Exploiting the NADPH pool for xylitol production using recombinant Saccharomyces cerevisiae

Abstract

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 in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH‐dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p 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‐preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH‐generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, 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.

The Pentose Phosphate Pathway in Yeasts–More Than a Poor Cousin of Glycolysis

“…Unlike the unwanted occurrence of xylitol as a by-product in bioethanol production from pentoses described above, xylitol is of value as a sweetener and a functional food additive, with proposed anticancerogenic effects. Heterologous expression of xylose reductase genes has been employed in this context for xylitol production in yeast [ 120 ].…”

Combining Xylose Reductase from Spathaspora arborariae with Xylitol Dehydrogenase from Spathaspora passalidarum to Promote Xylose Consumption and Fermentation into Xylitol by Saccharomyces cerevisiae

“…Although the recombinant strain produced no acetate (and consequently the pH of the medium dropped just to pH 4.5) and less xylitol and ethanol, the production of glycerol was only slightly increased ( Figure 2, Table 6), indicating that other factors may limit xylose utilization by our engineered yeast strains. For example, the bottleneck may be a consequence of the relatively low affinity of the cloned SpXYL2.2 xylitol dehydrogenase for both NAD + and xylitol (Table 4), or the intracellular pools of reduced or oxidized and NADH/NADPH ratio of co-substrates [49,50], or even the low affinity of yeast sugar permeases for xylose transport [5,14,51]. Nevertheless, the ASY-2 strain transformed with the pPGK-SaXYL1 and pTEF-SpXYL2.2 plasmids (Table 6) showed xylitol yields (Y p/s = 0.614 g xylitol/g xylose) and volumetric productivities (Q p = 0.513 g/L/h) as good as or superior to those reported by other naturally xylose fermenting yeasts [52][53][54][55] or even engineered S. cerevisiae strains [38,49,50].…”

“…For example, the bottleneck may be a consequence of the relatively low affinity of the cloned SpXYL2.2 xylitol dehydrogenase for both NAD + and xylitol (Table 4), or the intracellular pools of reduced or oxidized and NADH/NADPH ratio of co-substrates [49,50], or even the low affinity of yeast sugar permeases for xylose transport [5,14,51]. Nevertheless, the ASY-2 strain transformed with the pPGK-SaXYL1 and pTEF-SpXYL2.2 plasmids (Table 6) showed xylitol yields (Y p/s = 0.614 g xylitol/g xylose) and volumetric productivities (Q p = 0.513 g/L/h) as good as or superior to those reported by other naturally xylose fermenting yeasts [52][53][54][55] or even engineered S. cerevisiae strains [38,49,50]. It is important to note that the maximal expected theoretical yield for the biotransformation of xylose into xylitol is 0.905-0.917 g xylitol/g xylose consumed, depending on how the cells will regenerate the NADH/NADPH consumed in the reduction of xylose, and that no carbon is used for cell growth or production of other metabolites [56].…”

Obtaining xylitol by hydrolysis-hydrogenation of liquors derived from sugarcane bagasse

“…It has recently been reported that accessory enzymes from hypercellulolytic Penicillium funiculosum facilitate the complete saccharification of sugarcane bagasse [39]. sugarcane bagasse [41,42]. In previous work, Vallejos et al [14] reported maximal xylitol concentration (32.0 g L -1 ) from hemicellulosic liquors obtained from sugarcane bagasse autohydrolysis using Candida tropicalis (104.1 g L -1 xylose, 0.46 g g -1 yield, 0.27 g L -1 h -1 productivity).…”