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Applied and Environmental Microbiology, October 2007, p. 6072-6077, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.00955-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered Saccharomyces cerevisiae Strain{triangledown} ,{dagger}

Chenfeng Lu1 and Thomas Jeffries1,2,3*

Department of Food Science,1 Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706,2 USDA Forest Service, Forest Products Laboratory, Madison, Wisconsin 537263

Received 28 April 2007/ Accepted 31 July 2007

We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. Selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentation mixture by shuffling the promoters for GND2 and HXK2 with the genes for transaldolase (TAL1), transketolase (TKL1), and pyruvate kinase (PYK1) in the Saccharomyces cerevisiae strain FPL-YSX3. This host strain has integrated xylose-metabolizing genes, including xylose reductase, xylitol dehydrogenase, and xylulose kinase. The optimal expression levels for TAL1, TKL1, and PYK1 were identified by analysis of volumetric ethanol production by transformed cells. We found the optimal combination for ethanol production to be GND2-TAL1-HXK2-TKL1-HXK2-PYK1. The MGPS method could easily be adapted for other eukaryotic and prokaryotic organisms to optimize expression of genes for industrial fermentation.

* Corresponding author. Mailing address: USDA, FS, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI 53726-2398. Phone: (608) 231-9453. Fax: (608) 231-9262. E-mail: twjeffri{at}wisc.edu

{triangledown} Published ahead of print on 10 August 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, October 2007, p. 6072-6077, Vol. 73, No. 19
0099-2240/07/$08.00+0     doi:10.1128/AEM.00955-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hartner, F. S., Ruth, C., Langenegger, D., Johnson, S. N., Hyka, P., Lin-Cereghino, G. P., Lin-Cereghino, J., Kovar, K., Cregg, J. M., Glieder, A. (2008). Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 36: e76-e76 [Abstract] [Full Text]  


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