SulfoSys

The gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate was compared at 30 C and at 70 C. At 30 C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 C, 1,3- bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. At both temperatures it was possible to convert 3-phosphoglycerate to glyceraldehyde
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An investigation in the central carbon metabolism of S. solfataricus with a focus on the unique temperature adaptations and regulation; using a combined modelling and experimental approach.

The Sulfolobus systems biology (‘‘SulfoSYS’’)-project represented the first (hyper-)thermophilic Systems Biology project, funded within the European trans-national research initiative ‘‘Systems Biology of Microorganisms’’. Within the SulfoSYS-project, focus lies on studying the effect of temperature variation on the central carbohydrate metabolism (CCM) of S. solfataricus that is characterized by the branched Entner–Doudoroff (ED)-like pathway for sugar (glucose, galactose) degradation and the
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PGK-GAPDH reactions were studied in vitro at 30 and 70 using yeast or Sso enzymes

SED-ML:
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig4b/simulate
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig4c/simulate

PGK reaction at 30 C. Yeast PGK was incubated at 30 C, in the presence or absence of the ATP recycling system, and the conversion of 3 PG to BPG was followed.
SED-ML simulations
Fig. 1A: https://jjj.bio.vu.nl/models/experiments/kouril2017_fig1a/simulate

PGK reaction at 70C. Sulfolobus solfataricus PGK was incubated at 70C in presence and absence of an ATP recycling system.
Changes in metabolite concentrations was followed via 31P NMR or enzymatic analyses.

SED-ML:
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig3b/simulate
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig3c/simulate
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig3d/simulate

BPG produced with yeast PGK was incubated at 70 C,upon which BPG rapidly dephosphorylates to 3PG.
SED-ML:
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig2b/simulate

Carbon loss due to instability of gluconeogenic pathway intermediates (BPG, GAP, DHAP) at high temperature in S. solfataricus

Mathematical model of a subset of reactions comprising the three most temperature sensitive intermediates of the gluconeogenic pathway in S. solfataricus

The pilot experiment has been set up in order to develop uniform SOPs for the Sulfosys consortium. It comprises proteomics, transcriptomics, metabolomics as well as enzymatic essays. Cells for all the members of the consortium have been obtained from the same batch fermentations according to fermentation SOP of Sulfosys. The pilot resulted in creating a procedures for all the techniques used in consortium.

Experimental data for the yeast PGK incubations at 30C, with and without recycling of ATP.

Changes in metabolite concentrations were either quantified via 31P NMR or enzymatically

BPG degradation at 70C

Kinetic characterisation of glyceraldehyde 3-phosphate dehydrogenase

Kinetic characterisation of triose phosphate isomerase

Kinetic characterisation of fructose 1,6-bisphosphate aldolase phosphatase

Temperature degradation of BPG, GAP and DHAP

Experimental data for the conversion of 3PG to F6P and the gluconeogenic pathway intermediates

Kinetic characterisation of phosphoglycerate kinase

The pilot experiment was conducted in order to create SOPs and to gain insight in transcriptome of cells grown at 70 and 80C

Samples obtained form the central fermentation facility of Sulfosys have been compared using iTRAQ (isobaric tag for relative and absolute quantification). A pilot experiment resulted in creation of SOP and initial data on cells grown at 70 and 80C

In order to get uniform data from all geographically separated partners in a consortium, central fermentation facility has been set-up. Based on empirical data, standard procedures for growing S. solfataricus cells have been developed.

Based on initial cell material, series of SOPs connected with assays of enzymes involved in Central Carbon Metabolism of S. solfataricus have been developed.

Pilot experiment concerning metabolome of S. solfataricus was conducted in order to acquire SOPs regarding the technique and gain insight on differences in metabolite concentrations at 70 and 80C

A model for the PGK reaction of yeast in presence or absence of the ATP recycling reactions

PGK 70C model

BPG stability analysis

Simulation results of TPI experimental data for GAP and DHAP saturation.

Simulation results of temperature degradation of gluconeogenic intermediates

Simulation results of experimental data of the reconstituted gluconeogenic system

Simulation results of PGK experimental data for ADP, ATP, 3PG and BPG saturation.

Simulation results of FBPAase of experimental data for DHAP and GAP saturation

Simulation results of GAPDH experimental data for BPG, NADPH, NADP, GAP, and Pi

incubations with yeast enzymes at 30C

metabolite quantification via enzymatic analysis

PGK yeast without recycling of ATP

PGK yeast with recycling of ATP

Experimental data for 3PG conversion to fructose-6-phosphase in reconstituted systems of gluconeogenesis of S. solfataricus

Kinetic characterisation of FBPAase. Expermental data for enzyme reaction rates with increasing concentrations of DHAP and GAP.

Kinetic characterisation of TPI. Experimental data for enzyme reaction rates with increasing concentrations of DHAP and GAP, and inhibition with 3PG and PEP.

Kinetic characterisation of GAPDH. Expermental data for enzyme reaction rates with increasing concentrations of BPG, NADPH, NADP, GAP and Pi.

BPG stability notebook

PGK model for S. solfataricus

PGK-GAPDH model Sulfolobus kouril8

PGK-GAPDH model yeast kouril7

PGK-GAPDH models yeast and Sulfolobus Fig. 4 in manuscript

PGK 70C SBML

PGK yeast Fig1a

PGK yeast with/without recycling

Model of reconstituted gluconeogenesis system in S. solfataricus based on the individual kinetic models for PGK, GAPDH, TPI, FBPAase.

Exponential decay model of gluconeogenic intermediates

Mathematical model for TPI kinetics, GAP and DHAP saturation, and inhibition with 3PG and PEP.

Mathematical model for GAPDH kinetics, BPG, NADPH, NADP, GAP and Pi saturation.

Mathematical model for FBPAase kinetics, saturation with DHAP and GAP

Mathematical model for PGK kinetics, ADP, ATP, 3PG and BPG saturation.

The model describes the Entner-Doudoroff pathway in Sulfolobus solfataricus under temperature variation. The package contains source code written in FORTRAN as well as binaries for Mac OSX, Linux, and Windows. If compiling from source code, a FORTRAN compiler is required.
On-line versions of the model are also available at:
http://bioinfo.ux.uis.no/sulfosys
http://jjj.biochem.sun.ac.za/sysmo/projects/Sulfo-Sys/index.html

Preparation of cell free extracts of the recombinant E. coli strains expressing the gluconeogenic S. solfataricus enzymes.

Cloning and heterologous expression of gluconeogenic enzymes from S. solfataricus in E. coli

Purification of gluconeogenic enzymes from S. solfataricus in recombinant E.coli extracts

SOPs related with medium composition, fermentation and stocking of S. solfataricus.

Standard procedure regarding intracellular metabolome analysis using GC-MS.

Protocol for RNA isolation, cDNA synthesis and labeling and hybridization and cleanup of Sulfolobus solfataricus microarray

Standard operating procedures regarding iTraq based proteomics.

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