DISCUSSION - “What is metabolic engineering ?”
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Transcript of DISCUSSION - “What is metabolic engineering ?”
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-DISCUSSION -
“What is metabolic engineering?”
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What is metabolic engineering?
Bailey, Toward a science of metabolic engineering, Science, 252, 1668, 1991
=> Manipulate unicellular organisms to use them as ‘factories’
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A number of characteristics of cells
Artist impression of E. coli by Goodsell, http://mgl.scripps.edu/people/goodsell/Relative dimensions are accurate
All these proteins are moving and engaging in reactions!
Escherichia coli
Cell volume ~1m3
Proteins/cell ~ 4*106
Mean size of protein ~5 nmDistance between proteins ~ 10 nmGenes/genome 45001 protein/cell 1 nMDiffusion time protein across cell ~0.1 sCell generation time 30 min – hours
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A cell is already some kind of factory
Input : nutrients glucose, ammonium, phosphates, etc.
Output : excretion products fermentation products: ethanol, acetatebiomass
A cell achieves this through metabolism
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-DISCUSSION -
“What is metabolism?”
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HexokinaseATP+glucose glucose6P+ADP
Metabolic map
Metabolism
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-DISCUSSION -
“How to make a cell overproduce compound ‘X’?”
We need to take enzyme kinetics and the structure of metabolism into account
?X
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Enzyme kinetics
The rate of an enzyme catalysing SP can be described by a reversible Michaelis-Menten rate equation
mPmS
mPMAX
mSMAX
KP
KS
KP
VKS
V
v
1
EXERCISEStudy this relationship in Excel to determine the operationalmeaning of the kinetic constants
S and P denoteconcentrations
The other coefficientsare kinetic constants
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Operational meaning of kinetic constants
1. v=Vmax+ if P=0 and S>>Kms,
2. v=1/2 Vmax+ if P=0 and S=Kms,AND VICE VERSA FOR Vmax- and KMP
NOTE: v=0 if P/S=Vmax+*KMP/(Vmax-*KMS)=Keq
(Keq = equilibrium constant of reaction SP)
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Mass balancesThe activity of reactions changes the concentration of molecular species; this is described by a mass-balance
k
kconsumingjki
iproducingjij vnvnxdt
d,,
Rate of change in the concentration of molecular species xj
Stoichiometric coefficient of xj in producing reaction i
Rate of producing reaction i
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Exercise
• Go to http://jjj.biochem.sun.ac.za/database/
chassagnole2/
• And determine the mass-balances for the metabolites in black font (the others are assumed constant)
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Exercise
• Study in Excel the following pathway
PS 21 X- S and P are fixed- both enzymes follow reversible Michaelis-Menten kinetics- rate constants Vmax1
+ 10, Vmax2+ 100, all V-’s 1, all Km’s 1
QUESTIONS- study the rate of the enzymes in one plot- what happens when the two enzymes have an equal rate?- which enzyme would you overproduce to change the flux, if you can only overproduce them by 10%?