bioreactor studies of heterologous protein production by ...

4.3.6. Continuous CuIture in a Seleetive Medium
Using a YNB selective medium with a glucose concentration of 10 gL, continuous culture
was perfomed in the airiift bioreactor to examine the effkct of selection pressm. After
the bioreactor was operated in batch mode for 24 hours, the continuous culture was
initiated at a dilution rate = 0.10 Y'. As shown in Fignre 4.25, a decline in enzyme activity
was observed pnor to reaching steady state condition This is probably due to the
adaptation of ceils from solely ethanol coosumption to a glucose utïüzation during the
stan-up period. The ethanol concentration increased from 1 g/L at the beginning of
continuous culture to 1.50 g/L at steady state (after 24 hours) and rernained nearly
constant thereafter. The total ceU concentration at steady state was around 2.90 g/L. The
enzyme concentration decreased during the culture! process even though the fraction of
plasruid-bearing celh rernained constant figure 4.25). Decoupiing between plasmid Los
and enzyme production was observed in the selective medium. In contrast, in the
nonselective medium. borh the Mon of plasmid-bearing cells and enzyme activity
decayed exponentially; thus, piasmid loss comlated directly with the deciine of enyme
production (Figure 4.26).
It appears that a population of œiis which were capable of leucine synthesis, but
were Iess efficient in glucoamylase production had gradudiy supplanted the original
population during the continuous culture in selective medium. The YNB seIective medium
imposes only one seiection pressure which is based on the leucine gene (LEUZ) inserted in
the plasmid. Carrying plasmids and expressing LEUZ gene for leucine synthesis were