# Patent

## Title
Termite enzymes and uses thereof for in vitro conversion of lignin-containing materials to fermentable products

## Abstract

The disclosure provides isolated nucleic acid molecules derived from the gut of the termite R flavipes, recombinant nucleic acid molecules comprising a vector and an isolated heterologous nucleic acid molecule operably inserted therein, whereby, when transformed into an appropriate host cell system, the heterologous nucleic acid sequence is expressed as a polypeptide having an activity similar to that when expressed in the gut of the termite R. flavipes. The recombinant nucleic acid molecules can comprise more than one heterologous nucleic acid molecule such that more than one polypeptide may be expressed by the host system. The expressed polypeptides may be substantially purified, or used in a substantially unpurified form, to be admixed with a lignocellulose source to be converted to a fermentable product such as a sugar or a mixture of sugars. One aspect of the present disclosure, therefore, encompasses methods of converting a lignified plant material to a fermentable product, the method comprising obtaining a series of isolated polypeptides of a termite, wherein the series of polypeptides cooperate to convert a plant lignocellulose to a fermentable product; and incubating the series of polypeptides with a source of lignified plant material, under conditions allowing the polypeptides to cooperatively produce a fermentable product from the lignified plant material.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application is the 35 U.S.C. §371 national stage of PCT application which claims priority to “TERMITE ENZYMES AND USES THEREOF FOR IN VITRO CONVERSION OF LIGNIN-CONTAINING MATERIALS TO FERMENTABLE PRODUCTS,” having serial number PCT/US2010/029342, filed on Mar. 31, 2010. This application also claims priority to and benefit of U.S. Provisional Application No. 61/168,275 filed on Apr. 10, 2009, which is incorporated by reference in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under Grant No. DE-FG36-02G012026 awarded by the Consortium for Plant Biotechnology Research/Department of Energy of the United States government DE-FG36-02G012026 awarded by the Consortium for Plant Biotechnology Research/Department of Energy of the United States government; CSREES-NRI 2007-35607-17777 awarded by the Department of Agriculture of the United States government; DE-FG02-08ER85063 awarded by the Department of Energy of the United States government; and 2009-05245 awarded by the Department of Agriculture of the United States government. The government has certain rights in the invention.


TECHNICAL FIELD
The present disclosure is generally related to enzymes, and to recombinant nucleic acid molecules encoding and/or expressing said enzymes, of the gut of the termite Reticulitermes flavipes. The disclosure further relates to a system combining said enzymes for substantially converting a plant lignocellulose to a fermentable sugar-based product.
BACKGROUND
Termites are social insects that subsist on sugars and other micronutrients obtained from nutritionally poor lignocellulose diets (Ohkuma M., (2006) Appl. Microbiol. Biotechnol. 61: 1-9; Scharf & Tartar (2008) Biofuels Bioprod. Birefin. 2: 540-552). Lignocellulose is a natural complex of the three biopolymers cellulose, hemicellulose and lignin. Cellulose is composed of long β-1,4-linked polymers of glucose that are held together in bundles by hemicellulose (Ljungdahl & Erickson (1985) Adv. Micro. Ecol. 8: 237-299; Lange J. P. (2007) Biofuels Bioprod. Bioref. 1: 39-48). Hemicellulose is composed of shorter β-1,4-linked polymers of mixed sugars such as mannose, xylose, galactose, rhamnose, arabinose, glucuronic acid, mannuronic acid, and galacturonic acid (Saha B. C., (2003) J. Indust. Microbiol. Biotechnol. 30: 279-291). Lignin is a 3-dimensional polymer of phenolic compounds that are linked to each other and to hemicellulose by ester bonds. Lignin is composed of three “mono-lignol” monomers (p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol), which are combined in different ratios depending on the plant species. Another important characteristic of hemicellulose is its esterification with monomers and dimers of phenolic acid esters, which are identical to the mono-lignols that compose lignin (Saha B. C., (2003) J. Indust. Microbiol. Biotechnol. 30: 279-291; Crepin et al., (2004) Appl. Microbiol. Biotechnol. 63, 647-652; Benoit et al., (2008) Biotechnol. Letters 30, 387-396).
Termites digest lignocellulose with the assistance of endogenous and symbiont-produced digestive enzymes and co-factors (Breznak and Brune (1995) Appl. Env. Microbiol. 61: 2681-2687; Watanabe et al., (1998) Nature 394: 330-331; Ohkuma et al., (2006) Appl. Microbiol. Biotechnol. 61: 1-9; Scharf & Tartar (2008) Biofuels Bioprod. Birefin. 2: 540-552). Termite gut endosymbionts include a diversity of microorganisms that include protozoa, bacteria, spirochetes, fungi, and yeast, among others (Breznak and Brune (1995) Appl. Env. Microbiol. 61: 2681-2687; Warnecke et al., (2007) Nature 450, 560-565). The order Isoptera is divided into the higher and lower termites based mostly on symbiont composition. Lower termites, including Reticulitermes flavipes, possess cellulolytic protozoa in addition to a host of hydrogenic, methanogenic, and nitrogen fixing bacteria and spirochetes. Higher termites lack protozoa altogether, but instead possess cellulolytic bacteria. The roles of endosymbiotic fungi in higher and lower termites are not well defined; however, some higher termites cultivate fungus gardens in their nests that assist in lignocellulose digestion by producing cellulases, hemicellulases and lignases (Taprab et al., (2005) Appl. Env. Microbiol. 71: 7696-7704; Okhuma M., (2006) Appl Microbiol Biotechnol 61, 1-9).
Esterases are hydrolytic enzymes that cleave ester bonds in a diversity of biomolecules (Oakeshott et al. (2005) in Gilbert, L. I., Iatrou, K., Gill, S. S. (eds.) Comprehensive molecular insect science, Vol. 5, Elsevier-Pergamon, New York, pp. 309-382). Some insect esterases have very well defined biological functions, such as those involved in xenobiotic, lipid, acetylcholine, and juvenile hormone metabolism. However, many other insect esterases have largely undefined functions, yet are extremely efficient at metabolizing model substrates such as naphthyl and p-nitrophenyl esters. This latter category of esterases is referred to as the “general esterases”. Because of the highly esterified structure of lignin, it is possible that some general esterases may also contribute to lignin depolymerization in wood feeding insects such as termites.
Lignocellulose is a sustainable global resource with a great deal of relevance to renewable energy production. It is a naturally occurring complex of plant derived materials that includes the β-1,4-linked sugar polymers cellulose and hemicellulose, and the phenolic polymer lignin. In plants, lignocellulose provides key structural support for cell walls. Because it is plant-derived, lignocellulose is the most abundant and widespread bioenergy feedstock available on Earth. However, a major limitation in plant biomass utilization as a renewable energy source is the inefficiency of industrial lignocellulose depolymerization. This inefficiency increases energy inputs, reduces product yields, drives production costs higher, encourages political skepticism, and ultimately limits acceptance of cellulose-based renewable bioenergy. With respect to the problem of lignocellulose recalcitrance, it is germane that a number of invertebrate animals, and to some extent, their symbiotic gut fauna, have evolved specialized enzymes that cooperate in lignocellulose processing. In particular, endogenous lignocellulases encoded in marine and terrestrial invertebrate genomes can often confer high degrees of digestion capabilities to these organisms. When endogenous insect lignocellulases work synergistically with symbiont-derived enzymes, this can confer extremely high efficiency in lignocellulose processing. Termites (order Isoptera) are one of the most well recognized examples of an organism that subsists on lignocellulose; and thus, lignocellulase enzymes from termites and their gut symbionts have many potential bioenergy applications that warrant consideration.
SUMMARY
Briefly described, embodiments of this disclosure, among others, encompass the use of polypeptides derived from the termite R. flavipes alone, or in combination, in systems for the in vitro reduction of biomaterial containing a lignin component to a product that may be fermented to provide a source of a biofuel. The present disclosure provides data that support that a wood (lignocellulose) source can provide glucose moieties when treated in vitro with a combination of termite-derived enzyme such as, but not limited to, an endoglucanase, an exoglucanase, a laccase, and a β-glucosidase. It is well known that free glucose may be fermented to such as ethanol for use as a bio-fuel.
Accordingly, the present disclosure provides isolated nucleic acid molecules derived from the gut of the termite R. flavipes, recombinant nucleic acid molecules comprising a vector and an isolated heterologous nucleic acid molecule operably inserted therein, whereby, when transformed into an appropriate host cell system, the heterologous nucleic acid sequence is expressed as a polypeptide having an activity similar to that when expressed in the gut of the termite R. flavipes. It is within the scope of the disclosure for the recombinant nucleic acid molecules to comprise more than one heterologous nucleic acid molecule such that more than one polypeptide may be expressed by the host system. The expressed polypeptides may be substantially purified, or used in a substantially unpurified form, to be admixed with a lignocellulose source to be converted to a fermentable product such as a sugar or a mixture of sugars.
One aspect of the present disclosure, therefore, encompasses methods of converting a lignified plant material to a fermentable product, the method comprising the steps of: (a) obtaining a series of isolated polypeptides of a termite, wherein the series of polypeptides cooperate to convert a plant lignocellulose to a fermentable product; and (b) incubating the series of polypeptides with a source of lignified plant material, under conditions allowing the polypeptides to cooperatively produce a fermentable product from the lignified plant material.
In embodiments of this aspect of the disclosure, the isolated polypeptides can be derived from the termite Reticulitermes flavipes. 
In the various embodiments of this aspect of the disclosure, the isolated polypeptides of the series of isolated polypeptides can be recombinant polypeptides, where each polypeptide is expressed from an expression vector of a recombinant expression system, and wherein the recombinant expression system is selected from a eukaryotic cell-based system and a prokaryotic cell-based system.
In one embodiment of the disclosure, the expression vector is a baculovirus expression vector and the recombinant expression system is a eukaryotic cell-based system.
In the various embodiments of the disclosure, the series of isolated polypeptides comprise an endoglucanase having an amino acid sequence having about 75% sequence identity with the amino acid sequence SEQ ID NO.: 2.
In one embodiment of the disclosure, the endoglucanase can comprise the amino acid sequence SEQ ID NO.: 2.
In the various embodiments of the disclosure, the series of isolated polypeptides comprises an exoglucanase having an amino acid sequence having about 75% sequence identity with the amino acid sequence SEQ ID NO.: 4.
In one embodiment of the disclosure, the series of isolated polypeptides comprises an exoglucanase having the amino acid sequence SEQ ID NO.: 4.
In the various embodiments of the disclosure, the series of isolated polypeptides comprises a laccase having an amino acid sequence having about 75% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NOs.: 8, 10, 12, 15, 17, 19, 21, and 23.
In one embodiment of the disclosure, the series of isolated polypeptides comprises a laccase having the amino acid sequence selected from the group consisting of: SEQ ID NOs.: 8, 10, 12, 15, 17, 19, 21, and 23.
In the various embodiments of the disclosure, the series of isolated polypeptides comprises an esterase having an amino acid sequence having about 75% sequence identity with the amino acid sequence selected from the group consisting of: SEQ ID NOs.: 27, 29, 31, and 33.
In one embodiment of the disclosure, the series of isolated polypeptides comprises an esterase having the amino acid sequence selected from the group consisting of: SEQ ID NOs.: 27, 29, 31, and 33.
In one embodiment of the disclosure, the series of isolated polypeptides comprises a glucosidase having the amino acid sequence SEQ ID NO.: 6.
Another aspect of the present disclosure encompasses systems for producing a fermentable product from a lignified plant material, wherein the system may comprise at least two isolated polypeptides selected from the group consisting of: an endoglucanase, an exoglucanase, a laccase, an esterase, and a glucosidase of the termite Reticulitermes flavipes and wherein two or more isolated polypeptides can cooperate to convert a constituent of the lignified plant material to a fermentable product or a precursor thereof.
In embodiments of this aspect of the disclosure, the nucleotide sequence encoding the endoglucanase may hybridize under high stringency conditions to a nucleotide sequence according to SEQ ID NO.: 1, the nucleotide sequence encoding the exoglucanase may hybridize under high stringency conditions to a nucleotide sequence according to SEQ ID NO.: 3, the nucleic acid molecule encoding the laccase may hybridize under high stringency conditions to a nucleotide sequence selected from the group consisting of: SEQ ID NOS.: 7, 9, 11, 13, 14, 16, 18, 20, and 22, the nucleic acid molecule encoding the esterase may hybridize under high stringency conditions to a nucleotide sequence selected from the group consisting of: SEQ ID NOS.: 26, 28, 30, and 32, and the nucleotide sequence encoding the glucosidase may hybridize under high stringency conditions to SEQ ID NO.: 5.
In embodiments of this aspect of the disclosure, each of the isolated nucleic acid molecules thereof may be operably inserted into an expression vector.
Still another aspect of the disclosure encompasses a recombinant cell comprising an isolated nucleic acid molecule hybridizing under high stringency conditions to a nucleotide sequence encoding a polypeptide selected from the group consisting of: an endoglucanase, an exoglucanase, a laccase, an esterase, and a glucosidase of the termite Reticulitermes flavipes, wherein the nucleotide sequence encoding the endoglucanase is according to SEQ ID NO.: 1, the nucleotide sequence encoding the exoglucanase is according to SEQ ID NOS.: 3, the nucleic acid molecule encoding the laccase is selected from the group consisting of: SEQ ID NOS.: 7, 9, 11, 13, 14, 16, 18, 20, and 22, the nucleic acid molecule encoding the esterase is selected from the group consisting of: SEQ ID NOS.: 26, 28, 30, and 32, and the nucleotide sequence encoding the glucosidase is according to SEQ ID NO.: 5.



