Recombinant protein production in E.coliMolecular and process strategies

Plasmid maintenance and copy numberPlasmid stability :

Segregational stability

Structural stability

Host cell mutation

Improving plasmid stability Programmed cell death upon loss of plasmid Integrate gene of interest in chromosome –

gene copy number ? Combination of selection pressure and growth

rate control by limiting the carbon source in the medium

Expression vector design consideration

High Cell Density cultivationHigh Cell Density cultivation Development of growth mediaDevelopment of growth media Acetate formationAcetate formation Oxygen concentrationOxygen concentration Mixing patterns in large scale fermentersMixing patterns in large scale fermenters Dissolved carbondioxideDissolved carbondioxide

Above the following concentrations Above the following concentrations E.coliE.coli is is inhibited.inhibited.

Glucose – 50 g/lGlucose – 50 g/lAmmonia – 3 g/lAmmonia – 3 g/lPhosphorus – 10 g/lPhosphorus – 10 g/lMagnesium – 8.7 g/lMagnesium – 8.7 g/lZinc -0.038 g/lZinc -0.038 g/lIron 1.15 g/lIron 1.15 g/l

Hence fed batch is the most preferable Hence fed batch is the most preferable methodmethod

General methods applied to reduce acetate formation in E. coli General methods applied to reduce acetate formation in E. coli

Add nutrient to culture in response Add nutrient to culture in response to pH (‘pH-stat’) to pH (‘pH-stat’)

Indirect method which tends to be Indirect method which tends to be conservativeconservative

Apply glucose pulses and observe Apply glucose pulses and observe dissolved oxygen response. dissolved oxygen response.

Responsive to changes in Responsive to changes in metabolism during process metabolism during process

Remove acetate from culture as it is Remove acetate from culture as it is formed . formed .

Does not prevent acetate formation Does not prevent acetate formation and ‘loss’ of carbon and ‘loss’ of carbon

Use carbon source other than Use carbon source other than glucose in mediumglucose in medium

Expensive but becoming cost-Expensive but becoming cost-effective for glycerol effective for glycerol

Use select amino acids in medium Use select amino acids in medium Can be strain dependent, requires Can be strain dependent, requires optimization optimization

General methods applied to reduce acetate formation in E. coli Use strain with low propensity for acetate formation

Can decrease maximum specific-growth rate Can lead to other products Does not prevent acetate formation and ‘loss’ of carbon

Disrupt phosphotransferase system for glucose uptake Disrupt acetate synthesis pathways Divert carbon to another product Divert carbon to TCA cycle Disrupt regulation of TCA cycle Provide outlet for ‘excess’ NADH

Oxygen concentration Pure oxygen can be sparged. But expensive Anaerobic conditions lead to acetate formation

Mixing Cells close to the injection port are exposed to

high concentrated nutrients.