We previously reported the construction of a recombinant Candida utilis strain expressing mXYL1, XYL2 and XYL3, which encode mutated Candida shehatae xylose reductase K275R/N277D, C. shehatae xylitol dehydrogenase and Pichia stipitis xylulokinase to produce ethanol from xylose. However, its productivity was low. In this study, to breed a strain with higher productivity of ethanol from xylose, we used a cocktail multicopy integration method to attain optimized gene dosage of the three enzymes. Gene expression cassettes of the xylose-metabolizing enzymes were simultaneously integrated into C. utilis chromosomes in one step. Measurement of integrated gene copy number and xylose fermentability in all of the resulting integrant strains revealed that the copy number ratio of XYL2/mXYL1 in strains with higher ethanol yield was higher than that in strains with lower ethanol yield, whereas the copy number ratio of mXYL1/XYL3 was lower in strains with higher ethanol yield. The resultant strain CIS35, which was found to be the best producer of ethanol from xylose produced 29.2 g/L of ethanol, yielding 0.402 g ethanol/g xylose. This result provides that C utilis may be a good candidate as a host for ethanol production from xylose. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.