METABOLIC ENGINEERING TO INCREASE THE ETHANOL FLUX AND YIELD IN ETHANOLOGENIC Escherichia coli

  • G. Huerta-Beristain
  • J. Utrilla-Carreri
  • G. Hernández-Chávez
  • F. Bolívar
  • G. Gosset
  • A. Martínez
Keywords: metabolic flux control, glycolytic flux, ethanol flux, pyruvate decarboxylase, xylose, and glucose

Abstract

Modification of ethanol flux, using glucose or xylose as carbon source, in ethanologenic Escherichia coli KO11 was studied, by increasing the activity of key carbon metabolism enzymes. KO11 strain contains, integrated into the chromosome, genes that code for pyruvate decarboxylase (PdcZm) and alcohol dehydrogenase (AdhBZm) from Zymomonas mobilis. Results indicate that KO11 has limited Pdc activity to channel carbon flux to ethanol formation from glucose or xylose. Hence, flux control is outside glycolysis and Pdc controls the ethanol flux. When intracellular activity of Pdc was increased 6 fold, the theoretical yield of ethanol on glucose or xylose was increased by 27%, during the stationary phase the ethanol flux was increased 42 and 44%, and the organic acid formation rate was reduced 46 and 76% for glucose or xylose, respectively. Furthermore, as a response to allosteric effects and a limited Pdc activity, an increase in the phosphofructokinase or pyruvatekinase enzymatic activity drastically reduces glucose consumption and ethanol formation flux, with a concomitant increase in organic acid formation.

References

Aristidou, A. y Penttilä, M. (2000). Metabolic engineering applications to renewable resource utilization. Current Opinion in Biotechnology 11, 187-198.

Bailey, E. J. (1991). Toward a science of metabolic engineering. Science 252, 1668-1674

Beall, D. S., Ohta, K. e Ingram, L. O. (1991). Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli. Biotechnology and Bioengineering 38, 296-303.

Bergmeyer, J. y Gawehn, K. (1974). Methods of enzymatic analysis. VCH. Weinheim, Alemania.

Böck. A. y Sawers, G. 1996. Fermentation. En: Escherichia coli and Salmonella, Celular and molecular biology, Cápitulo 18, (Neidhardt et al., eds.), Pp. 262-282. American Society for Microbiology Press, Washington D.C.

Bothast R. J., Nichols, N. N. y Dien, B. S. (1999). Fermentations with new recombinant organisms. Biotechnology Progress 15, 867-875.

Chassagnole, C., Noisommil-Rizzi, N., Schmid, J.W., Mauch, K. y Reuss, M. (2002). Dynamic modeling of the central carbon metabolism of Escherichia coli Biotechnology and Bioengineering 79, 53- 73.

Emmerling, M., Bailey, J. E. y Sauer, U. (1999). Glucose catabolism of Escherichia coli strains with increased activity and altered regulation of key glycolytic enzymes. Metabolic Engineering 1, 117-127.

Gonzalez R., Tao, H., Shanmugam K. T., York, S. W. e Ingram, L. O. (2002). Global gene expression differences associated with changes in glycolytic flux and growth rate in Escherichia coli during the fermentation of glucose and xylose. Biotechnology Progress 18, 6-20.

Hernández-Montalvo, V., Martínez, A., Hernández-Chávez, G., Bolívar, F., Valle, F. y Gosset, G. (2003). Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products. Biotechnology and Bioengineering 83, 687-694.

Hoppner, T.C. y Doelle, H.W. (1983). Purification and kinetic characteristics of pyruvate decarboxylase and ethanol dehydrogenase from Zymomonas mobilis in relation to ethanol production. European Journal of Applied Microbiology and Biotechnology 17, 152-157.

Huerta-Beristain, G. (2004). Manipulación del metabolismo central de Escherichia coli para incrementar la productividad de etanol. Tesis de Maestría. Instituto de Biotecnología, UNAM, México.

Ingram, L.O., Aldrich, H.C., Borges, A.C.C., Causey, T.B., Martínez, A., Morales, F., Saleh, A., Underwood, S.A., Yomano, L.P., York, S.W., Zaldivar, J. y Zhou, S. (1999). Enteric bacterial catalyst for fuel ethanol production. Biotechnology Progress 15, 855-866.

Maitra, P. K. y Lobo, Z. (1971). Control of glycolytic enzyme synthesis in yeast by products of the hexokinase reaction. Journal of Biological Chemistry 246, 489-99.

Martínez, A., York, S.W., Yomano, L.P., Pineda, V.L., Davis, F.C., Shelton, J.C. e Ingram, L.O. (1999). Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli. Biotechnology Progress 15, 891–897.

Martínez, A., Rodríguez, M.E., York, S.W., Preston, J.F. e Ingram, L.O. (2000). Effects of Ca(OH)2 treatments (“overliming”) on the composition and toxicity of bagasse hemicellulose hydrolysate. Biotechnology Bioengineering 69, 526-536.

Martínez, A., Rodríguez, M.E., Wells, M.L., York, S.W., Preston, J.F. e Ingram, L.O. (2001). Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnology Progress 17, 287-293.

Martínez, A., Bolívar, F. y Gosset, G. (2002). Biotecnología energética sustentable: etanol carburante para el transporte. Revista Universidad de México 617, páginas centrales.

Mielenz, J.R. (2001). Ethanol production from biomass: technology and commercialization status. Current Opinion Microbiology 4, 324-329.

Nielsen, J. (2001). Metabolic engineering. Applied Microbiology and Biotechnology 55, 263-283.

Ohta, K., Beall, D.S., Shanmugam, K.T. e Ingram, L. O. (1991). Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. Applied Environmental Microbiology 57, 893-900.

Sakai, H., Susuki, K. e Imahori, K. (1986). Purification and properties of piruvate kinase from Bacillus stearothermophilus. Journal of Biochemistry 99, 1157-1167.

Sambrook, J., Fritsch, E. F. y Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2a ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NuevaYork.

Snoep, J.L., Yomano, L.P., Westerhoff, H.V. e Ingram, L.O. (1995). Protein burden in Zymomonas mobilis: Negative flux and growth control due to overproduction of glycolytic enzymes. Microbiology 141, 2329-2337.

Stephanopoulos, G. (1999). Metabolic fluxes and metabolic engineering. Metabolic Engineering 1, 1-11.

Stryer L. (1999). Biochemistry. 4a Ed. W.H. Freeman and Company, NuevaYork.

Tao H., González, R., Martínez, A., Rodríguez, M.E., Ingram, L.O., Preston, J.F. y Shanmugam, K.T. (2001). Engineering a homo-ethanol pathway in Escherichia coli: Increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation. Journal of Bacteriology 183, 2979-2988.
Published
2020-09-30
How to Cite
Huerta-Beristain, G., Utrilla-Carreri, J., Hernández-Chávez, G., Bolívar, F., Gosset, G., & Martínez, A. (2020). METABOLIC ENGINEERING TO INCREASE THE ETHANOL FLUX AND YIELD IN ETHANOLOGENIC Escherichia coli. Revista Mexicana De Ingeniería Química, 4(1), 25-36. Retrieved from http://www.rmiq.org/ojs311/index.php/rmiq/article/view/2074

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