Transgenic plants as an alternative source of lignocellulosic-degrading enzymes

This is a continuation-in-part of application Ser. No. 08/883,495, filed Jun. 26, 1997 now U.S. Pat. No. 5,981,835.

This invention was made with United States government support awarded by the following agencies: DOE Grant No. DE-FC05-92OR22072 and USDA Grant Nos. 94-34190-1204 and 92-34190-6941. The United States has certain rights in this invention.

The present invention is directed to the production of cellulose-degrading enzymes in genetically recombinant plants and the recombinant plants themselves.

Complete bibliographic citations for the non-patent references discussed hereinbelow are included in the Bibliography section, immediately preceding the claims. All of the references cited below are incorporated herein by reference.

Lignocellulosic plant matter, such as agricultural and forestry waste, as well as energy crops produced specifically for biomass, offer tremendous potential for the renewable production of fuel and as chemical feedstocks. However, production cost for desired products such as alcohols from lignocellulosic material is significantly higher than the production cost of equivalent alternatives. However, the prospect, either real or perceived, of limited fossil fuel reserves, along with the geo-political issues which swirl about petroleum-producing countries and regions, renders the production of basic chemical feedstocks and fuels from local, renewable sources an attractive alternative to fossil fuels.

For instance, alcohols have the potential to be excellent alternative transportation fuels if their production costs can be lowered. Brazil has sponsored several programs to replace car engines which run on gasoline alone to engines which run on ethanol or a gasoline-ethanol mix.

Unfortunately, the production of ethanol and other feedstock chemicals from lignocellulosic material is far more complex than an analogous production utilizing a starch-based starting material. Compared to lignocellulosic materials, starch is a simple polymer which is readily hydrolyzed to glucose. Yeasts can then be used to convert the glucose to ethanol.

In contrast, lignocellulosic biomass is a much more complex substrate in which crystalline cellulose is embedded within a matrix of hemicellulose and lignin. The intricate structure and relative inaccessibility of these substrates requires pre-treatment for the disruption of the lignocellulosic material, as well as hydrolysis of hemicellulose and lignin into xylose and phenolic compounds, respectively. (See, for instance, Micelli et al. (1996), Belkacemi et al. (1996), and Grohmann et al. (1992).)

Several enzymes which degrade lignocellulosic material, commonly referred to as “cellulases,” are known. The term “cellulase” shall be used herein to refer to any and all enzymes which catalyze the cleavage of cellulosic or lignocellulosic materials. Explicitly, but not exclusively, included within this definition are those cellulases which fall under the Enzyme Classification heading EC 3.2.1.x. Various genes encoding cellulases have also been isolated and characterized.

For instance, genes which encode endoglucanases from the fungus Trichoderma reesei are known and have been successfully incorporated and expressed in yeast. See, for instance, Pentilla et al. (1987). Likewise, cellulase E2 (EC 3.2.1.4) and cellulase E3 (EC 3.2.1.91) from the thermo-tolerant bacterium Thermomonospora fusca are known. See Lao et al. (1991), Spezio et al. (1993) and Zhang et al. (1995).

From a functional viewpoint, cellulases are catagorized into two large sub-groups based upon whether they catalyze cleavage from the cellulose chain ends (exocellulases) or if they catalyze cleavage in the middle of the cellulose chain (endocellulases). For instance, cellobiohydrolase I of T. reesei (CBH I, EC 3.2.1.91) is an exocellulase, which degrades crystalline cellulose by cleavage from the chain ends. By way of further illustration, CBH I is a 68 kDa protein with a two-domain architecture which is shared by many cellulases. In this chemical architecture, a large catalytic domain is joined to a cellulose-binding domain (CBD) through a flexible linker region. See Divne et al. (1994). Similarly, cellulase E3 of T. fusca is also an exocellulase.

Different types of cellulases exhibit synergistic activity on complex substrates. This synergism, especially between exocellulases, is believed to be due to differences in their patterns of absorption to and hydrolysis of complex cellulose substrates. See Henrissat et al. (1995).

Illustratively, cellulase E2 of T. fusca is a 40 kDa endocellulase which cleaves the cellulose chain internally. Such cleavage generates more chain ends for attack by exocellulases. Consequently when CBH I, E2, and E3 cellulases are combined, their activity together is approximately 5-fold greater than their additive individual activities. (See, for instance, Irwin et al. (1993) and WO 94/26880.) It is important to note that proteolytic fragments of cellulases can substitute for the intact enzymes in synergistic mixtures. For example, when combined with T. fusca E3 and CBH I, the catalytic domain of T. fusca E2 (“E2cd”) is as active as the intact enzyme in the digestion of filter paper substrate, Irwin et al. (1993).

A wide range of compositions containing cellulases are described in the patent literature. For instance, Evans et al., U.S. Pat. No. 5,432,074, describe the use of a formulation consisting essentially of a combination of xylanase and xylosidase, but being essentially free of glucanase and cellobiohydrolase. The formulation also contains a lactic acid-producing bacteria. The formulation is used to treat silage to increase its nutritive value. In operation, the action of the xylanase and xylosidase enzymes degrades non-cellulosic polysaccharides found in the silage material thereby producing sugars for fermentation.

Heterodimers of different types of cellulose-degrading enzymes are described in WO 94/29460. Here, a β-glucosidase molecule and a cellobiohydrolase molecule (i.e., an exocellulase) are chemically bonded to one another by a crosslinking reagent to yield a single molecule which retains the enzymatic activities of the two separate molecules.

Expression constructs which contain cellulase genes for the transformation of yeast have been constructed. For example, Knowles et al., U.S. Pat. No. 5,529,919, describe the transformation of S. cerevisiae to contain and express a thermostable β-endoglucanase (EG I) of T. reesei.

Likewise, attempts have been made to produce transgenic plants which express cellulose-degrading enzymes. Aspegren et al. (1995) describe transgenic suspension-cultured barley cells which express EG I of T. reesei . The cells were transformed by particle bombardment and transformed cells selected by a co-transformed antibiotic resistance marker. However, no attempt was made to regenerate complete plants from the cultured cells. Of particular note, this reference states that the production of β-glucanases in plant cells may be hampered by the fact that these enzymes catalyze the hydrolysis of essential cell wall components. Attempts by these authors to stably transform tobacco cells with the same construct used to successfully transform the suspended barley cells failed. Here, the authors observed that after transient expression in tobacco protoplasts, cell wall synthesis never resumed.

The present invention is drawn to genetically recombinant plants which contain one or more exogenous gene sequences which encode one or more cellulose-degrading gene products. The gene product or products are expressed in recoverable quantities in the recombinant plants and can be isolated from the plants, if desired. In the preferred embodiment, the genetically recombinant plant expresses the gene product constituitively.

However, the invention also encompasses recombinant plants which express the gene product stage-specifically or tissue-specifically. For example, the gene product or products can be expressed in a plant tissue such as the seeds, fruit, leaves, or tubers of the transformed plant host.

The invention is further drawn to recombinant plants as noted above, wherein the plant contains two exogenous genes whose respective gene products are expressed independently of one another. This allows for different types of cellulases to be expressed in different locations within the same recombinant plant. For example, the plant host can be transformed to express two or more heterologous cellulases in different sub-cellular compartments such as the plastid, cytosol, endoplasmic reticulum, mitochondrion, inclusion body, or vacuole. In addition, chloroplast targeting can also be accomplished through the use of direct chloroplast transformation, an approach that circumvents many of the problems associated with expression of heterologous genes in the nuclear genome. Carrer et al. (1993), McBride et al. (1994).

The invention is further drawn to a method for producing cellulose-degrading enzymes. The method comprises transforming a plant host with one or more exogenous genes which encode one or more cellulose-degrading gene products such that the gene product or products are expressed in recoverable quantities. The plant matter containing the expressed protein can be used directly as a feedstock for biomass conversion, or, if desired, the exogenous enzymes so produced can be isolated and purified.

The cellulases produced by the transgenic plants of the present invention can be utilized in the same manner as conventionally-derived cellulases. For instance, cellulases produced by the transgenic plants of the present invention can be isolated and used in fermentation processes such as brewing and wine-making. Here, the cellulases function to hydrolyze cellulose and β-glucans during fermentation. Or, as described in Example 4, below, whole plants transformed to express cellulases can be used directly or added to ensiled plant matter to increase the extent of fermentation of the ensiled matter. Plants transformed to express functional cellulases may also be fed directly to livestock, where the cellulase activity aids in the digestion of lignocellulosic substrates.

Cellulases produced in the transgenic plants of the present invention can also be utilized in the production of ethanol and other feedstock chemicals from lignocellulosic substrates.

Cellulases produced by transgenic plants of the present invention can also be used in the textile, pulping, and paper-making industries. For instance, cellulases are conventionally used to treat denim fabrics to give them a “stone-washed” appearance. Cellulases are also used to modify paper pulps by digesting the cellulose fibers contained within the pulp. The cellulases produced by the transgenic plants described herein can be used in this fashion.

FIG. 1 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain cellulase E2 of T. fusca . Promoters and structural genes are depicted as arrows which indicate the direction of transcription. Terminators are depicted as boxes. NPT II=neomycin phosphotransferase; Met-E2m= T. fusca E2 (mature form with N-terminal methionine added); MAS-ter=mannopine synthetase terminator; TML-ter=tumor morphology left terminator; MAC-pro=hybrid “MAC” promoter.

FIG. 2 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain cellulase E3 of T. fusca . Promoters and structural genes are depicted as arrows which indicate the direction of transcription. Terminators are depicted as boxes. NPT II=neomycin phosphotransferase; Met-E3m= T. fusca E3 (mature form with N-terminal methionine added); MAS-ter=mannopine synthetase terminator; TML-ter=tumor morphology left terminator; MAC-pro=hybrid “MAC” promoter.

FIG. 3 is a western blot analysis evidencing the expression of T. fusca E2 cellulase in tobacco transformed to contain the expression construct depicted in FIG. 1 .

FIG. 4 is a western blot analysis evidencing the expression of T. fusca E3 cellulase in tobacco transformed to contain the expression construct depicted in FIG. 2 .

FIG. 5 is a zymogram gel assay evidencing the expression of active T. fusca E2 cellulase in alfalfa transformed to contain the expression construct depicted in FIG. 1 .

FIG. 6A is a schematic diagram of a binary vector T-DNA for an expression construct to transform plants to contain cellulase E1 of A. cellulolyticus.

FIG. 6B is a schematic diagram of a binary vector T-DNA for an expression construct to transform plants to contain the catalytic domain (E1cd) of cellulase E1 of A. cellulolyticus.

FIG. 7 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain cellulase CBH I of T. Reesei.

FIG. 8A is a plot of data from an activity assay evidencing the expression of A. cellulolyticus E1 cellulase in tobacco transformed with Agrobacterium strain PZA8.

At FIG. 8B is a plot of data from an activity assay evidencing the expression of A. cellulolyticus E1 cellulase in tobacco transformed with Agrobacterium strain PZA9.

FIG. 9 is a western blot analysis evidencing the expression of CBH I cellulase of T. Reesei in tobacco transformed to contain the expression construct depicted in FIG. 7 .

FIG. 10 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain the CenA endoglucanase of Cellulomonas fimi.

FIG. 11 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain endoglucanase D of Clostridium thermocellum.

FIG. 12 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain exoglucanase S of Clostridium cellulovorans.

FIG. 13 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain exocellulase E6 of Thermobifida fusca.

