Stimulation of glucose catabolism through the pentose pathway by the absence of the two pyruvate kinase isoenzymes inEscherichia coli

Ponce, Elizabeth; Martı́nez, Alfredo; Bolívar, Francisco; Valle, Fernando

doi:10.1002/(sici)1097-0290(19980420)58:2/3<292::aid-bit25>3.0.co;2-d

Cited by 42 publications

(23 citation statements)

References 20 publications

(23 reference statements)

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“…It also exhibits a modest 15 to 25% reduction in growth rate (31,41), indicating that the isozymes are not essential for growth on glucose. In accord with predictions, PB25 exhibits increased HMP pathway and Ppc fluxes (9,33), as does a pykF single mutant (36). For example, the HMP/glycolysis flux ratio in the pykF knockout was found to increase from the wild-type value of 0.5 to 4.0, and its Ppc flux increased 2.6-fold (36).…”

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confidence: 73%

Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

et al. 2009

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Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37°C. At 42°C, its plasmid content was ninefold higher than JM101 at 37°C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp uv , under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.

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confidence: 73%

Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

et al. 2009

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“…Thus, it seems that stimulation of glucose catabolism via the pentose phosphate pathway in PB25 occurs only under some conditions, such as ammonia limitation (Fig. 6), or in nongrowing cells (36). Second, the TCA cycle flux response is inversely correlated with pentose phosphate pathway fluxes, i.e., ammonia-limited PB25 exhibits significantly reduced TCA cycle activity and also reduced acetate formation compared to JM101.…”

Section: Discussionmentioning

confidence: 97%

Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

et al. 2002

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The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13 C labeling experiments with [U-13 C 6 ]glucose in glucoseor 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.The central carbon pathways fulfill anabolic and catabolic functions by providing cofactors and building blocks for macromolecular synthesis as well as energy. While some singlegene knockout mutations in central metabolism preclude growth on glucose, a majority of such variations can potentially be compensated for either by isoenzymes (39) or by a rerouting of carbon fluxes through alternative pathways (7,15,20,42). Usually, the phenotypes of knockout mutants is characterized by quantitative physiological analysis, and conclusions on intracellular metabolism are then based on qualitative interpretation of these results. To reveal cause and effect relationships, however, it is important to gain deeper insight into the complex metabolic responses at the level of intracellular metabolite concentrations and fluxes. These intracellular carbon fluxes, or in vivo reaction rates, are per se nonmeasurable quantities that cannot usually be inferred directly from in vitro enzyme activities because not all in vivo effector concentrations are known. Hence, the intracellular reaction rates are commonly estimated by methods of metabolic flux analysis, which provide a holistic perspective on metabolism (52, 54).The most common approach is based on flux balancing analysis within a stoichiometric model of cellular metabolism (26,41, 50, 52). In this approach, uptake and secretion rates and biosynthetic requirements are balanced in a stoichiometric model, assuming quasi-steady-state material balances on the intermediates. In many cases, however, limited extracellular data and stoichiometric constraints lead to underdetermined systems, so that addition...

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“…Several attempts have been made to increase the in vivo pools of PEP in E. coli. They include the deletion of the PEP-consuming reaction PEP carboxylase (19) or pyruvate kinase (22) and use of either an alternative transport system utilizing ATP as the phosphate donor rather than PEP for the phosphotransferase (PTS) system (10) or an alternative carbon source, such as xylose, which does not rely on the PTS system (20). Although these strategies appeared to increase the level of PEP, the increase was not substantial (still remaining below the detection limit) or was not measured (10).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Growth and Acid Formation in a Bacillus subtilis Pyruvate Kinase Mutant

Fry

Zhu

Domach

et al. 2000

Appl Environ Microbiol

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Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO 2 production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.Acid production is among the important factors that limit process stability and cell concentration and thus cell-based biotechnological processes (e.g., see references 14, 23, and 25). Numerous approaches have been used in an attempt to reduce acid formation. One mechanism involves maintaining low levels of glucose in fed batch reactors (24,27). While this can lead to increased cell mass and product formation, it is a capitalintensive method. Manipulation of the growth media might also be used to reduce acid formation relative to the amount of glucose consumed (13,24).Majewski and Domach (17) used a constrained network analysis of the main metabolic pathways in conjunction with reported measurements of enzymatic activity levels to explain acid production by bacterial cells. This analysis suggested that Escherichia coli and Bacillus subtilis have excess glycolytic capacity relative to the Krebs cycle. This idea is consistent with the notion that given all the anabolic and catabolic tasks that metabolic networks must perform, stoichiometric conflicts and other conflicts arise, leading to imperfect coordination of all tasks. It is thus an overflow or "spilling" of excess carbon that leads to acid production.In experiments using B. subtilis to test the overflow hypothesis, it was found that a small amount of citrate added to glucose minimal medium (0.1 mol of citrate/1 mol of glucose) caused the rate of glucose (or total carbon) use per cell to decline several-fold, while the maximal growth rate was not diminished (12). Further, acid production was undetectable in the glucose-citrate cultures. Subsequent work showed that productivity of recombinant protein (units o...

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Stimulation of glucose catabolism through the pentose pathway by the absence of the two pyruvate kinase isoenzymes inEscherichia coli

Cited by 42 publications

References 20 publications

Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

Characterization of Growth and Acid Formation in a Bacillus subtilis Pyruvate Kinase Mutant

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