Effect of nutrient deficiency on plasmid stability and hepatitis B surface antigen production in recombinant yeast

Abstract

For the industrial scale production of a recombinant protein, a welldesigned medium is required, especially in the growth-associated recombinant protein production. However, it seems that bioprocess engineers as well as microbiologists do not pay attention to the nutrient deficiency in the commercially available medium. In this research, Hepatitis B surface antigen (HBsAg)-producing recombinant yeast was cultivated to thoroughly investigate the effect of the nutrient deficiency, resulted from the use of Bacto-yeast nitrogen base without amino acids (Difco, USA), on plasmid stability and HBsAg production. In Bacto-yeast nitrogen base without amino acids-containing medium for HBsAg-producing recombinant yeast, the nutrient deficiency, especially vitamins and trace elements, could be found. The nutrient deficiency resulted in the plasmid instability and the decrease in HBsAg production as well as the decrease in the growth yield and the maximum HBsAg production. Using the medium shift experiment, it could be observed that the medium based on the chemically defined medium for Baker``s yeast was more suitable for the cultivation of HBsAg-producing recombinant yeast. Those results were confirmed in batch cultivation and growth rate-controlled fed-batch cultivation of HBaAg-producing recombinant yeast as well. To investigate the effect of the nutrient deficiency in Bacto-yeast nitrogen base without amino acids on plasmid stability and HBsAg production, the chemically defined medium, in which the concentrate of nine amino acids was used as a substitute for Bacto-casamino acid, was developed. Only when Bacto-yeast nitrogen base without amino acids was added in the culture medium for HBsAg-producing recombinant yeast, did the plasmid instability and the decrease in HBsAg production occur in the repeated shaker flask culture. Among ingredients in Bacto-yeast nitrogen base without amino acids, the deficiency in Fe or Zn especially resulted in the plasmid instability an...