Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockout.
The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C(6)]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild-type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed.
Yang
Chen
C
Metabolome Unit, Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan.
Hua
Qiang
Q
Baba
Tomoya
T
Mori
Hirotada
H
Shimizu
Kazuyuki
K
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000227:Adenine Nucleotides / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D002952:Citric Acid Cycle; D004734:Energy Metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D006868:Hydrolysis; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007522:Isocitrate Lyase / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D009005:Monosaccharides / Q000378:metabolism; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010427:Pentose Phosphate Pathway; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism; D015219:Substrate Cycling
D000227:Adenine Nucleotides; D000596:Amino Acids; D002247:Carbon Isotopes; D002264:Carboxylic Acids; D009005:Monosaccharides; D002245:Carbon Dioxide; D000105:Acetyl Coenzyme A; D002244:Carbon; D000255:Adenosine Triphosphate; D007521:Isocitrate Dehydrogenase; D010730:Phosphoenolpyruvate Carboxylase; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D007522:Isocitrate Lyase; D005947:Glucose; D010100:Oxygen