Xylitol production from xylose was studied using recombinant Saccharomyces cerevisiae 2805 containing xylose reductase genes (XYLI) of Pichia stipitis at chromosomal delta-sequences. S. cerevisiae 2805-39-40, which contains about 40 copies of the XYL1 gene on the chromosome, was obtained by a sequential transformation using a dominant selection marker neo(r) and an auxotrophic marker URA3. The multiple XYLI genes were stably maintained on the chromosome even after 21 and IO days in the non-selective sequential batch and chemostat cultures, respectively, whereas S. cerevisiae 2805:pVTXR, which harbors the episomal plasmid pVTXR having the XYLI gene, showed mitotic plasmid instability and more than 95% of the cells lost the plasmid under the same culture conditions. In the first batch (3 days) of the sequential batch culture, volumetric xylitol productivity was 0.18 g l(-1) h(-1) for S. cerevisiae 2805-39-40, as compared to 0.21 g l(-1) h(-1) for S. cerevisiae 2805:pVTXR. However, the xylitol productivity of the latter started to decrease rapidly in the third batch and dropped to 0.04 g l(-1) h(-1) in the seventh batch, whereas the former maintained the stable xylitol productivity at 0.18 g l(-1) h(-1) through the entire sequential batch culture. The xylitol production level in the chemostat culture was about 8 g l(-1) for S. cerevisiae 2805-39-40, as compared to 2.0 g l(-1) for S. cerevisiae 2805:pVTXR after 10 days of cultures even though the xylitol production level of the latter was higher than that of the former for the first 5 days. The results of this experiment indicate that S. cerevisiae containing the multiple XYLI genes on the chromosome is much more efficient for the xylitol production in the long-term non-selective culture than S. cerevisiae harboring the episomal plasmid containing the XYLI gene. (C) 1999 Elsevier Science B.V. All rights reserved.