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Applied and Environmental Microbiology, January 2001, p. 6-14, Vol. 67, No. 1
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.1.6-14.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in Ethanologenic Klebsiella oxytoca P2†

Shengde Zhou, F. C. Davis, and L. O. Ingram*

Institute of Food and Agricultural Sciences, Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611

Received 1 August 2000/Accepted 8 October 2000

The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of Klebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental beta -glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes celY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.


* Corresponding author. Mailing address: Department of Microbiology and Cell Science, IFAS, P.O. Box 110700, University of Florida, Gainesville, FL 32611. Phone: (352) 392-8176. Fax: (352) 846-0969. E-mail: ingram{at}ufl.edu.

dagger Florida Agricultural Experiment Station journal series no. R-07702.


Applied and Environmental Microbiology, January 2001, p. 6-14, Vol. 67, No. 1
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.1.6-14.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.



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Copyright © 2001 by the American Society for Microbiology. All rights reserved.