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The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose-and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-13 C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.The central carbon pathways constitute the backbone of cell metabolism by providing energy, building blocks, and reducing power for biomass synthesis. Due to the extensive redundancy and the presence of isozymes, most single-gene knockout mutations in central metabolism do not block cell growth on glucose (13, 18). To reveal gene-phenotype relationships, it is important to gain insight into the complex responses of the metabolic network in its entirety to these mutations. The most important properties of biochemical networks are the per se nonmeasurable in vivo reaction rates, which may be estimated by metabolic flux analysis (41).The most common approach is based on flux balancing of extracellular uptake and secretion rates within a stoichiometric reaction model (26,45). This approach usually requires assumptions about redox or energy balances, and the validity of these assumptions strongly affects the flux estimates. To increase the reliability and resolution of such flux balance analyses, additional information may be obtained from 13 C labeling experiments (43,48). In this approach, the isotope labeling patterns of intracellular metabolites are analyzed by either nuclear magnetic resonance (NMR) or mass spectrometry. The data are then used for identification of the metabolic network structure or for quantification of the intracellular carbon fluxes.Direct analytical interpretation of 13 C labeling patterns may provide direct evidence for a particular flux or reaction (24,34). Recently, a more general method of flux ratio analysis has been developed...

Studies of steady-state metabolic fluxes in.8% of a total of 4,071 genes investigated, especially those involved in amino acid metabolism, central carbon and energy metabolism, transport system and cell envelope, were observed to be differentially expressed between the two nutrient-limited cultures. One important characteristic of E. coli grown under nutrient limitation was its capacity to scavenge carbon or nitrogen from the medium through elevating the expression of the corresponding transport and assimilation genes. The number of differentially expressed genes in faster-growing cells (specific growth rate of 0.55 h ؊1 ), however, decreased to below half of that in slow-growing cells, which could be explained by diverse transcriptional responses to the growth rate under different nutrient limitations. Independent of the growth rate, 92 genes were identified as being differentially expressed. Genes tightly related to the culture conditions were highlighted, some of which may be used to characterize nutrient-limited growth.Microorganisms are capable of varying their metabolic fluxes and sizes of intermediate pools over a wide range in response to changes in environmental conditions. These metabolic responses are usually studied in chemostat cultures, where growth conditions can be well controlled. By designing the composition of the feeding medium carefully, growth in chemostats may be limited by a single or multiple nutrients. Carbon limitation is the most widely used type and has been applied to investigate cell metabolism (9,17,18,30,37), metabolic differences among knockout mutants (11,30,38), metabolic reaction models (7), and so on. Growth in carbondeficient medium may be regarded as limiting energy or catabolism. On the other hand, although energy generation rates are high in carbon-replete cultures, cell yields are usually low, resulting in anabolism limitation and overflow metabolism (9, 18, 23). Other nutrient-limited chemostat cultures include limitation of the nitrogen source, phosphorus, or sulfur, among which nitrogen limitation is the most frequently studied (9, 18, 30, 37). Growth, metabolism and energetics in chemostat cultures under carbon-limited and nitrogen-limited conditions have been investigated for Saccharomyces cerevisiae, Bacillus subtilis, and other microorganisms (5,9,13,17,18). For Escherichia coli, the influences of nutrient limitation on metabolic responses has been investigated in recent studies by using information on isotopomer distribution (11,30). Metabolic changes and kinetic properties during the transition from glucose-excess to glucose-limited growth conditions have also been studied (37).Recently, we developed the method of flux ratio analysis (30) to identify central reaction networks and obtained reliable metabolic fluxes in wild-type W3110 and some knockout mutants of Escherichia coli grown in either glucose-limited or ammonia-limited chemostats (16,38). Steady-state flux information suggested remarkable metabolic alterations in these strains in response to changes of...

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.