Metabolic Engineering of the Pentose Phosphate Pathway of Xylose Fermenting Saccharomyces cerevisiae

Abstract: The aim of the work presented in this thesis is the improvement of xylose fermentation performance of recombinant Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH). A recombinant strain of S. cerevisiae, fermenting both xylose and glucose to ethanol could decrease the production cost of ethanol from ligno-cellulosic biomass. The xylose fermentation efficiency of recombinant S. cerevisiae is limited by a slow xylose fermentation rate and high xylitol by-product formation. Xylulose fermentation was used as a model of xylose fermentation, where the effect of the expression of the heterologous XYL1 and XYL2 is omitted. Xylulokinase over-production by over-expression of the native XKS1 improved xylulose consumption rate of S. cerevisiae, but decreased xylose consumption rate by S. cerevisiae expressing XYL1 and XYL2. This phenomenon was suggested to result from too high over-expression of the XKS1 gene in the strain expressing XYL1 and XYL2. XKS1 over-expression decreased xylitol yield from both xylulose and xylose. S. cerevisiae strains with a partly or completely blocked oxidative PPP, had a higher xylulose consumption rate than the control strains. This was interpreted as limiting capacity of the non-oxidative PPP, since blocking the oxidative PPP prevents the metabolic flux from this pathway from competing with the xylulose flux for the non-oxidative PPP capacity. Blocking the oxidative PPP had a very different effect on xylose fermentation. Recombinant S. cerevisiae expressing XYL1, XYL2, over-expressing the native XKS1 and blocked or partly blocked oxidative PPP fermented xylose slower than the control strains. Xylitol production also ceased as a result. The lower xylitol production was interpreted as a more NADH than NADPH dependent xylose reduction by the XR enzyme, and thus a better co-factor balanced xylose metabolism. The decrease in xylose consumption rate was probably the result of the absence of NADPH-dependent xylose reduction combined with inhibition of XR by higher intracellular levels of NADP+. Over-expression of XYL1 as well as allowing a low oxidative PPP activity increased the xylose consumption rate. S.cerevisiae TMB3026 over-expressing the entire non-oxidative PPP, including the genes RPE1, RKI1, TAL1 and TKL1, was constructed. This strain, also expressing the XYL1, XYL2 and over-expressing XKS1, fermented xylose at the same rate as the control, S.cerevisiae TMB3001. This suggests that the non-oxidative PPP does not limit the xylose consumption rate in xylose-fermenting S. cerevisiae TMB3001. A new expression vector was developed to achieve the multiple over-expressions of the non-oxidative pentose phosphate pathway (PPP) genes. This vector allows marker recycling, so the number of genetic modifications to a strain is independent of the number of available markers. Dominant markers were used to construct a xylose-fermenting wild-type S. cerevisiae strain.