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Metabolic Flux Analysis

Metabolic Flux Analysis. Metabolic Flux Analysis of Citric Acid Fermentation by Candida lipolytica Presentation by: Miles Beamguard and Wade Mack September 19, 2001. Case Study.

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Metabolic Flux Analysis

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  1. Metabolic Flux Analysis Metabolic Flux Analysis of Citric Acid Fermentation by Candida lipolytica Presentation by: Miles Beamguard and Wade Mack September 19, 2001

  2. Case Study • Aiba, S. & Matsuoka, M. (1979). Identification of metabolic model: Citrate production from glucose by Candida lipolytica. Biotechnology and Bioengineering. 21, 1373-1386. • Considered the first application of metabolite balancing to fermentation data

  3. Objectives of Presentation • Outline Objectives of Case Study • Analyze their reaction equations using matrix algebra calculations • Discuss the relevance of the matrix analysis approach to metabolite modeling

  4. Objectives of Presentation • Outline Objectives of Case Study • Analyze their reaction equations using matrix algebra calculations • Discuss the relevance of the matrix analysis approach to metabolite modeling

  5. Objectives of Case Study • Analyze the metabolic network • Form reaction equations • Determine some variables through experimental data • Reduce unknowns by a selected model

  6. Metabolic Network Glucose v1 v3 Glucose-6-P Carbohydrates v2 v4 v16 Pyruvate CO2 CO2 CO2 v14 Lipid AcCoA v5 v6 v17 OAA CIT Citrate v7 v11 v13 v12 v18 ICT Isocitrate MAL GOX v10 CO2 v8 SUC OGT v15 v9 Protein CO2

  7. G6P : v1 - v2/2 – v3 = 0 Pyr : v2 – v4 – v5 = 0 AcCoA : v4 – v6 – v13 – v14 = 0 CIT : v6 – v7 – v17 = 0 ICT : v7 – v8 – v12 – v18 = 0 OGT : v8 – v9 – v15 = 0 SUC : v9 – v10 + v12 = 0 MAL : v10 – v11 + v13 = 0 GOX : v12 - v13 = 0 OOA : v5 + v11 – v6 = 0 CO2 : v4 + v8 + v9 – v16 = 0 Reaction Rate Equations

  8. Determining Known Variables • Elimination of v13 due to glyoxylate reaction equal to v12 • 18 reaction rates but only 11 balance equations resulting in 7 degrees of freedom • Measurement within network led to empirical solving for 6 reaction rates.

  9. 6 Measured Reaction Rates • Glucose Uptake Rate (rglc) = v1 • Carbon Dioxide Production Rate (rc) = v16 • Citric Acid Production Rate (rcit) = v17 • Isocitrate Production Rate(rict) = v18 • Protein Synthesis Rate (rprot) = v15 • Carbohydrate Synthesis Rate (rcar) = v3

  10. Select A Model • With 12 unknown reaction rates and 11 balance equations we have 1 degree of freedom, so a model must be assumed. • Model 1 – The glyoxylate shunt is inactive, v12 = 0

  11. Metabolic Network Glucose v1 v3 Glucose-6-P Carbohydrates v2 v4 v16 Pyruvate CO2 CO2 CO2 v14 Lipid AcCoA v5 v6 v17 OAA CIT Citrate v7 v11 v13 v12 v18 ICT Isocitrate MAL GOX v10 CO2 v8 SUC OGT v15 v9 Protein CO2

  12. Select A Model • With 12 unknown reaction rates and 11 balance equations we have 1 degree of freedom, so a model must be assumed. • Model 1 – The glyoxylate shunt is inactive, v12 = 0 • Model 2 – Pyruvate carboxylation is inactive, v5 = 0

  13. Metabolic Network Glucose v1 v3 Glucose-6-P Carbohydrates v2 v4 v16 Pyruvate CO2 CO2 CO2 v14 Lipid AcCoA v5 v6 v17 OAA CIT Citrate v7 v11 v13 v12 v18 ICT Isocitrate MAL GOX v10 CO2 v8 SUC OGT v15 v9 Protein CO2

  14. Select A Model • With 12 unknown reaction rates and 11 balance equations we have 1 degree of freedom, so a model must be assumed. • Model 1 – The glyoxylate shunt is inactive, v12 = 0 • Model 2 – Pyruvate carboxylation is inactive, v5 = 0 • Model 3 – The Tricarboxylic Acid cycle was nullified, v9 = 0

  15. Metabolic Network Glucose v1 v3 Glucose-6-P Carbohydrates v2 v4 v16 Pyruvate CO2 CO2 CO2 v14 Lipid AcCoA v5 v6 v17 OAA CIT Citrate v7 v11 v13 v12 v18 ICT Isocitrate MAL GOX v10 CO2 v8 SUC OGT v15 v9 Protein CO2

  16. Which Model????? • Verification of Carbon Fluxes • Examination of the free-energy change at the biochemical standard state • After review, both models 2 and 3 resulted in a negative carbon flux and free energy change and thus were discarded.

