MXPA06010197A - Self-processing plants and plant parts - Google Patents

Abstract

The invention provides polynucleotides, preferably synthetic polynucleotides, which encode processing enzymes that are optimized for expression in plants. The polynucleotides encode mesophilic, thermophilic, or hyperthermophilic processing enzymes, which are activated under suitable activating conditions to act upon the desired substrate. Also provided are“self-processing”transgenic plants, and plant parts, e.g., grain, which express one or more of these enzymes and have an altered composition that facilitates plant and grain processing. Methods for making and using these plants, e.g., to produce food products having improved taste and to produce fermentable substrates for the production of ethanol and fermented beverages are also provided.

Description

SELF-PROCESSING PLANTS AND PLANT PARTS Field of. The Invention The present invention relates generally to the field of plant molecular biology, and more specifically, to the creation of plants that express a processing enzyme that provides a desirable characteristic to the plant or products thereof.

Background of the Invention Enzymes are used to process a variety of agricultural products such as woods, fruits and vegetables, starches, juices and the like. Typically, processing enzymes are produced and recovered, on an industrial scale, "from several sources, such as microbial fermentation (α-amylase). of Bacillus), or isolation of plants (ß-galactosidase from coffee or papain from plant parts). Enzyme preparations are used in different processing applications by mixing the enzyme and the substrate under the appropriate conditions of humidity, temperature, time and mechanical mixing such that the enzymatic reaction is achieved in a commercially viable manner. The methods comprise the separate steps of enzyme production, preparation of an enzyme preparation, mixing of the enzyme and substrate, and subjecting the mixture to the appropriate conditions to facilitate REF: 175505 the enzymatic reaction. A method that reduces or eliminates time, energy, mixing, capital expenditures, and / or enzyme production costs, or resulting in improved or novel products would be useful and beneficial. An example of where these improvements are needed is in the area of corn grinding. Currently, corn is milled to obtain corn starch and other co-products from corn milling such as corn gluten feed, corn gluten meal, and corn oil. The starch obtained from the process is often- processed further into other products such as starches and derivatized sugars, or fermented to make a variety of products including alcohols or lactic acid. The processing of corn starch frequently involves the use of enzymes, in particular, enzymes that hydrolyze and convert starch into fermentable sugars or fructose (a- and gluco-amylase, α-glucosidase, glucoisomerase, and the like). The process currently used commercially is capital intensive since it requires the construction of very large mills to process the corn at the scales required for reasonable monetary effectiveness. In addition, the process requires the separate elaboration of starch hydrolyzing or modifying enzymes and then the machinery for mixing the enzyme and the substrate to produce hydrolyzed starch products. The starch recovery process from corn kernels is well known and comprises a wet milling process. The wet milling of starch includes the steps of impregnating the corn kernel, grinding the corn kernel and separating the grain components. The grains are impregnated in an impregnation tank with a countercurrent flow of water at approximately 48.8 ° C (120 ° F) and the grains remain in the impregnation tank for 24 to 48 hours. This impregnation water typically comprises sulfur dioxide at a concentration of about 0.2% by weight. Sulfur dioxide is used in the process to help reduce microbial growth and also to reduce disulfide bonds in endosperm proteins to facilitate a more efficient separation of starch-protein. Normally, approximately 0.59 gallons of impregnation water is used per bushel of corn. Impregnation water is considered waste and often contains undesirable levels of residual sulfur dioxide. The impregnated grains are then drained and subjected to sets of wear type mills. The first set of wear-type mills breaks the kernels, releasing the germ from the rest of the grain. A commercial type wear mill suitable for the wet milling issue is sold under the Bauer trade name. Centrifugation is used to separate the germ from the rest of the grain. A commercial centrifuge separator, typical is the Merco centrifugal separator. Attrition mills and centrifugal separators are expensive large points that use energy to operate. In the next step of the process, the remaining components of the grain including the starch, husk, fiber and gluten are subjected to another set of wear mills and are passed through a set of washing sieves to separate the components of the fiber of starch and gluten (endosperm protein). "Starch and gluten pass through the sieves in so far as the fiber does not pass in. Centrifugation or a third" milling followed by centrifugation is used to separate the starch from the endosperm protein. The centrifugation produces a slurry of starch that is drained, then washed with fresh water and dried at approximately 12% humidity. The substantially pure starch is typically processed further by the use of enzymes. The separation of starch from the other grain components is carried out because the coating of the seed, embryo and endosperm proteins allows to efficiently contact the starch with processing enzymes, and the products resulting from hydrolysis are relatively free of contaminants from the other components of the grain.

The separation also ensures that other components of the grain are effectively recovered and can be sold subsequently as co-products to increase mill revenues. After the starch is recovered from the wet milling process, it is typically subjected to the processing steps of gelatinization, liquefaction and dextrinization for the production of maltodextrin, and the subsequent steps of saccharification, isomerization and refining for the production of glucose, maltose and fructose. Gelatinization is used in the hydrolysis of starch because the currently available enzymes can not rapidly hydrolyze the crystalline starch. To make the starch available to the hydrolytic enzymes, the starch is typically made in a suspension slurry with water (20-40% dry solids) and heated to the appropriate gelling temperatures. For corn starch, that temperature is between 105-110 ° C. The gelatinized starch is typically very viscous and therefore thins in the next step called liquefaction. Liquefaction breaks some of the bonds between the starch glucose molecules and is achieved enzymatically or through the use of acid. Endo-a-amylase enzymes are used, stable to heat in this step, and in the subsequent dextrinization step. The degree of hydrolysis is controlled in the dextrinization step to produce hydrolysis products of the desired percentage of dextrose. The additional hydrolysis of the dextrin products of the liquefaction step is carried out by several different exo-amylases and de-branching enzymes, depending on the products that are desired. And finally, if fructose is desired, then immobilized glucose isozyme enzyme is used to convert glucose into fructose. The process of dry milling to produce fermentable sugars (and then ethanol, as an example) of corn starch facilitates the efficient putting in contact of exogenous enzymes. with starch. These processes are less capital intensive than wet milling but significant cost advantages are still desirable, since often the co-products derived from these processes are not as valuable as those derived from wet milling. For example, in the dry milling of corn, the grain is milled to a powder to facilitate the efficient contact of starch by degrading enzymes. After the enzymatic hydrolysis of corn flour, the residual solids have some nutritional value since they contain proteins and some other components. Eckhoff described the potential for improvements and relevant issues related to dry grinding in an article entitled "Fermentation and costs of fuel ethanol from corn with quick germ process" (Appl. Biochem. Biotechnol., 94: 41 (2001)). The "quick germ" method allows the separation of the starch-containing germ with a high time of impregnation. An example where the regulation and / or level of endogenous processing enzymes in a plant can result in a desirable product is corn, tender. Typical varieties of young corn are distinguished from corn varieties of extensive cultivation by the fact that young corn is not capable of normal levels of starch biosynthesis. Genetic mutations in the genes encoding the enzymes comprised in starch biosynthesis are typically employed in varieties of tender maize starch to limit starch biosynthesis. These mutations are in the genes that code for starch synthases and ADP-glucose-pyrophosphorylases (such as sugary and super-sweet mutations). Fructose, glucose and sucrose, which are the simple sugars needed to produce the tasty sweetness that consumers of fresh edible corn desire, accumulate in the developing endosperm of these mutants. However, if the level of starch accumulation is too high, such as when the corn is left to mature too much (after harvesting) or the corn is stored for a prolonged period before it is consumed, the product loses its sweetness and takes a taste and sensation to the mouth, starched. The harvest advantage for tender corn is therefore quite narrow, and is limited to shelf life. Another significant disadvantage to the farmer who sows varieties of sweet starch is that the utility of these varieties is limited exclusively to edible food. If a farmer wishes to give up harvesting his sweet corn for use as edible food during the development of the seed, the crop will essentially be a waste. The yield of grain and the quality of the tender corn is poor for two fundamental reasons. The first reason is that mutations in the starch biosynthetic pathway prevent the starch biosynthesis machinery and the grains from being completely fulfilled, causing the performance and quality to be complemented. Second, due to the high levels of sugars present in the grain and the inability to sequester these sugars such as starch, the total concentration of seed accumulation is reduced, which exacerbates the reduction of nutrient storage in the grain. The endosperms of the seeds of the young corn variety shrink and collapse, suffering proper drying, and are susceptible to diseases. The poor quality of sweet corn grain has additional agronomic implications; such as poor seed viability, poor germination, susceptibility to seedling disease, and poor vigor of young seedlings resulting from the combination of factors caused by inadequate starch accumulation. In this way, issues of poor quality of the soft starch impact the consumer, farmer / grower, distributor and producer of seeds. In this way, for dry grinding, there is a need for a method that improves the efficiency of the process and / or increases the value of the co-products. For wet milling, there is a need for a process for processing starch that does not require the equipment necessary for prolonged immersion, grinding, milling and / or separation of grain components. For example, there is a need to modify or eliminate the immersion step in wet milling since this will reduce the amount of waste water that requires disposal, thus saving energy and time, and increasing the capacity of the mill (grains) they will spend less time in the immersion tanks). There is also a need to eliminate or improve the process for separating the starch-containing endosperm from the embryo.

