Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

ABSTRACT The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.

[1]  A. Harden.  Bacterial Metabolism , 1930, Nature.

[2]  C. H. Werkman.  Bacterial Metabolism (2nd ed.) , 1940 .

[3]  H. Katsuki,et al.  Physiological functions of NAD- and NADP-linked malic enzymes in Escherichia coli. , 1971, Biochemical and biophysical research communications.

[4]  H W Doelle,et al.  Effect of specific growth rate and glucose concentration on growth and glucose metabolism of Escherichia coli K-12. , 1976, Microbios.

[5]  D. Fraenkel,et al.  The use of 6-labeled glucose to assess futile cycling in Escherichia coli. , 1980, The Journal of biological chemistry.

[6]  F. Daldal,et al.  Assessment of a futile cycle involving reconversion of fructose 6-phosphate to fructose 1,6-bisphosphate during gluconeogenic growth of Escherichia coli , 1983, Journal of bacteriology.

[7]  D E Koshland,et al.  Determination of flux through the branch point of two metabolic cycles. The tricarboxylic acid cycle and the glyoxylate shunt. , 1984, The Journal of biological chemistry.

[8]  P. Postma,et al.  Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. , 1985, Microbiological reviews.

[9]  F. Neidhardt,et al.  Escherichia Coli and Salmonella: Typhimurium Cellular and Molecular Biology , 1987 .

[10]  F. A. Seiler,et al.  Numerical Recipes in C: The Art of Scientific Computing , 1989 .

[11]  William H. Press,et al.  The Art of Scientific Computing Second Edition , 1998 .

[12]  D. Fraenkel.  Genetics and intermediary metabolism. , 1992, Annual review of genetics.

[13]  G R Jacobson,et al.  Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. , 1993, Microbiological reviews.

[14]  G. Stephanopoulos,et al.  Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction , 2000, Biotechnology and bioengineering.

[15]  Thomas Bäck,et al.  An Overview of Evolutionary Algorithms for Parameter Optimization , 1993, Evolutionary Computation.

[16]  B. Palsson,et al.  Metabolic Flux Balancing: Basic Concepts, Scientific and Practical Use , 1994, Bio/Technology.

[17]  C Hellinga,et al.  Linear constrain relations in biochemical reaction systems III. Sequential application of data reconciliation for sensitive detection of systematic errors , 1994, Biotechnology and bioengineering.

[18]  J. Liao,et al.  Metabolic responses to substrate futile cycling in Escherichia coli. , 1994, The Journal of biological chemistry.

[19]  A Martinez,et al.  Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis , 1995, Journal of bacteriology.

[20]  T. Szyperski.  Biosynthetically Directed Fractional 13C‐labeling of Proteinogenic Amino Acids , 1995 .

[21]  T. Szyperski.  Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism. , 1995, European journal of biochemistry.

[22]  U. Sauer,et al.  Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis , 1996, Applied and environmental microbiology.

[23]  H. Holms,et al.  Flux analysis and control of the central metabolic pathways in Escherichia coli. , 1996, FEMS microbiology reviews.

[24]  M. J. Teixeira De Mattos,et al.  Growth yield and energy distribution , 1996 .

[25]  T. Ferenci,et al.  Adaptation to life at micromolar nutrient levels: the regulation of Escherichia coli glucose transport by endoinduction and cAMP. , 1996, FEMS microbiology reviews.

[26]  M. Yarmush,et al.  Numerical isotopomer analysis: estimation of metabolic activity. , 1997, Analytical biochemistry.

[27]  J. Villadsen,et al.  Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices. , 1997, Biotechnology and bioengineering.

[28]  W. Wiechert,et al.  Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments. , 1997, Biotechnology and bioengineering.

[29]  U. Sauer,et al.  Metabolic fluxes in riboflavin-producing Bacillus subtilis , 1997, Nature Biotechnology.

[30]  W. Wiechert,et al.  Bidirectional reaction steps in metabolic networks: II. Flux estimation and statistical analysis. , 1997, Biotechnology and bioengineering.

