Engineering highly functional thermostable proteins using ancestral sequence reconstruction


Commercial biocatalysis requires robust enzymes that can withstand elevated temperatures and long incubations. Ancestral reconstruction has shown that pre-Cambrian enzymes were often much more thermostable than extant forms. Here, we resurrect ancestral enzymes that withstand ~30 °C higher temperatures and ≥100 times longer incubations than their extant forms. This is demonstrated on animal cytochromes P450 that stereo- and regioselectively functionalize unactivated C–H bonds for the synthesis of valuable chemicals, and bacterial ketol-acid reductoisomerases that are used to make butanol-based biofuels. The vertebrate CYP3 P450 ancestor showed a 60T50 of 66 °C and enhanced solvent tolerance compared with the human drug-metabolizing CYP3A4, yet comparable activity towards a similarly broad range of substrates. The ancestral ketol-acid reductoisomerase showed an eight-fold higher specific activity than the cognate Escherichia coli form at 25 °C, which increased 3.5-fold at 50 °C. Thus, thermostable proteins can be devised using sequence data alone from even recent ancestors.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Ancestral reconstruction of the CYP3 family reveals a thermostable monooxygenase.
Fig. 2: The ancestral CYP3 shows comparable substrate and ligand-binding promiscuity to the major human drug-metabolizing P450, CYP3A4.
Fig. 3: Strategy for CLADE using site-directed mutagenesis.
Fig. 4: The ancestral enzyme shows increased tolerance to organic solvents.
Fig. 5: Ancestral reconstruction generates a thermostable and more active KARI.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.


  1. 1.

    Giver, L., Gershenson, A., Freskgard, P. O. & Arnold, F. H. Directed evolution of a thermostable esterase. Proc. Natl Acad. Sci. USA 95, 12809–12813 (1998).

    CAS  Article  Google Scholar 

  2. 2.

    Lehmann, M., Pasamontes, L., Lassen, S. F. & Wyss, M. The consensus concept for thermostability engineering of proteins. Biochim. Biophys. Acta 1543, 408–415 (2000).

    CAS  Article  Google Scholar 

  3. 3.

    Salazar, O., Cirino, P. C. & Arnold, F. H. Thermostabilization of a cytochrome P450 peroxygenase. ChemBioChem 4, 891–893 (2003).

    CAS  Article  Google Scholar 

  4. 4.

    Kim, J. H. et al. Enhanced thermostability and tolerance of high substrate concentration of an esterase by directed evolution. J. Mol. Catal. B Enzym. 27, 169–175 (2004).

    CAS  Article  Google Scholar 

  5. 5.

    Eiben, S., Bartelmas, H. & Urlacher, V. B. Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents. Appl. Microbiol. Biotechnol. 75, 1055–1061 (2007).

    CAS  Article  Google Scholar 

  6. 6.

    Li, Y. G. et al. A diverse family of thermostable cytochrome P450s created by recombination of stabilizing fragments. Nat. Biotechnol. 25, 1051–1056 (2007).

    CAS  Article  Google Scholar 

  7. 7.

    Kumar, S., Sun, L., Liu, H., Muralidhara, B. K. & Halpert, J. R. Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide. Protein Eng. Des. Sel. 19, 547–554 (2006).

    CAS  Article  Google Scholar 

  8. 8.

    Watanabe, K., Ohkuri, T., Yokobori, S. & Yamagishi, A. Designing thermostable proteins: ancestral mutants of 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree. J. Mol. Biol. 355, 664–674 (2006).

    CAS  Article  Google Scholar 

  9. 9.

    Heinzelman, P. et al. A family of thermostable fungal cellulases created by structure-guided recombination. Proc. Natl Acad. Sci. USA 106, 5610–5615 (2009).

    CAS  Article  Google Scholar 

  10. 10.

    Cole, M. F. & Gaucher, E. A. Utilizing natural diversity to evolve protein function: applications towards thermostability. Curr. Opin. Chem. Biol. 15, 399–406 (2011).

    CAS  Article  Google Scholar 

  11. 11.

    Khersonsky, O. et al. Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59. Proc. Natl Acad. Sci. USA 109, 10358–10363 (2012).

    CAS  Article  Google Scholar 

  12. 12.

    Gaucher, E. A., Thomson, J. M., Burgan, M. F. & Benner, S. A. Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins. Nature 425, 285–288 (2003).

