Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics

Abstract

BackgroundThe metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris.ResultsUsing whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-d-xylosidase (EC 3.2.1.37) or α-l-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens.ConclusionsThis study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the termite mound, although further investigation will be needed to clarify the complex synergies that might exist between the different microbiomes that constitute the termitosphere of fungus-growing termites. This study exemplifies the power of functional metagenomics for the discovery of biomass-active enzymes and has provided a collection of potentially interesting biocatalysts for further study.

A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes

“…However, Pm23 cannot at present be classified in any existing CAZy family and thus represents a module of "unknown" function. The three ␤-glucanases were annotated as GH8 (27). Therefore, apart from Pm23, P. militaris microbiome protein CAZy classification was consistent with the activities we recorded.…”

“…A previously described functional metagenomics approach (27) was used to investigate the gut microbiome of P. militaris for identification of hemicellulases. In function-driven metagenomics, only clones that respond positively to the screen are selected and further analyzed, making this an attractive alternative to less targeted conventional global metagenomic studies that typically generate vast data sets that are cumbersome to FIGURE 11.…”

“…Cells were incubated at 37°C for 5 h and at 17°C overnight in baffled flasks until an A 600 nm of ϳ11 was reached. Immobilized metal ion affinity chromatography was performed following the protocol described previously (27) for purification of the recombinant proteins. The concentration of purified proteins was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and applying the Beer-Lambert law.…”

“…11A). Based on amino acid sequence, the xylanases Pm12 and Pm25 were previously classified in CAZy glycoside hydrolase family GH10 (27). Pm23 (which is the N-terminal module of a putative multimodular xylanase, GH11) could be attributed from BLAST analysis to the DUF303 superfamily, which is a family of unknown function but with putative acetylesterase activities.…”

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

“…Functional metagenomic mining can be targeted to highly specific functional properties, while genome or sequence-based metagenome mining only accesses enzymes by class, without explicit specificity of properties. Few reported metagenomic screening studies have specifically targeted AcXEs, with most focusing on other CAZy classes or failing to classify identified CEs into families [116][117][118][119]. The limited focus on AcXEs is due, in part, to a lack of high throughput (HTP) metagenomic screening methods for direct detection of AcXE activity.…”