Plasmid instability kinetics in continuous culture of a recombinant Saccharomyces cerevisiae in airlift bioreactor

Plasmid instability of a recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) was examined during continuous culture in a nonselective medium in an airlift bioreactor. The recombinant strain contained a 2-mu m based shuttle vector pGAC9 and expresses Aspergillus awamori glucoamylase gene under the control of the yeast enolase I (ENO1) promoter. The changes in the fraction of plasmid-bearing cells and glucoamylase activity followed first-order kinetics. Expressed as a function of time, the decay rates of both the plasmid-bearing cell fraction and glucoamylase expression increased with increasing dilution rates. If expressed as a function of cell generations, the decay rates were nearly constant over the dilution rates tested. The results indicated that the growth rate difference between plasmid-bearing and plasmid-free cells was negligible. This was probably due to the low copy number of the 2-mu m based yeast shuttle vector. Thus the contribution of preferential growth to apparent plasmid instability was negligible. A novel numerical method is proposed to evaluate the parameters related to plasmid stability. The estimated values of probability of plasmid loss (P = 0.0499) were nearly constant at different dilution rates. No significant effect of growth rates on plasmid instability was observed. The proposed kinetics agreed well with experimental observations. (C) 1997 Elsevier Science B.V.