Question

Recombinant protein is produced by a genetically engineered strain of Escherichia coli during cell growth. The recombinant protein can be considered a product of cell culture even though it is not secreted from the cells; it is synthesized in addition to normal E. coli biomass. Ammonia is used as the nitrogen source for aerobic respiration of glucose. The recombinant protein has an overall formula of CH1.55O0.31N0.25. The yield of biomass (excluding recombinant protein) from glucose is measured as 0.48 g g-1 ; the yield of recombinant protein from glucose is about 20% of that for cells.

a) How much ammonia is required?

b) What is the oxygen demand?

c) If the biomass yield remains at 0.48 g g-1 , how much different are the ammonia and oxygen requirements for wild-type E. coli that is unable to synthesize recombinant protein?

Answer 1

Given: Biomass yield, Y_XS=0.48 g/g

Yield of recombinant protein=20% of that of the cells=0.20*0.48 g/g=0.096 g/g

a) Stoichiometric equation for the formation of recombinant protein is

where C₆H₁₂O₆ is Glucose

C_1.77H_0.49N_0.24 is E.Coli

CH_1.55O_0.31N_0.25 is Recombinant protein

a, b, c, d and e are the Stoichiometric coefficients

f is the Stoichiometric coefficient of product

The ammonia requirement is determined using the elemental N balance

Value of  stoichiometric coefficient, c, is obtained using the expression

where Y_XS is the Biomass yield

MW_susbtrate is the Molecular weight of substrate, i.e., glucose.

MW_substrate=6(12.001)+12(1.00797)+6(16)=180.2 g/mol

MW_cells is the Molecular weight of biomass, ie., E.Coli.

MW_cell=1.77(12.001)+0.49(1.00797)+0.24(14.0067)=25 g/mol

Stoichiometric coefficient of product, f, is obtained using the expression

where Y_PS is the Yield of recombinant protein

MW_product is the Molecular weight of recombinant protein

MW_product=12.001+1.55(1.00797)+0.31(16)+0.25(14.0067)=22.03 g/mol

Thus the ammonia requirement is

Thus, ammonia required is 1.028 gmol/gmol glucose.

b) The oxygen demand is obtained using the expression for electron balance

where w is the Number of atoms of Carbon present in substrate. For glucose, w=6

_S is the Degree of reduction of substrate

_B is the Degree of reduction of biomass

_P is the Degree of reduction of product

j is the Number of atoms of carbon present in product. For recombinant protein, j=1.

Degree of reduction of E.Coli relative to NH₃, _B, is obtained from the Elemental Composition and Degree of reduction for selected organisms table.

_B=4.07

Refer Degree of reduction of biological materials table for degree of reduction of product and substrate.

Degree of reduction of glucose relative to NH₃ is _S=4

Degree of reduction of recombinant protein relative to NH₃ is obtained using the degree of reduction of C, H, O and N.

Thus oxygen demand is

Therefore, oxygen demand is 1.654 gmol/gmol glucose

c) When the recombinant protein is not synthesized by wild E.Coli, f=0.

Ammonia requirement is

For wild E.Coli, the ammonia requirement has decreased from 1.028 gmol/gmol glucose to 0.8304 gmol/gmol glucose, which is a decrease of 19.22%.

Oxygen demand for wild E.Coli is

Oxygen demand has increased from 1.654 gmol/gmol glucose to 2.48 gmol/gmol glucose, which is an increase of 49.93%.

Thus the ammonia requirement for wild E.coli is 0.8304 gmol/gmol glucose, which is a decrease of 19.22% when compared to the requirement of genetically engineered strain of E.Coli. The oxygen demand for wild E.Coli is 2.48 gmol/gmol glucose, which is an increase of 49.93% when compared to the requirement of genetically engineered strain of E.Coli.