Engineering highly functional thermostable proteins using ancestral sequence reconstruction

Abstract

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.

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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.

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Acknowledgements

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.

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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.

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Correspondence to Elizabeth M. J. Gillam.

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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.

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Gumulya, Y., Baek, J., Wun, S. 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

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