MXPA00002971A - Expression of enzymes involved in cellulose modification - Google Patents

Abstract

Novel compositions and methods useful for genetic engineering of plant cells to provide expression of cellulose degrading enzymes in the plants or plant cells.

Description

EXPRESSION OF ENZYMES INVOLVED IN THE MODIFICATION OF CELLULOSE INTRODUCTION This application is a continuation in part of the Application Serial No. 09 / 122,533 filed July 24, 1998. Technical Field This invention relates to the application of plant genetic design techniques. More specifically, the invention relates to compositions and methods for expressing the hydrolyzing enzymes of polysaccharides (cellulases, cellobio idrolases, xylanases, hemicellulases) in plant plastids. BACKGROUND Polysaccharide hydrolyzing enzymes are a family of enzymes that work together to break down cellulose into its simple sugar components. Cellulose can also be degraded industrially through acidic hydrolysis using conditions more severe than those required by enzymes. In addition, polysaccharide hydrolyzing enzymes catalyze highly specific reactions, producing specific products, and are required in much smaller amounts compared to acidic hydrolysis reactions. Cellulose degrading enzymes are used for a wide variety of industrial applications. One of the main potential uses of such enzymes is found in the conversion of cellulosic biomass into industrially important end products (for example, sugars, which can be fermented to produce a variety of products). For example, in fuel production, ethanol can be produced from grains such as corn. A similar process is currently under development that uses highly cellulose rice straws. Unfortunately, the ethanol produced by such methods is still too expensive to compete commercially with gasoline. However, improvements in technology for using wood, grass and other biomass containing high cellulose for ethanol production would be valuable in the art for producing a cleaner and less expensive fuel source. In addition to the conversion of biomass, cellulose degrading enzymes find utility in a variety of other industrial processes and products including: textile finishing, detergent additive production, food and beverage processing, food additives, and fermentation and silage processes . It is generally believed that current methods for the production of cellulose degrading enzymes are limited to the further development of a lignocellulosic ethanol industry. Filamentous fungi are well known for the production of industrial cellulases. However, the economic production of cellulose degrading enzymes is composed by the relatively slow growth rates of the cellulose-degrading enzyme that produces fungi, the long time periods required for the induction of the enzyme and the high value of the ethanol product. , the genes encoding the cellulose degrading enzymes have been cloned from a variety of fungi and celuiitic bacteria. Cloned genes encoding celluloses that have highly specific activities over a wide pH range in addition to elevated thermostability are considered more desirable for the biotenanol derivative process. Fungal or recombinant bacterial hosts that produce cellulose degrading enzymes have been the focus of recent efforts for the production of various cellulase preparations. However, methods for the production of polysaccharide degrading enzymes in plant cells are necessary. Such methods will provide a less expensive and abundant source of cellulose degrading enzymes.

SUMMARY OF THE INVENTION The present invention provides methods and compositions for the production of polysaccharide hydrolyzing enzymes in plant cells. The methods generally involve the use of an expression construct having a promoter, a nucleic acid sequence encoding a polysaccharide hydrolyzing enzyme, and a transcription termination region for the transformation of the plant. Preferably, the promoter is functional in a plant cell plastid.

