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Applied and Environmental Microbiology, January 2001, p. 6-14, Vol. 67, No. 1
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
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
-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.
Florida Agricultural Experiment Station journal series no.
R-07702.
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