Presentation on theme: "Engineering of Biological Processes Lecture 6: Modeling metabolism Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University."— Presentation transcript:
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 Evaluate branch rigidity
These two curves have the same v max, but their K m values differ by a factor of 2. Low K m High K m r 1 = vmax 1 S K m1 + S Low K m will be the path with the higher flux (all other factors being equal). Low K m also means a strong interaction between substrate and enzyme. Michaelis Menten kinetics
Example: Enhancement of ethanol production Want to decrease the cost Cheaper substrates –Greater number of substrates Not just glucose Higher rates of production Y p/s Yield of product per substrate consumed Y p/x Yield of product per cell
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
Ethanol production 1 st 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 K m = 0.4 mM Acetate K m = 2.0 mM Lactate K m = 7.2 mM K m = 0.4 mM This approach worked because of the large differences in K m ’s
Some definitions F tot = vmax 1 S K m1 + S + vmax 2 S K m2 + S Total flux Selectivity F1F1 F2F2 vmax 2 S K m2 + S vmax 1 S K m1 + S =
Selectivity So, to enhance r 1, we want a small value of K m1
Reaction branch nodes P1P1 P2P2 I S J1 J2J3 J1 = J2 + J3 Flux of carbon Product yields are often a function of the split ratio in branch points (i.e., 20% / 80% left / right).
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
Types of reaction branch nodes P1P1 P2P2 I S - - Regulation Negative feedback
Flexible nodes The split ratio will depend on the cellular demands for the 2 products Can have substantial changes in the flux partitioning
Rigid nodes Partitioning is strongly regulated by end product activation and inhibition Deregulation of such a node can be very difficult to perform
P1P1 P2P2 I S - - Flexible node P1P1 P2P2 I S - - ++ Strongly rigid node P1P1 P2P2 I S - - + Weakly rigid node Regulation Negative feedback Regulation Positive feedback
Branch point effect Citrate Glyoxylate -Ketoglutarate Isocitrate Lyase (IL) K m =604 M V max =389 mM/min Isocitrate Dehydrogenase (IDH) K m =8 M V max =126 mM/min Glyoxylate shunt (cells grown on acetate) For growth on acetate, Isocitrate = 160 M
Flux is very sensitive to [isocitrate] first order in IL, zero order in IDH 160 M 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
Branch point effect Citrate Glyoxylate -Ketoglutarate Isocitrate Lyase (IL) K m =604 M V max =389 mM/min Dehydrogenase (IDH) K m =8 M V max =625 mM/min Glyoxylate shunt (cells grown on glucose) For growth on glucose, Isocitrate = 1 M V max had been =126 mM/min
Flux is not sensitive to [isocitrate] first order (but very low) in IL, first order in IDH 1 M Note that [S] is much lower than before.
Which path controls the branch ratio? Citrate Glyoxylate -Ketoglutarate Isocitrate Lyase (IL) K m =604 M V max =389 mM/min Dehydrogenase (IDH) K m =8 M V max =625 mM/min Glyoxylate shunt (cells grown on glucose) Under growth by glucose, Isocitrate = 1 M
Which path controls the branch ratio? The one that adapts to the available substrate controls the branch. This depends on the values of v max, K m, and [S] for each reaction.