1-28: Optimization of xylose-assimilating pathway for constructing efficient xylose-fermenting Saccharomyces cerevisiae

In order to switch raw materials from food crops to energy crops for producing biofuels, microbial strains capable of fermenting cellulosic hydrolyzates containing glucose and xylose need to be developed.  As such, metabolic engineering approaches for constructing efficient xylose-fermenting yeast strains have been actively attempted for decades.  Introduction of a heterologous xylose-assimilating pathway consisting of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XK) has yielded engineered Saccharomyces cerevisiae strains which ferment xylose.  However, the ethanol yields and productivities are less efficient than naturally occurring xylose-fermenting yeast, Pichia stipitis.  In this study, we systematically investigated how the expression levels of three genes (XYL1, XYL2, and XYL3 coding for XR, XDH, and XK of P. stipitis, respectively) affect on xylose fermentation by the engineered S. cerevisiae strains.  First, we found that high expression levels of XYL1 increased the amounts of xylitol accumulation and inhibited the ethanol production.  Nevertheless, the undesirable phenotypes were suppressed when expression levels of XYL2 also increased accordingly.  In addition, high expression levels of XYL3 improved ethanol yields and productivities only when the expression levels of XYL1 and XYL2 were balanced.  These results suggest that optimization of expression levels of three genes is the most important prerequisite for developing efficient xylose-fermenting S. cerevisiae.