Brief Description of the Invention The present invention relates to self-processing plants and plant parts and methods using same. The self-processing plant and plant parts of the present invention are capable of expressing and activating enzymes (mesophilic, thermophilic and / or hyperthermophilic). In the activation of enzymes (mesophilic, thermophilic or hyperthermophilic), the plant or part of the plant is capable of self-processing the substrate in which it acts to obtain the desired result. The present invention relates to an isolated polynucleotide, a) comprising SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or ' 5-9.- or the complement thereof, or a complement that hybridizes "to the - ^ complement of any of SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 under conditions of low stringency hybridization and encodes a polypeptide having α-amylase, pullulanase, α-glucosidase, glucose-isomerase, or glucoamylase, or b) that codes for a polypeptide comprising SEQ ID NO: 10, 13, 14, 15, 16, 18 , 20, 24, 26, 27, 28, 29, 30, 33, 34, 35, 36, 38, 40, 42, 44, 45, 47, 49, or 51 or an enzymatically active fragment thereof. In a preferred embodiment, the isolated polynucleotide encodes a fusion polypeptide comprising a first polypeptide and a second peptide, wherein the first polypeptide has α-amylase, pullulanase, α-glucosidase, glucose-isomerase, or glucoamylase activity. More preferably, the second peptide comprises a peptide of signal sequence, which can direct the first polypeptide to a vacuole, attic reticulum, atlas, chloroplast, starch granules, seed or cell wall of a plant. For example, the signal sequence may be a N-terminal waxy signal sequence, a N-terminal signal sequence of? -zein, a starch binding domain, or a C-terminal starch binding domain. Polynucleotides that hybridize to the complement of any of SEQ ID NO: 2, 9, or 52 are further encompassed under low stringency hybridization conditions and encode a polypeptide having α-amylase activity; the complement of SEQ ID NO: 4 or 25 under low stringency hybridization conditions and encodes a polypeptide having "pullulanase activity" to the complement of SEQ ID NO: 6 and codes for a polypeptide having α-glucosidase activity to the complement of any of SEQ ID NO: 19, 21, 37, 39, 41, or 43 under conditions of low stringency hybridization and encodes a polypeptide having glucose isomerase activity to the complement of any of SEQ ID NO. : 46, 48, 50, or 59 under conditions of low stringency hybridization and encodes a polypeptide having glucoamylase activity The present invention also relates to an isolated polynucleotide, a) comprising SEQ ID NO: 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108, and 110 or the complement thereof, or a polynucleotide that hybridizes to the complement of either SEQ. ID NO: 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108, or 110 under or low stringency hybridization conditions and encodes a polypeptide having xylanase, cellulase, glucanase, beta-glucosidase, esterase or phytase activity, b) which codes for a polypeptide comprising SEQ ID NO: 62, 64,.-66 , 70, 80, 82, 84, 86, 88, 90, 92, 109, or 111 or an enzymatically active fragment thereof. The isolated polynucleotide can encode a fusion polypeptide comprising a first polypeptide and a second peptide, wherein the first polypeptide has xylanase, cellulase, glucanase, beta-glucosidase, pro-tease, or phytase activity. The second peptide may comprise a signal sequence peptide, which directs the first polypeptide to a vacuole, endoplasmic reticulum, chloroplast, starch granule, seed or cell wall of a plant. For example, the signal sequence may be a waxy N-terminal signal sequence, a? -zein N-terminal signal sequence, a starch binding domain, or a C-terminal starch binding domain. Exemplary xylanases provided and useful in the invention include those encoded by SEQ ID NO: 61, 63, or 65. An exemplary protease, specifically bromelain, encoded by SEQ ID NO: 69 is also provided. Exemplary cellulases include cellobiohydrolase I and II as provided herein and encoded by SEQ ID NO: 79, 81, 93, and 94. An exemplary glucanase is provided as 6GP1 described herein as encoded by SEQ ID NO: 85 Exemplary beta-glucosidases include beta-glucosidase 2 and D, as described herein and encoded by SEQ ID NO: 96 and 97. An exemplary esterase, specifically ferulic esterase-acid as encoded by SEQ, is also provided. ID NO: 99. And, an example phytase, Nov9X as it is encoded by SEQ ID NO: 109-112, is also provided. Also included are expression cartridges comprising a polynucleotide a) having SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 or a complement thereof, or a polynucleotide that hybridizes to complement any of SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 , or 59 or under conditions of low stringency hybridization- and encodes a polypeptide having α-amylase, pullulanase, α-glucosidase, glucose-isomerase, or glucoamylase, or b) that codes for a polypeptide comprising SEQ ID NO : 10, 13, 14, 15, 16, 18, 20, 24, 26, 27, 28, 29, 30, 33, 34, 35, 36, 38, 40, 42, 44, 45, 47, 49, or 51, or an enzymatically active fragment thereof. The expression cassette further comprises a promoter operably linked to the polynucleotide, such as an inducible promoter, specific tissue promoter, or preferentially an endosperm-specific promoter. Preferably, the endosperm specific promoter is a corn? -zein promoter or a corn ADP-gpp promoter or a corn Q promoter promoter or a rice glutelin-1 promoter. In a preferred embodiment, the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 67 or SEQ ID NO: 98. In addition, in another preferred embodiment, the polynucleotide is oriented in homo-sense orientation relative to the promoter. The expression cassette of the present invention can additionally encode a signal sequence that is operably linked to the polypeptide encoded by the polynucleotide. The signal sequence preferably directs the polypeptide operably linked to a vacuole, endoplasmic reticulum, chloroplast, starch granule, seed or cell wall of a plant. The signal sequences include a N-terminal signal sequence of waxy, an N-terminal signal sequence of? -zein, or a starch binding domain. Additionally, an expression cassette comprising a polynucleotide a) having SEQ ID NO: 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 and 110 or a complement thereof, or a polynucleotide that hybridizes to the complement of any of SEQ ID NO: 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 and 110 or under conditions of low stringency hybridization and encodes a polypeptide having xylanase, cellulase, glucanase, beta-glucosidase, esterase or phytase activity, or b) encoding a polypeptide comprising SEQ. ID NO: 62, 64, 66, 70, 80, 82, -84, 86, 88, 90, 92, 109, or 111, or an enzymatically active fragment thereof. The expression cassette further comprises a promoter operably linked to the polynucleotide, such as an inducible promoter, specific tissue promoter, or preferentially an endosperm specific promoter. The endosperm-specific promoter may be a corn? -zein promoter or a corn ADP-gpp promoter or a corn Q promoter promoter or a rice glutelin-1 promoter. In one embodiment, the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 67 or SEQ ID NO: 98. Additionally, in another embodiment, the polynucleotide is oriented in homo-sense orientation in relation to the promoter. The expression cartridge of the present invention may additionally encode a signal sequence that is operably linked to the polypeptide encoded by the polynucleotide. The signal sequence preferably directs the polypeptide operably linked to a vacuole, endoplasmic reticulum, chloroplast, starch granule, seed or cell wall of a plant. The signal sequences include a N-terminal signal sequence of waxy, an N-terminal signal sequence of? -zein, or a starch binding domain. The present invention further relates to a vector or cell comprising the expression cartridges of the present invention. The cell can be selected from the group consisting of "an Agrobacterium, a monocotyledonous cell, a dicotyledonous cell, a Liliopsida cell, a Panicoideae cell, a maize cell, a cereal cell, such as a rice cell. In addition, the present invention encompasses a plant that is stably transformed with the vectors of the present invention, A plant that is stably transformed with a vector comprising an α-amylase having an amino acid sequence of any of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33, 35 or 88 or encoded by a polynucleotide comprising any of SEQ ID NO: 2, 9 or 87. In another embodiment, a plant transformed in a stable manner with a vector comprising a pullulanase having an amino acid sequence of any of SEQ ID NO: 24 or 34, or encoded by a polynucleotide comprising any of SEQ ID NO: 4 or 25. In addition a plant is provided stably transformed with a vector comprising an α-glucosidase having an amino acid sequence of any of SEQ ID NO: 26 or 27, or encoded by a polynucleotide comprising SEQ ID NO: 6. plant stably transformed with a vector comprising a glucose isomerase having an amino acid sequence of any of SEQ ID NO: 18, 20, 28, 29, 30, 38, 40, 42, or 44, or encoded by a polynucleotide comprising any of SEQ ID NO: 19, 21, 37, 39, 41, or 43. In another embodiment, a plant transformed stably with a vector comprising a glucose amylase having a sequence of amino acids of any of SEQ ID NO: 45, 4-7, or 49, or encoded by a polynucleotide comprising any of SEQ ID NO: 46, 48, 50, or 59. A further embodiment provides a plant stably transformed with a vector comprising a xylanase having a sequence of amino acids of any of SEQ ID NO: 62, 64 or 66, or encoded by a polynucleotide comprising any of SEQ ID NOS: 61, 63, or 65. A stably transformed plant is also provided with a vector comprising a protease. The protease can be a bromelain having an amino acid sequence as set forth in SEQ ID NO: 70, or encoded by a polynucleotide having SEQ ID NO: 69. In another embodiment, a plant transformed stably with a vector is provided. which comprises a cellulase. The cellulase can be a cellobiohydrolase encoded by a polynucleotide comprising any of SEQ ID NO: 79, 80, 81, 82, 93A 94. A further embodiment provides a plant stably transformed with a vector comprising a glucanase, such as endoglucanase. The endoglucanase can be endoglucanase I having an amino acid sequence as in SEQ ID NO: 84, or encoded by a polynucleotide comprising SEQ ID NO: 83. A stable transformed plant is also provided with a vector comprising a beta- glucosidase The beta-glucosidase can be a beta-glucosidase 2 or beta-glucosidase D, having an amino acid sequence set forth in SEQ ID NO: 90 or 92, or encoded by a polynucleotide having SEQ ID NO: 89 or 91. In another embodiment, a plant transformed in a stable manner with a vector comprising an esterase is provided. The esterase may be an esterase of ferulic acid encoded by a polynucleotide comprising SEQ ID NO: 99. In addition, plant products, such as seed, fruit or grain, of the stably transformed plants of the present invention are provided. In another embodiment, the invention relates to a transformed plant, the genome of which is augmented with a recombinant polynucleotide encoding at least one processing enzyme operably linked to a promoter sequence, polynucleotide of the sequence from which it is optimized for expression in the plant. The plant can be a monocot, such as corn or rice, or a dicot. The plant can be a cereal plant or a commercially grown plant. The processing enzyme is selected from the group consisting of α-amylase, glucoamylase, glucose-isomerase, glucanase, β-amylase, α-glucosidase, -isoamylase, pullulanase, neo-pullulanase, iso-pullulanase, amylopululanase, cellulase, exo-1 , 4-β-cellobiohydrolase, exo-1,3-β-D-glucanase, "β-glucosidase, endoglucanase, L-arabinase, α-arabinosidase, galactanase, galactosidase, mannanase, mannosidase, xylanase, xylosidase, protease, glucanase, xylanase, esterase, - phytase, and lipase The processing enzyme is a starch processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose-isomerase, β-amylase, α-glucosidase, isoamylase, pullulanase, neo-pullulanase , iso-pullulanase, and amilopululanase The enzyme can be selected from α-amylase, glucoamylase, glucose-isomerase, glucose-isomerase, α-glucosidase, and pullulanase The processing enzyme can be hyperthermophilic According to this aspect of the invention , the enzyme can be a non-degrading enzyme of selected midon of the group consisting of protease, glucanase, xylanase, esterase, phytase, cellulase, beta-glucosidase, and lipase. These enzymes can be hyperthermophilic. In one embodiment, the enzyme accumulates in the vacuole, endoplasmic reticulum, chloroplast, starch granule, seed or cell wall of a plant. In addition, in another embodiment, the genome of the plant can be further increased with a second recombinant polypeptide comprising a non-hyperthermophilic enzyme. In another aspect of the invention, a transformed plant is provided, the genome of which is augmented with a recombinant polynucleotide encoding at least one processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose-isomerase, a- glucosidase, pullulanase, xylanase, cellulase, protease, glucanase, beta-glucosidase, esterase, phytase or lipase operably linked to a promoter sequence, sequence polynucleotide of which is optimized for expression in the plant. Another embodiment refers to a corn transformed plant, the genome of which is augmented with a recombinant polynucleotide that codes for at least one processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose-isomerase, α-glucosidase, pullulanase, xylanase, cellulase, protease, glucanase, phytase, beta-glucosidase, esterase or lipase operably linked to a promoter sequence, polynucleotide sequence of which is optimized for expression in the corn plant. A transformed plant is provided, the genome of which is increased with a recombinant polynucleotide having SEQ ID NO: 83 operably linked to the promoter and to a signal sequence. Additionally, a transformed plant is described, the genome of which is augmented with a recombinant polynucleotide having SEQ ID NO: 93 or 94 operably linked to a promoter and a signal sequence. In another embodiment, a transformed plant, the genome, of which is augmented with a recombinant polynucleotide having SEQ ID NO: 95, operably linked to a promoter and a signal sequence, In addition, a transformed plant is described. , the genome of which is increased with a recombinant polynucleotide having SEQ ID NO: 96. A transformed plant is also described, the genome of which is augmented with a recombinant polynucleotide having SEQ ID NO: 97. transformed plant, the genome of which is augmented with a recombinant polypeptide having SEQ ID NO: 99. Additional products of the transformed plants are contemplated in the present example, the product includes, for example, seed, fruit or grain. Alternatively, a plant obtained from a stably transformed plant of the present invention is described in an alternative manner as a processing enzyme, starch or sugar. it can be a hybrid plant or a congenital plant. A starch composition of a further embodiment of the invention comprising at least one processing enzyme that is a protease, glucanase, or esterase. The grain is another embodiment of the invention comprising at least one processing enzyme, which is an α-amylase, pullulanase, α-glucosidase, glucoamylase, glucose isomerase, xylanase, cellulase, glucanase, beta-glucosidase, β-esterase. , protease, lipase or phytase. In another embodiment, there is provided a method for preparing starch granules, which comprises treating the grain comprising at least one non-starch processing enzyme under conditions that activate at least one enzyme, producing a mixture comprising starch granules and non-starch products. degradation of starch, where the grain is obtained from a transformed plant, the genome of which is increased with an expression cassette that encodes at least one enzyme; and separating the starch granules from the mixture. In the present, the enzyme can be a protease, glucanase, xylanase, phytase, lipase, beta-glucosidase, cellulase or esterase. Still further, the enzyme is preferably hyperthermophilic. The grain can be fractionated grain and / or can be treated under conditions of low or high humidity. Alternatively, the grain can be treated with sulfur dioxide. The present invention further comprises separating the non-starch products from the mixture. Additionally, the starch products and non-starch products obtained by this method are described. In yet another embodiment, there is provided a method for producing hyperdulceous corn comprising treating transformed corn or a part thereof, the genome of which is increased with and expresses in the endosperm an expression cassette that encodes at least one starch degrading enzyme or isomerizadora of starch, under conditions that activate at least one enzyme to convert polysaccharides in corn into sugar, producing hyperdulce corn. The expression cassette may further comprise a promoter operably linked to the polynucleotide encoding the enzyme. The promoter can be a constitutive promoter, seed specific promoter, or specific endosperm promoter, by way of example. The enzyme can be hyperthermophilic and can be an alpha-amylase. The expression cassette used herein may further comprise a polynucleotide that codes for a signal sequence operably linked to at least one enzyme. The signal sequence can direct the enzyme to the apoplast or the endoplasmic reticulum, by way of example. The enzyme comprises any of SEQ ID No: 13, 14, 15, 16, 33 or 35. The enzyme may also comprise SEQ ID No: 87. In a more preferred embodiment, a method for producing hyperdulce corn comprising treating corn is described. transformed or a part thereof, the genome of which is increased with and expresses in the endosperm an expression cassette that codes for a -milas, under conditions that activate at least one enzyme to convert the polysaccharides in the corn into sugar, producing corn hyperdulce The enzyme may be hyperthermophilic and the hyperthermophilic α-amylase may comprise the amino acid sequence of any of SEQ. ID. Nos .: 10, 13, 14, 15, 16, 33 or 35, or an enzymatically active fragment thereof having a-amylase activity. The enzyme comprises SEQ ID No: 87. A method for preparing a hydrolyzed starch product solution comprising; treating a plant part comprising starch granules and at least one processing enzyme under conditions that activate at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising a hydrolyzed starch product, wherein the plant is obtained from a transformed plant, the genome of which is augmented with an expression cassette that codes for at least one starch processing enzyme; and collecting the aqueous solution comprising the hydrolyzed starch product. The hydrolyzed starch product may comprise a dextrin, malto-oligosaccharide, glucose and / or mixtures thereof. The enzyme can be α-amylase, α-glucosidase, glucoamylase, pullulanase, amylopululanase, glucose-isomerase, or any combination thereof. In addition, the enzyme can be hyperthermophilic. In another aspect, the genome of the plant part can be further increased with an expression cassette that encodes a non-hyperthermophilic starch processing enzyme. The non-hyperthermophilic starch processing enzyme can be selected from the group consisting of amylase, glucoamylase, α-glucosidase, pullulanase, glucose-isomerase, or a combination thereof. In yet another aspect, the processing enzyme is preferably expressed in the endosperm. The plant part can be grain, and corn, wheat, barley, rye, oats, sugar cane or rice. At least one processing enzyme is operably linked to a promoter and to a signal sequence that directs the enzyme to the starch granule or the endoplasmic reticulum, or to the cell wall. The method may further comprise isolating the hydrolyzed starch product and / or fermenting the hydrolyzed starch product. In another aspect of the invention, there is disclosed a method for preparing a hydrolyzed starch product comprising treating a plant part comprising starch granules and at least one starch processing enzyme under conditions that activate at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising a hydrolyzed starch product, wherein the plant part extends from a transformed plant, the genome of which is augmented with an expression cassette encoding at least one amylase; and collecting the aqueous solution comprising the hydrolyzed starch product. The α-amylase can be hyperthermophilic and the hyperthermophilic α-amylase comprises the amino acid sequence of any of SEQ ID No: 1, 10, 13, 14, 15, 16, 33 or 35, or an active fragment thereof having a-amylase activity. The expression cassette may comprise a polynucleotide selected from any of SEQ ID No: 2, 9, 46 or 52, a complement thereof, or a polynucleotide that hybridizes to any of SEQ ID No: 2, 9, 46 or 52 under "Hybridization conditions of low stringency and encodes a polypeptide having α-amylase activity In addition, the invention further provides the genome of the transformed plant further comprising a polynucleotide that encodes a non-thermophilic starch processing enzyme. Alternatively, the plant part can be treated with a non-hyperthermophilic starch processing enzyme The present invention further relates to a portion of transformed plant comprising at least one starch processing enzyme present in the cells of the plant, wherein the plant part is obtained from a transformed plant, the genome of which is increased with an expression cassette that codes for at least one enzyme processed The enzyme is preferably a starch processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose-isomerase, β-amylase, α-glucosidase, isoamylase, pullulanase, neo-pullulanase, isoform, pullulanase, and amilopululanase. Still further, the enzyme can be hyperthermophilic. The plant can be any plant, such as corn or rice, as an example. Another embodiment of the invention is a transformed plant part comprising at least one starch non-processing enzyme present in the cell wall or the plant cell, wherein the plant part is obtained from a transformed plant, the genome of the which is increased with an expression cassette that codes for at least one non-starch processing enzyme or at least one non-processing starch polysaccharide enzyme. The enzyme can be hyperthermophilous. In addition, the non-starch processing enzyme may be a protease, gluconase, xylanase, esterase, phytase, beta-glucosidase, cellulose or lipase. The plant part can be any part of plant, but preferably it is a spike, seed, fruit, grain, fodder, shredded straw, or bagasse. The present invention also relates to parts of transformed plant. For example, a transformed plant part comprising an α-amylase having an amino acid sequence of any of SEQ ID No: 1, 10, 13, 14, 15, 16, 33 or 35, or encoded by a polynucleotide is described. comprising any of SEQ ID No: 2, 9, 46 or 52, a transformed plant part comprising an α-glucosidase having an amino acid sequence of any of SEQ ID No: 5, 26 or 47, or encoded by a polynucleotide comprising SEQ ID No: 6, a transformed plant part comprising a glucose isomerase having the amino acid sequence of any of SEQ ID No: 28, 29, 30, 38, 40, 42 or 44, or encodes a polynucleotide comprising any of SEQ ID No: 19, 21, 37, 39, 41 or 43, a transformed plant part comprising a glucoamylase having the amino acid sequence of SEQ ID NO: 45 or SEQ ID No: 47 or SEQ ID No: 49, or encoded by a polynucleotide comprising any of SEQ ID No: 46, 48, 50 or 59, and a transformed plant part comprising a pullulanase encoded by a polynucleotide comprising any of SEQ ID No: 4 or 25. The present invention also relates to parts of transformed plant. For example, a transformed plant part comprising a xylanase having an amino acid sequence of any of SEQ ID No: 62, 64 or 66, or encoded by a polynucleotide comprising any of SEQ ID No: 61, 63 or 65. A part of transformed plant comprising a protease is also provided. The protease can be a bromelain having an amino acid sequence as set forth in SEQ ID NO: 70, or encoded by a polynucleotide having SEQ ID NO: 69. In another embodiment, a transformed plant part comprising a cellulase is provided. . The cellulose can be a cellobiohydrolase encoded by a polynucleotide comprising any of SEQ ID No: 79, 80, 81, 82, 93 or 94. A further embodiment provides a transformed plant part comprising a glucanase, such as an endoglucanase. The endoglucanase can be endoglucanase I having an amino acid sequence as in SEQ ID No: 84, or encoded by a polynucleotide comprising SEQ ID No: 83. A part of transformed plant part comprising a beta-glucosidase is also provided. The beta-glucosidase may be a beta-glucosidase 2 or beta-glucosidase D, having an amino acid sequence set forth in SEQ ID No: 90 or 92, or encoded by a polynucleotide having SEQ ID No: 89 or 91. In another embodiment, a portion of transformed plant comprising an esterase is provided, the esterase may be a ferulic acid esterase encoded by a polynucleotide comprising SEQ ID No: 99. Another embodiment is a method for converting starch into the transformed plant part it comprises activating the processed enzyme of starch contained therein. Additionally the starch, dextrin, malto-oligosaccharide or sugar produced according to this method is described. The present invention further discloses a method for using a transformed plant part comprising at least one non-starch enzyme in the cell wall or the cell of the plant part, which comprises treating a transformed plant part comprising at least one non-processing enzyme of starch polysaccharide under conditions to activate at least one enzyme which thus digests the non-starch polysaccharide to form an aqueous solution comprising oligosaccharide and / or sugars, wherein the plant part is obtained from a part of transformed plant, the genome of which is augmented with an expression cassette that codes for at least one non-starch polysaccharide processing enzyme; and collecting the aqueous solution comprising the oligosaccharides and / or sugars. The non-starch polysaccharide processing enzyme can be hyperthermophilic. A method for using seeds comprising at least one process enzyme is described, which comprises treating transformed seeds comprising at least one protease or lipase under conditions to activate at least one enzyme, producing an aqueous mixture comprising amino acids and fatty acids, in where the seeds are obtained from a transformed plant, the genome of which is increased with an expression cassette that codes for at least one enzyme; and collect the aqueous mixture. Preferably amino acids, fatty acids or both are isolated. The at least one protease or lipase can be hyperthermophilic. A method for preparing ethanol comprising treating a plant part comprising at least one polysaccharide processing enzyme is described under conditions for activating at least one enzyme, thereby digesting the polysaccharide to form fermentable sugar or oligosaccharide, wherein the part of plant is obtained from a transformed plant, the genome of which is increased with an expression cassette that codes for at least one polysaccharide processing enzyme; and incubating the fermentable sugar under conditions that promote the conversion of the fermentable sugar or oligosaccharide into ethanol. The plant part can be a grain, fruit, seed, stems, wood, vegetable or root. The plant part can be obtained from a plant selected from the group consisting of oats, barley, wheat, berry, grapes, rice, corn, rye, potato, sugar beet, sugar cane, pineapple, lawns and trees. In another preferred embodiment, the polysaccharide processing enzyme is α-amylase, glucoamylase, glucoamylase, α-glucosidase, glucose-isomerase, pullulanase, or a combination thereof. A method for preparing ethanol is provided which comprises treating a plant part comprising at least one enzyme selected from the group consisting of α-amylase, glucoamylase, α-glucosidase, glucose-isomerase or pullulanase, or a combination thereof, with heat for a quantity of time and under conditions to activate at least one enzyme, thereby digesting the polysaccharide to form fermentable sugar, wherein the plant part is obtained from a transformed plant, the genome of which is augmented with a Cassette of expression encoding at least one enzyme; and incubate the fermentable sugar under conditions that promote the conversion of the fermentable sugar into ethanol. The at least one enzyme can be hyperthermophilic or mesophilic. In another embodiment, there is provided a method for preparing ethanol which comprises treating a plant part comprising at least one starch non-processing enzyme under conditions to activate at least one enzyme, thereby digesting the polysaccharide not from starch to oligosaccharide and sugar fermentable, wherein the plant part is obtained from a transformed plant, the genome of which is augmented with an expression cassette that codes for at least one enzyme; and incubate the fermentable sugar under conditions that promote the conversion of the fermentable sugar into ethanol. The non-starch processing enzyme may be a xylanase, cellulose, glucanase, beta-glucosidase, protease, esterase, lipase or phytase. There is further provided a method for preparing ethanol which comprises treating a plant part comprising at least one enzyme selected from the group consisting of α-amylase, glucoamylase, α-glucosidase, glucose isomerase or pullulanase, or a combination thereof, under conditions to activate at least one enzyme, thereby digesting the polysaccharide to form fermentable sugar, wherein the plant part is obtained from a transformed plant, the genome of which is augmented with a Expression cassette that codes for at least one enzyme; and incubate fermentable sugar under conditions that promote the conversion of fermentable sugar into ethanol.

