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Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae

Authors

  • JingHong M. DeJong,

    1. eXegenics, Inc., 2110 Research Row, Dallas, Texas 75235
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  • Yule Liu,

    1. eXegenics, Inc., 2110 Research Row, Dallas, Texas 75235
    Current affiliation:
    1. Inhibitex, Inc., 8995 Westside Parkway, Alpharetta, Georgia 30004.
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  • Arthur P. Bollon,

    1. eXegenics, Inc., 2110 Research Row, Dallas, Texas 75235
    Current affiliation:
    1. HemoBiotech, Inc., 2110 Research Row, Dallas, Texas 75235.
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  • Robert M. Long,

    1. Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340; telephone: (509) 335-1790; fax: (509) 335-7643
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  • Stefan Jennewein,

    1. Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340; telephone: (509) 335-1790; fax: (509) 335-7643
    Current affiliation:
    1. Institute for Chemistry and Biochemistry, Technical University-Darmstadt, Petersenstr. 22, G45287 Darmstadt, Germany.
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  • David Williams,

    1. Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340; telephone: (509) 335-1790; fax: (509) 335-7643
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  • Rodney B. Croteau

    Corresponding author
    1. Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340; telephone: (509) 335-1790; fax: (509) 335-7643
    • Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340; telephone: (509) 335-1790; fax: (509) 335-7643
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Abstract

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5α- acetoxy-10β-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast. © 2005 Wiley Periodicals, Inc.

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