Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or 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.     

Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli

ABSTRACT: BACKGROUND: In Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS-) and pyruvate kinase isozymes PykA and PykF (PTS- pykA- and PTS- pykF -). To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined. RESULTS: When compared to reference strain W3110, strain VH33 (PTS-) displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc) and PEP carboxykinase (Pck) was detected in all strains. In strains W3110, VH33 (PTS-) and VH35 (PTS-, pykF-), the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation. CONCLUSIONS: Metabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high degree of flexibility to perturbations of the central metabolic network in E. coli. The observed responses to reduced glucose uptake and PEP to pyruvate rate of conversion caused by PTS, PykA and PykF inactivation included flux rerouting in several central metabolism nodes towards anabolic biosynthetic reactions, thus compensating for carbon limitation in these mutant strains. The detected cycle involving Ppc and Pck was found to be required for maintaining the specific growth and glucose consumption rates in all studied strains. Strains VH33 (PTS-), VH34 (PTS- pykA-) and VH35 (PTS- pykF-) have useful properties for biotechnological processes, such as increased PEP availability and high biomass yields from glucose, making them useful for the production of aromatic compounds or recombinant proteins.     

Comparative study on central metabolic fluxes of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains in continuous culture using <Superscript>13</Superscript>C labelled substrates

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by ¹³C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.     

Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis

Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h(-1)) and high (0.44 h(-1)) dilution rates. Using a mixture of 10% [U-(13)C] and 90% glucose labeled at natural abundance, (13)C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [(13)C, (1)H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a chi(2) test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.     

Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments

The lipoamide dehydrogenase (LPD) encoded by lpdA gene is a component of the pyruvate dehydrogenase complex (PDHc), alpha-ketoglutarate dehydrogenase (AKGDH) and the glycine cleavage multi-enzyme (GCV) systems. In the present study, cell growth characteristics, enzyme activities and intracellular metabolite concentrations were compared between the parent strain Escherichia coli BW25113 and its lpdA knockout mutant in batch and continuous cultures. The lpdA knockout mutant produced significantly more pyruvate and L-glutamate under aerobiosis. Some D-lactate and succinate also accumulated in the culture broth. Based on the investigation of enzyme activities and intracellular metabolite concentrations, acetyl-CoA was considered to be formed by the combined reactions through pyruvate oxidase (PoxB), acetyl-CoA synthetase (Acs) and acetate kinase (Ack)-phosphoacetyltransferase (Pta) in the lpdA mutant. The effect of the lpdA gene knockout on the intracellular metabolic flux distributions was investigated based on 1H-13C NMR spectra and GC-MS signals obtained from 13C-labeling experiment using the mixture of [U-13C] glucose, [1-13C] glucose, and naturally labeled glucose. Flux analysis of the lpdA mutant indicated that the Entner-Doudoroff (ED) pathway and the glyoxylate shunt were activated. The fluxes through glycolysis and oxidative pentose phosphate (PP) pathway (except for the flux through glucose-6-phosphate dehydrogenase) were slightly downregulated. The TCA cycle was also downregulated in the mutant strain. On the other hand, the fluxes through the anaplerotic reactions of PEP carboxylase, PEP carboxykinase and malic enzyme were upregulated, which were consistent with the results of enzyme activities. Furthermore, the influence of the poxB gene knockout on the growth of E. coli was also studied because of its similar function to PDHc which connects the glycolysis to the TCA cycle. Under aerobiosis, a comparison of lpdA mutant and poxB mutant indicated that PDHc is the main enzyme which catalyzes the reaction from pyruvate to acetyl-CoA in the parent strain, while PoxB plays a very important role in the PDHc-deficient strain.     

