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质酶。然而，在当前的细胞表面显示系统中，为了进行复杂的反应，必须控制在细胞表面显示的蛋白质的比例。为了解决这一问题，我们利用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，以及来自梭状芽胞杆菌的纤维素体的2对内聚蛋白和dockerin，构建了一种新的细胞表面展示系统。在该系统中，支架蛋白（ZZ-Coh-Coh）在细胞表面显示，并分泌与Fc或Dockerin融合的靶蛋白。利用该系统，重组里氏木霉内切葡聚糖酶II在酵母细胞表面得到了表达。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，并在细胞颗粒中检测了EGII的活性。随后的实验表明，EGII和aculeatus Aspergillus β-glucosidase 1在细胞表面共同显示。产生的酵母细胞将β-葡聚糖水解为葡萄糖。本研究表明，利用该细胞表面显示系统可以实现两种酶在酵母细胞表面的同时显示。

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.