(719b) Targeted Discovery of Enzymes From Enriched Microbial Consortia for High Temperature Saccharification of Ionic-Liquid Pre-Treated Biomass

Singer, S. W., Joint Bioenergy Institute
Gladden, J. M., Joint Bioenergy Institute
D'haeseleer, P., Joint Bioenergy Institute
Allgaier, M., Joint Bioenergy Institute
Chivian, D. C., Joint BioEnergy Institute
Hazen, T. C., Joint BioEnergy Institute
Vandergheynst, J. S., Joint BioEnergy Institute
Hugenholtz, P., Joint Bioenergy Institute
Simmons, B. A., Joint BioEnergy Institute

Plant biomass is an enormous potential resource for generating renewable biofuels, however it is a recalcitrant substrate that requires pre-treatment and enzymatic hydrolysis to access the carbohydrates that will be transformed to biofuels. The enzyme cocktails used for saccharification of pre-treated biomass need to be robust and able to withstand inhibitors produced during pre-treatment. Enzymes secreted by filamentous fungi have traditionally been used for saccharification, however bacterial enzymes may be more suitable for saccharification under the harsh conditions encountered in an industrial process. To identify these enzymes, we have focused on adapting thermophilic bacterial microbial communities to specific feedstocks and pre-treatment conditions, followed by microbial community analysis and functional characterization of secreted glycosyl hydrolases. To select for thermophilic enzymes, microbial communities derived from compost were adapted to intact and ionic-liquid pre-treated switchgrass at 60°C. Phylogenetic profiling of these communities show that they consist of simple mixed consortia with representatives of the Firmicutes, Bacteriodetes, Thermi, Gemmatimonadetes and Chloroflexi phyla. Metagenomic sequencing of these consortia has allowed us to reconstruct the genomes of the important members of these microbial communities, resolve strain populations and assign functions to individual consortial members. Secretomes of these microbial communities were used to hydrolyze ionic-liquid pre-treated switchgrass at 80ºC. These assays indicated that the complement of glycosyl hydrolases in the secretomes could hydrolyze a broad range of polysaccharide linkages and were more active than commercial fungal enzyme cocktails at elevated temperature. Individual enzymes in the secretomes were identified by a combination of mass-spectrometry-based proteomics, using both a shotgun approach and in-gel digests of SDS-PAGE bands chosen through zymography. Synergistic effects on biomass hydrolysis have been demonstrated by adding exogenous thermophilic cellulases to the bacterial secretomes and individual enzymes identified in the proteogenomic anaylsis have been expressed in heterologous hosts (Escherichia coli, Thermus thermophilus) and characterized biochemically.