Fibrobacter succinogenes is a predominant 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 Fsu was finished by the DOE Joint Genome Institute in late 2009; preliminary analysis indicates that F. succinogenes contains ~ 133 glycosyl hydrolase and 63 CBM-containing genes, the most of any microbe when expressed as a percent of the total gene number.

One of the goals of this work is to functionally characterize all the putative glycosyl hydrolase genes from Fsu, as bioinformatic analysis is an inadequate proxy for actual activity results. Before the genome sequence was available we developed a robust method to enzymatically capture functionally active CAZymes in E. coli. Using new expression tools developed at Lucigen and C5-6 Technologies and a multi-substrate screen, we generated and screened 5760 random shotgun expression clones. This represents ~ 2 X genome expression coverage. 169 positive hits were recorded and 33 were unambiguously identified by sequence analysis of the inserts. Eliminating duplicates, 24 unique CAZyme genes were found by functional screening, or 40% of the ~60 genes present in this genome potentially detectable by the multiplex assay. Several previously uncharacterized enzymes were discovered using this approach. With the full genome sequence available we will attempt to express and characterize all of the recognizable CAZymes, as well as the CBM-containing genes. The active enzymes will also be sent to partners in the GLBRC to assess their ability to deconstruct plant biomass.