The phosphoenolpyruvate carboxykinase also catalyzes C3 carboxylation at the interface of glycolysis and the TCA cycle of Bacillus subtilis

Quantitative physiological characterization and isotopic tracer experiments revealed that pyruvate kinase mutants of Bacillus subtilis produced significantly more CO(2) from glucose in the tricarboxylic acid cycle than is explained by the remaining conversion of phosphoenolpyruvate (PEP) to pyruvate catalyzed by the phosphotransferase system. We show here that this additional catabolic flux into the tricarboxylic acid cycle was catalyzed by the PEP carboxykinase. In contrast to its normal role in gluconeogenesis, PEP carboxykinase can operate in the reverse direction from PEP to oxaloacetate upon knockout of pyruvate kinase in a riboflavin-producing B. subtilis strain and in wild-type 168. At least in the industrial strain, we demonstrate the additional capacity of PEP carboxykinase to function as a substitute anaplerotic reaction when the normal pyruvate carboxylase is inactivated. Presumably as a consequence of the unfavorable kinetics of an ATP-synthesizing anaplerotic PEP carboxykinase reaction, such pyruvate carboxylase mutants grow slowly or, as in the case of wild-type 168, not at all.     

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe – A quantitative approach using 13C-based metabolic flux analysis

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying ¹³C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.     

Gluconeogenesis is associated with high rates of tricarboxylic acid and pyruvate cycling in fasting northern elephant seals

Animals that endure prolonged periods of food deprivation preserve vital organ function by sparing protein from catabolism. Much of this protein sparing is achieved by reducing metabolic rate and suppressing gluconeogenesis while fasting. Northern elephant seals (Mirounga angustirostris) endure prolonged fasts of up to 3 mo at multiple life stages. During these fasts, elephant seals maintain high levels of activity and energy expenditure associated with breeding, reproduction, lactation, and development while maintaining rates of glucose production typical of a postabsorptive mammal. Therefore, we investigated how fasting elephant seals meet the requirements of glucose-dependent tissues while suppressing protein catabolism by measuring the contribution of glycogenolysis, glycerol, and phosphoenolpyruvate (PEP) to endogenous glucose production (EGP) during their natural 2-mo postweaning fast. Additionally, pathway flux rates associated with the tricarboxylic acid (TCA) cycle were measured specifically, flux through phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate cycling. The rate of glucose production decreased during the fast (F(1,13) = 5.7, P = 0.04) but remained similar to that of postabsorptive mammals. The fractional contributions of glycogen, glycerol, and PEP did not change with fasting; PEP was the primary gluconeogenic precursor and accounted for ～95% of EGP. This large contribution of PEP to glucose production occurred without substantial protein loss. Fluxes through the TCA cycle, PEPCK, and pyruvate cycling were higher than reported in other species and were the most energetically costly component of hepatic carbohydrate metabolism. The active pyruvate recycling fluxes detected in elephant seals may serve to rectify gluconeogeneic PEP production during restricted anaplerotic inflow in these fasting-adapted animals.     

Dynamics of intracellular metabolites of glycolysis and TCA cycle during cell-cycle-related oscillation in Saccharomyces cerevisiae

In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycolysis and TCA cycle during autonomous, cell-cycle synchronized oscillations in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. This study complements previously reported oscillations in carbon dioxide production rate, intracellular concentrations of trehalose and various free amino acids, and extracellular acetate and pyruvate in the same culture. Of the glycolytic intermediates, fructose 1,6-bisphosphate, 2- and 3-phosphoglycerate, and phosphoenolpyruvate show the most pronounced oscillatory behavior, the latter three compounds oscillating out of phase with the former. This agrees with previously observed metabolic control by phosphofructokinase and pyruvate kinase. Although individually not clearly oscillating, several intermediates of the TCA cycle, i.e., alpha-ketoglutarate, succinate, fumarate, and malate, exhibited increasing concentration during the cell cycle phase with high carbon flux through glycolysis and TCA cycle. The average mass action ratios of beta-phosphoglucomutase and fumarase agreed well with previously determined in vitro equilibrium constants. Minor differences resulted for phosphoglucose isomerase and enolase. Together with the observed close correlation of the pool sizes of the involved metabolites, this might indicate that, in vivo, these reactions are operating close to equilibrium, whereby care must be taken due to possible differences between in vivo and in vitro conditions. Combining the data with previously determined intracellular amino acid levels from the same culture, a few clear correlations between catabolism and anabolism could be identified: phosphoglycerate/serine and alpha-ketoglutarate/lysine exhibited correlated oscillatory behavior, albeit with different phase shifts. Oscillations in intracellular amino acids might therefore be, at least partly, following oscillations of their anabolic precursors.     

