2-30: Sugar cane bagasse and paper utilization by fosmids identified by functional metagenomic

Two fosmid libraries of 30.000 and 100.000 clones were constructed from metagenomic DNA isolated from the microorganisms inhabiting the rumen of Uruguayan cows and the sludge of an anaerobic digester, respectively. Fragments of approximately 40kb were purified and cloned into fosmids, packaged into phages and used to infect E.coli strain EPI300T1R. The library was screened for the expression of cellulases and xylanases using carboximethyl-cellulose, avicel and xylan as substrates. Twenty seven clones showed cellulolytic activity, eleven showed xylanase activity while eleven showed both activities in the screening media.

Two of these clones conferred E.coli the ability to grow on minimal media supplemented with cellobiose or filter paper (FP) as sole carbon sources. This ability was also conferred to the ethanologenic strain LY180 which was able to grow on FP and to produce detectable amounts of ethanol.  Similar assays were also performed with sugar cane bagasse. Both clones were able to grow on this feedstock as a sole carbon source. Degradation of the plant fibers was evidenced by ultrastructural studies. 

The full fosmid sequences were obtained for both clones. No obvious cellulase, glycosidase or xilanase genes were detected. One of the identified operons encodes ORFs with homologies for known glycosyl-transferases. Ongoing sub-cloning and mutagenesis will confirm the involvement of these genes in the observed phenotypes.