Production of xylitol by recombinant yeasts expressing XYL1 gene from pichia stipitis

Abstract

Saccharomyces cerevisiae transformants expressing the XYL1 gene encoding xylose reductase (XR) from Pichia stipitis were constructed using δ-sequence mediated multicopy integration vector or a YEp-type vector for the production of xylitol from xylose. Through the sequential transformations using a dominant selection marker, neo‘ and URA3 auxotrophic marker, transformants integrated chromosomally more than 30 copies of XYL1 gene were obtained. Tandem integration into a single site was most commonly observed with a maximum of three different insertion sites. The selected integrant with the highest activity showed the volumetric productivity of xylitol of 0.16-0.11 g/l/hr in sequential batch cultures and the stable xylitol production at the level of 5.5g/l in chemostat due to the stable maintenance of δ-integrated vector. In contrast, the transformant harboring a YEp-type plasmid showed mitotic plasmid instability. Xylitol production by recombinant Saccharomyces cerevisiae strains expressing the XYL1 gene through the glucose-repressible ADH2 promoter was investigated with glucose, glycerol, acetate and ethanol as cosubstrate under oxygen-limited conditions. Among the used cosubstrates, xylitol production was the best with glucose. With glycerol, acetate and ethanol as cosubstrate, both the expression level of XYL1 gene and the cellular specific productivity were relatively high, but volumetric xylitol productivity was lower than that with glucose. When glucose was used with supplement of non-sugar cosubstrates (1% or 2% glycerol, acetate and ethanol, respectively), both the xylitol productivity and the cellular specific productivity (0.27g xylitol/l/h and 0.060 g xylitol/g cells/h with 2% glycerol, respectively) were substantially increased compared with those with only glucose (0.19 g xylitol/l/h and 0.029 g xylitol/g cells/h, respectively).