High-Cell-Density Fermentation of Recombinant Saccharomyces cerevisiae Using Glycerol

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc I strain bearing a 2 ^m plasmid, pYES2 containing a GALI promoter for expression of the (beta)-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 d: O.I was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5—3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.