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

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.

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Entner–Doudoroff pathway and Non-OxPPP bypasses glycolysis and OxPPP in Ralstonia solanacearum

10.1101/2020.01.31.929778 ◽

2020 ◽

Author(s):

Poonam Jyoti ◽

Manu Shree ◽

Chandrakant Joshi ◽

Tulika Prakash ◽

Suvendra Kumar Ray ◽

...

Keyword(s):

Network Analysis ◽

Metabolic Network ◽

Ralstonia Solanacearum ◽

Metabolic Flux ◽

Pentose Phosphate ◽

Flux Analysis ◽

Flux Balance ◽

Metabolic Network Analysis ◽

Central Carbon ◽

Balance Analysis

AbstractIn Ralstonia solanacearum, a devastating phytopathogen whose metabolism is poorly understood, we observed that Entner-Doudoroff (ED) pathway and NonOxidative pentose phosphate pathway (OxPPP) bypasses glycolysis and OxPPP under glucose oxidation. Evidences derived from 13C stable isotopes 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 also including the 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]- or 99%[1,2-13C]- or 40%[13C6]-glucose) followed by GC-MS based labelling analysis of fragments from amino acids, glycerol and ribose provided clear evidence that rather than Glycolysis and OxPPP, ED pathway and NonOxPPP are the main routes sustaining metabolism in R. solanacearum. The 13C incorporation in the mass ions of alanine (m/z 260, m/z 232); valine (m/z 288, m/z 260), glycine (m/z 218), serine (m/z 390, m/z 362), histidine (m/z 440, m/z 412), tyrosine (m/z 466, m/z 438), phenylalanine (m/z 336, m/z 308), glycerol (m/z 377) and ribose (m/z 160) mapped the pathways supporting the observations. The outcomes help better defining 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.ImportanceUnderstanding the metabolic versatility of Ralstonia solanacearum is important as it regulates the tradeoff 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 potential roles of Glycolysis and Oxidative pentose phosphate pathways (OxPPP) in R. solanacearum (3, 4). This work provided evidence from 13C stable isotopes feeding and genome annotation based comparative metabolic network analysis that Entner-Doudoroff pathway and Non-OxPPP bypasses glycolysis and OxPPP during the oxidation of Glucose, one of the host xylem pool that serves as a potential carbon source (5). The outcomes help better defining 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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.03414-12 ◽

2013 ◽

Vol 79 (7) ◽

pp. 2336-2348 ◽

Cited By ~ 35

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02084-18 ◽

2018 ◽

Vol 85 (1) ◽

Cited By ~ 7

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.

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Novel Drivers of Virulence in Clostridioides difficile Identified via Context-Specific Metabolic Network Analysis

mSystems ◽

10.1128/msystems.00919-21 ◽

2021 ◽

Author(s):

Matthew L. Jenior ◽

Jhansi L. Leslie ◽

Deborah A. Powers ◽

Elizabeth M. Garrett ◽

Kimberly A. Walker ◽

...

Keyword(s):

Network Analysis ◽

Metabolic Network ◽

Virulence Factors ◽

Metabolic Pathways ◽

Content Type ◽

Hospital Acquired Infections ◽

Metabolic Network Analysis ◽

Clostridioides Difficile ◽

Context Specific ◽

Hospital Acquired

Clostridioides difficile has become the leading single cause of hospital-acquired infections. Numerous studies have demonstrated the importance of specific metabolic pathways in aspects of C. difficile pathophysiology, from initial colonization to regulation of virulence factors.

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Genealogy Profiling through Strain Improvement by Using Metabolic Network Analysis: Metabolic Flux Genealogy of Several Generations of Lysine-Producing Corynebacteria

Applied and Environmental Microbiology ◽

10.1128/aem.68.12.5843-5859.2002 ◽

2002 ◽

Vol 68 (12) ◽

pp. 5843-5859 ◽

Cited By ~ 136

Author(s):

Christoph Wittmann ◽

Elmar Heinzle

Keyword(s):

Mass Spectrometry ◽

Network Analysis ◽

Metabolic Network ◽

Metabolic Flux ◽

Tricarboxylic Acid Cycle ◽

Strain Improvement ◽

Gas Chromatography Mass Spectrometry ◽

Limiting Factor ◽

Strain Optimization ◽

Metabolic Network Analysis

ABSTRACT A comprehensive approach of metabolite balancing, 13C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C3 carboxylation and C4 decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C3 carboxylation and C4 decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.

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Metabolic flux and metabolic network analysis ofPenicillium chrysogenum using 2D [13C,1H] COSY NMR measurements and cumulative bondomer simulation

Biotechnology and Bioengineering ◽

10.1002/bit.10648 ◽

2003 ◽

Vol 83 (1) ◽

pp. 75-92 ◽

Cited By ~ 27

Author(s):

Wouter A. van Winden ◽

Walter M. van Gulik ◽

Dick Schipper ◽

Peter J.T. Verheijen ◽

Preben Krabben ◽

...

Keyword(s):

Network Analysis ◽

Metabolic Network ◽

Metabolic Flux ◽

Metabolic Network Analysis ◽

Cosy Nmr

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

Applied and Environmental Microbiology ◽

10.1128/aem.00575-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6644-6652 ◽

Cited By ~ 51

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.

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Amplified Expression of Fructose 1,6-Bisphosphatase in Corynebacterium glutamicum Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8587-8596.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8587-8596 ◽

Cited By ~ 140

Author(s):

Judith Becker ◽

Corinna Klopprogge ◽

Oskar Zelder ◽

Elmar Heinzle ◽

Christoph Wittmann

Keyword(s):

Corynebacterium Glutamicum ◽

Pentose Phosphate Pathway ◽

Metabolic Flux ◽

Carbon Sources ◽

Pentose Phosphate ◽

Metabolic Effects ◽

Flux Analysis ◽

Lysine Production ◽

Fructose 1,6 Bisphosphatase

ABSTRACT The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfb r . The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.

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

Metabolites ◽

10.3390/metabo11040226 ◽

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.

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