Metabolic Engineering of Xylose-Utilising Saccharomyces cerevisiae Strains. A Closer Look at Recombinant Strains Based on the Xylose Reductase-Xylitol Dehydrogenase Pathway.

Saccharomyces cerevisiae produces ethanol efficiently from the hexose sugars in lignocellulose hydrolysates, but it can not utilise pentose sugars such as xylose and arabinose. Stable xylose-utilising S. cerevisiae strains obtained by integration of the Pichia stipitis genes encoding xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase, exhibit low ethanol yields when utilising xylose, mainly because of xylitol formation. Xylitol results from a different cofactor usage in the NAD(P)H-dependent XR and the NAD+-dependent XDH reactions. By disruption of genes in the NADPH-producing oxidative pentose phosphate pathway, less NADPH becomes available for the reduction of xylose. As a result the XR utilises mainly NADH, and the strains show enhanced ethanol yields and decreased xylitol yields, however, at the expense of the xylose fermentation rate. The xylose fermentation rate can be partly restored by enhancing the XR activity and/or fine-tuning the glucose-6-phosphate dehydrogenase activity. The overproduction of a transhydrogenase, capable of converting NADPH and NAD+ into NADP+ and NADH, also lowers the xylitol yield but has no positive effect on the ethanol yield. Not only the ethanol yield but also the rate of xylose utilisation must be enhanced in recombinant S. cerevisiae strains. The transport of xylose, the xylose pathway and the pentose phosphate pathway are possible bottlenecks during xylose fermentation. Increased XR activity results in enhanced xylose flux, but also in enhanced glycerol yield, which can be explained by the dihydroxyacetone phosphate-reducing activity of XR. The genomic changes responsible for enhanced growth on xylose have not yet been identified in xylose-growing S. cerevisiae mutants. However, genome-wide transcription analysis on a number of xylose-growing strains points towards enhanced expression levels of genes in galactose metabolism and in the pentose phosphate pathway.