Oral Presentation 2-06

Minimal Metabolic Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Xylose Fermentation and Anaerobic Growth on Xylose: A Proof of Principle

Marko Kuyper,¹ Aaron A. Winkler,² Johannes P. van Dijken^1,2 and Jack T. Pronk¹*

¹Kluyver Laboratory of Biotechnology

Delft University of Technology

Julianalaan 67, 2628 BC Delft, The Netherlands

Phone: 31 15 2783214

Fax: 31 15 2133141

E-mail: j.t.pronk@tnw.tudelft.nl

²Bird Engineering B.V.

Vlaardingweg 62, 3044 CK Rotterdam, The Netherlands

Homoethanolic fermentation of xylose in yeasts utilizing NADPH-specific xylose reductase (XR) and NAD-specific xylitol dehydrogenase (XDH) is intrinsically impossible. Only if XR has a dual specificity for both NADPH and NADH is anaerobic alcoholic fermentation feasible, but large amounts of xylitol must be produced to obtain a closed redox balance. Theoretically, anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in S. cerevisiae via a heterologous xylose isomerase (EC 5.3.1.5). When the gene encoding the xylose isomerase from the anaerobic fungus Piromyces sp. E2 was functionally expressed in Saccharomyces cerevisiae, xylose isomerase activities of 1 μmol min^-1 (mg protein)^-1 were measured. The expression of this single gene enabled S. cerevisiae to grow very slowly on xylose. Prolonged selection for faster growing mutants of this strain followed by selection for growth under oxygen limitation resulted in a strain that can ferment and grow on xylose as the sole carbon source under fully anaerobic conditions. Alcohol yields on xylose were comparable to these on glucose.

These results provide evidence that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in S. cerevisiae. Our findings provide a gateway to a commercially viable ethanol production from xylose with Saccharomyces cerevisiae.