In this study, four engineered Saccharomyces cerevisiae carrying xylanase, beta-xylosidase and xylose reductase genes by different transcriptional regulations were constructed to directly convert xylan to xylitol. According to the results, the high-copy number plasmid required a rigid selection for promoter characteristics, on the contrast, the selection of promoters could be more flexible for low-copy number plasmid. For cell growth and xylitol production, glucose and galactose were found more efficient than other sugars. The semi-aerobic condition and feeding of co-substrates were taken to improve the yield of xylitol. It was found that the strain containing high-copy number plasmid had the highest xylitol yield, but it was sensitive to the change of fermentation. However, the strain carrying low-copy number plasmid was more adaptable to different processes. By optimization of the transcriptional regulation and fermentation processes, the xylitol concentration could be increased of 1.7 folds and the yield was 0.71 g xylitol/g xylan. (C) 2013 Elsevier Ltd. All rights reserved.