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Applied and Environmental Microbiology, November 2001, p. 5127-5133, Vol. 67, No. 11
Laboratory of
Microbiology1 and Agrotechnological
Research Institute (ATO),2 Wageningen
University and Research Centre, Wageningen, The Netherlands
Received 16 April 2001/Accepted 1 August 2001
Growth and the production of acetone, butanol, and ethanol by
Clostridium beijerinckii NCIMB 8052 on
several polysaccharides and sugars were analyzed. On
crystalline cellulose, growth and solvent production were observed only
when a mixture of fungal cellulases was added to the medium. On
lichenan growth and solvent production occurred, but this polymer was
only partially utilized. To increase utilization of these polymers and
subsequent solvent production, the genes for two new glycoside
hydrolases, celA and celD from the
fungus Neocallimastix patriciarum, were cloned
separately into C. beijerinckii. To do this, a secretion
vector based on the pMTL500E shuttle vector and containing the promoter
and signal sequence coding region of the Clostridium
saccharobutylicum NCP262 eglA gene was
constructed and fused either to the celA gene or the
celD gene. Stable C. beijerinckii
transformants were obtained with the resulting plasmids, pWUR3
(celA) and pWUR4 (celD). The recombinant
strains showed clear halos on agar plates containing carboxymethyl
cellulose upon staining with Congo red. In addition, their culture
supernatants had significant endoglucanase activities (123 U/mg of
protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although
C. beijerinckii harboring either celA or
celD was not able to grow, separately or in mixed
culture, on carboxymethyl cellulose or microcrystalline cellulose, both
transformants showed a significant increase in solvent production
during growth on lichenan and more extensive degradation of
this polymer than that exhibited by the wild-type strain.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5127-5133.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Clostridium beijerinckii Cells Expressing
Neocallimastix patriciarum Glycoside Hydrolases Show
Enhanced Lichenan Utilization and Solvent Production
*
Corresponding author. Mailing address: Laboratory of
Microbiology, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The
Netherlands. Phone: 31 317 483 110. Fax: 31 317 483 829. E-mail:
ana.lopez-contreras{at}algemeen.micr.wau.nl.
Applied and Environmental Microbiology, November 2001, p. 5127-5133, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5127-5133.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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