Letter | Published:

Designed divergent evolution of enzyme function

Nature volume 440, pages 10781082 (20 April 2006) | Download Citation



It is generally believed that proteins with promiscuous functions divergently evolved to acquire higher specificity and activity1,2,3,4,5, and that this process was highly dependent on the ability of proteins to alter their functions with a small number of amino acid substitutions (plasticity)6. The application of this theory of divergent molecular evolution to promiscuous enzymes may allow us to design enzymes with more specificity and higher activity. Many structural and biochemical analyses have identified the active or binding site residues important for functional plasticity (plasticity residues)6,7,8,9,10. To understand how these residues contribute to molecular evolution, and thereby formulate a design methodology, plasticity residues were probed in the active site of the promiscuous sesquiterpene synthase γ-humulene synthase11,12. Identified plasticity residues were systematically recombined based on a mathematical model in order to construct novel terpene synthases, each catalysing the synthesis of one or a few very different sesquiterpenes. Here we present the construction of seven specific and active synthases that use different reaction pathways to produce the specific and very different products. Creation of these enzymes demonstrates the feasibility of exploiting the underlying evolvability of this scaffold, and provides evidence that rational approaches based on these ideas are useful for enzyme design.

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We would like to thank P. C. Babbitt, J. D. Newman, M. C. Chang and S. C.-H. Pegg for discussions and critical reading of the manuscript. We are also grateful for D. Herschlag for critical comments. This research was funded by the Bill & Melinda Gates Foundation, the US Department of Agriculture, and the National Science Foundation.Author Contributions Y.Y. and J.D.K. conceived the project; Y.Y., J.D.K. and T.E.F. designed the experiments; and Y.Y. and J.D.K. wrote the paper.

Author information


  1. UCSF/UCB Joint Graduate Group in Bioengineering,

    • Yasuo Yoshikuni
    • , Thomas E. Ferrin
    •  & Jay D. Keasling
  2. Department of Chemical Engineering, and

    • Jay D. Keasling
  3. California Institute for Quantitative Biomedical Research (QB3), University of California at Berkeley, Berkeley, California 94720, USA

    • Jay D. Keasling
  4. Synthetic Biology Department, Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California 94710, USA

    • Yasuo Yoshikuni
    •  & Jay D. Keasling
  5. Department of Pharmaceutical Chemistry and Biopharmaceutical Sciences, University of California at San Francisco, San Francisco, California 94143, USA

    • Thomas E. Ferrin


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Competing interests

J.D.K. owns stock in, and is a founder of, Amyris Biotechnologies. Amyris may use this technology to produce terpenes. However, Amyris currently has no plans to use the technology.

Corresponding author

Correspondence to Jay D. Keasling.

Supplementary information

PDF files

  1. 1.

    Supplementary Tables

    This file contains Supplementary Tables 1–9.

  2. 2.

    Supplementary Data 1

    Three-dimensional coordinates for the homology structure of γ-humulene synthase

  3. 3.

    Supplementary Data 2

    DNA sequence of designed γ-humulene synthase

  4. 4.

    Supplementary Methods 1

    Additional methods used in this study.

  5. 5.

    Supplementary Methods 2

    Detailed protocols for systematic remodeling of plasticity residues

Word documents

  1. 1.

    Supplementary Figures

    This file contains Supplementary Figures 1–11. *In Figure 1 and Supplementary Figure 1, the structure of longifolene (4) has been corrected (it was missing a methyl group). In Supplementary Figure 1, the longipinene structure was also wrongly labelled 'longifolene' and vice versa. This correction was made on 27 February 2006.

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