Effect of zwf gene knockout on the metabolism of Escherichia coli grown on glucose or acetate.

The mutant deficient in glucose-6-phosphate dehydrogenase (G6PDH) was constructed by disrupting zwf gene by one-step inactivation protocol using polymerase chain reaction primers. The knockout of zwf gene was shown to have different influence on the metabolism of Escherichia coli grown on glucose or acetate. The decreased rates of substrate uptake and CO(2) production were found for the mutant grown on acetate, whereas these two rates were increased during the growth on glucose. The metabolic flux analysis based on (13)C-labeling experiments indicates that the metabolism of the mutant grown on glucose is related to the higher flux via tricorboxylic acid (TCA) cycle to generate anabolic reducing equivalents normally provided by the oxidative pentose phosphate pathway. However, the metabolism of the mutant grown on acetate shows a lower flux towards the TCA cycle as compared with the parent strain. The decreased flux through TCA cycle is associated with an increased flux via the glyoxylate shunt, by which the carbon source can bypass the two decarboxylative steps of TCA cycle in which CO(2) is released, thus conserving more carbon for biosynthesis in response to the decreased uptake rate of the carbon source.

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

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

[3]  F. Neidhardt,et al.  Physiology of the bacterial cell : a molecular approach , 1990 .

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

[5]  D. Fraenkel,et al.  Sugar Metabolism in Transketolase Mutants of Escherichia coli , 1974, Journal of bacteriology.

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

[7]  J. Nielsen,et al.  Quantitative analysis of metabolic fluxes in Escherichia coli, using two-dimensional NMR spectroscopy and complete isotopomer models. , 1999, Journal of biotechnology.

[8]  D. Fraenkel,et al.  Selection of Escherichia coli Mutants Lacking Glucose-6-Phosphate Dehydrogenase or Gluconate-6-Phosphate Dehydrogenase , 1968, Journal of bacteriology.

[9]  K. Shimizu,et al.  Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method. , 2003, Journal of biotechnology.

[10]  U. Sauer,et al.  Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli , 2002, Journal of bacteriology.

[11]  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.

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

[13]  W. Lee,et al.  Mass isotopomer analysis: theoretical and practical considerations. , 1991, Biological mass spectrometry.

[14]  A J Sinskey,et al.  Metabolic and physiological studies of Corynebacterium glutamicum mutants. , 1997, Biotechnology and bioengineering.

[15]  G. Sprenger.  Genetics of pentose-phosphate pathway enzymes ofEscherichia coli K-12 , 1995, Archives of Microbiology.

[16]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[17]  Katy C. Kao,et al.  Global Expression Profiling of Acetate-grown Escherichia coli * , 2002, The Journal of Biological Chemistry.

[18]  P. Kötter,et al.  Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress , 1996, Molecular and General Genetics MGG.

[19]  Ronald W. Davis,et al.  Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. , 1999, Science.

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

[21]  H Sahm,et al.  Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing , 1996, Biotechnology and bioengineering.

[22]  D. Fraenkel,et al.  Transketolase Mutants of Escherichia coli , 1969, Journal of bacteriology.

[23]  M. Winkler,et al.  An Escherichia coli K-12 tktA tktB mutant deficient in transketolase activity requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth , 1994, Journal of bacteriology.

[24]  D. Koshland,et al.  Branch point control by the phosphorylation state of isocitrate dehydrogenase. A quantitative examination of fluxes during a regulatory transition. , 1985, The Journal of biological chemistry.

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

[26]  E. Heinzle,et al.  Mass spectrometry for metabolic flux analysis. , 1999, Biotechnology and bioengineering.

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