    CAS  Article  Google Scholar 

  13. 13.

    Cole, M. F. & Gaucher, E. A. Exploiting models of molecular evolution to efficiently direct protein engineering. J. Mol. Evol. 72, 193–203 (2011).

    CAS  Article  Google Scholar 

  14. 14.

    Hartwig, J. F. Evolution of C–H bond functionalization from methane to methodology. J. Am. Chem. Soc. 138, 2–24 (2016).

    CAS  Article  Google Scholar 

  15. 15.

    Urlacher, V. B. & Girhard, M. Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol. 30, 26–36 (2012).

    CAS  Article  Google Scholar 

  16. 16.

    Romero, P. A., Krause, A. & Arnold, F. H. Navigating the protein fitness landscape with Gaussian processes. Proc. Natl Acad. Sci. USA 110, E193–E201 (2013).

    CAS  Article  Google Scholar 

  17. 17.

    Abécassis, V., Pompon, D. & Truan, G. High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Res. 28, e88 (2000).

    Article  Google Scholar 

  18. 18.

    Crameri, A., Raillard, S. A., Bermudez, E. & Stemmer, W. P. C. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288–291 (1998).

    CAS  Article  Google Scholar 

  19. 19.

    Voigt, C. A., Martinez, C., Wang, Z. G., Mayo, S. L. & Arnold, F. H. Protein building blocks preserved by recombination. Nat. Struct. Biol. 9, 553–558 (2002).

    CAS  PubMed  Google Scholar 

  20. 20.

    Rendic, S. & Guengerich, F. P. Survey of human oxidoreductases and cytochrome P450 enzymes involved in the metabolism of xenobiotic and natural chemicals. Chem. Res. Toxicol. 28, 38–42 (2015).

    CAS  Article  Google Scholar 

  21. 21.

    Guterl, J. K. et al. Cell-free metabolic engineering: production of chemicals by minimized reaction cascades. ChemSusChem 5, 2165–2172 (2012).

    CAS  Article  Google Scholar 

  22. 22.

    Tadrowski, S. et al. Metal ions play an essential catalytic role in the mechanism of ketol-acid reductoisomerase. Chemistry 22, 7427–7436 (2016).

    CAS  Article  Google Scholar 

  23. 23.

    Gillam, E. M. J., Baba, T., Kim, B.-R., Ohmori, S. & Guengerich, F. P. Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. Arch. Biochem. Biophys. 305, 123–131 (1993).

    CAS  Article  Google Scholar 

  24. 24.

    Wong, L. L. P450BM3 on steroids: the Swiss army knife P450 enzyme just gets better. ChemBioChem 12, 2537–2539 (2011).

    CAS  Article  Google Scholar 

  25. 25.

    Romero, P. A. & Arnold, F. H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).

    CAS  Article  Google Scholar 

  26. 26.

    Malcolm, B. A., Wilson, K. P., Matthews, B. W., Kirsch, J. F. & Wilson, A. C. Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing. Nature 345, 86–89 (1990).

    CAS  Article  Google Scholar 

  27. 27.

    Yamashiro, K., Yokobori, S., Koikeda, S. & Yamagishi, A. Improvement of Bacillus circulans beta-amylase activity attained using the ancestral mutation method. Protein Eng. Des. Sel. 23, 519–528 (2010).

    CAS  Article  Google Scholar 

  28. 28.

    Romero-Romero, M. L. et al. Selection for protein kinetic stability connects denaturation temperatures to organismal temperatures and provides clues to archaean life. PLoS ONE 11, e0156657 (2016).

    Article  Google Scholar 

  29. 29.

    Devamani, T. et al. Catalytic promiscuity of ancestral esterases and hydroxynitrile lyases. J. Am. Chem. Soc. 138, 1046–1056 (2016).

    CAS  Article  Google Scholar 

  30. 30.

    Alcolombri, U., Elias, M. & Tawfik, D. S. Directed evolution of sulfotransferases and paraoxonases by ancestral libraries. J. Mol. Biol. 411, 837–853 (2011).

    CAS  Article  Google Scholar 

  31. 31.

    Battistuzzi, F. U., Feijao, A. & Hedges, S. B. A genomic timescale of prokaryote evolution: insights into the origin of methanogenesis, phototrophy, and the colonization of land. BMC Evol. Biol. 4, 44 (2004).

