Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Improving catalytic function by ProSAR-driven enzyme evolution

Nature Biotechnology volume 25, pages 338–344 (2007)Cite this article

Abstract

We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process ∼4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: HHDH interconverts halohydrins and epoxides18 and can accept alternative nucleophiles17.
Figure 2: Multivariate optimization of enzymes.
Figure 3: Reaction progress curves for an expression mutant of the wild-type enzyme and representative variants obtained over the course of the evolution at a substrate loading of 130 g/l, substrate/biocatalyst at 100:1 (wt/wt), 40 °C and pH 7.3 (sequences in Supplementary Notes online).
Figure 4: Positions of mutations in the HHDH structure.
Figure 5: Function contribution by individual mutations under different screening conditions.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

Protein Data Bank

References

  1. Schmid, A. et al. Industrial biocatalysis today and tomorrow. Nature 409, 258–268 (2001).

    Article  CAS  Google Scholar 

  2. Schoemaker, H.E., Mink, D. & Wubbolts, M.G. Dispelling the myths—biocatalysis in industrial synthesis. Science 299, 1694–1697 (2003).

    Article  CAS  Google Scholar 

  3. Hibbert, E.G. & Dalby, P.A. Directed evolution strategies for improved enzymatic performance. Microb. Cell Fact. 4, 29 (2005).

    Article  Google Scholar 

  4. Tawfik, D.S. Biochemistry. Loop grafting and the origins of enzyme species. Science 311, 475–476 (2006).

    Article  CAS  Google Scholar 

  5. Castle, L.A. et al. Discovery and directed evolution of a glyphosate tolerance gene. Science 304, 1151–1154 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Ness, J.E. et al. Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Nat. Biotechnol. 20, 1251–1255 (2002).

    Article  CAS  Google Scholar 

  8. Ness, J.E. et al. DNA shuffling of subgenomic sequences of subtilisin. Nat. Biotechnol. 17, 893–896 (1999).

    Article  CAS  Google Scholar 

  9. Stemmer, W.P.C. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Kubinyi, H. QSAR and 3D QSAR in drug design Part1: methodology. Drug Disc. Today 2, 457–467 (1997).

    Article  CAS  Google Scholar 

  12. Hellberg, S., Sjostrom, M. & Wold, S. The prediction of bradykinin potentiating potency of pentapeptides. An example of a peptide quantitative structure-activity relationship. Acta Chem. Scand. B 40, 135–140 (1986).

    Article  CAS  Google Scholar 

  13. Eroshkin, A.M., Fomin, V.I., Zhilkin, P.A., Ivanisenko, V.A. & Kondrakhin, Y.V. PROANAL version 2: multifunctional program for analysis of multiple protein sequence alignments and studying structure-activity relationships in protein families. Comp. Appl. Biosci. 11, 39–44 (1995).

    CAS  PubMed  Google Scholar 

  14. Eroshkin, A.M., Zhilkin, P.A. & Fomin, V.I. Algorithm and computer program Pro__Anal for analysis of relationship between structure and activity in a family of proteins or peptides. Comput. Appl. Biosci. 9, 491–497 (1993).

    CAS  PubMed  Google Scholar 

  15. Fox, R. Directed molecular evolution by machine learning and the influence of nonlinear interactions. J. Theor. Biol. 234, 187–199 (2005).

    Article  CAS  Google Scholar 

  16. Fox, R. et al. Optimizing the search algorithm for protein engineering by directed evolution. Protein Eng. 16, 589–597 (2003).

    Article  CAS  Google Scholar 

  17. Nakamura, T., Nagasawa, T., Yu, F., Watanabe, I. & Yamada, H. A new catalytic function of halohydrin hydrogen-halide-lyase, synthesis of beta-hydroxynitriles from epoxides and cyanide. Biochem. Biophys. Res. Commun. 180, 124–130 (1991).

    Article  CAS  Google Scholar 

  18. van den Wijngaard, A.J., Reuvekamp, P.T. & Janssen, D.B. Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2. J. Bacteriol. 173, 124–129 (1991).

    Article  CAS  Google Scholar 

  19. Matsuda, H., Shibata, T., Hashimoto, H. & Kitai, M. Method for producing (R)-4-cyano-3-hydroxybutyric acid lower alkyl ester. US patent 5,908,953 (1999).

  20. Stemmer, W.P. DNA in vitro shuffling by random fragmentation and reassembly in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 (1994).