The enzyme can be hyperthermophilous. On the other hand, a method for producing a sweetened farinaceous food product without additional sweetener is described which comprises treating a plant part comprising at least one starch processing enzyme under conditions that activate at least one enzyme, thereby processing the granules of starch in the plant part to sugars to form a sweetened product, wherein the plant part is obtained from a transformed plant, the genome of which is augmented with an expression cassette that encodes at least one enzyme; and process the sweetened product in a farinaceous food product. The farinaceous food product can be formed from the sweetened product and water. Still further, the farinaceous food product may contain malt, flavors, vitamins, minerals, coloring agents or any combination thereof, or at least one enzyme may be hyperthermophilic, the enzyme may be selected from α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose-isomerase or any combination thereof. The plant can be selected in addition to the group consisting of soybeans, rye, oats, barley, wheat, corn, rice and sugar cane. The farinaceous food product can be a cereal food, a breakfast food, or a ready-to-eat food, or a cooked food. The processing may include cooking, boiling, heating, steam cooking, electric discharge or any combination thereof. The present invention further relates to a method for sweetening a product containing starch without adding sweetener comprising treating the starch comprising at least one starch processing enzyme under conditions to activate at least one enzyme, thereby digesting the starch to form a sugar to form sweetened starch, wherein the starch is obtained from a transformed plant, the genome of which is augmented with an expression cassette encoding at least one enzyme; and adding the sweetened starch to a product to produce a product containing sweetened starch. The transformed plant can be selected from the group consisting of corn, soybeans, rye, oats, barley, wheat, rice and sugar cane. At least one enzyme can be hyperthermophilic. The at least one enzyme can be α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose-isomerase or any combination thereof. A farinaceous food product and product containing sweetened starch is provided herein. . The invention also relates to a method for sweetening a fruit containing a polysaccharide or vegetable, which comprises treating a fruit or vegetable comprising at least one polysaccharide processing enzyme under conditions that activate at least one enzyme, thereby processing the polysaccharide in the fruit or vegetable to form sugar, producing a sweetened fruit or vegetable, where the fruit or vegetable is obtained from a transformed plant, the genome of which is increased with an expression cassette that codes for at least one enzyme polysaccharide processor. The fruit or vegetable is selected from the group consisting of potato, tomato, banana, chayote, peas and beans. The at least one enzyme can be hyperthermophilic. The present invention further relates to a method for preparing an aqueous solution comprising sugar comprising treating starch granules obtained from the plant part under conditions that activate at least one enzyme, thereby producing an aqueous solution comprising sugar. Another embodiment relates to a method for preparing grain starch products that do not comprise wet or dry milling of the grain prior to recovery of the starch derivative products comprising treating a plant part comprising starch granules and at least a starch processing enzyme under conditions that activate at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising dextrins or sugars, wherein the planting part obtains from a transformed plant, the genome of which is augmented with an expression cassette encoding at least a starch processing enzyme; and collecting the aqueous solution comprising the product derived from starch. The at least one starch processing enzyme can be hyperthermophilic. Further provided is a method for isolating an α-amylase, glucoamylase, glucose isomerase α-glucosidase, and pullulanase comprising culturing a transformed plant containing the α-amylase, glucoamylase, glucose-isomerase, α-glucosidase or pullulanase and isolating a-amylase, glucoamylase, glucose-isomerase, a-glucosidase or pullulanase thereof. Also provided is a method for isolating a xylanase, cellulase, glucanase, beta-glucosidase, protease, esterase, phytase or lipase comprising culturing a transformed plant containing the xylanase, cellulose, glucanase, beta-glucosidase, protease, esterase, phytase or lipase and isolate the xylanase, cellulase, glucanase, esterase, beta-glucosidase, protease, esterase, phytase or lipase. A method for preparing maltodextrin comprising mixing the transgenic grain with water, heating the mixture, separating the solid from the generated dextrin syrup, and collecting the maltodextrin. The transgenic grain comprises at least one starch processing enzyme. The starch processing enzyme can be α-amylase, glucoamylase, α-glucosidase, and glucose-isomerase. In addition, the maltodextrin produced by the method as well as the composition produced by this method is provided. A method is provided for preparing dextrins, grain sugars that do not comprise mechanical breakdown of the grain prior to recovery derived from starch, which comprises: treating a plant part comprising starch granules and at least one starch processing enzyme under conditions which activate at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising dextrins or sugars, wherein the plant part is obtained from a transformed plant, the genome of which is augmented with an expression cassette. which codes for at least one processing enzyme; and collecting the aqueous solution comprising sugar and / or dextrins. The present invention further relates to a method for producing fermentable sugar comprising treating a plant part comprising granules and starch and at least one starch processing enzyme under conditions that activate at least one enzyme, thereby processing the granules of starch to form an aqueous solution comprising dextrins or sugars, wherein the plant part is obtained from a transformed plant, the genome of which is augmented with an expression cassette encoding at least one processing enzyme, and collecting the In addition, a corn plant stably transformed with a vector comprising a hyperthermophilic α-amylase is provided herein, for example, a maize plant transformed from stable form with a vector comprising a polynucleotide sequence encoding α-amylase that is more than 60% identical to SEQ ID No: l or SEQ ID No: 51.

