Impact of PpSpi1, a glycosylphosphatidylinositol-anchored cell wall glycoprotein, on cell wall defects of N-glycosylation-engineered Pichia pastoris

In order to produce therapeutic glycoproteins modified with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. In this study, a Pichia pastoris mutant Glyco4, which has a humanized N-glycosylation pathway and could successfully generate the human-like N-glycans, was carefully characterized. Glyco4 displays a significant growth delay and cell wall defects. Comparative transcriptomics reveals that manipulating the N-glycosylation pathway could notably affect the expression pattern of numerous biological pathways in Glyco4. Among the differentially expressed genes, the down-regulation of PpSPI1 was proven to be the main cause of the cell wall defects in Glyco4. Deletion of PpSPI1 in P. pastoris GS115 strain presented growth delay and weaker resistance to cell wall/membrane perturbing agents. PpSpi1 was shown to be a glycosylphosphatidylinositol-anchored cell wall glycoprotein and involved in the formation of the mannoprotein layer on the outer surface of cell wall. Overexpressing PpSPI1 in Glyco4 could partially recover the cell wall defects and also improve its resistance to cell wall perturbing agents and osmotic stress. Thus, overexpression of PpSpi1 is a useful strategy to facilitate P. pastoris industrial applications in the manufacture of human glycoproteins. IMPORTANCE: Engineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.