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

Abstract

In recent years, many novel xylose-fermenting yeasts belonging to the new genus Spathaspora have been isolated from the gut of wood-feeding insects and/or wood-decaying substrates. We have cloned and expressed, in Saccharomyces cerevisiae, a Spathaspora arborariae xylose reductase gene (SaXYL1) that accepts both NADH and NADPH as co-substrates, as well as a Spathaspora passalidarum NADPH-dependent xylose reductase (SpXYL1.1 gene) and the SpXYL2.2 gene encoding for a NAD+-dependent xylitol dehydrogenase. These enzymes were co-expressed in a S. cerevisiae strain over-expressing the native XKS1 gene encoding xylulokinase, as well as being deleted in the alkaline phosphatase encoded by the PHO13 gene. The S. cerevisiae strains expressing the Spathaspora enzymes consumed xylose, and xylitol was the major fermentation product. Higher specific growth rates, xylose consumption and xylitol volumetric productivities were obtained by the co-expression of the SaXYL1 and SpXYL2.2 genes, when compared with the co-expression of the NADPH-dependent SpXYL1.1 xylose reductase. During glucose-xylose co-fermentation by the strain with co-expression of the SaXYL1 and SpXYL2.2 genes, both ethanol and xylitol were produced efficiently. Our results open up the possibility of using the advantageous Saccharomyces yeasts for xylitol production, a commodity with wide commercial applications in pharmaceuticals, nutraceuticals, food and beverage industries.

Valorisation of xylose to renewable fuels and chemicals, an essential step in augmenting the commercial viability of lignocellulosic biorefineries

“… 49–52 The xylose fermenting yeasts ( Candida shehatae , Scheffersomyces ( Pichia ) stipitis , Pichia fermentans , Spathaspora sp., etc ) employ the XR–XDH pathway for assimilation of xylose, and most of the yeasts utilize xylose under aerobic conditions. 53–55 …”

“…In the presence of xylose as a substrate, the affinity was observed to strengthen with K m of 29.5 (NADH) and 57.5 (NADPH) mM, respectively. 53,57 …”

New Strategies for Efficient Expression of Heterologous Sugar Transporters in Saccharomyces cerevisiae

“…Sp. passalidarum UFMG-CM-Y474 Isolated from rotting wood in Roraima, Brazil [17] S. cerevisiae DLG-K1 MATa gal2 ura3-52 his3-11,15 leu2-3,112 hxt2::HIS3 hxt5::LEU2 hxt7::HIS3 hxt3::LEU2::hxt6 hxt1::HIS3::hxt4 MAL2 SUC2 AUR1::pAUR-XKXDHXR Plasmids:…”

“…While glucose can be easily fermented by industrial Saccharomyces cerevisiae strains [11], this yeast is not naturally able to ferment the pentoses xylose or arabinose, unless it is genetically modified to express the assimilation routes for these sugars [12][13][14]. In the case of xylose, genes encoding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis or Spathaspora passalidarum, or xylose isomerase (XI) from bacteria and fungi have been extensively used [14][15][16][17][18]. Since both pathways transform xylose into xylulose, it is also required to overexpress the endogenous xylulokinase (XK) gene that will enhance the entrance of this sugar into the pentose-phosphate pathway.…”

Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae

“…While glucose can be easily fermented by industrial Saccharomyces cerevisiae strains [11], this yeast is not naturally able to ferment the pentoses, xylose and arabinose, unless it is genetically modified to express the assimilation routes for these sugars [12][13][14]. In the case of xylose (Figure 1), genes encoding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis or Spathaspora passalidarum, or xylose isomerase (XI) from bacteria and fungi, have been extensively used [14][15][16][17][18]. Since both pathways transform xylose into xylulose (Figure 1), it is also necessary to overexpress the endogenous xylulokinase (XK) gene that will enhance the entrance of this sugar into the pentose phosphate pathway.…”