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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Acknowledgements
This work was funded in part by DOE grant DOE DE-FG36-06GO16106, “Novel enzyme products for the conversion of defatted soybean meal to ethanol” and funded in part by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). The work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231.
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An erratum to this article can be found at http://dx.doi.org/10.1007/s12010-010-9125-7
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Brumm, P., Mead, D., Boyum, J. et al. Functional Annotation of Fibrobacter succinogenes S85 Carbohydrate Active Enzymes. Appl Biochem Biotechnol 163, 649–657 (2011). https://doi.org/10.1007/s12010-010-9070-5
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DOI: https://doi.org/10.1007/s12010-010-9070-5