By this invention, a method is described by which constructs encoding a polysaccharide hydrolyzing enzyme can be produced in plant cells. In one embodiment of the present invention, methods for expression of the elevated level of a polysaccharide hydrolyzing enzyme in plant plastids are described. The method provided for the expression of the high level of the polysaccharide hydrolyzing enzyme in plant cells offers a novel means for the production of cellulose degrading enzymes (cellulases, cellobiohydrolases, xylanases, hemicellulases) in plant cells. The method generally comprises the growth of a plant having integrated into its genome a construct comprising as components operably linked in the direction of transcription from 5 'to 3', a region of transcription initiation, functional in a plant cell , a DNA sequence encoding a polysaccharide hydrolyzing enzyme and a transcription termination sequence.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic representation of the primary DNA vector pCGN6115 for the transformation of the plant plastid. Figure 1 also shows a schematic representation of the homologous recombination site in the plastid genome. The upper line represents the transgene coding for cellulase E1, the aadA marker gene (strep / spec) for the selection of plastid transformers and plastid homology sequences. The intermediate line represents the region of the chloroplast genome for the integration of the transgene, and the lower line represents the nucleic acid probe used in the Southern hybridization for the determination of the plastid transformers. Figure 2 shows the result of Southern hybridization using the probe depicted. The wild-type band is found in passage 1. A plasmid control (pCGN6115) is found in passage 3. Several subclones of the same transgenic events were analyzed in the same blood analysis. Each subclone was regenerated from the same initial transformer. For example, in event 6115-4, there are 4 subclones of the same event, all are homplasmic for the inserted genes. Some subclones are wild type in other events. Figure 3 shows the results of the hybridization Western of the total soluble leaf protein extracted from homoplasmic tobacco lines transformed with the pCGN6115 construct using elevated monoclonal antibodies to purified β1-β4-endoglucanase of E1 from Acidotherm? S cellulycticus.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the subject invention, plastid expression constructs are provided, which generally comprise a functional promoter in a plant cell, a DNA sequence encoding a polysaccharide hydrolyzing enzyme and a region of transcription termination capable of terminating transcription in a plant cell. These elements are provided as components operably linked in the direction of transcription from 5 'to 3'. The polysaccharide hydrolyzing enzyme of the present invention can be obtained from any source. The polysaccharide hydrolyzing enzyme of the present invention is preferably obtained from a source other than plant. Preferably, the polysaccharide hydrolyzing enzyme is a cellulase, hemicellulase and ligninase. Polysaccharide hydrolyzing enzymes are a family of enzymes that break down cellulose into its simple sugar components. The family includes several classes of enzymes, including but not limited to cellulase, hemicellulase and ligninase. Polysaccharides, as used herein, refer to polymers of more than about ten monosaccharide residues glycosidically linked in branched or unbranched chains. A monosaccharide refers to a simple sugar that can not be hydrolyzed into smaller units. The empirical formula is (CH2O) n and varies in size from triose (n = 3) to heptose (n = 7). A cellulase enzyme, as used herein, includes but is not limited to endocellulases and exocellulases. The endocellulases, for example, include enzymes such as cellulase E1 from Acidothermus cellulycticus (described in U.S. Patent No. 5,536,655). Exocellulases, for example, include enzymes such as cellobiohydrolase 1 (CBH1) from Trichoderma reesei (Shoemarker, et al (1983) Biotechnology, 1: 691-696). A hemicellulase, as used herein, includes but is not limited to xylanase. Nucleic acid sequences encoding the xylanases are known in the art, and include xynk from Acidobacterium capsulatum (Inagaki, et al. (1998) Biosci, Biotechnol. Biochem., 62 (6): 1061-1067 and xynk from Clostridium. thermocellum (Hayashi, et al. (1999), Appl. Microbiol. Biotech., 51 (3): 348-357.) A ligninase, as used herein, includes but is not limited to laccase. nucleic acids for use in the methods of the present invention include, but are not limited to / acAl of Schizophyllum commune (Hatamoto, et al. (1999) Biosci, Biotechnol. Biochem., 63 (1): 58-64), / cd from Pichia pastoris (Jonsson, et al. (1997) Curr Genet, 32 (6): 425-530) and lcc \ y / cclV from Trametes versicolor (Ong, et al. (1997) Gene 196 (1-2): 113-119) In one aspect of the present invention, expression constructs are prepared to direct the expression of polysaccharide hydrolyzing enzymes in a plant cell. a functional promoter in a plant cell, a nucleic acid sequence encoding the polysaccharide hydrolyzing enzyme and a functional transcription termination region in a plant cell. Promoters for use in the constructions of the present invention include functional promoters in a plant cell nucleus, or functional promoters in plant cell organelles, such as mitochondria and plastids. Those skilled in the art will recognize that there are a number of promoters that are functional in plant cells, and have been described in the literature. The specific promoters of chloroplast and plastid, functional promoters of chloroplast and plastid and the operable promoters of chloroplast or plastid are also envisaged. A set of promoters are the constitutive promoters such as the CaMV35S or FMV35S promoters that produce high expression levels in most organs of the plant. Improved or duplicate versions of the CaMV35S and FMV35S promoters are useful in the practice of this invention (Odell, et al. (1985) Nature 313: 810-812; Rogers, U.S. Patent Number 5,378,619). In addition, the expression of the cellulose degrading enzyme in specific tissues of the plant, such as leaf, stem, root, tuber, seed, fruit, etc., may also be preferred, and the chosen promoter must have the desired tissue and development specificity. It may be advantageous to direct the location of the polysaccharide degrading enzymes to a particular subcellular compartment, for example, to the mitochondrion, endoplasmic reticulum, vacuoles, chloroplast or other plastidic compartment. For example, wherein the genes of interest of the present invention will be directed to plastids, such as chloroplasts, for their expression, the constructs will also employ the use of sequences to direct the gene to the plastid. Such sequences will be referred to herein as chloroplast transit peptides (CTP) or plastid transit peptides (PTP). In this way, where the gene of interest is not inserted directly into the plastid, the expression construct will additionally contain a gene encoding a transit peptide to direct the gene of interest to the plastid. Chloroplast transit peptides can be derived from the gene of interest, or can be derived from a heterologous sequence having a CTP. Such transit peptides are known in the art. See, for example, Von Heijne er al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res Commun. 796: 1414-1421; and Shah et al. (1986) Science 233: 478-481. Additional transit peptides for translocation of the protein to the endoplasmic reticulum (ER), or vacuole, may also find use in the constructions of the present invention. Preferably, the promoter used in the constructions of the present invention is functional in a plant cell plastid. A number of functional promoters in plastids are known in the art, and include, but are not limited to, the region of the 16S rRNA promoter, Prrn (Svab, et al. (1993), Proc. Nati. Acad. Sci. USA, 90 : 913-917) and the promoter region of thylakoid membrane protein D1, PpsbA (Staub, et al. (1993), EMBO J., 12 (2): 601-606).

