bioreactor studies of heterologous protein production by ...

phmid instabiiity paiticuiarly in nonselective media @iBiasio and Sadonini, 1986;
hpoolsup et al., 1989; Mosmti et al., 1993). A two-sfage continuous culture system
based on inducible operators bas been &vised in atîempts to overcome the instabiüty
pmblem (Ryu and Sugel. 1986; Lee and Ryu, 1988; Sayadi et al., 1987; Fu et ai., 1993).
In Mis scheme, a reprcssor protein binds to the operator of the plasrnid to block
transcription untii needed. When necessary, an inducer is added, to bind to the repressor,
and prevent its interaction with the operator, thus aiiowing transcription to begki. In the
fint stage of the two-stage systern, the ceils are grown in the repnssed state (no foreign
protein is synttiesized) or under selective pressure in order to prevent piasmid loss, and
obtain a high fraftion of plasmid-containing cells The ceh are then continuously
~ferred to the second stage, where the inducer is added to enable expression. Another
possibility for maintaining plasmid-harboring cells in continuous culture is the use of
selective recycle (Ogden and Davis, 1991). In such a process, plasmid-bearing ceiis
lehg the reactor are selectively concentrated and subsequently recycled back to the
reactor, resulting in increased plasmid-bearing ceii concentration and product synthesis.
Fearibiüty of seleaive recycle depends on the ability to easily separate the plasmid-
containing cells from the mixed population; therefore, the phenotype of the plasmid-
bearing ceh must include a property that can be the basis of aparation. Such a property
may be ceil size. density, or floaulation behavior.
Another promising stabilizing option is cycling of operational variables. Using a
theoreticai anaiysis, Stephens and Lyberatos (1988) showed that cycling of subsaate