Chasing the flux

915 views

Published on

Chasing the flux: selecting target pathways through flux analysis of carbon metabolism

My presentation @ COBRA 2011 conference

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
915
On SlideShare
0
From Embeds
0
Number of Embeds
5
Actions
Shares
0
Downloads
14
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide
  • Setpoints from the exometabolomic measurements used for FBA results
  • A sample of what kind of results we are getting and how these are used to select pathways
  • A sample of what kind of results we are getting and how these are used to select pathways
  • We will also use C13 measuremements to measure internal flux
  • The graph of No of enzymes relative to Carbon flux
  • The graph of No of enzymes relative to Carbon flux
  • The graph of No of enzymes relative to Carbon flux
  • Chasing the flux

    1. 1. Chasing the flux: selecting target pathways through flux analysis of carbon metabolism Vangelis Simeonidis COBRA 2011, Reykjavik 26 June 2011
    2. 2. Luxembourg Centre for Systems Biomedicine
    3. 3. LCSB new building
    4. 4. Mechanisms of Parkinson´s Disease Disease networks Gene-environment interactions Personalized Medicine Transcriptomics Proteomics Metabolomics Animal models Human genetics Chemical biology Imaging LCSB strategy http://wwwen.uni.lu/lcsb/ Bioinformatics Modelling and simulation Computational biology
    5. 5. Cell growth Protein purification Enzyme kinetics Quantitative proteomics Quantitative metabolomics SBML model Parameters (K M , K cat ) Variables (metabolite and protein concentrations) MCISB
    6. 6. Kinetic modelling Teusink et al. glycolysis model (Eur J Biochem 267:5313, 2000) aims to characterize fully the mechanics of each enzymatic reaction
    7. 7. Consensus yeast model Nature Biotechnology 26, 1155 - 1160 (2008) A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology Markus J Herrgård, Neil Swainston et al.
    8. 8. Challenge Which enzymes?
    9. 9. General strategy <ul><li>Brute-force approach: </li></ul><ul><li>Study all enzymes </li></ul><ul><li>Create complete map </li></ul><ul><li>But: </li></ul><ul><li>Gaps in the network </li></ul><ul><li>Might end up with eg: >90% of enzymes, but <10% of flux </li></ul>
    10. 10. General strategy <ul><li>Flux-centric approach: </li></ul><ul><li>Identify where the carbon flux goes </li></ul><ul><li>Prioritize pathways by ranking higher ones that carry the most flux </li></ul><ul><li>Will end up with eg: 20-30% of enzymes, but >90% of flux </li></ul>
    11. 11. Flux Balance Analysis Stoichiometric Matrix : signifies if and how a metabolite takes part in a certain reaction A B … G r 1 r 2 …. r n a 1 b 1 … . g 1 a 2 b 2 … . g 2 … . … . … . … . a n b n … . g n Flux Vector : Each component represents the flux through the corresponding reaction v 1 v 2 … . v n v dA/dt dB/dt … . dG/dt = Steady State condition 0 0 … . 0 = R n space R m Space Ker( S ) null vector 0 L 1 ≤ v 1 ≤ U 1 L 2 ≤ v 2 ≤ U 2 … .............. L n ≤ v n ≤ U n
    12. 12. Exometabolome measurements Experimental measurements (mmoles/hr/g DW) 1 2 3 AVE C% input flux for FBA Carbon input flux as glucose 67.5 42.1 74.1 61.2 100.00 1.0000 qBiomass 10.3 9.4 8.7 9.5 15.46 0.0234 qCO2 (offgas) 16.0 12.3 20.9 16.4 26.78 1.6070 qEthanol (exometabolome) 29.5 16.9 34.8 27.1 44.20 1.3261 qAcetate (exometabolome) 0.5 0.4 0.5 0.5 0.76 0.0229 qAcetaldehyde (exometabolome) 0.2 0.1 0.2 0.2 0.27 0.0082 qGlycerol (exometabolome) 5.9 6.2 8.1 6.7 11.00 0.2199 qTrehalose (exometabolome) 0.2 0.2 0.2 0.2 0.33 0.0016
    13. 13. Results Name flux glucose_transport__uniport 1.000 hexokinase_D_glucoseATP 1.000 glucose_6_phosphate_isomerase 0.957 fructose_bisphosphate_aldolase 0.931 phosphofructokinase 0.931 glyceraldehyde_3_phosphate_dehydrogenase 1.646 phosphoglycerate_kinase -1.646 enolase 1.437 phosphoglycerate_mutase -1.437 pyruvate_kinase 1.425 pyruvate_decarboxylase 1.349 glycerol_3_phosphate_dehydrogenase_NAD 1.059 glycerol_3_phosphate_dehydrogenase_FAD_mitochondrial 0.850 ethanol_reversible_transport -1.260 alcohol_dehydrogenase_ethanol -1.260 triose_phosphate_isomerase 0.721 CO2_transporter_via_diffusion -1.930 glycine_cleavage_complex_lipoamide_mitochondrial 0.183 phosphoserine_transaminase 0.209 glutamate_dehydrogenase_NADP -0.300 glycine_cleavage_system_lipoamide_irreversible_mitochondrial 0.183 glycine_cleavage_complex_lipoamide_mitochondrial 0.183 acetyl_CoA_hydrolase 0.051
    14. 14. Results biomass production 15.55% D-Glucose exchange 100.00% Glucose-6-phosphate isomerase 100.00% Glucokinase 100.00% glucose-6-phosphate isomerase 95.67% fructose bisphosphate aldolase 93.09% phosphofructokinase 93.09% glyceraldehyde-3-phosphate dehydrogenase 82.32% phosphoglycerate kinase 82.32% enolase 71.93% phosphoglycerate mutase 71.93% pyruvate kinase 71.30% pyruvate decarboxylase 67.39% alcohol dehydrogenase 41.99% Ethanol exchange 41.99% triose phosphate isomerase 36.07% CO2 exchange 32.16% glycine cleavage complex lipoamide 30.70%   .........................................................   ............   .........................................................   ............
    15. 15. Elementary Flux Mode (EFM) analysis the smallest sub-networks that allow a metabolic reconstruction network to function (in steady state)
    16. 16. Which enzymes? GLC DHAP G6P F6P FDP G3P 13PG 3PG 2PG PYR PEP ACALD CO2 ETOH AKG 3PHP PSEP GLU SER GLY CO2 GLYC3P GLYC OAA ASP G1P UDPG 13BDGLCN AC MAN6P MAN1P GDPMANN DOLMANP MANNAN 14GLUN GLYCOGEN
    17. 17. Flux percentage coverage
    18. 18. Targeted selection allows rapid coverage
    19. 19. Future plans <ul><li>The method was used to select/prioritize the enzymes for kinetic models of yeast metabolism in MCISB </li></ul><ul><li>13C measurements of intracellular flux will constrain the problem further and match it to our growing conditions </li></ul><ul><li>Paper in preparation </li></ul><ul><li>Such crucial improvements are a necessary stepping stone for experimental design and to verify predictions </li></ul>
    20. 20. Acknowledgments <ul><li>The MCISB team </li></ul>
    21. 21. Acknowledgments <ul><li>The LCSB team </li></ul>
    22. 22. <ul><li>Thank you! </li></ul><ul><li>[email_address] </li></ul><ul><li>@vangos </li></ul>

    ×