BRIEF DESCRIPTION OF THE DRAWINGS
Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.
FIGS. 1A and 1B illustrate a Clustal W alignment of translated termite gut esterases RfEst 1, 2, 3 and 4 (SEQ ID NOs.: 27, 29, 31, and 33). Boxes denote conserved esterase catalytic site and substrate recognition motifs. Arrows indicate amino acid residues that are 100% conserved across all proteins.
FIG. 2 illustrates a graph showing the average±std. error band intensities determined from densitometric analyses of five replicate gels from workers of two termite colonies (30 cycles of amplification). Bars for individual genes with the same letter are not significantly different by LSD t-tests (p<0.05). Abbreviations: FG (foregut/salivary gland), MG (midgut), HG (hindgut), bp (nucleotide base pairs).
FIG. 3 is a digital image of an analysis of R. flavipes worker gut and carcass esterases by native (non-denaturing) polyacrylamide gel electrophoresis (PAGE) using α-naphthyl acetate as a substrate. Two colonies were examined using 14,000×g supernatants from homogenized tissues of the foregut/salivary gland (FG), midgut (MG), hindgut (HG), and headless carcass that remained after gut removal (carcass).
FIG. 4 illustrates the analysis of R. flavipes worker gut esterases by native PAGE.
FIG. 5 shows a series of graphs illustrating the results of colorimetric microplate assays using various α-naphthyl ester substrates. 14,000×g supernatants from homogenized tissues of the foregut/salivary gland (FG), midgut (MG), hindgut (HG) were examined. Substrates were naphthyl acetate (ace), propionate (prop), butyrate (but), valerate (val), and caprylate (cap). These substrates contain esteratic chain lengths of, respectively, 2, 3, 4, 5 and 6 carbon molecules. Bars within graphs with the same letter are not significantly different by LSD t-tests (p<0.05).
FIG. 6 is a digital image of a Coomassie-stained SDS PAGE gel showing the purified recombinant Cell-1 protein in relation to various endogenous protein fractions from R. flavipes termites.
FIG. 7 is a graph illustrating Lineweaver-Burke kinetic plots comparing the relative activities of Cell-1 produced in a bacterial (E. coli) or a eukaryotic (insect) system.
FIG. 8 shows a pair of graphs illustrating the determination of optimal pH for carboxymethyl cellulose digestion by the Cell-1 protein (optimal activity is about the known termite gut pH of approximately 7). Optimal pH and temperature stability for the purified BEVS-expressed Cell-1. (FIG. 8, Left) pH dependence of CMC activity at 30° C. in sodium acetate buffer, pH 3.0-6.5. (Right) pH dependence of CMC activity at 30° C. in sodium phosphate buffer, pH 7.0-10.5.
FIGS. 9A-9C show a series of graphs illustrating the impacts of EDTA and calcium chloride on temperature stability of the purified BEVS-expressed Cell-1. (FIG. 9A) CMC activity after pre-incubation at 25° C. for 0 through 5 days with buffer alone (sodium phosphate, pH 7), buffer+5 mM EDTA, or buffer+30 mM calcium chloride. (FIG. 9B and FIG. 9C) CMC activity after pre-incubation at 60° C. or 70° C., respectively, for 0 through 60 min with buffer alone, buffer+5 mM EDTA, or buffer+30 mM calcium chloride. All assays were conducted at 25° C. Results shown are the average of three independent replicates is a graph of data showing that the presence of calcium improves Cell-1 temperature stability, while the calcium chelator EDTA reduces temperature stability.
FIG. 10 shows a pair of digital images of agarose gels showing RfBGluc-1, RfBGluc-2, NADH-dehydrogenase, and β-actin relative transcript abundance after 35 cycles of conventional PCR amplification. G, whole gut; C, carcass; (−), negative control reaction conducted without template.
FIGS. 11A and 11B show graphs illustrating the relative expression of RfBGluc-1 (FIG. 11A) and RfBGluc-2 (FIG. 11B) by quantitative real-time PCR. Analyses were performed using the 2−ΔΔCT method. Results are normalized to NADH-dh as a reference gene and midgut as a reference tissue. Bars within graphs with different letters are significantly different by ANOVA and Tukey's HSD tests (p<0.05). Abbreviations: SG, salivary gland; FG, foregut; MG, midgut; HG, hindgut.
FIG. 12A is a digital image of an SDS PAGE analysis showing purified recombinant RfBGluc-1 protein with and without deglycosylation treatment by PNGase F. Lanes 1 and 6, MW markers in kilodaltons; lanes 2 and 3, purified RfBGluc-1; lane 4, PNGase-treated RfBGluc-1; and lane 5, PNGase control.
FIG. 12B is a digital image of a native PAGE analyses showing purified recombinant RfBGluc-1 protein shown in relation to native R. flavipes tissue preparations and recombinant RfCell-1. Lane 7, MW markers in kilodaltons; lane 1, foregut+salivary gland tissue; lane 2, midgut tissue; lane 3; hindgut tissue; lane 4, remaining carcass after gut removal; lane 5, recombinant RfCell-1 endoglucanase (shown for reference); lane 6, purified recombinant RfBGluc-1. Arrows labeled with BG and C1 indicate putative RfBGluc-1 and RfCell-1 proteins in the foregut+salivary gland preparations.
FIGS. 13A-13D illustrates the concentration-dependent effects of chemicals that either inhibit or enhance RfLacA oxidation of 2,6-dimethoxyphenol: (FIG. 13A) EDTA, (FIG. 13B) sodium cyanide, (FIG. 13C) thioglycolic acid, (FIG. 13D) sodium azide. Bars with different letters are significantly different at p<0.05.
FIG. 14 is a graph illustrating an HPLC analysis of lignin alkali degradation by RfLacA. Two superimposed HPLC chromatograms are shown; the gray and black traces represent lignin alkali incubated without and with enzyme, respectively. Arrows and numbers indicate peaks with differential abundance between treatments. Peaks 1, 3, 4, 5 and 6 represent potential parent compounds acted upon by laccase action. Peaks 2 and 7 represent potential degradation products.
FIG. 15 is a graph illustrating that homogenates of recombinant Cell-3 infected larvae show higher exoglucanase (cellulase) activity than do uninfected homogenates when p-nitrophenyl cellobioside is used as a substrate. The reduced activity in filtrates suggests reduced stability of the mature protein through the enrichment process.
FIG. 16 is a graph illustrating measures of glucose release from saw-dust after incubation for 18 hrs at 37° C. with mixtures of recombinant lignocellulases. Enzymes were: Cell-1 endoglucanase, Cell-3 exoglucanase, Lacc6 laccase, and β-glucosidase. The mixture of four enzymes was required for quantifiable glucose release. 1×, 2× and 3×=1, 2 and 3 μL of enzyme stock.
FIG. 17 shows a graph illustrating substrate specificity by recombinant RfBGluc-1 towards natural and synthetic substrates. See Tables 3 and 4 for substrate information.
FIG. 18 shows a graph illustrating optimal pH determination for recombinant RfBGluc-1 using two buffer systems and 2 mM p-nitrophenyl-B-D-glucopyranoside as a substrate.
FIG. 19 shows a graph illustrating concentration-dependent inhibition of recombinant RfBGluc-1 by cellobioimidazole (CBI) and fluoromethyl cellobiose (FMCB). Assays were conducted at 25° C. in sodium acetate buffer (0.1M, pH 7) using 2 mM p-nitrophenyl-B-D-glucopyranoside as the substrate.
FIG. 20 is a graph illustrating the quantification of glucose release from pine sawdust. Treatments are shown across the x-axis are (1) Cell-1 endoglucanase, (2) the β-glucosidase, and (3) the Lac6 laccase, and their two—(1+2), (1+3), and (2+3) and three-way (1+2+3) combinations. Bars within graphs with the same letters are not significantly different by Tukey's HSD tests (p<0.05). Both ANOVA models were significant as well, with the parameters as shown.
FIG. 21 is a graph illustrating the quantification of glucose release from beechwood xylan. Treatments are shown across the x-axis are (1) Cell-1 endoglucanase, (2) the β-glucosidase, and (3) the Lac6 laccase, and their two—(1+2), (1+3), and (2+3) and three-way (1+2+3) combinations. Bars within graphs with the same letters are not significantly different by Tukey's HSD tests (p<0.05). Both ANOVA models were significant as well, with the parameters as shown.



