Angela Cintolesi and James M. Clomburg contributed equally to this work.
Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli†
Article first published online: 31 AUG 2011
Copyright © 2011 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 109, Issue 1, pages 187–198, January 2012
How to Cite
Cintolesi, A., Clomburg, J. M., Rigou, V., Zygourakis, K. and Gonzalez, R. (2012), Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli. Biotechnol. Bioeng., 109: 187–198. doi: 10.1002/bit.23309
- Issue published online: 17 NOV 2011
- Article first published online: 31 AUG 2011
- Accepted manuscript online: 19 AUG 2011 09:58AM EST
- Manuscript Accepted: 12 AUG 2011
- Manuscript Revised: 9 AUG 2011
- Manuscript Received: 3 JUN 2011
- U.S. National Science Foundation. Grant Number: CBET-0645188
- glycerol fermentation;
- Escherichia coli;
- kinetic modeling;
- metabolic control analysis;
Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions. Biotechnol. Bioeng. 2012;109: 187–198. © 2011 Wiley Periodicals, Inc.