The invention is directed to genetically recombinant plants which express one or more exogenous cellulose-degrading (cellulase) enzymes. The invention is further drawn to a method of producing cellulases in plants. The invention allows the production of cellulases using the means and methods of large-scale agriculture rather than the conventional route of large-scale fermentation of the bacteria or fungi which are native producers of the cellulases.

The recombinant plants are produced by incorporating into a plant host genome one or more expression constructs comprising a DNA sequence which encodes a protein having cellulose-degrading activity. Introduction of the exogenous gene or genes into the plant is accomplished by any means known to the art. The expression constructs described hereinbelow enable the stable transformation of plants with one or more genes which encode cellulose-degrading enzymes. The constructs include a DNA coding sequence which encodes a cellulase (as that term is described herein) which is operatively linked to regulatory sequences which direct constituitive, stage-specific, or tissue-specific expression of the cellulase DNA.

Cellulose-Degrading Enzymes (Cellulases) and Genes:

As noted above, the term “cellulase” shall be used herein to refer to any and all enzymes which catalyze the cleavage of cellulosic or lignocellulosic materials. As used herein, “cellulase” is synonymous with “cellulose-degrading enzymes.” Explicitly, but not exclusively, included within the term cellulases are those enzymes which fall under the Enzyme Classification heading EC 3.2.1.x. A non-exhaustive list of these enzymes, the genes for all of which can be used in the present invention, includes the following:

DNA sequences encoding enzymes having any of the above-described functionalities can be obtained from several microbial sources, including bacterial and fungal sources. Cloning the gene or cDNA sequence of the desired enzyme can be achieved by several well-known methods. A preferred method is to purify the cellulase of interest (or purchase a sample if commercially available) and determine its N-terminal amino acid sequence, as well as several internal amino acid sequences, using known methods. Oligonucleotide probes corresponding to the amino acid sequence are then constructed (again using known methods) and used to screen a genomic or cDNA library of the organism from which the cellulase was isolated. Positive hybrids are identified, characterized using known methods (restriction enzyme analysis, etc.), and cloned by known means to yield DNA fragments containing the coding sequence for the desired cellulase activity. (See, for instance, Current Protocols in Molecular Biology , Chapters 5 and 6.)

If a partial nucleotide sequence of the cellulase of choice is already known, this information can be used to construct suitable primers to directly clone the corresponding cDNA using the polymerase chain reaction (PCR). (See Current Protocols in Molecular Biology , Chapter 15.)

Particularly preferred for use in the present invention are those enzymes falling within the classifications EC 3.2.1.4; EC 3.2.1.6; EC 3.2.1.21; and EC 3.2.1.91. The functionality of these particular enzymes is summarized as follows:

EC 3.2.1.4 enzymes (β-1,4-endoglucanases) hydrolyze internal 1,4 glycosidic bonds of the polysaccharide chain, thereby yielding new chain ends at the surface of cellulose crystals.

EC 3.2.1.6 enzymes (β-1,3-endoglucanases) hydrolyze internal 1,3 glycosidic bonds of the polysaccharide chain, which also results in the formation of new chain ends at the surface of cellulose crystals.

EC 3.2.1.21 enzymes (β-glucosidases) hydrolyze cellobiose into glucose, a readily fermentable substrate.

EC 3.2.1.91 enzymes (β-1,4-exocellulases) cleave cellobiosyl residues (cellobiose is a glucose dimer) from the chain ends of cellulose.

Particularly preferred enzymes (and hence particularly preferred genes) for use in the present invention are cellulase E2 and cellulase E3 of T. fusca and CBH I of T. reesei.

Expression Constructs:

Once the protein coding sequence (i.e., the cellulase gene) has been identified and isolated, it must be inserted into an appropriate expression construct containing regulatory elements to direct the expression of the gene and to direct secretion of the gene product or targeting of the gene product to a particular sub-cellular location or organelle. Manipulation of oligonucleotide sequences using restriction endonucleases to cleave DNA molecules into fragments and DNA ligase enzymes to unite compatible fragments into a single DNA molecule with subsequent incorporation into a suitable plasmid, cosmid, or other transformation vector are well-known to the art.

A transcription regulatory sequence must be included in the expression construct in order to direct the transformed plant cells to transcribe the inserted cellulase coding sequence. Transcriptional regulators may be inducible or constituitive. Inducible transcription regulators direct transcription of the downstream coding sequences in a tissue-specific or growth-stage specific manner. Constituitive regulators provide for sustained transcription in all cell tissues. For purposes of the present invention, constructs which provide constituitive expression of the coding sequence are preferred.

It is also preferred that the expression construct contain a transcription initiation sequence from the tumor-inducing plasmid (Ti) of Agrobacterium. Several T-DNA transcription initiation sequences are well known and include, without limitation, the octopine synthase, nopaline synthase, and mannopine synthase initiators.

Downstream of the initiation sequence and fused to the coding sequence, the expression construct may be manipulated to contain a leader signal sequence which directs the resulting polypeptide to a particular organelle or targets the expressed product for secretion (or to signal post-transcriptional or post-translational modification of the gene product).

Likewise, the expression construct should also include a termination sequence to signal transcription termination.

To facilitate selection of successfully transformed plants, the expression construct should also include one or more selectable markers. The neomycin phosphotransferase gene (NPT II), a well-characterized and widely employed antibiotic resistance selection marker is preferred. This marker provides resistance to kanamycin. A large number of other markers are known and can be used with equal success (e.g., other antibiotic resistance markers, dihydrofolate reductase, luciferase, β-glucuronidase, and the like).

For example, FIGS. 1 and 2 depict schematic representations of suitable expression constructs for transformation of plants. These constructs are intended for use with Agrobacterium-mediated transformation using the binary vector approach. However, these same constructs can be coated onto micro-projectiles for transformation by particle bombardment. With the exception of the coding sequence, these two constructs are essentially identical: FIG. 1 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain cellulase E2 of T. fusca.

FIG. 2 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain cellulase E3 of T. fusca.

In both FIG. 1 and FIG. 2, promoters and structural genes are depicted as arrows which indicate the direction of transcription and terminators are depicted as boxes. See the “Brief Description of the Figures” for a legend to the abbreviations. In the expression constructs depicted in FIGS. 1 and 2, the “MAC” hybrid promoter drives the transcription of the recombinant cellulase genes. Both constructs also contain a constituitive NPT II expression cassette to allow for antibiotic resistance selection using kanamycin. The coding sequence of the construct shown in FIG. 1 (Met-E2m) encodes cellulase E2 from T. fusca . (See SEQ. ID. NO: 1; ATG start codon at nt's 255-257, TGA stop codon at nt's 1578-80, first codon of mature E2 protein (AAT) at nt's 348-350.) This sequence encodes the mature form of the enzyme with an N-terminal methionine added. In the same fashion, the coding sequence of the construct shown in FIG. 2 (Met-E3 m) encodes cellulase E3 from T. fusca . (See SEQ. ID. NO: 2; ATG start codon at nt's 575-577, TAA stop codon at nt's 2363-65, first codon of mature E3 protein (GCC) at nt's 689-692.) This sequence also encodes the mature form of the enzyme with an N-terminal methionine added.

Further examples of constructs which drive targetted expression of cellulose-degrading enzymes are provided in the Examples hereinbelow. Specifically included in the Examples are transformations illustrating apoplastic targeting and accumulation of two additional cellulases. The first of these is the endoglucanase E1 of Acidothermus cellulolyticus (EC 3.2.1.4, SEQ. ID. NO: 8). Also included is the cellobiohydrolase CBH I of T. Reesei (EC 3.2.1.91, SEQ. ID. NO: 9). In addition, further Examples of plant expression constructs containing cellulase genes encoding both endoglucanases and cellobiohydrolases (exoglucanase, exocellulase) are provided.

Transformation of Plants:

Transformation of the plants can be accomplished by any means known to the art, including Agrobacterium-mediated transformation, particle bombardment, electroporation, and virus-mediated transformation. The method of transformation is not critical to the functionality of the present invention insofar as the method chosen successfully incorporates the oligonucleotide construct containing the cellulase-encoding region and any accompanying regulatory sequences into the plant host. The nature of the plant host to be transformed has some bearing on the preferred transformation protocol. For dicots, Agrobacterium-mediated transformation utilizing protoplasts or leaf disks is most preferred. Although the Examples disclose the use of tobacco and alfalfa as bioreactors for cellulase production, any crop plant, including monocots, can be utilized. Transformation of monocots is typically achieved by particle bombardment of embryogenic cell lines or cultured embryos. See, for instance, Vasil et al. (1993) and Castillo et al. (1994). Recent developments in “super-binary” vectors, however, also allow for the use of Agrobacterium-mediated gene transfer in most of the major cereal crops. See, for instance, Ishida et al. (1996). In this case, the explant source is typically immature embryos.

Agrobacterium-mediated transformation of the plant host using explants is preferred for its relative ease, efficiency, and speed as compared to other methods of plant transformation. For example, disks are punched from the leaves of the plant host and cultured in a suitable medium where they are then exposed to Agrobacterium containing the expression construct and (preferably) a disarmed tumor-inducing (Ti) plasmid. Agrobacterium tumefaciens LBA 4404 is the preferred strain for transformation. The preferred binary vector is the pCGN1578 binary vector (McBride and Summerfelt (1990)).

The binary vector transformation method is well known and needs only be briefly described herein. See Zambryski et al. (1989) for a complete review. The Ti plasmid of Agrobacterium contains virulence genes (vir) which encode trans-acting proteins that enable the transfer of a portion of the plasmid (the T-DNA) to a plant cell. The T-DNA portion of the Ti plasmid is flanked by two border regions (the right and left borders) which act as recognition sites for the excision of the T-DNA from the plasmid prior to its transfer to the plant host. Excision of the T-DNA is mediated by the vir genes of the Ti plasmid and involves nicking of the right and left borders of the T-DNA, which frees a single-stranded oligonucleotide fragment. This fragment is then mobilized out of the Agrobacterium and into the plant host target.

In the binary vector method, the T-DNA with its right and left border regions is cloned into E. coli in known fashion, and the wild-type genes normally found between the two border regions is excised. The expression construct encoding the cellulase of interest is inserted between the right and left border regions. This construct is designated the “binary plasmid.” Construction of the binary plasmid is accomplished utilizing the well-characterized recombinant genetic methods applicable to E. coli . Successful transformants are selected utilizing a co-transformed marker appropriate for E. coli.

The binary plasmid is then mobilized back into Agrobacterium. This is accomplished by direct transformation procedures well known to those skilled in the art.

The Agrobacterium itself, such as the preferred LBA 4404 strain, is genetically manipulated to contain a Ti plasmid (called the helper plasmid) which lacks the T-DNA and the tumor-inducing regions (i.e., the Ti plasmid is “disarmed”) but which still encodes the virulence proteins necessary for DNA transfer. By cooperation between the helper plasmid and the binary plasmid, the length of DNA between the two border regions of the binary plasmid is excised and mobilized into the plant host, where it is incorporated into the plant host genome. The binary method derives its name from the fact that the plasmid containing the expression construct to be transferred is maintained within Agrobacterium as a distinct and independently replicating vector from the Ti plasmid itself.

Selection of successful transformants is accomplished using the co-transformed selection marker discussed above. If the marker is NPT II, selection is accomplished by treatment with kanamycin.

