ARS | Publication request: Functional annotation of Fibrobacter succinogenes carbohydrate active enzymes

Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Proceedings
Publication Acceptance Date: March 18, 2010
Publication Date: April 19, 2010
Citation: Brumm, P., Mead, D., Boyum, J., Drinkwater, C., Gowda, K., Stevenson, D.M., Weimer, P.J. 2010. Functional annotation of Fibrobacter succinogenes carbohydrate active enzymes. Applied Biochemistry and Biotechnology. 163:649-657.

Technical Abstract: Fibrobacter succinogenes (Fisuc) 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, 5760 random shotgun expression clones were screened for biomass-degrading enzymes, representing 2X 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 misannotated enzymes were functionally characterized. To complement this approach, a high throughput system was developed to clone and express all the annotated CAZymes in the genome. Using this method, 6 previously described and 5 novel CAZy enzymes were cloned, expressed, and purified in milligram quantities.