High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol.
To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 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 +/- 0.1 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.
D019149:Bioreactors; D002452:Cell Count; D005246:Feedback; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010710:Phosphates / Q000378:metabolism*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D010710:Phosphates; C024788:ammonium phosphate; D005947:Glucose; D005990:Glycerol