For the present invention, the most preferred plants for transformation are alfalfa and tobacco. However, any plant species will function with comparable success. Included among the plant species which can be utilized in the present invention are cauliflowers, artichokes, apples, bananas, cherries, cucumbers, grapes, lemons, melons, nuts, oranges, peaches, pears, plums, strawberries, tomatoes, cabbages, endive, leeks, lettuce, spinach, arrowroot, beets, carrots, cassava, turnips, radishes, yams, sweet potatoes, beans, peas, soya, wheat, barley, corn, rice, rapeseed, millet, sunflower, oats, tubers, kohlrabi, potatoes, and the like.

The plants to be transformed are preferably common green field plants, such as the preferred alfalfa and tobacco, as well as soya, corn, and the like. Equally preferred are plant hosts which are grown specifically for “biomass energy,” such as switchgrass, poplar, and the like. In this instance, the enzymes would not be recovered from the plants. The plants are then transformed and regenerated into whole plants which express fully-functional, cellulose-degrading enzymes in economically significant quantities. Alfalfa is one of the most preferred plant species for use in the present invention because alfalfa is a hardy, perennial plant, which grows well with minimal fertilization and irrigation. Alfalfa is also a very prolific plant. In temperate areas such as those found in the Midwestern United States, alfalfa will yield three or more harvests per growing season. Methods have also been developed for wet fractionation of the herbage matter to recover value-added products therefrom.

Tobacco is equally preferred for its prolific growth, ease of transformation, and its well-characterized genetics. Both alfalfa and tobacco are widely cultivated throughout the United States and in other parts of the world.

In the most preferred embodiment, alfalfa or tobacco plants are stably transformed to express, constituitively, enzymatically active E2 or E3 cellulases from T. fusca . Also preferred are alfalfa or tobacco which express enzymatically active CBH I from T. reesei or combinations of E2, E3, and CBH I. The T. fusca cellulases are most preferred because they are native to thermo-tolerant bacteria and are relatively heat stable. This allows isolation of the cellulase from plant material using relatively rigorous heat precipitation without adversely effecting the activity of the cellulase.

Stage-Specific and Tissue-Specific Expression of Cellulases:

Because the enzymes to be expressed by the transformed plant hosts hydrolyze components of the plant cell wall, high levels of expression might have a deleterious effect on the plant host. Therefore, targeting of the expressed enzyme to particular sub-cellular compartments may be preferred. Targeting of the expressed enzyme may also be preferred to avoid expression of the enzyme in sub-cellular compartments where proteolytic activity is high. Targeting of the expressed enzyme may also be preferred if the exogenous cellulase activity interferes with the normal cellular metabolism of certain compartments.

For instance, targeting expression to the apoplast allows the exogenous protein to avoid the more active protein-degrading systems of other cellular compartments, such as in plant leaf vacuoles.

Several signal sequences are known and can be utilized in the present invention. For example, signal sequences for targeting to the secretory pathway are known, Wandelt et al. (1992), Bednarek (1991), Mason et al. (1988), as are sequences for targeting to the chloroplast, Keegstra et al. (1993), and the mitochondrion, de Castro Silva Filho et al. (1996).

For apoplast targeting, the VSP leader is preferred. The VSP leader comprises the amino acid sequence: NH 3 -Met-Lys-Leu-Phe-Val-Phe-Phe-Val-Ala-Ala-Val-Val-Leu-Val-Ala-Trp-Pro-Cys-His-Gly-Ala- (SEQ. ID. NO: 3). See Mason et al. (1988).

Additionally, bacterial secretory sequences found in the wild-type cellulase gene may be removed to afford cytoplasmic expression of the enzyme in the recombinant plant host.

Targeting can be achieved by fusing combinations of mitochondrial and chloroplast targeting signals to the N-terminus of the desired cellulase, as has been demonstrated for the reporter genes chloramphenicol acetyl transferase and β-glucuronidase, de Castro Silva Filho et al. (1996). In some cases, efficient translocation requires the presence of both signal peptides, with the amino terminal peptide being crucial in specifying import into a particular organelle. In addition, vacuole targeting can be achieved by fusing the sequence encoding the N-terminal 146 amino acids of the vacuolar patatin protein between a secretory leader and structural gene for the cellulase, as has been demonstrated for the yeast invertase gene, Sonnewald et. al. (1991).

Regeneration of Mature Transgenic Plants:

Transgenic tobacco and alfalfa were produced by Agrobacterium-mediated transformation using explants as source material. This is a routine method easily followed by those skilled in the art. The production methods for transgenic tobacco and alfalfa are given as non-limiting illustrations of the practice of the invention.

The transformation procedure for tobacco is essentially the explant method developed by Horsh et al. (1985). Leaf explants are taken from the second and third fully expanded leaves of three-week old in vitro shoot cultures of Nicotiana tabacum W38 maintained on MS medium, Murashige and Skoog (1962). The leaf pieces are cut into 1 cm squares and pre-cultured on MS medium with 2.0 mg/L 6-benzyl-aminopurine (BAP) and 0.1 mg/L alpha-naphthalene acetic acid (NAA) for 24 hours at 25° C. with a 16 hour photo period of 70-90 μE m −2 s −1 . After pre-culture, explants are placed into a suspension of Agrobacterium cells. After 30 minutes, leaf explants are blotted on filter paper and placed abaxial-side down on MS medium with 1.0 mg/L BAP and 0.1 mg/L NAA and co-cultivated for four days under the same conditions as given above. Leaf pieces are then rinsed three times in sterile water, blotted on filter paper, and transferred to the media used for co-cultivation but containing 100 mg/L kanamycin and 400 mg/L carbenicillin. Plantlets (typically 2-3) develop 10-14 days later from callus formed along cut leaf edges. If desired, further plantlet formation can be achieved by transfer of explants to fresh medium at two week intervals. Plantlets are excised and rooted on MS media containing 100 mg/L kanamycin and 400 mg/L carbenicillin.

To transform alfalfa, new-growth trifoliates are taken from alfalfa plants (regenerable genotypes, Bingham et al. (1975)) maintained in a growth room and sterilized using alcohol and bleach washes (30 seconds in 70% alcohol, 90 seconds in 20% hypochlorite+0.1% SDS, followed by three rinses in sterile distilled water). Leaf edges are cut on moist filter paper and tissue then placed into liquid SH-II medium. (Bingham et al., supra.) When sufficient explants have been taken, the explants are moved to a suspension of Agrobacterium cells containing the engineered plasmid. (The Agrobacterium suspension is taken from an overnight culture grown in liquid YEP selection medium.) Cell density is adjusted to fall between about 0.6 to about 0.8 at A 660 . After 30 minutes inoculation, the explants are gently blotted on filter paper and placed on B5H medium, Brown and Atanassov (1985), for four days. They are then rinsed twice in sterile water and cultured on B5H for a further four days. At the end of this period, they are rinsed three times and transferred to B5H containing 25 mg/L kanamycin and 250 mg/L carbenicillin. Plates are maintained at 24° C., 16 hour photo period, light intensity 60-80 μE m −2 s −1 . Explant-derived calli (and occasionally embryoids) which form within 3 weeks on this medium are moved to B5H with antibiotics but without hormones to allow for further embryoid production and development of existing embryoids. After three to four weeks, embryos are transferred to MS medium including the two antibiotics to allow for development into plantlets. Callus forms on untreated explants in the presence of 25 mg/L kanamycin but embryos are never produced. Each explant piece can give rise to multiple (up to 40) embryos. Plantlets are rooted on MS medium lacking antibiotics.

Monitoring Cellulase Expression:

Cellulase expression can be monitored using a number of different methods, the two most common being western blot analysis (which detects cellulase protein using antibodies specific for the cellulase of interest) and zymographic analysis or enzyme assay (both of which measure the ability of the expressed cellulase to degrade a cellulosic substrate).

Briefly, in the western blot technique, whole plant samples (or root tips, leaves, etc.) are ground in an extraction buffer (preferably 50 mM sodium acetate (pH 5.5) and 10 mM dithiothreitol) and an aliquot of the extract loaded onto an electrophoresis gel (e.g., polyacrylamide containing SDS). Preferably, identical extractions are performed on non-transformed plants and aliquots of these extractions are then loaded onto parallel lanes of the gel to act as negative controls. Serial dilutions of purified cellulase standards can be also electrophoresed to act as positive controls. The gel is then subjected to electrophoresis in standard and well known fashion.

After electrophoresis is complete, the separated proteins are electro-transferred to a nitrocellulose, PVDF, or nylon membrane, in well known fashion. The membrane containing the immobilized proteins is then immersed in a non-specific blocking buffer or detergent (e.g., “TWEEN 20”), and then placed in a solution containing an antibody (the primary antibody) which is specifically reactive with the particular cellulase under investigation. The membrane is then washed and exposed to an enzyme-antibody conjugate directed against the primary antibody (e.g., goat anti-rabbit IgG). The membrane is then exposed to a chromogenic or luminescent substrate to visualize cellulase hybridization on the membrane.

Zymograms in which the cellulase of interest is resolved in a gel system and then assayed for activity within the gel provide a relatively simple way to assess the activity of cellulases in crude cell lysates. See Coughlan (1988). In this approach, plant tissue is ground in the presence of an appropriate grinding buffer (100 mM Tris-HCl pH 9.0, 5 mM 2-mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride, 0.5 mM ethylenediamine-tetraacetic acid, for example). After grinding of the tissue, an equal volume of a 50% (v/v) slurry of washed polyvinylpolypyrrolidone (suspended in grinding buffer) is added and mixed thoroughly. After centrifugation of the mixture, a sample of the cleared extract is subjected to electrophoresis through a non-denaturing (8%, w/v) polyacrylamide gel. The resulting gel is used to prepare a sandwich with a thin film (<2 mm) of agarose (0.7% agarose, 0.5% Sigma medium viscosity carboxymethycellulose) bonded to “GELBOND” film (FMC Corporation). After incubation for 1.5 hours at 50° C., the agarose film is stained with “CONGO RED” dye for 30 minutes followed by a 1M NaCl wash. After several minutes, it is possible to visualize cellulase activity as a clear zone within a background of red staining.

Cellulase activity is most commonly assayed in aqueous solution, using a cellulosic substrate and monitoring the reaction for either the release of a chromophore/fluorophore or release of cellobiose (“reducing sugar”). For example, T. fusca E2 activity can be measured by incubating a sample of the enzyme in a 0.4 ml reaction containing 1% (w/v) low viscosity carboxymethylcellulose (Sigma C-5678) and 50 mM NaOAc pH 5.5 at 55° C. for 2-20 hours. 1.0 ml of DNS solution, see Irwin et al. (1993), is then added and the mixture is boiled for 15 minutes. Measurement of absorbance values at 600 nm for each reaction can then be correlated to values determined for a known series of glucose standards to determine the extent of carboxymethylcellulose hydrolysis. For plant extracts, background values are determined by preparing parallel reaction samples which contain no substrate and subtracting this value from that obtained in the presence of 1% carboxymethylcellulose.

For a more complete discussion of cellulase assays, see Adney et al. (1994), Baker et al. (1992), Tucker et al. (1989) and Irwin et al. (1993).

Isolation of Cellulase Activity from Plants:

It is most preferred that, where applicable, the enzyme not be purified from the plant material, but rather that the plant material containing the cellulase activity be used directly. This is demonstrated in the Examples, below, where transgenic alfalfa which expressed cellulase activity is added directly to silage materials to further the extent of fermentation.

If isolation of the cellulase activity is desired, this can be accomplished by any means known to the art. For example, the preferred T. fusca E2, E3, and CBH I enzymes are taken from thermo-tolerant bacteria. The activity of these enzymes remains unchanged by treatments up to about 55-60° C. Therefore, these enzymes can be isolated by gently heating the plant material in aqueous buffered solution (100 mM Tris/HCl pH 9.0, for example) to precipitate the bulk of plant proteins. The soluble cellulase enzymes are then recovered and further purified by any means known to the art, including HPLC, affinity chromatography, and the like. To facilitate downstream processing of the enzyme, a purification tag may optionally be incorporated into the expressed cellulase.

Since the above-mentioned enzymes are well-characterized, the preferred purification scheme is based on established protocols already in existence. For example, T. fusca E2 from a heat-treated plant extract is further purified by adsorption to a phenyl “SEPHAROSE” column in the presence if 0.8 M ammonium sulfate. Successive column washes using ammonium sulfate concentrations of 0.6 M and 0.3 M in a buffer containing 5 mM KPi, pH 6.0 and 5 mM NaCl are followed by a final wash with 0.1 M KPi, pH 6.0. Elution of E2 is accomplished using 5 mM KPi, pH 6.0. Peak fractions are loaded on a hydroxylapatite column (equilibrated with 1 mM KPi, pH 6.0) and the flow-through fractions collected and pooled. The pooled fractions are loaded on a “Q-SEPHAROSE” column (pre-equilibrated with 10 mM BisTris, pH 6.0) and eluted with a continuous buffer gradient from 5 mM BisTris, pH 6.0 to 150 mM NaCl, 5 mM BisTris, pH 6.0. Peak fractions from the “Q-SEPHAROSE” column are then pooled, concentrated and stored frozen in convenient aliquots. Similarly detailed protocols exist for both T. fusca E3 and T. reesei CBH I, see Irwin et al. (1993).

The following Examples are included solely to aid in a more complete understanding of the manufacture and use of the transgenic plants disclosed and claimed herein. The Examples do not limit the scope of the invention in any fashion.

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Divne et al. (1994), The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. Science 265:524-528.

Ghangas & Wilson (1988), Cloning of the Thermomonospora fusca endoglucanase E2 gene in Streptomyces lividans : Affinity purification and functional domains of the cloned gene product. Appl. Envir. Microbiol . 54:2521-2526.

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Horsh et al. (1985), A simple and general method for transferring genes into plants. Science 227:1229-1231.

Irwin et al. (1993), Activity studies of eight purified cellulases: Specificity, synergism, and binding domain effects. Biotechnol. Bioeng . 42:1002-1013.

Irwin et al. (1999), Characterization of a Thermomonospora fusca family 48 exocellulase E6. Direct Genbank submission AF144563.

Ishida et al. (1996), High efficiency transformation of maize mediated by Agrobacterium tumefaciens. Nature Biotechnology 14:745-750.

Joliff et al. (1986), Nucleotide sequence of the cellulase gene celD encoding endoglucanse D of Clostridium thermocellum. Nucleic Acids Res . 14:8605-8613.

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<160> NUMBER OF SEQ ID NOS: 32

