The methylotrophic yeastPichia pastorishas been widely exploited for its high-level expression of heterologous proteins by recombination of gene sequences of interest with the methanol-inducible alcohol oxidase gene (AOX1) promoter (1 –3 ). Secreted and cytoplasmic expression of heterologous proteins at levels equivalent toEscherichia coliand significantly higher than inSaccharomyces cerevisiaehas been achieved (4 ). In addition,P. pastoriscultures can be easily scaled up to high cell densities, and as a result, yields are also high on a volumetric basis (e.g., 12 g/L, for tetanus toxin fragment C [5 ], 2.5 g/L for invertase [6 ], 2.5 g/L for α-amylase [7 ]). Secreted products can comprise more than 80% of the protein in the medium (6 ). However, secretion is a complex process that is not only dependent on gene dosage, but also on other factors, such as signal sequence recognition and processing, proteolysis, and glycosylation.
