1-27: High expression levels of XYL2 coding for xylitol dehydrogenase promise efficient xylose fermentation by engineered Saccharomyces cerevisiae

Engineering of xylose metabolism in yeast is necessary for producing cellulosic ethanol from lignocellulosic biomass because the traditional ethanologenic yeast, Saccharomyces cerevisiae cannot ferment xylose.  One of the most widely employed strategies is to introduce a xylose-assimilating pathway, implemented by expressing the XYL1, XYL2, and XYL3 genes from Pichia stipitis, into S.  cerevisiae.  However, the resulting engineered strains are reported to exhibit large variations in xylitol and ethanol yields, generating many hypotheses and arguments for elucidating the phenomena.  Here, we demonstrate low expression levels of XYL2, coding for xylitol dehydrogenase (XDH) might be a major bottleneck of efficient xylose fermentation.  Through an inverse metabolic engineering approach, we fortuitously identified XYL2 as an overexpression target for improving the xylose metabolism.  Specifically, we performed serial sub-culture experiments after transforming a genomic library of wild type S. cerevisiae into an engineered strain harboring integrated copies of XYL1, XYL2 and XYL3.  The isolated plasmids from efficient xylose-fermenting transformants contained XYL2, suggesting that the integrated XYL2 migrated into a multi-copy plasmid through homologous recombination.  We also found that the additional overexpression of XYL2 under the control of constitutive and strong promoters in various xylose-fermenting strains not only reduced xylitol accumulation, but also increased ethanol yields. This result suggests that a high expression level of XYL2 is a necessary condition for developing efficient xylose-fermenting strains.