超糖基化缺陷型酿酒酵母生产人源化糖蛋白N-聚糖位点占有率定向调控

Protein glycosylation process plays important roles in its chemical activity, pharmacokinetics and immunogenicity characteristics. In order to produce humanized glycoproteins in Saccharomyces cerevisiae, it is essential to knockout some specific glycosyltransferases, which result in an ultra-glycosylation deficient yeast strain. Previous works showed that the ultra-glycosylation deficient yeast strain performed both weak cell growth and low N-glycan site occupancy efficiency, thus hindered its application for the efficient production of functional glycoproteins. Therefore, in this project, we plan to recover the cell growth through the combination of rational engineering and error-prone DNA polymerase aided adaptive evolution. Besides, a series of yeast strains with enhanced N-glycan site occupancy efficiency would be obtained by a high-throughput screening process established in this work. With these strains, the mechanisms for the regulation of N-glycan site occupancy efficiency would be also investigated. The research presents here will facilitate the industrial production of humanized glycoproteins by yeast.

蛋白糖基化对于其化学活性、药代动力学特性和免疫原性均有重要影响。利用酿酒酵母生产人源化的糖蛋白必须敲除一些酵母N-糖基化途径中的特异性糖基转移酶，使其成为超糖基化缺陷型。但是，超糖基化缺陷型酵母存在细胞生长慢、重组糖蛋白N-聚糖位点占有率低等问题。为了克服这一困难，本研究拟采用理性改造结合易错DNA聚合酶辅助的适应性进化策略，获得细胞生长回复的超糖基化缺陷型酵母。在此基础上，拟利用本研究建立的一种新型高通量筛选策略，得到一系列重组糖蛋白N-聚糖位点占有率提高的突变株，并研究导致N-聚糖位点占有率改变的分子机制。本项目的顺利实施将为实现利用酿酒酵母大量生产人源化糖蛋白提供理论依据和技术支持。

蛋白糖基化对于其化学活性、药代动力学特性和免疫原性均有重要影响。利用酿酒酵母生产人源化的糖蛋白必须敲除一些酵母N-糖基化途径中的特异性糖基转移酶，使其成为超糖基化缺陷型。但是，超糖基化缺陷型酵母存在细胞生长慢、重组糖蛋白N-聚糖位点占有率低等问题。在研究中，我们利用生长适应性进化，构建二倍体酿酒酵母和过量表达RHO1等技术，回复了其生长缺陷和N-糖基化占有率等问题。本项目的顺利实施将为实现利用酿酒酵母大量生产人源化糖蛋白提供理论依据和技术支持。

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