-DISCUSSION -

“What is metabolic engineering?”

What is metabolic engineering?

Bailey, Toward a science of metabolic engineering, Science, 252, 1668, 1991

=> Manipulate unicellular organisms to use them as ‘factories’

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

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

-DISCUSSION -

“What is metabolism?”

HexokinaseATP+glucose glucose6P+ADP

Metabolic map

Metabolism

-DISCUSSION -

“How to make a cell overproduce compound ‘X’?”

We need to take enzyme kinetics and the structure of metabolism into account

?X

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

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)

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

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)

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%?