The increasing need of oil and the negative impacts of fossil fuels on climate change have motivated the research towards environmental friendly alternatives to diminish the use of fossil resources. Until today, bioethanol production and use has increased worldwide. The raw materials used today compete with food industry and conquently, the ethanol production from lignocellulose has been developed intensively. However, to make the processes economically competitive, also pentoses in lignocellulose should be fermented at high rate. Saccharomyces cerevisiae, alias baker’s yeast has an excellent ethanol production capacity but it can not naturally utilize xylose. Several xylose-utilizing S. cerevisiae strains have been constructed; however the fermentation rate is still lower than that of hexose sugars. The major obstacles are thought to be the xylose transport, ATP depletion during the fermentation and the redox imbalance due to the different cofactor specifities of xylose reductase (XR
) and xylitol dehydrogenase (XDH) enzymes in the xylose utilization pathway.
This thesis includes three separate studies related to xylose metabolism of recombinant S. cerevisiae. In the first part the aim was to set up a high throughput xylose uptake assay by overexpressing a gene encoding β-xylosidase enzyme from Tricoderma reesei in S. cerevisiae. The experimental part included the enzyme activity measurements by using commercial xylose analogue p-nitrophenyl-β-d-xylopyranoside, which creates yellow colour when hydrolyzed with β-xylosidase. In the second study Trypanosoma brucei’s NADH dependent fumarate reductase (FRDg) encoding gene was overexpressed in xylose utilizing S. cerevisiae in order to improve ethanol fermentation by providing intracellular NAD+ for the XDH reaction. Aerobic and anaerobic shake flask cultivations were carried out with the constructed strains. The xylose utilization and metabolite and ethanol productivities were measured with high pressure liquid chromatography (HPLC). In the third part of the study, XR and XDH activities were measured spectrophotometrically from the xylose-utilizing yeast strains from the VTT Culture collection. The aim of the study was to find new enzymes to be exploited in the engineering of xylose metabolism of S. cerevisiea.
The xylose transporter assay was not functional. The BXL1 gene was expressed in yeast, but no β-xylosidase activity was detected. The expression of FRDg slightly improved the xylose fermentation in xylose-utizing S. cerevisiae but the activity measurements did not show FRDg activity. In the XR and XDH activity measurements of strains from the VTT Culture collection a couple of strains with potentially interesting XR and XDH activities were found. These enzymes offer alternatives for S. stipitis XR and XDH enzymes in engineering of baker’s yeast strains for ethanol or xylitol production.
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