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.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.
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).
Lehmann, M., Pasamontes, L., Lassen, S. F. & Wyss, M. The consensus concept for thermostability engineering of proteins. Biochim. Biophys. Acta 1543, 408–415 (2000).
Salazar, O., Cirino, P. C. & Arnold, F. H. Thermostabilization of a cytochrome P450 peroxygenase. ChemBioChem 4, 891–893 (2003).
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).
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).
Li, Y. G. et al. A diverse family of thermostable cytochrome P450s created by recombination of stabilizing fragments. Nat. Biotechnol. 25, 1051–1056 (2007).
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).
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).
Heinzelman, P. et al. A family of thermostable fungal cellulases created by structure-guided recombination. Proc. Natl Acad. Sci. USA 106, 5610–5615 (2009).
Cole, M. F. & Gaucher, E. A. Utilizing natural diversity to evolve protein function: applications towards thermostability. Curr. Opin. Chem. Biol. 15, 399–406 (2011).
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).
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).
Cole, M. F. & Gaucher, E. A. Exploiting models of molecular evolution to efficiently direct protein engineering. J. Mol. Evol. 72, 193–203 (2011).
Hartwig, J. F. Evolution of C–H bond functionalization from methane to methodology. J. Am. Chem. Soc. 138, 2–24 (2016).
Urlacher, V. B. & Girhard, M. Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol. 30, 26–36 (2012).
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).
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).
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).
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).
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).
Guterl, J. K. et al. Cell-free metabolic engineering: production of chemicals by minimized reaction cascades. ChemSusChem 5, 2165–2172 (2012).
Tadrowski, S. et al. Metal ions play an essential catalytic role in the mechanism of ketol-acid reductoisomerase. Chemistry 22, 7427–7436 (2016).
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).
Wong, L. L. P450BM3 on steroids: the Swiss army knife P450 enzyme just gets better. ChemBioChem 12, 2537–2539 (2011).
Romero, P. A. & Arnold, F. H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).
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).
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).
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).
Devamani, T. et al. Catalytic promiscuity of ancestral esterases and hydroxynitrile lyases. J. Am. Chem. Soc. 138, 1046–1056 (2016).
Alcolombri, U., Elias, M. & Tawfik, D. S. Directed evolution of sulfotransferases and paraoxonases by ancestral libraries. J. Mol. Biol. 411, 837–853 (2011).
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).
Gaucher, E. A., Govindarajan, S. & Ganesh, O. K. Palaeotemperature trend for Precambrian life inferred from resurrected proteins. Nature 451, 704–707 (2008).
Perez-Jimenez, R. et al. Single-molecule paleoenzymology probes the chemistry of resurrected enzymes. Nat. Struct. Mol. Biol. 18, 592–596 (2011).
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).
Nguyen, V. et al. Evolutionary drivers of thermoadaptation in enzyme catalysis. Science 355, 289–293 (2017).
Akanuma, S. et al. Experimental evidence for the thermophilicity of ancestral life. Proc. Natl Acad. Sci. USA 110, 11067–11072 (2013).
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).
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).
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).
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).
Stepankova, V. et al. Strategies for stabilization of enzymes in organic solvents. ACS Catal. 3, 2823–2836 (2013).
Zakas, P. M. et al. Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction. Nat. Biotechnol. 35, 35–37 (2017).
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).
Paddon, C. J. et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528–532 (2013).
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).
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).
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).
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).
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).
Perdigao, N. et al. Unexpected features of the dark proteome. Proc. Natl Acad. Sci. USA 112, 15898–15903 (2015).
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).
Parikh, A., Gillam, E. M. J. & Guengerich, F. P. Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat. Biotechnol. 15, 784–788 (1997).
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).
Shukla, A., Huang, W., Depaz, I. M. & Gillam, E. M. J. Membrane integration of recombinant human P450 forms. Xenobiotica 39, 495–507 (2009).
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).
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).
Qiu, H. et al. CYP3 phylogenomics: evidence for positive selection of CYP3A4 and CYP3A7. Pharmacogenet. Genomics 18, 53–66 (2008).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Chang, T. K. & Waxman, D. J. in Cytochrome P450 Protocols Vol. 320 (eds Phillips, I. R. & Shephard, E. A.) 85–90 (Humana, Totowa, 2006).
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).
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).
Isin, E. M. & Guengerich, F. P. Substrate binding to cytochromes P450. Anal. Bioanal. Chem. 392, 1019–1030 (2008).
Isin, E. M. & Guengerich, F. P. Kinetics and thermodynamics of ligand binding by cytochrome P450 3A4. J. Biol. Chem. 281, 9127–9136 (2006).
Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).
Gill, S. C. & von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326 (1989).
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.
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.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Gumulya, Y., Baek, JM., Wun, SJ. et al. Engineering highly functional thermostable proteins using ancestral sequence reconstruction. Nat Catal 1, 878–888 (2018). https://doi.org/10.1038/s41929-018-0159-5
Nature Catalysis (2021)
Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties
Scientific Reports (2020)
Nature Chemical Biology (2020)
Nature Structural & Molecular Biology (2020)
Communications Materials (2020)