High Cell-Density Fermentation

The purpose of this chapter is to provide sufficient instruction for the reader to implement fermentation strategies to produce recombinant proteins in the yeast Pichia pastoris. P. pastoris utilizes the highly efficient alcohol oxidase 1 gene (AOX1 ) promoter for high-level expression of foreign genes. The AOX1 promoter is induced by methanol, but repressed in the presence of excess glycerol. Glycerol is readily metabolized by P. pastoris , and high-cell densities can be obtained utilizing this carbon source. The standard fermentation protocol is to grow P. pastoris in excess glycerol to repress expression followed by induction of protein production by the addition of methanol after the glycerol is exhausted.