6-01: Metagenomics for mining new deconstructive enzymes, exploring enzyme diversity and screening cellulolytic activities

Plant biomass is the most abundant biopolymer on earth and has long been recognized as a potential source of mixed sugars for bioenergy production.  Our goals are to understand the diversity and metabolic capabilities of the complex microbial communities, and to exploit their dynamics for converting plant biomass into feedstock for biofuels production.  Metagenomics allows the discovery of new enzymes from microbial communities, especially from organisms that are unknown or have never been cultivated.  From an anaerobic microbial community actively decaying poplar biomass, metagenomic DNA was isolated and microbial species distribution was investigated via 16S and 18S rRNA sequencing.  Saccharomycetes composed the major group among the Eukaryotes, and Clostridiales composed the major group among the Bacteria.  No major population of Archaea was found in this microbial community.  Using the 454-GS-FLX Titanium pyrosequencing, approximately 580Mbp metagenomic DNA was sequenced.  Preliminary blastx searches identified approximately 4,000 glycosyl hydrolase homologues.  Five candidates were selected for further investigation based on homology to enzyme families of interest (families 5, 9, 48, and 51 representing cellulase, hemicellulase, and xylanase activities) and quality of sequences.  Full-length open reading frames were obtained using inverse PCR and DNA walking, and gene cloning is presently in process.  A lambda-based expression library of one isolated strain from the community was also constructed and enzyme activity screening is in process.  Our metagenomic studies successfully provided insight into the microbial community composition as well as a resource of diverse, community-encoded glycosyl hydrolases, for mining new deconstructive enzymes and screening cellulolytic activities.