Sunday, April 29, 2007 - 4:45 PM

Overexpression of bacterial xylose isomerase and own xylulokinase improves alcoholic fermentation of xylose in the thermotolerant yeast Hansenula polymorpha

Andriv A. Sibirny1, Olena V. Verba1, Kostyantyn V. Dmytruk1, Olena P. Ishchuk1, and Charles Abbas2. (1) Institute of Cell Biology, NAS of Ukraine, Drahomanov St. 14/16, Lviv 79005, Ukraine, (2) Archer Daniels Midland, 1001 Brush College Road, Decatur, IL 62521-1656

The thermotolerant yeast Hansenula polymorpha is able to ferment xylose to ethanol at high temperatures (48–50 °C), however, yield and productivity of ethanol synthesis are too low. To improve characteristics of xylose fermentation, the recombinant strain Δxyl1 Δxyl2-A Δxyl2-B with deletions of genes encoding first enzymes of xylose utilization (NAD(P)H-dependent xylose reductase and NAD-dependent xylitol dehydrogenases, respectively) was constructed and used as a recipient for coexpression of the Escherichia coli xylA gene coding for xylose isomerase and endogenous XYL3 gene coding for xylulokinase. The expression of both genes was driven by the H. polymorpha strong constitutive glyceraldehyde-3-phosphate dehydrogenase (HpGAP) promoter. Xylose isomerase activities of obtained transformants amounted to ~ 80% of that of the bacterial host strain. Xylulokinase activities of the transformants were twice higher than that in the parental strain. The recombinant strains with simultaneous overexpression of bacterial xylose isomerase and own xylulokinase accumulated 50-150% more ethanol during xylose fermentation as compared to the wild-type strain. Perspectives of metabolic engineering of H. polymoprha for further improvement of the yield and productivity of xylose alcoholic fermentation will be discussed.