Preferably, the transcription termination region employed in the constructions of the present invention is functional in a plant cell plastid. A number of such termination regions is available in the art, and includes, but is not limited to, the tilacoid membrane protein termination region D1, TpsbA (Staub, et al., (1993) EMBO J., 12 ( 2): 601 -606). In the examples described herein, the nucleic acid sequences encoding a thermophilic cellulase E1 of Acidothermus cellulolyticus, as well as the sequences encoding ceiobiohydrolase 1 (CBH 1), laccase and xylanase, are used in constructs to direct the expression of the Plastid plant cells. In addition, transplastomic tobacco plants expressing cellulase E1 demonstrate a high expression level of the cellulase enzyme. In addition, the expressed enzyme demonstrates enzymatic characteristics similar to the extracted wild-type enzyme. For example, in the examples provided below, crude protein extracts containing the expressed cellulase from the homoplasmic tobacco plants show higher activity at 80 ° C than at 55 ° C. In this way, a thermophilic cellulase expressed from the plant plastid as described in the present invention, shows the same thermophilic properties as the wild-type cellulase having an optimum temperature of 83 ° C (described in the US Pat. No. 5,536,655, the entirety of which is incorporated herein by reference). A thermophilic cellulase with increased activity above 45 ° C provides a safeguard against the activity of the cellulase during the cultivation of the transformed plant in a production field. Cellulase with activities optimized at or above 55CC would be preferable. One skilled in the art to which the present invention pertains will recognize that enzymes from other sources can be used in plastid expression constructs of the present invention. For example, DNA sequences encoding other hydrolyzing enzymes of polysaccharides such as those of Thermomonospora fusca (See, for example, Wilson (1992) Crit. Rev. Biotechnol.12: 45-63), can be used in the constructions of expression of the present invention. The constructs of the present invention can be integrated into the nuclear genome of host plant cells and the enzyme is directed to a cellular organelle. For example, the sequences directing the expressed enzyme towards the vacuole can be used, as well as the sequences that direct transport to the plastid. Such plastid transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark er al. (1989) J. Biol. Chem. 264: 17544-17550; della-Cioppa er al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res Commun. 96; 1414-1421; and Shah er al. (1986) Science 233: 478-481. The protein signal for targeting the vacuoles can be provided from a plant gene that is normally transported through the rough endoplasmic reticulum, such as the 32 amino acid N-terminal region of the metallocarboxypeptidase inhibitor gene from the tomato (Martineau et al. (1991) Mol Gen Genet 228: 281: 286). In addition to the signal sequence, the vacuolar targeting constructs also encode a vacuolar localization signal (VLS) placed at the carboxy terminus of the encoded protein. The appropriate VLS signal sequences and regions can be obtained from various other plant genes and can be used in a similar manner in the constructions of this invention. Numerous vacuolar targeting peptides are known in the art, as are envisioned in Chrispeels et al, Cell (1992) 68: 613-616. In developing the constructions, the various fragments comprising the regulatory regions and the open reading structure may be subjected to different processing conditions, such as ligation, assimilation of restriction enzymes, PCR, in vitro mutagenesis for improved enzymes, addition of linkers and adapters and the like. In this way, transitions, transversions, insertions, omissions or the like of nucleotides can be carried out in the DNA that is used in the regulatory regions or in the DNA sequences of interest for their expression in the plastids. Methods for restriction assimilations, terminal Klenow end removal treatments, ligations and the like are well known to those skilled in the art and are described, for example, by Maniatis et al. (in Molecular Cloning: A Laboratory Manual (1989 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) Often, during the preparation of the constructs, the various DNA fragments are cloned into an appropriate cloning vector, which allows DNA amplification, DNA modification or DNA manipulation by binding or removal of sequences, linkers or the like Preferably, the vectors will be capable of reproducing to at least a relatively high number of copies in E. coli. A number of vectors are readily available for cloning, including vectors such as pBR322, vectors from the pUC series, vectors from the M13 series and pBluescript vectors (Stratagene; La Jolla, CA). In order to provide a means for selecting the desired plant cells, the vectors for plastid transformation typically contain a construct that provides for the expression of a selectable marker gene. The marker genes are DNA sequences expressible in plants, which express a polypeptide that overcomes a natural inhibition by attenuating or inactivating a selective substance, ie, antibiotic, herbicide, etc ... Alternatively, a marker gene can provide some other visibly reactive responses, i.e., may originate a distinct appearance or pattern of development in relation to plants or plant cells that do not express the selectable marker gene in the presence of any substance, either by applying directly to the plant or plant cells or when presented in the medium of plant or plant cell development. In any case, plants or plant cells containing such selectable marker genes will have a distinctive phenotype for identification purposes, i.e., they will be distinguishable from non-transformed cells. The characteristic phenotype allows the identification of cells, groups of cells, tissues, organs, parts of plants or complete plants that contain the construction. The detection of the marker phenotype makes possible the selection of cells having a second gene to which the marker gene has been physically bound. This second gene typically comprises a desirable phenotype that can not be readily identified in transformed cells, but which occurs when the plant cell or derivative thereof develops to maturity, even under conditions where the selectable marker phenotype does not It is apparent by itself. The use of such a marker for the identification of plant cells containing a plastid construct has been described by Svab et al. (1993, supra). In the examples provided below, a bacterial gene aao? it is expressed as the marker under the regulatory control of the chloroplast 5 'promoter and the 3' transcription termination regions, specifically the regulatory regions of the theps & A gene (described in Staub et al., (1993), supra). Numerous additional promoter regions can also be used to drive the expression of the selectable marker gene, including various plastid promoters and bacterial promoters that have been shown to function in plant plastids. The expression of the aadA gene confers resistance to spectinomycin and streptomycin and allows, therefore, the identification of plant cells expressing this marker. The product of the aadA gene allows the continuous development and greening of cells whose chloroplasts produce the product of the selectable marker gene. Cells that do not contain the selectable marker gene product are discolored. Therefore, the selection of the aadA gene marker is based on the identification of plant cells that are not discolored by the presence of streptomycin, or more preferably spectinomycin, in the plant's growth medium. A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, aminoglycoside G418, hygromycin or the like. Other genes that code for a product involved in chloroplast metabolism can also be used as selectable markers. For example, genes that provide resistance to herbicides from plants such as glyphosate, bromoxynil or imidazolinone, may find a particular use. Such genes have been reported (Staiker et al., J. Biol. Chem. (1985) 260: 4724-4728 (glyphosate-resistant EPSP); Staiker et al., J. Biol. Chem. (1985) 263: 6310- 6314 (bromoxynil resistant nitrilase gene) and Sathasivan er al., Nucí Acids Res. (1990) 18: 2188 (imidazolinone resistance gene AHAS)). In the present invention, methods of nuclear transformation and selection of plants employing a biolistic or bombardment method can also be used to transfer the target DNA constructs to the plant cells. Such methods are particularly useful in the transformation of plant cells that are less susceptible to Agrobacterium-mediated transformation methods. Bombardment transformation methods are described in Sanford et al. (1991) Technique 3: 3-16; Klein et al. (1992) Bio / Technology 70: 286-291. Generally, in the transformation of plant cells, the target explants are incubated with the transformed Agrobacterium, as described, for example, by Horsch et al. (Science (1985) 227: 1229-1232), or are bombarded with DNA coated particles. The plant cells are then grown in an appropriate medium to selectively culture those plant cells which have obtained the desired constructions. Once in the form of callus, shoot formation can be encouraged by employing the appropriate plant hormones according to known methods and shoots can be transferred to the rooting medium for regeneration of the plants. The plants can then be grown and either pollinated with the same transformed strain or with different strains. For the production of a homozygous line, self pollination is used. The stable transformation of tobacco plastid genomes is reported by bombardment of particles (Svab er al. (1990), supra) and Svab et al. (1993), supra). The methods described herein can be used to obtain homoplasmic plants for plastid expression constructs. Generally, the bombarded tissue is cultured for about two days in a cell-dividing promoter medium after which the tissue is transferred to a selective medium containing an inhibitory amount of the particular selective agent, as well as the particular hormones and other substances. necessary to obtain the regeneration of that particular plant species. The shoots are then subcultured in