Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae |
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Authors: | Yong-Su Jin Thomas W Jeffries |
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Institution: | (1) Department of Food Science, University of Wisconsin, 1605 Linden Drive, 53706 Madison, WI;(2) Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, 53706 Madison, WI;(3) USDA Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive, 53705 Madison, WI |
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Abstract: | We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes (XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into the chromosome or by transforming cells with XYL2 in a multicopy vector. Genes in all three constructs were under control of the strong constitutive glyceraldehyde-3-phosphate
dehydrogenase promoter. Enzymatic activity of XR and XDH in the recombinant strains increased with the copy number of XYL1 and XYL2. XR activity was not detected in the parent but was present at a nearly constant level in all of the transformants. XDH activity
increased 12-fold when XYL2 was on a multicopy vector compared with when it was present in an integrated single copy. Product formation during xylose
fermentation was affected by XDH activity and by aeration in recombinant S. cerevisiae. Higher XDH activity and more aeration resulted in less xylitol and more xylulose accumulation during xylose fermentation.
Secretion of xylulose by strains with multicopy XYL2 and elevated XDH supports the hypothesis that d-xylulokinase limits metabolic flux in recombinant S. cerevisiae. |
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Keywords: | Metabolic flux metabolic engineering xylose xylose reductase xylitol dehydrogenase |
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