    Article  Google Scholar 

  32. 32.

    Gaucher, E. A., Govindarajan, S. & Ganesh, O. K. Palaeotemperature trend for Precambrian life inferred from resurrected proteins. Nature 451, 704–707 (2008).

    CAS  Article  Google Scholar 

  33. 33.

    Perez-Jimenez, R. et al. Single-molecule paleoenzymology probes the chemistry of resurrected enzymes. Nat. Struct. Mol. Biol. 18, 592–596 (2011).

    CAS  Article  Google Scholar 

  34. 34.

    Risso, V. A., Gavira, J. A., Mejia-Carmona, D. F., Gaucher, E. A. & Sanchez-Ruiz, J. M. Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian beta-lactamases. J. Am. Chem. Soc. 135, 2899–2902 (2013).

    CAS  Article  Google Scholar 

  35. 35.

    Nguyen, V. et al. Evolutionary drivers of thermoadaptation in enzyme catalysis. Science 355, 289–293 (2017).

    CAS  Article  Google Scholar 

  36. 36.

    Akanuma, S. et al. Experimental evidence for the thermophilicity of ancestral life. Proc. Natl Acad. Sci. USA 110, 11067–11072 (2013).

    CAS  Article  Google Scholar 

  37. 37.

    Williams, P. D., Pollock, D. D., Blackburne, B. P. & Goldstein, R. A. Assessing the accuracy of ancestral protein reconstruction methods. PLoS Comput. Biol. 2, e69 (2006).

    Article  Google Scholar 

  38. 38.

    Trudeau, D. L., Kaltenbach, M. & Tawfik, D. S. On the potential origins of the high stability of reconstructed ancestral proteins. Mol. Biol. Evol. 33, 2633–2641 (2016).

    CAS  Article  Google Scholar 

  39. 39.

    Tartese, R., Chaussidon, M., Gurenko, A., Delarue, F. & Robert, F. Warm Archean oceans reconstructed from oxygen isotope composition of early-life remnants. Geochem. Perspect. Lett. 3, 55–65 (2017).

    Article  Google Scholar 

  40. 40.

    Hao, J. J. & Berry, A. A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents. Protein Eng. Des. Sel. 17, 689–697 (2004).

    CAS  Article  Google Scholar 

  41. 41.

    Stepankova, V. et al. Strategies for stabilization of enzymes in organic solvents. ACS Catal. 3, 2823–2836 (2013).

    CAS  Article  Google Scholar 

  42. 42.

    Zakas, P. M. et al. Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction. Nat. Biotechnol. 35, 35–37 (2017).

    CAS  Article  Google Scholar 

  43. 43.

    Zhang, Y. H. P., Myung, S., You, C., Zhu, Z. G. & Rollin, J. A. Toward low-cost biomanufacturing through in vitro synthetic biology: bottom-up design. J. Mater. Chem. 21, 18877–18886 (2011).

    CAS  Article  Google Scholar 

  44. 44.

    Paddon, C. J. et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528–532 (2013).

    CAS  Article  Google Scholar 

  45. 45.

    Ba, L. N., Li, P., Zhang, H., Duan, Y. & Lin, Z. L. Engineering of a hybrid biotransformation system for cytochrome P450sca-2 in Escherichia coli. Biotechnol. J. 8, 785–793 (2013).

    CAS  Article  Google Scholar 

  46. 46.

    Brinkmann-Chen, S. et al. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH. Proc. Natl Acad. Sci. USA 110, 10946–10951 (2013).

    CAS  Article  Google Scholar 

  47. 47.

    Lv, Y. et al. Crystal structure of Mycobacterium tuberculosis ketol-acid reductoisomerase at 1.0 Å resolution—a potential target for anti-tuberculosis drug discovery. FEBS J. 283, 1–13 (2016).

    Article  Google Scholar 

  48. 48.

    Reisse, S., Garbe, D. & Bruck, T. Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis. Biochimie 108, 76–84 (2015).

    CAS  Article  Google Scholar 

  49. 49.

    Eng, W. S., Keough, D. T., Hockova, D., Winzor, D. J. & Guddat, L. W. Oligomeric state of hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis. Biochimie 135, 6–14 (2017).

    CAS  Article  Google Scholar 

  50. 50.

