scholarly journals Metabolic Flux Hierarchy Prioritizes the Entner-Doudoroff Pathway for Carbohydrate Co-Utilization inPseudomonas protegensPf-5

2018 ◽  
Author(s):  
Rebecca A. Wilkes ◽  
Caroll M. Mendonca ◽  
Ludmilla Aristilde
Keyword(s):  
Metabolic Network ◽  
Carbohydrate Metabolism ◽  
Metabolic Pathways ◽  
Metabolic Flux ◽  
Gene Annotation ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Environmental Matrices ◽  
Fructose 1,6 Bisphosphate ◽  
Kinase Phosphorylation

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. Similar to otherPseudomonasspecies,P. protegensPf-5 relied primarily on the Entner-Doudoroff (ED) pathway to connect initial glucose catabolism to downstream metabolic pathways. Flux quantitation determined that, in lieu of the direct phosphorylation of glucose by glucose kinase, phosphorylation of oxidized products of glucose (gluconate and 2-ketogluconate) towards the ED pathway accounted for over 90% of consumed glucose and greater than 35% of consumed glucose was secreted as gluconate and 2-ketogluconate. Consistent with the lack of annotated pathways for the initial catabolism of pentoses and galactose inP. protegensPf-5, 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 the hexoses was evident but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism toward fructose 6-phosphate, metabolite labeling patterns revealed that mannose-derived carbons specifically entered central carbon metabolism via fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were found to cycle backward through the upper Emden-Meyerhof-Parnas pathway to feed into the ED pathway. Therefore, the operational metabolic network for processing carbohydrates inP. protegensPf-5 prioritizes flux through the ED pathway to channel carbons to downstream metabolic 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 inP. protegensPf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through the network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the gene annotation of two different pathways 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 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 Phosphoketolase Pathway Dominates in Lactobacillus reuteri ATCC 55730 Containing Dual Pathways for Glycolysis

2007 ◽  
Vol 190 (1) ◽  
pp. 206-212 ◽  
Author(s):  
Emma Årsköld ◽  
Elke Lohmeier-Vogel ◽  
Rong Cao ◽  
Stefan Roos ◽  
Peter Rådström ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Metabolic Flux ◽  
Lactobacillus Reuteri ◽  
Intermediate Stage ◽  
Exponential Growth Phase ◽  
Flux Analysis ◽  
Atp Production ◽  
Phosphoketolase Pathway ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic flux analysis indicated that the heterofermentative Lactobacillus reuteri strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of L. reuteri ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-1,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro 31P NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.

scholarly journals Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

2004 ◽  
Vol 70 (12) ◽  
pp. 7277-7287 ◽  
Author(s):  
Christoph Wittmann ◽  
Patrick Kiefer ◽  
Oskar Zelder
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

scholarly journals Atom Identifiers Generated by a Neighborhood-Specific Graph Coloring Method Enable Compound Harmonization across Metabolic Databases

Metabolites ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 368
Author(s):  
Huan Jin ◽  
Joshua M. Mitchell ◽  
Hunter N. B. Moseley
Keyword(s):  
Metabolic Network ◽  
Graph Coloring ◽  
Metabolic Networks ◽  
Metabolic Flux ◽  
Enzyme Commission ◽  
Metabolic Model ◽  
Metabolic Reprogramming ◽  
Automatic Identification ◽  
Flux Analysis ◽  
Database Curation

Metabolic flux analysis requires both a reliable metabolic model and reliable metabolic profiles in characterizing metabolic reprogramming. Advances in analytic methodologies enable production of high-quality metabolomics datasets capturing isotopic flux. However, useful metabolic models can be difficult to derive due to the lack of relatively complete atom-resolved metabolic networks for a variety of organisms, including human. Here, we developed a neighborhood-specific graph coloring method that creates unique identifiers for each atom in a compound facilitating construction of an atom-resolved metabolic network. What is more, this method is guaranteed to generate the same identifier for symmetric atoms, enabling automatic identification of possible additional mappings caused by molecular symmetry. Furthermore, a compound coloring identifier derived from the corresponding atom coloring identifiers can be used for compound harmonization across various metabolic network databases, which is an essential first step in network integration. With the compound coloring identifiers, 8865 correspondences between KEGG (Kyoto Encyclopedia of Genes and Genomes) and MetaCyc compounds are detected, with 5451 of them confirmed by other identifiers provided by the two databases. In addition, we found that the Enzyme Commission numbers (EC) of reactions can be used to validate possible correspondence pairs, with 1848 unconfirmed pairs validated by commonality in reaction ECs. Moreover, we were able to detect various issues and errors with compound representation in KEGG and MetaCyc databases by compound coloring identifiers, demonstrating the usefulness of this methodology for database curation.

