Measuring non-steady-state metabolic fluxes in starch-converting faecal microbiota in vitro

2010 ◽  
Vol 1 (4) ◽  
pp. 391-405 ◽  
Author(s):  
T. Binsl ◽  
A. De Graaf ◽  
K. Venema ◽  
J. Heringa ◽  
A. Maathuis ◽  
...  
Keyword(s):  
Steady State ◽  
Metabolic Flux ◽  
Flux Distribution ◽  
Computational Modelling ◽  
Total Carbon ◽  
Flux Analysis ◽  
Small Contribution ◽  
Faecal Microbiota ◽  
Isotope Labelling

This paper explores human gut bacterial metabolism of starch using a combined analytical and computational modelling approach for metabolite and flux analysis. Non-steady-state isotopic labelling experiments were performed with human faecal microbiota in a well-established in vitro model of the human colon. After culture stabilisation, [U-13C] starch was added and samples were taken at regular intervals. Metabolite concentrations and 13C isotopomeric distributions were measured amongst other things for acetate, propionate and butyrate by mass spectrometry and NMR. The vast majority of metabolic flux analysis methods based on isotopomer analysis published to date are not applicable to metabolic non-steady-state experiments. We therefore developed a new ordinary differential equation-based representation of a metabolic model of human faecal microbiota to determine eleven metabolic parameters that characterised the metabolic flux distribution in the isotope labelling experiment. The feasibility of the model parameter quantification was demonstrated on noisy in silico data using a downhill simplex optimisation, matching simulated labelling patterns of isotopically labelled metabolites with measured metabolite and isotope labelling data. Using the experimental data, we determined an increasing net label influx from starch during the experiment from 94±1 µmol/l/min to 133±3 µmol/l/min. Only about 12% of the total carbon flux from starch reached propionate. Propionate production mainly proceeded via succinate with a small contribution via acrylate. The remaining flux from starch yielded acetate (35%) and butyrate (53%). Interpretation of 13C NMR multiplet signals further revealed that butyrate, valerate and caproate were mainly synthesised via cross-feeding, using acetate as a co-substrate. This study demonstrates for the first time that the experimental design and the analysis of the results by computational modelling allows the determination of time-resolved effects of nutrition on the flux distribution within human faecal microbiota in metabolic non-steady-state.

Metabolic flux analysis of hybridoma continuous culture steady state multiplicity

1999 ◽  
Vol 63 (6) ◽  
pp. 675-683 ◽  
Author(s):  
Brian D. Follstad ◽  
R. Robert Balcarcel ◽  
Gregory Stephanopoulos ◽  
Daniel I. C. Wang
Keyword(s):  
Steady State ◽  
Continuous Culture ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Flux Analysis ◽  
State Multiplicity

Strategies for metabolic flux analysis in plants using isotope labelling

2000 ◽  
Vol 77 (1) ◽  
pp. 81-102 ◽  
Author(s):  
Albrecht Roscher ◽  
Nicholas J. Kruger ◽  
R.George Ratcliffe
Keyword(s):  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Flux Analysis ◽  
Isotope Labelling

scholarly journals Bayesian metabolic flux analysis reveals intracellular flux couplings

2019 ◽  
Vol 35 (14) ◽  
pp. i548-i557 ◽  
Author(s):  
Markus Heinonen ◽  
Maria Osmala ◽  
Henrik Mannerström ◽  
Janne Wallenius ◽  
Samuel Kaski ◽  
...  
Keyword(s):  
Steady State ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Reaction Network ◽  
Reaction Rates ◽  
Supplementary Information ◽  
Flux Analysis ◽  
Metabolic Reaction ◽  
Metabolic Systems ◽  
Genome Scale

AbstractMotivationMetabolic flux balance analysis (FBA) is a standard tool in analyzing metabolic reaction rates compatible with measurements, steady-state and the metabolic reaction network stoichiometry. Flux analysis methods commonly place model assumptions on fluxes due to the convenience of formulating the problem as a linear programing model, while many methods do not consider the inherent uncertainty in flux estimates.ResultsWe introduce a novel paradigm of Bayesian metabolic flux analysis that models the reactions of the whole genome-scale cellular system in probabilistic terms, and can infer the full flux vector distribution of genome-scale metabolic systems based on exchange and intracellular (e.g. 13C) flux measurements, steady-state assumptions, and objective function assumptions. The Bayesian model couples all fluxes jointly together in a simple truncated multivariate posterior distribution, which reveals informative flux couplings. Our model is a plug-in replacement to conventional metabolic balance methods, such as FBA. Our experiments indicate that we can characterize the genome-scale flux covariances, reveal flux couplings, and determine more intracellular unobserved fluxes in Clostridium acetobutylicum from 13C data than flux variability analysis.Availability and implementationThe COBRA compatible software is available at github.com/markusheinonen/bamfa.Supplementary informationSupplementary data are available at Bioinformatics online.

scholarly journals Compartmentalized Glucose Metabolism in Pseudomonas putida Is Controlled by the PtxS Repressor

2010 ◽  
Vol 192 (17) ◽  
pp. 4357-4366 ◽  
Author(s):  
Abdelali Daddaoua ◽  
Tino Krell ◽  
Carlos Alfonso ◽  
Bertrand Morel ◽  
Juan-Luis Ramos
Keyword(s):  
Pseudomonas Putida ◽  
Metabolic Flux ◽  
Glucose Dehydrogenase ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Titration Calorimetry ◽  
Flux Analysis ◽  
Scanning Calorimetry ◽  
Host Chromosome

ABSTRACT Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent KD (equilibrium dissociation constant) of 15 μM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1°C and 57.6°C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 μM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5′-TGAAACCGGTTTCA-3′. This palindrome overlaps with the RNA polymerase binding site.

