1. Cloning and Expression in Saccharomyces cerevisiae of The NAD(P)H-dependent Xylose Reductase- Encoding Gene (XYLI) From The Xylose-assimilating Yeast Pichia stipitis Bianca Peixoto Correia Ohio University Biotechnology and Genetic Engineering - PBIO4500 René Amore, Peter KOtter, Christina Kiister, Michael Ciriacy and Cornelis P. Hoilenberg 11/25/14 1

2. Introduction  Population growth - Fuel (petroleum) - Biofuel (ethanol)  Bioethanol production - Sugar fermentation - Microorganisms - Saccharomyces cerevisiae 2

3. Introduction  Biomass - Corn - Sugar cane 3

4. Introduction  Xylose metabolism - Bacterias - Yeasts Pichia stipitis XYL1 XYL2 4

5. Methods  Construction of a Pichia stipitis genomic library and isolation of genes required in biossynthetic pathways P. stipitis DNA XR fragments YEp13 vector BamHI SauA3 E. coli DH5 α F Cleaved vectors 5

6. Methods  Isolation and subcloning of the XR-encoding gene pXRa cDNA pR1 / pR7 / pR20 EcoRI S. cerevisiae 6

7. Methods  Expression of the Pichia stipitis XYLI gene in S. cerevisiae  pR1 and pR20 plasmids introduced into S. cerevisiae  Selected by using leucine  Western Blot G = Glucose X = Xylose 7

8. Methods  The XR activity in Saccharomyces cerevisiae transformants  XYL1 gene is induced by xylose in P. stipitis (purple box) 8

9. Conclusion  XR is a important enzyme in the xylose fermentation and it was high expressed in S. cerevisiae  However, the amount of XR protein and activity was 20 times less than in P. stipitis  The lack of xylose fermentation of S. cerevisiae is due to the absence of a xylose to xylulose converting pathway  This deficiency can be overcome by the introduction and expression of the genes for XYL1 and XYL2 9

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