scholarly journals 13C-Metabolic Flux Analysis in Developing Flaxseed Embryos to Understand Storage Lipid Biosynthesis

2019 ◽  
Author(s):  
Sebastien Acket ◽  
Anthony Degournay ◽  
Yannick Rossez ◽  
Stephane Mottelet ◽  
Pierre Villon ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Metabolic Network ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Fatty Acid Biosynthesis ◽  
Lipid Biosynthesis ◽  
Flux Analysis ◽  
13C Metabolic Flux Analysis ◽  
Storage Lipid ◽  
Metabolic Systems

Flaxseed (Linum usitatissinum L.) oil is an important source of α-linolenic (C18:3 ω-3), this polyunsaturated fatty acid is well known for its nutritional role in human and animal diet. Understanding storage lipid biosynthesis in developing flaxseed embryos can lead to an increase in seed yield. While a tremendous amount of work has been done on different plant species to highlight their metabolism during embryos development, flaxseed metabolic flux analysis is still lacking. In this context, we have developed an in vitro cultured developing embryos of flaxseed and determined net fluxes by performing three complementary parallel labeling experiments with 13C-labeled glucose and glutamine. Metabolic fluxes were estimated by computer- aided modeling of the central metabolic network including 11 cofactors of 118 reactions of the central metabolism, 12 pseudo fluxes. A focus on lipid storage biosynthesis and the associated pathways was done in comparison with rapeseed, arabidopsis, maize and sunflower embryos. In our conditions, glucose was the main source of carbone of flaxseed embryos, leading to the conversion of phosphoenolpyruvate to pyruvate. The oxidative pentose phosphate pathway (OPPP) was identified as the producer of NADPH for fatty acid biosynthesis. Overall, the use of 13C-metabolic flux analysis provided new insight into flaxseed embryos metabolic processes involved in storage lipids biosynthesis. The elucidation of the metabolic network of this important crop plant reinforces the relevance of the application of this technique to the analysis of complex plant metabolic systems.

scholarly journals Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis

2021 ◽  
Author(s):  
Khushboo Borah ◽  
Tom A. Mendum ◽  
Nathaniel D. Hawkins ◽  
Jane L. Ward ◽  
Michael H. Beale ◽  
...  
Keyword(s):  
Metabolic Network ◽  
Metabolic Flux ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Flux Analysis ◽  
Glyoxylate Shunt ◽  
Citrate Cycle ◽  
Metabolic Fluxes ◽  
The Tca Cycle ◽  
Carbon Substrates

AbstractThe utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates.ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

scholarly journals Quantitative analysis of the physiological contributions of glucose to the TCA cycle

2019 ◽  
Author(s):  
Shiyu Liu ◽  
Ziwei Dai ◽  
Daniel E. Cooper ◽  
David G. Kirsch ◽  
Jason W. Locasale
Keyword(s):  
Quantitative Analysis ◽  
Metabolic Flux ◽  
Isotope Labeling ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Experimental Conditions ◽  
The Tca Cycle ◽  
Metabolic Physiology

ABSTRACTThe carbon source for catabolism in vivo is a fundamental question in metabolic physiology. Limited by data and rigorous mathematical analysis, controversy exists over the nutritional sources for carbon in the tricarboxylic acid (TCA) cycle under physiological settings. Using isotope-labeling data in vivo across several experimental conditions, we construct multiple models of central carbon metabolism and develop methods based on metabolic flux analysis (MFA) to solve for the preferences of glucose, lactate, and other nutrients used in the TCA cycle across many tissues. We show that in nearly all circumstances, glucose contributes more than lactate as a nutrient source for the TCA cycle. This conclusion is verified in different animal strains from different studies, different administrations of 13C glucose, and is extended to multiple tissue types. Thus, this quantitative analysis of organismal metabolism defines the relative contributions of nutrient fluxes in physiology, provides a resource for analysis of in vivo isotope tracing data, and concludes that glucose is the major nutrient used for catabolism in mammals.

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