Brief Description of the Figures Figures IA and IB illustrate the activity of α-amylase expressed in corn grains and in the endosperm of segregating IT grains, plants pNOV6201, and six lines pNOV6200. Figure 2 illustrates the α-amylase activity in segregating IT grains of lines pNOV6201. Figure 3 depicts the amount of ethanol produced in the fermentation of transgenic corn masses containing thermostable α-amylase 797GL3 which were subjected to liquefaction times of up to 60 minutes at 85 ° C and 95 ° C.

The figure illustrates that the yield of ethanol at 72 hours of fermentation was almost unchanged from 15 minutes to 60 minutes of liquefaction. Furthermore, it shows that the mass produced by liquefaction at 95 ° C produced more ethanol at each point of time than the mass produced by liquefaction at 85 ° C. Figure 4 represents the amount of residual starch (%) remaining after fermentation of the transgenic corn masses containing thermostable α-amylase that were subjected to a liquefaction time: of 'up to' 60 minutes at 85 ° C and 95 ° C. This figure illustrates that the yield of ethanol at 72 hours of fermentation was almost unchanged from 15 minutes to 60 minutes of liquefaction.Furthermore, it shows that the mass produced by liquefaction at 95 ° C produced more ethanol in each point of time that the mass produced by liquefaction at 85 ° C. Figure 5 represents the ethanol yields for masses of a transgenic corn, control corn and various mixtures thereof prepared at 85 ° C and 95 ° C. Figure illustrates that the transgenic maize comprising α-amylase results in significant improvement in the preparation of starch available for fermentation since there was a reduction in starch left after fermentation. ura 6 represents the amount of residual starch measured in dried spill after fermentation for masses of a transgenic maize, control maize and various mixtures thereof prepared at 85 ° C and 95 ° C. Figure 7 depicts the ethanol yields as a function of the fermentation time of a sample comprising 3% transgenic corn during a period of -80 hours at various pH ranges from 5.2-6.4. The figure illustrates that the fermentation carried out at a lower pH proceeds faster than at a pH of 6.0 or higher. Figure 8 depicts the ethanol yields during the fermentation of a dough comprising various percentages by weight of transgenic corn from 0-12% by weight at various pH ranges from 5.2-6.4. This figure illustrates that the ethanol yield was independent of the amount of transgenic grain included in the sample. Figure 9 shows the analysis of T2 seeds of different events transformed with pNOV7005. High expression of pullulanase activity can be detected in several events, compared to non-transgenic controls. Figures 10A and 10B show the results of the HPLC analysis of hydrolytic products generated by expressed pullulanase of starch in the transgenic corn flour. Incubation of the starch meal expressing pullulanase in reaction buffer at 75 ° C for 30 minutes results in the production of medium chain oligosaccharides (degree of polymerization (DP) approximately 10-30) and short amylose chains (DP approximately 100-200) of corn starch. Figures 10A and 10B also show the effect of added calcium ions on the pullulanase activity. Figures HA and 11B represent the data generated from the HPLC analysis of the starch hydrolysis product of two reaction mixtures. The first reaction indicated as "Amylase" contains a mixture [1: 1 (w / w)] of samples of transgenic corn flour expressing α-amylase and non-transgenic corn A188; and the second reaction mixture "Amylase + Pullulanase" contains a mixture [1: 1 (w / w)] of corn flour samples of transgenic maize expressing α-amylase and transgenic maize expressing pullulanase. Figure 12 represents the amount of the sugar product in μg in 25 μl of reaction mixture for two reaction mixtures. The first reaction indicated, "Amylase" contains a mixture [1: 1 (w / w)] of corn flour samples from transgenic maize expressing α-amylase and non-transgenic corn A188; and the second reaction mixture "Amylase + Pullulanase" contains a mixture [1: 1 (w / w)] of corn flour samples of transgenic maize expressing α-amylase and transgenic corn -jr that expresses pullulanase. Figures 13A and 13B show the product of starch hydrolysis of two sets of reaction mixture at the end of 30 minutes of incubation at 85 ° C and 95 ° C. For each set there are two reaction mixtures; The first reaction indicated as "Amylase X Pullulanase" contains corn or transgenic flour (generated by cross-pollination) that expresses both α-amylase and pullulanase, and the second reaction indicated, as a mixture of "Amylase" from corn flour samples of Transgenic corn expressing α-amylase and non-transgenic corn A188 in a ratio to obtain the same amount of α-amylase as observed in the cross (Amylase X Pullulanase). Figure 14 depicts the degradation of starch to glucose using non-transgenic maize seeds (control), transgenic maize seeds comprising the a-amylase 797GL3 and a combination of the transgenic maize seed 797GL3 with α-glucosidase Mal A. The Figure 15 represents the conversion of starch to natural at room temperature or 30 ° C. In this figure, reaction mixtures 1 and 2 are a combination of water and starch at room temperature and 30 ° C, phase responses, and reaction mixtures 3 and 4 are a combination of barley α-amylase and starch. ambient temperature and at 30 ° C, respectively Reaction mixtures 5 and 6 are combinations of Thermoanaerobacterium glucoamylase and starch at room temperature and 30 ° C, respectively, and reaction mixtures 7 and 8 are barley α-amylase combinations ( sigma) and Thermoanaerobacterium glucoamylase and starch at room temperature and 30 ° C, respectively, Reaction mixtures 9 and 10 are combinations of barley alpha (amylase) (sigma), control, and starch at room temperature and 30 ° C, The degree of polymerization (DP) of the Thermoanaerobacterium glucoamylase products is indicated Figure 16 depicts the production of fructose from transgenic maize flour amylase using a combination of and alf-amylase, alpha-glucosidase, and glucose-isomerase as described in Example 19. Amylase corn flour is mixed with enzymatic solutions plus water or buffer. All reactions contained 60 mg of amylase flour and a total of 600 μl of liquid and were incubated for 2 hours at 90 ° C. Figure 17 depicts the peak areas of the reaction products with 100% amylase flour of a self-processing grain as a function of the incubation time of 0-1200 minutes at 90 ° C. "Figure 18 represents the peak areas of the reaction products with 10% transgenic amylase flour from a self-processing grain and 90% control corn meal as a function of incubation time from 0-1200 minutes to 90 minutes. C. Figure 19 provides the results of HPLC analysis of transgenic amylase flour incubated at 70 °, 80 °, 90 ° or 100 ° C for up to 90 minutes to assess the effect of temperature on the hydrolysis of starch. Figure 20 represents the ELSD peak area for samples containing 60 mg of transgenic amylase flour mixed with enzyme solutions plus water or buffer under various reaction conditions A set of reactions was buffered with 50 mM MOPS, pH 7.0 at room temperature, plus 10 mM MgSO4 and 1 mM CoCl2, in a second set of metal-containing buffer reactions was replaced by water.All reactions were incubated for 2 hours at 90 ° C.

Detailed Description of the Invention According to the present invention, a plant "self-processing" or part of plant has incorporated in it an isolated polynucleotide that codes for a processing enzyme capable of processing, for example, modifying, starches, polysaccharides, lipids, proteins, and the like, in plants, wherein the processing enzyme can be mesophilic, thermophilic, or hyperthermophilic, and can be activated by grinding, adding water, heating, or providing another mode favorable conditions for enzyme function. The isolated polynucleotide encoding the processing enzyme is integrated into a plant or part of the plant for the expression thereof. In the expression and activation of the processing enzyme, the plant or part of the plant of the present invention processes the substrate in which the processing enzyme acts. Therefore, the plant or plant part of the present invention are capable of self-processing the substrate of the enzyme in the activation of the processing enzyme, contained therein in the absence of, or with reduced external sources, normally required to process these substrates. As such, the transformed plants, the transformed plant cells, and the transformed plant parts have "integrated" processing capabilities to process the desired substrates by the incorporated enzymes thereof according to this invention. Preferably, the polynucleotide encoding the processing enzyme is "genetically stable" ie the polynucleotide is stably maintained in the transformed plant or plant parts of the present invention and is stably inherited by the progeny through successive generations. In accordance with the present invention, the methods employing these plants and plant parts can eliminate the need to grind or otherwise physically break the integrity of the plant parts prior to the recovery of the starch products. For example, the invention provides improved methods for processing corn and other grains to recover products derived from starch. The invention also provides a method that allows the recovery of starch granules containing levels of starch degrading enzymes, in or on the granules, which are suitable for the hydrolysis of specific bonds within the starch without the requirement of adding hydrolyzing enzymes of the starch. starch, exogenously produced. The invention also provides improved products of the self-processing plant, or plant parts, obtained by methods of the invention. In addition, the part of the transformed plant "self-processing", for example, a grain and the transformed plant avoid the problems with the existing technology, that is, the processing enzymes are typically produced by fermentation of microbes, which require isolation of the enzymes of the culture supernatants, which is expensive; the isolated enzyme needs to be formulated for the particular application, and processes and machinery must be developed to add, mix and react the enzyme with its substrate. The transformed plant of the invention or a part thereof is also a source of the same processing enzyme as well as substrates and products of that enzyme, such as sugars, amino acids, fatty acids and starch and not starch polysaccharides. The plant of the invention can also be used to prepare progeny plants such as hybrids and inbreds.