    Perdigao, N. et al. Unexpected features of the dark proteome. Proc. Natl Acad. Sci. USA 112, 15898–15903 (2015).

    CAS  Article  Google Scholar 

  51. 51.

    Gillam, E. M. J., Guo, Z. Y., Martin, M. V., Jenkins, C. M. & Guengerich, F. P. Expression of cytochrome P450 2D6 in Escherichia coli: purification, and spectral and catalytic characterization. Arch. Biochem. Biophys. 319, 540–550 (1995).

    CAS  Article  Google Scholar 

  52. 52.

    Parikh, A., Gillam, E. M. J. & Guengerich, F. P. Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat. Biotechnol. 15, 784–788 (1997).

    CAS  Article  Google Scholar 

  53. 53.

    Kinobe, R. T., Parkinson, O. T., Mitchell, D. J. & Gillam, E. M. P450 2C18 catalyzes the metabolic bioactivation of phenytoin. Chem. Res. Toxicol. 18, 1868–1875 (2005).

    CAS  Article  Google Scholar 

  54. 54.

    Shukla, A., Huang, W., Depaz, I. M. & Gillam, E. M. J. Membrane integration of recombinant human P450 forms. Xenobiotica 39, 495–507 (2009).

    CAS  Article  Google Scholar 

  55. 55.

    Tyagi, R., Lee, Y. T., Guddat, L. W. & Duggleby, R. G. Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site. FEBS J. 272, 593–602 (2005).

    CAS  Article  Google Scholar 

  56. 56.

    Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).

    CAS  Article  Google Scholar 

  57. 57.

    Qiu, H. et al. CYP3 phylogenomics: evidence for positive selection of CYP3A4 and CYP3A7. Pharmacogenet. Genomics 18, 53–66 (2008).

    CAS  Article  Google Scholar 

  58. 58.

    Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282 (1992).

    CAS  PubMed  Google Scholar 

  59. 59.

    Alix, B., Boubacar, D. A. & Vladimir, M. T-REX: a web server for inferring, validating and visualizing phylogenetic trees and networks. Nucleic Acids Res. 40, W573–W579 (2012).

    CAS  Article  Google Scholar 

  60. 60.

    Gascuel, O. BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol. Biol. Evol. 14, 685–695 (1997).

    CAS  Article  Google Scholar 

  61. 61.

    Pupko, T., Pe’er, I., Shamir, R. & Graur, D. A fast algorithm for joint reconstruction of ancestral amino acid sequences. Mol. Biol. Evol. 17, 890–896 (2000).

    CAS  Article  Google Scholar 

  62. 62.

    Muchmore, D. C., McIntosh, L. P., Russell, C. B., Anderson, D. E. & Dahlquist, F. W. Expression and N-15 labeling of proteins for proton and N-15 nuclear magnetic resonance. Methods Enzymol. 177, 44–73 (1989).

    CAS  Article  Google Scholar 

  63. 63.

    Li, W. Z. & Godzik, A. CD-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 1658–1659 (2006).

    CAS  Article  Google Scholar 

  64. 64.

    Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009).

    CAS  Article  Google Scholar 

  65. 65.

    Yano, J. K. et al. The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-A resolution. J. Biol. Chem. 279, 38091–38094 (2004).

    CAS  Article  Google Scholar 

  66. 66.

    Williams, E. M., Copp, J. N. & Ackerley, D. F. in Directed Evolution Library Creation Vol. 1179, 2nd edn (eds Gillam, E. M. J., Copp, J. N. & Ackerley, D. F.) 83–101 (Humana Press, Totowa, 2014).

  67. 67.

    Notley, L. M., de Wolf, C. J. F., Wunsch, R. M., Lancaster, R. G. & Gillam, E. M. J. Bioactivation of tamoxifen by recombinant human cytochrome P450 enzymes. Chem. Res. Toxicol. 15, 614–622 (2002).

    CAS  Article  Google Scholar 

  68. 68.

    Gillam, E. M. J. et al. Expression of cytochrome P450 3A5 in Escherichia coli: effects of 5′ modification, purification, spectral characterization, reconstitution conditions, and catalytic activities. Arch. Biochem. Biophys. 317, 374–384 (1995).

    CAS  Article  Google Scholar 

  69. 69.

    Rawal, S., Yip, S. S. M. & Coulombe, R. A. Cloning, expression and functional characterization of cytochrome P450 3A37 from turkey liver with high aflatoxin B-1 epoxidation activity. Chem. Res. Toxicol. 23, 1322–1329 (2010).

