title: Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  Using 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, Toulouse F-31077, France. michael.odonohue@insa-toulouse.fr.
authors.forename: Géraldine
authors.initials: G
authors.lastname: Bastien
authors.forename: Grégory
authors.initials: G
authors.lastname: Arnal
authors.forename: Sophie
authors.initials: S
authors.lastname: Bozonnet
authors.forename: Sandrine
authors.initials: S
authors.lastname: Laguerre
authors.forename: Fernando
authors.initials: F
authors.lastname: Ferreira
authors.forename: Régis
authors.initials: R
authors.lastname: Fauré
authors.forename: Bernard
authors.initials: B
authors.lastname: Henrissat
authors.forename: Fabrice
authors.initials: F
authors.lastname: Lefèvre
authors.forename: Patrick
authors.initials: P
authors.lastname: Robe
authors.forename: Olivier
authors.initials: O
authors.lastname: Bouchez
authors.forename: Céline
authors.initials: C
authors.lastname: Noirot
authors.forename: Claire
authors.initials: C
authors.lastname: Dumon
authors.forename: Michael
authors.initials: M
authors.lastname: O'Donohue