1 / 39

Engineering of Biological Processes Lecture 6: Modeling metabolism

Engineering of Biological Processes Lecture 6: Modeling metabolism. Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University of Arizona, Tucson, AZ 2007. Objectives: Lecture 6. Model metabolic reactions to shift carbon and resources down certain paths

may
Download Presentation

Engineering of Biological Processes Lecture 6: Modeling metabolism

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Engineering of Biological ProcessesLecture 6: Modeling metabolism Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University of Arizona, Tucson, AZ 2007

  2. Objectives: Lecture 6 • Model metabolic reactions to shift carbon and resources down certain paths • Evaluate branch rigidity

  3. r1 = vmax1 S Low Km High Km Km1 + S Michaelis Menten kinetics Low Km will be the path with the higher flux (all other factors being equal). Low Km also means a strong interaction between substrate and enzyme. These two curves have the same vmax, but their Km values differ by a factor of 2.

  4. Example: Enhancement of ethanol production • Want to decrease the cost • Cheaper substrates • Greater number of substrates • Not just glucose • Higher rates of production • Yp/s Yield of product per substrate consumed • Yp/x Yield of product per cell

  5. Species used • Saccharomyces cerevisiae • Produces a moderate amount of ethanol • Narrow substrate specificity (glucose) • Zymomonas mobilis • Produces a large amount of ethanol • Narrow substrate specificity (glucose) • Escherichia coli • Broad substrate specificity • Low ethanol production • Much is known about its genetics

  6. Goal Combine the advantages of ZM + EC

  7. 1st attempt: amplify PDC activity Resulted in accumulation of acetaldehyde. No significant increase in EtOH. Increase in byproducts from acetaldehyde 2nd attempt: amplify PDC activity & ADH (alcohol dehydrogenase) Gave a significant increase in EtOH Ethanol production

  8. Km = 0.4 mM Ethanol Km = 0.4 mM Acetate Km = 2.0 mM Lactate Km = 7.2 mM This approach worked because of the large differences in Km’s

  9. F1 F2 + vmax2 S vmax2 S = Km2 + S Km2 + S Ftot = vmax1 S vmax1 S Km1 + S Km1 + S Some definitions Total flux Selectivity

  10. Selectivity So, to enhance r1, we want a small value of Km1

  11. Model conversion of pyruvate

  12. Model conversion of pyruvate

  13. Model production of ethanol

  14. Ethanol Km = 0.4 mM

  15. Ethanol Km = 1 mM

  16. Ethanol Km = 10 mM

  17. NADH NADH CO2+NADH GTP CO2+NADH GDP+Pi FADH2 2-Keto-3-deoxy-6- phosphogluconate Glucose Glucose 6-Phosphate Phosphogluconate Fructose 6-Phosphate Fructose 1,6-Bisphosphate Glyceraldehyde 3-Phosphate Glyceraldehyde 3-Phosphate + Pyruvate Glyceraldehyde 3-Phosphate Phosphoenolpyruvate Acetaldehyde Pyruvate Lactate Acetyl CoA Acetate Ethanol Citrate Oxaloacetate Isocitrate Malate a-Ketoglutarate Fumarate Succinate

  18. Glucose Glucose 6-Phosphate Phosphogluconate Fructose 6-Phosphate Fructose 1,6-Bisphosphate Glyceraldehyde 3-Phosphate Phosphoenolpyruvate Pyruvate

  19. v6 ADP ATP v7 ATP ADP v8 ATP + AMP 2 ADP Simplified metabolism - upstream end of glycolysis ADP ADP ATP ATP v1 v2 Glucose Glucose 6-Phosphate v3 Additional reactions Fructose 6-Phosphate ATP v4 ADP Fructose 1,6-Bisphosphate v5 Pyruvate

  20. How do you model this? • What information is needed? • equations for each v • initial concentrations of each metabolite

  21. Mass balances

  22. Mass balances

  23. Metabolite profiles

  24. Rates of reaction

  25. S I P1 P2 Reaction branch nodes Flux of carbon J1 J1 = J2 + J3 J2 J3 Product yields are often a function of the split ratio in branch points (i.e., 20% / 80% left / right).

  26. Types of reaction branch nodes (rigidity) • Flexible nodes • Flux partitioning can be easily changed • Weakly rigid nodes • Flux partitioning is dominated by one branch of the pathway • Deregulation of supporting pathway has little effect on flux • Deregulation of dominant pathway has large effect on flux • Strongly rigid nodes • Flux partitioning is tightly controlled • Highly sensitive to regulation

  27. S I - - P1 P2 Types of reaction branch nodes Regulation Negative feedback

  28. Flexible nodes • The split ratio will depend on the cellular demands for the 2 products • Can have substantial changes in the flux partitioning

  29. Rigid nodes • Partitioning is strongly regulated by end product activation and inhibition • Deregulation of such a node can be very difficult to perform

  30. S S I - - I + - - P1 P2 Weakly rigid node P1 P2 Flexible node S I - - + + P1 P2 Strongly rigid node Regulation Negative feedback Regulation Positive feedback

  31. Branch point effect Citrate Glyoxylate shunt (cells grown on acetate) For growth on acetate, Isocitrate = 160 mM Isocitrate Isocitrate Dehydrogenase (IDH) Km=8 mM Vmax=126 mM/min Lyase (IL) Km=604 mM Vmax=389 mM/min Glyoxylate a-Ketoglutarate

  32. Flux is very sensitive to [isocitrate] first order in IL, zero order in IDH 160 mM When [S] = 50 uM, r IL = 110 uM/min r IDH = 20 uM/min When [S] = 160 uM, r IL = 120 uM/min r IDH = 60 uM/min

  33. Branch point effect Citrate Glyoxylate shunt (cells grown on glucose) For growth on glucose, Isocitrate = 1 mM Isocitrate Dehydrogenase (IDH) Km=8 mM Vmax=625 mM/min Lyase (IL) Km=604 mM Vmax=389 mM/min Vmax had been =126 mM/min Glyoxylate a-Ketoglutarate

  34. Flux is not sensitive to [isocitrate] first order (but very low) in IL, first order in IDH 1 mM Note that [S] is much lower than before.

  35. Which path controls the branch ratio? Citrate Under growth by glucose, Isocitrate = 1 mM Glyoxylate shunt (cells grown on glucose) Isocitrate Dehydrogenase (IDH) Km=8 mM Vmax=625 mM/min Lyase (IL) Km=604 mM Vmax=389 mM/min Glyoxylate a-Ketoglutarate

  36. Which path controls the branch ratio? • The one that adapts to the available substrate controls the branch. • This depends on the values of vmax, Km, and [S] for each reaction.

More Related