Construction of yeast secretion production system of soybean proteins using mutant strains and their utilization for food

本课题的目的是利用缺乏液泡分选系统的突变株，建立大豆蛋白酵母分泌生产体系。为了达到这一目的，我们从以下四个角度进行了调查。以转基因拟南芥种子为材料，研究了大豆β-伴大豆球蛋白α‘亚单位含有液泡分选决定簇的区域。结果表明，空泡分选决定簇存在于C-末端10个残基中，足以将GFP分选为空泡。对裂解酵母羧基肽酶Y在芽殖酵母中的转运行为分析表明，羧基肽酶Y在芽殖酵母中的转运方式与芽殖酵母相同。研究了大豆球蛋白A1aB1b亚基在各种空泡分选系统中发生突变的芽殖酵母和分裂酵母中的表达行为，但不分泌该蛋白。激光共聚焦显微镜分析裂解酵母中球蛋白A1aB1b亚基的积累表明，AlaB1b亚基-GFP主要定位于内质网，高尔基体和泡囊中不存在。此外，在裂解酵母中共表达球蛋白-GFP和共生球蛋白-RFP导致共定位在同一隔室。这些事实表明，萌芽酵母和分裂酵母几乎不可能分泌大豆蛋白。结果表明，利用α因子信号肽，巴氏毕赤酵母能有效地分泌球蛋白。如上所述，我们确定了利用酵母分泌生产大豆蛋白所需的基本条件。

Object of this project is to establish yeast secretion production system of soybean proteins using mutant strains which are deficient in vacuolar sorting systems. We approached this purpose by investigating from the following four viewpoints.1. The region of soybean β-conglycinin α'subunit containing a vacuolar sorting determinant was investigated using transgenic Arabidopsis seeds. The results indicated that the vacuolar sorting determinant is present in the C-terminal 10 residues, which is sufficient for sorting GFP to vacuole.2. Analysis of transportation behavior of fission yeast carboxypeptidase Y in budding yeast indicated that the carboxypeptidase Y is transported to vacaoles in the same way as that of budding yeast.3. Expression behavior of glycinin A1aB1b subunit in various budding and fission yeasts having mutation in vacuolar sorting systems was examined, but the protein was not secreated. Analysis of accumulation of glycinin A1aB1b subunit in fission yeast using con focal laser microscope indicated that AlaB1b subunit-GFP mainly located in the endoplasmic reticulum but not in the Golgi body nor vaccoles. In addition, coexpression of glycinin-GFP and conglycinin-RFP in fission yeast resulted in colocation in the same compartment. These facts suggest that secretion of soybean proteins in budding and fission yeasts is almost impossible4. We ovsserved that Pichia pastoris efficiently secreated glycinin, as using signal peptide of α factor. As described, we established basic things required for secretion production of soybean proteins using yeast.