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Continuous directed evolution of aminoacyl-tRNA synthetases

An Erratum to this article was published on 16 January 2018

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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.

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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.

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  • 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.


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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).

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Authors and Affiliations



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.

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

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

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Bryson, D., Fan, C., Guo, LT. et al. Continuous directed evolution of aminoacyl-tRNA synthetases. Nat Chem Biol 13, 1253–1260 (2017).

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