the same selective medium to ensure the production and selection of homoplasmic shoots. Homoplasmy is verified by Southern analysis with respect to transplastomic plants for the gene encoding the protein of interest. In the examples provided below, the total cellular DNA assimilated by Xba I is examined with a radiolabelled probe, specifically, a portion of the plastid targeting fragment, including the aadA marker gene and the sequence of the integration region containing the sequence of acetyl CoA carboxylase DNA. Southern blood analysis with this probe confirms the integration of the chimeric E1 cellulase gene into the plastid tobacco genome to produce transplastoma lines. When the transformation and regeneration methods have been adapted for a given plant species, either by Agrobacterium-mediated transformation, bombardment or some other method, the established techniques can be modified for use in selection and regeneration methods to produce plants transformed into plastids. For example, the methods described herein for tobacco are readily adaptable to other Solanaceous species, such as tomato, petunia and potato. For the transformation of soybeans and other plant species, particle bombardment protocols as well as nuclear transformation and regeneration mediated by Agrobacterium have been described (Hinchee et al, U.S. Patent No. 5,416,011 and Christou et al. from U.S. No. 5,015,580). The skilled artisan will recognize that the described protocols for the transformation of the soybean seed can be used and adapted to other plant species. Generally, in the transformation and regeneration protocols mediated by Brassica, Agrobacterium involves the use of hypocotyl tissue, a non-green tissue that could contain a low plastid content. Thus, for Brassica, the preferred target tissues would include hypocotyl or cotyledonary tissues derived from microspores (which are green and therefore contain numerous plastids) or leaf tissue explants. Although the regeneration rates of such tissues may be low, positional effects are not expected, such as with the Agrobacterium-mediated transformation., therefore, it will not be necessary to select numerous successfully transformed plants in order to obtain a desired phenotype. For cotton, the transformation of L. cotyledons Gossypium hirsutum by co-culture with Agrobacterium tumefaciens, by Firoozabady er al. , Plant Mol. Bio. (1987) 70: 105-116 and Umbeck er al. , Bio / Technology (1987) 5: 263-266. Again, as for Brassica, this tissue may have an insuient plastid content for the transformation of chloroplasts. Therefore, as for Brassica, an alternative method for the transformation and regeneration of chloroplasts containing alternative target tissue, for example green embryogenic targeting tissue, may be desirable. Other plant species can be transformed in a similar way by the use of related techniques. Alternatively, methods of bombardment by microprojectiles, such as that described by Klein et al. . { Bio / Technology 70: 286-291), can also be used to obtain nuclear transformed plants comprising the unique viral subunit RNA polymerase expression constructs described herein. The transformation of cotton by particle bombardment is reported in WO 92/15675, published on September 17, 1992. Plants for practicing the present invention include, but are not limited to, soybean seed, cotton, alfalfa, oil of rapeseed, flax, tomato, sugar beet, sunflower, potato, tobacco, corn, wheat, rice and lettuce. Methods for processing corn, wheat and rice are well known in the art and are described, for example, in US Patents. Numbers 5,538,877 and 5,538,880, the U.S. Patent. Number 5,610,042 and European Patent Number EP539563. Vectors for use in plastid transformation preferably include means for providing a stable transfer of the plastid expression construct and construction of the selectable marker towards the plastid genome. This is most conveniently provided by regions of homology to the target plastid genome. The regions of homology distinguish the construction by transfer and provide the transfer of the plastid genome through homologous recombination, through a double step towards the genome. The complete DNA sequence of the tobacco plastid genome has been reported (Shinozaki er al., EMBO J. (1986) 5: 2043-2049). Complete DNA sequences of plastid genomes from hepatic (Ohyama er al., Nature (1986) 322: 572-574) and rice (Hiratsuka et al., Mol. Gen. Genet. (1989) 277) have also been reported. : 185-194). When regions of homology occur in the repeating regions of the plastid genome (known as IRA and IRB), two copies of the transformed plastid transgene are expected. The regions of homology within the plastid genome are approximately 1 kb in size. Smaller homology regions can also be used, and as little as 100 bp can provide homologous recombination in the plastid genome. However, the frequency of recombination and therefore the frequency of obtaining plants that have transformed plastids decreases with the decrease in size of the regions of homology. Examples of constructs having regions of plastid genome homology are described in Svab et al. (1990 supra), Svab et al. (1993 supra) and Zoubenko et al. . { Nuc Acid Res (1994) 22 (19): 3819-3824). In the examples provided herein, the plastid-distinctive tobacco homology regions of the plastid expression construct direct the insertion of an E1 cellulase transgene into the tobacco chloroplast genome between the acetyl CoA carboxylase (ORF512). and the large subunit of RuBisCo (rbcL). Such regions of homology are described in Svab and Maliga (1993) supra. Since the integration in the plastid genome occurs through homologous recombination and the selected site is not found in an inverted repeat region of the plastid genome, a copy of the transgene is expected by plastid genome. The selection is made for the spectinomycin resistance marker phenotype expressed by the aadA gene. Since one of the main components of plants is cellulose, it is expected that the production of cellulose degrading enzymes in plant cells may have harmful effects on the host organism. However, by compartmentalizing the cellulose degrading enzyme expressed in a plant organelle, for example in a plastid, any deleterious effect of the expression of the cellulase enzyme can be overcome. In addition, the use of a cellulase degrading enzyme with a high temperature and / or an optimum pH can also provide safeguards to the expression of such enzymes in plants that develop at ambient temperatures. Plant plastids (chloroplasts, amyloplasts, elaioplast, chromoplasts, etc.) are the main biosynthetic centers that, in addition to photosynthesis, are responsible for the production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids and pigments. The plastids are derived from a common precursor known as proplástida and, therefore, the plastids present in a given plant species all have the same genetic content. The plant cells contain 500-10,000 copies of a small circular genome of 120-160 kilobases, each molecule of which has a large inverted repeat (approximately 25 kb). Therefore, it is possible to design plant cells containing up to 20,000 copies of a particular gene of interest that can potentially result in very high levels of foreign gene expression. In addition, the plastids of most plants are maternally inherited. Accordingly, heterologous genes expressed in plastids are not disseminated by pollen, therefore, a trait introduced into a plant plastid will not be transmitted to wild-type relatives by cross-fertilization. Therefore, plastids of older plants are an attractive target for genetic design. The present invention also provides methods for the production of thermally stable industrial enzymes in a host plant cell. Such methods generally include the use of the expression constructs of the present invention in the production of transgenic plants. Preferably, the methods involve the expression of a thermostable, polysaccharide degrading enzyme in a plant cell, the development of a plant having transformed cells, the harvesting of plants or plant parts and the subjection of the harvested plant material to conditions that promote the optimal activity of the enzyme. The present invention further provides methods for encapsulating cell wall degrading enzymes in organelles, such as vacuoles, plastids and the like. The methods generally involve the expression of a polysaccharide degrading enzyme in a plant cell nucleus and the location of the enzyme in a plant cell organelle by the use of an organocellular targeting sequence. Alternatively, the enzyme can be expressed directly from a plant cell organelle, for example, a plant cell plastid. The methods of the present invention may also include sequences and additional constructs involved in the modification of polysaccharides in plant cells. Such sequences and constructions are known in the art and include, but are not limited to, those described in PCT Publication WO 98/18949, all of which is incorporated herein by reference. The methods of the present invention may also include the expression of one or more expression constructs that provide for the expression of one or more sequences encoding the polysaccharide degrading enzymes. For example, an expression cassette can be introduced into a plant cell that provides expression of an endocellulase and a second expression cassette can also be introduced for the expression of a ligninase. In this manner, the present invention provides novel methods for the production of thermophilic polysaccharide hydrolyzing enzymes (ie, cellobiohydrolase, xylanase, hemicellulase) in plant cells. The expression of such enzymes in plant cells provides an alternative source for the production of polysaccharide degrading enzymes used in industrial processes / products (such as textile finishing, detergents, food and beverage processing, food additives, silage, pulp reduction, papermaking and biomass conversions). Also, the expression of thermophilic cellulases and related cellulose degrading enzymes in plant plastids provides an alternative or complementary method to degrade endogenous cellulose contained in plant tissues and to release stored carbon (such as monosaccharides) for subsequent fermentation processes. .