The YfiD protein contributes to the pyruvate formate-lyase flux in an Escherichia coli arcA mutant strain

The product of yfiD gene is similar to pyruvate formate-lyase (PFL) activase and it has been reported to activate PFL by replacing the glycyl radical domain. To quantitate the effect of YfiD on the cell metabolism in microaerobic cultures, glucose-limited chemostat cultures were conducted with Escherichia coli yfiD mutant and yfiDarcA mutant strains. The microaerobic condition was controlled by purging the culture media with 2.5% O(2) in N(2). The intracellular metabolic flux distributions in these cultures were estimated based on C-13 labeling experiments. By comparing with the flux distributions in wild-type E. coli and the arcA mutant, it was shown that YfiD contributes to about 18% of the PFL flux in the arcA mutant, but it did not contribute to the PFL flux in wild-type E. coli. It was also shown that the cell used both PFL and pyruvate dehydrogenase (PDH) to supplement the acetyl-coenzyme A (AcCoA) pool under microaerobic conditions. The flux through PDH was about 22-30% of the total flux toward AcCoA in the wild-type, the yfiD mutant and yfiDarcA mutant strains. Relatively higher lactate production was seen in the yfiDarcA mutant than the other strains, which was due to the lower total flux through PFL and PDH toward AcCoA in this strain.     

Effect of the global redox sensing/regulation networks on Escherichia coli and metabolic flux distribution based on C-13 labeling experiments

Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. Among them, the one component Fnr protein and the two-component Arc system coordinate the adaptive responses to oxygen availability. To systematically investigate the contribution of Arc- and Fnr-dependent regulation in catabolism, glucose-limited chemostat cultures were conducted on wild-type E. coli, an arcA mutant, an fnr mutant, and an arcAfnr double mutant strains under a well-defined semi-aerobic condition. The metabolic flux distributions of the cultures of these strains were estimated based on C-13 labeling experiments. It was shown that the oxidative pentose phosphate (PP) pathway was functioning at low level under semi-aerobic condition. The fluxes through pyruvate dehydrogenase (PDH) and tricarboxylic acid (TCA) cycle were found to be lower in the arcA mutant and the arcAfnr double mutant strains than that in the wild-type strain, although the expression of the genes involved in these pathways have been proved to be derepressed in the mutant strains ([Shalel-Levanon, S., San, K.Y., Bennett, G.N., 2005a. Effect of ArcA and FNR on the expression of genes related to the oxygen regulation and the glycolysis pathway in Escherichia coli under microaerobic growth conditions. Biotechnol. Bioeng. 92, 147-159; Shalel-Levanon, S., San, K.Y., Bennett, G.N., 2005c. Effect of oxygen, and ArcA and FNR regulators on the expression of genes related to the electron transfer chain and the TCA cycle in Escherichia coli. Metab. Eng. 7, 364-374]). The significantly higher lactate production in the arcAfnr double mutant strain was shown to be an indirect effect caused by the reduced pyruvate formate-lyase (PFL) and PDH fluxes as well as the intracellular redox state.     

Oxygen supply strongly influences metabolic fluxes,the production of poly(3-hydroxybutyrate) and alginate,and the degree of acetylation of alginate in Azotobacter vinelandii

The aim of this study was to evaluate carbon flux in Azotobacter vinelandii using metabolic flux analysis (MFA) under high and low aeration conditions to achieve an improved understanding of how these changes could be related to alginate acetylation and PHB production. Changes in oxygen availability had a considerable impact on the metabolic fluxes and were reflected in the growth rate, the specific glucose consumption rate, and the alginate and PHB yields. The main differences at the metabolic flux level were observed in three important pathways. The first important difference was consistent with respiratory protection; an increase in the flux generated through the tricarboxylic acid (TCA) cycle for cultures grown under high aeration conditions (up to 2.61 times higher) was observed. In the second important difference, the fluxes generated through pyruvate dehydrogenase, phosphoenol pyruvate carboxykinase and pyruvate kinase, all of which are involved in acetyl-CoA metabolism, increased by 10, 43.9 and 17.5%, respectively, in cultures grown under low aeration conditions compared with those grown under high aeration conditions. These changes were related to alginate acetylation, which was 2.6 times higher in the cultures with limited oxygen, and the changes were also related to a drastic increase in PHB production. Finally, the glyoxylate shunt was active under both of the conditions that were tested, and a 2.79-fold increase was observed in cultures that were grown under the low aeration condition.     

