Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
Department of Life Science, Faculty of Science and Engineering, Setsunan University, 17-8 Ikeda-Nakamachi, Neyagawa, Osaka, 572-8508, Japan.
Appl Microbiol Biotechnol. 2021 Dec;105(23):8771-8781. doi: 10.1007/s00253-021-11649-5. Epub 2021 Nov 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 (och1Δ), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1Δ 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 och1Δ 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 och1Δ 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.
酵母的糖蛋白含有大的 N 连接寡糖的外壳链；因此，酵母不适合用于生产人类用的治疗性糖蛋白。我们之前使用 α1,6-甘露糖基转移酶（och1Δ）的缺失突变株在裂殖酵母中产生了人源化 N-糖链；然而，裂殖酵母 och1Δ 细胞即使在营养生长过程中也表现出生长延迟，导致异源蛋白的产量降低。为了克服这个问题，我们在这里进行了全基因组筛选，以寻找能够抑制温度敏感型 och1Δ 细胞生长缺陷的基因。我们使用基因组文库并筛选了 18000 个转化体，鉴定了两个基因（pwp1，SPBC1E8.05），它们都编码糖基磷脂酰肌醇（GPI）锚定蛋白，能够提高缺乏外壳链的 och1Δ 细胞的生长速度。我们进一步表明，高拷贝数的基因需要提高生长速度。对 Pwp1p 的突变分析表明，Pwp1p 的 GPI 锚定区域在减弱生长缺陷方面很重要。pwp1 和 SPBC1E8.05 缺失突变体的分析表明，这两个基因都不是细胞存活所必需的；然而，两个突变体都对β-葡聚糖酶敏感，表明 Pwp1p 和 SPBC1E8.05 编码的蛋白非酶促地支持裂殖酵母细胞表面的β-葡聚糖。总之，我们的工作不仅阐明了裂殖酵母糖蛋白的 GPI 锚定蛋白和 N 连接寡糖之间的功能关系，而且支持裂殖酵母在人类糖蛋白生产中的应用。关键点：• 我们筛选了能够抑制裂殖酵母 och1Δ 细胞生长缺陷的基因。• 适当表达 GPI 锚定蛋白可缓解 och1Δ 细胞的生长延迟。• Pwp1p 的 GPI 锚定域对于抑制 och1Δ 细胞的生长缺陷很重要。
