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Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli

  1. Guyton Durnin1,2,
  2. James Clomburg1,
  3. Zeno Yeates1,
  4. Pedro J.J. Alvarez2,
  5. Kyriacos Zygourakis1,3,
  6. Paul Campbell4,
  7. Ramon Gonzalez1,3,*

Article first published online: 30 DEC 2008

DOI: 10.1002/bit.22246

Issue

Biotechnology and Bioengineering

Biotechnology and Bioengineering

Volume 103, Issue 1, pages 148–161, 1 May 2009

Additional Information

How to Cite

Durnin, G., Clomburg, J., Yeates, Z., Alvarez, P. J.J., Zygourakis, K., Campbell, P. and Gonzalez, R. (2009), Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli. Biotechnol. Bioeng., 103: 148–161. doi: 10.1002/bit.22246

Author Information

  1. 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. 2

    Department of Civil and Environmental Engineering, Rice University, Houston, Texas

  3. 3

    Department of Bioengineering, Rice University, Houston, Texas

  4. 4

    Glycos Biotechnologies Inc., 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.

Publication History

  1. Issue published online: 23 MAR 2009
  2. Article first published online: 30 DEC 2008
  3. Accepted manuscript online: 30 DEC 2008 12:00AM EST
  4. Manuscript Accepted: 24 NOV 2008
  5. Manuscript Revised: 8 NOV 2008
  6. Manuscript Received: 18 SEP 2008

Funded by

  • U.S. National Science Foundation. Grant Number: CBET-0645188
  • National Research Initiative of the U.S. Department of Agriculture Cooperative State Research, Education and Extension Service. Grant Number: 2005-35504-16698
  • Glycos Biotechnologies, Inc.

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Keywords:

  • glycerol metabolism;
  • Escherichia coli;
  • metabolic engineering;
  • biofuels and biochemicals

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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