A GHF7 CELLULASE FROM THE PROTIST SYMBIONT COMMUNITY OF Reticulitermes flavipes ENABLES MORE EFFICIENT LIGNOCELLULOSE PROCESSING BY HOST ENZYMES

Abstract

Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7-3, GHF7-5, and GHF7-6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full-length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus-insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7-3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and β-glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell-1), beta-glucosidase (β-glu), and laccase (LacA). GHF7-3 was the only GHF7 to enhance glucose release by Cell-1 and β-glu. Finally, GHF7-3, Cell-1, and β-glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only β-glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7-3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases.

Protozoacidal Trojan-Horse: Use of a Ligand-Lytic Peptide for Selective Destruction of Symbiotic Protozoa within Termite Guts

“…However, the autofluorescence was easy to distinguish from specific binding of the ligands, since there is no autofluorescence of the surface and the axostyle region (Fig. S2A–C) These binding sites are likely to be present in all species of protozoa, as both the ligands also bound to all eleven species of protozoa [27] found in the hindgut of another termite species Reticulitermes flavipes (Fig. 2A–H) and the four free-living aerobic protozoa species tested ( Tetrahymena pyriformis , Amoeba sp., Euglena sp., and Paramecium sp.)…”

A meta-analysis testing eusocial co-option theories in termite gut physiology and symbiosis

“…This enzyme family confers cellobiohydrolase and endoglucanase activity against cellulose polymers, 25,26 and it was downregulated up to ∼1000x before the onset of mortality after lethal imidacloprid + fungal pathogen challenges 19 . This enzyme family is absent from higher termite metagenomes and is poorly represented in the tri-partite symbiotic system associated with fungus-farming higher termites 27–28 .…”

Cloning, Production and Characterization of a Glycoside Hydrolase Family 7 Enzyme from the Gut Microbiota of the Termite Coptotermes curvignathus

“…A phylogenetic tree of termite and fungal cellulases was constructed using Molecular Evolutionary Genetic Analysis (MEGA6) software [25]. Three GHF7 sequences (KC751534, KC751535 and KC751536) from the termite R. flavipes [16] along with others well characterized fungal CBHs were included. The cellulase of Ruminococcus sp.…”

“…Both EGLs and BGLs are found in the midgut and hindgut of lower termites [6]. Using a baculovirus-insect expression system, Sethi et al [16] Nevertheless, due to lower expression levels and specific activities, expression of recombinant cellobiohydrolases has been challenging (17,18). To increase both expression levels and specific activities, we synthesized, sub-cloned and expressed the gene encoding a putative cellobiohydrolase (Gh1254) from C. curvignathus in the methylotrophic yeast P. pastoris which strongly over-expresses foreign proteins [19] and can serve as an expression system for insect proteins [20].…”

“…Since successful heterologous expression of recombinant genes is dependent on the complexity of post-translational modifications including disulfide bond formation and glycosylation[32], one transcript (HPK2EODRW_1254) was selected due to its relatively low number of disulfide bonds (6 disulfide bonds as predicted by the DiANNA Server, http://clavius.bc.edu/~clotelab/DiANNA/) and the lack of N-//www.cbs.dtu.dk/services/NetNGlyc/).Together with KC751534, GH1254 diverges from other cellulases forming a distinct clade within the GHF7 group(Fig.1). KC751534 is a GHF7 cellulase from the termite R. flavipes Sethi et al (2013). demonstrated that KC751534 exhibited a combination of cellobiohydrolase, endoglucanase and β-glucosidase activities[16].Two other termite GHF7 cellulases of R. flavipes (KC751535, KC751536) also diverge from fungal cellulases, but less profoundly than GH1254.…”