The details of some exemplary embodiments of the methods and systems of the present disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent to one of skill in the art upon examination of the following description, drawings, examples and embodiments. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure.
DETAILED DESCRIPTION
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
It must be noted that, as used in the specification and the appended embodiments, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the embodiments that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. “Consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.
DEFINITIONS
In describing the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.
The term “termite gut” as used herein refers to the gut of R. flavipes workers. The gut of R. flavipes workers is composed of three main regions: foregut, midgut, and hindgut. The foregut region includes the esophagus, crop, and attached salivary gland. The salivary glands secrete endogenous (termite-derived) digestive factors and enzymes into the digestive tract. The midgut is a slender, tubular region that secretes a peritrophic matrix around food materials and, presumably, is a location where some lignocellulose degradation occurs. The Malpighian tubules connect at the junction of the midgut and hindgut and participate in waste excretion. The hindgut includes a fermentation chamber that is generally anaerobic in its core, but it does possess a micro-oxic zone around its periphery. The hindgut houses gut symbionts, and it is the location where most lignocellulose degradation, as well as fermentation and nutrient assimilation, are thought to occur.
The fermentation chamber of the hindgut is a source of microbial diversity. Microorganisms from various taxa present in the termite gut include bacteria/spirochetes and protozoans. In lower termites such as R. flavipes, protozoan symbionts are considered to be primarily involved in cellulose/hemicellulose degradation, while bacteria are considered important to nitrogen economy and simple sugar fermentation. Spirochetes, which are difficult to culture, are found in the hindguts of all termites. Spirochetes play roles in acetogenesis and nitrogen fixation, and they and other endomicrobionts also occur as cytoplasmic symbionts of hindgut protozoa.
The term “lignocellulose” as used herein refers to a natural complex of the three biopolymers: cellulose, hemicellulose and lignin. Cellulose is composed of rigid, high-molecular-weight, β-1,4-linked polymers of glucose that are held together in bundles by hemicellulose. Hemicellulose is composed of shorter β-1,4-linked polymers of mixed sugars. Mannose is usually the dominant sugar present in hemicelluloses of softwoods fed upon by termites, with lesser amounts of xylose, galactose, rhamnose, arabinose, glucuronic acid, mannuronic acid and galacturonic acid.
The term “lignin” as used herein refers to a 3-dimensional polymer of phenolic compounds that are linked to each other and to hemicellulose by ester bonds. Lignin is composed of the three mono-lignol monomers p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol combined in different ratios depending on the plant species. Another noteworthy aspect of hemicellulose is its high degree of esterification with monomers and dimers of phenolic acid esters, which are analogous to the mono-lignols noted above. Phenolic acid esters are derived mostly from the mono-lignols p-coumaryl and coniferyl alcohol (i.e., coumaric acid and ferulic acid). The three individual lignocellulose components, cellulose, hemicellulose and lignin, compose approximately 40%, 25%, and 20%, respectively, of lignocellulose (Lange 2007).
The term “nucleic acid” as used herein refers to any natural and synthetic linear and sequential arrays of nucleotides and nucleosides, for example cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives thereof. For ease of discussion, such nucleic acids may be collectively referred to herein as “constructs,” “plasmids,” or “vectors.” Representative examples of the nucleic acids of the present disclosure include bacterial plasmid vectors including expression, cloning, cosmid and transformation vectors such as, but not limited to, pBR322, animal viral vectors such as, but not limited to, modified adenovirus, influenza virus, polio virus, pox virus, retrovirus, insect viruses (baculovirus), and the like, vectors derived from bacteriophage nucleic acid, and synthetic oligonucleotides like chemically synthesized DNA or RNA. The term “nucleic acid” further includes modified or derivatized nucleotides and nucleosides such as, but not limited to, halogenated nucleotides such as, but not only, 5-bromouracil, and derivatized nucleotides such as biotin-labeled nucleotides.
The term “isolated nucleic acid” as used herein refers to a nucleic acid with a structure (a) not identical to that of any naturally occurring nucleic acid or (b) not identical to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes, and includes DNA, RNA, or derivatives or variants thereof. The term covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic molecule but is not flanked by at least one of the coding sequences that flank that part of the molecule in the genome of the species in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic nucleic acid of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any vector or naturally occurring genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein, and (e) a recombinant nucleotide sequence that is part of a hybrid sequence that is not naturally occurring. Isolated nucleic acid molecules of the present disclosure can include, for example, natural allelic variants as well as nucleic acid molecules modified by nucleotide deletions, insertions, inversions, or substitutions.
The term “enriched” as used herein in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. Enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. The other DNA may, for example, be derived from a yeast or bacterial genome, or a cloning vector, such as a plasmid or a viral vector. The term “significant” as used herein is used to indicate that the level of increase is useful to the person making such an increase.
It is advantageous for some purposes that a nucleotide sequence is in purified form. The term “purified” in reference to nucleic acid represents that the sequence has increased purity relative to the natural environment.
The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid sequence” are used interchangeably herein and include, but are not limited to, coding sequences (polynucleotide(s) or nucleic acid sequence(s) which are transcribed and translated into polypeptide in vitro or in vivo when placed under the control of appropriate regulatory or control sequences); control sequences (e.g., translational start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, and the like); and regulatory sequences (DNA sequences to which a transcription factor(s) binds and alters the activity of a gene's promoter either positively (induction) or negatively (repression)). No limitation as to length or to synthetic origin is suggested by the terms described herein.
The terms “polypeptide” and “protein” as used herein refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. The term “polypeptide” includes proteins, protein fragments, protein analogues, oligopeptides and the like. The term “polypeptides” contemplates polypeptides as defined above that are encoded by nucleic acids, produced through recombinant technology (isolated from an appropriate source such as a bird), or synthesized. The term “polypeptides” further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids covalently or non-covalently linked to labeling ligands.
The term “fragment” as used herein to refer to a nucleic acid (e.g., cDNA) refers to an isolated portion of the subject nucleic acid constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or a portion of a nucleic acid synthesized by PCR, DNA polymerase or any other polymerizing technique well known in the art, or expressed in a host cell by recombinant nucleic acid technology well known to one of skill in the art. The term “fragment” as used herein may also refer to an isolated portion of a polypeptide, wherein the portion of the polypeptide is cleaved from a naturally occurring polypeptide by proteolytic cleavage by at least one protease, or is a portion of the naturally occurring polypeptide synthesized by chemical methods well known to one of skill in the art.
The term “gene” or “genes” as used herein refers to nucleic acid sequences (including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any portion of such whole RNA or whole protein. Genes that are not naturally part of a particular organism's genome are referred to as “foreign genes,” “heterologous genes” or “exogenous genes” and genes that are naturally a part of a particular organism's genome are referred to as “endogenous genes”. The term “gene product” refers to RNAs or proteins that are encoded by the gene. “Foreign gene products” are RNA or proteins encoded by “foreign genes” and “endogenous gene products” are RNA or proteins encoded by endogenous genes. “Heterologous gene products” are RNAs or proteins encoded by “foreign, heterologous or exogenous genes” and are, therefore, not naturally expressed in the cell.
The term “expressed” or “expression” as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene. The term “expressed” or “expression” as used herein also refers to the translation from said RNA nucleic acid molecule to give a protein, a polypeptide, or a portion or fragment thereof.
The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
The terms “transcription regulatory sequences” and “gene expression control regions” as used herein refer to nucleotide sequences that are associated with a gene nucleic acid sequence and which regulate the transcriptional expression of the gene. Exemplary transcription regulatory sequences include enhancer elements, hormone response elements, steroid response elements, negative regulatory elements, and the like. The “transcription regulatory sequences” may be isolated and incorporated into a vector nucleic acid to enable regulated transcription in appropriate cells of portions of the vector DNA. The “transcription regulatory sequence” may precede, but is not limited to, the region of a nucleic acid sequence that is in the region 5′ of the end of a protein coding sequence that may be transcribed into mRNA. Transcriptional regulatory sequences may also be located within a protein coding region, in regions of a gene that are identified as “intron” regions, or may be in regions of nucleic acid sequence that are in the region of nucleic acid.
The term “promoter” as used herein refers to the DNA sequence that determines the site of transcription initiation from an RNA polymerase. A “promoter-proximal element” may be a regulatory sequence within about 200 base pairs of the transcription start site.
The term “coding region” as used herein refers to a continuous linear arrangement of nucleotides that may be translated into a protein. A full length coding region is translated into a full length protein; that is, a complete protein as would be translated in its natural state absent any post-translational modifications. A full length coding region may also include any leader protein sequence or any other region of the protein that may be excised naturally from the translated protein.
The term “complementary” as used herein refers to two nucleic acid molecules that can form specific interactions with one another. In the specific interactions, an adenine base within one strand of a nucleic acid can form two hydrogen bonds with thymine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities. Also in the specific interactions, a guanine base within one strand of a nucleic acid can form three hydrogen bonds with cytosine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities. Complementary nucleic acids as referred to herein, may further comprise modified bases wherein a modified adenine may form hydrogen bonds with a thymine or modified thymine, and a modified cytosine may form hydrogen bonds with a guanine or a modified guanine.
The term “probe” as used herein, when referring to a nucleic acid, refers to a nucleotide sequence that can be used to hybridize with and thereby identify the presence of a complementary sequence, or a complementary sequence differing from the probe sequence but not to a degree that prevents hybridization under the hybridization stringency conditions used. The probe may be modified with labels such as, but not only, radioactive groups, chemiluminescent moieties, biotin, and the like that are well known in the art.
The term “capable of hybridizing under stringent conditions” as used herein refers to annealing a first nucleic acid to a second nucleic acid under stringent conditions as defined below. Stringent hybridization conditions typically permit the hybridization of nucleic acid molecules having at least 70% nucleic acid sequence identity with the nucleic acid molecule being used as a probe in the hybridization reaction. For example, the first nucleic acid may be a test sample or probe, and the second nucleic acid may be the sense or antisense strand of an gene expression control region or a fragment thereof. Hybridization of the first and second nucleic acids may be conducted under stringent conditions, e.g., high temperature and/or low salt content that tend to disfavor hybridization of dissimilar nucleotide sequences. Alternatively, hybridization of the first and second nucleic acid may be conducted under reduced stringency conditions, e.g., low temperature and/or high salt content that tend to favor hybridization of dissimilar nucleotide sequences. Low stringency hybridization conditions may be followed by high stringency conditions or intermediate medium stringency conditions to increase the selectivity of the binding of the first and second nucleic acids. The hybridization conditions may further include reagents such as, but not limited to, dimethyl sulfoxide (DMSO) or formamide to disfavor still further the hybridization of dissimilar nucleotide sequences. A suitable hybridization protocol may, for example, involve hybridization in 6×SSC (wherein 1×SSC comprises 0.015 M sodium citrate and 0.15 M sodium chloride), at 65° Celsius in an aqueous solution, followed by washing with 1×SSC at 65° Celsius. Formulae to calculate appropriate hybridization and wash conditions to achieve hybridization permitting 30% or less mismatch between two nucleic acid molecules are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138: 267-284; the contents of which is incorporated herein by reference in its entirety. Protocols for hybridization techniques are well known to those of skill in the art and standard molecular biology manuals may be consulted to select a suitable hybridization protocol without undue experimentation. See, for example, Sambrook et al., 1989, “Molecular Cloning: A Laboratory Manual,” 2nd ed., Cold Spring Harbor Press: the contents of which is incorporated herein by reference in its entirety.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) from about pH 7.0 to about pH 8.3 and the temperature is at least about 30° Celsius for short probes (e.g., 10 to 50 nucleotides) and at least about 60° Celsius for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° Celsius and a wash in 1-2×SSC at 50 to 55° Celsius. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° Celsius and a wash in 0.5-1×SSC at 55 to 60° Celsius. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° Celsius.
The terms “unique nucleic acid region” and “unique protein (polypeptide) region” as used herein refer to sequences present in a nucleic acid or protein (polypeptide) respectively that is not present in any other nucleic acid or protein sequence. The terms “conserved nucleic acid region” as referred to herein is a nucleotide sequence present in two or more nucleic acid sequences, to which a particular nucleic acid sequence can hybridize under low, medium or high stringency conditions. The greater the degree of conservation between the conserved regions of two or more nucleic acid sequences, the higher the hybridization stringency that will allow hybridization between the conserved region and a particular nucleic acid sequence.
The term “sense strand” as used herein refers to a single stranded DNA molecule from a genomic DNA that may be transcribed into RNA and translated into the natural polypeptide product of the gene. The term “antisense strand” as used herein refers to the single strand DNA molecule of a genomic DNA that is complementary with the sense strand of the gene.
The term “nucleic acid vector” as used herein refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule that can be transfected or transformed into cells and replicate independently of, or within, the host cell genome. A circular double stranded plasmid can be linearized by treatment with an appropriate restriction enzyme based on the nucleotide sequence of the plasmid vector. A nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together. The nucleic acid molecule can be RNA or DNA.
The term “expression vector” as used herein refers to a nucleic acid vector that comprises a gene expression control region operably linked to a nucleotide sequence coding at least one polypeptide. As used herein, the term “regulatory sequences” includes promoters, enhancers, and other elements that may control gene expression. Standard molecular biology textbooks (for example, Sambrook et al., eds., 1989, “Molecular Cloning: A Laboratory Manual,” 2nd ed., Cold Spring Harbor Press) may be consulted to design suitable expression vectors that may further include an origin of replication and selectable gene markers. It should be recognized, however, that the choice of a suitable expression vector and the combination of functional elements therein depends upon multiple factors including the choice of the host cell to be transformed and/or the type of protein to be expressed.
The terms “transformation” and “transfection” as used herein refer to the process of inserting a nucleic acid into a host. Many techniques are well known to those skilled in the art to facilitate transformation or transfection of a nucleic acid into a prokaryotic or eukaryotic organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt such as, but not only, a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection, to render the host cell competent for the uptake of the nucleic acid molecules, and by such methods as sperm-mediated and restriction-mediated integration.
The term “transfecting agent” as used herein refers to a composition of matter added to the genetic material for enhancing the uptake of heterologous DNA segment(s) into a eukaryotic cell including, but not limited to, an insect host cell. The enhancement is measured relative to the uptake in the absence of the transfecting agent. Examples of transfecting agents include adenovirus-transferrin-polylysine-DNA complexes. These complexes generally augment the uptake of DNA into the cell and reduce its breakdown during its passage through the cytoplasm to the nucleus of the cell. Other preferred transfecting agents include, but are not limited to, lipofectin, lipofectamine, DIMRIE C, Supeffect, and Effectin (Qiagen), unifectin, maxifectin, DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecytammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB (N-n-hexadecylN,N-dihydroxyethylammoniurn bromide), polybrene, poly(ethylenimine) (PEI) and the like.
The term “recombinant cell” refers to a cell that has a new combination of nucleic acid segments that are not covalently linked to each other in nature. A new combination of nucleic acid segments can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. A recombinant cell can be a single eukaryotic cell, or a single prokaryotic cell, or a mammalian cell. The recombinant cell may harbor a vector that is extragenomic. An extragenomic nucleic acid vector does not insert into the cell's genome. A recombinant cell may further harbor a vector or a portion thereof that is intragenomic. The term intragenomic defines a nucleic acid construct incorporated within the recombinant cell's genome.
The terms “recombinant nucleic acid” and “recombinant DNA” as used herein refer to combinations of at least two nucleic acid sequences that are not naturally found in a eukaryotic or prokaryotic cell. The nucleic acid sequences include, but are not limited to, nucleic acid vectors, gene expression regulatory elements, origins of replication, suitable gene sequences that when expressed confer antibiotic resistance, protein-encoding sequences, and the like. The term “recombinant polypeptide” is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location, purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
The techniques used to isolate and characterize the nucleic acids and proteins of the present disclosure are well known to those of skill in the art and standard molecular biology and biochemical manuals may be consulted to select suitable protocols without undue experimentation (see, for example, Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ed., 1989, Cold Spring Harbor Press; the contents of which is incorporated herein by reference in its entirety).
The term “glycosylation site” as used herein refers to a location on a polypeptide that has a glycosylation chain attached thereto. The “site” may be an amino acid side-chain, or a plurality of side-chains (either contiguous in the amino acid sequence or in cooperative vicinity to one another to define a specific site associated with at least one glycosylation chain).
Many cell surface and secretory proteins produced by eukaryotic cells are modified with one or more oligosaccharide groups. This modification, referred to as “glycosylation” can dramatically affect the physical properties of proteins and can also be important in protein stability, secretion, and subcellular localization. Proper glycosylation can be essential for biological activity. In fact, some genes from eukaryotic organisms, when expressed in bacteria (e.g., E. coli) which lack cellular processes for glycosylating proteins, yield proteins that are recovered with little or no activity by virtue of their lack of glycosylation.
Glycosylation occurs at specific locations along the polypeptide backbone and is usually of two types: O-linked oligosaccharides are attached to serine or threonine residues while N-linked oligosaccharides are attached to asparagine residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (hereafter referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycoprotein.
A “cyclic polymerase-mediated reaction” refers to a biochemical reaction in which a template molecule or a population of template molecules is periodically and repeatedly copied to create a complementary template molecule or complementary template molecules, thereby increasing the number of the template molecules over time.
“Denaturation” of a template molecule refers to the unfolding or other alteration of the structure of a template so as to make the template accessible to duplication. In the case of DNA, “denaturation” refers to the separation of the two complementary strands of the double helix, thereby creating two complementary, single stranded template molecules. “Denaturation” can be accomplished in any of a variety of ways, including by heat or by treatment of the DNA with a base or other denaturant.
“DNA amplification” as used herein refers to any process that increases the number of copies of a specific DNA sequence by enzymatically amplifying the nucleic acid sequence. A variety of processes are known. One of the most commonly used is the polymerase chain reaction (PCR), which is defined and described in later sections below. The PCR process of Mullis is described in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR involves the use of a thermostable DNA polymerase, known sequences as primers, and heating cycles, which separate the replicating deoxyribonucleic acid (DNA), strands and exponentially amplify a gene of interest. Any type of PCR, such as quantitative PCR, RT-PCR, hot start PCR, LAPCR, multiplex PCR, touchdown PCR, etc., may be used. Advantageously, real-time PCR is used. In general, the PCR amplification process involves an enzymatic chain reaction for preparing exponential quantities of a specific nucleic acid sequence. It requires a small amount of a sequence to initiate the chain reaction and oligonucleotide primers that will hybridize to the sequence. In PCR the primers are annealed to denatured nucleic acid followed by extension with an inducing agent (enzyme) and nucleotides. This results in newly synthesized extension products. Since these newly synthesized sequences become templates for the primers, repeated cycles of denaturing, primer annealing, and extension results in exponential accumulation of the specific sequence being amplified. The extension product of the chain reaction will be a discrete nucleic acid duplex with a termini corresponding to the ends of the specific primers employed.
“DNA” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form, or as a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
By the terms “enzymatically amplify” or “amplify” is meant, for the purposes of the specification or embodiments, DNA amplification, i.e., a process by which nucleic acid sequences are amplified in number. There are several means for enzymatically amplifying nucleic acid sequences. Currently the most commonly used method is the polymerase chain reaction (PCR). Other amplification methods include LCR (ligase chain reaction) which utilizes DNA ligase, and a probe consisting of two halves of a DNA segment that is complementary to the sequence of the DNA to be amplified, enzyme Qβ replicase and a ribonucleic acid (RNA) sequence template attached to a probe complementary to the DNA to be copied which is used to make a DNA template for exponential production of complementary RNA; strand displacement amplification (SDA); Qβ replicase amplification (QβRA); self-sustained replication (3SR); and NASBA (nucleic acid sequence-based amplification), which can be performed on RNA or DNA as the nucleic acid sequence to be amplified.