<210> SEQ ID NO 1

<211> LENGTH: 1621

<212> TYPE: DNA

<213> ORGANISM: Thermomonospora fusca

<400> SEQUENCE: 1

cgatatggat gatctgacgt ctgaatcccc ttgtcaccct agacattcac ccattttgtc 60

gcttttacgg ctttctttgg gagttctccg tttcaccaag gaacaaaacc gcaacggaga 120

gtaggcgcgg tctttacagc tcccttgcca atggttatcg tccgaacgga aaacgatctg 180

ggagcgctcc cagccatgcg ctcctcttcg tgcccctcac ttcttttgag ccttgtgctc 240

gttaggagcc ccgaatgtcc cccagacctc ttcgcgctct tctgggcgcc gcggcggcgg 300

ccttggtcag cgcggctgct ctggccttcc ggtcgcaagc ggcggccaat gattctccgt 360

tctacgtcaa ccccaacatg tcctccgccg aatgggtgcg gaacaacccc aacgacccgc 420

gtaccccggt aatccgcgac cggatcgcca gcgtgccgca gggcacctgg ttcgcccacc 480

acaaccccgg gcagatcacc ggccagatcg acgcgctcat gagcgccgcc caggccgccg 540

gcaagatccc gatcctggtc gtgtacaacg ccccgggccg cgactgcggc aaccacagca 600

gcggcggcgc ccccagtcac agcgcctacc ggtcctggat cgacgaattc gctgccggac 660

tgaagaaccg tcccgcccac atcatcgtcg ggccggacct gatctcgctg atgtcgagct 720

gcatccagca cgtccagcag gaagtcctgg agacgatggc gtacgcgggc aaggccctca 780

aggccgggtc ctcgcaggcg cggatctact tcgacgccgg ccactccgcg tggcactcgc 840

ccgcacagat ggcttcctgg ctccagcagg ccgacatctc caacagcgcg cacggtatcg 900

ccaccaacac ctccaactac cggtggaccg ctgacgaggt cgcctacgcc aaggcggtgc 960

tctcggccat cggcaacccg tccctgcgcg cggtcatcga caccagccgc aacggcaacg 1020

gccccgccgg taacgagtgg tgcgacccca gcggacgcgc catcggcacg cccagcacca 1080

ccaacaccgg cgacccgatg atcgacgcct tcctgtggat caagctgccg ggtgaggccg 1140

acggctgcat cgccggcgcc ggccagttcg tcccgcaggc ggcctacgag atggcgatcg 1200

ccgcgggcgg caccaacccc aacccgaacc ccaacccgac gcccaccccc actccgaccc 1260

ccacgccgcc tcccggctcc tcgggggcgt gcacggcgac gtacacgatc gccaacgagt 1320

ggaacgacgg cttccaggcg accgtgacgg tcaccgcgaa ccagaacatc accggctgga 1380

ccgtgacatg gaccttcacc gacggccaga ccatcaccaa cgcctggaac gccgacgtgt 1440

ccaccagcgg ctcctcggtg accgcgcgga acgtcggcca caacggaacg ctctcccagg 1500

gagcccccac agagttcggc ttcgtcggct ctaagggcaa ctccaactct gttccgaccc 1560

ttacctgcgc cgccagctga cccctcctgg cagtgcactg ggtggcttag gcgtgctggg 1620

g 1621

<210> SEQ ID NO 2

<211> LENGTH: 3503

<212> TYPE: DNA

<213> ORGANISM: Thermomonospora fusca

<400> SEQUENCE: 2

cggcgatccc cctcatcatt caggtgcggt tagttccccc aggctaccga ggaccgaatt 60

tcggtccgtt tttcttgcgg cgagccctga gaccgtttcc tgttccgttc cgtcaccatc 120

cttgcgcgtc ccggcggagg ggggaagcac cccgcgagat ggctccgcca cggcctgttt 180

ccgacccccg tcacaaaagc ccatttaacg cggtatttac aaccggtcat gaagtggcta 240

ctctcttttg ggagcgctcc cgtgccgcta gtcacactgg gacgtgaatg gcgtcacggt 300

agggctcgtc gtgtgacacg cattttcgac cctgctttaa gtccctaagt gggagcgctc 360

ccagccttcg ggagaactcc cacacaacca accgtccgac gccactctcc cagcgctcaa 420

acggaggcag cagtgttcac catcccccgc tcccctccgg ggcgcccggc cgtcgtccgc 480

gcaaccaccc cgaccggtcg gctgaacact gcagcgtccg gttctcgacc atccccttgc 540

gagagaacat cctccaacca aggaagacac cgatatgagt aaagttcgtg ccacgaacag 600

acgttcgtgg atgcggcgcg ggctggcagc cgcctctgga ctggcgcttg gcgcctccat 660

ggtggcgttc gctgctccgg ccaacgccgc cggctgctcg gtgtactaca cggtcaactc 720

ctggggtacc gggttcaccg ccaacgtcac catcaccaac ctcggcagtg cgatcaacgg 780

ctggaccctg gagtgggact tccccggcaa ccagcaggtg accaacctgt ggaacgggac 840

ctacacccag tccgggcagc acgtgtcggt cagcaacgcc ccgtacaacg cctccatccc 900

ggccaacgga acggttgagt tcgggttcaa cggctcctac tcgggcagca acgacatccc 960

ctcctccttc aagctgaacg gggttacctg cgacggctcg gacgaccccg accccgagcc 1020

cagcccctcc cccagccctt cccccagccc cacagacccg gatgagccgg gcggcccgac 1080

caacccgccc accaaccccg gcgagaaggt cgacaacccg ttcgagggcg ccaagctgta 1140

cgtgaacccg gtctggtcgg ccaaggccgc cgctgagccg ggcggttccg cggtcgccaa 1200

cgagtccacc gctgtctggc tggaccgtat cggcgggatc gagggcaacg acagcccgac 1260

caccggctcc atgggtctgc gcgaccacct ggaggaggcc gtccgccagt ccggtggcga 1320

cccgctgacc atccaggtcg tcatctacca cctgcccggc cgcgactgcg ccgcgctggc 1380

ctccaacggt gagctgggtc ccgatgaact cgaccgctac aagagcgagt acatcgaccc 1440

gatcgccgac atcatgtggg acttcgcaga ctacgagaac ctgcggatcg tcgccatcat 1500

cgagatcgac tccctgccca acctcgtcac caacgtgggc gggaacggcg gcaccgagct 1560

ctgcgcctac atgaagcaga acggcggcta cgtcaacggt gtcggctacg ccctccgcaa 1620

gctgggcgag atcccgaacg tctacaacta catcgacgcc gcccaccacg gctggatcgg 1680

ctgggactcc aacttcggcc cctcggtgga catcttctac gaggccgcca acgcctccgg 1740

ctccaccgtg gactacgtgc acggcttcat ctccaacacg gccaactact cggccactgt 1800

ggagccgtac ctggacgtca acggcaccgt taacggccag ctcatccgcc agtccaagtg 1860

ggttgactgg aaccagtacg tcgacgagct ctccttcgtc caggacctgc gtcaggccct 1920

gatcgccaag ggcttccggt ccgacatcgg tatgctcatc gacacctccc gcaacggctg 1980

gggtggcccg aaccgtccga ccggaccgag ctcctccacc gacctcaaca cctacgttga 2040

cgagagccgt atcgaccgcc gtatccaccc cggtaactgg tgcaaccagg ccggtgcggg 2100

cctcggcgag cggcccacgg tcaacccggc tcccggtgtt gacgcctacg tctgggtgaa 2160

gcccccgggt gagtccgacg gcgccagcga ggagatcccg aacgacgagg gcaagggctt 2220

cgaccgcatg tgcgacccga cctaccaggg caacgcccgc aacggcaaca acccctcgga 2280

tgcgctgccc aacgccccca tctccggcca ctggttctct gcccagttcc gcgagctgct 2340

ggccaacgcc tacccgcctc tgtaaagcgg agtgaggcaa cggctgacag cctcaacgag 2400

gaactgatca gcacctccta gccggagacg gcgcccgtcc actccccgtg ggcgggcgcc 2460

gcttttatgc cgacccgtgc cccagccgca aggggcacgg gtcggcctat tccggcgatg 2520

tcggtcacgt cgccctagca cccggaaacg ccgagaaaga ctgccccgaa acggtcctct 2580

cccatccctg cattaggttg gccgagtccg cctatggctt cgtgggccgg aacccaaccc 2640

accatcaacg agaggtatca ccatggccag tgtggtgaaa ttcaatgtgc tgacggttcc 2700

tcccggtgcc ggcgccaccc cggaggacgt ttgccaagcg cgcaggcctc gtggagaacc 2760

gggccgggtt tcacgagttc caactgccgg cgcccggcga cgggacggac aagtacatcg 2820

tctacacgcg ctggcgctcc ggagaggact accagaactg gctgaacagc gaggccttcc 2880

agcgcggaca cgcccaggcc tctgaagact cccgccgcag cagccagggc ggcccggccg 2940

cgtccgcgag tgaactctgg tccttcgaag tcgtccagca cgtccaggcc caggactgat 3000

cccggtgcgg ccctcggttc tttaccgggg gccgcccacc cccttcatcc cttttcttct 3060

cccccgcacc ccttttgatc tgcaatgatg gaattcgcga ttcttgagaa ggccgatcgt 3120

gtccatgacc gcgcagaagg caggacgacc acgcgtaccg gtcgacatcg aaggagtcaa 3180

ctgacagtgg ggactatcgc ggggctgatt gtcgcgctgt caggcgtggg gatggtctcg 3240

gccaacgtgc tcccgtggga accgtcggac ccggcatccg tggtccccgc cacctcgcag 3300

ggcagcagtt ctcccatgac gccggagccc tcgcgtcccc ggtaccccca ctcgtgcgct 3360

ccgtggtcga agaggtgccc agcgcaagcg gagaactgcg ggtcgtcgaa ggtgacgggg 3420

aggtcgtcgg cgaaggcacg ctcctgcgct acctggtgga ggtcgaagaa gggcttcccg 3480

gagaccccgc cgacttcgct gca 3503

<210> SEQ ID NO 3

<211> LENGTH: 21

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: VSP Leader

Sequence

<400> SEQUENCE: 3

Met Lys Leu Phe Val Phe Phe Val Ala Ala Val Val Leu Val Ala Trp

1 5 10 15

Pro Cys His Gly Ala

20

<210> SEQ ID NO 4

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Xba E2 PCR

Primer

<400> SEQUENCE: 4

gctctagatg aatgattctc cgttc 25

<210> SEQ ID NO 5

<211> LENGTH: 17

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Xba E2 PCR

Primer

<400> SEQUENCE: 5

tgaccggcag caaaatg 17

<210> SEQ ID NO 6

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Xba E3 PCR

Primer

<400> SEQUENCE: 6

gctctagatg gccggctgct cggtg 25

<210> SEQ ID NO 7

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: RIE3 PCR

Primer

<400> SEQUENCE: 7

ggaattctta cagaggcggg tag 23

<210> SEQ ID NO 8

<211> LENGTH: 3004

<212> TYPE: DNA

<213> ORGANISM: Acidothermus cellulolyticus

<300> PUBLICATION INFORMATION:

<400> SEQUENCE: 8

ggatccacgt tgtacaaggt cacctgtccg tcgttctggt agagcggcgg gatggtcacc 60

cgcacgatct ctcctttgtt gatgtcgacg gtcacgtggt tacggtttgc ctcggccgcg 120

attttcgcgc tcgggcttgc tccggctgtc gggttcggtt tggcgtggtg tgcggagcac 180

gccgaggcga tcccaatgag ggcaagggca agagcggagc cgatggcacg tcgggtggcc 240

gatggggtac gccgatgggg cgtggcgtcc ccgccgcgga cagaaccgga tgcggaatag 300

gtcacggtgc gacatgttgc cgtaccgcgg acccggatga caagggtggg tgcgcgggtc 360

gcctgtgagc tgccggctgg cgtctggatc atgggaacga tcccaccatt ccccgcaatc 420

gacgcgatcg ggagcagggc ggcgcgagcc ggaccgtgtg gtcgagccgg acgattcgcc 480

catacggtgc tgcaatgccc agcgccatgt tgtcaatccg ccaaatgcag caatgcacac 540

atggacaggg attgtgactc tgagtaatga ttggattgcc ttcttgccgc ctacgcgtta 600

cgcagagtag gcgactgtat gcggtaggtt ggcgctccag ccgtgggctg gacatgcctg 660

ctgcgaactc ttgacacgtc tggttgaacg cgcaatactc ccaacaccga tgggatcgtt 720

cccataagtt tccgtctcac aacagaatcg gtgcgccctc atgatcaacg tgaaaggagt 780

acgggggaga acagacgggg gagaaaccaa cgggggattg gcggtgccgc gcgcattgcg 840

gcgagtgcct ggctcgcggg tgatgctgcg ggtcggcgtc gtcgtcgcgg tgctggcatt 900

ggttgccgca ctcgccaacc tagccgtgcc gcggccggct cgcgccgcgg gcggcggcta 960

ttggcacacg agcggccggg agatcctgga cgcgaacaac gtgccggtac ggatcgccgg 1020

catcaactgg tttgggttcg aaacctgcaa ttacgtcgtg cacggtctct ggtcacgcga 1080

ctaccgcagc atgctcgacc agataaagtc gctcggctac aacacaatcc ggctgccgta 1140

ctctgacgac attctcaagc cgggcaccat gccgaacagc atcaattttt accagatgaa 1200

tcaggacctg cagggtctga cgtccttgca ggtcatggac aaaatcgtcg cgtacgccgg 1260

tcagatcggc ctgcgcatca ttcttgaccg ccaccgaccg gattgcagcg ggcagtcggc 1320

gctgtggtac acgagcagcg tctcggaggc tacgtggatt tccgacctgc aagcgctggc 1380

gcagcgctac aagggaaacc cgacggtcgt cggctttgac ttgcacaacg agccgcatga 1440

cccggcctgc tggggctgcg gcgatccgag catcgactgg cgattggccg ccgagcgggc 1500

cggaaacgcc gtgctctcgg tgaatccgaa cctgctcatt ttcgtcgaag gtgtgcagag 1560

ctacaacgga gactcctact ggtggggcgg caacctgcaa ggagccggcc agtacccggt 1620

cgtgctgaac gtgccgaacc gcctggtgta ctcggcgcac gactacgcga cgagcgtcta 1680

cccgcagacg tggttcagcg atccgacctt ccccaacaac atgcccggca tctggaacaa 1740

gaactgggga tacctcttca atcagaacat tgcaccggta tggctgggcg aattcggtac 1800

gacactgcaa tccacgaccg accagacgtg gctgaagacg ctcgtccagt acctacggcc 1860

gaccgcgcaa tacggtgcgg acagcttcca gtggaccttc tggtcctgga accccgattc 1920

cggcgacaca ggaggaattc tcaaggatga ctggcagacg gtcgacacag taaaagacgg 1980

ctatctcgcg ccgatcaagt cgtcgatttt cgatcctgtc ggcgcgtctg catcgcctag 2040

cagtcaaccg tccccgtcgg tgtcgccgtc tccgtcgccg agcccgtcgg cgagtcggac 2100

gccgacgcct actccgacgc cgacagccag cccgacgcca acgctgaccc ctactgctac 2160

gcccacgccc acggcaagcc cgacgccgtc accgacggca gcctccggag cccgctgcac 2220

cgcgagttac caggtcaaca gcgattgggg caatggcttc acggtaacgg tggccgtgac 2280

aaattccgga tccgtcgcga ccaagacatg gacggtcagt tggacattcg gcggaaatca 2340

gacgattacc aattcgtgga atgcagcggt cacgcagaac ggtcagtcgg taacggctcg 2400

gaatatgagt tataacaacg tgattcagcc tggtcagaac accacgttcg gattccaggc 2460

gagctatacc ggaagcaacg cggcaccgac agtcgcctgc gcagcaagtt aatacgtcgg 2520

ggagccgacg ggagggtccg gaccgtcggt tccccggctt ccacctatgg agcgaaccca 2580

acaatccgga cggaactgca ggtaccagag aggaacgaca cgaatgcccg ccatctcaaa 2640

acggctgcga gccggcgtcc tcgccggggc ggtgagcatc gcagcctcca tcgtgccgct 2700

ggcgatgcag catcctgcca tcgccgcgac gcacgtcgac aatccctatg cgggagcgac 2760

cttcttcgtc aacccgtact gggcgcaaga agtacagagc gaacggcgaa ccagaccaat 2820

gccactctcg cagcgaaaat gcgcgtcgtt tccacatatt cgacggccgt ctggatggac 2880

cgcatcgctg cgatcaacgg cgtcaacggc ggacccggct tgacgacata tctggacgcc 2940

gccctctccc agcagcaggg aaccacccct gaagtcattg agattgtcat ctacgatctg 3000

ccgg 3004

<210> SEQ ID NO 9

<211> LENGTH: 2220

<212> TYPE: DNA

<213> ORGANISM: Trichoderma reesei

<300> PUBLICATION INFORMATION:

<308> DATABASE ACCESSION NUMBER: Genbank E00389

<309> DATABASE ENTRY DATE: 1997-09-29

<310> PATENT DOCUMENT NUMBER: JP 1985149387-A1

<312> PUBLICATION DATE: 1985-08-06

<400> SEQUENCE: 9

aaggttagcc aagaacaata gccgataaag atagcctcat taaacggaat gagctagtag 60

gcaaagtcag cgaatgtgta tatataaagg ttcgaggtcc gtgcctccct catgctctcc 120

ccatctactc atcaactcag atcctccagg agacttgtac accatctttt gaggcacaga 180

aacccaatag tcaaccgcgg actggcatca tgtatcggaa gttggccgtc atcacggcct 240

tcttggccac agctcgtgct cagtcggcct gcactctcca atcggagact cacccgcctc 300

tgacatggca gaaatgctcg tctggtggca cttgcactca acagacaggc tccgtggtca 360

tcgacgccaa ctggcgctgg actcacgcta cgaacagcag cacgaactgc tacgatggca 420

acacttggag ctcgacccta tgtcctgaca acgagacctg cgcgaagaac tgctgtctgg 480

acggtgccgc ctacgcgtcc acgtacggag ttaccacgag cggtaacagc ctctccattg 540

gctttgtcac ccagtctgcg cagaagaacg ttggcgctcg cctttacctt atggcgagcg 600

acacgaccta ccaggaattc accctgcttg gcaacgagtt ctctttcgat gttgatgttt 660

cgcagctgcc gtaagtgact taccatgaac ccctgacgta tcttcttgtg ggctcccagc 720

tgactggcca atttaaggtg cggcttgaac ggagctctct acttcgtgtc catggacgcg 780

gatggtggcg tgagcaagta tcccaccaac aacgctggcg ccaagtacgg cacggggtac 840

tgtgacagcc agtgtccccg cgatctgaag ttcatcaatg gccaggccaa cgttgagggc 900

tgggagccgt catccaacaa cgcaaacacg ggcattggag gacacggaag ctgctgctct 960

gagatggata tctgggaggc caactccatc tccgaggctc ttacccccca cccttgcacg 1020

actgtcggcc aggagatctg cgagggtgat gggtgcggcg gaacttactc cgataacaga 1080

tatggcggca cttgcgatcc cgatggctgc gactggaacc cataccgcct gggcaacacc 1140

agcttctacg gccctggctc aagctttacc ctcgatacca ccaagaaatt gaccgttgtc 1200

acccagttcg agacgtcggg tgccatcaac cgatactatg tccagaatgg cgtcactttc 1260

cagcagccca acgccgagct tggtagttac tctggcaacg agctcaacga tgattactgc 1320

acagctgagg agacagaatt cggcggatct ctttctcaga caagggcggc ctgactcagt 1380

tcaagaaggc tacctctggc ggcatggttc tggtcatgag tctgtgggat gatgtgagtt 1440

tgatggacaa acatgcgcgt tgacaaagag tcaagcagct gactgagatg ttacagtact 1500

acgccaacat gctgtggctg gactccacct acccgacaaa cgagacctcc tccacacccg 1560

gtgccgtgcg cggaagctgc tccaccagct ccggtgtccc tgctcaggtc gaatctcagt 1620

ctcccaacgc caaggtcacc ttctccaaca tcaagttcgg acccattggc agcaccggca 1680

accctagcgg cggcaaccct cccggcggaa accgtggcac caccaccacc cgccgcccag 1740

ccactaccac tggaagctct cccggaccta cccagtctca ctacggccag tgcggcggta 1800

ttggctacag cggccccacg gtctgcgcca gcggcacaac ttgccaggtc ctgaaccctt 1860

actactctca gtgcctgtaa agctccgtgc gaaagcctga cgcaccggta gattcttggt 1920

gagcccgtat catgacggcg gcgggagcta catggccccg ggtgatttat tttttttgta 1980

tctacttctg acccttttca aatatacggt caactcatct ttcactggag atgcggcctg 2040

cttggtattg cgatgttgtc agcttggcaa attgtggctt tcgaaaacac aaaacgattc 2100

cttagtagcc atgcatttta agataacgga atagaagaaa gaggaaatta aaaaaaaaaa 2160

aaaaacaaac atcccgttca taacccgtag aatcgccgct cttcgtgtat cccagtacca 2220

<210> SEQ ID NO 10

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Nar E1 PCR

Primer

<400> SEQUENCE: 10

cggggcgccg gcggcggcta t 21

<210> SEQ ID NO 11

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Sac E1 PCR

Primer

<400> SEQUENCE: 11

ccgagctctt aacttgctgc 20

<210> SEQ ID NO 12

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Sac E1cd

PCR Primer

<400> SEQUENCE: 12

tggagctcta gacaggatcg aaaat 25

<210> SEQ ID NO 13

<211> LENGTH: 76

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: VSP leader

coding sequence

<400> SEQUENCE: 13

tctagagtcg accatgaagt tgtttgtttt ctttgttgct gcagtagttt tggtagcatg 60

gccatgccat ggcgcc 76

<210> SEQ ID NO 14

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: CBH2-2 PCR

Primer

<400> SEQUENCE: 14

gctctagatg tatcggaagt tggc 24

<210> SEQ ID NO 15

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: CBH3-1 PCR

Primer

<400> SEQUENCE: 15

cccccgggtt acaggcactg agag 24

<210> SEQ ID NO 16

<211> LENGTH: 2199

<212> TYPE: DNA

<213> ORGANISM: Cellulomonas fimi

<300> PUBLICATION INFORMATION:

<303> JOURNAL: Gene

<304> VOLUME: 44

<306> PAGES: 315-342

<307> DATE: 1986

<308> DATABASE ACCESSION NUMBER: Genbank M15823

<309> DATABASE ENTRY DATE: 1993-06-04

<400> SEQUENCE: 16

ggatccggac ggtgggcgtc gtggccgaca ccgacgcgct ggagacgacc ttcgcggacg 60

tcgcggacct cgcgcggcag tgccggttcg gcgactgccg gcacgagcgg gagccggggt 120

gcgcggtgcg ggcggccgtc gagtcgggcg acctgccggc ccggcggctg gactcgtggc 180

ggcgcctgga gcgcgaggcg gcctaccagg cacggcgcag cgacggcggc tggccgcgga 240

ggagcgcgca cgctggaaga agatcaccaa ggagtaccag cgggggatgc gcgggccggg 300

gcgtccgcgg agctgacggg cccgggaggc ccgcagccgg gcggtgggga gtccgctcgg 360

cgccagcggg tgtcgaagcg acgggtcgaa gcgcgccaac gtcgcccgat ccggaactga 420

agcgattagg aaatcctcat ccgctcgcgc cgtggggcat tcgtcgggtt tcctcgtcgg 480

gacccgcacg agcgtgccac gaggcccgaa cccagggagc tccttgatgt ccacccgcag 540

aaccgccgca gcgctgctgg cggccgcggc cgtcgccgtc ggcggtctga ccgccctcac 600

caccaccgcc gcgcaggcgg ctcccggctg ccgcgtcgac tacgccgtca ccaaccagtg 660

gcccggcggc ttcggcgcca acgtcacgat caccaacctc ggcgaccccg tctcgtcgtg 720

gaagctcgac tggacctaca ccgcaggcca gcggatccag cagctgtgga acggcaccgc 780

gtcgaccaac ggcggccagg tctccgtcac cagcctgccc tggaacggca gcatcccgac 840

cggcggcacg gcgtcgttcg ggttcaacgg ctcgtgggcc gggtccaacc cgacgccggc 900

gtcgttctcg ctcaacggca ccacctgcac gggcaccgtg ccgacgacca gccccacgcc 960

gaccccgacg ccgacgaccc ccacgccgac gccgaccccg acccccaccc ccacgccgac 1020

ggtcacgccg cagccgacca gcggcttcta cgtcgacccg acgacgcagg gctaccgcgc 1080

gtggcaggcc gcgtccggca cggacaaggc gctgctcgag aagatcgcgc tcaccccgca 1140

ggcgtactgg gtcggcaact gggccgacgc gtcgcacgcg caggccgagg tcgccgacta 1200

caccggccgc gccgtcgcgg ccgggaagac gccgatgctc gtcgtctacg cgatcccggg 1260

ccgcgactgc ggctcgcact ccggcggtgg tgtgtccgag tccgagtacg cgcgctgggt 1320

cgacaccgtc gcgcagggca tcaagggcaa cccgatcgtg atcctcgagc ccgacgcgct 1380

cgcgcagctc ggcgactgct ccggccaggg tgaccgcgtc ggcttcctca agtacgccgc 1440

caagtcgctc accctcaagg gcgcgcgcgt ctacatcgac gcgggccacg cgaagtggct 1500

gtcggtcgac acgccggtga accgcctcaa ccaggtcggc ttcgagtacg cggtgggctt 1560

cgcgctcaac acgtcgaact accagacgac ggcggacagc aaggcgtacg gccagcagat 1620

ctcgcagcgg ctgggcggca agaagttcgt catcgacacc tcgcgcaacg gcaacggctc 1680

gaacggcgag tggtgcaacc cgcgcggccg cgcgctcggc gaacgcccgg tcgcggtgaa 1740

cgacggctcc ggcctggacg cgctcctgtg ggtcaagctg cccggcgagt ccgacggcgc 1800

gtgcaacggc ggcccggccg ccggccagtg gtggcaggag atcgccctgg agatggcgcg 1860

caacgccagg tggtgagctg agacctcgcc cacgacgagc ccgcggacgg cgcacgtgcg 1920

tccgcgggct cgtccgtccg gccgcgggcg cccggacgtc ggggcggcgg ggacaatggg 1980

gcggtggcag ggcagacgac ggaccgcacc cgacgacgga cgcgccgcgc tcgacgtgtg 2040

gcgcgccgac cccgcaggcg tgccgacccc ggcgcggcgg accgcggtcc ggttcacgct 2100

cgaggagctc gccgacgtgg cccccggcaa cgcggtcgag gtgcgcgtcc cgccggacgg 2160

cggccgtgca ggccgtgcag ggcccgcggc acacccggg 2199

<210> SEQ ID NO 17

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: cenApst PCR

Primer

<400> SEQUENCE: 17

ggctgcaggc ggctgccgcg tcgac 25

<210> SEQ ID NO 18

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: cemAsac PCR

Primer

<400> SEQUENCE: 18

ccgagctctc accacctggc gtt 23

<210> SEQ ID NO 19

<211> LENGTH: 82

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: VSP leader

coding sequence

<400> SEQUENCE: 19

tctagagtcg accatgaagt tgtttgtttt ctttgttgca gcagtagttt tggtagcttg 60

gccttgccac ggcgctgcag tc 82

<210> SEQ ID NO 20

<211> LENGTH: 2286

<212> TYPE: DNA

<213> ORGANISM: Clostridium thermocellum

<300> PUBLICATION INFORMATION:

<303> JOURNAL: Nucleic Acids Res.

<304> VOLUME: 14

<305> ISSUE: 21

<306> PAGES: 8605-8613

<307> DATE: 1986

<308> DATABASE ACCESSION NUMBER: Genbank X04584

<309> DATABASE ENTRY DATE: 1999-02-10

<400> SEQUENCE: 20

aaactaaaac tcctatccaa tactttagtt cagttccagc atacgtctgt attcaaaatg 60

cctgtattta taactgcatt tataatacct gaagcaaata ataattaaac ttgtggaaga 120

aaggaggttg caacaggttt taaattatct taattcaggt attttacaat ttttaataaa 180

aagggggata aaggtaaaaa atgagtagaa tgaccttgaa aagcagcatg aaaaaacgtg 240

tgttatcttt gctcattgct gtagtgtttc taagcttgac cggagtattt ccttcgggat 300

tgattgagac caaagtgtca gctgcaaaaa taacggagaa ttatcaattt gattcacgaa 360

tccgtttaaa ctcaataggt tttataccga accacagcaa aaaggcgact atagctgcaa 420

attgttcaac cttttatgtt gttaaagaag acggaacaat agtgtatacc ggaacggcaa 480

cttcaatgtt tgacaatgat acaaaagaaa ctgtttatat tgctgatttt tcatctgtta 540

atgaagaagg aacgtactat cttgccgtgc cgggagtagg aaaaagcgta aactttaaaa 600

ttgcaatgaa tgtatatgag gatgctttta aaacagcaat gctgggaatg tatttgctgc 660

gctgcggcac cagtgtgtcg gccacataca acggaataca ctattcccat ggaccgtgcc 720

atactaatga tgcatatctt gattatataa acggacagca tactaaaaaa gacagtacaa 780

aaggctggca tgatgcgggc gactacaaca aatatgtggt aaacgccggc ataaccgttg 840

gttcaatgtt cctggcgtgg gagcatttta aagaccagtt ggagcctgtg gcattggaga 900

ttcccgaaaa gaacaattca ataccggatt ttcttgatga attaaaatat gagatagact 960

ggattcttac catgcaatac cctgacggga gcggaagggt ggctcataaa gtttcgacaa 1020

ggaactttgg cggctttatc atgcctgaga acgaacacga cgaaagattt ttcgtgccct 1080

ggagcagtgc cgcaacggca gactttgttg ccatgacggc catggctgca agaatattca 1140

ggccttatga tcctcaatat gctgaaaaat gtataaatgc ggcaaaagta agctatgagt 1200

ttttgaagaa caatcctgcg aatgtttttg caaaccagag tggattctca acaggagaat 1260

atgccactgt cagtgatgca gatgacagat tgtgggcggc ggctgaaatg tgggagaccc 1320

tgggagatga agaatacctt agagattttg aaaacagggc ggcgcaattc tcgaaaaaaa 1380

tagaagccga ttttgactgg gataatgttg caaacttagg tatgtttaca tatcttttgt 1440

cagaaagacc gggcaagaat cctgctttgg tgcagtcaat aaaggatagt ctcctttcca 1500

ctgcggattc aattgtgagg accagccaaa accatggcta tggcagaacc cttggtacaa 1560

catattactg gggatgcaac ggcacggttg taagacagac tatgatactt caggttgcga 1620

acaagatttc acccaacaat gattatgtaa atgctgctct cgatgcgatt tcacatgtat 1680

ttggaagaaa ctattacaac aggtcttatg taacaggcct tggtataaat cctcctatga 1740

atcctcatga cagacgttca ggggctgacg gaatatggga gccgtggccc ggttaccttg 1800

taggaggagg atggcccgga ccgaaggatt gggtggatat tcaggacagt tatcagacca 1860

atgaaattgc tataaactgg aatgcggcat tgatttatgc ccttgccgga tttgtcaact 1920

ataattctcc tcaaaatgaa gtactgtacg gagatgtgaa tgatgacgga aaagtaaact 1980

ccactgactt gactttgtta aaaagatatg ttcttaaagc cgtctcaact ctcccttctt 2040

ccaaagctga aaagaacgca gatgtaaatc gtgacggaag agttaattcc agtgatgtca 2100

caatactttc aagatatttg ataagggtaa tcgagaaatt accaatataa attctgataa 2160

atattgataa acactaatat ataagtgttt aatcggtaaa agagccctgt ggcaaaaact 2220

gccgcaggct gtttttatca attccggcgc agacgaaaat agcagacgta aatattaatt 2280

actgaa 2286

<210> SEQ ID NO 21

<211> LENGTH: 17

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: celDpst PCR

Primer

<400> SEQUENCE: 21

agctgcagaa ataacgg 17

<210> SEQ ID NO 22

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: celDsac PCR

Primer

<400> SEQUENCE: 22

ccgagctctt atattggtaa tttctc 26

<210> SEQ ID NO 23

<211> LENGTH: 4578

<212> TYPE: DNA

<213> ORGANISM: Clostridium cellulovorans

<300> PUBLICATION INFORMATION:

