scholarly journals Comparative13C Metabolic Flux Analysis of Pyruvate Dehydrogenase Complex-Deficient, l-Valine-Producing Corynebacterium glutamicum

2011 ◽  
Vol 77 (18) ◽  
pp. 6644-6652 ◽  
Author(s):  
Tobias Bartek ◽  
Bastian Blombach ◽  
Siegmund Lang ◽  
Bernhard J. Eikmanns ◽  
Wolfgang Wiechert ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Pyruvate Dehydrogenase Complex ◽  
Pentose Phosphate ◽  
Strong Increase ◽  
Flux Analysis ◽  
Wild Type ◽  
Content Type ◽  
Split Ratio

ABSTRACTl-Valine can be formed successfully usingC. glutamicumstrains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-typeC. glutamicumand four PDHC-deficient strains were compared by13C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH forl-valine formation. In accordance, the introduction of theEscherichia colitranshydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into anl-valine-producingC. glutamicumstrain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated forl-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

scholarly journals Combined Fluxomics and Transcriptomics Analysis of Glucose Catabolism via a Partially Cyclic Pentose Phosphate Pathway in Gluconobacter oxydans 621H

2013 ◽  
Vol 79 (7) ◽  
pp. 2336-2348 ◽  
Author(s):  
Tanja Hanke ◽  
Katharina Nöh ◽  
Stephan Noack ◽  
Tino Polen ◽  
Stephanie Bringer ◽  
...  
Keyword(s):  
Phase Ii ◽  
Growth Phase ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Glucose Dehydrogenase ◽  
Gluconobacter Oxydans ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Content Type ◽  
Activity Measurements

ABSTRACTIn this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes inGluconobacter oxydans621H with glucose were studied by13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For13C-MFA, cells were cultivated with specifically13C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. SinceG. oxydanslacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP).13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.

scholarly journals The Entner-Doudoroff and Nonoxidative Pentose Phosphate Pathways Bypass Glycolysis and the Oxidative Pentose Phosphate Pathway in Ralstonia solanacearum

mSystems ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Poonam Jyoti ◽  
Manu Shree ◽  
Chandrakant Joshi ◽  
Tulika Prakash ◽  
Suvendra Kumar Ray ◽  
...  
Keyword(s):  
Network Analysis ◽  
Metabolic Network ◽  
Pentose Phosphate Pathway ◽  
Ralstonia Solanacearum ◽  
Metabolic Flux ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Content Type ◽  
Metabolic Network Analysis ◽  
Balance Analysis

ABSTRACT In Ralstonia solanacearum, a devastating phytopathogen whose metabolism is poorly understood, we observed that the Entner-Doudoroff (ED) pathway and nonoxidative pentose phosphate pathway (non-OxPPP) bypass glycolysis and OxPPP under glucose oxidation. Evidence derived from 13C stable isotope feeding and genome annotation-based comparative metabolic network analysis supported the observations. Comparative metabolic network analysis derived from the currently available 53 annotated R. solanacearum strains, including a recently reported strain (F1C1), representing the four phylotypes, confirmed the lack of key genes coding for phosphofructokinase (pfk-1) and phosphogluconate dehydrogenase (gnd) enzymes that are relevant for glycolysis and OxPPP, respectively. R. solanacearum F1C1 cells fed with [13C]glucose (99% [1-13C]glucose or 99% [1,2-13C]glucose or 40% [13C6]glucose) followed by gas chromatography-mass spectrometry (GC-MS)-based labeling analysis of fragments from amino acids, glycerol, and ribose provided clear evidence that rather than glycolysis and the OxPPP, the ED pathway and non-OxPPP are the main routes sustaining metabolism in R. solanacearum. The 13C incorporation in the mass ions of alanine (m/z 260 and m/z 232), valine (m/z 288 and m/z 260), glycine (m/z 218), serine (m/z 390 and m/z 362), histidine (m/z 440 and m/z 412), tyrosine (m/z 466 and m/z 438), phenylalanine (m/z 336 and m/z 308), glycerol (m/z 377), and ribose (m/z 160) mapped the pathways supporting the observations. The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. IMPORTANCE Understanding the metabolic versatility of Ralstonia solanacearum is important, as it regulates the trade-off between virulence and metabolism (1, 2) in a wide range of plant hosts. Due to a lack of clear evidence until this work, several published research papers reported on the potential roles of glycolysis and the oxidative pentose phosphate pathway (OxPPP) in R. solanacearum (3, 4). This work provided evidence from 13C stable isotope feeding and genome annotation-based comparative metabolic network analysis that the Entner-Doudoroff pathway and non-OxPPP bypass glycolysis and OxPPP during the oxidation of glucose, a component of the host xylem pool that serves as a potential carbon source (5). The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. The study highlights the need to critically examine phytopathogens whose metabolism is poorly understood.

scholarly journals Metabolic flux analysis of wild-type Escherichia coli and mutants deficient in pyruvate-dissimilating enzymes during the fermentative metabolism of glucuronate

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1860-1872 ◽  
Author(s):  
Abhishek Murarka ◽  
James M. Clomburg ◽  
Ramon Gonzalez
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Reducing Power ◽  
Building Blocks ◽  
Exponential Phase ◽  
Flux Analysis ◽  
Wild Type ◽  
Fermentative Metabolism

The fermentative metabolism of d-glucuronic acid (glucuronate) in Escherichia coli was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDH-deficient strain (Pdh−) exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH- and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh− were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : NAD+ ratio supported the proposed role of PDH. Moreover, the NADH : NAD+ ratio in a strain deficient in both PDH and the ox-PPP (Pdh−Zwf−) was even lower than that observed for Pdh−. Strain Pdh−Zwf− also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh−. Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh− mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : NAD+ ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules.

