A GHF7 cellulase from the protist symbiont community of Reticulitermes flavipes enables more efficient lignocellulose processing by host enzymes.

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 ß-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 (ß-glu), and laccase (LacA). GHF7-3 was the only GHF7 to enhance glucose release by Cell-1 and ß-glu. Finally, GHF7-3, Cell-1, and ß-glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only ß-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.

Production and characterization of a recombinant beta-1,4-endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes.

Cell-1 is a host-derived beta-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell-1 using eukaryotic (Baculovirus Expression Vector System; BEVS) and prokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily obtained in solubilized form and more active than the E. coli-expressed enzyme. K(m) and V(max) values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993% w/v and 1.056 micromol/min/mg. Additional characterization studies on the BEVS-expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5-7.5 and 50-60 degrees C, is inhibited by EDTA, and displays enhanced activity up to 70 degrees C in the presence of CaCl(2). These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol-oxidizing laccases, and others.

Recombinant protein production in insect larvae: host choice, tissue distribution, and heterologous gene instability.

The expression of the fluorescent protein, DsRed, facilitates the optimization of protein production in orally-infected whole larvae. Trichoplusia ni was shown to be a much better host for recombinant AcMNPV compared to four other noctuid Lepidopteran species achieving 100% infectivity at the minimal tested dose. The highest density of marker protein was found in endothelial and tracheal cells, fat body, and hemocytes. Trichoplusia ni larvae possessed visually detected color over sequential passages of oral infection until the sixth round. Western blot analysis confirmed the progressive decrease of both tetramer and monomer forms of DsRed. The intact DsRed gene and promoter region was present in late passages, but viral population carrying the heterologous gene had dropped more than 2-logs after the fifth round while the amount of total viral DNA remained unchanged over sequential passages.

Human proprotein convertase 2 homologue from a plant nematode: cloning, characterization, and comparison with other species.

Proprotein convertases (PCs) are evolutionarily conserved enzymes responsible for processing the precursors of many bioactive peptides in mammals. The invertebrate homologues of PC2 play important roles during development that makes the enzyme a good target for practical applications in pest management. Screening of a plant nematode Heterodera glycines cDNA library resulted in isolation of a full-length clone encoding a PC2-like precursor. The deduced protein (74.2 kD) exhibits strong amino acid homology to all known PC2s, including human, and shares the main structural characteristics: signal peptide; prosegment; catalytic domain, with D/H/S catalytic triad, PC2-specific residues, and 7B2 binding sites; P domain (with RRGDT pentapeptide); and carboxyl terminus. Comparative analysis of PC2s from 15 species discloses the presence of an insert in the catalytic domain unique to nematodes. Expression of PC2-like mRNA found in eggs and juveniles was undetectable in adult stages of H. glycines. Nucleotide analysis reveals distinctive differences in base composition and codon usage between H. glycines and Caenorhabditis elegans PC2s. The H. glycines cDNA clone encoding PC2 is the first one isolated from plant-parasitic nematodes.

Lignin-associated metagene expression in a lignocellulose-digesting termite.

Lignin is a component of plant biomass that presents a significant obstacle to biofuel production. It is composed of a highly stable phenylpropanoid matrix that upon degradation, releases toxic metabolites. Termites have specialized digestive systems that overcome the lignin barrier in wood lignocellulose to efficiently release fermentable simple sugars; however, how termites specifically degrade lignin and tolerate its toxic byproducts remains unknown. Here, using the termite Reticulitermes flavipes and its symbiotic (protozoan) gut fauna as a model system, we used high throughput Roche 454-titanium pyrosequencing and proteomics approaches to (i) experimentally compare the effects of diets containing varying degrees of lignin complexity on host-symbiont digestome composition, (ii) deeply sample host and symbiont lignocellulase diversity, and (iii) identify promising lignocellulase candidates for functional characterization. In addition to revealing over 9500 differentially expressed transcripts related to a wide range of physiological processes, our findings reveal two detoxification enzyme families not generally considered in connection with lignocellulose digestion: aldo-keto reductases and catalases. Recombinant versions of two host enzymes from these enzyme families, which apparently play no roles in cellulose or hemicellulose digestion, significantly enhance lignocellulose saccharification by cocktails of host and symbiont cellulases. These hypothesis-driven results provide important new insights into (i) dietary lignin as a xenobiotic challenge, (ii) the complex mechanisms used by termites to cope with their lignin-rich diets, and (iii) novel lignin-targeted enzymatic approaches to enhance biofuel and biomaterial production.

Functional and translational analyses of a beta-glucosidase gene (glycosyl hydrolase family 1) isolated from the gut of the lower termite Reticulitermes flavipes.

This research focused on digestive beta-glucosidases from glycosyl hydrolase family (GHF) 1 from the gut of the lower termite Reticulitermes flavipes. In preceding studies on R. flavipes, we characterized beta-glucosidase activity across the gut and its inhibition by carbohydrate-based inhibitors, and subsequently we identified two partial beta-glucosidase cDNA sequences from a host gut cDNA library. Here, we report on the full-length cDNA sequence for one of the R. flavipes beta-glucosidases (RfBGluc-1), the expression of its mRNA in the salivary gland and foregut, the production of recombinant protein using a baculovirus-insect expression system, optimal recombinant substrate specificity profiles and parameters, and significant inhibition by the established beta-glucosidase inhibitor cellobioimidazole. We also report the partial cDNA sequence for a second gut beta-glucosidase (RfBGluc-2), and show that like RfBGluc-1 its mRNA is localized mainly in the salivary gland. Other results for RfBGluc-1 showing activity against laminaribose, a component of microbial cell walls, suggest that RfBGluc-1 may serve dual functions in cellulose digestion and immunity. These findings provide important information that will enable the testing of hypotheses related to collaborative host-symbiont lignocellulose digestion, and that contributes to the development of next-generation termiticides and novel biocatalyst cocktails for use in biomass-to-bioethanol applications.