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-micron based shuttle vector pGAC9 and expresses Aspergillus awatnori 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-micron 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.
Zhang
Z
Z
Department of Chemical Engineering, University of Waterloo, Ontario, Canada.
Scharer
J M
JM
Moo-Young
M
M
D019149:Bioreactors; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D008957:Models, Genetic; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development
D005656:Fungal Proteins; D011994:Recombinant Proteins; D005087:Glucan 1,4-alpha-Glucosidase