Engineering of Biological Processes Lecture 6: Modeling metabolism

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Engineering of Biological ProcessesLecture 6
Modeling metabolism

• Mark Riley, Associate Professor
• Department of Ag and Biosystems Engineering
• The University of Arizona, Tucson, AZ
• 2007

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Objectives Lecture 6

• Model metabolic reactions to shift carbon and
  resources down certain paths
• Evaluate branch rigidity

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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.
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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

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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

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Goal

• Combine the advantages of ZM EC

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Ethanol production
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This approach worked because of the large
differences in Kms
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Some definitions
Total flux
Selectivity
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Selectivity
So, to enhance r1, we want a small value of Km1
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Model conversion of pyruvate
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Model conversion of pyruvate
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Model production of ethanol
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Ethanol Km 0.4 mM
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Ethanol Km 1 mM
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Ethanol Km 10 mM
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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
Acetate
Acetyl CoA
Ethanol
Citrate
Oxaloacetate
Isocitrate
Malate
a-Ketoglutarate
Fumarate
Succinate
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Glucose
Glucose 6-Phosphate
Phosphogluconate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Phosphoenolpyruvate
Pyruvate
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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
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How do you model this?

• What information is needed?
• equations for each v
• initial concentrations of each metabolite

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Mass balances
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Mass balances
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Metabolite profiles
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Rates of reaction
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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).
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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

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Types of reaction branch nodes
Regulation Negative feedback
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Flexible nodes

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

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Rigid nodes

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

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Regulation Negative feedback
Regulation Positive feedback
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Branch point effect
Citrate
Glyoxylate shunt (cells grown on acetate)
For growth on acetate, Isocitrate 160 mM
Isocitrate
Isocitrate Dehydrogenase (IDH) Km8 mM Vmax126
mM/min
Lyase (IL) Km604 mM Vmax389 mM/min
Glyoxylate
a-Ketoglutarate
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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
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Branch point effect
Citrate
Glyoxylate shunt (cells grown on glucose)
For growth on glucose, Isocitrate 1 mM
Isocitrate
Dehydrogenase (IDH) Km8 mM Vmax625 mM/min
Lyase (IL) Km604 mM Vmax389 mM/min
Vmax had been 126 mM/min
Glyoxylate
a-Ketoglutarate
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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.
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Which path controls the branch ratio?
Citrate
Under growth by glucose, Isocitrate 1 mM
Glyoxylate shunt (cells grown on glucose)
Isocitrate
Dehydrogenase (IDH) Km8 mM Vmax625 mM/min
Lyase (IL) Km604 mM Vmax389 mM/min
Glyoxylate
a-Ketoglutarate
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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.