Comparison of dynamic responses of cellular metabolites in <Emphasis Type="Italic">Escherichia coli</Emphasis> to pulse addition of substrates

We conducted an integrated study of cell growth parameters, product formation, and the dynamics of intracellular metabolite concentrations using Escherichia coli with genes knocked out in the glycolytic and oxidative pentose phosphate pathway (PPP) for glucose catabolism. We investigated the same characteristics in the wild-type strain, using acetate or pyruvate as the sole carbon source. Dramatic effects on growth parameters and extracellular and intracellular metabolite concentrations were observed after blocking either glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (disruption of pgi gene) or pentose phosphate breakdown of glucose by inactivation of glucose-6-phosphate dehydrogenase (disruption of zwf gene). Reducing power (NADPH) was mainly produced through PPP when the pgi gene was knocked out, while NADPH was produced through the tricarboxylic acid (TCA) cycle by isocitrate dehydrogenase or NADP-linked malic enzyme when the zwf gene was knocked out. As expected, when the pgi gene was knocked out, intracellular concentrations of PPP metabolites were high and glycolytic and concentrations of TCA cycle pathway metabolites were low. In the zwf gene knockout, concentrations of PPP metabolites were low and concentrations of intracellular glycolytic and TCA cycle metabolites were high.     

Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15

Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-d-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735 mg cytidine/l using glucose as substrate, which was approx. 128 % higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2 %) slightly and increased glucose uptake (72 %). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli.     

Improved oxytetracycline production in Streptomyces rimosus M4018 by metabolic engineering of the G6PDH gene in the pentose phosphate pathway

The aromatic polyketide antibiotic, oxytetracycline (OTC), is produced by Streptomyces rimosus as an important secondary metabolite. High level production of antibiotics in Streptomycetes requires precursors and cofactors which are derived from primary metabolism; therefore it is exigent to engineer the primary metabolism. This has been demonstrated by targeting a key enzyme in the oxidative pentose phosphate pathway (PPP) and nicotinamide adenine dinucleotide phosphate (NADPH) generation, glucose-6-phosphate dehydrogenase (G6PDH), which is encoded by zwf1 and zwf2. Disruption of zwf1 or zwf2 resulted in a higher production of OTC. The disrupted strain had an increased carbon flux through glycolysis and a decreased carbon flux through PPP, as measured by the enzyme activities of G6PDH and phosphoglucose isomerase (PGI), and by the levels of ATP, which establishes G6PDH as a key player in determining carbon flux distribution. The increased production of OTC appeared to be largely due to the generation of more malonyl-CoA, one of the OTC precursors, as observed in the disrupted mutants. We have studied the effect of zwf modification on metabolite levels, gene expression, and secondary metabolite production to gain greater insight into flux distribution and the link between the fluxes in the primary and secondary metabolisms.     

Comparative Metabolic Flux Analysis of Lysine-Producing Corynebacterium glutamicum Cultured on Glucose or Fructose

A comprehensive approach to ¹³C tracer studies, labeling measurements by gas chromatography-mass spectrometry, metabolite balancing, and isotopomer modeling, was applied for comparative metabolic network analysis of lysine-producing Corynebacterium glutamicum on glucose or fructose. Significantly reduced yields of lysine and biomass and enhanced formation of dihydroxyacetone, glycerol, and lactate in comparison to those for glucose resulted on fructose. Metabolic flux analysis revealed drastic differences in intracellular flux depending on the carbon source applied. On fructose, flux through the pentose phosphate pathway (PPP) was only 14.4% of the total substrate uptake flux and therefore markedly decreased compared to that for glucose (62.0%). This result is due mainly to (i) the predominance of phosphoenolpyruvate-dependent phosphotransferase systems for fructose uptake (PTS_Fructose) (92.3%), resulting in a major entry of fructose via fructose 1,6-bisphosphate, and (ii) the inactivity of fructose 1,6-bisphosphatase (0.0%). The uptake of fructose during flux via PTS_Mannose was only 7.7%. In glucose-grown cells, the flux through pyruvate dehydrogenase (70.9%) was much less than that in fructose-grown cells (95.2%). Accordingly, flux through the tricarboxylic acid cycle was decreased on glucose. Normalized to that for glucose uptake, the supply of NADPH during flux was only 112.4% on fructose compared to 176.9% on glucose, which might explain the substantially lower lysine yield of C. glutamicum on fructose. Balancing NADPH levels even revealed an apparent deficiency of NADPH on fructose, which is probably overcome by in vivo activity of malic enzyme. Based on these results, potential targets could be identified for optimization of lysine production by C. glutamicum on fructose, involving (i) modification of flux through the two PTS for fructose uptake, (ii) amplification of fructose 1,6-bisphosphatase to increase flux through the PPP, and (iii) knockout of a not-yet-annotated gene encoding dihydroxyacetone phosphatase or kinase activity to suppress overflow metabolism. Statistical evaluation revealed high precision of the estimates of flux, so the observed differences for metabolic flux are clearly substrate specific.     

