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![Page 1: Engineering of Biological Processes Lecture 6: Modeling metabolism Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649c7b5503460f9492f80d/html5/thumbnails/1.jpg)
Engineering of Biological Processes
Lecture 6: Modeling metabolism
Mark Riley, Associate ProfessorDepartment of Ag and Biosystems
EngineeringThe University of Arizona, Tucson, AZ
2007
![Page 2: Engineering of Biological Processes Lecture 6: Modeling metabolism Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649c7b5503460f9492f80d/html5/thumbnails/2.jpg)
Objectives: Lecture 6
• Model metabolic reactions to shift carbon and resources down certain paths
• Evaluate branch rigidity
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These two curves have the same vmax, but their Km values
differ by a factor of 2.
0
5
10
15
20
0 10 20 30 40 50
[S]
r
Low Km High Km
r1 = vmax1 S
Km1 + S
Low Km will be the path with the higher flux (all other factorsbeing equal).
Low Km also means a strong interaction between substrate and enzyme.
Michaelis Menten kinetics
![Page 4: Engineering of Biological Processes Lecture 6: Modeling metabolism Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649c7b5503460f9492f80d/html5/thumbnails/4.jpg)
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
![Page 5: Engineering of Biological Processes Lecture 6: Modeling metabolism Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649c7b5503460f9492f80d/html5/thumbnails/5.jpg)
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
![Page 6: Engineering of Biological Processes Lecture 6: Modeling metabolism Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649c7b5503460f9492f80d/html5/thumbnails/6.jpg)
Goal
Combine the advantages of ZM + EC
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Ethanol production
1st attempt: amplify PDC activityResulted 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
![Page 8: Engineering of Biological Processes Lecture 6: Modeling metabolism Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649c7b5503460f9492f80d/html5/thumbnails/8.jpg)
EthanolKm = 0.4 mM
AcetateKm = 2.0 mM
LactateKm = 7.2 mM
Km = 0.4 mM
This approach worked because of the large differences in Km’s
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Some definitions
Ftot = vmax1 S
Km1 + S
+ vmax2 S
Km2 + S
Total flux
Selectivity
F1
F2 vmax2 S
Km2 + S
vmax1 S
Km1 + S=
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Selectivity
SK
SK
vmax
vmax
r
r
m1
m2
2
1
2
1
So, to enhance r1, we want a small value of Km1
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Model conversion of pyruvate
]pyr[K
]pyr[vv
1m
1max1
]pyr[K
]pyr[v
dt
]pyr[d
1m
1max
4321 vvvvdt
]pyr[d
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Model conversion of pyruvate
]pyr[K
]pyr[v
]pyr[K
]pyr[v
]pyr[K
]pyr[v
]pyr[K
]pyr[v
dt
]pyr[d
4m
4max
3m
3max
2m
2max
1m
1max
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Model production of ethanol
]pyr[K
]pyr[v
dt
]ethanol[d
4m
4max
dt]pyr[K
]pyr[v]ethanol[]ethanol[
4m
4maxo
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Ethanol Km = 0.4 mM
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
0 0.02 0.04 0.06 0.08 0.1
Time (sec)
Ra
tio
of
rate
s
Eth / TCA
Eth / Lac
Eth / Act
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Ethanol Km = 1 mM
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
0 0.02 0.04 0.06 0.08 0.1
Time (sec)
Ra
tio
of
rate
s
Eth / TCA
Eth / Lac
Eth / Act
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Ethanol Km = 10 mM
0
0.2
0.4
0.6
0.8
1
1.2
0 0.02 0.04 0.06 0.08 0.1
Time (sec)
Ra
tio
of
rate
s
Eth / TCA
Eth / Lac
Eth / Act
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Glucose Glucose 6-Phosphate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Pyruvate
Acetate Acetyl CoA
Citrate
-Ketoglutarate
Succinate
Fumarate
Oxaloacetate
Phosphogluconate
Glyceraldehyde 3-Phosphate
Acetaldehyde
