Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose.
Gene libraries ("zoolibraries") were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic digesters, which were in continuous operation with lignocellulosic feedstocks for over 10 years. Clones expressing cellulase and xylosidase were readily recovered from these libraries. Four clones that hydrolyzed carboxymethylcellulose and methylumbelliferyl-beta-D-cellobiopyranoside were characterized. All four cellulases exhibited temperature optima (60-65 degrees C) and pH optima (pH 6-7) in accordance with conditions of the enrichment. The DNA sequence of the insert in one clone (plasmid pFGH1) was determined. This plasmid encoded an endoglucanase (celA) and part of a putative beta-glucosidase (celB), both of which were distinctly different from all previously reported homologues. CelA protein shared limited homology with members of the A3 subfamily of cellulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity). The N-terminal part of CelB protein was most similar to beta-glucosidase from Pseudomonas fluorescens subsp. cellulosa (28% homology). The use of zoolibraries constructed from natural or laboratory enrichment cultures offers the potential to discover many new enzymes for biotechnological applications.
Healy
F G
FG
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA.
Ray
R M
RM
Aldrich
H C
HC
Wilkie
A C
AC
Ingram
L O
LO
Shanmugam
K T
KT
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose; D004247:DNA / Q000737:chemistry; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008031:Lignin; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; C036909:lignocellulose; D002482:Cellulose; D008031:Lignin; D004247:DNA; D002480:Cellulase