Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli

Authors

  • Guyton Durnin,

    1. Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, P.O. Box 1892, Houston, Texas 77251-1892; telephone: 713-348-4893; fax: 713-348-5478
    2. Department of Civil and Environmental Engineering, Rice University, Houston, Texas
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  • James Clomburg,

    1. Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, P.O. Box 1892, Houston, Texas 77251-1892; telephone: 713-348-4893; fax: 713-348-5478
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  • Zeno Yeates,

    1. Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, P.O. Box 1892, Houston, Texas 77251-1892; telephone: 713-348-4893; fax: 713-348-5478
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  • Pedro J.J. Alvarez,

    1. Department of Civil and Environmental Engineering, Rice University, Houston, Texas
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  • Kyriacos Zygourakis,

    1. Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, P.O. Box 1892, Houston, Texas 77251-1892; telephone: 713-348-4893; fax: 713-348-5478
    2. Department of Bioengineering, Rice University, Houston, Texas
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  • Paul Campbell,

    1. Glycos Biotechnologies Inc., Houston, Texas
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  • Ramon Gonzalez

    Corresponding author
    1. Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, P.O. Box 1892, Houston, Texas 77251-1892; telephone: 713-348-4893; fax: 713-348-5478
    2. Department of Bioengineering, Rice University, Houston, Texas
    • Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main Street, MS-362, P.O. Box 1892, Houston, Texas 77251-1892; telephone: 713-348-4893; fax: 713-348-5478.
    Search for more papers by this author

Abstract

Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed ∼95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK-GlpD/GlpABC) and fermentative (GldA-DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate-lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl-CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co-production of ethanol and hydrogen (EH05 [pZSKLMgldA]), and ethanol and formate (EF06 [pZSKLMgldA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of by-products succinate, acetate, and lactate. Co-production of hydrogen required the use of acidic pH while formate co-production was facilitated by inactivation of the enzyme formate-hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements. Biotechnol. Bioeng. 2009;103: 148–161. © 2008 Wiley Periodicals, Inc.

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