Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain
Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain
复制标题
DOI：
10.1007/s00253-021-11440-6
复制
发表时间：
2021-07-17
影响因子：
5
通讯作者：
Hasunuma, Tomohisa
中科院分区：
文献类型：
--
作者：
Inokuma, Kentaro;Kitada, Yuki;Hasunuma, Tomohisa
利用糖基磷脂酰肌醇（GPI）锚定技术在细胞表面表达功能蛋白，是构建具有特殊功能的酵母细胞的一种很有前途的方法。表面工程酵母菌株的功能性很大程度上取决于其细胞表面上展示的功能蛋白的量。另一方面，由于酵母细胞壁空间有限，细胞壁的异源蛋白携带能力有限。在这里，我们报告的影响CCW 12和CCW 14敲除，编码主要的非酶GPI锚定的细胞壁蛋白（GPI-CWP）参与细胞壁组织，对酵母细胞壁的异源蛋白携带能力。以棘孢曲霉β-葡萄糖苷酶（BGL）为报告基因，研究了酿酒酵母的蛋白质携带能力。CCW 12和CCW 14基因敲除菌株与其对照菌株之间的细胞壁相关BGL的量和细胞表面BGL活性没有观察到显著差异。相反，在CCW 12和CCW 14共敲除菌株中，细胞壁结合BGL的量及其活性比对照菌株和CCW 12或CCW 14敲除菌株高约1.4倍。电子显微镜观察显示，与亲本菌株相比，CCW 12和CCW 14共敲除菌株的总细胞壁厚度增加，表明细胞壁的异源蛋白携带能力潜在增加。这些结果表明，CCW 12和CCW 14共敲除菌株是一个很有前途的宿主，用于构建高功能的重组酵母菌，利用细胞表面展示技术。
The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus beta-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology.