bioreactor studies of heterologous protein production by ...

ate depended on activities of the pacemaker enzyme pools of the individual pathways.
The pacemaker enzyme pools were regulated by the specifc glucose uptake rate. The
effect of substmte concentration on the specinc growth rate was described by a modified
Monod equation. It was assumed that the recombinant protein formation is only
associated with oxidative pathways Plasmid loss kinetics was fomulated based on
segregational instability during ceil division by assuming a constant probability of plaunid
loss. When applied to batch and continuous fermentations, the model successfuiiy
predicted the dynamics of ceil growth (diauxic growth), glucose consurnption (Cnbtree
effect). ethanol metabolism. glucoamylase production and plasmid instability. Good
agreement beween model simulations and the experimental data was achieved. Using
published experimentd da& model agreement was ako found for other recombinant yeast
strains The proposed model seems to be genedy applicable to the design, opention,
conuol and optimization of recombinant yeast bioprocesses.
The novel immobilized-ceil-ftlm airlift bioreactor w u based on Cotton cloth sheers
to immobilize the yeast cells by attachment Continuous culture experiments were
performed in it rt different dilution rates in the YPG nonselective medium. The
irnmobilized celi systerns gave higher giucoamylase concentration (about 608) and
maintained recombinant protein production for longer periods of time (more than 100%)
compared with the corresponding free suspension systems. The more stable glucomylase
production was due to a reduced plasmid los in the immobiüzed ceU system. By
openting the immobilized-ceM-film bioreactor in repeated batch mode, further reduction
of plasmid instability of the recombinant yeast was obtained. An mathematical mode1 was
developed to describe! ee kinetics of piasmid ioss and enzyme production decay based on
a biofilm concept. The proposed mode1 successfuiiy described the experimental results
and provided useful information for be~r understanding of the stabiüzing mechanisms of
the UNnobilized recombinant celis. It ma) be concluded that a reduced sped?c growth
nte accompanied by ûn increased plasmid copy number is the basic explmation for the
effective enhanced plasmid stability in the immobiüred cell system. In the present case,
the attached yeast fîim could be a dynvnic zeseme of highly concentraieci plamid-bearing