This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Underwood, S. A. Articles by Ingram, L. O. Search for Related Content PubMed PubMed Citation Articles by Underwood, S. A. Articles by Ingram, L. O. Agricola Articles by Underwood, S. A. Articles by Ingram, L. O.

Flux through Citrate Synthase Limits the Growth of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

Institute of Food and Agricultural Sciences, Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611

Received 17 September 2001/ Accepted 19 December 2001

Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD⁺. Expression of low-K_m Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.

Florida Agricultural Experiment Station publication number R08310.

This article has been cited by other articles:

• PETERSON, J. D., INGRAM, L. O. (2008). Anaerobic Respiration in Engineered Escherichia coli with an Internal Electron Acceptor to Produce Fuel Ethanol. Ann. N. Y. Acad. Sci. 1125: 363-372 [Abstract] [Full Text]  
• Vemuri, G. N., Altman, E., Sangurdekar, D. P., Khodursky, A. B., Eiteman, M. A. (2006). Overflow Metabolism in Escherichia coli during Steady-State Growth: Transcriptional Regulation and Effect of the Redox Ratio.. Appl. Environ. Microbiol. 72: 3653-3661 [Abstract] [Full Text]  
• Underwood, S. A., Buszko, M. L., Shanmugam, K. T., Ingram, L. O. (2004). Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation. Appl. Environ. Microbiol. 70: 2734-2740 [Abstract] [Full Text]  
• Zhou, S., Shanmugam, K. T., Ingram, L. O. (2003). Functional Replacement of the Escherichia coliD-(-)-Lactate Dehydrogenase Gene (ldhA) with the L-(+)-Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici. Appl. Environ. Microbiol. 69: 2237-2244 [Abstract] [Full Text]  
• Causey, T. B., Zhou, S., Shanmugam, K. T., Ingram, L. O. (2003). Inaugural Article: Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: Homoacetate production. Proc. Natl. Acad. Sci. USA 100: 825-832 [Abstract] [Full Text]  
• Zhou, S., Causey, T. B., Hasona, A., Shanmugam, K. T., Ingram, L. O. (2003). Production of Optically Pure D-Lactic Acid in Mineral Salts Medium by Metabolically Engineered Escherichia coli W3110. Appl. Environ. Microbiol. 69: 399-407 [Abstract] [Full Text]  
• Underwood, S. A., Zhou, S., Causey, T. B., Yomano, L. P., Shanmugam, K. T., Ingram, L. O. (2002). Genetic Changes To Optimize Carbon Partitioning between Ethanol and Biosynthesis in Ethanologenic Escherichia coli. Appl. Environ. Microbiol. 68: 6263-6272 [Abstract] [Full Text]