Xylose is the second major fermentable sugar present in hard woods and herbs (after D-glucose). Therefore, efficient conversion of xylose to ethanol is essential for the commercialization of lignocellulosic ethanol, which may provide an ideal alternative to fossil fuels in the future. ZLYRHZ7 is a fusant produced by protoplast fusion between two different yeast species, Saccharomyces cerevisiae W5 and Candida shehatae 20335, which is able to utilize xylose to produce ethanol. To improve ethanol production and to quantitatively analyze metabolic pathway in ZLYRHZ7, we used high performance liquid chromatography (HPLC) to assess the utilization rates of xylose, xylitol, and xylulose, and to measure ethanol yields using xylose, xylitol, and xylulose as sole carbon sources. The ethanol yields reached 0.549 +/- 0.003, 0.567 +/- 0.003 and 0.544 +/- 0.005 g/g in 72 h, which indicated that the metabolic pathways from xylose to xylitol, xylitol to xylulose, and xylulose to ethanol, respectively, were functional. In addition, enzyme activity and qRT-PCR analyses showed that the xylose metabolism-related enzymes xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XK) and their respective genes were expressed at significantly higher levels in ZLYRHZ7 than in both S. cerevisiae W5 and C. shehatae 20335 at 24, 48, and 72 h of fermentation. These results clearly show that the fusant ZLYRHZ7, obtained by protoplast fusion of two different yeast species, has the ability to ferment xylose to produce ethanol. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.