High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli

Authors

  • Jack D. Newman,

    1. Department of Chemical Engineering, University of California, Berkeley, California
    Current affiliation:
    1. Amyris Biotechnologies Inc., 5980 Horton St., Suite 450, Emeryville, California.
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  • Jessica Marshall,

    1. Department of Chemical Engineering, University of California, Berkeley, California
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  • Michelle Chang,

    1. Department of Chemical Engineering, University of California, Berkeley, California
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  • Farnaz Nowroozi,

    1. Department of Bioengineering, University of California, Berkeley, California
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  • Eric Paradise,

    1. Department of Chemical Engineering, University of California, Berkeley, California
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  • Douglas Pitera,

    1. Department of Chemical Engineering, University of California, Berkeley, California
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  • Karyn L. Newman,

    1. California Institute of Quantitative Biomedical Research, University of California, Berkeley, California
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  • Jay D. Keasling

    Corresponding author
    1. Department of Chemical Engineering, University of California, Berkeley, California
    2. Department of Bioengineering, University of California, Berkeley, California
    3. California Institute of Quantitative Biomedical Research, University of California, Berkeley, California
    4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California
    5. Berkeley Center for Synthetic Biology, University of California, 717 Potter St., MC 3224, Berkeley, California 94720-3224; telephone: 510-642-4862; fax: 510-495-2630
    • Berkeley Center for Synthetic Biology, University of California, 717 Potter St., MC 3224, Berkeley, California 94720-3224; telephone: 510-642-4862; fax: 510-495-2630
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Abstract

Reconstructing synthetic metabolic pathways in microbes holds great promise for the production of pharmaceuticals in large-scale fermentations. By recreating biosynthetic pathways in bacteria, complex molecules traditionally harvested from scarce natural resources can be produced in microbial cultures. Here we report on a strain of Escherichia coli containing a heterologous, nine-gene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the anti-malarial drug artemisinin. Previous reports have underestimated the productivity of this strain due to the volatility of amorphadiene. Here we show that amorphadiene evaporates from a fermentor with a half-life of about 50 min. Using a condenser, we take advantage of this volatility by trapping the amorphadiene in the off-gas. Amorphadiene was positively identified using nuclear magnetic resonance spectroscopy and determined to be 89% pure as collected. We captured amorphadiene as it was produced in situ by employing a two-phase partitioning bioreactor with a dodecane organic phase. Using a previously characterized caryophyllene standard to calibrate amorphadiene production and capture, the concentration of amorphadiene produced was determined to be 0.5 g/L of culture medium. A standard of amorphadiene collected from the off-gas showed that the caryophyllene standard overestimated amorphadiene production by approximately 30%. © 2006 Wiley Periodicals, Inc.

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