Overexpression of cell-wall GPI-anchored proteins restores cell growth of N-glycosylation-defective och1 mutants in Schizosaccharomyces pombe

• Published in: Applied microbiology and biotechnology 2021-12, Vol.105 (23), p.8771-8781
• Main Authors: Fukunaga, Takamasa, Sakurai, Yuki, Ohashi, Takao, Higuchi, Yujiro, Maekawa, Hiromi, Takegawa, Kaoru
• Format: Article
• Language: English
• Subjects: Analysis; Applied Genetics and Molecular Biotechnology; Biomedical and Life Sciences; Biotechnology; Cell surface; Cell viability; Chains; Copy number; Deletion mutant; Fission; Genes; Genetic aspects; Genetic screening; Genome-wide association studies; Genomics; Glucan; Glycoproteins; Glycosylation; Glycosylphosphatidylinositols; GPI-Linked Proteins - biosynthesis; Growth rate; Identification and classification; Life Sciences; Mannosyltransferases - genetics; Mannosyltransferases - metabolism; Microbial Genetics and Genomics; Microbiology; Mutants; N-glycans; Oligosaccharides; Polysaccharides; Properties; Proteins; Schizosaccharomyces - genetics; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism; Yeast; Yeast fungi; Yeasts; β-Glucan
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-021-11649-5

The glycoproteins of yeast contain a large outer chain on N -linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of α1,6-mannosyltransferase ( och1 Δ), we previously produced humanized N -glycans in fission yeast; however, the Schizosaccharomyces pombe och1 Δ cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1 Δ cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes ( pwp1 + , SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1 Δ cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1 + and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive β-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support β-glucan on the cell-surface of S. pombe . Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N -linked oligosaccharides of glycoproteins in S. pombe , but also supports the application of S. pombe to the production of human glycoprotein. Key points • We screened for genes that suppress the growth defect of fission yeast och1Δ cells. • Appropriate expression of GPI-anchored proteins alleviates the growth delay of och1Δ cells. • The GPI-anchor domain of Pwp1p is important for suppressing the growth defect of och1Δ cells.