bioreactor studies of heterologous protein production by ...

necessary for host ce& to grow in the selective medium. However, the expression of
glucoamylse gene was not needed for survival, In order to rednce the metabolic load, the
expression of glucoamylase rnight have ben tumed off. In this case, the plasmids would
be maiBtained for the synthesis of leucine without producing glucoamylase. It was also
possible that the plasmids lost a part or tk whole giucoamylase gene by deletion. The
reduced plasmid size was an advantage for recombinant cells since increased plasmid size
required increased arnount of energy (Cheah et al., 1987). Furthemore, since the host ceii
was a LEU2 auxonophic suain, it was iikely that an integration of the LEU2 gene
encoded on the plasmid into the host chromosome occnrred as a resuit of a homologous
recombination event (Kuriyama et aL, 1992). The host cells which reacquired the Ieucine
gene did not need plasmids to swive. They could grow in the selective medium and
appeared to contain the plasrnids; but they were not able to synthesize glucoamylase. The
decoupling between phsrnid Ioss and enzyme production in the selective medium may be
explained by the above mechanism. The decay in enzyme production in selective medium
was mainly caused by the structural instability of the plasmids. ui contrast, the plasrnid
instabiüty in the nonselective medium was mainiy segregationai, plasrnid los occurred
because of the bias putitionhg of plasrnids beween mother and daughter cells.
'Ihe m o n of the piasmid-bearing ce& never reached 100%. It remained at 80-
86% during cuitun. In a sdective medium the plasmid-fiee œlls are expected to be
washed out, nevertheles, many researchers reponed the existence of plasmid-negative
cells in a selective medium. It was shown that phsrnid-free cells codd swive in the