Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and alpha-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the alpha-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast alpha factor, respectively, were fused with the gene encoding the C-terminal half of the yeast alpha-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and alpha-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and alpha-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase.