马克斯克鲁维酵母细胞壁改构对其表面展示系统的调节作用

The yeast surface display system for adding new protein functions to the surfaces of cell wall has been developed, and applications of the system to various fields have been proposed. But we still know little about the correlation between the display efficiency and the cell wall structure. The regulation mechanisms of the yeast surface display is also not very clear. As a kind of newly approved food material, Kluyveromyces marxianus shows the abilities of outstanding temperature tolerance, wider substrate utilization and favorable fermentative performance, which is potential for various fields. And the surface display system of K. marxianus has already constructed by the project applicants. In this study, the project applicants will reconstructing the cell wall of K. marxianus and try to find the suitable cell wall structure for protein display and prove the display efficiency of foreign proteins. The research results will show the effect of the cell wall structure on the yeast surface display and reveal the regulation mechanism of K. marxianus surface display system to enrich the yeast surface display theory.

酵母表面展示系统是近年来发展最为迅速的真核表达系统，广泛应用于生命、医药等众多领域。然而，如何提高酵母表面展示系统的展示效率是其进一步发展应用的关键。目前酵母菌细胞壁结构对表面展示系统展示效率的影响及相关机制仍不明确。马克斯克鲁维酵母（Kluyveromyces marxianus）作为外源表达宿主具有更独特的优势。它生长速度快、可以利用各种非粮生物质原料，能够适应高温的发酵条件，在现代工业生物技术领域具有广阔的应用前景。本项目申请人成功构建了K. marxianus表面展示系统，拟以其为研究对象，以改变细胞壁“骨架”构成和甘露糖蛋白含量的方式，完善现有细胞壁的改构方案，构建不同类型的细胞壁改构模型，监测细胞壁改构前后表面展示效率的变化。揭示细胞壁改构对K. marxianus表面展示系统调节的机理，预测适合表面展示系统的细胞壁结构，为更进一步提高表面展示效率奠定基础。

本项目以改变细胞壁骨架构成和甘露糖蛋白含量的方式，进行酵母细胞壁的改构，构建不同类型的细胞壁改构模型，监测细胞壁改构前后表面展示效率的变化。筛选得到展示效率提高的菌株big1_02和Δmnn2_35，酶活力较原始菌株分别提高了21.6%和33.6%。明确改变β-1,6-葡聚糖和甘露糖的含量可以提高表面展示的效率。在今后的研究中可以构建更复杂的细胞壁改构模型，获得表面展示效率更高的改构菌，进一步推进酵母菌表面展示的应用。