In addition, enzyme sequestration (substrate isolation) and / or optimal high temperature (against low activity at ambient temperatures) of the thermophilic cellulase provide two internal safeguards to protect the plant from intrinsic enzyme activity during critical development of the plant and the stages of development. Having now described the invention in general, it will be more readily understood in relation to the following examples, which are included for purposes of illustration only and are not intended to limit the present invention.

EXAMPLES Example 1 Preparation of the Construction The constructions and methods to be used in the transformation of plastids of larger plants are described in Zoubenko et al. . { Nuc Acid Res (1994) 22 (19): 3819-3824), Svab er al. (Proc. Nati, Acad. Sci. (1990) 87: 8526-8530 and Proc. Nati, Acad. Sci. (1993) 90: 913-917) and Staub et al. (EMBO J. (1993) 12: 601-606). The complete DNA sequences of the tobacco plastid genome are reported by Shinozaki er al. (EMBO J. (1986) 5: 2043-2049). All plastid references of DNA in the following description are for the nucleotide number of tobacco. A vector was prepared to direct the expression of β-1, 4-endoglucanase Acidothermus in plant plastids. The pMPT4 plasmid, a derivative of pGEM (Clonetech) containing the coding sequence of cellulase E1 from whole Acidothermus (EU Patent Number 5,536,655) and distinguishing the regions on a 3.7 kb Pvu I genomic DNA fragment, was assimilated with the restriction endonuclease sites Sacll and Asp718 to remove the coding sequence of the mature E1 cellulase protein. This fragment was cloned into the same restriction sites of plasmid pBCSK + (Stratagene) to create the vector pCGN6063. This plasmid was assimilated with Sacl and Sacll and a double-stranded oligonucleotide sequence, 5'-GGAGCTGCGTCACCATGGCGGGA-3 ', was inserted to introduce an ATG translation initiation codon derived from the? / Cab site, fused to the mature sequence of 60,000 mol by weight (minus the peptide amino acid sequence of endogenous bacterial signal) of the polypeptide of β-1, 4-endoglucanase E1, which creates the construct pCGN6067. The E1 gene was separated from pCGN6067 as a DNA segment of? col to A sel and cloned into the T7 promoter expression cassette pCGN5063 to create the pCGN6108 construct. This plasmid contains the plastid expression regulatory elements of the bacteriophage T7 promoter operably linked to the mature protein portion of the E1 coding sequence and the transcription termination region psbA. The chimeric cellulase expression cassette was separated as a Hind III to Not I DNA fragment and cloned into the tobacco chloroplast homology vector, pCGN6043 at the same restriction sites, to create the pCGN6115 construct. The homology sequences employed in the vector direct the integration of the E1 cellulase gene and the aadA marker transgene into the region between the rbcL and ORF512 sequences (described in Svab et al., (1993) supra). The construction of pCGN6115 (figure 1) was used to transform the tobacco plants in order to direct the transformation towards homoplasmy and the plastid expression of the β-1,4-endoglucanase E1 encoding the gene in the plastid of the plant. In order to express the nucleic acid sequence encoding the xylanase from Dictyoglomus thermophilum (Gibbs, er al. (1995) Appl. Environ. Microbiology 61 (12): 4403-4408), the oligonucleotide primers can be synthesized for use in Polymerase Chain Reactions to amplify the entire coding region of the XynA gene. The two primers, 5'-ATGGTACCATGCTTAACCAAAGGTTTTCTATC (the underlined sequence represents the ATG of initiation) which contain a? / Cab site at the 5 'terminus for cloning into the T7 promoter, and 5'-CCTATAGGCGCGCCAAAACTTTACAATCTCCC containing a sel site at the 5 'end for cloning, can be used to amplify the entire coding region of the XynA sequence. The 1300 base pair amplification product can then be cloned into the T7 promoter expression cassette pCGN5063 to create the pCGN6581 construct. This plasmid contains the regulatory elements of the plastid expression of the bacteriophage T7 promoter operably linked to the mature protein portion of the XynA coding sequence and the transcription termination region psbA. The chimeric xylanase expression cassette can be separated as a Hind III to Not I DNA fragment and cloned into the tobacco chloroplast homology vector, pCGN6043 at the same restriction sites to create the plastid transformation construct pCGN6582. In order to express the nucleic acid sequence encoding CBH1 from Trichoderma reesei (Shoemaker, et al (1983) Biotechnology, 1: 691-696), oligonucleotide primers can be synthesized for use in Polymerase Chain Reactions in order to amplify the coding region entirely of the CBH1 gene. The two primers, d'-TGGCACCAJGCATCGGAAGTTGGCCGTCA (the underlined sequence represents the initiation ATG) containing a? / Cab site at the 5 'end for cloning at the T7 promoter, and 5'- CCTATAGGCGCGCCCAGGCACTGAGAGTAGTAAGG containing an Asc site at the 5 'end for cloning, can be used to amplify the coding region entirely of the CBH1 sequence. The amplification product of approximately 1700 base pairs can then be cloned into the T7 promoter expression cassette pCGN5063 to create the pCGN6583 construct. This plasmid contains the plastid expression regulatory elements of the bacteriophage T7 promoter operably linked to the mature protein portion of the CBH1 coding sequence and the transcription termination region psbA. The chimeric CBH1 expression cassette can be separated as a Hind III to Not I DNA fragment and cloned into the tobacco chloroplast homology vector, pCGN6043 at the same restriction sites in order to create the plastid transformation construct pCGN6584. In order to express the nucleic acid sequence coding for laccasel from Aspergillus nidulans (Aramayo, et al (1990) Nucleic Acids Research, 18 (11): 3415), oligonucleotide primers can be synthesized for use in Chain Reactions Polymerase to amplify the entire coding region of the yA gene. The two primers, 5'- ATCCAGCCATGCACCTCTCCACGGTCCTCTTCCA (the underlined sequence represents the initiation ATG) containing a? / Cab site in the 5 'terminus for cloning into the T7 promoter, and 5'-TATGAGGGCGCGCCCTAAGAATCCCAAACATCAACCCCG containing an Asc site in the 5 'end for cloning can be used to amplify the coding region entirely of the sequence yA. The amplification product of approximately 2100 base pairs can then be cloned into the T7 promoter expression cassette pCGN5063 to create the pCGN6585 construct. This plasmid contains the plastid expression regulatory elements of the bacteriophage T7 promoter operably linked to the mature protein portion of the yA coding sequence and the transcription termination region psbA. The chimaeric laccase expression cassette can be separated as a Hind III to Not I DNA fragment and cloned into the tobacco chloroplast homology vector, pCGN6043 at the same restriction sites in order to create the plastid transformation construct pCGN6586.

