Engineering of biological processes lecture 6 modeling metabolism
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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

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Engineering of biological processes lecture 6 modeling metabolism

Engineering of Biological ProcessesLecture 6: Modeling metabolism

Mark Riley, Associate Professor

Department of Ag and Biosystems Engineering

The University of Arizona, Tucson, AZ

2007


Engineering of biological processes lecture 6 modeling metabolism

Objectives: Lecture 6

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

  • Evaluate branch rigidity


Engineering of biological processes lecture 6 modeling metabolism

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.


Example enhancement of ethanol production

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


Species used

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


Engineering of biological processes lecture 6 modeling metabolism

Goal

Combine the advantages of ZM + EC


Ethanol production

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


Engineering of biological processes lecture 6 modeling metabolism

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


Some definitions

F1

F2

+ vmax2 S

vmax2 S

=

Km2 + S

Km2 + S

Ftot = vmax1 S

vmax1 S

Km1 + S

Km1 + S

Some definitions

Total flux

Selectivity


Selectivity

Selectivity

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


Model conversion of pyruvate

Model conversion of pyruvate


Model conversion of pyruvate1

Model conversion of pyruvate


Model production of ethanol

Model production of ethanol


Ethanol k m 0 4 mm

Ethanol Km = 0.4 mM


Engineering of biological processes lecture 6 modeling metabolism

Ethanol Km = 1 mM


Ethanol k m 10 mm

Ethanol Km = 10 mM


Engineering of biological processes lecture 6 modeling metabolism

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


Engineering of biological processes lecture 6 modeling metabolism

Glucose

Glucose 6-Phosphate

Phosphogluconate

Fructose 6-Phosphate

Fructose 1,6-Bisphosphate

Glyceraldehyde 3-Phosphate

Phosphoenolpyruvate

Pyruvate


Engineering of biological processes lecture 6 modeling metabolism

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


How do you model this

How do you model this?

  • What information is needed?

    • equations for each v

    • initial concentrations of each metabolite


Mass balances

Mass balances


Engineering of biological processes lecture 6 modeling metabolism

Mass balances


Metabolite profiles

Metabolite profiles


Rates of reaction

Rates of reaction


Reaction branch nodes

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).


Types of reaction branch nodes rigidity

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

S

I

-

-

P1

P2

Types of reaction branch nodes

Regulation

Negative feedback


Flexible nodes

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

Rigid nodes

  • Partitioning is strongly regulated by end product activation and inhibition

  • Deregulation of such a node can be very difficult to perform


Engineering of biological processes lecture 6 modeling metabolism

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


Branch point effect

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


Engineering of biological processes lecture 6 modeling metabolism

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


Engineering of biological processes lecture 6 modeling metabolism

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


Engineering of biological processes lecture 6 modeling metabolism

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.


Which path controls the branch ratio

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


Which path controls the branch ratio1

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.


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