Strategy Study On The Cultivation Of Recombinant Yeast Plasmid Stability And Protein Yield

In this study the recombinant yeast, S.cerevisiae 20B-12, carrying plasmid pNA3 for production of a -amylase was used as model system to demonstrate the effects of fermentation strategy on enhanced plasmid stability and heterologous protein production. A simple mathematical model was also developed to simulate the fermentation kinetics.Experimental data in continuous propagating flask cultivation showed plasmid stability of recombinant yeast was above 90% under selective pressure, and the plasmid lost immediately when the selective pressure was removed. After 130 hours, no plasmid-carrying cells were detected.The optimization of culture condition was carried out with uniform experimental design method in flask cultivation. The optimal condition was found for flask cultivation with 34.8g/L glucose concentration, 6.6g/L yeast nitrogen base w/o amino acids, initial pH of 5.8 and the favorite feeding time of yeast for inducing enzyme with the glucose concentration below 0.299g/L.The effects of temperature ,dissolved oxygen and pH on the production of a -amylase in the inducing stage were studied in the 2L fermenter. Further studies were carried to demonstrate the feeding strategy for enhanced plasmid stability and protein production. It showed that the plasmid stability increased in the stationary phase with nutrient starvation. With application of this to the inducement and expression of heterologous proteins, the results showed it is effective to enhance the plasmid stability and protein production by pulse-addition of yeast extract.Considering the combination of needs to cell growth, plasmid stability and protein expression, a novel feeding strategy was developed to achieve high plasmid stability and protein production for recombinant yeast fermentation by using constant-addition of SM and pulsing-addition of yeast at the same time. The cell density and the final enzyme activity reached 36.4g/l and 207.6U/mL respectively.A simple mathematical model was also developed to simulate the fermentation kinetics and all parameters in the model were estimated either from direct calculation of the batch fermentation data or model fitting.Since the SUC2 promoter is commonly used in recombinant yeast, the method proposed in this paper could be used for a reference in a scale-up cultivation with a similar recombinant yeast system.