Analysis of metabolic pathways and fluxes in a newly discovered thermophilic and ethanol-tolerant Geobacillus strain

Authors

  • Yinjie J. Tang,

    1. Virtual Institute for Microbial Stress and Survival, Berkeley, California; telephone: 510-642-4862; fax: 510-643-1228
    2. Department of Energy, Environmental and Chemical Engineering, Washington University, St Louis, Missouri
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  • Rajat Sapra,

    1. Joint Bio-Energy Institute, Emeryville, California 94608
    2. Sandia National Laboratories, PO Box 969, Livermore, California
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  • Dominique Joyner,

    1. Virtual Institute for Microbial Stress and Survival, Berkeley, California; telephone: 510-642-4862; fax: 510-643-1228
    2. Ecology Department, Lawrence Berkeley National Lab, Berkeley, California
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  • Terry C. Hazen,

    1. Virtual Institute for Microbial Stress and Survival, Berkeley, California; telephone: 510-642-4862; fax: 510-643-1228
    2. Ecology Department, Lawrence Berkeley National Lab, Berkeley, California
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  • Samuel Myers,

    1. Department of Chemical Engineering, University of California, Berkeley, California 94720
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  • David Reichmuth,

    1. Sandia National Laboratories, PO Box 969, Livermore, California
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  • Harvey Blanch,

    1. Joint Bio-Energy Institute, Emeryville, California 94608
    2. Department of Chemical Engineering, University of California, Berkeley, California 94720
    3. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
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  • Jay D. Keasling

    Corresponding author
    1. Virtual Institute for Microbial Stress and Survival, Berkeley, California; telephone: 510-642-4862; fax: 510-643-1228
    2. Joint Bio-Energy Institute, Emeryville, California 94608
    3. Department of Chemical Engineering, University of California, Berkeley, California 94720
    4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
    5. Department of Bioengineering, University of California, Berkeley, California 94720
    • Virtual Institute for Microbial Stress and Survival, Berkeley, California; telephone: 510-642-4862; fax: 510-643-1228.
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Abstract

A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and is tolerant to high ethanol concentrations (10%, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner–Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accurately determined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)−1 h−1) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64 ± 3 to 25 ± 2 and from 30 ± 2 to 19 ± 2, respectively. The carbon flux under micro-aerobic growth was directed to ethanol, L-lactate (>99% optical purity), acetate, and formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38 ± 0.07 mol mol−1 glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yield by approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production. Biotechnol. Bioeng. 2009;102: 1377–1386. © 2008 Wiley Periodicals, Inc.

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