PhysioFit: a software to quantify cell growth parameters and extracellular fluxes

Le Grégam, Loïc; Guitton, Yann; Bellvert, Floriant; Heux, Stéphanie; Jourdan, Fabien; Portais, Jean-Charles; Millard, Pierre

doi:10.1101/2023.10.12.561695

Cited by 4 publications

(4 citation statements)

References 14 publications

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“…The exometabolome was analysed by 1 H-NMR and extracellular uptake and production fluxes were calculated from these data using PhysioFit 41 (Table 3; Fig.S6C-D). With NH 4 Cl as sole N source, glycerol was fully assimilated and no by-products were detected.…”

Section: Resultsmentioning

confidence: 99%

“…Glycerol and ethanolamine uptake fluxes, acetate and ethanol production fluxes, and growth rates were calculated from glycerol, ethanolamine, acetate, ethanol, and biomass concentrationtime profiles using PhysioFit (v1.0.1 41 , https://github.com/MetaSys-LISBP/PhysioFit). Ethanol evaporation was considered when calculating ethanol production flux, as detailed in Peiro et al 93 using an evaporation constant of 0.0379 h -1 that was determined experimentally.…”

Section: Methodsmentioning

confidence: 99%

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Integrativein vivoanalysis of the ethanolamine utilization bacterial microcompartment inEscherichia coli.

Jallet,

Soldan,

Shayan

et al. 2024

Preprint

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Bacterial microcompartments (BMCs) are self-assembling protein megacomplexes that encapsulate metabolic pathways. Although approximately 20% of sequenced bacterial genomes contain operons encoding putative BMCs, few have been thoroughly characterized, nor any in the most studiedEscherichia colistrains. We used an interdisciplinary approach to gain deep molecular and functional insights into the ethanolamine utilization (Eut) BMC system encoded by the eut operon inE. coliK-12. Theeutgenotype was linked with the ethanolamine utilization phenotype using deletion and overexpression mutants. The subcellular dynamics and morphology of theE. coliEut BMC were characterized in cellula by fluorescence microscopy and electron (cryo)microscopy. The minimal proteome reorganization required for ethanolamine utilization and thein vivostochiometric composition of the Eut BMC were determined by quantitative proteomics. Finally, the first flux map connecting the Eut BMC with central metabolism in cellula was obtained by genome scale modelling and 13C-fluxomics. Our results reveal that, contrary to previous suggestions, ethanolamine serves both as a nitrogen and a carbon source inE. coliK-12, while also contributing significant metabolic overflow. Overall, this study provides a quantitative molecular and functional understanding of the BMCs involved in ethanolamine assimilation by E. coli.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Integrativein vivoanalysis of the ethanolamine utilization bacterial microcompartment inEscherichia coli.

Jallet,

Soldan,

Shayan

et al. 2024

Preprint

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“…Extracellular fluxes ( i.e. glucose uptake, GNT accumulation and growth rates) were determined from the time course concentrations of biomass, substrates, and products using PhysioFit v2.0.4 39 (https://github.com/MetaSys-LISBP/PhysioFit).…”

Section: Methodsmentioning

confidence: 99%

“…-1 ) 7.77 ± 0.17 8.52 ± 0 39. 10.11 ± 0.83 7.60 ± 0.08 qAce (mmol • [gCDW • h] -1 ) 2.37 ± 0.15 4.56 ± 0.16 7.60 ± 0.31 3.95 ± 0.16 BY-NC-ND 4.0 International license perpetuity.…”

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confidence: 99%

6-Phosphogluconolactonase is critical for the efficient functioning of the pentose phosphate pathway

Phégnon,

Pérochon,

Uttenweiler-Joseph

et al. 2023

Preprint

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The metabolic networks of microorganisms are remarkably robust to genetic and environmental perturbations. This robustness stems from redundancies such as gene duplications, isoenzymes, alternative metabolic pathways, and also from non-enzymatic reactions. In the oxidative branch of the pentose-phosphate pathway (oxPPP), 6-phosphogluconolactone hydrolysis into 6-phosphogluconate is catalysed by 6-phosphogluconolactonase (Pgl) but in the absence of the latter, the oxPPP flux is thought to be maintained by spontaneous hydrolysis. However, in 11pgl Escherichia coli, an extracellular pathway can also contribute to pentose-phosphate synthesis. This raises question as to whether the non-enzymatic reaction can compensate for the absence of 6-phosphogluconolactonase and, ultimately, on the role of 6-phosphogluconolactonase in central metabolism. Our results indicate that in the absence of Pgl, this bypass pathway accounts for the entire flux into the oxPPP, suggesting that non-enzymatic hydrolysis does not compensate for the absence of Pgl and demonstrating that Pgl is critical for an efficiently functioning oxPPP.

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6‐Phosphogluconolactonase is critical for the efficient functioning of the pentose phosphate pathway

Phégnon,

Pérochon,

Uttenweiler‐Joseph

et al. 2024

The FEBS Journal

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The metabolic networks of microorganisms are remarkably robust to genetic and environmental perturbations. This robustness stems from redundancies such as gene duplications, isoenzymes, alternative metabolic pathways, and also from non‐enzymatic reactions. In the oxidative branch of the pentose phosphate pathway (oxPPP), 6‐phosphogluconolactone hydrolysis into 6‐phosphogluconate is catalysed by 6‐phosphogluconolactonase (Pgl) but in the absence of the latter, the oxPPP flux is thought to be maintained by spontaneous hydrolysis. However, in Δpgl Escherichia coli, an extracellular pathway can also contribute to pentose phosphate synthesis. This raises question as to whether the intracellular non‐enzymatic reaction can compensate for the absence of 6‐phosphogluconolactonase and, ultimately, on the role of 6‐phosphogluconolactonase in central metabolism. Our results validate that the bypass pathway is active in the absence of Pgl, specifically involving the extracellular spontaneous hydrolysis of gluconolactones to gluconate. Under these conditions, metabolic flux analysis reveals that this bypass pathway accounts for the entire flux into the oxPPP. This alternative metabolic route—partially extracellular—sustains the flux through the oxPPP necessary for cell growth, albeit at a reduced rate in the absence of Pgl. Importantly, these findings imply that intracellular non‐enzymatic hydrolysis of 6‐phosphogluconolactone does not compensate for the absence of Pgl. This underscores the crucial role of Pgl in ensuring the efficient functioning of the oxPPP.

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PhysioFit: a software to quantify cell growth parameters and extracellular fluxes

Cited by 4 publications

References 14 publications

Integrativein vivoanalysis of the ethanolamine utilization bacterial microcompartment inEscherichia coli.

Integrativein vivoanalysis of the ethanolamine utilization bacterial microcompartment inEscherichia coli.

6-Phosphogluconolactonase is critical for the efficient functioning of the pentose phosphate pathway

6‐Phosphogluconolactonase is critical for the efficient functioning of the pentose phosphate pathway

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