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

为了降低固定化酶的成本，许多研究人员开发了显示重组微生物的异源酶。然而，在目前的细胞-表面展示系统中，为了执行复杂的反应，需要控制在细胞表面展示的蛋白质的比例。为了解决这一问题，我们构建了一种新型的细胞表面展示系统，它使用两对相互组装的蛋白质，一对是金黄色葡萄球菌蛋白A的ZZ结构域和人免疫球蛋白G的Fc部分，另一对是来自纤维素梭菌的纤维小体的2对粘附素和Dockerin。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融合的目的蛋白。利用该体系，将重组里氏木霉内切葡聚糖酶II展示在酵母细胞表面。以β-葡聚糖为底物的水解实验证实了EGII在细胞表面的显示，并在细胞颗粒组分中检测到EGII活性。后续实验表明，EGII和尖锐曲霉β-葡萄糖苷酶1共显示在细胞表面。由此产生的酵母细胞将β-葡聚糖水解成葡萄糖。这项研究表明，利用该细胞表面展示系统可以同时在酵母细胞表面展示两种酶。

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.