毕赤酵母蛋白质糖基化途径的改造

Modification of Pichia kudriavzevii cells for humanized glycosylation

摘要:

【背景】以酵母为宿主生产的蛋白往往发生过糖基化，形成高甘露糖型的N-糖基化。高甘露糖型的结构易在人体中引起免疫反应，这是酵母不能用于绝大部分糖蛋白药物生产的主要限制因素。因此，构建表达人源糖基化糖蛋白的酵母底盘细胞将为糖蛋白药物的生产提供强有力的工具。库德里阿兹威氏毕赤酵母(Pichia kudriavzevii)具有极强的抗逆性且生长迅速，是一种近年来备受关注的非典型性酵母，对其进行糖基化途径的改造将具有巨大的应用前景。【目的】对酵母 N-糖基化途径的改造，首先要使其N-糖基化转变为Man₅GlcNAc₂核心结构，本研究对P. kudriavzevii的och1基因进行敲除并引入源自曲霉的msdS基因，以改变其分泌糖蛋白N-糖链的糖型结构。【方法】通过基因编辑对P. kudriavzevii的N-糖基化途径进行改造，获得P. kudriavzevii Δura3Δoch1::msdS菌株，分析P. kudriavzevii Δura3Δoch1::msdS菌株分泌糖蛋白上N-糖组的变化。【结果】与野生型P. kudriavzevii相比，P. kudriavzevii Δura3Δoch1::msdS菌株分泌糖蛋白上的N-糖链中可检测到Man₅GlcNAc₂型糖链结构。【结论】通过敲除och1基因和引入曲霉msdS基因，可使P. kudriavzevii合成Man₅GlcNAc₂糖链结构，为后续糖基化途径的改造奠定了基础。

Abstract:

[Background] Proteins produced by yeast usually undergo hyper glycosylation and form high mannose-type N-glycans, which can induce immune reactions in humans. As a result, the use of yeast is limited in the production of glycoprotein in drugs. Modifying the glycosylation pathway in yeast is a feasible approach for the production of glycoproteins used in drugs. Pichia kudriavzevii, a non-typical yeast species with strong stress resistance and rapid growth, has attracted much attention in recent years. By humanized glycosylation, P. kudriavzevii will serve as a cell factory to produce glycoproteins. [Objective] To obtain Man₅GlcNAc₂, the core structure of human N-glycan, is the key step to modify the glycosylation pathway in yeast. To achieve this goal, we deleted och1 of P. kudriavzevii and introduced msdS of Aspergillus into P. kudriavzevii. [Methods] The CRISPR-Cas9 system was employed to modify the N-glycosylation pathway of P. kudriavzevii, and the modified strain Δura3Δoch1::msdS was obtained. Furthermore, the N-glycocome of the glycoproteins secreted by Δura3Δoch1::msdS was analyzed. [Results] The N-linked Man₅GlcNAc₂ was detected on the glycoproteins secreted by Δura3Δoch1::msdS. [Conclusion] After the deletion of och1 and the introduction of msdS from Aspergillus,P. kudriavzevii cells can secrete the proteins with N-linked Man₅GlcNAc₂, which underpins the further humanized glycosylation in P. kudriavzevii.