A “fragment” of a molecule such as a protein or nucleic acid is meant to refer to any portion of the amino acid or nucleotide genetic sequence.
As used herein, the term “genome” refers to all the genetic material in the chromosomes of a particular organism. Its size is generally given as its total number of base pairs. Within the genome, the term “gene” refers to an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product (e.g., a protein or RNA molecule). In general, a patient's genetic characteristics, as defined by the nucleotide sequence of its genome, are known as its “genotype,” while the patient's physical traits are described as its “phenotype.”
The term “polymerase chain reaction” or “PCR” refers to a thermocyclic, polymerase-mediated, DNA amplification reaction. A PCR typically includes template molecules, oligonucleotide primers complementary to each strand of the template molecules, a thermostable DNA polymerase, and deoxyribonucleotides, and involves three distinct processes that are multiply repeated to effect the amplification of the original nucleic acid. The three processes (denaturation, hybridization, and primer extension) are often performed at distinct temperatures, and in distinct temporal steps. In many embodiments, however, the hybridization and primer extension processes can be performed concurrently. The nucleotide sample to be analyzed may be PCR amplification products provided using the rapid cycling techniques described in U.S. Pat. Nos. 6,569,672; 6,569,627; 6,562,298; 6,556,940; 6,569,672; 6,569,627; 6,562,298; 6,556,940; 6,489,112; 6,482,615; 6,472,156; 6,413,766; 6,387,621; 6,300,124; 6,270,723; 6,245,514; 6,232,079; 6,228,634; 6,218,193; 6,210,882; 6,197,520; 6,174,670; 6,132,996; 6,126,899; 6,124,138; 6,074,868; 6,036,923; 5,985,651; 5,958,763; 5,942,432; 5,935,522; 5,897,842; 5,882,918; 5,840,573; 5,795,784; 5,795,547; 5,785,926; 5,783,439; 5,736,106; 5,720,923; 5,720,406; 5,675,700; 5,616,301; 5,576,218 and 5,455,175, the disclosures of which are incorporated by reference in their entireties. Other methods of amplification include, without limitation, NASBR, SDA, 3SR, TSA and rolling circle replication. It is understood that, in any method for producing a polynucleotide containing given modified nucleotides, one or several polymerases or amplification methods may be used. The selection of optimal polymerization conditions depends on the application.
A “polymerase” is an enzyme that catalyzes the sequential addition of monomeric units to a polymeric chain, or links two or more monomeric units to initiate a polymeric chain. In advantageous embodiments of this disclosure, the “polymerase” will work by adding monomeric units whose identity is determined by and which is complementary to a template molecule of a specific sequence. For example, DNA polymerases such as DNA pol 1 and Taq polymerase add deoxyribonucleotides to the 3′ end of a polynucleotide chain in a template-dependent manner, thereby synthesizing a nucleic acid that is complementary to the template molecule. Polymerases may be used either to extend a primer once or repetitively or to amplify a polynucleotide by repetitive priming of two complementary strands using two primers.
A “primer” is an oligonucleotide, the sequence of at least a portion of which is complementary to a segment of a template DNA which to be amplified or replicated. Typically primers are used in performing the polymerase chain reaction (PCR). A primer hybridizes with (or “anneals” to) the template DNA and is used by the polymerase enzyme as the starting point for the replication/amplification process. By “complementary” is meant that the nucleotide sequence of a primer is such that the primer can form a stable hydrogen bond complex with the template; i.e., the primer can hybridize or anneal to the template by virtue of the formation of base-pairs over a length of at least ten consecutive base pairs.
The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
As used herein, the term “protein” refers to a large molecule composed of one or more chains of amino acids in a specific order. The order is determined by the base sequence of nucleotides in the gene coding for the protein. Proteins are required for the structure, function, and regulation of the body's cells, tissues, and organs. Each protein has a unique function.
As used herein, a “template” refers to a target polynucleotide strand, for example, without limitation, an unmodified naturally-occurring DNA strand, which a polymerase uses as a means of recognizing which nucleotide it should next incorporate into a growing strand to polymerize the complement of the naturally-occurring strand. Such DNA strand may be single-stranded or it may be part of a double-stranded DNA template. In applications of the present disclosure requiring repeated cycles of polymerization, e.g., the polymerase chain reaction (PCR), the template strand itself may become modified by incorporation of modified nucleotides, yet still serve as a template for a polymerase to synthesize additional polynucleotides.
A “thermocyclic reaction” is a multi-step reaction wherein at least two steps are accomplished by changing the temperature of the reaction.
A “thermostable polymerase” refers to a DNA or RNA polymerase enzyme that can withstand extremely high temperatures, such as those approaching 100° C. Often, thermostable polymerases are derived from organisms that live in extreme temperatures, such as Thermus aquaticus. Examples of thermostable polymerases include Taq, Tth, Pfu, Vent, deep vent, UITma, and variations and derivatives thereof.
Typically, the annealing of the primers to the target DNA sequence is carried out for about 2 minutes at about 37-55° C., extension of the primer sequence by the polymerase enzyme (such as Taq polymerase) in the presence of nucleoside triphosphates is carried out for about 3 minutes at about 70-75° C., and the denaturing step to release the extended primer is carried out for about 1 minute at about 90-95° C. However, these parameters can be varied, and one of skill in the art would readily know how to adjust the temperature and time parameters of the reaction to achieve the desired results. For example, cycles may be as short as 10, 8, 6, 5, 4.5, 4, 2, 1, 0.5 minutes or less.
Also, “two temperature” techniques can be used where the annealing and extension steps may both be carried out at the same temperature, typically between about 60-65° C., thus reducing the length of each amplification cycle and resulting in a shorter assay time.
Typically, the reactions described herein are repeated until a detectable amount of product is generated. Often, such detectable amounts of product are between about 10 ng and about 100 ng, although larger quantities, e.g. 200 ng, 500 ng, 1 mg or more can also, of course, be detected. In terms of concentration, the amount of detectable product can be from about 0.01 pmol, 0.1 pmol, 1 pmol, 10 pmol, or more. Thus, the number of cycles of the reaction that are performed can be varied, the more cycles are performed, the more amplified product is produced. In certain embodiments, the reaction comprises 2, 5, 10, 15, 20, 30, 40, 50, or more cycles.
For example, the PCR reaction may be carried out using about 25-50 μl samples containing about 0.01 to 1.0 ng of template amplification sequence, about 10 to 100 pmol of each generic primer, about 1.5 units of Taq DNA polymerase (Promega Corp.), about 0.2 mM dDATP, about 0.2 mM dCTP, about 0.2 mM dGTP, about 0.2 mM dTTP, about 15 mM MgCl2 about 10 mM Tris-HCl (pH 9.0), about 50 mM KCl, about 1 μg/ml gelatin, and about 10 μl/ml Triton X-100 (Saiki, 1988).
Those of skill in the art are aware of the variety of nucleotides available for use in the cyclic polymerase mediated reactions. Typically, the nucleotides will consist at least in part of deoxynucleotide triphosphates (dNTPs), which are readily commercially available. Parameters for optimal use of dNTPs are also known to those of skill, and are described in the literature. In addition, a large number of nucleotide derivatives are known to those of skill and can be used in the present reaction. Such derivatives include fluorescently labeled nucleotides, allowing the detection of the product including such labeled nucleotides, as described below. Also included in this group are nucleotides that allow the sequencing of nucleic acids including such nucleotides, such as chain-terminating nucleotides, dideoxynucleotides and boronated nuclease-resistant nucleotides. Commercial kits containing the reagents most typically used for these methods of DNA sequencing are available and widely used. Other nucleotide analogs include nucleotides with bromo-, iodo-, or other modifying groups, which affect numerous properties of resulting nucleic acids including their antigenicity, their replicatability, their melting temperatures, their binding properties, etc. In addition, certain nucleotides include reactive side groups, such as sulfhydryl groups, amino groups, or N-hydroxysuccinimidyl groups, that allow the further modification of nucleic acids comprising them.
For the purposes of the present disclosure, sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithms. A non-limiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268, modified as in Karlin & Altschul, (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.
Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, (1988) Proc. Natl. Acad. Sci. USA 85: 2444-2448.
Advantageous for use according to the present disclosure is the WU-BLAST (Washington University BLAST) version 2.0 software. This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., (1990) J. Mol. Biol. 215: 403-410; Gish & States (1993); Nature Genetics 3: 266-272; Karlin & Altschul, (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein).
In all search programs in the suite the gapped alignment routines are integral to the database search itself. Gapping can be turned off if desired. The default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any integer. The default per-residue penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer. Any combination of values for Q and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.
Alternatively or additionally, the term “homology” or “identity”, for instance, with respect to a nucleotide or amino acid sequence, can indicate a quantitative measure of homology between two sequences. The percent sequence homology can be calculated as (Nref−Ndif)*100/Nref, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Nref=8; Ndif=2). “Homology” or “identity” can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur & Lipman, (1983) Proc Natl Acad Sci USA 80:726, incorporated herein by reference), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., IntelligeneticsTM Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the disclosure and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences. Without undue experimentation, the skilled artisan can consult with many other programs or references for determining percent homology.
Further definitions are provided in context below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein.
Producing the Primers and Probes of the Disclosure
The primers and probes described herein may be readily prepared by, for example, directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production. Methods for making a vector or recombinants or plasmid for amplification of the fragment either in vivo or in vitro can be any desired method, e.g., a method which is by or analogous to the methods disclosed in, or disclosed in documents cited in: U.S. Pat. Nos. 4,603,112; 4,769,330; 4,394,448; 4,722,848; 4,745,051; 4,769,331; 4,945,050; 5,494,807; 5,514,375; 5,744,140; 5,744,141; 5,756,103; 5,762,938; 5,766,599; 5,990,091; 5,174,993; 5,505,941; 5,338,683; 5,494,807; 5,591,639; 5,589,466; 5,677,178; 5,591,439; 5,552,143; 5,580,859; 6,130,066; 6,004,777; 6,130,066; 6,497,883; 6,464,984; 6,451,770; 6,391,314; 6,387,376; 6,376,473; 6,368,603; 6,348,196; 6,306,400; 6,228,846; 6,221,362; 6,217,883; 6,207,166; 6,207,165; 6,159,477; 6,153,199; 6,090,393; 6,074,649; 6,045,803; 6,033,670; 6,485,729; 6,103,526; 6,224,882; 6,312,682; 6,348,450 and 6; 312,683; U.S. patent application Ser. No. 920,197, filed Oct. 16, 1986; WO 90/01543; WO91/11525; WO 94/16716; WO 96/39491; WO 98/33510; EP 265785; EP 0 370 573; Andreansky et al., Proc. Natl. Acad. Sci. USA 1996; 93:11313-11318; Ballay et al., EMBO J. 1993; 4:3861-65; Feigner et al., J. Biol. Chem. 1994; 269:2550-2561; Frolov et al., Proc. Natl. Acad. Sci. USA 1996; 93:11371-11377; Graham, Tibtech 1990; 8:85-87; Grunhaus et al., Sem. Virol. 1992; 3:237-52; Ju et al., Diabetologia 1998; 41:736-739; Kitson et al., J. Virol. 1991; 65:3068-3075; McClements et al., Proc. Natl. Acad. Sci. USA 1996; 93:11414-11420; Moss, Proc. Natl. Acad. Sci. USA 1996; 93:11341-11348; Paoletti, Proc. Natl. Acad. Sci. USA 1996; 93:11349-11353; Pennock et al., Mol. Cell. Biol. 1984; 4:399-406; Richardson (Ed), Methods in Molecular Biology 1995; 39, “Baculovirus Expression Protocols,” Humana Press Inc.; Smith et al. (1983) Mol. Cell. Biol. 1983; 3:2156-2165; Robertson et al., Proc. Natl. Acad. Sci. USA 1996; 93:11334-11340; Robinson et al., Sem. Immunol. 1997; 9:271; and Roizman, Proc. Natl. Acad. Sci. USA 1996; 93:11307-11312.
DISCUSSION
The embodiments of the present disclosure encompass the use of isolated polypeptides derived from the termite R. flavipes alone, or in combination, in systems for the in vitro conversion of biomaterial containing a lignin component to a fermentable product useful as a source of a biofuel. Accordingly, the embodiments of the present disclosure encompass compositions comprising combinations of termite-derived enzymes, such as but not limited to, an endoglucanase, an exoglucanase, a laccase, and a β-glucosidase and/or expressible nucleic acid elements encoding such enzymes, and in vitro methods of use thereof for converting a wood (lignocellulose) source to sugar and other moieties. It is well known that free glucose may then, for example, be fermented to ethanol for use as a bio-fuel.
Lignocellulose Processing by Termites
The biologically mediated degradation of lignocellulose into fermentable or otherwise utilizable sugars is a multistep process that requires many types of enzymes. The first step of lignin depolymerization enables hemicellulose degradation. Lignin breakdown requires oxygen, which is supported by evidence that termite guts are not completely anaerobic environments. Lignin degradation and/or modification have been documented in termite guts (see, for example: Esenther & Kirk (1974) Ann. Entom. Soc. Am. 67: 989-991; Breznak & Brune (1994) Ann. Rev. Entomol. 39: 453-487; Brune et al., (1995) Appl. Environ. Microbiol. 61: 2681-2687). Although no enzymes responsible for termite lignin oxidation have been conclusively identified, enzymes such as the laccases and peroxidases are known to degrade lignin in fungi. Other relevant enzymes may include, but are not limited to, those involved in xenobiotic detoxification, such as alcohol dehydrogenase, catalase, superoxide dismutase, cytochrome P450, epoxide hydrolase, reductase, glutathione-S-transferase, esterase, and the like. Over 25 candidate termite genes associated with lignin degradation encoding for such as a salivary laccase, have been identified from R. flavipes through the sequencing work as encompassed in the present disclosure.
The second step in the conversion of wood to a fermentable product is hemicellulose degradation, is important for making cellulose accessible for depolymerization. Complete biodegradation of hemicellulose requires the combined activity of endo- and exo-β-1,4-xylanases, β-xylosidases, α-arabinofuranosidases, α-uronidases, and esterases such as acetylxylan esterase, ferulic acid esterase, and p-coumaric acid esterase. Specific enzymatic conversions are recognized as follows: (1) endo-xylanases hydrolyze the β-1,4-xylose linkages in the xylan backbone; (2) exo-xylanases hydrolyze reduced β-1,4-xylan linkages releasing xylobiose; (3) β-xylosidases act on xylobiose to liberate xylose and other short-chain oligosaccharides; (4) α-arabinofuranosidases hydrolyze terminal non-reducing α-arabinofuranose from arabinoxylans; (5) α-uronidases release α-glucuronic, α-mannuronic and α-galacturonic acids; and (6) esterases hydrolyze phenolic ester bonds, namely those associated with acetyl xylans, ferulic acid xylans and p-coumaric xylans. Several esterases and over 50 candidate termite and symbiont hemicellulase genes from glycosyl hydrolase families 2, 3, 5, 10, 11, 26, and 43 have been identified from R. flavipes through the screening of libraries, as described in the present disclosure.
The third step, cellulose depolymerization, requires the action of three primary enzymes: endo-β-1,4-glucanases, exo-β-1,4-glucanases, and β-glucosidases. Complete or nearly complete hydrolysis of cellulose typically requires synergistic collaboration by each of these three types of cellulases. Specific enzymatic conversions include: (1) endoglucanases that hydrolyze β-1,4-glycosyl linkages in the primary cellulose backbone, releasing glucose, cellobiose, cellotriose or other longer oligomers, (2) exoglucanases or “cellobiohydrolases” that target the terminal regions of polymeric chains to liberate either glucose or cellobiose, and (3) β-glucosidases hydrolyze cellobiose and cellotriose to liberate glucose monomers. In R. flavipes, over 55 candidate termite and symbiont cellulase genes from glycosyl hydrolase families 1, 7, 9, and 45 have been identified from R. flavipes through sequencing of the EST libraries of the present disclosure. The biological degradation/depolymerization of lignocellulose, therefore, is a highly complex process that involves many diverse enzymes. Because of the complexity of these enzyme systems, it has generally been considered impractical to characterize lignocellulose degradation from termites by enzyme biochemistry alone.
cDNA Libraries and Sequence Statistics
Two R. flavipes cDNA libraries were initially created, one representing symbiont-free termite gut tissue, and the other a combination of prokaryotic and eukaryotic hindgut symbionts. A total of 10,610 high-quality Expressed Sequence Tags (ESTs) were generated from the two libraries. The 5,871 ESTs produced from the termite gut library were assembled into 875 contigs and 2,169 singlets to produce 3,044 putatively unique transcripts. In the symbiont EST database, a contig assembly performed using the same parameters identified 358 contigs and 3,153 singlets (for a total of 3,511 putative transcripts). Similarity searches showed that a high proportion of the transcripts from both libraries (38% and 48%, respectively) did not produce significant matches in the NCBI nr database
The majority of the sequences generated from the termite gut tissue libraries had sequence similarity to genes previously identified from insects or from other invertebrates. The analysis also revealed a number of gut library sequences that may be attributed to residual endosymbiotic protists within the guts that had not been completely cleared during the dissection and cleaning process.
Similarly, the symbiont-specific library contained a small fraction of transcripts matching insect and invertebrate sequences. Despite this contamination, more than 80% of the annotated sequences from the symbiont-specific EST library were found to be most similar to predicted proteins from microbes. BLAST analyses demonstrated a significant difference between the two libraries and emphasized the utility of the two libraries for novel gene discovery.
Glycosyl Hydrolase Sequence Composition
The EST databases described in the preceding section were searched for cellulases, hemicellulases, β-glucosidases, and all other carbohydrate-active, enzyme-coding sequences as listed in the CAZy database. The CAZy database maintained by the CNRS-Universites Aix-Marseilles 1 & 11 describes the families of structurally-related catalytic and carbohydrate-binding modules (or functional domains) of enzymes that degrade, modify, or create glycosidic bonds.
For the termite gut library, 298 total clones were compiled that corresponded to 127 potential transcripts and 42 different carbohydrate-active domains. The symbiont library revealed 261 clones associated with carbohydrate catabolism, composing 116 unique sequences that included 26 different carbohydrate-active domains. In the host and symbiont databases, the majority of recognized protein domains corresponded to glycoside hydrolase families (GHFs), including various cellulases, hemicellulases, α-carbohydrolases, and chitinases.
Regarding cellulases, over 55 candidate termite and symbiont cellulase genes from GHF 1 (β-glucosidase), 7 (exoglucanase), 9 and 45 (endoglucanase) have been identified. With respect to hemicellulases, over 50 candidate termite and symbiont hemicellulase genes from glycosyl hydrolase families 2 (β-1,4-galactosidase/mannosidase/glucuronidase), 3 (β-1,4-xylosidase), 5 (β-1,4-endoxylosidase), 10 (β-1,3-endoxylanase), 11 (β-1,4-xylanase), 26 (β-1,3-xylanase), and 43 (β-1,3-endoxylosidase) now have been identified.
Lignase, Antioxidant, and Detoxification Genes
For the termite gut library, a total of 49 enzyme-coding genes were identified that could have a role in lignin degradation and/or possibly protection from toxic lignin degradation products. The putative lignases identified include laccases, peroxidases, and glutathione peroxidases. This identification of candidate lignase genes also agrees with earlier biochemical findings showing various degrees of modification of lignin, mono-lignol, and/or phenolic compounds in the guts of diverse termites, including R. flavipes. Also, numerous genes encoding detoxification and antioxidant enzymes (cytochrome P450, esterases/carboxylesterases, glutathione-S-transferase, alcohol dehydrogenases, epoxide hydrolase, glutathione transferase, superoxide dismutase, catalase, and alcohol dehydrogenase) were identified from sequencing of the EST libraries of the present disclosure. Because lignin degradation is known to result in free radical generation, the expression of antioxidant enzymes in the R. flavipes gut provides supporting evidence of lignin degradation.
Production and Characterization of Recombinant Lignocellulases
Three recombinant digestive enzymes were obtained from cloned R. flavipes genes. These enzymes include three salivary gland-expressed genes identified through cDNA library sequencing. The first protein, Cell-1, is an endoglucanase; it was produced in both baculovirus-infected moth larvae (C-PERLTM system) and E. coli (PETTM system; Novagen). The recombinant Cell-1 enzyme is more active toward the model substrate carboxy-methyl (CM) cellulose when the enzyme is expressed using the baculovirus/insect-expressed form than when in the E. coli-expressed form, as shown in FIG. 33A. While not wishing to be bound by any one theory, the greater Cell-1 activity of the enzyme expressed in the baculovirus system is likely attributable to more correct post-translational processing of the protein, including the glycosylation state thereof, by insect cells. The second protein, a β-glucosidase, shows very strong activity toward its natural substrate cellobiose, as shown in FIG. 33B. The third recombinant protein, a salivary laccase potentially involved in lignin degradation, showed strong activity toward the model laccase substrates pyrogallol, as shown in FIG. 33C, guaiacol, dimethoxyphenol, and syringaldazine, and ABTS.
Four esterase genes were characterized for esterase gene expression, isoform composition, subcellular distribution, and activity across the gut of a lower termite. Analyses of translated amino acid sequences revealed one sequence (RfEst1) with significant sequence similarity to insect juvenile hormone (JH) esterases, and three esterases (RfEst2, RfEst 3 and RfEst 4) with similarities to fungal phenolic acid esterases. Gene expression studies revealed ubiquitous midgut expression for all four genes, significant hindgut expression for RfEst1 and RfEst4, and generally lowest overall expression in the foregut/salivary gland.
Non-denaturing PAGE of the esterases revealed that, in relation to isoform composition outside the gut, there is greater isoform diversity inside the gut. Moreover, in agreement with gene expression findings, highest isoform diversity and total esterase activity both occurred in the midgut. With respect to substrate specificity, α-naphthyl esters with aliphatic chain lengths of three (propionate) and four (butyrate) carbons were metabolized at the greatest rates, followed by the two carbon (acetate) ester. Additionally, of the three subcellular fractions investigated by esterase native PAGE (mitochondrial, cytosolic and microsomal), all showed expression of a number of isoforms. Finally, a substantial band of soluble esterase activity was observed in hindgut lumen supernatant, suggesting that esterases and other enzymes can be readily transferred from donor to recipient termites by proctodeal trophallaxis.
These findings indicate that the esterase genes of the present disclosure, and corresponding enzyme activities, are encoded in the termite genome, and are specifically expressed by gut and/or salivary gland tissue, rather than being derived from gut endosymbionts. The results further suggest hormone and hemicellulose hydrolysis occurs in the termite gut.
Juvenile Hormone (JH) Esterases and Semiochemical/Hormone Processing.
JH esterases are members of the carboxylesterase family. They are typically synthesized in the fat body and secreted into hemolymph where they catabolize JH with high affinity (Goodman & Granger, 2005 (eds.) Comprehensive molecular insect science, Vol. 3, Elsevier-Pergamon, New York, pp. 319-408). In termites, JH has established links to worker-to-soldier caste differentiation (Park & Raina (2004) J. Insect Physiol. 50: 561-566; Park & Raina (2005) J. Insect Physiol. 51: 385-391; Mao et al., (2005) Ann. Entomol. Soc. Am. 98: 340-345; Elliott & Stay (2008) Gen. Comp. Endocrinol. 152: 102-110; Cornette et al., (2008) J. Insect Physiol. 54: 922-930) and vitellogenesis in adult female reproductives (Elliott & Stay (2007) Gen. Comp. Endocrinol. 152: 102-110; Cornette et al., (2008) J. Insect Physiol. 54: 922-930).