Network identification and flux quantification of glucose metabolism in Rhodobacter sphaeroides under photoheterotrophic H(2)-producing conditions

The nonsulfur purple bacteria that exhibit unusual metabolic versatility can produce hydrogen gas (H(2)) using the electrons derived from metabolism of organic compounds during photoheterotrophic growth. Here, based on (13)C tracer experiments, we identified the network of glucose metabolism and quantified intracellular carbon fluxes in Rhodobacter sphaeroides KD131 grown under H(2)-producing conditions. Moreover, we investigated how the intracellular fluxes in R. sphaeroides responded to knockout mutations in hydrogenase and poly-β-hydroxybutyrate synthase genes, which led to increased H(2) yield. The relative contribution of the Entner-Doudoroff pathway and Calvin-Benson-Bassham cycle to glucose metabolism differed significantly in hydrogenase-deficient mutants, and this flux change contributed to the increased formation of the redox equivalent NADH. Disruption of hydrogenase and poly-β-hydroxybutyrate synthase resulted in a significantly increased flux through the phosphoenolpyruvate carboxykinase and a reduced flux through the malic enzyme. A remarkable increase in the flux through the tricarboxylic acid cycle, a major NADH producer, was observed for the mutant strains. The in vivo regulation of the tricarboxylic acid cycle flux in photoheterotrophic R. sphaeroides was discussed based on the measurements of in vitro enzyme activities and intracellular concentrations of NADH and NAD(+). Overall, our results provide quantitative insights into how photoheterotrophic cells manipulate the metabolic network and redistribute intracellular fluxes to generate more electrons for increased H(2) production.     

Comparative metabolic flux analysis of lysine-producing Corynebacterium glutamicum cultured on glucose or fructose

A comprehensive approach to (13)C tracer studies, labeling measurements by gas chromatography-mass spectrometry, metabolite balancing, and isotopomer modeling, was applied for comparative metabolic network analysis of lysine-producing Corynebacterium glutamicum on glucose or fructose. Significantly reduced yields of lysine and biomass and enhanced formation of dihydroxyacetone, glycerol, and lactate in comparison to those for glucose resulted on fructose. Metabolic flux analysis revealed drastic differences in intracellular flux depending on the carbon source applied. On fructose, flux through the pentose phosphate pathway (PPP) was only 14.4% of the total substrate uptake flux and therefore markedly decreased compared to that for glucose (62.0%). This result is due mainly to (i) the predominance of phosphoenolpyruvate-dependent phosphotransferase systems for fructose uptake (PTS(Fructose)) (92.3%), resulting in a major entry of fructose via fructose 1,6-bisphosphate, and (ii) the inactivity of fructose 1,6-bisphosphatase (0.0%). The uptake of fructose during flux via PTS(Mannose) was only 7.7%. In glucose-grown cells, the flux through pyruvate dehydrogenase (70.9%) was much less than that in fructose-grown cells (95.2%). Accordingly, flux through the tricarboxylic acid cycle was decreased on glucose. Normalized to that for glucose uptake, the supply of NADPH during flux was only 112.4% on fructose compared to 176.9% on glucose, which might explain the substantially lower lysine yield of C. glutamicum on fructose. Balancing NADPH levels even revealed an apparent deficiency of NADPH on fructose, which is probably overcome by in vivo activity of malic enzyme. Based on these results, potential targets could be identified for optimization of lysine production by C. glutamicum on fructose, involving (i) modification of flux through the two PTS for fructose uptake, (ii) amplification of fructose 1,6-bisphosphatase to increase flux through the PPP, and (iii) knockout of a not-yet-annotated gene encoding dihydroxyacetone phosphatase or kinase activity to suppress overflow metabolism. Statistical evaluation revealed high precision of the estimates of flux, so the observed differences for metabolic flux are clearly substrate specific.     

Metabolic regulation of carbon and electron flow as a function of pH during growth of Sarcina ventriculi

The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO₂ reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent K _m of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S_0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.     

Metabolic flux analysis of<Emphasis Type="Italic"> pykF</Emphasis> gene knockout<Emphasis Type="Italic"> Escherichia coli</Emphasis> based on <Superscript>13</Superscript>C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations

Metabolic flux analysis based on ¹³C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture. In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture. It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF^– mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant. In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant. This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6-phosphate and 6-phosphogluconate, etc. It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF^– mutant. To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracellular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.     

Role of the pentose phosphate pathway and the Entner–Doudoroff pathway in glucose metabolism of Gluconobacter oxydans 621H

Glucose catabolism by the obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H proceeds in two phases comprising rapid periplasmic oxidation of glucose to gluconate (phase I) and oxidation of gluconate to 2-ketogluconate or 5-ketogluconate (phase II). Only a small amount of glucose and part of the gluconate is taken up into the cells. To determine the roles of the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP) for intracellular glucose and gluconate catabolism, mutants defective in either the PPP (Δgnd, Δgnd zwf*) or the EDP (Δedd–eda) were characterized under defined conditions of pH 6 and 15 % dissolved oxygen. In the presence of yeast extract, neither of the two pathways was essential for growth with glucose. However, the PPP mutants showed a reduced growth rate in phase I and completely lacked growth in phase II. In contrast, the EDP mutant showed the same growth behavior as the reference strain. These results demonstrate that the PPP is of major importance for cytoplasmic glucose and gluconate catabolism, whereas the EDP is dispensable. Reasons for this difference are discussed.     

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 effect of<Emphasis Type="Italic"> pfl</Emphasis> gene knockout on the metabolism for optically pure <Emphasis Type="SmallCaps">d</Emphasis>-lactate production by<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The effect of gene knockout on metabolism in the pflA^–, pflB^–, pflC^–, and pflD^– mutants of Escherichia coli was investigated. Batch cultivations of the pfl ^– mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA^– and pflB^– mutants, but not pflC^– and pflD^– mutants, produced large amounts of d-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD⁺ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA^– and pflB^– mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD⁺ ratio in pfl ^– mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.     

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.     

Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum

Zwf (code for glucose-6-phosphate dehydrogenase) and gnd (code for 6-phosphogluconate dehydrogenase) genes from Corynebacterium glutamicum were firstly cloned, and then site-directed mutagenesis was successfully introduced to remove allosteric inhibition by intracellular metabolites. Expression of the mutant zwf and gnd in Bacillus subtilis RH33 resulted in significant enhancement of riboflavin productivity, while the specific growth rate decreased slightly and the specific glucose uptake rate was unchanged. Introduction of the mutant zwf and gnd led to approximately 18% and 22% increased riboflavin production, respectively. An improvement by 31% and 39% of the riboflavin production was obtained by co-expression of the mutated dehydrogenases in shaker flask and fed-batch cultivation. Intracellular metabolites analysis indicated that metabolites detected in pentose phosphate pathway or riboflavin synthesis pathway of engineered strains showed higher concentration, while TCA cycle and glycolysis metabolites detected were lower abundance than that of parent strain.     

Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts

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.     

Effect of <Emphasis Type="Italic">poxB</Emphasis> gene knockout on metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on growth characteristics and enzyme activities

The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O₂ consumption and CO₂ evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.     

Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry

The metabolic fluxes of central carbon metabolism were measured in chemostat-grown cultures of Methylobacterium extorquens AM1 with methanol as the sole organic carbon and energy source and growth-limiting substrate. Label tracing experiments were carried out using 70% (13)C-methanol in the feed, and the steady-state mass isotopomer distributions of amino acids derived from total cell protein were measured by gas chromatography coupled to mass spectrometry. Fluxes were calculated from the isotopomer distribution data using an isotopomer balance model and evolutionary error minimization algorithm. The combination of labeled methanol with unlabeled CO(2), which enters central metabolism in two different reactions, provided the discriminatory power necessary to allow quantification of the unknown fluxes within a reasonably small confidence interval. In wild-type M. extorquens AM1, no measurable flux was detected through pyruvate dehydrogenase or malic enzyme, and very little flux through alpha-ketoglutarate dehydrogenase (1.4% of total carbon). In contrast, the alpha-ketoglutarate dehydrogenase flux was 25.5% of total carbon in the regulatory mutant strain phaR, while the pyruvate dehydrogenase and malic enzyme fluxes remained insignificant. The success of this technique with growth on C(1) compounds suggests that it can be applied to help characterize the effects of other regulatory mutations, and serve as a diagnostic tool in the metabolic engineering of methylotrophic bacteria.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Coordinated reprogramming of metabolism and cell function in adipocytes from proliferation to differentiation

Adipose tissue plays a major role in regulating lipid and energy homeostasis by storing excess nutrients, releasing energetic substrates through lipolysis, and regulating metabolism of other tissues and organs through endocrine and paracrine signaling. Adipocytes within fat tissues store excess nutrients through increased cell number (hyperplasia), increased cell size (hypertrophy), or both. The differentiation of pre-adipocytes into mature lipid-accumulating adipocytes requires a complex interaction of metabolic pathways that is still incompletely understood. Here, we applied parallel labeling experiments and ¹³C-metabolic flux analysis to quantify precise metabolic fluxes in proliferating and differentiated 3T3-L1 cells, a widely used model to study adipogenesis. We found that morphological and biomass composition changes in adipocytes were accompanied by significant shifts in metabolic fluxes, encompassing all major metabolic pathways. In contrast to proliferating cells, differentiated adipocytes 1) increased glucose uptake and redirected glucose utilization from lactate production to lipogenesis and energy generation; 2) increased pathway fluxes through glycolysis, oxidative pentose phosphate pathway and citric acid cycle; 3) reduced lactate secretion, resulting in increased ATP generation via oxidative phosphorylation; 4) rewired glutamine metabolism, from glutaminolysis to de novo glutamine synthesis; 5) increased cytosolic NADPH production, driven mostly by increased cytosolic malic enzyme flux; 6) increased production of monounsaturated C16:1; and 7) activated a mitochondrial pyruvate cycle through simultaneous activity of pyruvate carboxylase, malate dehydrogenase and malic enzyme. Taken together, these results quantitatively highlight the complex interplay between pathway fluxes and cell function in adipocytes, and suggest a functional role for metabolic reprogramming in adipose differentiation and lipogenesis.     

Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement

The physiological and metabolic responses to gnd knockout in Escherichia coli K-12 was quantitatively investigated by using the (13)C tracer experiment (GC-MS/NMR) together with the enzyme activity analysis. It was shown that the general response to the gene knockout was the local flux rerouting via Entner-Doudoroff pathway and the direction reversing via non-oxidative pentose phosphate pathway (PPP). The mutant was found to direct higher flux to phosphoglucose isomerase reaction as compared to the wild-type, but the respiratory metabolism was comparable in both strains. The anaplerotic pathway catalyzed by malic enzyme was identified in the mutant, which was accompanied with an up-regulation of phosphoenolpyruvate carboxylase and down-regulation of phosphoenolpyruvate carboxykinase. The presented results provide first evidence that compensatory mechanism existed in PPP and anaplerotic pathway in response to the gnd deletion.