Exploiting the NADPH pool for xylitol production using recombinantSaccharomyces cerevisiae

Xylitol is a five-carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H-dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed inSaccharomyces cerevisiaeto screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH-dependent enzymes (Candida tropicalisXR andS.cerevisiaeGre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH-preferringC.tropicalisXR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of theZWF1gene, coding for the first enzyme of the NADPH-generating pentose phosphate pathway, was replaced with the constitutiveGPDpromoter in a strain expressingC.tropicalisXR. This change led to a ~12% increase in xylitol yield. Deletion ofXYL2andSOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach.