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DISCUSSION - “What is metabolic engineering ?”

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. Escherichia coli

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DISCUSSION - “What is metabolic engineering ?”

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  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 4500 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, http://mgl.scripps.edu/people/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’?” ? 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 denoteconcentrations The other coefficientsare kinetic constants EXERCISEStudy this relationship in Excel to determine the operationalmeaning 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. The activity of reactions changes the concentration of molecular species; this is described by a mass-balance Mass balances 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%?

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