Development of nanofabrication method of supermacromolecules on the yeast cell surface and its application to bioprocesses

酵母细胞表面超高分子纳米加工方法的发展及其在生物过程中的应用

• 批准号: 16360413
• 负责人: KONDO Akihiko
• 金额: $ 9.41万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16360413/
• 关键词: Yeast; Cell Surface Engineering; Dockerin; Cohesin; Cellulosome; Faburication; Biomass; Cellulase

To reduce the cost of immobilized enzymes, heterogonous enzymes displaying recombinant microorganisms have been developed by many researchers. However, in a current cell-surface display system to perform the complicated reaction, it is necessary to control the ration of the proteins displayed on the cell surface. To overcome this problem, we constructed a novel cell-surface display system using two pairs of proteins that assemble each other, 1 pair namely ZZ domain of protein A derived from Staphylococcus aureus and Fc part of human immunoglobulin G. And 2 pair cohesin and dockerin of cellulosome from Clostridium cellulovorans. In this system, scaffolding protein (ZZ-Coh-Coh) is displayed on the cell surface and the target protein fused to the Fc or Dockerin are secreted. Using this system, recombinant Trichoderma reesei endoglucanase II was displayed on the yeast cell surface. Display of the EGII on the cell surface was confirmed by hydrolysis experiment using β-glucan as a substrate and EGII activity was detected in the cell-pellet fraction. Following experiment showed that, EGII and Aspergillus aculeatus β-glucosidase 1 were co-displayed on the cell surface. The resulting yeast cells hydrolyzed β-glucan to glucose. This study demonstrated simultaneous display of two enzymes on the yeast cell surface can be accomplished using this cell surface display system.

为了降低固定化酶的成本，许多研究人员开发了展示重组微生物的异源酶。然而，在当前的细胞表面展示系统中，为了进行复杂的反应，需要控制展示在细胞表面上的蛋白质的比例。为了克服这个问题，我们使用两对相互组装的蛋白质构建了一种新型细胞表面展示系统，其中一对即来自金黄色葡萄球菌的蛋白 A 的 ZZ 结构域和人免疫球蛋白 G 的 Fc 部分。以及两对来自纤维素梭菌的多纤维素酶体的粘连蛋白和坞蛋白。在该系统中，支架蛋白（ZZ-Coh-Coh）展示在细胞表面，并分泌与Fc或Dockerin融合的目标蛋白。使用该系统，重组里氏木霉内切葡聚糖酶 II 被展示在酵母细胞表面。通过使用β-葡聚糖作为底物的水解实验证实了EGII在细胞表面上的展示，并在细胞沉淀部分中检测到了EGII活性。后续实验表明，EGII与棘孢曲霉β-葡萄糖苷酶1共展示在细胞表面。所得酵母细胞将 β-葡聚糖水解为葡萄糖。这项研究证明，使用这种细胞表面展示系统可以在酵母细胞表面同时展示两种酶。

项目成果

Ethanol fermentation from lignocellulosic hydrolysate by a recom binant xylose-and celloolingosaccharide-assimilationg veast strain.

通过重组木糖和纤维糖同化酵母菌株从木质纤维素水解产物中发酵乙醇。

• 期刊: Applied Microbiology and Biotechnology (In press)
• 作者: Katahira;S.;Mizuike;A.;Fukuda;H.;Kondo;A.
• 通讯作者: A.