The methylotrophic yeast Pichia pastoris is used as a host system to produce different types of proteins for a variety of applications, from cheese-making enzymes to anti-tumorigenesis pharmaceuticals. The popularity of this system can be attributed to several characteristics, most notably the ability of P. pastoris to perform many eukaryotic post-translational modifications, and the capability of this yeast to produce heterologous proteins at high levels, either intracellularly or extracellularly. Because P. pastoris secretes only a small amount of native protein, extracellular targeting of the heterologous proteins serves as a major purification step. However, despite the success of the Pichia pastoris system, relatively little is known about the molecular biology of the secretion machinery of this particular yeast. In order to better understand the mechanisms by which P. pastoris produces, modifies and localizes proteins, we propose to pursue alterations of the yeast system which change the secretion of heterologously expressed proteins. To that end, we propose to search for supersecreting strains of P. pastoris using two approaches, 1) by developing a genetic screen for mutant strains which secrete higher levels of heterologously expressed proteins and 2) by isolating and characterizing the P. pastoris homolog of PMRI, a gene implicated to cause supersecretion in related yeasts. In addition, we will isolate new selectable markers along with corresponding auxotrophic strains in order to construct P. pastoris strains with multiple supersecretion properties. These studies will identify cis- and trans-acting factors that dictate the secretory efficiency of proteins expressed in P. pastoris, illuminating ways in which the system can be improved. The elucidation and optimization of the secretory mechanism of P. pastoris will benefit the growing number of biomedical scientists who employ this heterologous protein expression system for their studies, accelerating the progress of their research.
