A system for the continuous directed evolution of biomolecules


Laboratory evolution has generated many biomolecules with desired properties, but a single round of mutation, gene expression, screening or selection, and replication typically requires days or longer with frequent human intervention1. Because evolutionary success is dependent on the total number of rounds performed2, a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness3. Although researchers have accelerated individual steps in the evolutionary cycle4,5,6,7,8,9, the only previous example of continuous directed evolution was the landmark study of Wright and Joyce10, who continuously evolved RNA ligase ribozymes with an in vitro replication cycle that unfortunately cannot be easily adapted to other biomolecules. Here we describe a system that enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in Escherichia coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle in a manner that is dependent on the activity of interest. Dozens of rounds of evolution can occur in a single day of PACE without human intervention. Using PACE, we evolved T7 RNA polymerase (RNAP) variants that recognize a distinct promoter, initiate transcripts with ATP instead of GTP, and initiate transcripts with CTP. In one example, PACE executed 200 rounds of protein evolution over the course of 8 days. Starting from undetectable activity levels in two of these cases, enzymes with each of the three target activities emerged in less than 1 week of PACE. In all three cases, PACE-evolved polymerase activities exceeded or were comparable to that of the wild-type T7 RNAP on its wild-type promoter, representing improvements of up to several hundred-fold. By greatly accelerating laboratory evolution, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.

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Figure 1: Overview of the PACE system.
Figure 2: Linkage of three protein activities to pIII production and phage infectivity using three distinct accessory plasmids.
Figure 3: Continuous evolution of T7 RNAP variants that recognize the T3 promoter.
Figure 4: Continuous evolution of T7 RNAP variants that initiate transcription with A.


  1. 1

    Yuan, L., Kurek, I., English, J. & Keenan, R. Laboratory-directed protein evolution. Microbiol. Mol. Biol. Rev 69, 373–392 (2005)

    CAS  Article  Google Scholar 

  2. 2

    Voigt, C. A., Kauffman, S. & Wang, Z. G. Rational evolutionary design: the theory of in vitro protein evolution. Adv. Protein Chem. 55, 79–160 (2000)

    CAS  Article  Google Scholar 

  3. 3

    Mills, D. R., Peterson, R. L. & Spiegelman, S. An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule. Proc. Natl Acad. Sci. USA 58, 217–224 (1967)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Wang, L., Jackson, W. C., Steinbach, P. A. & Tsien, R. Y. Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl Acad. Sci. USA 101, 16745–16749 (2004)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Camps, M., Naukkarinen, J., Johnson, B. P. & Loeb, L. A. Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I. Proc. Natl Acad. Sci. USA 100, 9727–9732 (2003)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Makeyev, E. V. & Bamford, D. H. Evolutionary potential of an RNA virus. J. Virol. 78, 2114–2120 (2004)

    CAS  Article  Google Scholar 

  7. 7

    Davis, J. N. & van den Pol, A. N. Viral mutagenesis as a means for generating novel proteins. J. Virol. 84, 1625–1630 (2009)

    Article  Google Scholar 

  8. 8

    Das, A. T. et al. Viral evolution as a tool to improve the tetracycline-regulated gene expression system. J. Biol. Chem. 279, 18776–18782 (2004)

    CAS  Article  Google Scholar 

  9. 9

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

    ADS  CAS  Article  Google Scholar 

  10. 10

    Wright, M. C. & Joyce, G. F. Continuous in vitro evolution of catalytic function. Science 276, 614–617 (1997)

    CAS  Article  Google Scholar 

  11. 11

    Husimi, Y. Selection and evolution of bacteriophages in cellstat. Adv. Biophys. 25, 1–43 (1989)

    CAS  Article  Google Scholar 

  12. 12

    Smith, G. P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317 (1985)

    ADS  CAS  Article  Google Scholar 

  13. 13

    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  Article  Google Scholar 

  14. 14

    Nelson, F. K., Friedman, S. M. & Smith, G. P. Filamentous phage DNA cloning vectors: a noninfective mutant with a nonpolar deletion in gene III. Virology 108, 338–350 (1981)

    CAS  Article  Google Scholar 

  15. 15

    Rakonjac, J. & Model, P. Roles of pIII in filamentous phage assembly. J. Mol. Biol. 282, 25–41 (1998)

