Metabolic Control Points in Ethanolic Fermentation of Xylose by Saccharomyces cerevisiae

Baker’s yeast, Saccharomyces cerevisae, has been engineered to utilize the pentose sugar xylose present in lignocellulose biomass for the production of ethanol. Currently ethanol production from xylose is slow which limits the implementation of said strains in industrial production. This thesis describes the investigation of several metabolic aspects that control the rate of xylose utilization by recombinant S. cerevisiae. The affinity of S. cerevisiae transporters for xylose is generally at least one order of magnitude lower than for glucose. It was therefore attempted to improve xylose uptake by expressing a heterologous xylose transporter in S. cerevisiae. Among the transporters investigated, the Gxf1 transporter from Candida intermedia improved xylose transport kinetics the most. The xylose uptake rate was however only increased at substrate concentration below 4 g/L, which indicated that other parts of the metabolism controlled xylose utilization to a greater extent. The enzyme xylose reductase (XR) was found to control xylose utilization in S. cerevisiae. Based on these results, XR was modified by random mutagenesis and the resulting S. cerevisiae library was selected in sequential anaerobic batch cultivation on xylose. At the end of the selection process a single mutant, XR N272D, was isolated. The XR N272D mutant displayed improved kinetics in terms of Vmax and cofactor specificity, and significantly increased ethanol productivity. The regulatory response to xylose was also investigated. It was observed that respiratory genes were up-regulated on xylose compared to glucose under both aerobic and anaerobic conditions. In addition, S. cerevisae expressed different glycolytic isozymes during growth on xylose compared to glucose. It still remains to be established whether the regulatory response to xylose is optimal for ethanol production.