    CAS  Article  Google Scholar 

  70. 70.

    Johnston, W. A., Huang, W., Hayes, M. A., De Voss, J. J. & Gillam, E. M. J. Quantitative whole cell cytochrome P450 measurement suitable for high throughput application. J. Biomol. Screen. 13, 135–141 (2008).

    CAS  Article  Google Scholar 

  71. 71.

    Huang, W., Johnston, W. A., Hayes, M. A., De Voss, J. J. & Gillam, E. M. J. A shuffled CYP2C library with a high degree of structural integrity and functional versatility. Arch. Biochem. Biophys. 467, 193–205 (2007).

    CAS  Article  Google Scholar 

  72. 72.

    Johnston, W. A., Huang, W., De Voss, J. J., Hayes, M. A. & Gillam, E. M. J. A shuffled CYP1A library shows both structural integrity and functional diversity. Drug Metab. Dispos. 35, 2177–2185 (2007).

    CAS  Article  Google Scholar 

  73. 73.

    Waxman, D. J. & Chang, T. K. H. in Cytochrome P450 Protocols Vol. 320 (eds Phillips, I. R. & Shephard, E. A.) 153–156 (Humana, Totowa, 2006).

  74. 74.

    Chang, T. K. & Waxman, D. J. in Cytochrome P450 Protocols Vol. 320 (eds Phillips, I. R. & Shephard, E. A.) 85–90 (Humana, Totowa, 2006).

  75. 75.

    Hunter, D. J. B. et al. Facile production of minor metabolites for drug development using a CYP3A shuffled library. Metab. Eng. 13, 682–693 (2011).

    CAS  Article  Google Scholar 

  76. 76.

    Guengerich, F. P. et al. Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem. 261, 5051–5060 (1986).

    CAS  PubMed  Google Scholar 

  77. 77.

    Isin, E. M. & Guengerich, F. P. Substrate binding to cytochromes P450. Anal. Bioanal. Chem. 392, 1019–1030 (2008).

    CAS  Article  Google Scholar 

  78. 78.

    Isin, E. M. & Guengerich, F. P. Kinetics and thermodynamics of ligand binding by cytochrome P450 3A4. J. Biol. Chem. 281, 9127–9136 (2006).

    CAS  Article  Google Scholar 

  79. 79.

    Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).

    CAS  Article  Google Scholar 

  80. 80.

    Gill, S. C. & von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326 (1989).

    CAS  Article  Google Scholar 

Download references


This work was supported by ARC Discovery Project grants DP120101772 and DP160100865, and by AstraZeneca Innovative Medicines and Early Development, Cardiovascular and Metabolic Diseases, Gothenburg, Sweden. The authors are grateful to R. Coulombe, D. Buhler and F. P. Guengerich for donation of complementary DNA used in this work, and to K. Alexandrov and P. Hugenholtz for critical reading of the manuscript.

Author information




E.M.J.G. and Y.G. conceived the project with input from M.B. and L.G. Y.G., J.-M.B., S.-J.W., R.E.S.T., D.J.B.H., K.L.H., S.Z., X.W., B.W., J.B.Y.H.B., E.M.J.G., J.E.S., J.K., E.M.I. and U.J. performed the experiments and/or metabolite analysis. S.A., J.J.D.V., G.S. and M.B. contributed specialist expertise and resources. Y.G., J.-M.B., E.M.J.G., S.-J.W., R.E.S.T., D.J.B.H., K.L.H., E.M.I., U.J., J.B.Y.H.B. and J.K. analysed the results. E.M.J.G. and Y.G. wrote the paper with input from L.G., G.S., S.-J.W., R.E.S.T., J.-M.B., S.A., E.M.I. and U.J.

Corresponding author

Correspondence to Elizabeth M. J. Gillam.

Ethics declarations

Competing interests

Some of the material included here forms the basis of a patent application by Y.G., K.L.H., M.B. and E.M.J.G—Australian Provisional Patent Application #2014905277. The other authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–12 and Supplementary Tables 1–10

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gumulya, Y., Baek, J., Wun, S. et al. Engineering highly functional thermostable proteins using ancestral sequence reconstruction. Nat Catal 1, 878–888 (2018).

Download citation

Further reading