[31]  F. Bolivar,et al.  Stimulation of glucose catabolism through the pentose pathway by the absence of the two pyruvate kinase isoenzymes in Escherichia coli. , 1998, Biotechnology and bioengineering.

[32]  T Szyperski,et al.  13C-NMR, MS and metabolic flux balancing in biotechnology research , 1998, Quarterly Reviews of Biophysics.

[33]  T. Conway,et al.  What’s for Dinner?: Entner-Doudoroff Metabolism inEscherichia coli , 1998, Journal of bacteriology.

[34]  U. Sauer,et al.  Bioreaction network topology and metabolic flux ratio analysis by biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy. , 1999, Metabolic engineering.

[35]  U. Sauer,et al.  Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism , 1999, Journal of bacteriology.

[36]  W Wiechert,et al.  Bidirectional reaction steps in metabolic networks: IV. Optimal design of isotopomer labeling experiments. , 1999, Biotechnology and bioengineering.

[37]  Jens Nielsen,et al.  Metabolic Network Analysis , 1999 .

[38]  J Villadsen,et al.  Quantification of intracellular metabolic fluxes from fractional enrichment and 13C-13C coupling constraints on the isotopomer distribution in labeled biomass components. , 1999, Metabolic engineering.

[39]  Thomas Szyperski,et al.  Amino Acid Biosynthesis in the Halophilic ArchaeonHaloarcula hispanica , 1999, Journal of bacteriology.

[40]  J E Bailey,et al.  Glucose catabolism of Escherichia coli strains with increased activity and altered regulation of key glycolytic enzymes. , 1999, Metabolic engineering.

[41]  N. Bruce,et al.  The udhA Gene of Escherichia coli Encodes a Soluble Pyridine Nucleotide Transhydrogenase , 1999, Journal of bacteriology.

[42]  G. Stephanopoulos.  Metabolic fluxes and metabolic engineering. , 1999, Metabolic engineering.

[43]  J. Portais,et al.  NMR in microbiology: theory and applications. , 2000 .

[44]  B. Palsson,et al.  The Escherichia coli MG1655 in silico metabolic genotype: its definition, characteristics, and capabilities. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Peter D. Karp,et al.  The EcoCyc and MetaCyc databases , 2000, Nucleic Acids Res..

[46]  W. Wiechert,et al.  In Vivo Quantification of Parallel and Bidirectional Fluxes in the Anaplerosis of Corynebacterium glutamicum * , 2000, The Journal of Biological Chemistry.

[47]  U. Sauer,et al.  Altered regulation of pyruvate kinase or co-overexpression of phosphofructokinase increases glycolytic fluxes in resting Escherichia coli. , 2000, Biotechnology and bioengineering.

[48]  M. Riley,et al.  Interim report on genomics of Escherichia coli. , 2000, Annual review of microbiology.

[49]  U. Sauer,et al.  Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA. , 2001, FEMS Microbiology Letters.

[50]  P. Verheijen,et al.  Possible pitfalls of flux calculations based on (13)C-labeling. , 2001, Metabolic engineering.

[51]  J. Guest,et al.  Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli. , 2001, Microbiology.

[52]  U. Sauer,et al.  Stoichiometric growth model for riboflavin-producing Bacillus subtilis. , 2001, Biotechnology and bioengineering.

[53]  W. Wiechert.  13C metabolic flux analysis. , 2001, Metabolic engineering.

[54]  J E Bailey,et al.  Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. , 2001, Biotechnology and bioengineering.

[55]  U. Sauer,et al.  Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids. , 2001, European journal of biochemistry.

[56]  Uwe Sauer,et al.  Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture , 2001, Journal of bacteriology.

[57]  R. Takors,et al.  Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques. , 2001, Analytical biochemistry.

[58]  A. D. de Graaf,et al.  Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13)C labeling and NMR spectroscopy. , 2002, Metabolic Engineering.

[59]  G. Stephanopoulos,et al.  Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction , 2000, Biotechnology and bioengineering.