Metabolic flux analysis of simultaneous production of vitamin B12 and propionic acid in a coupled fermentation process by Propionibacterium freudenreichii

2021 ◽  
Author(s):  
Yuhan Zhang ◽  
Xiaolian Li ◽  
Ziqiang Wang ◽  
Yunshan Wang ◽  
Yuanyuan Ma ◽  
...  
Keyword(s):  
Propionic Acid ◽  
Fermentation Process ◽  
Metabolic Flux ◽  
Flux Distribution ◽  
Vitamin B ◽  
Flux Analysis ◽  
Propionibacterium Freudenreichii ◽  
Simultaneous Production ◽  
Metabolic Flux Distribution ◽  
New Strategy

Abstract The metabolic processes involved in simultaneous production of vitamin B12 and propionic acid by Propionibacterium freudenreichii are very complicated. To further investigate the regulatory mechanisms of this metabolism, a simplified metabolic network was established. The effects of glucose feeding, propionic acid removal, and 5,6-dimethylbenzimidazole (DMB) addition on the metabolic flux distribution were investigated. The results showed that synthesis of propionic acid can be increased by increasing the metabolic flux through the oxaloacetate and methylmalonyl-CoA branches in the early and middle stages of the coupled fermentation. After DMB addition, the synthesis of vitamin B12 was significantly enhanced via increased metabolic flux through the δ-aminolevulinate branch, which promoted the synthesis of uroporphyrinogen III, a precursor of vitamin B12. Therefore, the analysis of metabolic flux at key nodes can provide theoretical guidance for the optimization of P. freudenreichii fermentation processes. In an experimental coupled fermentation process, the concentrations of vitamin B12 and propionic acid reached 21.6 and 50.12 g/L respectively, increased by 105.71% and 73.91% compared with batch fermentation, which provides a new strategy for industrial production.

scholarly journals STAT3 governs the HIF-1α response in IL-15 primed human NK cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna Coulibaly ◽  
Sonia Y. Velásquez ◽  
Nina Kassner ◽  
Jutta Schulte ◽  
Maria Vittoria Barbarossa ◽  
...  
Keyword(s):  
Nk Cells ◽  
Metabolic Flux ◽  
Hypoxia Inducible Factor ◽  
Magnetic Bead ◽  
Cytokine Response ◽  
Cell Contact ◽  
Flux Analysis ◽  
Microbial Infection ◽  
Hypoxia Response

AbstractNatural killer (NK) cells mediate innate host defense against microbial infection and cancer. Hypoxia and low glucose are characteristic for these tissue lesions but do not affect early interferon (IFN) γ and CC chemokine release by interleukin 15 (IL-15) primed human NK cells in vitro. Hypoxia inducible factor 1α (HIF-1α) mediates cellular adaption to hypoxia. Its production is supported by mechanistic target of rapamycin complex 1 (mTORC1) and signal transducer and activator of transcription 3 (STAT3). We used chemical inhibition to probe the importance of mTORC1 and STAT3 for the hypoxia response and of STAT3 for the cytokine response in isolated and IL-15 primed human NK cells. Cellular responses were assayed by magnetic bead array, RT-PCR, western blotting, flow cytometry, and metabolic flux analysis. STAT3 but not mTORC1 activation was essential for HIF-1α accumulation, glycolysis, and oxygen consumption. In both primed normoxic and hypoxic NK cells, STAT3 inhibition reduced the secretion of CCL3, CCL4 and CCL5, and it interfered with IL-12/IL-18 stimulated IFNγ production, but it did not affect cytotoxic granule degranulation up on target cell contact. We conclude that IL-15 priming promotes the HIF-1α dependent hypoxia response and the early cytokine response in NK cells predominantly through STAT3 signaling.

scholarly journals Lipid availability influences the metabolic maturation of human pluripotent stem cell-derived cardiomyocytes

2020 ◽  
Author(s):  
Hui Zhang ◽  
Mehmet G. Badur ◽  
Sean Spiering ◽  
Ajit Divakaruni ◽  
Noah E. Meurs ◽  
...  
Keyword(s):  
Stem Cell ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Pluripotent Stem Cell ◽  
Metabolic Flux ◽  
Acid Oxidation ◽  
Flux Analysis ◽  
Human Pluripotent Stem Cell

AbstractObjectivesPluripotent stem cell-derived cardiomyocytes are phenotypically immature, which limits their utility in downstream applications. Metabolism is dramatically reprogramed during cardiac maturation in vivo and presents a potential avenue to drive in vitro maturation. We aimed to identify and address metabolic bottlenecks in the generation of human pluripotent stem cell (hPSC)-derived cardiomyocytes.MethodshPSCs were differentiated into cardiomyocytes using an established, chemically-defined differentiation protocol. We applied 13C metabolic flux analysis (MFA) and targeted transcriptomics to characterize cardiomyocyte metabolism in during differentiation in the presence or absence of exogenous lipids.ResultshPSC-derived cardiomyocytes induced some cardiometabolic pathways (i.e. ketone body and branched-chain amino acid oxidation) but failed to effectively activate fatty acid oxidation. MFA studies indicated that lipid availability in cultures became limited during differentiation, suggesting potential issues with nutrient availability. Exogenous supplementation of lipids improved cardiomyocyte morphology, mitochondrial function, and promoted increased fatty acid oxidation in hPSC-derivatives.ConclusionhPSC-derived cardiomyocytes are dependent upon exogenous sources of lipids for metabolic maturation. Proper supplementation removes a potential roadblock in the generation of metabolically mature cardiomyocytes. These studies further highlight the importance of considering and exploiting metabolic phenotypes in the in vitro production and utilization of functional hPSC-derivatives.

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