tsao, M.L., Summerer, D., Ryu, Y. & Schultz, P.G. The genetic incorporation of a distance probe into proteins in Escherichia coli. J. Am. Chem. Soc. 128, 4572–4573 (2006).

    CAS  PubMed  Google Scholar 

  15. Grünewald, J. et al. Immunochemical termination of self-tolerance. Proc. Natl. Acad. Sci. USA 105, 11276–11280 (2008).

    PubMed  PubMed Central  Google Scholar 

  16. Yanagisawa, T. et al. Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode Nε-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem. Biol. 15, 1187–1197 (2008).

    CAS  PubMed  Google Scholar 

  17. Lubkowski, J., Hennecke, F., Plückthun, A. & Wlodawer, A. The structural basis of phage display elucidated by the crystal structure of the N-terminal domains of g3p. Nat. Struct. Biol. 5, 140–147 (1998).

    CAS  PubMed  Google Scholar 

  18. Lubkowski, J., Hennecke, F., Plückthun, A. & Wlodawer, A. Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA. Structure 7, 711–722 (1999).

    CAS  PubMed  Google Scholar 

  19. Deng, L.W. & Perham, R.N. Delineating the site of interaction on the pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli. J. Mol. Biol. 319, 603–614 (2002).

    CAS  PubMed  Google Scholar 

  20. Boeke, J.D. & Model, P. A prokaryotic membrane anchor sequence: carboxyl terminus of bacteriophage f1 gene III protein retains it in the membrane. Proc. Natl. Acad. Sci. USA 79, 5200–5204 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Boeke, J.D., Model, P. & Zinder, N.D. Effects of bacteriophage f1 gene III protein on the host cell membrane. Mol. Gen. Genet. 186, 185–192 (1982).

    CAS  PubMed  Google Scholar 

  22. Rapoza, M.P. & Webster, R.E. The filamentous bacteriophage assembly proteins require the bacterial SecA protein for correct localization to the membrane. J. Bacteriol. 175, 1856–1859 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Badran, A.H. & Liu, D.R. Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Nat. Commun. 6, 8425 (2015).

    CAS  PubMed  Google Scholar 

  24. Guo, L.T. et al. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution. Proc. Natl. Acad. Sci. USA 111, 16724–16729 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kavran, J.M. et al. Structure of pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic code innovation. Proc. Natl. Acad. Sci. USA 104, 11268–11273 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Wong, M.L., Guzei, I.A. & Kiessling, L.L. An asymmetric synthesis of l-pyrrolysine. Org. Lett. 14, 1378–1381 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Jiang, R. & Krzycki, J.A. PylSn and the homologous N-terminal domain of pyrrolysyl-tRNA synthetase bind the tRNA that is essential for the genetic encoding of pyrrolysine. J. Biol. Chem. 287, 32738–32746 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Herring, S. et al. The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity. FEBS Lett. 581, 3197–3203 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Neumann, H. et al. A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation. Mol. Cell 36, 153–163 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Neumann, H., Peak-Chew, S.Y. & Chin, J.W. Genetically encoding Nε-acetyllysine in recombinant proteins. Nat. Chem. Biol. 4, 232–234 (2008).

    CAS  PubMed  Google Scholar 

  31. Ho, J.M. et al. Efficient reassignment of a frequent serine codon in wild-type Escherichia coli. ACS Synth. Biol. 5, 163–171 (2016).

    CAS  PubMed  Google Scholar 

  32. Adhin, M.R. & van Duin, J. Scanning model for translational reinitiation in eubacteria. J. Mol. Biol. 213, 811–818 (1990).

    CAS  PubMed  Google Scholar 

  33. Andrè, A. et al. Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis-elements unrelated to canonical translation initiation signals. FEBS Lett. 468, 73–78 (2000).

    PubMed  Google Scholar 

  34. Cabantous, S., Rogers, Y., Terwilliger, T.C. & Waldo, G.S. New molecular reporters for rapid protein folding assays. PLoS One 3, e2387 (2008).

