Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

Abstract

Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ∼10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50°C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

Metabolic interdependencies between phylogenetically novel fermenters and respiratory organisms in an unconfined aquifer

“…The lower incidence of pathways for complete cellulose breakdown relative to those for utilization of cellobiose is consistent with a bioinformatic study that found genomes with only cello-oligosaccharide utilization outnumbered cellulasecontaining genomes two to one (Berlemont and Martiny, 2013). Compared with compost and peat wetland soil metagenomic data sets (Allgaier et al, 2010;Tveit et al, 2013), the ratios of hemicellulose: cellulose genes were less, suggesting different carbon sources between the systems selectively enrich at the functional level (Figure 2). Genes for chitin and N-acetyl-glucosaminidase (almost in a 1:1 ratio), suggest the capacity to completely degrade this biopolymer to C-N residues is encoded in up to 30 different organisms within the community (Figure 2).…”

Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

“…Metagenomics and metaproteomics have previously been shown to be invaluable tools for analyzing microbial communities (Ram et al, 2005;Gill et al, 2006;Woyke et al, 2006;Kalyuzhnaya et al, 2008;Wilmes et al, 2008;Allgaier et al, 2009;Verberkmoes et al, 2009;Burnum et al, 2011), including those associated with herbivores (Warnecke et al, 2007;Brulc et al, 2009;Pope et al, 2010;Burnum et al, 2011). Here we use these techniques to provide insight into the fungus gardens of leaf-cutter ants.…”

Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium,Enterobacter lignolyticus

“…Utilization of microorganisms isolated from natural environments, such as tropical rain forest (23) soil, can greatly improve the current biofuels strategy. Natural microbial communities that degrade biomass are often exposed to fluctuating environmental conditions, and thus represent a vast resource for both stress-tolerant organisms and highly efficient, stable lignocellulolytic enzymes (23)(24)(25).…”