Processing enzymes and polynucleotides that encode them A polynucleotide that codes for a processing enzyme (mesophilic, thermophilic or hyperthermophilic) is introduced into a plant or part of a plant. The processing enzyme is selected based on the desired substrate in which it acts as it is found in transgenic plants or plants and / or the desired final product. For example, the processing enzyme may be a starch processing enzyme, such as a starch degrading enzyme or starch isomerizer, or a non-starch processing enzyme. Suitable processing enzymes include, without limitation, degrading enzymes or starch isomerizers including, for example, α-amylase, endo- or exo-1,4 or 1,6-αD, glucoamylase, glucose isomerase, β-amylases, α-glucosidases and other exo-amylases; and starch de-branching enzymes, such as isoamylase, pullulanase, neo-pullulanase, iso-pullulanase, amylopululanase and the like, glycosyl-transferases such as cyclodextrin-glycosyltransferase and the like, cellulase such as exo-l, 4-β-cellobiohydrolase , exo-1, 3-ß-D-glucanase, hemicellulosase, ß-glucosidase and the like; endoglucanases such as endo-l, 3-β-glucanase and endo-1,4-β-glucanase and the like; L-arabinases, such as endo-1, 5-a-L-arabinase, a-arabinosidases and the like; galactanases such as. endo-1, 4-β-D-galactanase, endo-l-3-β-D-galactanase, β-galactosidases, α-glucosidase and the like; mannanases, such as endo-1, 4-β-D-mannanase, β-mannnosidase, α-mannosidases and the like; xylanases, such as endo-1, 4-β-xylanase, β-D-xylosidase, 1,3-β-D-xylanase and the like; and pectinases; and non-starch processing enzymes, including protease, glucanase, xylanase, thioredoxin / thioredoxin-reductase, esterase, phytase, and lipase. In one embodiment, the processing enzyme is a starch-degrading enzyme selected from the group α-amylase, pullulanase, α-glucosidase, glucoamylase, amylopululanase, glucose-isomerase, or combinations thereof. According to this embodiment, the starch degrading enzyme is capable of allowing the self-processing plant or part of the plant to degrade starch in the activation of the enzyme contained in the plant or part of the plant, as will be described further in the present. The starch degrading enzyme (s) is selected based on the desired final products. For example, a glucose isomerase can be selected to convert glucose (hexose) and fructose. Alternatively, the enzymes may be selected based on the desired end product, derived from starch, with various chain lengths based on eg a function of the degree of processing or with various desired branching patterns. For example, an α-amylase, glucoamylase or amylopululanase can be used under short incubation times to produce dextrin products and under prolonged incubation times to produce products or short chain sugars. A pullulanase can be used to specifically hydrolyze branching points in the starch producing a starch :, with high amylase content, or a neo-pullulanase can be used to produce starch with stretches of at-1,4 bonds with "linkages" intercalated a-1,6: Glucosidases can be used to produce limiting dextrins, or a combination of different enzymes to make other starch derivatives In another embodiment, the processing enzyme is a starch non-processing enzyme selected from protease, glucanase, xylanase , phytase, lipase, cellulase, beta-glucosidase and esterase These non-degrading starch enzymes allow the autoprocessing plant or part of the plant of the present invention to incorporate a desired area of the plant and, on activation, destabilize the plant as which leave the starch granule intact therein For example, in a preferred embodiment, the non-degrading enzymes of starch are aimed at the mat endosperm rizum of the plant cell, in activation, destabilize or break the endosperm matrix while leaving the starch granule intact therein and more easily retrievable the resulting material. Combinations of processing enzymes are additionally contemplated by the present invention. For example, starch processing enzymes and non-starch processing enzymes may be used in combination. Combinations of processing enzymes can be obtained by employing the use of multiple gene constructs that code for each of the enzymes. Alternatively, the individual transgenic plants stably transformed with the enzymes can be crossed by known methods to obtain a plant containing both enzymes. Another method includes the use of exogenous enzymes with the transgenic plant. Processing enzymes can be isolated or derived from any source and the polynucleotides corresponding thereto can be determined by one skilled in the art. For example, the processing enzyme, such as α-amylase, is derived from Pyrococcus (e.g., Pyrococcus furiosus), Thermus, Termococcus (e.g., Thermococcus hydrothermalis), Sulfolobus (e.g., Sulfolobus solfataricus) Thermotoga (e.g. Thermotoga marí tima and Thermotoga neapoli tana), Thermoanaerobacterium (for example, Thermoanaerobacter tengcongensis), Aspergillus (for example, Aspergillus shirousami and Aspergillus niger), Rhizopus (for example, Rhizopus oryzae), Thermoproteales, Desulfurococcus (for example Desulfurococcus amylolyticus), Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Methanopyrus kandleri, Thermosynechococcus elongatus, Thermoplasma acidophilum, Thermoplasma volcanium, Aeropyrum pernix and plants such as corn, barley and rice. The processing enzymes of the present invention are capable of being activated after they are introduced and expressed in the genome of a plant. The conditions for activating the enzyme are determined for each individual enzyme and may include various conditions such as - temperature, pH, hydration, presence of metals, activating compounds, inactivating compounds, etc. For example, temperature-dependent enzymes may include mesophilic, thermophilic, and hyperthermophilic enzymes. Mesophilic enzymes typically have normal activity at temperatures between 20 ° and 65 ° C and are activated "at temperatures above 70 ° C. Mesophilic enzymes have significant activity of 30 to 37 ° C, activity at 30 ° C is preferably at least 10% of the maximum activity, more preferably at least 20% of the maximum activity.The thermophilic enzymes have a maximum activity at temperatures between 50 and 80 ° C and are inactivated at temperatures above 80 ° C. The thermophilic enzyme will preferably have less than 20% maximum activity at 30 ° C, more preferably less than 10% maximum activity.A "hyperthermophilic" enzyme has activity even at higher temperatures.Highermophilic enzymes have a maximum activity at temperatures greater than 80 ° C and retain activity at temperatures of at least 80 ° C, more preferably retain activity at temperatures of at least 90 ° C and more preferably retain activity at temperatures below 95 ° C. The hyperthermophilic enzymes also have reduced activity at low temperatures. A hyperthermophilic enzyme can have activity at 30 ° C which is less than 10% maximum activity, and preferably less than 5% maximum activity. The polynucleotide encoding the processing enzyme is preferably modified to include codons that are optimized for expression in a selected organism such as a plant (see, for example, Wada et al., Nucí Acids Res., 18 .: 2367 (1990), Murria et al., Nucí.

Acids Res.-, 17477 (1989), Patents of the United States Nos. 5,096,825, 5,625,136, 5,670,356 and 5,874,304). The sequences optimized by codons are synthetic sequences, that is, they do not occur in nature and preferentially code for the identical polypeptide (or an enzymatically active fragment of a full-length polysaccharide having substantially the same activity as the full-length polypeptide) encoded by the polynucleotide of non-optimized origin with codons encoding a processing enzyme. It is preferred that the polypeptide be biochemically distinct or enhanced, for example, by recursive mutagenesis of DNA encoding a particular processing enzyme, of the source source polypeptide such that its performance in the application of the process is improved. Preferred polynucleotides are optimized for expression in a target host plant and code for a processing enzyme. Methods for preparing these enzymes include mutagenesis, for example, mutates recursive genesis and selection. Methods for mutagenesis and nucleotide sequence alterations are well known. See, for example Kunkel, Proc. Nati Acad. Sci. USA, 82.:488, (1985); Kunkel et al., Methods in Enzymol. , .154: 367 (1987); U.S. Patent No. 4,873,192; Walker and Gastar, eds. (198) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein and Arnold et al., Chem. Eng. Sci., 51: 5091 (1996)). Methods for optimizing the expression of a nucleic acid segment in a target plant or target organism are well known in the art. Concisely; a codon usage table is obtained indicating the optimal codons used by the target organism and the optimal codons are selected to replace. to those in the target polynucleotide and then the optimized sequence is synthesized chemically. Preferred codons for corn are described in U.S. Patent No. 5,625,136. Additionally nucleic acids complementary to the polynucleotides of the present invention are contemplated. An example of low stringency conditions for hybridization of complementary nucleic acids having more than 100 complementary residues in a filter in a Southern Blot or Northern blot is 50% formamide, eg, hybridization in 50% formamide, 1 M NaCl, SDS 1% at 37 ° C, and a wash in 0.1 X SSC at 60 ° C up to 65 ° C. Exemplary low stringency conditions include hybridization with a formamide buffer solution of to 35%, 1M NaCl, SDS (sodium dodecyl sulfate) at 1% 37 ° C, and a wash in 1 X to 2 X SSC (20X SSC = NaCl 3.0 M / 0.3 M sodium citrate) at 50 to 55 ° C. Moderately severe conditions of example include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37 ° C, and a 0.5X to IX SSC wash at 55 to 60 ° C. Still further, polynucleotides encoding an "enzymatically active" fragment of the processing enzymes are contemplated. As used herein "enzymatically active" means a polypeptide fragment of the processing enzyme having substantially the same biological activity as the processing enzyme to modify the substrate in which the processing enzyme normally acts under appropriate conditions. In a preferred embodiment, the polynucleotide of the present invention is a polynucleotide optimized for maize that codes for α-amylase, as provided in SEQ.

ID Nos: 2, 9, 46 and 52. In another preferred embodiment, the polynucleotide is a polynucleotide optimized for maize coding for pullulanase, as provided in SEQ ID Nos: 4 and 25. In yet another preferred embodiment, the polynucleotide is a corn-optimized polynucleotide that codes for a-glucosidase as provided in SEQ ID No: 6. Another preferred polynucleotide is the corn-optimized polynucleotide encoding glucose isomerase having SEQ. ID. Nos .: 19, 21, 37, 39, 41 or 43. In another embodiment, the corn optimized polynucleotide encoding glucoamylase is preferred as set forth in SEQ ID No: 46, 48 or 50. Further, a polynucleotide optimized for corn is provided. for the glucanase / mannanase fusion polypeptide in SEQ ID No: 57. The invention further provides complements of these polynucleotides, which hybridize under hybridization conditions of moderate stringency or preferably under low stringency and which encode a polypeptide having activity of α-amylase, pullulanase, α-glucosidase, glucose-isomerase, glucoamylase, glucanase or mannanase, as may be the case. The polynucleotide can be used interchangeably with "nucleic acid" or "polynucleic acid" if it refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single or double strand form, composed of monomers (nucleotides) containing a sugar, phosphate and a base, which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to nucleotides that occur naturally . Unless indicated otherwise, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with residues with mixed bases and / or deoxyinosine. In addition, "variants" or substantially similar sequences are encompassed herein. For nucleotide sequences, the variants include those sequences which, due to the degeneracy of the genetic code, code for the identical amino acid sequences of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques such as, for example, polymerase chain reaction (PCR), "hybridization" techniques, and refractive techniques. ligation assembly Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis, which code for the native protein, as well as those encoding a polypeptide having substitutions In general, the variants of the nucleotide sequences of the invention will have at least 40%, 50%, 60%, preferably 70%, more preferably 80%, more preferably 90%, more preferably preferential 90%, and individual unit percentage of identity to the native nucleotide sequence based on these classes, for example, 71%, 72%, 73%, and Imilar, up to at least 90% of class.

Variants may also include a full-length gene corresponding to an identified gene fragment.

