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High-Throughput Screening for Terpene-Synthase-Cyclization Activity and Directed Evolution of a Terpene Synthase

Authors

  • Dr. Ryan Lauchli,

    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)
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  • Dr. Kersten S. Rabe,

    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)
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  • Karolina Z. Kalbarczyk,

    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)
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  • Amulya Tata,

    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)
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  • Dr. Thomas Heel,

    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)
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  • Rebekah Z. Kitto,

    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)
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  • Prof. Dr. Frances H. Arnold

    Corresponding author
    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)
    • Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)

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  • We are grateful to David Cane, Scott Virgil, Robert M. Coates, and David Christianson for their pioneering studies, invaluable advice, encouragement, and materials. We thank Indira Wu, Chris Farwell, and Jack Zhang for assistance. R.L. acknowledges the support of NIH fellowship F32GM095061. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. K.S.R. thanks the Deutscher Akademischer Austauschdienst (DAAD) for a postdoctoral fellowship. K.Z.K. and R.Z.K. acknowledge the support of Summer Undergraduate Research Fellowships from the California Institute of Technology. T.H. was funded by the FWF grant number: J3327-B21.

Abstract

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An easy assay: A synthetic substrate enables a colorimetric screen for terpene synthase cyclization activity, thereby facilitating the engineering of these enzymes. By using directed evolution, the thermostability of a sesquiterpene synthase was increased without the loss of other properties. The technique also enabled rapid optimization of conditions for expression and stabilization in lysate of another terpene synthase. PPO=diphosphate.

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