Repeated batch fermentation from raw starch using a maltose transporter and amylase expressing diploid yeast strain.

We successfully demonstrated batch ethanol fermentation repeated ten times from raw starch with high ethanol productivity. We constructed a yeast diploid strain coexpressing the maltose transporter AGT1, alpha-amylase, and glucoamylase. The introduction of AGT1 allows maltose and maltotriose fermentation as well as the improvement of amylase activities. We also found that alpha-amylase activity during fermentation was retained by the addition of 10 mM calcium ion and that the highest alpha-amylase activity was 9.26 U/ml during repeated fermentation. The highest ethanol productivity was 2.22 g/l/h at the fourth batch, and after ten cycles, ethanol productivity of more than 1.43 g/l/h was retained, as was alpha-amylase activity at 6.43 U/ml.