scholarly journals 13C Metabolic Flux Analysis Indicates Endothelial Cells Attenuate Metabolic Perturbations by Modulating TCA Activity

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 226
Author(s):  
Bilal Moiz ◽  
Jonathan Garcia ◽  
Sarah Basehore ◽  
Angela Sun ◽  
Andrew Li ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Isotope Labeling ◽  
Umbilical Vein ◽  
Pentose Phosphate ◽  
Metabolic Inhibitors ◽  
Flux Analysis ◽  
Therapeutic Effects ◽  
13C Metabolic Flux Analysis

Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.

scholarly journals A Cyclic Metabolic Network inPseudomonas protegensPf-5 Prioritizes the Entner-Doudoroff Pathway and Exhibits Substrate Hierarchy during Carbohydrate Co-Utilization

2018 ◽  
Vol 85 (1) ◽  
Author(s):  
Rebecca A. Wilkes ◽  
Caroll M. Mendonca ◽  
Ludmilla Aristilde
Keyword(s):  
Metabolic Network ◽  
Carbohydrate Metabolism ◽  
Metabolic Flux ◽  
Gene Annotation ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Environmental Matrices ◽  
Content Type ◽  
Phosphofructokinase Activity ◽  
Fructose 1,6 Bisphosphate

ABSTRACTThe genetic characterization ofPseudomonas protegensPf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here, we employed13C-tracers and quantitative flux analysis to investigate the intracellular network for carbohydrate metabolism. In lieu of the direct phosphorylation of glucose by glucose kinase, glucose catabolism was characterized primarily by the oxidation of glucose to gluconate and 2-ketogluconate before the phosphorylation of these metabolites to feed the Entner-Doudoroff (ED) pathway. In the absence of phosphofructokinase activity, a cyclic flux from the ED pathway to the upper Embden-Meyerhof-Parnas (EMP) pathway was responsible for routing glucose-derived carbons to the non-oxidative pentose phosphate (PP) pathway. Consistent with the lack of annotated genes inP. protegensPf-5 for the transport or initial catabolism of pentoses and galactose, only glucose was assimilated into intracellular metabolites in the presence of xylose, arabinose, or galactose. However, when glucose was fed simultaneously with fructose or mannose, co-uptake of these hexoses was evident, but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism to fructose-6-phosphate, metabolite labeling patterns revealed that mannose was assimilated into fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were also found to cycle backward through the upper EMP pathway toward the ED pathway. Therefore, the operational metabolic network for processing carbohydrates inP. protegensPf-5 prioritizes flux through the ED pathway to channel carbons to EMP, PP, and downstream pathways.IMPORTANCESpecies of thePseudomonasgenus thrive in various nutritional environments and have strong biocatalytic potential due to their diverse metabolic capabilities. Carbohydrate substrates are ubiquitous both in environmental matrices and in feedstocks for engineered bioconversion. Here, we investigated the metabolic network for carbohydrate metabolism inPseudomonas protegensPf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through a cyclic metabolic network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the annotation of a different pathway in the genome. Elucidation of the constitutive metabolic network inP. protegensis important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untappedPseudomonasspecies.

scholarly journals Impact of CO2/HCO3– Availability on Anaplerotic Flux in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Aileen Krüger ◽  
Johanna Wiechert ◽  
Cornelia Gätgens ◽  
Tino Polen ◽  
Regina Mahr ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Carboxylase ◽  
Metabolic Flux ◽  
Pyruvate Dehydrogenase Complex ◽  
Lag Phase ◽  
Production Rates ◽  
Content Type ◽  
The Impact ◽  
Dehydrogenase Complex

ABSTRACT The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate, yielding acetyl coenzyme A (acetyl-CoA) and CO2. The PDHC-deficient Corynebacterium glutamicum ΔaceE strain therefore lacks an important decarboxylation step in its central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15-h lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates, such as acetate. Growth was successfully restored by deletion of ptsG, encoding the glucose-specific permease of the phosphotransferase system (PTS), thereby linking the observed phenotype to the increased sensitivity of the ΔaceE Δpyc strain to glucose catabolism. In this work, the ΔaceE Δpyc strain was used to systematically study the impact of perturbations of the intracellular CO2/HCO3– pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3– levels, such as external addition of HCO3–, increasing the pH, or rerouting metabolic flux via the pentose phosphate pathway, at least partially eliminated the lag phase of the ΔaceE Δpyc strain on glucose medium. In accordance with these results, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3– pool, led to an even longer lag phase, accompanied by the excretion of l-valine and l-alanine. Transcriptome analysis, as well as an adaptive laboratory evolution experiment with the ΔaceE Δpyc strain, revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose-acetate mixtures. Taken together, our results highlight the significant impact of the intracellular CO2/HCO3– pool on metabolic flux distribution, which becomes especially evident in engineered strains exhibiting low endogenous CO2 production rates, as exemplified by PDHC-deficient strains. IMPORTANCE CO2 is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3– pool in many organisms. In this study, we used a PDHC-deficient strain of Corynebacterium glutamicum, which additionally lacked pyruvate carboxylase (ΔaceE Δpyc). This strain featured a >15-h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3– pool on growth in glucose-acetate medium. Remarkably, all measures enhancing CO2/HCO3– levels successfully restored growth. These results emphasize the strong impact of the intracellular CO2/HCO3– pool on metabolic flux, especially in strains exhibiting low endogenous CO2 production rates.

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