Abstract
BackgroundNew lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched on rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes.ResultsDifferential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora.ConclusionsCoupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production.
Highlights
New lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass
While enzymatic hydrolysis using lignocellulolytic enzymes is a standard approach for digestion, enzymes must be tolerant to several potentially inhibitory conditions including high temperatures associated with biomass pretreatment or heating to decrease the viscosity and required mixing energy of the biomass suspension [2,3] and high-solids environments necessary for minimizing water use [4]
While the representation of certain glycoside hydrolases in enriched deconstructive microbial communities provides promising gene targets, it offers no insight into whether communities express these genes. Metatranscriptomic analysis of these communities may refine the array of target genes identified via metagenomics by highlighting deconstructive enzymes expressed within the community. This is important for deconstructive microbial communities, where digestion of the various components of lignocellulose may result from enzymes spanning different species, some of which may not be abundant within the community and not readily identified through metagenome analysis
Summary
New lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Inhibitors derived from the biomass itself present additional challenges for deconstructive enzymes [5] Given these constraints and the costs of enzymes, using enzymes optimized for industrial deconstruction processes that maintain activity under harsh industrial conditions is economically important [6]. While the representation of certain glycoside hydrolases in enriched deconstructive microbial communities provides promising gene targets, it offers no insight into whether communities express these genes Metatranscriptomic analysis of these communities may refine the array of target genes identified via metagenomics by highlighting deconstructive enzymes expressed within the community. This is important for deconstructive microbial communities, where digestion of the various components of lignocellulose may result from enzymes spanning different species, some of which may not be abundant within the community and not readily identified through metagenome analysis
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