bioreactor studies of heterologous protein production by ...

ecause glucose fermentation dominated and most of the glucose was used for ethanol
production due to the Crabtree effect. The Crabtree effect was induced by high glucose
concentration in batch culture and high glucose flux in continuous culture.
3. Results of the continuous culaires confimied the high instability of the model
recombinant yeast in free suspension when growing in the YPG nonselective medium.
The changes in the fraction of phmid-bedng cells and glucoamylase activity followed an
exponentid decay pattern. Expressed as a function of time, the decay ntes of both the
plasmid-bearing ceii fraction and glucoamylase expression increased with increâsing
dilution ntes. IT expressed as a function of ceil generations, the decay ntes were nearly
constant over the dilution ntes tested. A novel method is proposed to evaluate the
instability panmeters p*, p* and p. The results indicated that the growth rate difference
between plasrnid-beaing and plasmid-free cells was negligible. This was probably due to
the low copy number of the 2 pm based yeast shunle vector. Thus, the contribution of
preferential growth to apparent plasmid h~bility was negligible. The estimated values of
probability of plamid los @ = 0.05) were nearly constant at different dilution ntes. No
signifcant effect of growth rates on plasmid instabüity was observed. When the model
recombinant yeast was cultured continuously in the YNB selective medium, no apparent
plasmid los was observed. However, the giucoamylase production stiü declined du~g
the culture period. The decoupling between the plasmid Ioss and enzyme production in
the seleetive medium was atuibuted to the smctural instability.