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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.

Construction and screening of a glycosylphosphatidylinositol protein deletion library in Pichia pastoris