Regulatory Sequences: Promoters / Sequences of Signal / Selectable Markers The polynucleotide sequences encoding the processing enzyme of the present invention can be operably linked to "polynucleotide sequences that encode localization signals or signal sequences (in the N or C-terminus of a polypeptide), for example, to direct the hyperthermophilic enzyme or a particular compartment within a plant.Examples of these objectives include, without limitation, the vacuole, endoplasmic reticulum, chloroplast, amyloplast, starch granule, or cell wall, or to a particular tissue, eg, seed The expression of a polypeptide encoding a processing enzyme having a signal sequence in a plant, in particular, in conjunction with the use of an inducible or tissue-specific promoter can produce high levels of processing enzyme located in the plant. They influence the expression or targeting of a polynucleotide to a particular compartment or outside a particular compartment. Suitable signal sequences and targeting promoters are known in the art and include, without limitation, those provided herein.

For example, where expression in specific tissues or organs is desired, tissue-specific promoters can be used. In contrast, where gene expression is desired in response to a stimulus, inducible promoters are the regulatory elements of choice. Where continuous expression is desired throughout the cells of a plant, constitutive promoters are used. Additional regulatory sequences in the 5 'and / or 3' direction of the core promoter sequence can be included in expression constructs of transformation vectors to give rise to varying levels of expression of heterologous nucleotide sequences in a transgenic plant. Several plant promoters with various expression characteristics have been described. Examples of some constitutive promoters that have been described include rice actin 1 (Wang et al., Mol Cell. Biol. 12: 3399 (1992), U.S. Patent No. 5,641,876), CaMV 35S (Odell et al. ., Nature, 313: 810 (1985)), CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987), sucrose synthase (Yang & Russell, 1990), and ubiquitin promoters. Vectors for use in target selection, tissue-specific, of genes in transgenic plants will typically include tissue-specific promoters and may also include other tissue-specific control elements, such as enhancer sequences. Promoters that direct specific or enhanced expression in certain plant tissues will be known to those skilled in the art in view of the present disclosure. These include, for example, the rbcS promoter, specific for green tissue; the promoters oes, nos and more that have greater activity in roots or tissue of injured leaves; 35S truncated promoter. (-90 to +8) that directs enhanced expression in roots, an a-tubulin gene that directs expression in roots and ~ promoters derived from storage protein genes "of zein that directs expression in the endosperm Tissue-specific expression can be achieved functionally by introducing a constitutively expressed gene (all tissues) in combination with an antisense gene that is expressed only in those tissues where the gene product is not desired. a gene encoding a lipase can be introduced such that it is expressed in all tissues using the 35S promoter of the Cauliflower Mosaic Virus.The expression of an antisense transcript of a lipase gene in a corn kernel, using for example A zein promoter will prevent the accumulation of lipase protein in the seed, therefore, the protein encoded by the introduced gene will be present in all tissues except grain. s, in plants have been reported several genes and / or tissue specific regulated promoters.

Some tissue-specific reported genes include genes encoding seed storage proteins (such as napin, cruciferin, beta-conglycimine, and phaseolin), zein or oil body proteins (such as oleosin), or genes included in the fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP-desaturase, and fatty acid desaturases (fad 2-1)), and other genes expressed during embryo development (such as Bce4, see, for example, EP 255378 and Kridl et al., Seed Science Research, 1: 209 (1991)). Examples of tissue-specific promoters, which have been described, include lectin (Vodkin, Proq. Clin. Biol. Res., 138: 87 (1983; Lindstrom et al., Der. Genet., 11: 160 (1990)), corn alcohol dehydrogenase 1 (Vogel et al., 1989; Dennis et al., Nucleic Acids Res., 12: 3983 (1984)), light maize harvesting complex (Simpson, 1986; Bansal et al., Proc. Nati, Acad. Sci. USA, _8_9: 3654 (1992)), corn technical shock protein (Odell et al., 1985, Rochester et al., 1986), small subunit of pea, RuBP-carboxylase (Poulsen et al. ., 1986, Cashmore et al., 1983), mannopine synthase from Ti plasmid (Langridge et al., 1989), nopaline synthase from Ti plasmid (Langridge et al., 1989), chalcone-isomerase from petunia (vanTunen et al. al., EMBQ J.: 1257 (1988)), protein 1 with high bean glycine content (Keller et al., Genes Dev., _3: 1639 (1989)), truncated CaMV 35s (Odell et al., Nature , 313: 810 (1985)), patatina de patato (Wenzler et al., Plant Mol. Biol., 13: 347 (1989)), root cell (Yamamoto et al., Nucleic Acids Res., 18.7449 (1990)), corn zein (Reina et al., Nucleic Acids Res., 18: 6425 (-1990), Kriz et al. ., Mol. Gen. Genet., 207: 90 (1987); Wandelt et al., Nucleic Acids Res., 17: 2354 (1989); Langridge et al., Cell, 3_4: 1015 (1983); Reina et al., Nucleic Acids Res., 18: 7449 (1990)), I-globulin Belanger et al., Genetics, 129: 863 (1991)), a-tubulin, cab (Sullivan et al., Mol. Genet., 215: 431 (1989)), PEPCase (Hudspeth &Gruía, 1989), promoters associated with the R gene complex (Chandler et al., Plant Cell, _1: 1175 (1989)), and chalcone promoters. - synthase (Franken et al., EMBO J., 10: 2605 (1991)). Particularly useful for seed specific expression is the pea vicilin promoter (Czako et al., Mol. Gen. Genet., 235: 33 (1992). See also U.S. Patent No. 5,625,136, incorporated herein by reference. reference). Other useful promoters for expression in mature leaves are those that are activated at the onset of senescence, such as the SAG promoter of Arabidopsis (Gan et al., Science, 270: 1986 (1995).) A class of fruit-specific promoters expressed in or during the anthesis or fluorescence through the development of the fruit, at least until the beginning of maturation, is analyzed in US 4,943,674, the description of which is incorporated in this way as reference.The cDNA clones have been isolated. which are expressed preferentially in cotton fiber (John et al., Proc. Nati, Acad. Sci. USA, 89: 5769 (1992). "Tomato cDNA clones exhibiting differential expression have been isolated and characterized. ", the development of the fruit (Mansson et al., Gen. Genet., 200: 356 (1985), Slater et al., Plant Mol. Biol., _5: 137 (1985)). The promoter for the gene of Polygalacturonase is active in the ripening of the fruit.The polygalacturonase gene is described in the patent of United States Number 4,535,060, United States Patent Number 4,769,061, United States Patent Number 4,801,590, and United States Patent Number 5,107,065, descriptions which are incorporated herein by reference. Other examples of tissue-specific promoters include those that direct expression in leaf cells after damage to the leaf (e.g., chewing insects), in tubers (e.g., patatin gene promoter), and in fiber cells (an example of a fiber cell protein, regulated in the development is E6 (John et al., Proc., Nati. Acad. Sci. USA, 89: 5769 (1992) .The E6 gene is more active in fiber, although low levels of sheet transcripts are found, ovule and flower The tissue specificity of some "tissue-specific" promoters can not be absolute and can be tested by one skilled in the art using the diphtheria toxin sequence, or tissue-specific expression can be achieved with expression. perforated "by a combination of different tissue-specific promoters (Beals et al., Plant Cell,: 1527 (1997).) Other tissue-specific promoters can be isolated by one skilled in the art (see US 5,589,379). , the product direction of a polysaccharide hydrolysis gene, such as α-amylase, can be directed to a particular organelle such as the apoplast instead of the cytoplasm.This is exemplified by the use of the N-terminal signal sequence of ? corn husk (SEQ ID NO: 17 ), which confers objective, apoplasto-specific, protein selection. Addressing the protein or enzyme to a specific compartment will allow the enzyme to be localized in a manner that will not come into contact with the substrate. In this way, the enzymatic action of the enzyme will not occur until the enzyme makes contact with its substrate. The enzyme can be contacted with its substrate by the grinding process (physical breakdown of cell integrity), or by heating the plant cells or tissues to break or destabilize the physical integrity of the cells or plant organs that contain the enzyme . For example, a starch hydrolyzing enzyme, mesophilic, can be directed to the apoplast or endoplasmic reticulum and not to contact the starch granules in the amyloplast. The grinding of the grain will break or destabilize the integrity of the grain and the starch hydrolyzing enzyme will then be in contact with the starch granules. In this way, the potential negative effects of co-localization of an enzyme and its substrate can be circumvented. In another embodiment, a specific tissue promoter includes endosperm-specific promoters such as the corn? -zein promoter (exemplified by SEQ ID NO: 12) or the corn ADP-gpp promoter (exemplified by SEQ ID NO: 11) , which includes a 5 'untranslated sequence and an intron sequence) or a Q protein promoter (exemplified by SEQ ID NO: 98) or a rice glutein 1 promoter (exemplified by SEQ ID NO: 67 ). Thus, the present invention includes an isolated polynucleotide comprising a promoter comprising SEQ ID NO: 11, 12, 67 or 98, a polynucleotide that hybridizes to the complement thereof under low stringency hybridization conditions, or a fragment thereof. having promoter activity, for example, at least 10% and preferably at least 50%, the activity of a promoter having SEQ ID NO: 11, 12, 67 or 98. In another embodiment of the invention, the polynucleotide encodes a hyperthermophilic processing enzyme that operably links to a chloroplast (amyloplasts) transit peptide (CTP) and a starch binding domain, for example, of the waxy gene. An exemplary polynucleotide in this embodiment codes for SEQ ID NO: 10 (α-amylase linked to the waxy starch binding domain). Other exemplary polynucleotides encode a hyperthermophilic processing enzyme linked to a signal sequence that directs the enzyme to the endoplasmic reticulum and secretion to the apoplast (exemplified by a polynucleotide encoding SEQ ID NO: 13, 27 or 30, comprising the N-sequence). -terminal of corn-zein operably linked to α-amylase, α-glucosidase, glucose-isomerase, respectively), a hyperthermophilic processing enzyme linked to a signal sequence that retains the enzyme in the endoplasmic reticulum (exemplified by a polynucleotide) which codes for SEQ ID NO: 14, 16, 28, 29, 33, 34, 35 or 36, which comprises the N-terminal sequence of corn? -zein operably linked to the hyperthermophilic enzyme, which is operably linked to SEKDEL, where the enzyme is α-amylase, malA α-glucosidase, glucose- isomerase of T. maritime, glucose-isomerase of T. neapolitana), a hyperthermophilic processing enzyme linked to an N-terminal sequence that directs the enzyme to the amyloplast (exemplified by a polynucleotide coding for SEQ ID NO: 15, which comprises the sequence N-terminal amyloplast target selection operably linked to α-amylase), a hyperthermophilic fusion polypeptide that directs an enzyme to starch granules (exemplified by a polynucleotide encoding SEQ ID NO: 16, comprising the sequence "N-terminal amyloplast targeting target waxy linked operably to an a-amylase / waxy fusion polypeptide comprising the waxy starch binding domain, a processing enzyme hyperthermophilus linked to an ER retention signal (exemplified by a polynucleotide coding for SEQ "ID NO: 38 and 39). In addition, a hyperthermophilic processing enzyme can be linked to a native starch binding site having the amino acid sequence (SEQ ID NO: 53), wherein the polynucleotide encoding the processing enzyme is linked to the nucleic acid sequence optimized for corn (SEQ ID NO: 54) that codes for this binding site. Several inducible promoters have been reported. Many are described in a review by Gatz, in Current Opinion in Biotechnology, 1_: 168 (1996) and Gatz, C, Annu. Rev. Plant Physiol, Plant Mol. Biol., £ 8: 89 (1997). Examples include tetracycline repressor system, Lac repressor system, copper inducible systems, salicylate-inducible systems (such as the PRla system), glucocorticoid-inducible systems (Aoyama T. et al., NH Plant Journal, 1: 605 (1997 )) and inducible by ecdysona. Other inducible promoters include the promoters inducible by ABA and turgor, the promoter of the gene of the bovine binding protein (Schwob et al., Plant J., _4: 423 (1993)), the promoter of the UDP-glucose-flavonoid gene -glycosyltransferase (Ralston et al., Genetics, 119: 185 (1988)), the MPI-proteinase inhibitor promoter (Cordero et al., Plant J., .6: 141 (1994)), and the promoter of the "glyceraldehyde-3-phosphate dehydrogenase gene (KohleM et al., Plant Mol. Biol., 29: 1293 (1995); Quigley et al., ~ J. '-'Mol. Evol., 29: 412 (1989); Martinez et al., J "Mol. Biol., 2.08: 551 (1989).) Benzene-sulfonamide (US 5,364,780) inducible and alcohol-inducible systems are also included (WO 97/06269 and WO 97/06268). and glutathione-S-transferase promoters Other studies have focused on genes inducibly regulated in response to stress or environmental stimuli such as increased salinity, drought, pathogens and wounds (Graham et al., J. Biol .. Chem., 260: 6555 (1985), Graham et al., J. Biol. Chem., 260: 6561 (1985), Smith et al., Planta, 168: 94 (1986).) The accumulation of the metallocarboxypeptidase inhibitory protein is has reported on leaves of injured potato plants (Graham et al., Biochem Biophys, Res. Comm., 101: 1164 (1981)). Other plant genes have been reported that are induced by methyl jasmonate, response producers , technical shock, anaerobic tension, or herbicide insurers The regulated expression of a viral replication protein Chimeric ivation can be further regulated by other genetic strategies, such as, for example, gene activation mediated by Cre (Odell-et al., Mol. Gen. Genet. , 113: 369 (1990)). In this manner, a DNA fragment containing the 3 'regulatory sequence linked by lox sites between the promoter and the coding sequence of 1-replication protein that blocks the expression of a generic replication gene of the promoter can be removed by mediated cleavage by It creates and results in the expression of the trans-activating replication gene. In this case, the chimeric Cre gene, the chimeric transaction replication gene, or both may be under the control of inducible or tissue-specific promoters and development. An alternative genetic strategy is the use of the AR suppressor gene t. For example, regulated expression of a tRNA suppressor gene can conditionally control the expression of a trans-acting replication protein coding sequence containing an appropriate stop codon (Ulmasov et al., Plant Mol. Biol., 3_5: 417 (1997)). Again, either the chimeric tRNA suppressor gene, the chimeric transaction replication gene, or both may be under the control of inducible or tissue-specific promoters and development.

Preferably, in the case of a multicellular organism, the promoter may also be specific to a tissue, organ or particular developmental state. Examples of these promoters include, without limitation, the ADP-gpp promoter of Zea mays and Zea mays? -zein and the Zea mays globulin promoter. The expression of a gene in a transgenic plant can be desired only at a certain period of time during the development of the plant. Frequently, the timing of development is correlated with tissue-specific gene expression. For example, the expression!:, Of zein storage proteins is initiated in the endosperm about 15 days after pollination. Additionally, vectors can be constructed and used in the intracellular targeting of a specific gene product within the cells of a transgenic plant or by targeting a protein to the extracellular environment. This will generally be achieved by attaching a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resulting transit or signal peptide will transport the protein to a particular intracellular or extracellular destination, respectively, and then be removed post-transductionally. The transit or signal peptides act to facilitate the transport of proteins through intracellular membranes, for example, vacuole, vesicle, plastid and mitochondrial membranes, while the signal peptides direct proteins through the extracellular membrane. of signal such as the N-terminal sequence of corn? -zein for the direction to the endoplasmic reticulum and secretion in the apoplast can be operably linked to a polynucleotide encoding a hyperthermophilic processing enzyme according to the present invention ( Torrent et al., 1997) For example, SEQ ID NOs: 13, 27 and 30 provides a polynucleotide that encodes a hyperthermophilic enzyme operably linked to the N-terminal sequence of the corn? -zein protein. is the SEKDEL amino acid sequence for retaining polypeptides in the endoplasmic reticulum (Munro and Pelham, 1987) For example, a polynucleotide which encodes SEQ ID NOS: 14, 26, 28, 29, 33, 34, 35 or 36, which comprises the N-terminal sequence of corn? -zein operably linked to a processing enzyme that is operably linked to SEKDEL. A polypeptide can also be targeted to the amyloplast by fusion to the waxy amyloplast target selection peptide (Klosgen et al., 1986) or to a starch granule. For example, the polynucleotide encoding a hyperthermophilic processing enzyme can be operably linked to a chloroplast (amyloplasts) transit peptide (CTP) and a starch binding domain, for example, of the waxy gene. SEQ ID NO: 10 exemplifies α-amylase linked to the waxy starch binding domain. SEQ ID NO: 15 exemplifies the selection sequence of amyloplast target of N-terminal sequence of waxy operably linked to α-amylase. In addition, the polynucleotide encoding the processing enzyme can be fused to target starch granules using the waxy starch binding domain. For example, SEQ ID NO: 16 exemplifies a fusion polypeptide comprising the waxy N-terminal amyloplast target selection sequence operably linked to an α-amylase / waxy fusion polypeptide comprising waxy starch binding domains. . The polynucleotides of the present invention, in addition to processing signals, may also include other regulatory sequences, as is known in the art. "Regulatory sequences" and "suitable regulatory sequences" each refer to nucleotide sequences located in the 5 'direction (5' non-coding sequences), within or in the 3 'direction (3' non-coding sequences) of a coding sequence , and that has influence on the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences, which can be a combination of synthetic and natural sequences. Selectable markers can also be used in the present invention to allow the selection of transformed plants and plant tissue, as is well known in the art. It may be desirable to employ a selectable or detectable marker gene as, or in addition to, the expressible gene of interest. "Marker genes" are genes that "impart a distinct phenotype to cells that" express the marker gene and thereby allow these transformed cells to be distinguished from cells that do not have the marker. These genes can encode either a selectable or detectable marker, depending on whether the marker confers a trait that can be selected by chemical means, that is, through the use of a selective agent (eg, a herbicide, antibiotic, or the like), or if it is simply a trait that can be identified through observation or testing, that is, by detection (eg, the R-locus trait). Of course, many examples of suitable marker genes are known in the art and can be used in the practice of the invention. Included within the terms selectable or detectable marker genes are also genes that code for a "secretable marker" whose secretion can be detected as a means to identify or select transformed cells. Examples include labels that code for a secretable antigen that can be identified by antibody interaction, or even segregatable enzymes that can be detected by their catalytic capacity. Segregatable proteins fall into several classes, including small, detectable, detectable proteins, for example, by ELISA; active small detectable enzymes in extracellular solution "(eg, α-amylase, β-lactamase, phosphinothricin-acetyltransferase), and proteins that are inserted or entrapped in the cell wall (eg, proteins that include a leader sequence such as that found in the expression unit of extensin or snuff PR-S.) With respect to selectable segregatable markers, the use of a gene that codes for a protein that becomes sequestered in the cell wall, and protein that includes a single epitope is considered which is particularly advantageous.This segregated antigen marker will ideally employ an epitope sequence that will provide underfloor in the plant tissue, a promoter-leader sequence that will impart efficient expression and efficient target selection through the plasma membrane, and will produce protein that binds to the cell wall and is still accessible to antibodies, a normally segregated wall protein modified a to include a unique epitope will satisfy all these requirements. An example of a protein suitable for modification in this manner is extensin, glycoprotein with high content of hydroxyproline (HPRG). For example, the corn HPRG molecule (Steifel et al., The Plant Cell, 2: 785 (1990)) is well characterized in terms of molecular biology, expression and protein structure. However, any of a variety of extensins and / or high glycine wall proteins (Keller et al., EMBO Journal, 8: 1309 (1989)) can be modified by the addition of an antigenic site to create a marker. detectable to. Selectable Markers Possible selectable markers for use in conjunction with the present invention include, without limitation, a neo or nptll gene (Potrykus et al., Mol. Gen. Genet., 199: 183 (1985)) which codes for kanamycin resistance and can be selected to use kanamycin, G418, and the like; a bar gene that confers resistance to the herbicide phosphinothricin; a gene encoding an altered EPSP-synthase protein (Hinchee et al., Biotech., 6: 915 (1988)) thus conferring glyphosate resistance; a nitrilase gene such as Klebsiella ozaenae bxn that confers resistance to bromoxynil (Stalker et al., Science, 242: 419 (1988)); an acetolactate synthase (ALS) mutant gene that confers resistance to i idazolinone, sulfonylurea or other ALS inhibitory chemicals (European Patent Application Number 154,204, 1985); a DHFR gene resistant to methotrexate (Thillet et al., J. Biol. Chem., 263: 12500 (1988)); a dalapon-dehalogenase gene that confers resistance to the dalapon herbicide; a phosphomannanase isomerase (PMI) gene; a mutated anthranilate-synthase gene that confers resistance to 5-methyl-tryptophan; the hph gene that confers resistance to the hygromycin antibiotic; or the mannose-6-phosphate isomerase gene (also referred to herein as the phosphomannanase-isomerase gene), which provides the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629). One skilled in the art is capable of selecting a selectable marker gene, suitable for use in the present invention. Where a mutant EPSP-synthase gene is employed, additional benefits can be realized through the incorporation of a suitable chloroplast transit peptide, CTP (European Patent Application 0,218,571, 1987). An illustrative mode of a selectable marker gene capable of being used in systems to select The transformants are the genes encoding the phosphinotricin-acetyltransferase enzyme, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes. The enzyme phosphinothricin-acetyltransferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., Mol.Gen. Genet., 205: 42 (1986); Twell et al., Plant Physiol., 91: 1270 (1989)) causing rapid accumulation of ammonia and cell death. . The success in the use of this selective system in conjunction with monocotyledons was particularly surprising due to the main difficulties that have been reported in the transformation of cereals (Potrykus, Trends Biotech., 7X269- (1989)). Where it is desired to employ a bialaphos resistance gene in the practice of the invention, a gene particularly useful for this purpose is the bar or pat gene obtainable from the Streptomyces species (eg, ATCC No. 21,705). The cloning of the bar gene (Murakami et al., Mol. Gen. Genet., 205: 42 (1986), Thompson et al., EMBO Journal, _6: 2519 (1987)) has been described as having the use of the gene. bar in the context of different monocotyledonous plants (De Block et al., EMBO Journal, _6: 2513 (1987), De Block et al., Plant Physiol., 91: 694 (1989)). b. Detectable Markers The detectable labels that may be employed include, without limitation, a ß-glucuronidase or uidA (GUS) gene encoding an enzyme for which several chromogenic substrates are known; an R-locus gene, which codes for a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., in Chromosome Structure and Function, pp. 263-282 (1988)); a ß-lactamase gene (Sutcliffe, PNAS USA, 75: 3737 (1978)), which codes for an enzyme for which several chromogenic substrates are known (for example PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., PNAS USA, 80: 1101 (1983)) coding for a catechol-dioxygenase that can convert chromogenic catechols; an α-amylase gene (Ikuta et al., Biotech., 8: 241 (1990)); a tyrosinase gene. (Katz et al., J. Gen. Microbiol., 129-2703 (1983)) which codes for an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily detectable compound, melanin; a β-galactosidase gene, which codes for an enzyme for which there are chromogenic substrates; a luciferase gene (lux) (Ow et al., Science, 234: 856 (1986)), which allows the detection of bioluminescence; an aequorin gene (Prasher et al., Biochem. Biophys. Res. Comm., 126: 1259 (1985)), which can be used in detection of bioluminescence sensitive to calcium, or a green fluorescent protein gene (Niedz et al. ., Plant Cell Reports, 14: 403 (1995)). It is contemplated that genes for the corn R gene complex are particularly useful as detectable markers. The R gene complex in corn codes for a protein that acts to regulate the production of anthocyanin pigments in most plant seeds and tissues.

