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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Academic Research Enhancement Awards (AREA) (R15)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-F05 (20))
Program Officer
Shapiro, Bert I
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of the Pacific-Stockton
Schools of Arts and Sciences
United States
Zip Code
Larsen, Sasha; Weaver, Jun; de Sa Campos, Katherine et al. (2013) Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins. Biotechnol Lett 35:1925-35
Lin-Cereghino, Geoff P; Stark, Carolyn M; Kim, Daniel et al. (2013) The effect of ?-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene 519:311-7
Staley, Chris A; Huang, Amy; Nattestad, Maria et al. (2012) Analysis of the 5' untranslated region (5'UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris. Gene 496:118-27
Li, Zhiguo; Moy, Allison; Gomez, Seth R et al. (2010) An improved method for enhanced production and biological activity of human secretory leukocyte protease inhibitor (SLPI) in Pichia pastoris. Biochem Biophys Res Commun 402:519-24
Li, Zhiguo; Leung, Wilson; Yon, Amy et al. (2010) Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris. Protein Expr Purif 72:113-24
Li, Zhiguo; Moy, Allison; Sohal, Kirti et al. (2009) Expression and characterization of recombinant human secretory leukocyte protease inhibitor (SLPI) protein from Pichia pastoris. Protein Expr Purif 67:175-81
Hartner, Franz S; Ruth, Claudia; Langenegger, David et al. (2008) Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 36:e76
Lin-Cereghino, Joan; Hashimoto, Matthew D; Moy, Allison et al. (2008) Direct selection of Pichia pastoris expression strains using new G418 resistance vectors. Yeast 25:293-9
Lin-Cereghino, Joan; Wong, William W; Xiong, See et al. (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques 38:44, 46, 48
Thor, Der; Xiong, See; Orazem, Claire C et al. (2005) Cloning and characterization of the Pichia pastoris MET2 gene as a selectable marker. FEMS Yeast Res 5:935-42