A Novel Metabolic Cycle Catalyzes Glucose Oxidation and Anaplerosis in Hungry Escherichia coli (original) (raw)
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Microbial Physiology, 2008
The ptsHIcrr operon was deleted from Escherichia coli wild-type JM101 to generate strain PB11 (PTS–). In a mutant derived from PB11 that partially recovered its growth capacity on glucose by an adaptive evolution process (PB12, PTS–Glc+), part of the phosphoenolpyruvate not used in glucose transport has been utilized for the synthesis of aromatic compounds. In this report, it is shown that on acetate as a carbon source, PB11 displayed a specific growth rate (μ) higher than PB12 (0.21 and 0.13 h–1, respectively) while JM101 had a μ of 0.28 h–1. To understand these growth differences on acetate, we compared the expression profiles of central metabolic genes by RT-PCR analysis. Obtained data revealed that some gluconeogenic genes were downregulated in both PTS– strains as compared to JM101, while most glycolytic genes were upregulated in PB12 in contrast to PB11 and JM101. Furthermore, inactivation of gluconeogenic genes, like ppsA, sfcA, and maeB,and poxB gene that codes for pyruvate ...
Journal of Biological Chemistry, 2010
Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of 13 C-labeled and-unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional 13 C, 1 H NMR spectroscopy. Metabolite balancing, biosynthetic 13 C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO 2 and the calculated CO 2 evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO 2 for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO 2 , thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO 2 and H 2) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO 2 generation via PDHC and ox-PPP revealed that the PDHC route is more carbon-and energy-efficient, in agreement with its beneficial role in cell growth.
The PEPâpyruvateâoxaloacetate node as the switch point for carbon flux distribution in bacteria
FEMS Microbiology Reviews, 2005
In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3-(PEP-and/or pyruvate-) carboxylation and C4-(oxaloacetate-and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.
Biotechnology Progress, 1990
Escherichia coli HB101 was grown in complex medium under anaerobic and aerobic conditions. Cells prepared under these two different conditions were characterized by two‐dimensional protein gel electrophoresis, by NMR measurements under identical (anaerobic) conditions, and by measuring the kinetics of glucose uptake and catabolite end‐product appearance in the medium under identical anaerobic conditions. Specific rates of glucose uptake and end‐product formation were significantly greater for the anaerobically grown cells, which also exhibited lower intracellular concentrations of sugar phosphates, nucleoside di‐and triphosphates, UDPG, and NAD(H). Two‐dimensional gel electrophoretic analyses reveal changes in the intracellular levels of proteins involved in pyruvate catabolism that have been observed previously for E. coli grown in minimal medium under aerobic and anaerobic conditions. Enzymes involved in the TCA cycle were not detected in cells grown aerobically or anaerobically i...
Biotechnology Letters, 2006
E. coli is still one of the most commonly used hosts for protein production. However, when it is grown with excess glucose, acetate accumulation occurs. Elevated acetate concentrations have an inhibitory effect on growth rate and recombinant protein yield, and thus elimination of acetate formation is an important aim towards industrial production of recombinant proteins. Here we examine if over-expression of citrate synthase (gltA) or phosphoenolpyruvate carboxylase (ppc) can eliminate acetate production. Knock-out as well as over-expression mutants were constructed and characterized. Knocking out ppc or gltA decreased the maximum cell density by 14% and increased the acetate excretion by 7%, respectively decreased it by 10%. Over-expression of ppc or gltA increased the maximum cell dry weight by 91% and 23%, respectively. No acetate excretion was detected at these increased cell densities (35 g/l and 23 g/l, respectively).
Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli
Journal of Bacteriology, 2002
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.
Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli
Journal of Bacteriology, 2002
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.