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

突变菌株大豆蛋白酵母分泌生产系统的构建及其食品应用

基本信息

批准号：
10556030
负责人：
UTSUMI Shigeru
金额：
$ 8.45万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10556030/
关键词：
soybean proteins glycinin conglysinin vacuolar sorting secretion production budding yeast fission yeast Pichia pastoris カルボキシペプチダーゼY ミュータント

项目摘要

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.

本课题的目的是利用空泡分选系统缺陷的突变株建立酵母分泌生产大豆蛋白的系统。我们从以下四个角度进行了研究，以达到这一目的。利用转基因拟南芥种子研究了大豆β-伴大豆球蛋白β '亚基中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端10个残基，足以将GFP分选到空泡中.对裂殖酵母羧肽酶Y在芽殖酵母中的转运行为分析表明，羧肽酶Y以芽殖酵母相同的方式转运到孔隙中.研究了大豆球蛋白A1 aB 1b亚基在不同的芽殖酵母和裂殖酵母中的表达行为，这些酵母在液泡分选系统中具有突变，但该蛋白质不分泌。用激光共聚焦显微镜分析裂殖酵母中大豆球蛋白A1 aB 1b亚基的积累，发现AlaB 1b亚基-GFP主要定位于内质网，而不在高尔基体和液泡中。此外，在裂殖酵母中共表达大豆球蛋白-GFP和伴大豆球蛋白-RFP导致在同一隔室中共定位。这些事实表明，大豆蛋白质在芽殖酵母和裂殖酵母中的分泌几乎是不可能的。我们观察到毕赤酵母利用α因子的信号肽，高效地分泌大豆球蛋白。如上所述，我们建立了使用酵母分泌生产大豆蛋白所需的基本事项。