EXAMPLE 2 Plastid Transformation of the Plant Tobacco plants transformed to express T7 polymerase from the nuclear genome and directed towards the plant plastid are obtained as described in McBride et al. Patent of E.U. Number 5,576, 198. The homozygous transgenic tobacco plants for the T7 polymerase directed to the plastid, are used for the plastid transformation by the use of particle bombardment. Tobacco plastids are transformed by the delivery of microprojectile particle shots. Since the integration in the plastid genome occurs through homologous recombination and the target site is between the acetyl CoA carboxylase and the large RuBisCo subunit (rbcL), a single copy of the transgene is expected per plastid genome (Svab er al (1993) supra). Tobacco seeds (.tabacum v. Xanthi N / C) homozygoses for the T-DNA of pCGN4026 (McBride et al., U.S. Patent Number 5,576, 198) are surface sterilized in a 50% chlorine solution (2.5% of sodium hypochlorite) for 20 minutes and rinsed 4 times in sterile H2O. The seeds are then planted aseptically in a 0.2X MS salt medium and allowed to germinate. The seedlings are grown on an agar solidified MS medium with 30 g / l of sucrose (Murashige and Skoog (1962) Physiol, Plan 75: 493-497).

The tungsten microprojectiles (1.0μm) are covered with DNA, such as the T7 / E1 cellulase expression construct, pCGN6115, and the covered microprojectiles, used to bombard mature leaves, are placed abaxially upwards in an RMOP medium (salts). MS, 1 mg / l BAP, 0.1 mg / l NAA, 30 g / l of sucrose and 0.7% of phyagege) (Svab et al (1990) supra) by using the 1000 / He bombardment system of Bio- Rad PDS (Sanford et al. (1991) Technique 3: 3-16; Klein et al. (1992) Bio / Technology 70: 286-291). The development of transformed plants in the RMOP medium supplemented with 500 mg / l of spectinomycin dihydrochloride and the subsequent subcloning in the same selective medium are conducted according to Svab er al. (1990); Svab and Maliga (1993); supra). The selected plants are rooted in the MS medium containing 1 mg / l of IBA, 500 mg / l of spectinomycin dihydrochloride and 0.6% of phytagaro.

EXAMPLE 3 Analysis of Cellulase Expression in Plastids Following the plastid transformation as described above, five independently separated homoplasmic lines generated in the nuclear-coded T7 RNA polymerase that produce the environment were generated. A schematic construction of pCGN6115 and an embodiment of Incorporation in the plastid tobacco genome is shown in Figure 1. The upper line represents the input DNA donated from pCGN61 15 and the middle line represents the target region of integration. Expected sizes for the Xbal fragments are shown in the input DNA as well as for the wild-type DNA. Since there is no Xbal site at the 5 'end of the input DNA, the combined size of the two chimeric genes is indicated. Also shown in Figure 1 is the location of the probe used for the Southern analysis. Homoplasmy was determined by Southern blood analysis as shown in Figure 2. To confirm homoplasmy by Southern hybridization, the total cell DNA of the plant is prepared as described by Bematzky and Tanskley ((1986) Theor Appl Genet. 72: 314-321). Approximately 3 μg of DNA are assimilated for each sample with Xibal, electrophoresed through 0.7% agarose, transferred to Nytran + (Schleicher and Schuell). The filters were hybridized in a compensator (50% formamide, 6x SSC, 5X Denhardt's solution, 0.5% SDS, 150 μg / ml salmon sperm DNA) at 42 ° C with a probe labeled alpha 32P-dCTP. The hybridization probe was prepared from nucleic acid sequences extending the integration zone. This DNA sequence contains approximately 50% of the acetylcoa carboxylase of the native chloroplast gene and some distinctive intergenic sequences, and is derived as a Notl / XhoI fragment from pCCN6042, a precursor plasmid containing only the plastid homology sequences of tobacco present in the plasmid pCGN6115. The results of Southern hybridization are shown in Figure 2. Homoplasmic lines are identified which contain the cellulase E1 and the aadA coding sequences. Untransformed control tobacco lines (wild-type Xanthi) examined with DNA fragment 6042 are hybridized with a 1.5 kb DNA fragment, while lines of homoplasmic tobacco containing cellulase E1 and aadA sequences are hybridized with a 2.5 kb DNA fragment. The transplastomic lines which are heteroplasmic demonstrate a hybridization pattern that contains both the 1.5 kb wild-type DNA fragment and the 2.5 kb homoplasmic DNA fragment. The difference in band size between the transgenic plants and the wild type is the presence of the aadA resistance gene and its regulatory sequences in the transgenic plants. This adds about 1 kb to the wild type band. To demonstrate that the homologous 6115 tobacco lines express the ß-1, 4-endoglucanase E1, Western blood analysis was performed using total soluble leaf protein. The leaf protein was extracted as follows: 200mg of mature leaf samples were frozen in liquid N2 and ground in 0.08 mi extraction buffer containing 0.1 M NaPO4 pH6.8, 0.15M NaCl, 0.01 M EDTA, 0.01 M DTT, 0.01 M thiourea, 0.3% Tween-20, 0.05% Triton-X100. Protein concentrations were determined by the Bradford assay. Protein samples were combined with an equal volume of 2x Laemmii sample compensator (Laemmii (1970) Nature 227: 680-685) and boiled before loading on 10% Laemmii gels. Approximately 40μg of the total leaf protein was loaded / passed. The results of the Western blood analysis (Figure 3) using monoclonal antibodies constructed against the cellulase Acidothermus E1 demonstrate that the E1 protein is expressed in all the 6115 homoplasmic lines examined. 250 ng of purified E1 cellulase was loaded from the Strepromyces in the first passage. This protein flows in multiple forms in a denaturing gel, the highest form being a molecular weight of 72,000 since this form includes the signal peptide for secretion. The mature form of the enzyme is approximately 60 kd. The second passage contains control tobacco tissue. The third passage contains an extract of the homologous plant 6115 which does not contain the T7 RNA polymerase to activate the expression of cellulase E1 in the plastid. Passages 4-8 are independent of homoplasmic lines 6115 in a xanthi 4026 environment. Construction 4026 expresses the T7 RNA polymerase directed at the plastid which activates the expression of cellulase E1 in the plastid. The main protein band at 60 kd constitutes the mature cellulase E1. Passage 9 shows 100 ng of the purified catalytic form E. coli of cellulase E1 minus the cellulose binding domain (CBD). The Acidothermus E1 cellulase purified from the recombinant Streptomyces flows in multiple forms in a denaturing gel, the highest form being a molecular weight of 72,000 since this form includes the endogenous signal peptide required for the secretion of the enzyme from the bacterium. The mature form of the enzyme is approximately 60 kilodaltons. Accordingly, as can be seen from Figure 3, cellulase E1 expressed in the plant plastids as a mature form of 60 kd forms and can be converted to the catalytic domain form of 40 kd, presumably by the proteolytic process in vivo . In addition, from the results of Western blood analysis it can be estimated that the protein expression of β-1,4-endoglucanase E1 is approximately 1% of the total soluble plant protein in leaves of transplastomic tobacco lines. The crude total soluble leaf protein of homologous tobacco 6115 lines expressing cellulase E1 was further analyzed with respect to cellulase activity. Because the Vmax of cellulase E1 of Acidothermus is close to the maximum point approaching 80 ° C, experiments were carried out at 55 ° C and 80 ° C. Protein extracts (approximately 12 μg of total leaf protein) were tested in reactions to measure the hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-β-D-cellobioside (MUC) as described in Laymon et al. (1996) Applied Biochem. Biotechnol. 57/58: 389-397. The results are listed in Table 1.

Table 1 The above results clearly indicate that the cellulase expressed in plant plastids has a higher level of activity at 80 ° C. Increases in enzyme activity from 5 to 6 bends are observed when crude extracts are incubated with MUC at 80 ° C when cellulase activities are obtained in incubations at 55 ° C. Accordingly, the cellulase expressed in the plant plastids demonstrates similar kinetic characteristics as the wild-type enzyme isolated from Acidothermus cellulolyticus. All publications and patent applications mentioned in this specification are indicative of the level of experience of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each publication or individual patent application was specifically and individually indicated in order to be incorporated for reference. Although the above invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious that some changes and modifications may be practiced within the scope of the appended claims.

