Abstract
BackgroundGlycosylphosphatidylinositol (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.ResultsThrough 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.ConclusionsThis 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.
Highlights
Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they have different uses in vitro
Construction of a GPI protein deletion library with the Cre/loxP system According to the amino acid sequence characteristics of GPI-anchored proteins, Zhang et al predicted and screened 50 potential GPI-anchored proteins in P. pastoris [11]
The glucan content of strain ku70Δ gcw8Δ increased by 40%, and the mannan content of strain gcw1Δ increased by 34%. These results suggest that the deletion of these GPI proteins leads to remodeling of the yeast cell wall polysaccharide
Summary
Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they have different uses in vitro. The functions of potential GPI proteins in Pichia pastoris were explored by gene knockout approaches. 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. 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
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