With respect to hormonal processing and caste regulation, previous sequencing from an R. flavipes gut library revealed a number of endogenous (i.e. termite-derived) genes encoding enzymes that participate in both JH biosynthesis and degradation. JH biosynthetic genes have been identified from the R. flavipes digestive tract. JH degradation genes, also from the gut, include P450s, epoxide hydrolases, and the putative JH esterase RfEst1.
The RfEst1 nucleotide sequence (SEQ ID NO.: 26) provided by the present disclosure shares significant translated identity across its entire length with several putative insect JH esterases, including an RNAi-validated JH esterase from the honey bee A. mellifera (Mackert et al., (2008) Comp. Biochem. Physiol. B Biochem. Mol. Bio. 150: 33-44). The RfEst1 protein shares several sequence features in common with other putative JH esterases. These common features include a signal peptide (MAETVTVTVAQ (SEQ ID NO.: 44), distinct JH esterase catalytic esterase motifs, such as a catalytic triad (G-SAG, E/D, and G-H-D-), and two putative substrate recognition motifs (RF, DQ) (Campbell et al., 2001 Insect Biochem. Mol. Bio. 31, 513-20; Goodman and Granger, 2005 in Gilbert et al., (eds.) Comprehensive molecular insect science, Vol. 3, Elsevier-Pergamon, New York, pp. 319-408, Munyiri & Ishikawa, (2007) Insect Biochem. Mol. Bio. 37: 497-505; Mackert et al., (2008) Comp. Biochem. Physiol. B Biochem. Mol. Bio. 150: 33-44).
Phenolic Acid Esterases
Phenolic acid esterases, also known as feruloyl, coumaryl or cinnamoyl esterases play roles in hemicellulose and pectin depolymerization by cleaving ester bonds that connect ferulic and coumaric acids to hemicellulose and pectin sugar monomers (Saha (2003) J. Indust. Microbiol. Biotechnol. 30: 279-291; Crepin et al., (2004) Appl. Microbiol. Biotechnol. 63: 647-652; Anderson & Akin (2008) J. Ind. Microbiol. Biotechnol. 35: 355-366, 2008; Benoit et al., (2008) Biotechnol. Letters 30: 387-396). Of these two potential substrates, hemicellulose is a significant component of the termite diet. Hemicellulose, also referred to as xylan, is a complex β-linked sugar polymer that composes approximately 25% of lignocellulose and represents a significant obstacle to cellulose digestion by cellulolytic organisms. Phenolic acid esterases catalyze a critical step in cellulose and hemicellulose depolymerization by dissociating both polymers from each other and lignin, making each polymer more accessible to hemicellulase and cellulase enzymes.
“General” esterases are a large group of insect esterases that are typically active toward naphthyl ester substrates. Accordingly, the isoforms identified by native PAGE analysis of the present disclosure can be classified as general esterases (Oakeshott et al., (2005) in Gilbert et al., (eds.) Comp. Mol. Insect. Sci. Vol. 5, Elsevier-Pergamon, New York, pp. 309-382), and possibly permethrin esterases (Valles et al., (2001) Insect Biochem. Mol. Biol. 31: 715-725). Aside from roles in lipid and xenobiotic metabolism, the roles of insect general esterases remain mostly undefined (Oakeshott et al., 2005). While not wishing to be bound by any one theory, general esterases from the termite gut may participate in hemicellulose depolymerization by acting as phenolic acid esterases.
Little ferulic/phenolic acid esterase activity has been reported from animals and insects, although a large number of such activities have been identified previously from various microbial sources (reviewed by Crepin et al., (2004) Appl. Microbiol. Biotechnol. 63: 647-652; Benoit et al., (2008) Biotechnol. Letters 30: 387-396). In amino acid alignments of two A. niger fae with the four translated termite esterases RfEst1, RfEst2, RfEst3 and RfEst4 (SEQ ID NOs.: 27, 29, 31, and 33, respectively) of the present disclosure, none showed statistically significant Blast homology; however, faeA does align at key positions with the short 20-23 kDa peptides encoded by RfEst2, RfEst 3 and RfEst 4 (SEQ ID NOs.: 29, 31, and 33, respectively), as shown in FIGS. 1A and 1B. In particular, five amino acids show 100% conservation across the alignment, and there is some conservation around the faeA catalytic site.
The termite esterases RfEst2, RfEst3 and RfEst4 are smaller proteins and, based on their lack of N-terminal homology with faeA, they may be subunits of larger functional multimeric proteins. A. niger faeA shows preferential activity toward hemicellulose side-chain esters (de Vries et al., (2002) Biochem. J. 363: 377-386), the termite diet is largely composed of hemicellulose, and there is a large complement of hemicellulase genes encoded within the R. flavipes gut transcriptome. Additional information supporting that phenolic acid esterases exist in the R. flavipes gut is the significantly elevated esterase activity toward α-naphthyl butyrate, indicating that RfEst2, RfEst3 and RfEst4 (SEQ ID Nos.: 26, 28, 30, and 32, respectively) encode termite-derived phenolic acid esterases.
Phenolic acid esterases have been identified from gut symbiotic bacteria of higher termites including from Clostridium xylanolyticum, a bacterial gut symbiont of the grass feeding higher termite Tumilitermes pastinator. Clostridium bacteria have also been sampled from R. flavipes. However, there has been no evidence suggesting that symbiotic bacteria of lower termites possess lignocellulolytic abilities (Watanabe & Tokuda (2007) Nature 394: 330-331). Other termite fungal symbiont esterases have been identified (Sreerama & Veerabhadrappa, (1993) Int. J. Biochem. 25: 1637-1651); however, their actual substrates are unknown, and they represent less than 5% of total gut esterase activity from their host termite. The results of the present disclosure do not indicate microbial origins for the four R. flavipes esterase genes RfEst1, RfEst2, RfEst3 and RfEst4, based on: (1) the genes were identified from a termite gut library substantially free of symbiont-derived nucleic acid sequences, (2) the genes were not sampled from a hindgut symbiont library, and (3) esterase activity is clearly abundant in symbiont-free termite gut regions.
Cell-1 is a host-derived β-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Embodiments of the present disclosure provide 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. Km and Vmax values for BEVS-expressed Cell-1 against the model substrate CMC were 1.496% w/v and 1.469 pmol/min/mg. The BEVS-expressed enzyme has activity comparable to the native enzyme, is optimally active around neutral pH and 50° C.-60° C., is inhibited by EDTA, and displays enhanced activity up to 70° C. in the presence of CaCl2. It is contemplated that GHF9 or a variant thereof may be combined with enzymes such as, but not limited to, host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1β-glucosidases, phenol-oxidizing laccases, and others.
cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Using a baculovirus-insect expression system, the two isoforms were functionally expressed and purified to near homogeneity. Both isoforms showed identically strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by RfLacA was also observed. These findings provide evidence that R. flavipes gut laccases are host-derived, produced in the salivary gland, secreted into the foregut, and involved in lignocellulose digestion.
Two apparent laccase isoform-coding cDNAs were sequenced from the termite R. flavipes; they were greater than 99% similar at the nucleic and amino acid levels. Both translated amino acids contain secretory signal peptides. This finding, in combination with gene expression and phenoloxidase activity in salivary gland tissue, demonstrates that laccases are secreted from the salivary gland into the R. flavipes digestive tract.
Using a baculovirus-insect expression system, recombinant versions of the RfLacA and RfLacB isoforms were functionally expressed and purified to near homogeneity. While both isoforms are clearly laccases based upon sequence homology and activity profiles, they are also clearly distinct from other laccases. In particular, RfLacA and RfLacB both (i) have a higher optimal pH of 7.0-7.5 that is within the known pH range of the R. flavipes gut (Brune et al., (1995) Appl. Environ. Microbiol. 61: 2681-2687), and (ii) apparently lack a T1 copper, and (iii) require hydrogen peroxide for activity. In addition, both laccases were unable to oxidize the non-specific laccase substrate ABTS, and the diagnostic substrate syringaldazine (Harkin et al., (1974) Mycologia 66: 469-476). Finally, RfLacA responds differently than other laccases to known laccase inhibitors, showing greater EDTA inhibition than most laccases and also showing enhanced activity in the presence of other usual inhibitors.
Both laccases showed good catalytic activity against the lignin monomer sinapinic acid, and against the four phenolic substrates hydroquinone, pyrocatechol, 2,6-DMP, and pyrogallol. Finally, experimental results showing modification of lignin alkali by RfLacA provide a parallel line of evidence showing activity against lignin itself. These findings suggest that, unlike many known insect laccases, RfLacA and RfLacB are not involved in integument formation and support that the two laccase isoforms play a role in lignocellulose-related phenol oxidation in the termite gut.
The data of the present disclosure verify that R. flavipes laccases are polymorphic (exist in at least two isoforms), evolutionarily distinct, host-derived, produced in secretory tissue, active on lignin, and do not participate in integument formation. These data also support RfLacA and RfLacB isoforms as new types of copper-limited white laccases, and that they are involved in lignocellulose-related phenol oxidation in the termite gut.
Relation to Previous Studies on Termite Esterase Biochemistry
Esterase biochemistry has been studied in several termite species, including R. flavipes. Sreerama & Veerabhadrappa (1991) Int. J. Biochem. 21: 833-844; (1993) Int. J. Biochem. 25: 1637-1651) provided the evidence of esterase activity in the termite gut. These studies investigated midgut and fungal symbiont esterases from the higher termite Odontotermes horni. Two approximately 78 kDa midgut esterases were purified to homogeneity by column chromatography; upon treatment with β-mercaptoethanol, each protein was split into two subunits of approximately 40 kDa. Both termite midgut esterases showed optimal catalytic activity at pH 7, and greater activity toward α-naphthyl acetate than propionate or butyrate.
Sreerama & Veerabhadrappa also purified esterases from the Odontotermes horni fungal symbiont Xylaria nigripes. Here, four esterases ranging in size from about 85 kDa to about 88 kDa were purified to homogeneity and found to have considerably different biochemical and catalytic properties than the endogenous midgut esterases. Starvation studies revealed little impact on host and symbiont esterase activity, while distribution studies revealed that more than 98% of total esterase activity was host-derived. These studies suggested little importance for fungal symbionts in the hydrolytic metabolism of carboxylester substrates in Odontotermes horni, which agrees with the identification of esterase genes from a symbiont-free R. flavipes gut cDNA library, and identification of esterase gene expression and activity from gut regions that do not house symbionts.
Other studies have examined esterases in other termite species, from either the hemolymph or whole-body preparations. Wyss-Huber (1981) (Insectes Soc. 28: 71-86) investigated hemolymph-soluble esterases in the higher and a lower termite species Macrotermes subhyalinus and Zootermopsis angusticollis, and showed caste- and phenotype-specific native PAGE banding patterns in both species, supporting a role for possible hemolymph JH esterases in caste differentiation and/or caste homeostasis. Ruvolo-Takasusuki & Collett (2000) (Biochem. Genet. 38: 367-375) investigated esterases from whole-body preparations of different castes of the higher termite Nasutitermes globiceps. Since whole-body preparations were used, it was not possible to determine which N. globiceps esterases, if any, are expressed in the gut. However, a diversity of esterase isoforms was found by native PAGE.
The present disclosure, therefore, encompasses a series of isolated nucleic acid molecules, the nucleotide sequences of which encode for enzymes of the gut of the termite R. flavipes that probably are associated with the digestion of lignocellulose by the insect. The isolated nucleic acid molecules of the disclosure have been operably inserted into expression vector systems and expressed in two types of host, namely a prokaryotic (bacterial) cell system, and a eukaryotic (insect cell) system. The expressed polypeptide products were characterized as to their respective enzymic activities, the data of which further showed that, in the termite, these enzymes probably have a role in lignocellulose digestion. It is contemplated that the isolated enzymes of the disclosure may be expressed from expression vectors transfected into a host cell system, whereby their activity (as found in the gut of the parent termite) may be substantially replicated in an in vitro system. It is also contemplated that an operable system of the expressed enzymes may be reconstructed in vitro to provide a method of converting a lignocellulose-containing plant material to a fermentable breakdown product, and in particular to sugars such as, but not limited to, glucose.
The present disclosure, therefore, further encompasses the use of the isolated polypeptides from the termite R. flavipes alone, or in combination, in systems for the in vitro reduction of biomaterial containing a lignocellulose component to a product that may be fermented to provide a source of a biofuel. Accordingly, the present disclosure further includes data that shows that a wood (lignocellulose) source can provide glucose moieties when treated in vitro with a combination of an endoglucanase, an exoglucanase, a laccase, and a β-glucosidase. It is well known that free glucose may be fermented to such as ethanol for use as a bio-fuel.
Accordingly, the present disclosure provides isolated nucleic acid molecules derived from the gut of the termite R flavipes, recombinant nucleic acid molecules comprising a vector and an isolated heterologous nucleic acid molecule operably inserted therein, whereby, when transformed into an appropriate host cell system, the heterologous nucleic acid sequence is expressed as a polypeptide having an activity similar to that when expressed in the gut of the termite R. flavipes. It is within the scope of the disclosure for the recombinant nucleic acid molecules to comprise more than one heterologous nucleic acid molecule such that more than one polypeptide may be expressed by the host system. The expressed polypeptides may be substantially purified, or used in a substantially unpurified form, to be admixed with a lignocellulose source to be converted to a fermentable product such as a sugar or a mixture of sugars.
One aspect of the present disclosure, therefore, encompasses methods of converting a lignified plant material to a fermentable product, the method comprising the steps of: (a) obtaining a series of isolated polypeptides of a termite, wherein the series of polypeptides cooperate to convert a plant lignocellulose to a fermentable product; and (b) incubating the series of polypeptides with a source of lignified plant material, under conditions allowing the polypeptides to cooperatively produce a fermentable product from the lignified plant material.
In embodiments of this aspect of the disclosure, the isolated polypeptides can be derived from the termite Reticulitermes flavipes. 
In embodiments of this aspect of the disclosure, the isolated polypeptides of the series of isolated polypeptides can be selected from the group consisting of: an endoglucanase, an exoglucanase, a laccase, an esterase, and a glucosidase. In one embodiment of this aspect of the disclosure, the series of the isolated polypeptides comprises an endoglucanase, an exoglucanase, a laccase, an esterase, and a glucosidase.
In the various embodiments of this aspect of the disclosure, the isolated polypeptides of the series of isolated polypeptides can be recombinant polypeptides, where each polypeptide is expressed from an expression vector of a recombinant expression system, and wherein the recombinant expression system is selected from a eukaryotic cell-based system and a prokaryotic cell-based system.
In one embodiment of the disclosure, the expression vector is a baculovirus expression vector and the recombinant expression system is a eukaryotic cell-based system.
In the various embodiments of the disclosure, the series of isolated polypeptides comprise an endoglucanase having an amino acid sequence having about 75% sequence identity with the amino acid sequence SEQ ID NO.: 2.
In one embodiment of the disclosure, the endoglucanase can comprise the amino acid sequence SEQ ID NO.: 2.
In the various embodiments of the disclosure, the series of isolated polypeptides comprises an exoglucanase having an amino acid sequence having about 75% sequence identity with the amino acid sequence SEQ ID NO.: 4.
In one embodiment of the disclosure, the series of isolated polypeptides comprises an exoglucanase having the amino acid sequence SEQ ID NO.: 4.
In the various embodiments of the disclosure, the series of isolated polypeptides comprises a laccase having an amino acid sequence having about 75% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NOs.: 8, 10, 12, 15, 17, 19, 21, and 23.
In one embodiment of the disclosure, the series of isolated polypeptides comprises a laccase having the amino acid sequence selected from the group consisting of: SEQ ID NOs.: 8, 10, 12, 15, 17, 19, 21, and 23.
In the various embodiments of the disclosure, the series of isolated polypeptides comprises an esterase having an amino acid sequence having about 75% sequence identity with the amino acid sequence selected from the group consisting of: SEQ ID NOs.: 27, 29, 31, and 33.
In one embodiment of the disclosure, the series of isolated polypeptides comprises an esterase having the amino acid sequence selected from the group consisting of: SEQ ID NOs.: 27, 29, 31, and 33.
In one embodiment of the disclosure, the series of isolated polypeptides comprises a glucosidase having the amino acid sequence SEQ ID NO.: 6.
In the various embodiments of the disclosure, the fermentable product may comprise at least one carbohydrate selected from the group consisting of: a glucose, a mannose, a xylose, a galactose, a rhamnose, an arabinose, a glucuronic acid, a mannuronic acid, and a galacturonic acid.
In the various embodiments of the disclosure, the fermentable product may comprise glucose.
Another aspect of the present disclosure encompasses systems for producing a fermentable product from a lignified plant material, wherein the system may comprise at least two isolated polypeptides selected from the group consisting of: an endoglucanase, an exoglucanase, a laccase, an esterase, and a glucosidase of the termite Reticulitermes flavipes and wherein the at least two isolated polypeptides can cooperate to convert a constituent of the lignified plant material to a fermentable product or a precursor thereof.
In embodiments of this aspect of the disclosure, the nucleotide sequence encoding the endoglucanase may hybridize under high stringency conditions to a nucleotide sequence according to SEQ ID NO.: 1, the nucleotide sequence encoding the exoglucanase may hybridize under high stringency conditions to a nucleotide sequence according to SEQ ID NO.: 3, the nucleic acid molecule encoding the laccase may hybridize under high stringency conditions to a nucleotide sequence selected from the group consisting of: SEQ ID NOS.: 7, 9, 11, 13, 14, 16, 18, 20, and 22, the nucleic acid molecule encoding the esterase may hybridize under high stringency conditions to a nucleotide sequence selected from the group consisting of: SEQ ID NOS.: 26, 28, 30, and 32, and the nucleotide sequence encoding the glucosidase may hybridize under high stringency conditions to SEQ ID NO.: 5. In embodiments of this aspect of the disclosure, each of the isolated nucleic acid molecules thereof may be operably inserted into an expression vector.
Still another aspect of the disclosure encompasses a recombinant cell comprising an isolated nucleic acid molecule hybridizing under high stringency conditions to a nucleotide sequence encoding a polypeptide selected from the group consisting of: an endoglucanase, an exoglucanase, a laccase, an esterase, and a glucosidase of the termite Reticulitermes flavipes, wherein the nucleotide sequence encoding the endoglucanase is according to SEQ ID NO.: 1, the nucleotide sequence encoding the exoglucanase is according to SEQ ID NOS.: 3, the nucleic acid molecule encoding the laccase is selected from the group consisting of: SEQ ID NOS.: 7, 9, 11, 13, 14, 16, 18, 20, and 22, the nucleic acid molecule encoding the esterase is selected from the group consisting of: SEQ ID NOS.: 26, 28, 30, and 32, and the nucleotide sequence encoding the glucosidase is according to SEQ ID NO.: 5.
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.
It should be emphasized that the embodiments of the present disclosure, particularly, any “preferred” embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure, and protected by the following embodiments.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere.
It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt % to about 5 wt %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. The term “about” can include±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%, or more of the numerical value(s) being modified.
EXAMPLES
Example 1
Termites: Reticulitermes flavipes colonies were collected from Gainesville, Fla., and maintained in sealed plastic boxes (30×24×10 cm) in complete darkness (L:D=0:24), at 22° C. and 69% relative humidity. Colonies were maintained without soil for more than six months and provisioned with moist brown paper towels and pine wood shims. The identity of colonies as R. flavipes was verified by a combination of soldier morphology and 16S-mt-rDNA gene sequencing. Worker termites were used in this study because of their significant lignocellulose digestion capability and developmental plasticity. Termites were considered workers if they did not possess any sign of wing buds or distended abdomens, and had pronotal widths wider than mesonotal widths (Laine et al., (2003) Bull. Entomol. Res. 93; 267-378).
Example 2
Esterase gene identification: A number of putative esterase genes were sequenced from a normalized R. flavipes gut cDNA library. Four esterase genes were deduced from three contigs (515, 493 and 275), as well as a single EST (TG—15_C3) representing a near full-length cDNA. The three contigs (515, 493 and 275) were assembled from 4, 4 and 3 individual EST sequences, respectively. The genes are referred to, respectively, as Rf (R. flavipes) Est (esterase) genes 1, 2, 3 and 4, and comprise the nucleotide sequences SEQ ID NOs.: 26, 28, 30, and 32, respectively. The ORF amino acid translations of the four genes RfEst1-4 have the amino acid sequences according to SEQ ID NOs.: 27, 29, 31, and 33, respectively.
Sequence alignments were made under default settings using MEGALIGNTM in the LASERGENETM software package (Madison, Wis.). Signal peptides were determined using PROTEANTM in the Lasergene software package. Glycosylation sites in translated proteins were predicted using an online prediction tool available at (Hamby & Hirst (2008) BMC Bioinformatics 9: 500). The sequence identities of the open reading frames of the four genes are less than 25%, and non-overlapping in their sequence composition, as shown in FIGS. 1A and 1B.
The deduced protein sequences (SEQ ID NOs.: 27, 29, 31, and 33) encoded by the four esterase genes segregate into two groups that include longer (i.e., RfEst1) and shorter open reading frames (i.e., RfEst2, RfEst 3 and RfEst 4). The alignment reveals conserved N-terminal esterase motifs in RfEst1 that are absent in RfEst2, RfEst 3 and RfEst 4, but greater degrees of conservation in the region homologous to the C-terminal end of RfEst1. In particular, beginning at consensus residue 346 there are ten completely conserved amino acids and a conserved G-H-D-esterase catalytic motif across all four esterases.
This RfEst1 group is composed of larger proteins with predicted molecular masses of about 60 kDa. The predicted RfEst1 protein (SEQ ID NO.: 27) has a molecular mass of 59 kDa, an 11 amino acid signal peptide (MAETVTVTVAQ (SEQ ID NO.: 44)), 30 N-glycosylation sites, and shares significant amino acid identity across its entire length with a number of putative insect Juvenile Hormone (JH) esterases, including representatives from the longicorn beetle Psacothea hilaris (GenBank Accession No: BAE94685), the red flour beetle Tribolium castaneum (GenBank Accession No: XP—967137), the honeybee Apis mellifera (GenBank Accession No: AAU81605), and the saw fly Athalia rosae (GenBank Accession No: BAD91555).
The second esterase group comprises RfEst2, RfEst3 and RfEst4 (SEQ ID NOs.: 29, 31, and 33). The deduced amino acid sequences for these three genes are about 18 kDa to about 3 kDa and only share similarity with the JH esterase-like proteins described above at their C-terminal ends. Other than the carboxylesterases/JH esterases noted above, there were no other full-length translated database matches for RfEst2, RfEst 3 and RfEst 4 (SEQ ID NOs.: 29, 31, and 33).
The translated RfEst2, RfEst 3 and RfEst 4 sequences were also aligned with the translated faeA sequence of Aspergillus niger, a lignocellulose digester (GenBank Accession No: Y09330). The G-H-D-catalytic sites in faeA and RfEst2, RfEst 3 and RfEst 4 are 100% conserved. Additionally, there are 5 completely conserved residues among all five proteins, and many other similar regions. There is also a degree of conservation in the N-terminal regions of faeA and RfEst2.
Example 3
Esterases
Quantitative PCR