    Article  CAS  Google Scholar 

  21. Wells, J.A. Additivity of mutational effects in proteins. Biochemistry 29, 8509–8517 (1990).

    Article  CAS  Google Scholar 

  22. Sandberg, W.S. & Terwilliger, T.C. Engineering multiple properties of a protein by combinatorial mutagenesis. Proc. Natl. Acad. Sci. USA 90, 8367–8371 (1993).

    Article  CAS  Google Scholar 

  23. de Jong, S. SIMPLS: an alternative approach to partial least squares regression. Chemomet. and Intell. Lab. Sys. 18, 251–263 (1993).

    Article  CAS  Google Scholar 

  24. Mee, R.P., Auton, T.R. & Morgan, P.J. Design of active analogues of a 15-residue peptide using D-optimal design, QSAR and a combinatorial search algorithm. J. Pept. Res. 49, 89–102 (1997).

    Article  CAS  Google Scholar 

  25. Bennett, K. & Embrechts, M. An Optimization Perspective on Partial Least Squares. Advances in Learning Theory: Methods, Models and Applications, NATO Science Series III: Computer & Systems Sciences vol. 190. (eds. Suykens, J., Horvath, G., Basu, S., Micchelli, J. & Vandewalle, J.) 227–250, (IOS Press, Amsterdam, 2003).

    Google Scholar 

  26. de Jong, R.M. et al. Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site. EMBO J. 22, 4933–4944 (2003).

    Article  CAS  Google Scholar 

  27. Li, W.H., Wu, C.I. & Luo, C.C. Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications. J. Mol. Evol. 21, 58–71 (1984).

    Article  CAS  Google Scholar 

  28. Hartl, D.L. & Taubes, C.H. Towards a theory of evolutionary adaptation. Genetica 102-103, 525–533 (1998).

    Article  CAS  Google Scholar 

  29. Hylckama Vlieg, J.E.T. et al. Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases. J. Bacteriol. 183, 5058–5066 (2001).

    Article  Google Scholar 

  30. Kauffman, S. . The Origins of Order (Oxford University Press, New York, 1993).

    Google Scholar 

  31. Thayer, A. Competitors want to get a piece of Lipitor. Chem. Eng. News 84, 26–27 (2006).

    Article  Google Scholar 

  32. Zhang, J.H., Dawes, G. & Stemmer, W.P. Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl. Acad. Sci. USA 94, 4504–4509 (1997).

    Article  CAS  Google Scholar 

  33. Stutzman-Engwall, K. et al. Semi-synthetic DNA shuffling of aveC leads to improved industrial scale production of doramectin by Streptomyces avermitilis. Metab. Eng. 7, 27–37 (2005).

    Article  CAS  Google Scholar 

  34. Reetz, M.T., Wang, L.W. & Bocola, M. Directed evolution of enantioselective enzymes: Iterative cycles of CASTing for probing protein-sequence space. Angew Chem. Int. Ed. Engl. 45, 1236–1241 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank David Gray, Alice Wang, Su Chen, John Peterson, Walter Heath, Tim Brandon, Jon Postlethwaite, Anjali Srivastava and Bill Dewhirst for help with bioprocess development and production; Malissa Jefferson and Susan Louie for additional assay support; Patricia Babbitt, Pim Stemmer, Lynne Gilson, Lori Giver, Birthe Borup, Anke Krebber, Stephen DelCardayre and Jonathan Blanding for careful reading of the manuscript and helpful suggestions; and three anonymous reviewers for insightful commentary and critical feedback.

Author information

Author notes

  1. Richard J Fox, S Christopher Davis and Emily C Mundorff: These authors contributed equally to this work.

Authors and Affiliations

  1. Codexis, Inc., 200 Penobscot Drive, Redwood City, 94063, California, USA

    Richard J Fox, S Christopher Davis, Emily C Mundorff, Lisa M Newman, Vesna Gavrilovic, Steven K Ma, Loleta M Chung, Charlene Ching, Sarena Tam, Sheela Muley, John Grate, John Gruber, John C Whitman & Gjalt W Huisman

  2. Department of Organic Chemistry, Delft University of Technology, Julianalaan 136, 2628 BL Delft, Netherlands

    Roger A Sheldon

Authors
  1. Richard J Fox
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Christopher Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Emily C Mundorff
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lisa M Newman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vesna Gavrilovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Steven K Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Loleta M Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Charlene Ching
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sarena Tam
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sheela Muley
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. John Grate
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. John Gruber
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. John C Whitman
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Roger A Sheldon
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Gjalt W Huisman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gjalt W Huisman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fox, R., Davis, S., Mundorff, E. et al. Improving catalytic function by ProSAR-driven enzyme evolution. Nat Biotechnol 25, 338–344 (2007). https://doi.org/10.1038/nbt1286

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1286

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing