Engineering xylose and arabinose metabolism in recombinant Saccharomyces cerevisiae

Abstract: Utilization of all sugars in lignocellulose hydrolysates is a prerequisite for economically feasible bioethanol production. The yeast commonly used for industrial ethanol production, Saccharomyces cerevisiae, is naturally unable to utilize pentose sugars xylose and arabinose, which constitute a large fraction of many lignocellulosic materials. Xylose utilization by S. cerevisiae can be achieved by heterologous expression of a xylose utilization pathway, consisting either of xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK), or alternatively, of xylose isomerase (XI) and XK. Xylitol formed by XR is a major by-product in xylose fermentation when using the XR-XDH pathway. In this thesis, high-level expression of both XR and XDH was shown to decrease xylitol formation. The influence of other genetic modifications was also evaluated. It was shown that the overexpression of the non-oxidative pentose phosphate pathway (PPP) genes enables efficient growth on xylose and xylose fermentation, provided that the initial xylose pathway is expressed at a high level. When comparing the two xylose utilization pathways, higher ethanol productivity was achieved using the XR-XDH pathway, whereas higher ethanol yield was achieved with the XI pathway. The industrial xylose-fermenting S. cerevisiae strain TMB 3400, which has been previously generated by mutagenesis and selection, was tested for fermentation of lignocellulose hydrolysate. TMB 3400 displayed significantly better fermentation performance compared to the laboratory strains tested, highlighting the need for robust industrial strains in lignocellulose fermentation. TMB 3400 was also characterized by proteome analysis using difference in-gel 2-D electrophoresis. Consistently with the results obtained in other studies, increased activities of XR, XDH and a PPP enzyme TKL were found. The bacterial arabinose utilization pathway was introduced into TMB 3400, which resulted in the novel glucose, xylose and arabinose co-fermenting strain TMB 3063, with ethanol, xylitol and arabitol as the main fermentation products.