TABLE 1







qPCR primer sequences for the esterases


RfEst1-4 and the control gene β-actin.













SEQ


Primer
Alternate

ID


Name
Name
Sequence (5′-3′)
NO.:





RfEst1 L
Contig515L-
ATGACAGAAGAGGAGCTTGACC
34



set 4




 


RfEst1 R
Contig515R-
CAGGAGTTCAAAGTCACTGTCG
35



set4




 


RfEst2 L
Contig493-
GGTTAGGCGTCATTTGTAGAGG
36



1R




 


RfEst2 R
Contig493-
GGCTCATGGTCATCTAAGAACC
37



1L




 


RfEst3 L
Contig275L-
AGAGACGGTTGAAGACTTGGTC
38



set5




 


RfEst3 R
Contig275R-
ACTGATACGTAGTGGCCTGACA
39



set5




 


RfEst4 L
TG_15_C3
GCCAGAATTCAAGCTGCTGT
40



1L




 


RfEst4 R
TG_15_C3
TGTCCTTGTCTTGCTGTGTCTC
41



1R




 


β-actin L
N/A
AGAGGGAAATCGTGCGTGAC
42


 


β-actin R
N/A
CAATAGTGATGACCTGGCCGT
43









The stable expression of the reference gene β-actin across gut regions was validated previously (Zhou et al., (2007) Gene 395: 29-39). qPCR primers were designed with specificity to sequence-unique regions of the target genes, and to produce products in the 100-300 bp size range, were designed using PRIMER3TM (Rozen & Skaletsky (2000) in Krawetz & Misener (eds) Bioinformatics Methods and Protocols, Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386). cDNA from the foregut and salivary gland, the midgut and the hindgut regions served as templates for qPCR. Gut dissections were performed as described in Example 4, below, using RNA lysis buffer (Promega; Madison, Wis.) in place of potassium phosphate. cDNA was synthesized from the total RNA of 25 individual gut regions per experimental replicate. Total RNA and cDNA were obtained using the SV total RNA isolation kit (Promega) and the ISCRIPTTM cDNA Synthesis Kit (Bio-Rad; Hercules, Calif.), respectively, following manufacturer protocols. qPCR reactions contained equal template loadings and proceeded for 30-35 cycles. PCR products were viewed on 1.5% agarose gels, imaged, and the images analyzed densitometrically.
Pooled densitometric analyses from five replicate gels on two colonies, with statistical support (p<0.05), are shown in FIG. 2. All four esterases are expressed in the midgut. RfEst1 is most strongly expressed in midgut and hindgut tissues, but also has foregut/salivary gland expression. The RfEst2 and RfEst 3 esterases are predominantly expressed in the midgut. RfEst4 is expressed mainly in midgut and hindgut tissues, but also has significant foregut/salivary gland expression.
Example 4
Dissections, protein preparation, centrifugation protocols, and protein assays: All manipulations were performed on ice. For tissue localization studies, 25 termite worker guts were removed and dissected into the three regions of foregut/salivary gland, midgut and hindgut. Each gut region preparation was homogenized using a Tenbroeck glass homogenizer in potassium phosphate (0.1M, pH 7.6), and then centrifuged for 15 min at 14,000×g and 4° C. The supernatant was saved for assays and the pellet discarded.
For isolation of subcellular gut protein fractions, the protocol of Kupfer & Levin (1972) Biochem. Biophys. Res. Comm. 47: 611-618 was followed with minor modifications. Fifty whole worker termite guts were homogenized in potassium phosphate as above. The homogenate was centrifuged at 1,000×g and 4° C. for 5 min and the pellet discarded. The supernatant was transferred to a fresh tube and centrifuged at 10,000×g for 10 min and 4° C. A small aliquot of the supernatant was retained and the remainder transferred to a fresh tube. The resulting 10,000×g mitochondrial pellet was washed and re-spun three times in potassium phosphate buffer before being re-suspended in potassium phosphate/0.1% Triton X-100 (Valles et al., (2001) Insect Biochem. Mol. Biol. 31, 715-725). Next, a volume of calcium chloride was added to the post-mitochondrial supernatant to a final concentration of 8 mM. The mixture was inverted several times, left on ice for 5 min, and then centrifuged at 10,000×g for 10 min and 4° C. The resulting supernatant was transferred to a fresh tube and used as the soluble/cytosolic enzyme source. The resulting microsomal pellet was washed and re-spun three times in potassium phosphate buffer before being re-suspended in potassium phosphate/0.1° A Triton X-100 (Valles et al., (2001) Insect Biochem. Mol. Biol. 31, 715-725).
Gut luminal contents were isolated from 100 worker termites by first holding them by the abdomen with soft larval forceps and gentle squeezing to induce defecation. The termite abdomen was then lowered into potassium phosphate buffer, where the fecal material was collected. Symbiont cells and other debris were pelleted by centrifugation for 10 min at 14,000×g and 4° C. and the remaining supernatant used as the enzyme source.
Protein content of protein preparations was estimated by a microplate Bradford assay (Bio-Rad; Hercules, Calif.) using bovine serum albumin as a standard and corresponding buffers as blanks.
Example 5
Statistical analyses: Native PAGE and colorimetric esterase assays were performed on three replicate protein preparations from two termite colonies. qPCR analyses were performed on five replicate RNA isolations and cDNA syntheses from two termite colonies. Statistical analyses consisted of one-way ANOVA followed by Tukey's multiple range test for colorimetric enzyme data and LSD t-tests for quantitative PCR data. With the exception of hindgut colorimetric data, all variances were equal and ANOVAs were verified as being significant before conducting mean separation testing.
Example 6
Distribution of naphthyl acetate esterase activity: Native PAGE was used in combination with hydrolysis of the model esterase substrate α-naphthyl acetate, as shown in FIG. 3. Although a significant proportion of total esterase activity is located in the termite carcass, a diverse number of esterase isoforms and activity are also present in different gut regions. Within gut tissues, naphthyl acetate staining was strongest in the midgut, followed by the foregut/salivary gland, and the hindgut regions. Minor differences in banding patterns occurred between the two termite colonies that were investigated. However, results were consistent across the two colonies. Colony differences were most evident in the foregut/salivary gland, the hindgut, and carcass, and may be the result of genetic variation between the two colonies.
Example 7
Gut esterase activity toward naphthyl esters with varying carbon chain lengths: Activities toward different naphthyl ester substrates were investigated in different regions of the termite gut using native PAGE, as above, the results being shown in FIGS. 4 and 5. Gut esterases are most active toward substrates with side-chains of 3 (α-naphthyl propionate) and 4 (α-naphthyl butyrate) carbons, as well as the 2-carbon substrate α-naphthyl acetate. This result was consistent for all three termite gut tissues, with the exception of the foregut/salivary gland, which did not show strong differences between butyrate (C4) and valerate (C5) substrates (as shown in FIG. 40).
For all three gut tissues, the lowest esterase staining activity occurred with α-naphthyl caprylate (C6). PAGE results were verified statistically using colorimetric microplate assays on the three gut regions, as shown in FIG. 5. Here, all three termite gut regions showed significantly greatest activity toward α-naphthyl propionate (p<0.05). Differences in activity between α-naphthyl butyrate and acetate were not as evident on native PAGE as in activity assays. Thus, the majority/greatest diversity of gut esterase activity in R. flavipes is located in the midgut, and that activity is greatest toward the 3-carbon ester naphthyl propionate. The results from using naphthyl propionate are similar to hemicellulose-degrading ferulic acid esterases that also show preferentially highest activity toward naphthyl propionate.
Example 8
Recombinant β-1,4-endoglucanase: protein expression: BEV system: The cDNA open-reading frame (GenBank Accession No.: AY572862; Zhou et al., (2007) Gene 395:29-39, SEQ ID NO.: 1 (encoding deduced amino acid sequence SEQ ID NO.: 2) was amplified without its signal sequence by PCR. Incorporated into the PCR amplicon were: (i) a heterologous signal sequence modeled after the Bombyx mori hormone bombyxin A-6 (GenBank Accession No.: 100169714 Bbx-a6), (ii) an XbaI restriction site, (iii) a C-terminal 6×His tag, and (iv) a NotI restriction site. These four features were incorporated into the amplicon via the primers: forward, 5′-CTAGTCTAGACTAGATGAAGATACTCCTTGCTATTGCATTAATGTTGTCAACAGTAATGT GGGTGTCAACAGCTGCTTACGACTATAAG-3′ (SEQ ID NO.: 45); reverse, 5′-TTTCCTTTTGCGGCCGCTTAGTGATGATGGTGATGATGCACGCCAGCCTTGAGGAG-3′ (SEQ ID NO.: 46). The PCR amplicon was cloned into the XbaI-NotI sites of the pVL1393 transfer vector.
The resulting plasmid DNA was verified by sequencing and used for the co-transfection with linearized baculovirus DNA (BD Biosciences Pharmigen; San Diego, Calif.) into Sf9 cells. Cells were incubated at 27° C. for 4 days (Passage 0) and the supernatant was collected and used for virus amplification in fresh cell culture (Passage 1). The cell pellet from Passage 0 was tested by Western blotting with anti-His antibody to confirm expression of His-tagged protein. After 2 days, the recombinant virus from Passage 1 was harvested and injected to T. ni larvae as described previously (Liu et al., (2007) in: D. Murhammer (Ed.), Baculovirus and insect cell expression protocols, 2nd ed, Humana Press, Totowa, pp 267-280; Kovaleva et al., (2009) Biotechnol Lett. 31: 381-386). Larvae were orally infected with active pre-occluded baculovirus, harvested in large scale, and stored at −80° C. for later processing.
Recombinant protein was recovered from clarified T. ni homogenates by tandem Ni-IMAC (nickel-immobilized metal affinity chromatography) followed by buffer exchange with Sephadex G-25 chromatography. Protein storage buffer consisted of 0.1 M sodium acetate, 0.15M sodium chloride, 0.5M calcium chloride, and 0.5 mM copper sulfate (pH 5.8). Purity was assessed by SDS-PAGE with Coomassie staining and Western blotting with anti-His tag antibody.
Example 9
β-1,4-endoglucanase gel electrophoresis, deglycosylation assay, and Western blotting: For Coomassie-stained Native PAGE, volumes of supernatant containing 10 μg of total protein were diluted 1:1 with Native PAGE sample buffer (Bio-Rad) and loaded onto native PAGE gels prepared with 7.5% resolving gels and 4% stacking gels. Electrophoresis was conducted in a discontinuous Tris-Glycine running buffer for 1.5 hr at 4° C. Gels were stained as above. For CMC-native PAGE, gels were prepared and run as described above, except that carboxymethyl cellulose (CMC; Sigma) was incorporated into gels at 0.5%. After running, CMC gels were incubated in sodium acetate (0.05M, pH 5.0) and stained with Congo Red as described previously (Nakashima et al., (2002) Cell Mol. Life. Sci. 59: 1554-1560; Zhang et al., (2009) Insect Biochem. Mol. Bio. 39: 516-522).
An approximately 48 kDa band was present in baculovirus-infected larvae, but absent from uninfected and blank virus controls. Following purification and concentration the recombinant Cell-1 protein migrated as a single band at approximately 48 kDa. Preliminary stability tests conducted by incubating the protein at 65° C. or 26° C. for 65 hr revealed no protein degradation, indicating that it is stable in purified form. In addition to endoglucanase activity (the expected activity for Cell-1), exoglucanase and β-glucosidase activities were also investigated using the substrates pNPC and pNPG. All three activities were present in both uninfected and blank-virus-infected T. ni larvae, with exoglucanase and β-glucosidase activity being the strongest. However, only endoglucanase activity remained for purified Cell-1. In agreement with SDS-PAGE results, it was enriched at least 10-fold relative to clarified supernatants of infected larvae.
Analyses with and without the deglycosylation enzyme PNGase-F was negative (as shown in FIG. 5); they did not show changes in migration characteristics for the recombinant Cell-1 protein after PNGase-F treatment. Thus, the baculovirus/insect-expressed Cell-1 protein is properly assembled and retained the expected endoglucanase activity.
Example 10
Colorimetric β-1,4-endoglucanase enzyme assays: Six total substrates were tested: CMC (carboxymethyl cellulose), pNPG (p-nitrophenyl-beta-D-glucopyranoside), pNPC2 (p-nitrophenyl-beta-D-cellobioside), pNPC3 (p-nitrophenyl-beta-D-cellotrioside), pNPC4 (p-nitrophenyl-beta-D-cellotetraoside), and pNPC5 (p-nitrophenyl-beta-D-cellopentaoside). All assay methods were carried out under optimal conditions (Zhou et al., (2007) Gene 395:29-39).
The kinetic constants Km and Vmax were determined in 100 mM sodium acetate buffer (pH 6.5) at 25° C. (FIG. 7). For subsequent characterizations, the 1.5% CMC concentration was used. pH studies were performed by dissolving 1.5% CMC in two different buffer systems: 100 mM sodium acetate (pH 3-6.5) and 100 mM sodium phosphate (pH 7-10.5), as shown in FIG. 8. Thermal stability tests were conducted using 1.5% CMC in 100 mM sodium phosphate at the optimal pH of 7. Temperature-cofactor studies were performed using 1.5% CMC dissolved in 100 mM sodium phosphate (pH 7) alone, plus 4 mM EDTA (final concentration), or plus 10 mM calcium chloride (final concentration). Pre-incubations took place for 0-5 days at 25° C. or 0-60 min at 60° C. or 70° C.; reactions were terminated by the combination of adding stop solution and boiling for 10 min, and then assays were read at 25° C. (FIG. 9). All reported activities are the average of 3-5 independent replicates.
Example 11
Production of Recombinant β-1,4-endoglucanase (Cell-1) in E. coli: R. flavipes Cell-1 was produced in E. coli to enable direct comparisons with the BEVS-expressed enzyme. Cell-1 was heterologously expressed with a heterologous signal peptide and C-terminal histidine tag in E. coli strain BL21 (DE3) pLysS, using the pET26 vector. IPTG induction of recombinant Cell-1 in E. coli at 37° C., 30° C., and 25° C. resulted in production of insoluble protein that readily precipitated after cell lysis and centrifugation. Recombinant Cell-1 was then partially solubilized with IPTG induction at 19° C.
Soluble fractions obtained after 19° C. IPTG induction were subjected to Ni-chromatography for purification. The His-tagged Cell-1 was retained on Ni columns and eluted in a single peak of CMC activity after the introduction of imidazole elution buffer. Active fractions also were assayed against the exoglucanase and β-glucosidase substrates pNPC and pNPG but showed no activity. After purification, pooling, and concentration the active Cell-1 fractions showed an enriched approximately 48 kDa protein band.
Example 12
pH and temperature stability of BEVS expressed β-1,4-endoglucanase Cell-1: Having thermostable enzymes for industrial lignocellulose processing is advantageous by enabling (i) greater activity with less enzyme, (ii) longer processing times due to higher stability, and (iii) increased flexibility for process configurations (Viikari et al., (2007) Adv. Biochem. Eng. Biotechnol. 108: 121-145). Maximal CMC activity was observed between pH 6.5 and 7.5 (FIG. 8). Temperature dependence of CMC activity in sodium phosphate buffer (pH 7) was maximal in the range of 50-60° C. but fairly constant across a wide temperature range (FIG. 10). There is, therefore, optimal activity toward the model substrate CMC around pH 7 and 50-60° C. BEVS-expressed recombinant Cell-1 shows slightly improved temperature stability at neutral pH relative to a number of other recombinant termite endoglucanases.
Example 13
Calcium as a cofactor in β-1,4-endoglucanase Cell-1 hydrolytic activity and temperature stability: The effects of calcium on recombinant Cell-1 activity were investigated using calcium chloride as a calcium source and EDTA as a potential calcium chelator. As determined from incubations conducted over a 5-day period at about 25° C. (FIG. 9), calcium chloride slightly enhanced CMC endoglucanase activity and EDTA was slightly inhibitory. With shorter incubations conducted at higher temperatures of 60° C. and 70° C., the same trends were apparent: calcium chloride stabilized/extended CMC hydrolysis activity over time, while EDTA rapidly reduced temperature stability (FIG. 9).
Example 14
β-glucosidase sequencing and sequence analyses: Original sequence tags were obtained by conventional Sanger sequencing from a host gut cDNA library as described in Tartar et al., ((2009) Biotechnol. Biofuels 2: 25). RfBGluc-1 was represented by eight library clones (GenBank Accession Nos. FL640173, FL639268, FL637619, FL637754, FL639498, FL636506, FL636125, FL635251) that aligned into a single contig (“Contig 771” having nucleotide sequence SEQ ID NO.: 5 comprising an ORF encoding the amino acid sequence SEQ ID NO.: 6). RfBGluc-2 was represented by one clone (GenBank Accession No. FL635576; having nucleotide sequence SEQ ID NO. 47).
The RfBGluc-1 cDNA nucleotide sequence SEQ ID NO.: 5 comprises 75 nucleotides of 5′ untranslated region (UTR) ahead of the ATG start codon, a 1483 bp open reading frame (ORF), and a 3′ UTR of 357 nucleotides after the “taa” termination codon. The 3′ UTR contains a putative polyadenylation signal “aataa” and a terminal poly-A tail. The translated RfBGluc-1 amino acid sequence SEQ ID NO.: 6 comprises contains 495 amino acids and several identifiable motifs, including a 17-amino acid signal peptide MRLQTVCFVIFVTAVFG (SEQ ID NO.: 48), indicating that the mature protein is soluble and secreted. Two motifs likely involved in substrate binding and catalysis are also present; “NEPL” with a GLU(E)190 residue as a proton donor, and “TENG” with a GLU(E)399 as a nucleophilic residue. Finally, two predicted N-glycosylation sites are N259 and N409.
The RfBGluc-2 partial cDNA sequence (GenBank Accession No.: FL635576 (SEQ ID NO.: 47)) is based on a single EST (Tartar et al., (2009) Biotechnol. Biofuels 2; 25)) composed of 496 nucleotides. It has no significant nucleotide identity with the full-length RfBGluc-1 cDNA; however, within a 165 amino acid residue overlap in the middle of their ORFs the translated RfBGluc-2 (SEQ ID NO.: 49) and RfBGluc-1 (SEQ ID NO.: 6) amino acid sequences share 53% identity. A proton donor component of the active site “TGNG” is included in the overlap region, as well as one N-glycosylation site (present in RfBGluc-1 but absent in RfBGluc-2).
Example 15
β-glucosidases-Quantitative PCR: RNA isolations were performed using the SV Total RNA kit (Promega; Madison, Wis.). Two replicate colonies were tested. RNA was isolated from whole gut, whole carcass (remaining body tissues after head and gut removal), and the four individual gut tissues foregut, salivary gland, midgut, and hindgut. cDNA was synthesized from total RNA using the iScript cDNA kit, which included a combination of oligo-dT and random hexamer primers (Bio-Rad; Hercules, Calif.). All PCR was performed in 20 μL volumes using SensiMix SYBR & Fluorescein one-step PCR reagent (Bioline; Taunton, Mass.), 1 μL cDNA template, and 0.5 μM primer concentrations. PCR primer sequences were as follows:









