Novel strategy for yeast construction using delta-integration and cell fusion to efficiently produce ethanol from raw starch.

We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining delta-integration and polyploidization through cell fusion. Streptococcus bovis alpha-amylase and Rhizopus oryzae glucoamylase/alpha-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The alpha-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts.