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

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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.