Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2010
Publication Date: 2/23/2011
Publication URL: http://naldc.nal.usda.gov/catalog/53697
Citation: Brumm, P., Mead, D., Boyum, J., Drinkwater, C., Gowda, K., Stevenson, D.M., Weimer, P.J. 2011. Functional annotation of Fibrobacter succinogenes S85 carbohydrate active enzymes. Applied Biochemistry and Biotechnology. 163:649-657. Interpretive Summary: Fibrobacter succinogenes is a bacterium from the rumen that shows one of the highest capacities for degrading cellulose in all of nature. To better understand the source of this capacity, we obtained the whole DNA sequence of this bacterium, then developed cloning and screening methods to characterize individual enzymes responsible for some of the plant fiber-degrading activity. The results will provide new insights to scientists and bioengineers seeking to develop improved enzymes for degradation of cellulose to produce fuels and chemicals from plant biomass.
Technical 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 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 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.