RfBGluc-1



(SEQ ID NO.: 50)



(“21C3 L” = TGCTTCTTCATGGCTCAGAGT;



 



(SEQ ID NO.: 51))



“21C3 R” = TGGTCTCCAGGTTGTGTATCC;



 



RfBGluc-2



(SEQ ID NO.: 52)



(“309K21 L” = GAGAGCTCCTCAACCAACGAT;



 



(SEQ ID NO.: 53))



“309K21 R” = CCTGTAACAAGCACCGGAGTA-3';



 



beta-actin



(SEQ ID NO.: 42)



(Forward = AGAGGGAAATCGTGCGTGAC;



 



(SEQ ID NO.: 43))



Reverse = CAATAGTGATGACCTGGCCGT,



and



 



NADH-dh



(SEQ ID NO.: 56)



(Forward = GCTGGGGGTGTTATTCATTCCTA;



 



(SEQ ID NO.: 57))



Reverse = GGCATACCACAAAGAGCAAAA. 






Whole gut versus carcass comparisons were made by viewing reaction products after 35 cycles of amplification on 2% agarose gels. Relative expression within gut tissues was determined by quantitative real-time PCR (QRT-PCR), using NADH-dh as the reference gene. QRT-PCR data were analyzed by the 2−ΔCT ΔCT method (Livak & Schmittgen (2001) Methods 25: 402-408), with normalization to the midgut. Statistical analysis of qRT-PCR data was performed by analyzing ΔCT data for all tissues. Mean separations were made using the Tukey HSD method (SAS software; Cary, N.C.).
Example 16
Recombinant β-glucosidase protein production and purification: Of three β-glucosidase-encoding cDNA clones, the entire RfBGluc-1 ORF was present in, and amplified from, clone TG—21_C3. Restriction sites and a C-terminal nucleotide sequence plus 6 histidine residues were introduced into the amplicon using the following primers: forward, 5′-GTCGACATGAGGTTACAGACGGTTTGC-3′ (SEQ ID NO.: 56); and reverse, 5′-CTGCAGTTAGTGATGATGGTGATGATGGTCTAGGAAGCGTTCTGGAA-3′ (SEQ ID NO.: 57). This PCR amplicon, which encoded RfBGluc-1 ORF sequence (amino acids 1 to 495; SEQ ID NO.: 6)) and a hexahistamine tag at the C-terminus, was cloned into Sal1-Pst1 site of pFastBac1 transfer vector (Invitrogen; Carlsbad, Calif.).
Example 17
β-Glucosidaseenzyme Activity, Optimization, and Inhibition Assays







TABLE 2







Activity of recombinant RfBGluc-1 β-glucosidase towards synthetic nitrophenol (NP)


substrates. Assays were read kinetically at 420 nm. Means ± standard errors are based on


3-5 independent replicates.














Sugar








Moiety/
Linkage/

Km [S]
Vmax
Activity


Substrate
Position
isomer
r2*
(mM)
(μmol/min/mg)
(%)**
















pNP-βD-
Glucose/p
β/D
0.99
1.66 ± 0.07
22.92 ± 0.14
100


Glucopyranoside


pNP-βD-
Cellotriose/p
β/D
0.99
1.63 ± 0.50
22.56 ± 5.56
98


Cellotrioside


pNP-βD-
Cellotetraose/p
β/D
0.99
0.59 ± 0.01
16.64 ± 0.08
73


Cellotetraoside


pNP-βD-
Cellobiose/p
β/D
0.99
1.56 ± 0.04
13.84 ± 0.35
60


Cellobioside


pNP-βD-
Fucose/p
β/D
0.99
0.90 ± 0.07
12.56 ± 1.54
55


Fucopyranoside


oNP-βD-
Xylose/o
β/D
0.99
7.57 ± 1.17
 8.77 ± 1.35
38


Xylopyranoside


oNP-βD-
Glucose/o
β/D
0.99
1.58 ± 0.19
 4.20 ± 0.13
18


Glucopyranoside


mNP-αD-
Galactose/m
α/D
ND
ND
ND
0


Galactopyranoside


pNP-αD-
Galactose/p
α/D
ND
ND
ND
0


Galactopyranoside


pNP-βD-
Galactose/p
β/D
ND
ND
ND
0


Galactopyranoside


pNP-βD-
Xylose/p
β/D
ND
ND
ND
0


Xylopyranoside


pNP-βL-
Arabinose/p
β/L
ND
ND
ND
0


Arabinopyranoside


pNP-βD-
glucuronic
β/D
ND
ND
ND
0


Glucuronide
acid/p


oNP-βD-
Mannose/o
β/D
ND
ND
ND
0


Mannopyranoside





*Correlation coefficients for Lineweaver-Burke plots;


**Percent activity rankings based on Vmax;


ND, Activity not detectable.













TABLE 3







Activity of recombinant RfBGluc-1 β-glucosidase


towards natural substrates. Assays were read as


endpoints at 505 nm using glucose detection reagent.


Means ± standard errors are based on 3-5 independent replicates.













Km [S]
Vmax
Activity


Substrate
r2*
(mM)
(μmol/min/mg)
(%)**














Cellobiose
0.99
1.44 ± 0.14
638.0 ± 39.0 
100


Salicin
0.99
34.30 ± 14.75
278.0 ± 11.00
44


Laminaribose
0.99
0.63 ± 0.02
218.1 ± 4.17 
34


Carboxymethyl cellulose
0.99
52.58 ± 24.35
9.98 ± 4.70
2


Microcrystalline cellulose
ND
ND
ND
0


Sucrose
ND
ND
ND
0





*Correlation coefficients for Lineweaver-Burke plots;


**Percent activity rankings based on Vmax;


ND, Activity not detectable.






All initial characterization assays took place in 0.1 M sodium acetate buffer (pH 6) using 2 μL of recombinant enzyme preparation (=1.58 μg protein per assay) in a total reaction volume of 250 μL. Synthetic p-nitrophenol substrates (Table 1) were tested in direct kinetic assays using a microplate reader. Reactions were initiated by adding 248 μL assay buffer to 2 μL protein. Assays were read at 420 nm every 20 sec for 5 min to yield mean velocity data in mOD/min. Specific activity was determined with the p-nitrophenol extinction coefficient of 0.6605 mM−1 cm−1. All results were averaged from three independent replicates, each conducted in triplicate.
Natural carbohydrate substrates (Table 2) were tested in endpoint assays in which color development was enabled using 439-90901 glucose-mutarose detection reagent (Wako Chemical; Richmond, Va.). Assays were conducted in 125 μL for 5 min at room temperature as described above and stopped with the addition of 125 μL glucose detection reagent. After 1 min, the absorbance was read at 505 nm.
Example 19
β-glucosidase gene expression in gut and carcass: RfBGluc-1 and RfBGluc-2 gene expression was compared in worker whole gut and carcass tissues, and different gut regions (FIG. 10). First, using 35 cycles of convention PCR and agarose electrophoresis, RfBGluc-1 and RfBGluc-2 only showed detectable expression in gut tissue (FIG. 10). Next, qRT-PCR revealed highest RfBGluc-1 and RfBGluc-2 expression in salivary gland and foregut, followed distantly by hindgut and midgut (FIGS. 11A and 11B). RfBGluc-1 had the highest overall expression levels, with more than 25,000- and 225-fold higher expression in the salivary gland and foregut than midgut. RfBGluc-2 expression showed the same distribution through the gut, but at much lower levels; specifically, RfBGluc-2 was expressed at greater than 8,000-fold lower levels in the salivary gland than RfBGluc-1. Because salivary gland and foregut tissues are symbiont-free, these results indicate that RfBGluc-1 and RfBGluc-2 are host-derived genes.
Example 20
Recombinant RfBGluc-1 production, purification, and tissue-PAGE analyses: The RfBGluc-1 cDNA was functionally expressed with a C-terminal His-tag in T. ni larvae. The recombinant His-tagged protein was enriched to more than 98% purity from infected T. ni homogenates by filtration, Ni-IMAC, and Sephadex G-25 chromatography (FIG. 12A). The presence of His-tag on the purified protein was verified by western blotting with anti-His antibody and no cross-reactivity was observed in non-infected T. ni homogenates. Consistent with bioinformatic predictions presented above, treatment with the deglycosylation enzyme PNGase-F resulted in a single protein band and mobility shift on SDS-PAGE gels, verifying that recombinant RfBGluc-1 is glycosylated. The mass of the purified recombinant protein, with its two apparent glycosylations and a C-terminal histidine tag, was estimated at about 55-60 kDa by SDS-PAGE. Comparison of recombinant RfBGluc-1 against the recombinant cellulase (RfCell-1), as well as foregut/salivary gland, midgut, hindgut and carcass tissue preps suggests foregut/salivary gland expression for both RfCell-1 and RfBGluc-1 (FIG. 12B). Consistent with gene expression analyses, SDS-PAGE results did not show RfBGluc-1 expression in midgut, hindgut or carcass.
Example 21
Laccase cDNA sequencing: Sequencing efforts were guided initially by six contiguous laccase ESTs obtained from a normalized gut cDNA library (Scharf & Tartar (2008) Biofuels Bioprod. Bioref. 2: 540-552; Tartar et al., (2009) Biotech. Biofuels 2: 25, incorporated herein by reference in their entireties). This library was prepared from host gut tissues and has yielded more than 6,000 ESTs. Total RNA for RACE was isolated from 20 worker termite thoracic segments using the SV Total RNA Isolation Kit (Promega; Madison, Wis.). The SMART RACE cDNA Amplification Kit (Clontech; Valencia, Calif.) was used. RNA was DNase-treated and used as a template to produce 5′ and 3′ cDNA. RACE PCR amplifications were performed using 5′ or 3′ cDNA as a template. The anchor primer used in these reactions was the universal “primer A” mix paired with “Laccase5-R—336” reverse primer (5′-GCTTGCTGGTTGCGGGAATCCGTCGT-3′ (SEQ ID NO.: 58)) or the “Laccase3-F—584” forward primer (5′-CGCCCCCATCTCCGCTCCTCTCACA-3′ (SEQ ID NO.: 59)) for 5′ and 3′-RACE reactions, respectively. These laccase primers were designed from the contiguous EST sequences noted above. The 5′-RACE product (approximately 1.3 kb) and the 3′-RACE reaction products (approximately 700 bp and 900 bp, respectively) were cloned into pCR8/GW/Topo. A second 5′-RACE reaction used the reverse primer 5′-ACTTCAGTTGGTGTTCACGGGAGG-3′ (SEQ ID NO.: 60), which included the putative consensus stop codon. The reaction products, which included the entire laccase ORFs and their 5′ untranslated regions, were cloned into pCR8/GW/Topo and clones Lac6, 7, 12, 13, 14, 15, 17, 19 and 20 were sequenced in both directions.







TABLE 4







Summary of laccase cDNA sequences


Nucleotide number or position



















Length






Start
Stop
ORF
(Amino
Accession
SEQ ID


RfLac No
Total length
(ATG)
(TAG)
length
acids)
No
NO.:

