2-Keto-3-deoxy-6-phosphogluconate
Glyceraldehyde 3-Phosphate
+Pyruvate
Lactate
Ethanol
MalateIsocitrate
CO2+NADHFADH2
CO2+NADH
NADH
NADH
GTP
GDP+Pi
Phosphoenolpyruvate
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Glucose Glucose 6-Phosphate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Pyruvate
Phosphogluconate
Phosphoenolpyruvate
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Glucose Glucose 6-Phosphate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Pyruvate
Simplified metabolism - upstream end of glycolysis
ATP ADPv1
ATP ADPv2
v3
ATP
ADP
v4
v5
ATPADPv6
ADPATPv7
2 ADPATP + AMPv8
Additional reactions
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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
dt
dAMPdt
dADPdt
dATPdt
2P6,1dFrucdt
P6dFrucdt
P6dGluc
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Mass balances
8vdt
dAMP
8v27v6v4v2v1vdt
dADP
8v7v6v4v2v1vdt
dATP
5v4vdt
2P6,1dFruc
4v3vdt
P6dFruc
3v2v1vdt
P6dGluc
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1ecosGlu1ATP1ecosGlu1ATP
1max,
K]ecosGlu[
K)]t(ATP[
K]ecosGlu[
K)]t(ATP[
1
]ecosGlu)][t(ATP[v1v
]P6Gluc)][t(ATP[k2v 2
]2P6,1Fruc[k5v 5
22
24max,
)t(P6Fruc)t(AMP)t(ATP
14P6KFruc
)t(P6Frucvv4
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Metabolite profiles
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 0.1 0.2 0.3 0.4 0.5
Time (min)
Co
nc
en
tra
tio
n (
mM
) Gluc 6PFruc 16P2Fruc 6PATPADPAMP
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Rates of reaction
-2
2
6
10
14
18
22
26
30
34
38
42
46
50
0 0.1 0.2 0.3 0.4 0.5
Time (min)
Ra
te o
f re
ac
tio
n (
mM
/ m
in)
v1
v2
v3
v4
v5
v6
v7
v8
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Reaction branch nodes
P1 P2
I
S
J1
J2 J3
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).
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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
P1 P2
I
S
- -
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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P1 P2
I
S
- -
Flexible node
P1 P2
I
S
- -
++
Strongly rigid node
P1 P2
I
S
- -
+
Weakly rigid node
RegulationNegative feedback
RegulationPositive feedback
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Branch point effect
Citrate
Glyoxylate -Ketoglutarate
IsocitrateLyase (IL)Km=604 MVmax=389 mM/min
IsocitrateDehydrogenase (IDH)Km=8 MVmax=126 mM/min
Glyoxylate shunt(cells grown on acetate) For growth on acetate,
Isocitrate = 160 M
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Growth on acetate
0
20
40
60
80
100
120
140
0 50 100 150 200 250 300
[S] uM
Rea
ctio
n r
ate
r (IL)
r (IDH)
Flux is very sensitive to [isocitrate] first order in IL, zero order in IDH
160 M
When [S] = 50 uM, r IL = 110 uM/minr IDH = 20 uM/min
When [S] = 160 uM, r IL = 120 uM/minr IDH = 60 uM/min
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Branch point effect
Citrate
Glyoxylate -Ketoglutarate
IsocitrateLyase (IL)Km=604 MVmax=389 mM/min
Dehydrogenase (IDH)Km=8 MVmax=625 mM/min
Glyoxylate shunt(cells grown on glucose) For growth on glucose,
Isocitrate = 1 M
Vmax had been=126 mM/min
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Growth on glucose
0
20
40
60
80
100
120
0 0.5 1 1.5 2
[S] uM
Rea
ctio
n r
ate
r (IL)
r (IDH)
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.
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Growth on glucose
0
20
40
60
80
100
120
0 0.5 1 1.5 2
[S] uM
Rea
ctio
n r
ate
r (IL)
r (IDH)
Growth on acetate
0
20
40
60
80
100
120
140
0 50 100 150 200 250 300
[S] uM
Rea
ctio
n r
ate
r (IL)
r (IDH)
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Which path controls the branch ratio?
Citrate
Glyoxylate -Ketoglutarate
IsocitrateLyase (IL)Km=604 MVmax=389 mM/min
Dehydrogenase (IDH)Km=8 MVmax=625 mM/min
Glyoxylate shunt(cells grown on glucose)
Under growth by glucose,Isocitrate = 1 M
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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.