    PubMed  PubMed Central  Google Scholar 

  35. Nozawa, K. et al. Pyrrolysyl-tRNA synthetase-tRNA(Pyl) structure reveals the molecular basis of orthogonality. Nature 457, 1163–1167 (2009).

    CAS  PubMed  Google Scholar 

  36. Xie, J. et al. The site-specific incorporation of p-iodo-l-phenylalanine into proteins for structure determination. Nat. Biotechnol. 22, 1297–1301 (2004).

    CAS  PubMed  Google Scholar 

  37. Turner, J.M., Graziano, J., Spraggon, G. & Schultz, P.G. Structural plasticity of an aminoacyl-tRNA synthetase active site. Proc. Natl. Acad. Sci. USA 103, 6483–6488 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Yanagisawa, T. et al. Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase. J. Mol. Biol. 378, 634–652 (2008).

    CAS  PubMed  Google Scholar 

  39. Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Amiram, M. et al. Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat. Biotechnol. 33, 1272–1279 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Lajoie, M.J. et al. Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Schmied, W.H., Elsässer, S.J., Uttamapinant, C. & Chin, J.W. Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1. J. Am. Chem. Soc. 136, 15577–15583 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Fan, C., Xiong, H., Reynolds, N.M. & Söll, D. Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids. Nucleic Acids Res. 43, e156 (2015).

    PubMed  PubMed Central  Google Scholar 

  44. Young, T.S., Ahmad, I., Yin, J.A. & Schultz, P.G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395, 361–374 (2010).

    CAS  PubMed  Google Scholar 

  45. Guo, J., Melançon, C.E. III, Lee, H.S., Groff, D. & Schultz, P.G. Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids. Angew. Chem. Int. Edn. Engl. 48, 9148–9151 (2009).

    CAS  Google Scholar 

  46. Li, Z. et al. Nitrilase-activatable noncanonical amino acid precursors for cell-selective metabolic labeling of proteomes. ACS Chem. Biol. 11, 3273–3277 (2016).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank S. Trauger at the Small Molecule Mass Spectrometry Laboratory at Harvard University for providing expertise with intact protein mass spectrometry analysis. This work was supported by the Defense Advanced Research Projects Agency N66001-12-C-4207 (D.R.L.), the US National Institutes of Health (NIH) R01 EB022376 (D.R.L.), R35 GM118062 (D.R.L.), R21 AI119813 (C.F.), R01 GM022854 (D.S.), and R35 GM122560 (D.S.), the Department of Energy FG02-98ER2031 (D.S.), and the Howard Hughes Medical Institute (D.R.L.). D.I.B. is supported by a Ruth L. Kirschstein National Research Service Award (F32 GM106621).

Author information

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    David I Bryson & David R Liu

  2. Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas, USA

    Chenguang Fan

  3. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Li-Tao Guo, Corwin Miller & Dieter Söll

  4. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    Dieter Söll

  5. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    David R Liu

  6. Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA

    David R Liu

Authors
  1. David I Bryson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chenguang Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Li-Tao Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Corwin Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dieter Söll
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David R Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.I.B. designed the research, performed experiments, analyzed data, and wrote the manuscript. D.R.L. designed and supervised the research and wrote the manuscript. D.S. designed and supervised the research. C.F. performed protein purification, in vitro aminoacylation assays, aided with in vivo amber suppression assays, and analyzed data. L.-T.G. designed the chimeric chPylRS variant for evolution in PACE, performed protein purification, performed in vitro aminoacylation assays, and analyzed data. C.M. aided in mutation analysis of evolved chPylRS variants from PACE. All authors contributed to editing the manuscript.

Corresponding author

Correspondence to David R Liu.

Ethics declarations

Competing interests

The authors have filed a provisional patent application on the PACE system and related improvements.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–19, Supplementary Tables 1–8 and Supplementary Note 1 (PDF 8892 kb)

Reporting Summary (PDF 129 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bryson, D., Fan, C., Guo, LT. et al. Continuous directed evolution of aminoacyl-tRNA synthetases. Nat Chem Biol 13, 1253–1260 (2017). https://doi.org/10.1038/nchembio.2474

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2474

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing