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Advances in glucose/fructose-selective ethanol production have successfully enhanced raw sugar extraction from sugarcane juice by converting inhibitory substances (i.e., glucose/fructose) into ethanol, which is removed by subsequent operations in cane sugar mills. However, the commercial implementation of this breakthrough process in existing cane sugar mills requires a yeast strain that (i) can be used in food production processes, (ii) exhibits stable saccharometabolic selectivity, and (iii) can be easily separated from the saccharide solution. In this study, we developed a suitable saccharometabolism-selective and flocculent strain, Saccharomyces cerevisiae GYK-10. We obtained a suitable yeast strain for selective fermentation in cane sugar mills using a yeast mating system. First, we crossed a haploid strain defective in sucrose utilization with a flocculent haploid strain. Next, we performed tetrad dissection of the resultant hybrid diploid strain and selected GYK-10 from various segregants by investigating the sucrose assimilation and flocculation capacity phenotypes. Ten consecutive fermentation tests of the GYK-10 strain using a bench-scale fermentor confirmed its suitability for the implementation of practical selective fermentation in a commercial sugar mill. The strain exhibited complete saccharometabolic selectivity and sustained flocculation, where it maintained a high ethanol yield and conversion rate throughout the test., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)