基于壳聚糖结合模块构建新型酿酒酵母孢子表面展示系统

Construction of a novel Saccharomyces cerevisiae spore surface display system based on chitosan-binding module

摘要:

【目的】基于当前细胞表面展示体系存在的问题，旨在构建一个新型的普适性强、抗逆性好、高效稳定的酿酒酵母孢子表面展示系统。【方法】首先，根据酵母孢子固定化酶的特性，通过查阅文献寻找潜在的与孢子壁壳聚糖层高度亲和的壳聚糖结合模块；其次，利用绿色荧光蛋白(green fluorescent protein，GFP)与结合模块融合表达，在体外和孢子内分别验证结合模块与孢子壁的亲和能力；之后，选择Saccharomyces cerevisiae AH109来源的α-半乳糖苷酶(α-galactosidase，MEL1)评估新型展示体系的效能。【结果】首先，选择Paenibacillussp. IK-5来源壳聚糖酶的碳水化合物结合模块32 (carbohydrate binding module 32，CBM32)作为壳聚糖结合模块。其次，将大肠杆菌表达纯化后的融合蛋白CBM32-GFP与dit1Δ孢子共孵育，通过GFP荧光定位以及荧光强度验证CBM32在体外与孢子壁具有较好的亲和能力；CBM32-GFP在dit1Δ孢子内的荧光定位与结合能力证明了其在孢子内能够与孢子壁紧密结合；最后，以MEL1替换GFP应用到新型展示体系中，与只表达MEL1的孢子相比，CBM32-MEL1孢子酶活不仅提高了68.65%，最高酶比活力达到460.59 U/g DCW (dry cell weight, 菌体干重)，重复使用能力也得到了显著提高；此外，该体系提高了MEL1的稳定性和可操作性。【结论】本研究首次提出利用结合模块来构建一个新型酿酒酵母孢子表面展示体系，为真核来源的多糖基化位点修饰以及多亚基结构蛋白提供了可靠的细胞表面展示平台，为实现工业化应用孢子固定化酶提供了理论依据。

Abstract:

[Objective] Based on the existing problems in the current cell surface display systems, we aimed to establish a novel Saccharomyces cerevisiae spore surface display system with strong universality, better stress resistance, and higher efficiency and stability. [Methods] Firstly, according to the characteristics of immobilized enzymes in S. cerevisiae spores, we searched the potential chitosan-binding modules with high affinity with the chitosan layer of spore wall by referring to literature. Then, the binding module was fused and expressed with green fluorescent protein (GFP) and the affinity ability of the binding module with the spore wall was verified in vitro and in vivo. Finally, we selected α-galactosidase (MEL1) derived from S. cerevisiae AH109 to evaluate the efficacy of the novel display system. [Results] Firstly, we selected a carbohydrate binding module 32 (CBM32) derived from Paenibacillus sp. IK-5 chitosanase as the chitosan-binding module. Next, the fusion protein CBM32-GFP, which was expressed in Escherichia coli and purified, was co-incubated with dit1Δ spores, and the result showed that CBM32 exhibited good affinity ability with the spore wall in vitro by the localization and intensity of GFP fluorescence. Furthermore, the fluorescence localization and binding ability of CBM32-GFP in dit1Δ spores proved that CBM32 was tightly bound to the spore wall in vivo. Finally, we replaced GFP with MEL1 in this display system. Compared with those of spores only expressing MEL1, the activity of spores displaying CBM32-MEL1 was increased by 68.65%, with the highest specific activity reaching 460.59 U/g DCW (dry cell weight), and the reusability was also significantly improved. Moreover, the stability and operability of MEL1 were enhanced. [Conclusion] We constructed a novel S. cerevisiae spore surface display system based on the chitosan-binding module for the first time, which provided a reliable cell surface display platform for the eukaryotic proteins with multi-glycosylation sites and multi-subunit structures. It also provided the theoretical basis for the industrial application of immobilized enzymes in spores.