title: Functional annotation of Fibrobacter succinogenes S85 carbohydrate active enzymes.
abstract: Fibrobacter succinogenes is a cellulolytic bacterium that degrades plant cell wall biomass in ruminant animals and is among the most rapidly fibrolytic of all mesophilic bacteria. The complete genome sequence of Fisuc was completed by the DOE Joint Genome Institute in late 2009. Using new expression tools developed at Lucigen and C5-6 Technologies and a multi-substrate screen, 5,760 random shotgun expression clones were screened for biomass-degrading enzymes, representing 2× genome expression coverage. From the screen, 169 positive hits were recorded and 33 were unambiguously identified by sequence analysis of the inserts as belonging to CAZy family genes. Eliminating duplicates, 24 unique CAZy genes were found by functional screening. Several previously uncharacterized enzymes were discovered using this approach and a number of potentially mis-annotated enzymes were functionally characterized. To complement this approach, a high-throughput system was developed to clone and express all the annotated glycosyl hydrolases and carbohydrate esterases in the genome. Using this method, six previously described and five novel CAZy enzymes were cloned, expressed, and purified in milligram quantities.
authors.affiliation: C5-6 Technologies and Great Lakes Bioenergy Research Center, 2120 W Greenview Drive, Middleton, WI 53511, USA. pbrumm@lucigen.com
authors.forename: Phillip
authors.initials: P
authors.lastname: Brumm
authors.forename: David
authors.initials: D
authors.lastname: Mead
authors.forename: Julie
authors.initials: J
authors.lastname: Boyum
authors.forename: Colleen
authors.initials: C
authors.lastname: Drinkwater
authors.forename: Jan
authors.initials: J
authors.lastname: Deneke
authors.forename: Krishne
authors.initials: K
authors.lastname: Gowda
authors.forename: David
authors.initials: D
authors.lastname: Stevenson
authors.forename: Paul
authors.initials: P
authors.lastname: Weimer
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002480:Cellulase / Q000378:metabolism; D019295:Computational Biology; D045848:Fibrobacter / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C023305:hemicellulase; D002480:Cellulase