scholarly journals Dissection of Central Carbon Metabolism of Hemoglobin-ExpressingEscherichia coli by 13C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

2001 ◽  
Vol 67 (2) ◽  
pp. 680-687 ◽  
Author(s):  
Alexander D. Frey ◽  
Jocelyne Fiaux ◽  
Thomas Szyperski ◽  
Kurt Wüthrich ◽  
James E. Bailey ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Carbon Metabolism ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
Distribution Analysis ◽  
E Coli ◽  
13C Nuclear Magnetic Resonance ◽  
Central Carbon ◽  
Nuclear Magnetic

ABSTRACT Escherichia coli MG1655 cells expressingVitreoscilla hemoglobin (VHb), Alcaligenes eutrophus flavohemoprotein (FHP), the N-terminal hemoglobin domain of FHP (FHPg), and a fusion protein which comprises VHb and theA. eutrophus C-terminal reductase domain (VHb-Red) were grown in a microaerobic bioreactor to study the effects of low oxygen concentrations on the central carbon metabolism, using fractional13C-labeling of the proteinogenic amino acids and two-dimensional [13C, 1H]-correlation nuclear magnetic resonance (NMR) spectroscopy. The NMR data revealed differences in the intracellular carbon fluxes between E. coli cells expressing either VHb or VHb-Red and cells expressingA. eutrophus FHP or the truncated heme domain (FHPg).E. coli MG1655 cells expressing either VHb or VHb-Red were found to function with a branched tricarboxylic acid (TCA) cycle. Furthermore, cellular demands for ATP and reduction equivalents in VHb- and VHb-Red-expressing cells were met by an increased flux through glycolysis. In contrast, in E. coli cells expressingA. eutrophus hemeproteins, the TCA cycle is running cyclically, indicating a shift towards a more aerobic regulation. Consistently, E. coli cells displaying FHP and FHPg activity showed lower production of the typical anaerobic by-products formate, acetate, and d-lactate. The implications of these observations for biotechnological applications are discussed.

scholarly journals Oxidative Glucose Metabolism in Rat Brain during Single Forepaw Stimulation: A Spatially Localized 1H[13C] Nuclear Magnetic Resonance Study

1997 ◽  
Vol 17 (10) ◽  
pp. 1040-1047 ◽  
Author(s):  
Fahmeed Hyder ◽  
Douglas L. Rothman ◽  
Graeme F. Mason ◽  
Anand Rangarajan ◽  
Kevin L. Behar ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Glucose Metabolism ◽  
Glucose Oxidation ◽  
Spectroscopic Method ◽  
Tca Cycle ◽  
Blood Oxygenation ◽  
13C Nuclear Magnetic Resonance ◽  
Activated State ◽  
Nuclear Magnetic

In the α-chloralose-anesthetized rat during single forepaw stimulation, a spatially localized 1H[13C] nuclear magnetic resonance spectroscopic method was used to measure the rate of cerebral [C4]-glutamate isotopic turnover from infused [1,6-13C]glucose. The glutamate turnover data were analyzed using a mathematical model of cerebral glucose metabolism to evaluate the tricarboxylic acid (TCA) cycle flux (VTCA). During stimulation the value of VTCA in the sensorimotor region increased from 0.47 ± 0.06 (at rest) to 1.44 ± 0.41 μmol·g−1 min−1 ( P < 0.01) in the contralateral hemispheric compartment (24 mm3) and to 0.65 ± 0.10 μmol·g−1min−1 ( P < 0.03) in the ipsilateral side. Each VTCA value was converted to the cerebral metabolic rates of glucose oxidation ( oxidative-CMRglC) and oxygen consumption (CMRO2). These rates were corrected for partial-volume based on activation maps obtained by blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI). The percent increase and the absolute value of oxidative-CMRglc in the activated regions are similar to values reported previously for total-CMRglc using the same activation paradigm. This indicates that the large majority of energy required for brain activation, in going from the resting to an activated state, is supplied by glucose oxidation. The level of activity during stimulation is relevant to awake animals because the oxidative-CMRglc (1.05 ± 0.28 μmol·g−1·min−1; current study) is in the range of total-CMRglc previously reported for awake rats undergoing physiologic activation (0.7–1.4 μmol·g−1 min−1). It is concluded that oxidative glycolysis is the main source of energy for increased brain activity and a positive BOLD fMRI signal-change occurs in conjunction with a large increase in CMRO2.

scholarly journals Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

1999 ◽  
Vol 181 (21) ◽  
pp. 6679-6688 ◽  
Author(s):  
Uwe Sauer ◽  
Daniel R. Lasko ◽  
Jocelyne Fiaux ◽  
Michel Hochuli ◽  
Ralf Glaser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Metabolism ◽  
Metabolic Flux ◽  
Flux Ratio ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
E Coli ◽  
Chemostat Cultures ◽  
Central Carbon ◽  
Pep Carboxykinase

ABSTRACT The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional13C-1H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. colicells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

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