Claims (37)

1. A construction characterized in that it comprises, as components operably linked in the direction of transcription 5 'to 3': (a) a functional promoter in a plant cell; (b) a DNA sequence encoding a polysaccharide hydrolyzing enzyme; and (c) a transcription termination region.

2. The construction according to claim 1, characterized in that said promoter is functional in a plant plastid.

3. The construction according to claim 1, characterized in that it further comprises a targeting sequence capable of directing the transport towards a cellular organelle.

4. The construction according to claim 3, characterized in that said targeting sequence directs transport to a vacuole.

5. The construction according to claim 3, characterized in that said addressing sequence directs the transport to a plastid.

The construction according to claim 1, characterized in that said enzyme is active at or above a temperature selected from the group consisting of approximately 45 ° C, approximately 55 ° C and approximately 80 ° C.

7. The construction according to claim 1 characterized in that said enzyme is selected from the group consisting of cellulases, cellobiohydrolases, xylanases, ligninases and hemicellulases.

The construction according to claim 1, characterized in that said enzyme is selected from the group consisting of endocellulases and exocellulases.

The construction according to claim 1, characterized in that said DNA sequence encodes an enzyme selected from the group consisting of CBH 1, xynA, lacAL, Icd, and IccIV.

10. A plant cell containing the construct according to claim 1.

11. The plant cell according to claim 10, characterized in that said construction is integrated into the nuclear genome of said cell ..

12. The plant cell according to claim 10 characterized in that said construction is integrated into the plastid genome of said cell.

A plant, plant seed or plant part comprising a plant cell selected from the group consisting of the plant cell according to claim 11 and the plant cell according to claim 12.

14. A method for producing a polysaccharide hydrolyzing enzyme comprising: the growth of a plant including a construct comprising, as components operably linked in the transcription direction 5 'to 3' i) a functional promoter in a plant cell; ii) a DNA sequence encoding a polysaccharide hydrolyzing enzyme; and iii) a transcription termination region; and the isolation of said enzyme from a cell of said plant.

15. A method for producing an optimal heat industrial enzyme comprising: the growth of a plant including a construct comprising, as components operably linked in the transcription direction 5 'to 3' i) a functional promoter in a plant cell; ii) a DNA sequence that encodes an enzyme that degrades a cell wall component; and iii) a transcription termination region; and the isolation of said enzyme from a cell of said plant.

16. A method for altering the cellulose content in the plant tissue characterized in that it comprises the steps of: the growth of a plant that includes a construction comprising, as components operably linked in the direction of transcription 5 'to 3' i) a functional promoter in a plant cell; ii) a DNA sequence encoding a polysaccharide hydrolyzing enzyme; and iii) a transcription termination region, wherein said plant grows under conditions for the expression of said enzyme in a cell of said plant.

17. The method according to claim 16 characterized in that said enzyme degrades cellulose of said plant cell and reduces said cellulose content.

18. The method according to claim 16, characterized in that the assimilation capacity of the plant material comprising said plant cell is improved.

19. The method according to claim 16 characterized in that said promoter is functional in a plant plastid.

The method according to claim 16 characterized in that said construction further comprises a targeting sequence capable of directing the transport towards a cellular organelle.

21. The method according to claim 20, characterized in that said targeting sequence directs transport to a vacuole.

22. The method according to claim 20, characterized in that said targeting sequence directs the transport to a plastid.

23. The method according to claim 16 characterized in that the cellulose content of plant material mixed with said plant cell is degraded

24. The method according to claim 16 characterized in that it further comprises the steps of: harvesting the plant material of said plant; and securing said harvested plant material to conditions where the activity of said enzyme is increased and the cellulose content of said plant material is reduced.

25. The method according to claim 16 characterized in that said enzyme is active at or above a temperature selected from the group consisting of about 45 ° C, about 55 ° C, and about 80 ° C.

26. The method according to claim 16 characterized in that said enzyme is selected from the group consisting of cellulases, cellobiohydrolases, xiianases, ligninases and hemiceiulases.

27. The method according to claim 16 characterized in that said enzyme is selected from the group consisting of endocellulases and exocellulases.

The method according to claim 16 characterized in that said DNA sequence encodes an enzyme selected from the group consisting of CBH1, xynA, lacAL, Icc1, and IccIV.

29. A method for encapsulating an enzyme that degrades a cell wall component in a plant cell organelle, characterized in that it comprises the steps of: the growth of a plant including a construct comprising, as components operably linked in the transcription direction 5 'to 3' i) a functional promoter in a plant cell; ii) a DNA sequence that encodes an enzyme that degrades a cell wall component; iii) a transcription termination region; and iv) a targeting sequence capable of directing transport to a cell organelle, characterized in that said plant cell grows under conditions for the expression of said enzyme in an organelle of said plant cell.

30. The method according to claim 29, characterized in that said targeting sequence directs transport to a vacuole.

31. The method according to claim 29, characterized in that said targeting sequence directs the transport to a plastid.

32. The method according to claim 29, characterized in that the cellulose content of the plant material mixed with said plant cell is degraded.

The method according to claim 29 characterized in that it further comprises the steps of: harvesting the plant material of said plant cell; and subjecting said harvested plant material to conditions wherein the activity of said enzyme is increased and the cellulose content of said plant material is reduced.

34. The method according to claim 29, characterized in that said enzyme is active at or above a temperature selected from the group consisting of about 45 ° C, about 55 ° C, and about 80 ° C.

35. The method according to claim 29 characterized in that said enzyme is selected from the group consisting of cellulases, cellobiohydrolases, xylanases, ligninases and hemicellulases.

36. The method according to claim 29 characterized in that said enzyme is selected from the group consisting of endocellulases and exocellulases.

37. The method according to claim 29, characterized in that said DNA sequence encodes an enzyme selected from the group consisting of CBH1, xynA, lacAL, Icc1, and IccIV.