    CAS  Article  Google Scholar 

  16. 16

    Calendar, R. The Bacteriophages (Oxford Univ. Press, 2006)

    Google Scholar 

  17. 17

    Vidal, M. & Legrain, P. Yeast forward and reverse ‘n’-hybrid systems. Nucleic Acids Res. 27, 919–929 (1999)

    CAS  Article  Google Scholar 

  18. 18

    Baker, K. et al. Chemical complementation: a reaction-independent genetic assay for enzyme catalysis. Proc. Natl Acad. Sci. USA 99, 16537–16542 (2002)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Fijalkowska, I. J. & Schaaper, R. M. Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe. Proc. Natl Acad. Sci. USA 93, 2856–2861 (1996)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Opperman, T., Murli, S., Smith, B. T. & Walker, G. C. A model for a umuDC-dependent prokaryotic DNA damage checkpoint. Proc. Natl Acad. Sci. USA 96, 9218–9223 (1999)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Raskin, C. A., Diaz, G., Joho, K. & McAllister, W. T. Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity. J. Mol. Biol. 228, 506–515 (1992)

    CAS  Article  Google Scholar 

  22. 22

    Ikeda, R. A., Chang, L. L. & Warshamana, G. S. Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences. Biochemistry 32, 9115–9124 (1993)

    CAS  Article  Google Scholar 

  23. 23

    Raskin, C. A., Diaz, G. A. & McAllister, W. T. T7 RNA polymerase mutants with altered promoter specificities. Proc. Natl Acad. Sci. USA 90, 3147–3151 (1993)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Vidal-Aroca, F. et al. One-step high-throughput assay for quantitative detection of beta-galactosidase activity in intact Gram-negative bacteria, yeast, and mammalian cells. Biotechniques 40, 433–438 (2006)

    CAS  Article  Google Scholar 

  25. 25

    Imburgio, D., Rong, M., Ma, K. & McAllister, W. T. Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Biochemistry 39, 10419–10430 (2000)

    CAS  Article  Google Scholar 

  26. 26

    Brieba, L. G., Padilla, R. & Sousa, R. Role of T7 RNA polymerase His784 in start site selection and initial transcription. Biochemistry 41, 5144–5149 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Kuzmine, I., Gottlieb, P. A. & Martin, C. T. Binding of the priming nucleotide in the initiation of transcription by T7 RNA polymerase. J. Biol. Chem. 278, 2819–2823 (2003)

    CAS  Article  Google Scholar 

  28. 28

    Cheetham, G. M., Jeruzalmi, D. & Steitz, T. A. Structural basis for initiation of transcription from an RNA polymerase-promoter complex. Nature 399, 80–83 (1999)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Martin, C. T. & Coleman, J. E. Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters. Biochemistry 26, 2690–2696 (1987)

    CAS  Article  Google Scholar 

  30. 30

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343–345 (2009)

    CAS  Article  Google Scholar 

  31. 31

    Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97, 6640–6645 (2000)

    ADS  CAS  Article  Google Scholar 

  32. 32

    Ichetovkin, I. E., Abramochkin, G. & Shrader, T. E. Substrate recognition by the leucyl/phenylalanyl-tRNA-protein transferase. Conservation within the enzyme family and localization to the trypsin-resistant domain. J Biol Chem 272, 33009–33014 (1997)

    CAS  Article  Google Scholar 

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This work was supported by National Institutes of Health/National Institute of General Medical Sciences R01 GM065400 and by HHMI. K.M.E. acknowledges graduate research fellowships from the Hertz Foundation and the National Science Foundation. J.C.C. was supported by the Harvard Chemical Biology Graduate Program. We thank B. Dorr for assistance with phage generation modelling, E. Curtis for suggestions and V. D’Souza for plasmid pT7-911Q.

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K.M.E., J.C.C. and D.R.L. designed the experiments. K.M.E. designed and built the PACE apparatus. K.M.E. and J.C.C. performed the experiments. All authors analysed the data and wrote the manuscript.

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Correspondence to David R. Liu.

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The authors declare no competing financial interests.

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Esvelt, K., Carlson, J. & Liu, D. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011). https://doi.org/10.1038/nature09929

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