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,
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 Glucose-6-P Carbohydrates Pyruvate CO2 CO2
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. Reaction Rate Equations
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

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 Glucose-6-P Carbohydrates Pyruvate CO2 CO2
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 Glucose-6-P Carbohydrates Pyruvate CO2 CO2
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 Glucose-6-P Carbohydrates Pyruvate CO2 CO2
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. Reaction Rate Equations
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

19. Reaction Rates in Matrix Form1
-0.5 -1
v =

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

21. Simplified Intracellular Flux MatrixV2 2 -2 V4 1 -1 V5
rglc V6
1.5
0.5
rcar V7 V8 =
rprot V9 rc
V10
rcit
V11
rict
V13
V14 3 -3
-2.5
-0.5

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,
Mathews, C. & Van Holde, K. E. (1996). Biochemistry, 2nd edition. Benjamin/Cummings Inc., Menlo Park, CA
Stephanopoulus, G., Aristidou, A., Nielson, J. (1998). Metabolic Engineering. Academic Press, San Diego, CA