Functional metagenomics reveals abundant polysaccharide-degrading gene clusters and cellobiose utilization pathways within gut microbiota of a wood-feeding higher termite

Abstract

Plant cell-wall polysaccharides constitute the most abundant but recalcitrant organic carbon source in nature. Microbes residing in the digestive tract of herbivorous bilaterians are particularly efficient at depolymerizing polysaccharides into fermentable sugars and play a significant support role towards their host's lifestyle. Here, we combine large-scale functional screening of fosmid libraries, shotgun sequencing, and biochemical assays to interrogate the gut microbiota of the wood-feeding "higher" termite Globitermes brachycerastes. A number of putative polysaccharide utilization gene clusters were identified with multiple fibrolytic genes. Our large-scale functional screening of 50,000 fosmid clones resulted in 464 clones demonstrating plant polysaccharide-degrading activities, including 267 endoglucanase-, 24 exoglucanase-, 72 β-glucosidase-, and 101 endoxylanase-positive clones. We sequenced 173 functionally active clones and identified ~219 genes encoding putative carbohydrate-active enzymes (CAZymes) targeting cellulose, hemicellulose and pectin. Further analyses revealed that 68 of 154 contigs encode one or more CAZyme, which includes 35 examples of putative saccharolytic operons, suggesting that clustering of CAZymes is common in termite gut microbial inhabitants. Biochemical characterization of a representative xylanase cluster demonstrated that constituent enzymes exhibited complementary physicochemical properties and saccharolytic capabilities. Furthermore, diverse cellobiose-metabolizing enzymes include β-glucosidases, cellobiose phosphorylases, and phopho-6-β-glucosidases were identified and functionally verified, indicating that the termite gut micro-ecosystem utilizes diverse metabolic pathways to interconnect hydrolysis and central metabolism. Collectively, these results provide an in-depth view of the adaptation and digestive strategies employed by gut microbiota within this tiny-yet-efficient host-associated ecosystem.

Fig. 1

Putatively representative fibrolytic gene clusters and a hypothetical model recovered from gut microbiome of G. brachycerastes. a Gene organization of the representative fibrolytic gene clusters targeting plant polysaccharides. Shading box indicates shared operon. b A hypothetical model of contig00026 depicts complete cellulose and hemicellulose cleavage pathways (see the text for more details). Proteins marked with an asterisk. OM outer membrane, IM inner membrane

Fig. 2

Biochemical characterization of the four xylanases on the fosmid contig00057. a Schematic organization of lignocellulase genes on contig00057. b 12% SDS–PAGE analysis of the four purified xylanases. Lanes: M, protein molecular weight marker; lane 1–4: purified recombinant proteins for Xyl-ORF7, Xyl-ORF19, Xyl-ORF20 and Xyl-ORF21, with molecular weights of 45.1, 51.3, 42.3 and 53.9 kDa, respectively. c TLC analysis. Marker (M), xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentose (X5), and xylohextose (X6). a hydrolytic products of 10 min of the four xylanases against birch wood xylan; b hydrolytic products of 12 h of the four xylanases against birch wood xylan. d pH and temperature profile. (i) Temperature range of Xyl-ORF7, Xyl-ORF19, Xyl-ORF20 and Xyl-ORF21 were assayed between 30 and 70 °C. (ii) Thermostability were measured by dectecting the residual activity after preincubation at 50 °C for 5 min, 10 min, 15 min, and 30 min respectively. (iii) pH range were assayed between pH 4.5 and 10. (iv) pH stability were examined after preincubation in buffers ranging from pH 4.5 to 10 at 4 °C for 5 days. All activity assays were obtained from triplicate experiments

Fig. 3

Identification and functional verifications of genes related to three well-known cellobiose-metabolizing pathways in gut microbiome of G. brachycerastes. T:transporter, Bgl:β-glucosidase, CP: cellobiose phosphorylase, Pbgl:6-phospho-β-glucosidase, Pi:inorganic phosphate, EII:sugar-specific membrane components of PEP-PTS, HPr and EI soluble components of PEP-PTS