6
1985
39
1980
1941
647
GQ421909
7


7
1987
41
1982
1941
647
GQ421910
9


12
2035
89
2030
1941
647
GQ421911
11


13
1584
16
1579
1563
521
GQ421912
13


14
1988
42
1983
1941
647
GQ421913
14


15
2038
91
2032
1941
647
GQ421914
16


17
2038
92
2033
1941
647
GQ421915
18


19
1986
40
1981
1941
647
GQ421916
19


20
1963
19
412
393
131
GQ421917
20









Seven of the nine clones encoded highly similar, full-length ORFs that had 22 variable nucleotide positions. All putative start codons were present at the first ATG following the 5′ RACE PCR primer sequence. Additionally, the 5′ end of the full length ORFs are translatable into identical signal peptides. Identifiable ORF amino acid sequences derived from the nucleotide sequences listed in Table 4 are according to SEQ ID NOs.: 8, 10, 12, 15, 17, 19, and 21, respectively.
Based on sequence alignments, three highly similar ORFs aligned well into one contig (Lac6, Lac7, and Lac19) (contig 1) and the remaining four into another (Lac12, Lac14, Lac15, and Lac17) (contig 2). Overall, the two contigs are 99.4% identical. Clones Lac6 (SEQ ID NO.: 7) and Lac12 (SEQ ID NO.: 11) were chosen as representative sequences for contigs (isoforms) 1 and 2, respectively (hereafter referred to as RfLacA and RfLacB).
Translations of RfLacA (SEQ ID NO.: 7) and RfLacB (SEQ ID NO.: 11) yielded the amino acid sequences SEQ ID NOs.: 8 and 12, respectively, which have 98.1% identity. A conserved signal peptide of 16 amino acids (MLPCVLLACAIGVASA (SEQ ID NO.: 61)) with a Thr17 cleavage site was identified. All conserved amino acids implicated in copper ion binding are present in the RfLacA and RfLacB translation products. The fourth axial residue of the putative T1 copper center is a Met, which is present in RfLacA and RfLacB, and has been associated with laccases with lower redox potentials.
Example 22
Recombinant laccase production: Recombinant laccase proteins were produced in whole T. ni larvae using the PERLXpress procedure described in O'Connell et al. ((2007) Mol. Biotechnol. 36: 44-51) and Kovaleva et al. ((2009) Biotechnol Lett. 31: 381-386). The RfLacA (SEQ ID NO.: 7) and RfLacB (SEQ ID NO.: 11) clones were used as templates for PCR amplification and cloning. A C-terminal tag composed of two GLY and six HIS residues, as well as XbaI and EagI restriction sites, were incorporated into the amplicons utilizing the following primers: forward, 5′-TCTAGAATGTTGCCTTGCGTCCTGCTTG-3′ (SEQ ID NO.: 62); reverse, 5′-CGGCCGTTAGTGATGATGGTGATGATGACCTCC-GTTGGTGTTCACGGGAGGTGT-3′ (SEQ ID NO.: 63). The PCR amplicons, which encoded full-length RfLacA (SEQ ID NO.: 8) and RfLacB (SEQ ID NO.: 12) (amino acids 1 to 647), plus the C-terminal Gly+His tag, were cloned into XbaI-EagI sites of the pVL1393 transfer vector, and recombinant baculoviruses were generated using a homologous recombination system in insect Sf9 cells.
The recombinant, His-tagged proteins were purified to greater than 90% purity from infected T. ni homogenates by filtration, Ni-IMAC, and Sephadex G-25 chromatography. The approximately 80 kDa recombinant proteins migrate as doublets on SDS-PAGE. Predicted sizes of the mature laccase peptides and histidine tags, respectively, are about 69.5 kDa and about 1 kDa.
Example 23
Lignin metabolism assays: Lignin alkali, a water soluble by-product of lignocellulose processing (Hernandez et al., (2001) J. Chromat. A 919: 389-394) was used as a model substrate dissolved at 1% w/v in sodium phosphate buffer (0.1M, pH 7.5). Reactions (176 μl volumes) contained: 150 μl buffer+substrate, 3 μl H2O2, 3 μl sodium azide, and 20 μl recombinant RfLacA. Control reactions contained all components except RfLacA. Reactions proceeded for 16 hr at 32° C. with shaking in unsealed 5 mL borosilicate tubes. Reactions were stopped with 1 mL acetic acid, followed by extraction with 5×−1 mL ethyl acetate (Humphreys et al., (1999) Proc. Natl. Acad. Sci. USA 96: 10045-10050). After mixing and partitioning by centrifugation, ethyl acetate extracts were pooled in fresh amber vials and stored at −20° C.
For HPLC analysis, samples and controls were dried to completeness under N2, reconstituted in 150 μL methanol, and transferred to fresh 1⁄2-dram autosampler vials. HPLC was conducted on an Agilent system with two 100×4.6 mm Onyx Monolithic C18 columns connected in parallel (Phenomenex; Torrance, Calif.). Solvent systems were: (A) 1% acetic acid in water, and (B) 1% acetic acid in acetonitrile. Samples were injected in 50 μL volumes and separated at flow rates of 0.8 mL/min with gradients of A:B (min): 95:5 (0-6.5) to 0:100 (25.5-35) to 95: 5 (45-50).
Example 24
Laccase
Quantitative Real-Time PCR
Four gut regions (salivary gland, foregut, midgut and hindgut) were dissected from workers of three R. flavipes colonies (n=25 workers) and placed directly into RNA lysis buffer (Promega). Total RNA was extracted using the SV Total RNA Isolation System Kit (Promega), according to instructions. Equal quantities of RNA (250 ng) were used as templates for cDNA synthesis using the iScript cDNA Synthesis kit (Bio-Rad). qRT-PCR reactions were conducted as described previously (Zhou et al., (2007) Gene 395: 29-39) using 1 μL cDNA template and 10 μL SensiMixPlus SYBR and Fluorescein PCR mix (Quantace; Taunton, Mass.). Laccase forward and reverse primers (1 μL each of 5 μM), respectively, were “RFLacc-exp-F1—52663” 5′-AATCAAACTGGGTGCTTTGG-3′ (SEQ ID NO.: 64) and “RfLacc-exp-R1—52664” 5′-AACTTGATGATCGCGTAGCC-3′ (SEQ ID NO.: 65). These primers targeted conserved regions of all seven full-length sequence variants identified in the current work at consensus sequence positions 1536-1778. The reference gene, NADH-dh (Zhou et al., (2007) Gene 395: 29-39), was amplified with the forward and reverse primers “NADHdhF—46415” (SEQ ID NO.: 54) and “NADHdhR—46416” (SEQ ID NO.: 55), respectively. PCR products were analyzed on 2% agarose gels. Relative expression levels were determined using the 2−ΔΔCT method (Livak & Schmittgen (2001) Methods 25, 402-408, incorporated herein by reference in its entirety) from three independent replicates, each conducted in triplicate. Expression data in the form of critical threshold (CT) values were statistically analyzed by ANOVA and post-hoc LSD t-tests (SAS software; Cary, N.C.).
Example 25
Recombinant Laccase RfLacA and RfLacB: optimal pH and substrate specificity determinations: Using 1 mM substrate concentrations, recombinant RfLacA and RfLacB were able to oxidize the following substrates in order of decreasing relative activity: hydroquinone, sinapinic acid, pyrocatechol, 2,6-DMP, pyrogallol, NADA, L-DOPA, and NBAD, as shown in Table 5 below.







TABLE 5







RfLacA substrate specificities at 1 mM substrate concentrations, and the kinetic


parameters Vmax and Km for hydroquinone, 2,6-DMP, and pyrogallol.











Specific Activity

Apparent



at 1 mM [S]
Relative
Km and Vmax
















Optimal
nmol

Activity
Km1

Vmax2



Substrate
pH
min mg−1
SEM
(%)
(mM)
SEM
(μm min−1)
SEM


















Hydroquinone
7  
8.373
0.019
100.00
2.306
0.118
0.824
0.065


Sinapinic Acid
7.5
3.383
0.197
40.40
5.022
0.879
0.247
0.012


Pyrocatechol
7.5
2.315
0.160
27.65
17.527
1.457
15.434
1.175


2,6-DMP
7.5
2.146
0.027
25.63
34.888
3.870
0.260
0.020


Pyrogallol
7  
1.482
0.072
17.70


NADA
 7.5*
0.760
0.036
9.08


L-DOPA
6.0, 7.5
0.676
0.016
8.07


NBAD
 7.5*
0.190
0.024
2.27


Guaiacol
ND
N/A
N/A
N/A


Caffeic Acid
ND
N/A
N/A
N/A


Ferulic Acid
ND
N/A
N/A
N/A


Syringaldazine
ND
N/A
N/A
N/A


ABTS
ND
N/A
N/A
N/A





*Optimal pH not determined;


ND Activity not detected;


N/A Not applicable due to lack of detectable activity;


1Substrate concentrations [S] in mM providing 1⁄2 maximal activity;


2Maximal specific activities in nmol/min/mg.






Specific activity against the melanin precursors L-DOPA, NADA, NBAD, and L-tyrosine relative to that of 2,6-DMP was low (2.27-9.08%). Assays using the recombinant RfLacB isoform provided statistically indistinguishable results to RfLacA against all substrates.
Example 26
Recombinant Laccase Lac6 inhibition and enhancement: RfLac6 inhibition assays were conducted using 1 mM 2,6-DMP as a substrate in B&R buffer at pH 7.5. Enzyme and inhibitors were pre-incubated 5 min at room temperature on an orbital shaker before adding substrate. The inhibitors and concentrations used were: ethylenediaminetetraacetate (EDTA, 0.01-5 mM), sodium azide (NaN3, 0.01-1000 mM), sodium cyanide (NaCN, 0.05-10 mM), and thioglycolic acid (TGA, 0.01-60 mM) (FIG. 13A-13D).
EDTA completely inhibited RfLac6 activity at concentrations as low as 0.01 mM (FIG. 13A). NaCN and TGA enhanced activity up to 0.2 and 10 mM, respectively, but became inhibitory above these concentrations (FIGS. 13B and 13C). NaN3 significantly enhanced RfLac6 activity against DMP (FIG. 13D), catechol, pyrogallol, and hydroquinone. No activity was observed with NaN3 and RfLac6, or NaN3 and substrate alone. Finally, the enhancers did not enable the enzyme to oxidize ABTS or syringaldazine, both of which are not acted upon by RfLacA alone.
Example 27
Laccase-Lignin alkali metabolism: Representative HPLC chromatograms from lignin alkali metabolism experiments are shown in FIG. 14. The chromatograms from control (buffer blank) and RfLacA incubations are superimposed to reveal peaks with differential abundance between treatments. Seven peaks showed differential abundance between control and laccase incubations. Peaks 1, 3, 4, 5 and 6 were higher in control incubations than laccase incubations, showing they contain lignin components degraded by laccase action. Conversely, peaks 2 and 7 are higher in laccase than control incubations, therefore containing degradation products. These results provide evidence of RfLacA action on lignin.
Example 32
Recombinant Cell-3 characterization: The recombinant Cell-3 protein (nucleotide sequence SEQ ID NO.: 3; amino acid sequence SEQ ID NO.: 4) was investigated in unpurified form. As shown in FIG. 16, relative to uninfected control larvae not exposed to recombinant baculovirus, Cell-3 homogenates from infected larvae had significantly higher activity toward the model exoglucanase substrate p-nitrophenyl cellobioside (pNPC). However, pNPC activity in filtrates (=clarified supernatants) was significantly diminished, indicating that the recombinant Cell-3 protein has reduced stability. Other experiments investigating activity in crude homogenates over time and after repeated freeze-thaw cycles further verified reduced Cell-3 stability.
Example 33
Assay to Characterize Mixtures of Recombinant Enzymes Against a Woody-Feedstock Substrate
All reactions were performed in 0.1 M sodium acetate buffer containing 10 mM calcium chloride (pH 7). The two feedstock substrates were pine sawdust (=lignin+cellulose+hemicellulose) and beechwood xylan (=lignin+hemicellulose). Feedstocks were tested at 2% w/v (10 mg in 0.5 mL buffer). Reactions were run for 18 hrs at 37° C. with shaking at 220 rpm in vented 1.5 mL plastic tubes.
Protein volumes used per assay were 10 μL of each of the Cell-1, β-glucosidase, and Laccase 6 recombinant protein preparations. These loading volumes provided 8.5, 7.9 and 2.0 μg of each protein, respectively, per reaction. The storage buffer for Cell-1 and β-glucosidase was 0.1 M sodium acetate, 0.15 M sodium chloride, and 0.5 M calcium chloride (pH 5.8). The storage buffer for Laccase 6 was 0.1M boric acid, phosphoric acid, and acetic acid (pH 7.0).
Reactions were stopped after 18 hours by the addition of 10 μL of 0.2 Methylene-diamine tetra-acetic acid and centrifuged for 5 min at 16,000×g. Glucose detection was performed in triplicate on 125 μL aliquots of each reaction supernatant. The entire experiment was replicated three times. Glucose release was measured using the Autokit Glucose reagent kit (Wako Chemical, Richmond, Va.) at a volume of 125 μL per determination. Glucose was quantified based on absorbance at 505 nm relative to glucose standard curves. Standard curves were prepared in eight serial dilutions downward from 0.6 mM in sodium acetate-calcium chloride reaction buffer.
FIG. 20 shows the results of assays using pine sawdust as a substrate, which is a complex lignocellulose substrate composed of approximately 20% lignin, approximately 40% cellulose and approximately 25% hemicellulose. No glucose release was detectable when the combinations of β-glucosidase and Laccase 6, Cell-1+Laccase 6 and p-glucosidase+Laccase 6 were used.
Cell-1 alone released a slight amount of glucose (3.70 pmol/min). Most notably, while the combination of Cell-1+β-glucosidase released 330.59 pmol glucose/min (greater that 300-fold synergy relative to either component alone), the three-enzyme combination showed synergy, but released significantly less glucose than the Cell-1+β-glucosidase combination. Without wishing to be bound by any one theory, this inhibitory effect by Laccase 6 may be due to laccase-mediated lignin degradation being detrimental to glucose release from cellulose. In the alternative, three-way combination may initially release large quantities of glucose that inhibit Cell-1 and β-glucosidase via a process known as “end-product inhibition” (Xiao et al., (2004) Applied Biochem. Biotechnol. 113: 1115-1126).
FIG. 21 shows the results of assays using beechwood xylan, a less complex substrate than pine sawdust, as a substrate. The beechwood xylan polymer contains a mix of β-1,4-linked pentose and hexose sugars that are esterified with polymeric lignin and its monomeric subunits. Beechwood xylan is composed of less than 10% glucose; thus, significantly lower glucose is available for release relative to sawdust (note differences in Y-axis scales between FIGS. 20 and 21).
The beechwood xylan results are significant because none of three recombinant enzymes when tested alone were able to release glucose. Conversely, the two enzyme combination of Cell-1+β-glucosidase showed an approximately 80-fold synergy, indicating that they can act on hemicellulose as well as cellulose. The combination of Cell-1+Laccase 6 was also able to release low levels of glucose, which is suggestive of a role for laccase in hemicellulose delignification. Most notably, the three enzyme combination produced significantly greater glucose release than did the Cell-1+β-glucosidase combination and thereby provides evidence that the Lac 6 laccase can delignify hemicellulose, and also suggests that delignification is more important in release of fermentable sugars from hemicellulose than from cellulose.

## Claims

1. A method of converting a lignified plant material to a fermentable product, the method comprising the steps of:
(a) obtaining a series of isolated polypeptides wherein said polypeptides are encoded by the genome of a termite, wherein the series of polypeptides cooperate to convert a plant lignocellulose to a fermentable product and wherein said series includes one or more polypeptides having at least 90% sequence identity to a polypeptide selected from the group consisting of (i) a laccase selected from the group the consisting of SEQ ID NOs: 8, 10, 12, 15, 17, 19, 21 and 23; (ii) an esterase selected from the group consisting of SEQ ID NO: 27, 29, 31 and 33 and (iii) a glucosidase comprising SEQ ID NO: 6, and wherein said series further comprises one or more polypeptides selected from the group consisting of an endoglucanase and an exoglucanase; and
(b) incubating the series of polypeptides with a source of lignified plant material, under conditions allowing the polypeptides to cooperatively produce a fermentable product from the lignified plant material.

2. The method according to claim 1, wherein the isolated polypeptides are derived from the termite Reticulitermes flavipes, and not from a symbiont thereof, or a protozoa or a fungus.

3. The method according to claim 1, wherein the isolated polypeptides of the series of isolated polypeptides comprise an endoglucanase, a laccase, an esterase, and a glucosidase.

4. The method according to claim 3, wherein the series of the isolated polypeptides consists of an endoglucanase, an exoglucanase, a laccase, an esterase, and a glucosidase.

5. The method according to claim 1, wherein the isolated polypeptides of the series of isolated polypeptides are recombinant polypeptides, and wherein each polypeptide is expressed from an expression vector of a recombinant expression system, and wherein the recombinant expression system is selected from a eukaryotic cell-based system and a prokaryotic cell-based system.

6. The method according to claim 5, wherein the expression vector is a baculovirus expression vector and the recombinant expression system is a eukaryotic cell-based system.

7. The method according to claim 1, wherein the endoglucanase has an amino acid sequence having about 75% sequence identity with the amino acid sequence SEQ ID NO: 2.

8. The method according to claim 7, wherein the endoglucanase has the amino acid sequence SEQ ID NO: 2.

9. The method according to claim 1, wherein the series of isolated polypeptides further comprises an exoglucanase having an amino acid sequence having about 75% sequence identity with the amino acid sequence SEQ ID NO: 4.

10. The method according to claim 9, wherein the exoglucanase has the amino acid sequence SEQ ID NO: 4.

11. The method according to claim 1, wherein the laccase has the amino acid sequence selected from the group consisting of: SEQ ID NOs: 8, 10, 12, 15, 17, 19, 21, and 23.

12. The method according to claim 1, wherein the esterase has the amino acid sequence selected from the group consisting of: SEQ ID NOs: 27, 29, 31, and 33.

13. The method according to claim 3, wherein the glucosidase has the amino acid sequence SEQ ID NO: 6.

14. The method according to claim 1, wherein the fermentable product comprises at least one carbohydrate selected from the group consisting of: a glucose, a mannose, a xylose, a galactose, a rhamnose, an arabinose, a glucuronic acid, a mannuronic acid, and a galacturonic acid.

15. The method according to claim 14, wherein the fermentable product comprises glucose.

16. A system for producing a fermentable product from a lignified plant material, wherein the system comprises at least two isolated polypeptides selected from the group consisting of: an endoglucanase, an exoglucanase, a laccase having at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOs: 8, 10, 12, 15, 17, 19, 21 and 23, an esterase having at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 27, 29, 31 and 33, and a glucosidase having at least 90% sequence identity to a polypeptide of SEQ ID NO: 6, from of the termite Reticulitermes flavipes and wherein the at least two isolated polypeptides can cooperate to convert a constituent of the lignified plant material to a fermentable product or a precursor thereof, and wherein said system comprises at least one of said laccase, said esterase or said glucosidase.

17. The system according to claim 16, wherein the nucleotide sequence encoding the endoglucanase hybridizes under high stringency conditions to a nucleotide sequence according to SEQ ID NO: 1, the nucleotide sequence encoding the exoglucanase hybridizes under high stringency conditions to a nucleotide sequence according to SEQ ID NO: 3, the nucleic acid molecule encoding the laccase hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 7, 9, 11, 13, 14, 16, 18, 20, and 22, the nucleic acid molecule encoding the esterase hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 26, 28, 30, and 32, and the nucleotide sequence encoding the glucosidase hybridizes under high stringency conditions to SEQ ID NO:5, wherein said high stringency conditions are 50% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.1×SSC at 60-65° C.

18. The system according to claim 16, wherein each of the isolated nucleic acid molecules thereof is operably inserted into an expression vector.

19. A recombinant cell comprising an isolated nucleic acid molecule hybridizing under high stringency conditions to a nucleotide sequence encoding a polypeptide selected from the group consisting of: an endoglucanase, an exoglucanase, a laccase having at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOs: 8, 10, 12, 15, 17, 19, 21 and 23, an esterase having at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 27, 29, 31 and 33, and a glucosidase having at least 90% sequence identity to a polypeptide of SEQ ID NO: 6 from of the termite Reticulitermes flavipes, wherein the nucleotide sequence encoding the endoglucanase is according to SEQ ID NO: 1, the nucleotide sequence encoding the exoglucanase is according to SEQ ID NOS: 3, wherein said recombinant cell comprises at least one nucleic acids encoding said laccase, said esterase or said glucosidase.