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS.

We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13C] and [U-13C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the Entner-Doudoroff pathway in wild-type E. coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover, glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.     

Pathway and sites for energy conservation in the metabolism of glucose by Selenomonas ruminantium.

On the basis of enzyme activities detected in extracts of Selenomonas ruminantium HD4 grown in glucose-limited continuous culture, at a slow (0.11 h-1) and a fast (0.52 h-1) dilution rate, a pathway of glucose catabolism to lactate, acetate, succinate, and propionate was constructed. Glucose was catabolized to phosphoenol pyruvate (PEP) via the Emden-Meyerhoff-Parnas pathway. PEP was converted to either pyruvate (via pyruvate kinase) or oxalacetate (via PEP carboxykinase). Pyruvate was reduced to L-lactate via a NAD-dependent lactate dehydrogenase or oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA) and CO2 by pyruvate:ferredoxin oxidoreductase. Acetyl-CoA was apparently converted in a single enzymatic step to acetate and CoA, with concomitant formation of 1 molecule of ATP; since acetyl-phosphate was not an intermediate, the enzyme catalyzing this reaction was identified as acetate thiokinase. Oxalacetate was converted to succinate via the activities of malate dehydrogenase, fumarase and a membrane-bound fumarate reductase. Succinate was then excreted or decarboxylated to propionate via a membrane-bound methylmalonyl-CoA decarboxylase. Pyruvate kinase was inhibited by Pi and activated by fructose 1,6-bisphosphate. PEP carboxykinase activity was found to be 0.054 mumol min-1 mg of protein-1 at a dilution rate of 0.11 h-1 but could not be detected in extracts of cells grown at a dilution rate of 0.52 h-1. Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase. Possession of these five sites for energy conservation may explain the high yields reported here (56 to 78 mg of cells [dry weight] mol of glucose-1) for S. ruminantium HD4 grown in glucose-limited continuous culture.     

Shewanella oneidensis MR-1 fluxome under various oxygen conditions

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.     

An integrated (2)H and (13)C NMR study of gluconeogenesis and TCA cycle flux in humans

Hepatic glucose synthesis from glycogen, glycerol, and the tricarboxylic acid (TCA) cycle was measured in five overnight-fasted subjects by (1)H, (2)H, and (13)C NMR analysis of blood glucose, urinary acetaminophen glucuronide, and urinary phenylacetylglutamine after administration of [1,6-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]propionate. This combination of tracers allows three separate elements of hepatic glucose production (GP) to be probed simultaneously in a single study: 1) endogenous GP, 2) the contribution of glycogen, phosphoenolpyruvate (PEP), and glycerol to GP, and 3) flux through PEP carboxykinase, pyruvate recycling, and the TCA cycle. Isotope-dilution measurements of [1,6-(13)C(2)] glucose by (1)H and (13)C NMR indicated that GP in 16-h-fasted humans was 10.7 +/- 0.9 micromol.kg(-1).min(-1). (2)H NMR spectra of monoacetone glucose (derived from plasma glucose) provided the relative (2)H enrichment at glucose H-2, H-5, and H-6S, which, in turn, reflects the contribution of glycogen, PEP, and glycerol to total GP (5.5 +/- 0.7, 4.8 +/- 1.0, and 0.4 +/- 0.3 micromol.kg(-1).min(-1), respectively). Interestingly, (13)C NMR isotopomer analysis of phenylacetylglutamine and acetaminophen glucuronide reported different values for PEP carboxykinase flux (68.8 +/- 9.8 vs. 37.5 +/- 7.9 micromol.kg(-1).min(-1)), PEP recycling flux (59.1 +/- 9.8 vs. 27.8 +/- 6.8 micromol.kg(-1).min(-1)), and TCA cycle flux (10.9 +/- 1.4 vs. 5.4 +/- 1.4 micromol.kg(-1).min(-1)). These differences may reflect zonation of propionate metabolism in the liver.     

Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using ¹³C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and ¹³C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with ¹³C NMR for metabolic flux analysis to reduce the use of ¹³C-labeled substrates and to obtain more-accurate flux values.