EFFECTS OF MEDIUM COMPOSITION AND NUTRIENT LIMITATION ON LOSS OF THE RECOMBINANT PLASMID PLG669-Z AND BETA-GALACTOSIDASE EXPRESSION BY SACCHAROMYCES-CEREVISIAE

The effects of medium composition, nutrient limitation and dilution ra te on the loss of the recombinant plasmid pLG669-z and plasmid-borne b eta-galactosidase expression were studied in batch and chemostat cultu res of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (Delta mu) an d the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were i dentified. Glucose-limited chemostat cultures of CGpLG grown on define d medium were more stable at higher dilution rates and exhibited Delta mu-dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-domin ated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was l east stable in magnesium-limited cultures grown on defined medium. Del ta mu decreased and R increased with increased dilution rate, irrespec tive of medium composition. Increased plasmid loss rates at high or lo w dilution rates would appear to be characteristic of loss kinetics do minated by R or Delta mu, respectively. Growth of glucose-limited chem ostat cultures on complex medium decreased Delta mu values but increas ed R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of Del ta mu may have conferred was counteracted by an increased R value. Inc reased beta-galactosidase productivity was correlated with increased p lasmid stability only in glucose-limited chemostat cultures grown on d efined medium and not in those grown on complex medium. Previous studi es have yielded contrasting responses with regard to the effect of dil ution rate on recombinant plasmid loss from S. cerevisiae. Our finding s can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid inst ability results in reduced culture productivity.