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Applied and Environmental Microbiology, March 2002, p. 1232-1239, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1232-1239.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Cloning of XYL3 (D-Xylulokinase) from Pichia stipitis and Characterization of Its Physiological Function

Yong-Su Jin,1 Sharon Jones,2,{dagger} Nian-Qing Shi,3,{ddagger} and Thomas W. Jeffries1,2,3*

Department of Food Science,1 Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706,3 Forest Products Laboratory, USDA Forest Service, Madison, Wisconsin 537052

Received 23 July 2001/ Accepted 23 November 2001

XYL3, which encodes a D-xylulokinase (EC 2.7.1.17), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated D-xylulokinase activity, but D-ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae D-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, D-xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae.

* Corresponding author. Mailing address: USDA Forest Service, Forest Products Laboratory, One Gifford Pinchot Dr., Madison, WI 53705. Phone: (608) 231-9453. Fax: (608) 231-9262. E-mail: tjeffri{at}facstaff.wisc.edu.

{dagger} Present address: Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright Patterson AFB, OH 45433, and Technical Management Concepts, Inc., Beavercreek, OH 45434.

{ddagger} Present address: Tate and Lyle North America, Decatur, IL 62521.

Applied and Environmental Microbiology, March 2002, p. 1232-1239, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1232-1239.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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