Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli
Article first published online: 30 DEC 2008
Copyright © 2008 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 103, Issue 1, pages 148–161, 1 May 2009
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
- Issue published online: 23 MAR 2009
- Article first published online: 30 DEC 2008
- Accepted manuscript online: 30 DEC 2008 12:00AM EST
- Manuscript Accepted: 24 NOV 2008
- Manuscript Revised: 8 NOV 2008
- Manuscript Received: 18 SEP 2008
- 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.
- glycerol metabolism;
- Escherichia coli;
- metabolic engineering;
- biofuels and biochemicals
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.