Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities.
An integrated study on cell growth, enzyme activities and carbon flux redistribution was made to investigate how the central metabolism of Escherichia coli changes with the knockout of genes in the oxidative pentose phosphate pathway (PPP). Mutants deficient in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were constructed by disrupting the zwf and gnd genes and were grown in minimal media with two different carbon sources, such as glucose or pyruvate. It was shown that the knockout of either gnd or zwf gene did not affect the cell growth rate significantly, but the cellular metabolism was changed. While the specific substrate uptake rate and the specific carbon dioxide evolution rate for either mutant grown on glucose were higher than those obtained for the parent strain, these two rates were markedly decreased in mutants grown on pyruvate. The measurement of enzyme activities implied a significant change in metabolism, when alternative pathways such as the Entner-Doudoroff pathway (EDP) and the malic enzyme pathway were activated in the gnd mutant grown on glucose. As compared with the parent strain, the activities of phosphoglucose isomerase were increased in mutants grown on glucose but decreased in mutants grown on pyruvate. The metabolic flux redistribution obtained based on 13C-labeling experiments further indicated that the direction of the flux through the non-oxidative PPP was reversed in response to the gene knockout. Moreover, the knockout of genes caused an increased flux through the tricarboxlic acid cycle in mutants grown on glucose but caused a decrease in the case of using pyruvate. There was also a negative correlation between the fluxes through malic enzyme and isocitrate dehydrogenase in the mutants; and a positive correlation was found between the fluxes through malic enzyme and phosphoenolpyruvate carboxylase.
Zhao
J
J
Metabolome Unit, Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
Baba
T
T
Mori
H
H
Shimizu
K
K
D050260:Carbohydrate Metabolism; D002245:Carbon Dioxide / Q000032:analysis / Q000378:metabolism; D002952:Citric Acid Cycle; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005947:Glucose / Q000378:metabolism; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D006019:Glycolysis; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D010427:Pentose Phosphate Pathway; D010734:Phosphogluconate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism
D003470:Culture Media; D002245:Carbon Dioxide; D019289:Pyruvic Acid; D008291:Malate Dehydrogenase; D007521:Isocitrate Dehydrogenase; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D005956:Glucose-6-Phosphate Isomerase; D005947:Glucose