  17. Objectives of Presentation • Outline Objectives of Case Study • Analyze their reaction equations using matrix algebra calculations • Discuss the relevance of the matrix analysis approach to metabolite modeling

  18. G6P : v1 - v2/2 – v3 = 0 Pyr : v2 – v4 – v5 = 0 AcCoA : v4 – v6 – v13 – v14 = 0 CIT : v6 – v7 – v17 = 0 ICT : v7 – v8 – v12 – v18 = 0 OGT : v8 – v9 – v15 = 0 SUC : v9 – v10 + v12 = 0 MAL : v10 – v11 + v13 = 0 GOX : v12 - v13 = 0 OOA : v5 + v11 – v6 = 0 CO2 : v4 + v8 + v9 – v16 = 0 Reaction Rate Equations

  19. 1 -0.5 -1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 -1 -1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 -1 0 0 0 0 0 0 -1 -1 0 0 0 0 0 0 0 0 0 0 1 -1 0 0 0 0 0 0 0 0 0 -1 0 0 0 0 0 0 0 0 1 -1 0 0 0 -1 0 0 0 0 0 -1 0 0 0 0 0 0 0 0 1 -1 0 0 0 0 0 -1 0 0 0 v = 0 0 0 0 0 0 0 0 0 1 -1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 -1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 -1 0 0 0 0 0 0 0 0 0 0 1 -1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 -1 0 0 1 1 0 0 0 0 0 0 -1 0 0 0 Reaction Rates in Matrix Form

  20. -1 V2 -0.5 0 0 0 0 0 0 0 0 0 0 1 -1 0 0 0 0 0 V4 1 -1 -1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 V5 0 1 0 -1 0 0 0 0 0 -1 -1 0 0 0 0 0 0 0 rglc V6 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 -1 0 rcar V7 0 0 0 0 1 1 0 0 0 0 0 0 0 -1 0 0 0 -1 0 V8 = - 0 0 0 0 0 1 -1 0 0 0 0 X 0 0 0 -1 0 0 0 rprot V9 0 0 0 0 0 0 1 -1 0 0 0 0 0 1 0 0 0 0 rc V10 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 rcit V11 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 rict V13 0 0 1 -1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 V14 0 1 -1 0 0 1 1 0 0 0 0 0 0 0 0 -1 0 0 Matrix Solution for Intracellular Fluxes

  21. V2 2 -2 0 0 0 0 0 V4 2 -2 1 -1 0 -1 -1 V5 0 0 -1 1 0 1 1 rglc V6 -1 1 0 1.5 0.5 2 2 rcar V7 -1 1 0 1.5 0.5 1 2 0 V8 = -1 1 -1 1.5 0.5 1 1 rprot V9 -1 1 -1 0.5 0.5 1 1 rc V10 -1 1 0 0.5 0.5 1 1 rcit V11 -1 1 1 0.5 0.5 1 1 rict V13 0 0 1 0 0 0 0 V14 3 -3 0 -2.5 -0.5 -3 -3 Simplified Intracellular Flux Matrix

  22. Objectives of Presentation • Outline Objectives of Case Study • Analyze their reaction equations using matrix algebra calculations • Discuss the relevance of the matrix analysis approach to metabolite modeling

  23. Relevance of Matrix Approach • Allows a simplified analysis of a complex metabolic network • Succinctly demonstrates 11 different reaction equations in relation to one another

  24. References • Aiba, S. & Matsuoka, M. (1979). Identification of metabolic model: Citrate production from glucose by Candida lipolytica. Biotechnology and Bioengineering. 21, 1373-1386. • Mathews, C. & Van Holde, K. E. (1996). Biochemistry, 2nd edition. Benjamin/Cummings Inc., Menlo Park, CA. 415-516. • Stephanopoulus, G., Aristidou, A., Nielson, J. (1998). Metabolic Engineering. Academic Press, San Diego, CA. 320-326.

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