<303> JOURNAL: Gene

<304> VOLUME: 211

<305> ISSUE: 1

<306> PAGES: 39-47

<307> DATE: 1998

<308> DATABASE ACCESSION NUMBER: Genbank U34793

<309> DATABASE ENTRY DATE: 1999-03-09

<400> SEQUENCE: 23

taatatataa ataatattta aaatcaataa atcaatcggg gaaaatttaa aaaaagagag 60

ggataatcaa tgagaaaaag attaaataag atcgttgctg ttgctttaac tgcaacaact 120

atatcatcag tagcagctac tgttaataca gctcaagttt cagctgcacc agtagtgcca 180

aataatgagt atgttcaaca ctttaaggat atgtacgcta agatccataa tgcaaacaat 240

ggatacttca gtgatgaagg aataccttat cacgcagttg aaacattaat ggttgaagca 300

ccagactatg gtcatgaaac tacaagtgaa gcttgggatg taactgaaaa gtacataatt 360

ccaggtgaga ctgatcaacc aagcgcaagt atgagcaatt atgatccaaa taagccagct 420

acatatgcag ctgaacatcc agatccaagc atgtacccat ctcaattaca atttggtgct 480

gctgtaggta aggatccatt atacaatgaa ttaaaatcta cttatggaac tagccaagta 540

tatggtatgc attggttact agatgttgat aactggtatg gttttggtgg tgcaacaagc 600

acaagcccag tatacatcaa cactttccaa agaggtgttc aagaatcttg ttgggaaact 660

gtgccacaac catgtaaaga cgaaatgaag tacggtggaa gaaacggttt cttagatcta 720

ttcactggtg attcacaata cgcaactcaa tttaaatata ctaacgctcc agacgcagat 780

gctcgtgcag ttcaagctac ttactatgca caattagctg ctaaagaatg gggagtagac 840

atcagctcat atgtagcaaa atctactaag atgggtgact tcttaagata ttcattcttt 900

gataaatact ttagaaaagt tggaaattca acacaagcag gaactggata tgattcagct 960

caatacctat taaactggta ctatgcttgg ggtggtggaa tcagctcaaa ctggtcttgg 1020

agaattggat caagccataa ccatttcgga taccaaaacc caatggcagc atggatatta 1080

tcaaatacat ctgactttaa accaaagtca ccaaatgctg ctacagattg gaataacagt 1140

ttaaagagac aaatagaatt ctatcaatgg ttacaatctg ctgaaggtgg tatcgctgga 1200

ggagctagta actcaaatgg aggaagctat caagcatggc cagcaggtac tcgaacattc 1260

tacggaatgg gatatactcc tcacccagta tacgaagatc caggtagtaa cgaatggttt 1320

ggtatgcaag catggtcaat gcaacgtgtg gctgaatact actacagttc aaaagatcca 1380

gcagctaaat cattacttga taaatgggct aaatgggctt gtgcaaatgt tcaattcgat 1440

gatgcagcta agaaatttaa gattcctgct aaattagtat ggactggaca accagatact 1500

tggactggat catatacagg aaattcaaat cttcatgtta aagttgaagc ttatggagaa 1560

gatcttggag tagcaggttc actttctaat gcattatcat attatgcaaa agctcttgaa 1620

tctagcacag atgctgcaga taaagtagca tataacactg caaaagaaac ttctagaaag 1680

atacttgatt acttatgggc aagctaccaa gatgataagg gtatagcagt tactgaaaca 1740

agaaatgatt tcaaacgttt caatcaatct gtatatattc catcaggttg gacaggaaaa 1800

atgcctaatg gagatgtaat ccaaagtgga gctactttct taagcatacg ttcaaaatac 1860

aaacaagatc catcatggcc aaatgttgaa gctgctttag caaatggtac tggtgttgat 1920

atgacatacc acagattctg gggtcaaagt gatatcgcta tagcatttgg aacatacggt 1980

acattattca cagaccctac tccaggatta aaaggtgatg ttaactctga tgctaaagta 2040

aatgctatag atttagctat attaaagaaa tacatcttag attcaacaac taaaattaac 2100

actgctaatt ctgatatgaa cggtgatgga aaagttaatg caatggattt agctttatta 2160

aagaaagcac ttctgcttaa gattaaataa ctttagatcg aaattgtaag gttatttaag 2220

gctggacaat atcaagtata ttgtccagct actttaaaaa atattgggaa acactgtgta 2280

aggtaaactt aaaccatgga tatgaaatat agtaagatta atgccattgc tatggcaaac 2340

ttaaaataaa tatattagag cataaacatg aaatttaagt aaaaggcgaa taaataattc 2400

cctaatcaaa aaaattaagg ggtggaacta gtgtttaaca tatctaagaa aaaagcgcaa 2460

gctcttcttt tatcaggaat cttgggtgca acttcattta caccagctgt attggtaaaa 2520

ggtgaaacaa cagcgactcc aacattcaat tatggagaag cattacaaaa gtcaataatg 2580

ttttatgaat tccaacgttc tggaaagtta ccaacggata ttcgtagtaa ttggcgtggt 2640

gattctggaa caaaagatgg ctctgatgta ggagttgatt taactggtgg atggtatgat 2700

gctggagacc acgttaaatt taatctgcca atgtcttata ctgtggcaat gcttgcatgg 2760

tcattaagtg aagacaaagc agcttacgaa aaaagcggcc aattagatta ccttgttaag 2820

gaaataaaat gggctacaga ttatctaatg aagtgccata cggcaccaaa tgaatactat 2880

tatcaagttg gtgatggtgg agctgatcac aaatggtggg gacctgcaga agtaatgcag 2940

atggcaagac cggcttataa agtagatttg caaaaaccag gatcatcagt tgtcgctgaa 3000

acagcagcag cattagcttc tacagctttt gcattaaaag acatagataa agcgtattca 3060

gaacaatgta ttcagcatgc aaaagaactt tataactttg ctgatacaac aaagagtgat 3120

gctggttata cagcagcaaa tacatattac aattcatgga gtggatacta tgatgaatta 3180

tcatgggctg cagcatggct ttacatggca acaaatgatg catcatatct agaaaaagcg 3240

gaatcatatg ttccattttg gaaggttgaa cagcaaacaa ccactatagc atatagatgg 3300

gcgcattgtt gggatgatgt acatttcgga gctcaattac tccttgccag attaacagga 3360

aaatcaatat acaaagaatc agttgaaaga aaccttgatt attggacaac tggttatgat 3420

ggaaataaaa taaagtacac tccaaaaggt ttagcttgga tggattcttg gggctcatta 3480

agatatgcaa ctacaacggc attccttgcc gatgtttatg caagctcaga tgtttgttct 3540

atttctaagg tagatacata taagaatttt gctaagagtc aagctgatta tgctttagga 3600

agtactggaa gaagttttgt ggtaggattt ggtgaaaatg ctccaaagaa accacatcat 3660

agaactgccc atagttcatg gtcagatcaa caagtaaatc caacagacca tagacatgtt 3720

ttatatggtg ctttagttgg aggaccagat gccagtgatg gttatactga tgctattgac 3780

aattttacta ataatgaggt ggcttgtgat tataatgcag gatttgtagg acttttagct 3840

agacaatatt ctaaatatgg cggagatcca atacctgatt ttaaagcgat agaaaagcca 3900

accaacgatg agttctttgt cgaagcagga gtaaattgta caggtccaaa ttttgtagaa 3960

attaaagctt tagttaataa tagaacagga tggccagcaa gaatgggaga taaactttca 4020

ttcaaatact tcataaatgt aagtgaattt gttaatgctg gttacagtgc agatgattta 4080

aaggttactg ttggttacaa tactggcgga actgtatcaa acctaatccc atgggataag 4140

gaaaataata tttattatgt aaatgttgat ttcacagggg taaagattta tccaggtgga 4200

caatcagatt ataaaaaaga aattcaattt agaatttcag gaattcaaaa tgttaatatt 4260

tgggataatt ctgatgactt ctcttatgag gggattacaa aaactccagg tgaaacacct 4320

gtgaaggtta caaacatccc agtttatgat aatggagtta aggtattcgg aaatgaacca 4380

ggaactacta agccacctgt tatagctggt gatgtaaaca ataatggtat cgtgaattca 4440

atggatttag cgatgttaaa gaaatatata cttggatacg aagtagaaat gaataaagag 4500

gcttcagatt taaataaaga tggtaagatt aatgccattg atttcgctct tttaaagaaa 4560

ctacttttat cacagtag 4578

<210> SEQ ID NO 24

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: exgSnar PCR

Primer

<400> SEQUENCE: 24

cggggcgccg caccagtagt gcca 24

<210> SEQ ID NO 25

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: exgSsac PCR

Primer

<400> SEQUENCE: 25

ccgagctctt atttaatctt aagc 24

<210> SEQ ID NO 26

<211> LENGTH: 5971

<212> TYPE: DNA

<213> ORGANISM: Thermobifida fusca

<300> PUBLICATION INFORMATION:

