Abstract
To identify carbohydrate-active enzymes (CAZymes) that might be particularly relevant for wood fiber processing, we performed a comparative metagenomic analysis of digestive systems from Canadian beaver (Castor canadensis) and North American moose (Alces americanus) following 3 years of enrichment on either microcrystalline cellulose or poplar hydrolysate. In total, 9,386 genes encoding CAZymes and carbohydrate-binding modules (CBMs) were identified, with up to half predicted to originate from Firmicutes, Bacteroidetes, Chloroflexi, and Proteobacteria phyla, and up to 17% from unknown phyla. Both PCA and hierarchical cluster analysis distinguished the annotated glycoside hydrolase (GH) distributions identified herein, from those previously reported for grass-feeding mammals and herbivorous foragers. The CAZyme profile of moose rumen enrichments also differed from a recently reported moose rumen metagenome, most notably by the absence of GH13-appended dockerins. Consistent with substrate-driven convergence, CAZyme profiles from both poplar hydrolysate-fed cultures differed from cellulose-fed cultures, most notably by increased numbers of unique sequences belonging to families GH3, GH5, GH43, GH53, and CE1. Moreover, pairwise comparisons of moose rumen enrichments further revealed higher counts of GH127 and CE15 families in cultures fed with poplar hydrolysate. To expand our scope to lesser known carbohydrate-active proteins, we identified and compared multi-domain proteins comprising both a CBM and domain of unknown function (DUF) as well as proteins with unknown function within the 416 predicted polysaccharide utilization loci (PULs). Interestingly, DUF362, identified in iron–sulfur proteins, was consistently appended to CBM9; on the other hand, proteins with unknown function from PULs shared little identity unless from identical PULs. Overall, this study sheds new light on the lignocellulose degrading capabilities of microbes originating from digestive systems of mammals known for fiber-rich diets, and highlights the value of enrichment to select new CAZymes from metagenome sequences for future biochemical characterization.
Highlights
Lignocellulose comprises the non-edible fraction of plant biomass and as such is a recognized resource for the production of renewable energy, chemicals, and materials
An ethics approval from an Animal Care and Use Committee was not required by the Office of Research Ethics of the University of Toronto, as the moose rumen sample was collected from a dead moose that was hunted in the wild for meat by a registered hunter with a license authorized by the Ministry of Natural Resources and Forestry under Government of Ontario, Canada
These sequences were assigned to 100 distinct families of glycoside hydrolase (GH), 13 families of carbohydrate esterase (CE), 15 families of polysaccharide lyases (PLs), and 39 families of GTs, as well as 43 families of associated carbohydrate-binding module (CBM)
Summary
Lignocellulose comprises the non-edible fraction of plant biomass and as such is a recognized resource for the production of renewable energy, chemicals, and materials. Metagenomic approaches to identify CAZymes relevant to the conversion of a given biomass feedstock have considered environmental samples persistently subjected to the targeted feedstock. Metagenomic analyses aimed at identifying CAZymes most relevant to bioconversion of nonwoody biomass have sampled digestive systems of animals that graze on straw, grasses, and lichens (Pope et al, 2010). Parallel metagenomic analyses to identify CAZymes contributing to bioconversion of woody biomass have included samples ranging from forest soils (Damon et al, 2012; Pold et al, 2016), to insects (Warnecke et al, 2007; He et al, 2013; Rossmassler et al, 2015), and wood-feeding mollusks (O’Connor et al, 2014). Enrichment of environmental samples on specific biomass feedstocks prior to metagenome sequencing
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