Functional Annotation of <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 Carbohydrate Active Enzymes |
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Authors: | Phillip Brumm David Mead Julie Boyum Colleen Drinkwater Krishne Gowda David Stevenson Paul Weimer |
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Institution: | (1) Lucigen and Great Lakes Bioenergy Research Center, 2120 W Greenview Drive, Middleton, WI 53511, USA;(2) US Dairy Forage Research Center, Agricultural Research Service, US Department of Agriculture, University of Wisconsin, 1925 Linden Drive West, Madison, WI 53706, USA;(3) C5-6 Technologies and Great Lakes Bioenergy Research Center, 2120 W Greenview Drive, Middleton, WI 53511, USA |
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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. |
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