Efficient co-displaying and artificial ratio control of α-amylase and glucoamylase on the yeast cell surface by using combinations of different anchoring domains

Recombinant yeast strains that display heterolo- gous amylolytic enzymes on their cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system are considered as promising biocatalysts for direct ethanol pro- duction from starchy materials. For the effective hydrolysis of these materials, the ratio optimization of multienzyme activity displayed on the cell surface is important. In this study, we have presented a ratio control system of multienzymes displayed on the yeast cell surface by using different GPI- anchoring domains. The novel gene cassettes for the cell- surface display of Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase were constructed using the Saccharomyces cerevisiae SED1 promoter and two different GPI-anchoring regions derived from Saccharomyces cerevisiae SED1 orSAG1. These gene cassettes were integrat- ed into the Saccharomyces cerevisiae genome in different combinations. Then, the cell-surface α-amylase and glucoamylase activities and ethanol productivity of these re- combinant strains were evaluated. The combinations of the gene cassettes of these enzymes affected the ratio of cell- surface α-amylase and glucoamylase activities and ethanol productivity of the recombinant strains. The highest ethanol productivity from raw starch was achieved by the strain har- boring one α-amylase gene cassette carrying the SED1-an- choring region and two glucoamylase gene cassettes carrying the SED1-anchoring region (BY-AASS/GASS/GASS). This strain yielded 22.5±0.6 g/L of ethanol from 100 g/L of raw starch in 120 h of fermentation.