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.

Figure 1

In vivo functional screening of metagenomic clones arabinofuranosidase. A. and B. xylosidase activities using the chromogenic substrates BI-Xylp and BCI-Araf respectively.

Figure 2

Secondary screening of 87 fosmid clones. A. pNP-Araf (pH 6, 40°C), B. pNP-xylopyranoside (pH 6, 40°C). C+ denotes positive controls. C. depicts the pH and temperature dependant activities of clones D2, F3 and G12 (from left to right) on pNP-Araf.

Figure 3

Schematic representation of gene clusters encoding putative carbohydrate-active enzymes. A. the endoxylanase cluster found in clone Xyn3. B. the gene cluster present in clone A10 displaying low arabinofuranosidase and β-xylosidase activity. C: the cluster present within clone G12 displaying arabinosidase activity. The abbreviation Ara denotes an arabinosidase; Xyl, a xylosidase; Xyl transp, a xylose transporter; Xyn, a endoxylanase; Xylan glu, a xylan glucuronidase; and Glc, a glucosidase.

Figure 4

Distribution pattern of COG-assigned proteins. The COG categories are: (C) Energy production and conversion; (D) Cell cycle control, mitosis, and meiosis; (E) Amino acid transport and metabolism. (F) Nucleotide transport and metabolism; (G) Carbohydrate transport and metabolism; (H) Coenzyme transport and metabolism; (I) Lipid transport and metabolism; (J) Translation; (K) Transcription; (L) Replication, recombination, and repair; (M) Cell wall/membrane biogenesis; (N) Cell motility; (O) Post-translational modification, protein turnover, chaperones; (P) Inorganic ion transport and metabolism; (Q) Secondary metabolite biosynthesis, transport, and catabolism; (R) General function prediction only; (S) Function unknown; (T) Signal transduction mechanisms; (U) Intracellular trafficking and secretion; (V) Defense mechanisms; (Z) Cytoskeleton. Sequences that could not be assigned to any of the above COGS are not included in the figure.