Investigating the function of an arabinan utilization locus isolated from a termite gut community

Abstract

Biocatalysts are essential for the development of bioprocesses efficient for plant biomass degradation. Previously, a metagenomic clone containing DNA from termite gut microbiota was pinpointed in a functional screening that revealed the presence of arabinofuranosidase activity. Subsequent genetic and bioinformatic analysis revealed that the DNA fragment belonged to a member of the genus Bacteroides and encoded 19 open reading frames (ORFs), and annotation suggested the presence of hypothetical transporter and regulator proteins and others involved in the catabolism of pentose sugar. In this respect and considering the phenotype of the metagenomic clone, it was noted that among the ORFs, there are four putative arabinose-specific glycoside hydrolases, two from family GH43 and two from GH51. In this study, a thorough bioinformatics analysis of the metagenomic clone gene cluster has been performed and the four aforementioned glycoside hydrolases have been characterized. Together, the results provide evidence that the gene cluster is a polysaccharide utilization locus dedicated to the breakdown of the arabinan component in pectin and related substrates. Characterization of the two GH43 and the two GH51 glycoside hydrolases has revealed that each of these enzymes displays specific catalytic capabilities and that when these are combined the enzymes act synergistically, increasing the efficiency of arabinan degradation.

FIG 1

Genetic map of the 41-kb fosmid containing arabinose and arabinan utilization genes from the G12 metagenomic clone. The letters P and Ω indicate, respectively, the putative promoter rho-independent transcription termination sites. Black segments represent ORFs encoding glycoside hydrolases, while gray segments correspond to ORFs encoding putative pentose metabolism enzymes. White boxes represent ORFs encoding proteins putatively involved in arabinan and arabinose recognition and utilization. No hypothetical function could be attributed to ORF16 and ORF17.

FIG 2

Schematic representation of the four ORFs encoding the glycoside hydrolases studied. Enzymatic modules (gray rectangles) were successfully cloned and expressed in E. coli, while attempts to express other modules (white rectangles) failed. The protein size in number of amino acids does not account for signal peptide sequences.

FIG 3

Degradation of arabinans by Abn43A and Abn43B. Time-dependent assays were performed using the DNS protocol to quantify reducing sugars at different time points. Solid and dotted lines represent SBA and LA hydrolysis, respectively, by Abn43A (▲) or Abn43B (●).

FIG 4

Hydrolysis of arabino-oligosaccharides by Abn43B. The time-dependent degradation of arabinotriose (A) and arabinohexaose (B) by Abn43B was analyzed using HPAEC-PAD. Symbols: ●, arabinose; +, arabinobiose; △, arabinotriose; □, arabinotetraose; ×, arabinopetaose; ◇, arabinohexaose.

FIG 5

Synergistic effect of enzymes in cocktail. The time-dependent release of reducing sugars from sugar beet arabinan (A) and α-1,5 linear arabinan (B) was quantified by using the DNS assay. Gray bars represent the theoretical sum of the individual action of the four enzymes, while white bars represent the experimentally measured impact of the four enzymes working together (2 μg/ml of each enzyme). The numbers above the bars indicate the synergistic index.

FIG 6

Degradation of sugar beet arabinan. HPAEC-PAD elution patterns of AOS after degradation of sugar beet arabinan by Abn43A (A), Abn43A + Abf51A (B), Abn43A + Abf51A + Abn43B (C), and Abn43A + Abf51A + Abn43B + Abf51B_trunc (D) during a 24-h reaction at 30°C and pH 6.5 are shown.