Engineering triterpene production in Saccharomyces cerevisiae–β-amyrin synthase from Artemisia annua

Authors

  • James Kirby,

    1.  California Institute for Quantitative Biomedical Research, University of California, Berkeley, CA, USA
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  • Dante W. Romanini,

    1.  Department of Chemistry, University of California, Berkeley, CA, USA
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  • Eric M. Paradise,

    1.  California Institute for Quantitative Biomedical Research, University of California, Berkeley, CA, USA
    2.  Department of Chemical Engineering, University of California, Berkeley, CA, USA
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  • Jay D. Keasling

    1.  California Institute for Quantitative Biomedical Research, University of California, Berkeley, CA, USA
    2.  Department of Chemical Engineering, University of California, Berkeley, CA, USA
    3.  Department of Bioengineering, University of California, Berkeley, CA, USA
    4.  Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
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J. D. Keasling, Berkeley Center for Synthetic Biology, 717 Potter Street, Building 977, Mail code 3224, University of California, Berkeley, CA 94720-3224, USA
Fax: +1 510 495 2630
Tel: +1 510 495 2620
E-mail: keasling@berkeley.edu

Abstract

Using a degenerate primer designed from triterpene synthase sequences, we have isolated a new gene from the medicinal plant Artemisia annua. The predicted protein is highly similar to β-amyrin synthases (EC 5.4.99.–), sharing amino acid sequence identities of up to 86%. Expression of the gene, designated AaBAS, in Saccharomyces cerevisiae, followed by GC/MS analysis, confirmed the encoded enzyme as a β-amyrin synthase. Through engineering the sterol pathway in S. cerevisiae, we explore strategies for increasing triterpene production, using AaBAS as a test case. By manipulation of two key enzymes in the pathway, 3-hydroxy-3-methylglutaryl-CoA reductase and lanosterol synthase, we have improved β-amyrin production by 50%, achieving levels of 6 mg·L−1 culture. As we have observed a 12-fold increase in squalene levels, it appears that this strain has the capacity for even higher β-amyrin production. Options for further engineering efforts are explored.

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