bioreactor studies of heterologous protein production by ...

4.3.4. Effixt of Dilution Rate On Recombinant Rotein Expression
Besides the iÏaction of plasmid-be&g ce&, concentrations of the recombinant prolein
(glucoamylase) were also followed dming coniinuous col- in the airlin bioreactor
using the YPG nonselective medium, nie effm of the diiution rate and the on
glucoamylase production are shown in Fignre 4.18. 'The data taken at the dilution rate
0.25 h-' was not included as glucose was not limiting at mis dilution rate and the residual
glucose level inteifered with glucoamylase measunment Like the fiaction of the
plasmid-bea~g tell$ the decline of recombinant protein concentrations &O followed
fit-order kinetics. Figure 4.18 also shows increased decay rate of recombyiant protein
expression (as function of tirne) at higher dilution rates However, the decay rates were
nearly constant at different dilution rates when the giucoamyiase activity was plotted
against the nurnber of generations (Figure 4.19). Thus, the recombinant protein
expression was directly related to the fiaction of plasmid-bearing cek. This mer
confis the assumption of fht-order kinetics for plasmid loss.
It is evident h m Figure 4.18 that the enzyme concentrations decreased with
increasing dilution rates. This is because of a metabolic shift At lower dilution rates the
metabolisrn of recombinant yeast was mainiy respiratory Such conditions gave efficient
conversion of carbon source to ceU and the biomass nmained at a higher level, resuiting in
higher enzyme production. But at higher diiution rates glucose fermentation dorninated as
most of the glucose was used for ethano1 production due to the Crabttee effm Under
these conditions biomass yield dropped to a lower kvel and ceIl concentrations were