1. DISCUSSION - “What is metabolic engineering?”

2. What is metabolic engineering?
Bailey, Toward a science of metabolic engineering, Science, 252, 1668, 1991
=> Manipulate unicellular organisms to use them as ‘factories’

3. A number of characteristics of cells
Escherichia coli
Cell volume ~1m3
Proteins/cell ~ 4*106
Mean size of protein ~5 nm
Distance between proteins ~ 10 nm
Genes/genome
1 protein/cell 1 nM
Diffusion time protein across cell ~0.1 s
Cell generation time 30 min – hours
Artist impression of E. coli by Goodsell,
Relative dimensions are accurate
All these proteins are moving and engaging in reactions!

4. A cell is already some kind of factory
Input : nutrients
glucose, ammonium, phosphates, etc.
Output : excretion products
fermentation products: ethanol, acetate
biomass
A cell achieves this through metabolism

5. DISCUSSION - “What is metabolism?”

6. Metabolism
Hexokinase
ATP+glucose  glucose6P+ADP
Metabolic map

7. ? DISCUSSION - “How to make a cell overproduce compound ‘X’?”
We need to take enzyme kinetics and the structure of metabolism into account

8. Enzyme kinetics
The rate of an enzyme catalysing SP can be described by a reversible Michaelis-Menten rate equation
S and P denote concentrations
The other coefficients are kinetic constants
EXERCISE Study this relationship in Excel to determine the operational meaning of the kinetic constants

9. Operational meaning of kinetic constants
v=Vmax+ if P=0 and S>>Kms,
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 SP)

10. Mass balances
The activity of reactions changes the concentration of molecular species; this is described by a mass-balance
Stoichiometric coefficient of xj in producing reaction i
Rate of producing reaction i
Rate of change in the concentration of molecular species xj

11. 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)

12. Exercise Study in Excel the following pathway 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%?