Plasmid stability in a recombinant S cerevisiae strain secreting a bifunctional fusion protein

The recombinant Saccharomyces cerevisiae strain, YPB-G, producing and secreting Bacillus subtilis α-amylase and Aspergillus awamori glucoamylase as a fusion protein yielded efficient utilisation of starch. A segregated population balance model has been used to determine the probability of plasmid loss and plasmid copy number. The kinetics of cell growth and product (fusion protein) formation were based on a genetically structured model. The predictions were compared with the experimental observations obtained for the unstable recombinant S cerevisiae cells in a 1.5 dm^-3 batch bioreactor with 30 g dm³ initial starch under non-aerated conditions. The main advantage of the present model is that three different genetic classes were defined on the basis of the existence of plasmid and of the expression of the enzymes, ie cells containing plasmids and expressing the gene product, x₁; cells containing plasmids and but not expressing the gene product, x₂; and cells without plasmids, x₃. It is confirmed by this model that the cells without plasmids outgrow and dominate in the fermentation medium (2.27 g dm^-3 vs 0.51 g dm^-3) as more and more glucose becomes available by the degradation of starch.