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菌株，建立大豆蛋白的酵母菌分泌生产系统。为了达到这个目的，我们从以下四个角度进行了调查。利用转基因拟南芥种子研究了大豆β-甘氨酸α′亚基中含有空泡分选决定因子的区域。结果表明，液泡分选决定因子存在于c端10个残基中，这足以将GFP分选到液泡中。对裂变酵母羧肽酶Y在出芽酵母中的运输行为分析表明，羧肽酶Y在出芽酵母中的运输方式与出芽酵母相同。研究了甘氨酸A1aB1b亚基在液泡分选系统突变的各种出芽和裂变酵母中的表达行为，但该蛋白不分泌。利用激光共聚焦显微镜对裂变酵母中甘氨酸A1aB1b亚基- gfp的积累进行分析，发现al1b亚基- gfp主要位于内质网，而不在高尔基体和裂孔中。此外，在裂变酵母中，glycine - gfp和conglycin - rfp的共表达导致了在同一胞室中的共存。这些事实表明，芽殖酵母和裂变酵母几乎不可能分泌大豆蛋白。我们观察到毕赤酵母利用α因子的信号肽有效地分泌甘氨酸。如上所述，我们建立了用酵母分泌大豆蛋白所需的基本物质。

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.