Mining biomass-degrading genes through Illumina-based de novo sequencing and metagenomic analysis of free-living bacteria in the gut of the lower termite Coptotermes gestroi harvested in Vietnam

Abstract

The 5.6 Gb metagenome of free-living microbial flora in the gut of the lower termite Coptotermes gestroi, harvested in Vietnam, was sequenced using Illumina technology. Genes related to biomass degradation were mined for a better understanding of biomass digestion in the termite gut and to identify lignocellulolytic enzymes applicable to biofuel production. The sequencing generated 5.4 Gb of useful reads, containing 125,431 ORFs spanning 78,271,365 bp, 80% of which was derived from bacteria. The 12 most abundant bacterial orders were Spirochaetales, Lactobacillales, Bacteroidales, Clostridiales, Enterobacteriales, Pseudomonades, Synergistales, Desulfovibrionales, Xanthomonadales, Burkholderiales, Bacillales, and Actinomycetales, and 1460 species were estimated. Of more than 12,000 ORFs with predicted functions related to carbohydrate metabolism, 587 encoding hydrolytic enzymes for cellulose, hemicellulose, and pectin were identified. Among them, 316 ORFs were related to cellulose degradation, and included β-glucosidases, 6-phospho-β-glucosidases, licheninases, glucan endo-1,3-β-D-glucosidases, endoglucanases, cellulose 1,4-β-cellobiosidases, glucan 1,3-β-glucosidases, and cellobiose phosphorylases. In addition, 259 ORFs were related to hemicellulose degradation, encoding endo-1,4-β-xylanases, α-galactosidases, α-N-arabinofuranosidases, xylan 1,4-β-xylosidases, arabinan endo-1,5-α-L-arabinosidases, endo-1,4-β-mannanases, and α-glucuronidases. Twelve ORFs encoding pectinesterases and pectate lyases were also obtained. To our knowledge, this is the first successful application of Illumina-based de novo sequencing for the analysis of a free-living bacterial community in the gut of a lower termite C. gestroi and for mining genes related to lignocellulose degradation from the gut bacteria.

Keywords: Cellulase; Coptotermes gestroi; Free-living gut bacterial community; Hemicellulase; Illumina de novo sequencing; Metagenome; Pectinesterase.