<400> SEQUENCE: 26

gcggccgcca ggtggggcgg ccgagtccga cggtggcgag gacttcgacc ccgtggtcgt 60

cagcgaagca tgcttcgctg acgttggcgg cggtgagcag gcctaccccg gtgccggtca 120

tcccgaagtg ggtggcgagt tcttcgaggt ggttgtccgg gtcggtccgc cggtagtcgc 180

cgtcgacttg ggcgttgatg acgtagtccc gtggtccgag gccgccgccg agcacgctgg 240

aggcggccat gcgccagccg gggccgcact gccagagcac tgcggcaagg gcgcggccgt 300

tgtcgcgccg ccagcgcagg tgcgcggtga ggagggtgcc gtcgccgatc gtctccgtgt 360

ccactccgct ctccagggtc ggttccgcgg ggccacttta acgagtccgc atcgggcggc 420

agcggggtca tgcttccatc tgggcgaatg ccacgcggag tactgcgagt ctcacgcttc 480

gtcccattgt ctttttacgg agggtttaca cggcacatcc ggaacgttac cctcctactg 540

ggagcgctcc cgtgactcag gccacaggcc cccttcccgg ggcgagcccc caccaccccg 600

ggaaggccac tgcgacccct gagctcaatt catgggagcg ctcccatgcc agtgctcccg 660

gcagaaagga gagaaccgga acccggtacc gctggtttca ctgatccccc tgttttcacc 720

cggcatcgcc ccgacaccag cgatgccacc cccgcggcgg ctgcagtcca cacccggcac 780

gccgcacacc cctctcctgt gtgacacgcg gttccgaacg gccttgccgg ttctcggtat 840

gccgacgaac ggcacggctg ctccggaacc ggaagatccg gaggttattt ccaagcatga 900

gatcgttact gtctccccgg cgctggcgca cgctggcctc gggggcgctc gcagcggccc 960

tggccgccgc tgtactctcc cccggcgtcg cgcacgccgc cgtcgcctgc tcggtggact 1020

acgacgactc caacgactgg ggtagcgggt tcgtcgccga agtcaaggtg accaacgaag 1080

gcagcgaccc catccagaac tggcaagtag gctggacctt ccccggtaac cagcagatca 1140

ccaacggctg gaacggcgtg ttcagccaga gcggcgccaa cgtcaccgtc cgctacccgg 1200

actggaaccc caatatcgcc cccggagcca ccatctcctt cggcttccag ggcacctaca 1260

gcggctccaa cgacgccccg accagcttca ccgtcaacgg cgtcacctgc agcggatccc 1320

agcccgccaa cctgccgccc gatgtcaccc tgacatcccc ggccaacaac tcgaccttcc 1380

tggtcaacga cccgatcgag ctgaccgcgg tcgcctccga ccccgacggc tcgatcgacc 1440

gggtggaatt cgccgccgac aacaccgtca tcggcatcga caccacctcc ccctacagct 1500

tcacctggac ggacgctgcc gccggctcct actcggtgac cgcgatcgcc tacgacgacc 1560

agggagccag gaccgtctcc gctcccatcg ccatccgagt gctggaccgg gccgccgtca 1620

tcgcctcacc gcccaccgtc cgcgtgccgc agggcggcac cgccgacttc gaggtgcggc 1680

tgtccaacca gccctccggc aacgtcacgg tcaccgtggc gcgcacgtcg ggcagctccg 1740

acctgaccgt ctccagcggc tcccaactcc agttcacctc cagcaactgg aaccagccgc 1800

agaaggtgac catcgcctcc gctgacaacg gcggaaacct ggccgaggcg gtcttcaccg 1860

tcagcgcccc cggccacgac tcggccgagg tgacggtccg ggagatcgac ccgaacacca 1920

gctcctacga ccaggccttc ctggagcagt acgagaagat caaggacccc gccagcggct 1980

acttccgcga attcaacggg ctcctggtcc cctaccactc ggtggagacc atgatcgtcg 2040

aggctccgga ccacggccac cagaccacgt ccgaggcgtt cagctactac ctgtggctgg 2100

aggcgtacta cggccgggtc accggtgact ggaagccgct ccacgacgcc tgggagtcga 2160

tggagacctt catcatcccc ggcaccaagg accagccgac caactccgcc tacaacccga 2220

actccccggc gacctacatc cccgagcagc ccaacgctga cggctacccg tcgcctctca 2280

tgaacaacgt cccggtgggt caagacccgc tcgcccagga gctgagctcc acctacggga 2340

ccaacgagat ctacggcatg cactggctgc tcgacgtgga caacgtctac ggcttcgggt 2400

tctgcggcga cggcaccgac gacgcccccg cctacatcaa cacctaccag cgtggtgcgc 2460

gcgagtcggt gtgggagacc attccgcacc cgtcctgcga cgacttcacg cacggcggcc 2520

ccaacggcta cctggacctg ttcaccgacg accagaacta cgccaagcag tggcgctaca 2580

ccaacgcccc cgacgctgac gcgcgggccg tccaggtgat gttctgggcg cacgaatggg 2640

ccaaggagca gggcaaggag aacgagatcg cgggcctgat ggacaaggcg tccaagatgg 2700

gcgactacct ccggtacgcg atgttcgaca agtacttcaa gaagatcggc aactgcgtcg 2760

gcgccacctc ctgcccgggt ggccaaggca aggacagcgc gcactacctg ctgtcctggt 2820

actactcctg gggcggctcg ctcgacacct cctctgcgtg ggcgtggcgt atcggctcca 2880

gctcctcgca ccagggctac cagaacgtgc tcgctgccta cgcgctctcg caggtgcccg 2940

aactgcagcc tgactccccg accggtgtcc aggactgggc caccagcttc gaccgccagt 3000

tggagttcct ccagtggctg cagtccgctg aaggtggtat cgccggtggc gccaccaaca 3060

gctggaaggg aagctacgac accccgccga ccggcctgtc gcagttctac ggcatgtact 3120

acgactggca gccggtctgg aacgacccgc cgtccaacaa ctggttcggc ttccaggtct 3180

ggaacatgga gcgcgtcgcc cagctctact acgtgaccgg cgacgcccgg gccgaggcca 3240

tcctcgacaa gtgggtgccg tgggccatcc agcacaccga cgtggacgcc gacaacggcg 3300

gccagaactt ccaggtcccc tccgacctgg agtggtcggg ccagcctgac acctggaccg 3360

gcacctacac cggcaacccg aacctgcacg tccaggtcgt ctcctacagc caggacgtcg 3420

gtgtgaccgc cgctctggcc aagaccctga tgtactacgc gaagcgttcg ggcgacacca 3480

ccgccctcgc caccgcggag ggtctgctgg acgccctgct ggcccaccgg gacagcatcg 3540

gtatcgccac ccccgagcag ccgagctggg accgtctgga cgacccgtgg gacggctccg 3600

agggcctgta cgtgccgccg ggctggtcgg gcaccatgcc caacggtgac cgcatcgagc 3660

cgggcgcgac cttcctgtcc atccgctcgt tctacaagaa cgacccgctg tggccgcagg 3720

tcgaggcaca cctgaacgac ccgcagaacg tcccggcgcc gatcgtggag cgccaccgct 3780

tctgggctca ggtggaaatc gcgaccgcgt tcgcagccca cgacgaactg ttcggggccg 3840

gagctccctg atcctgaaca gtccgtctcc tgaccggcgg tccgtggcgg cgtagtgctc 3900

ccccgccgcg gaccgccctc caccaccccc ctggggccgt ccggcgcacc aacaccggac 3960

ggccccagac tcctttcacg gcctgctact cggcccgacg ccccgcccta tccagcccga 4020

tcaaccaggt cagcgtagtc cgggtgccgc tcgagccacg ccttgacata ggggcagacc 4080

gcacgcaccc gcagcccgcg ggcccgcaca tcatccaacg cctcccgcac cagccggctg 4140

cccatcccct tcccctggaa agagccgtcc acccgagtgt ggaagaagac cacccgatca 4200

ccggcaacag gctggtattc cgtgaaaccg gcgattttct ccccggcacg gatctcgtac 4260

cggtgctgtg ccggattgtc gatcaccgtg acatccatgg cgttcccctc ccgttcccca 4320

ggcggcggcg catctctcat ccggcggcct gcccaggact gtcctcttcc ccaggacagc 4380

ctgagcacgc gcccgcacgg cgaaccccag ccacagccct agcatggtga acggccacgg 4440

tcgcagggcg accacgacac cgaggaggag tgatgggcga cggcggcaca gtacgccacc 4500

cttccgtacg tgccgtatca ccggcagcgt tggacgaggc ggtacgcgtc ctgcgggcag 4560

gcggcctcgt agctttcccc accgaaaccg tctacggcct aggcgcggac gccgcgaacc 4620

cggccgcggt agcgcgcatt ttcgccgcca aaggccgtcc cgctgaccac cccctgatcg 4680

tgcacgtcgc ttccgcggag agcgcgcgcg actgggctgc tacctttcct cctctcgccc 4740

gcacgctggc ggacgcgttc tggcccggcc cgctgaccct catcctcccc cgctccgccc 4800

aagtgccgga cgcagtgacc ggtggacgcg ccacggtagg gctgcgggtc cccgaccagc 4860

cggtcgcgct cgcactcctg gaacgcttcg gcggcggaat cgccgcacct tccgcgaacc 4920

ggttcggccg agtgagcccg accacggccg cgcacgttgc cgctgacctc ggggaccggg 4980

tcgacctggt gttggacggc ggaccgtgca cggtcggcgt ggaatcgacg atcgtcgaag 5040

tggccgacgg ccggctcacc gtgctccgca ccggaggcat cacccccgac gaccttgccg 5100

cggtcaccgg agcccccgtc gacaccaccc ccaccggacc ggcccgggca cccggcatgc 5160

tcgccgccca ctacgcaccc gccgcacggg tcgtgctagc cgaagcagca gaagccgcgg 5220

acacggtcgc ccagtgggtg gagaaaggac accgggtggc tgtgctggcg gagaccgcta 5280

ccgtgcccga aaacctgccg gaaggcgtgg tggtgctacc gtcccctgct tcggctcggg 5340

actatgcccg cgtgctgtac cagcggctgc gggacgtgga cgcggcggga gccgacgtgg 5400

tcgtcgcgat cccccccgaa cccgcgggga tcggcttggc ggtacgggac cggctgctgc 5460

gcgcatcccg ggcgcactga cctctccccc tggggcaagg gatttttccg catagacgag 5520

cccgtttccg ggatctcttt ctgtggagac agaaagagag caccgacacc agggagggcc 5580

gatgaaagct caagccggag accggatcgt tgtggaacgc ccccgcgatg acctgcccgc 5640

gcgcaaaggc gtcgtgctca aagtgcaggg ggacaacggg ggcccgccct actgggtgcg 5700

gtgggaggac gaaggccggg aaaccctcgt ctacccggga ccggacgccc gtatcgagcc 5760

ccgccacccc gtcccccaag cccgccagga gcatacggag gcccgtcaac cgcagccggg 5820

acagtccctg aaacggatac agatcgacgt cgcagtgtcg gaggtgcacg agaacggctc 5880

cgtgcgcacc ctcgccgaag cgcaactgcc gtccaccaag tggaacctgc gcggccacgg 5940

agaagcacgc aagcatccca ccgacgccga t 5971

<210> SEQ ID NO 27

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: celFpst

PCR Primer

<400> SEQUENCE: 27

acgctgcagt cgcctgctcg g 21

<210> SEQ ID NO 28

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: celFxma

PCR Primer

<400> SEQUENCE: 28

cccccgggtc agggagctcc ggc 23

<210> SEQ ID NO 29

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: 2777 PCR

Primer

<400> SEQUENCE: 29

ggccacctgg gcagg 15

<210> SEQ ID NO 30

<211> LENGTH: 17

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: M13-20 PCR

Primer

<400> SEQUENCE: 30

gtaaaacgac ggccagt 17

<210> SEQ ID NO 31

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: 3227 PCR

Primer

<400> SEQUENCE: 31

gcgacgctcg ggccg 15

<210> SEQ ID NO 32

<211> LENGTH: 16

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Reverse

3227 PCR Primer

<400> SEQUENCE: 32

aacagctatg accatg 16