Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

Abstract

BackgroundTo efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases) from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms.ResultsFrom the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-α-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-β-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-β-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids.ConclusionsMetagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate). Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

Proteogenomic Analysis of a Thermophilic Bacterial Consortium Adapted to Deconstruct Switchgrass

“…In natural environments, plant biomass is deconstructed by complex microbial communities that employ hydrolytic and oxidative enzymes to depolymerize polysaccharides and lignin [11]. Studying lignocellulose deconstruction by microbial communities, rather than isolates, may provide a more comprehensive view of lignocellulose deconstruction and uncover new microbial groups and deconstruction mechanisms.…”

Towards a metagenomic understanding on enhanced biomethane production from waste activated sludge after pH 10 pretreatment

“…Conventional sequence homology-based enzyme discovery introduces a bias towards the identification of candidates similar to known enzymes, rather than enzymes with low sequence identity and potentially divergent biochemical properties [66]. The entries in the CAZy database contains both experimentally verified and putative carbohydrate-active enzyme domains, hence this search strategy would be able to provide a better insight into the catalysis of biochemical reactions [68,70]. …”

Bioprospecting Metagenomics for New Glycoside Hydrolases

“…We successfully applied the following protocol ( 38 ) : for inverse PCR, puri fi ed metagenome DNA was partially digested with restriction endonuclease BamHI or EcoRI and subsequently diluted and treated with T4 DNA ligase. Two sets of primers that are speci fi c to each candidate gene were used successively to amplify fl anking regions from self-ligated metagenome DNA.…”