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

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.

Light-Driven CO2 Reduction with a Surface-Displayed Enzyme Cascade–C3N4 Hybrid

“…Activity assay of the displayed enzymes showed that the Cb FDH-Pir1 and YADH-Agα1 constructs exhibited functional enzymes on the cell surface (Figure S16), while the constructs with Pc FaldDH failed to show activity after displaying it on the cell surface. Other six FaldDHs from different organisms were also displayed with three other GPI anchor motifs (Table S1), and these constructs failed to generate a viable strain with the activity of FaldDH. Therefore, free Pc FaldDH was used with displayed Cb FDH and YADH to construct a multienzyme assembly.…”