Construction and screening of a glycosylphosphatidylinositol protein deletion library in Pichia pastoris
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RESEARCH ARTICLE
Open Access
Construction and screening of a glycosylphosphatidylinositol protein deletion library in Pichia pastoris Pan Wang, Ying Lin, Chengjuan Zou, Fengguang Zhao, Shuli Liang, Suiping Zheng and Shuangyan Han*
Abstract Background: Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. In this study, the functions of potential GPI proteins in Pichia pastoris were explored by gene knockout approaches. Results: Through an extensive knockout of GPI proteins in P. pastoris, a single-gene deletion library was constructed for 45 predicted GPI proteins. The knockout of proteins may lead to the activation of a cellular response named the ‘compensatory mechanism’, which is characterized by changes in the content and relationship between cell wall polysaccharides and surface proteins. Among the 45 deletion strains, five showed obvious methanol tolerance, four owned high content of cell wall polysaccharides, and four had a high surface hydrophobicity. Some advantages of these strains as production hosts were revealed. Furthermore, the deletion strains with high surface hydrophobicity were used as hosts to display Candida antarctica lipase B (CALB). The strain gcw22Δ/CALB-GCW61 showed excellent fermentation characteristics, including a faster growth rate and higher hydrolytic activity. Conclusions: This GPI deletion library has some potential applications for production strains and offers a valuable resource for studying the precise functions of GPI proteins, especially their putative functions. Keywords: GPI protein, Deletion, Phenotypic screen, Pichia pastoris
Background Glycosylphosphatidylinositol (GPI)-anchored proteins are found in all eukaryotic cells. They harbor GPIanchoring machinery and utilize the anchor to express proteins on the cell surface. Precursors of GPI anchored proteins contain an N-terminal signal sequence for import into the ER and a C-terminal signal for GPI anchoring [1]. In yeast, the GPI anchor is essential for viability and maintenance of normal cell morphology [2, 3]. These GPI-cell wall proteins (CWPs) can be grouped into different classes based on their functions. Some GPI proteins play a structural role and may provide stretch * Correspondence: [email protected] Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China
resistance by interacting with glucans and other wall components or by interacting with each other through noncovalent bonds and disulfide bridges. Other GPI proteins may act as enzymes that make and break glycosidic linkages, and the rest are required for elaboration of the cell wall and its reshaping during bud emergence, cell separation, mating or entry into stationary phase. Yeast cells elicit a rescue mechanism called the ‘compensatory salvage response’, which provides compensatory synthesis of cell wall material and changes the cross-li
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