A gene of the R gene complex is suitable for corn transformation, because the expression of this gene in transformed cells does not harm the cells. In this way, an R gene introduced into these cells will cause the expression of a red pigment, and if it is incorporated stably, it can be marked visibly as a red sector. If a corn line has dominant alleles for genes that code for the intermediate-enzymatic compounds in the anthocyanin biosynthetic pathway (C2, Al, A2, Bzl, Bz2), but has a recessive allele at the R locus, the transformation of Any cell of that line with R will result in the formation of red pigment.The example lines include Wisconsin 22 containing the rg-Stadler allele and TR112, a K55 derivative that is rg, b, Pl. Alternatively, it can be use any maize genotype if Cl and R alleles are co-introduced. An additional detectable marker contemplated for use in the present invention is firefly luciferase., encoded by the lux gene. The presence of the lux gene in transformed cells can be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low light video cameras, photon count cameras or multiple cavity luminometry. It is also contemplated that this system can be developed for population detection by bioluminescence, such as in tissue culture plates, or even for detection of whole plants. The polynucleotides used to transform the plant include, without limitation, DNA of plant genes and non-plant genes, such as those of bacteria, yeast, "animals or viruses." The introduced DNA may include modified genes, portions of genes, or chimeric genes, including genes from the same or different maize genotype The term "chimeric gene" or "chimeric DNA" is defined as a gene or DNA sequence or segment comprising at least two DNA sequences or segments of species that do not combine the DNA under natural conditions, or DNA sequences or segments that are placed or linked in a manner that does not occur normally in the native genome of the non-transformed plant. polynucleotide encoding a hyperthermophilic processing enzyme, and preferably a polynucleotide optimized by codons It is preferred that the polynucleotide in the cassette The expression protein (the first polynucleotide) is operably linked to regulatory sequences, such as a promoter, an enhancer, an intron, a terminator sequence, or any combination thereof, and optionally, to a second polynucleotide encoding a designed sequence (N- or C-terminal) that directs the enzyme encodes the first polynucleotide to a particular cellular or subcellular location. In this manner, a promoter and one or more signal sequence can provide high levels of expression of the enzyme at particular locations in a plant, plant tissue or plant cell. The promoters may be constitutive promoters, inducible promoters (conditional) or tissue-specific promoters, for example, specific endosperm promoters such as the corn? -zein promoter (exemplified by SEQ.ID.NO:12) or the corn ADP-gpp promoter (exemplified by SEQ ID NO: 11, which includes a 5 'intron and untranslated sequence.) The invention also provides an isolated polynucleotide comprising a promoter comprising SEQ ID NO. .: 11 or 12, a polynucleotide that hybridizes the complement thereof under low stringency hybridization conditions, or a fragment thereof having promoter activity, for example at least 10%, and preferably at least 50% , the activity of a promoter having SEQ ID NO: 11 or 12. Also provided are vectors comprising the expression cassette or polynucleotide of the invention and transformed cells comprising the polynucleotide, expression cassette. or vector of the invention. A vector of the invention may comprise a polynucleotide sequence encoding more than one hyperthermophilic processing enzyme of the invention, - sequence which may be in homosense or antisense orientation, and a transformed cell may comprise one or more vectors of the invention. Preferred vectors are those useful for introducing nucleic acids into plant cells.

Transformation The expression cassette, or a vector construct containing the expression cassette can be inserted into a cell. The cassette of expression or construction of the vector can be carried episomally or integrated into the genome of the cell. The transformed cell can then be grown in a transgenic plant. Accordingly, the invention provides the products of the transgenic plant. These products may include, without limitation, the seeds, fruit, progeny and products of the "progeny of the transgenic plant." A variety of techniques for the introduction of constructions are available and known to those skilled in the art. in a cellular host. The transformation of bacteria and many eukaryotic cells can be achieved through the use of polyethylene glycol, calcium chloride, viral infection, phage infection, electroporation and other methods known in the art. Techniques for transforming plant cells or plant tissue include transformation with DNA using A. A. tumefaciens or A. rhizogenes as the transforming agent, electroporation, DNA injection, bombardment and micropoyectiles, acceleration of particles, etc. (See, for example EP 295959 and EP 138341). In one embodiment, the binary type vectors of the Ti and Ri plasmids of Agrobacterium spp. The vectors derived from Ti are used to transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soy, cotton, colsa, tobacco and rice (Pacciotti et al., Bio / Technology, 3: 241 (1985): Byrne et al, Plant Cell Tissue and Organ Culture, 8: 3 (1987); Sukhapinda et al., Plant Mol. Biol., 8: 209 (1987); Lorz et al. Mol. Gen. Genet. , 199: 178 (1985); Potrykus Mol. Gen. Genet .. 199: 183 (1985); Park et al., J. Plant. Biol .. 38: 365 (1985): Hiei et al., Plant J., 6: 271 (1994)). The use of DNA to "" transform plant cells has received extensive study and is widely described (EP 120516; "? Oekema, In: The Binary Plant Vector System, offset-drukkerij Kanters BV; Alblasserdam (1985), Chapter V; Knauf , et al., Genetic Analysis of Host Range Expression by Agrobacterium In: Molecular Genetics of the Bacteria-Plant "Interaction, Puhler, A. ed., Springer-Verlag, New York, 1983, p.245 and An et al., EMBO J., 4: 277 (1985)). Other transformation methods are available to those skilled in the art such as direct uptake of foreign DNA constructs (see, EP 295959), electroporation techniques (Fromm et al., Nature (London), 319: 791 (1986), or high velocity ballistic bombardment with metallic particles coated with the nucleic acid constructs (Kline et al., Nature (London) 327: 70 (1987), and U.S. Patent No. 4,945,050). can be regenerated by those skilled in the art. "Of particular relevance are the methods just described for transforming foreign genes into commercially important crops, such as turnip, (from Block et al., Plant Physiol. 91: 694-701 (1989)), sunflower (Everett et al., Bio / Technology, 5: 1201 (1987)), soy (McCabe et al., Bio / Technology, 6: 923 (1988); Hinchee et al.

Bio / Tecnology, 6: 915 (1988); Chee et al. Plant Physiol, 91: 1212 (1989); Christou et al., Proc. Nati Acad. Sci. USA, 86: 7500 (1989) EP 301749), rice (Hici et al., Plant J. 6: 271 (1994)), and corn (Gordon Kamm et al., Plant Cell, 2: 603 (1990); Fromm et al., Biotechnology, 8: 833, (1990)). Expression vectors containing genomic or synthetic fragments can be introduced into protoplasts or into intact tissues or isolated cells. Preferably, the expression vectors are introduced into intact tissue. "General methods for cultivating plant tissues are provided, for example by Maki et al.," Procedures for Introducing Foreign DNA into Plants "in Methods in Plant Molecular Biology &Biotechnology, Glich et al. (Eds.), Pp. 67 -88 CRC Press (1993); and by Phillips et al. "Cell-Tissue Culture and In-Vitro Manipulation" in Corn & Corn Improvement, 3rd. Edition 10, Sprague et al. (Eds.) Pp. 345-387- American Society of Agronomy Inc. (1988). In one embodiment, expression vectors can be introduced into corn tissues or another plant using a direct gene transfer method such as microprojectile-mediated distribution, DNA injection, electroporation and the like. Expression vectors are introduced into plant tissue using the microprojectile-mediated distribution with the biological device.

See, for example, Tomes et al. "Direct DNA transfer into intact plant cells via microprojectile bombardment" in Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ culture: Fundamental Methods, springer Verlag, Berlin (nineteen ninety five) . However, the present invention contemplates the transformation of plants with a hyperthermophilic processing enzyme according to known methods of transformation. See also, Weissinger et al., Annual Rev. Genet. 22: 421 (1988); Sanford et al., Particulate Science and Technology, 5:27 (1987) (onion); Christou et al., Plant Physiol., 87: 671 (1988) (soybean); McCabe et al, Bio / Technology, 6: 923 (1988) (soybean); Datta et al., Bio / Technology, 8: 736 (1990) (rice); Klein et al., Proc. Nati Acad. Sci. USA, 85: 4305 (1988) (corn); Klein et al., Bio / Technology, 6: 559 (1988) (corn); Klein et al., Plant Physiol, 91: 440 (1988) (corn); Fromm et al., Bio / Technology, 8: 833 (1990) (corn); and Gordon-Kamm et al., Plant Cell, 2, 603 (1990) (corn); Svab et al., Proc. Nati Acad. Sci. EUSA, 87: 8526 (1990) (Tobacco chloroplast); Koziel et al., Biotechnology, 11: 194 (1993) (corn); Shimamoto et al, Nature, 338: 274 (1989) (rice); Christou et al., Biotechnology, 9: 957 (1991) (rice); European Patent Application EP 0,332,581 (garden grass and other Pooideae); Vasil et al., Biotechnology, 11: 1553 (1993) (wheat); Weeks et al., Plant Physiol., 102: 1077 (1993) (wheat). Methods in Molecular Biology, 82. Arabidopsis Protocols Ed. Martinez-Zapater and Salinas 1998 Humana Press (Arabidopsis). The transformation of plants can be undertaken with a single DNA molecule or multiple DNA molecules (ie, co-transformation) and both of these techniques are suitable for use with the expression cartridges and constructions of the present invention. Numerous transformation vectors are available for the transformation of plants, and the expression cartridges of this invention can be used in conjunction with any of these vectors. The selection of the vector will depend on the preferred transformation technique and on the target species for the transformation. Finally, the most desirable DNA segments for introduction into a monocot genome can be homologous genes or gene families that code for a desired trait (eg, hydrolysis of proteins, lipids or polysaccharides) and that are introduced under the control of new promoters or enhancers, for example, or perhaps even homologous or tissue-specific promoters (eg, root specific, collar / sheath, spiral, stem, spike, grain or leaves) or control elements. In fact, it is contemplated that a particular use of the present invention will be the targeting of a gene in a constitutive manner or in an inducible manner.

Examples of suitable transformation vectors Numerous vectors available for plant transformation are known to those skilled in the plant transformation art, and the relevant genes of this invention can be used in conjunction with any of these vectors known in the art. The selection of the vector will depend on the preferred transformation technique and on the target species for the transformation. to. Vectors suitable for transformation of Agrobacterium Many vectors are available for transformation using Agrobacterium tumefaciens. These typically have at least one T-DNA borderline sequence and include vectors such as pBIN19 (Bevan, Nuci, Acids, Res. (1984)). Below, the construction of two typical vectors suitable for transformation of Agrobacterium is described. Y -pCIB200 and pCIB2001 The binary vectors pcIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner. PTJS75kan is created by digestion with NarI from pTJS75 (Schimidhauser &Helinski, J. Bacterial., 164: 446 (1985)) which allows cleavage of the tetracycline resistance gene, followed by insertion of the Accl fragment of pUC4K having NPTII ( Messing & Vierra, Gene, 19: 259 (1982): Bevan et al., Nature, 304: 184 (1983): McBride et al., Plant Molecular Biology, 14: 266 (1990)). The Xhol linkers are ligated to the EcoRV fragment of PCIB7 containing the left and right T-DNA boundaries, a chimeric us / nptll gene selectable from the plant and the pUC polylinker (Rothstein et al., Gene, 53: 153 (1987)) , and the fragment digested with XhoI are cloned into pTJS75kan digested with SalI to create pCIB200 (see also EP 0,332,104, example 19). pCIB200 contains the following unique restriction sites of EcoRI, SstI, • Kpnl, BglII, Xbal and Salí polylinker. pCIB2001 is a derivative of pCIB200 created by the insertion in the polylinker of additional restriction sites. The unique restriction sites in the pCIB2001 polylinker are JEcoRI, SstI, Kpnl, BglII, Xbal, Sali, Mlul, BcII, Avrll, Apal, Hpal, and Stul, pCIB2001, as well as containing these "unique" sites of The restriction also has selection of bacterial and vegetal kanamycin, the limits of left or right T-DNA for transformation measured by agrobacterium, the trfA function derived from RK2 for mobilization between E. coli and other hosts, and the OriT and OriV functions also of RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cartridges containing their own regulatory signals. pCIBlO and Hygromycin Selection Derivatives thereof: The binary vector pCIBlO contains a gene coding for kanamycin resistance for selection in plants and the right and left border sequences of T-DNA and incorporates sequences from the pRK252 plasmid of wide range of hosts that allows it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene, 53: 152 (1997)). Several pCIBlO derivatives are constructed that incorporate the gene for hygromycin-B-phosphotransferase described by Gritz et al. (Gene, .25 .: 179 (1983)). These derivatives allow the selection of transgenic plant cells in hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717). b. Vectors Suitable for Transformation not in Agroba cterium Transformation without the use of Agrobacterium tumefaciens circumvents or evades the requirement of T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be used in addition to vectors such as those described above containing sequences of T-DNA Transformation techniques that do not depend on Agrobacterium include transformation by particle bombardment, protoplast uptake (e.g., PEG and electroporation) and micromyecium. The choice of the vector depends for the most part on the preferred selection of the species that is transformed. Additionally, non-limiting examples of construction of typical vectors suitable for transformation not in AGROBACTERIUM are described. pCIB3064 pCIB3064 is a vector derived from pUC suitable for direct gene transfer techniques in combination with selection by the coarse herbicide (or phosphinothricin). Plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the GUS gene of E. coli and the transcriptional terminator CaMV 35S and is described in published PCT application WO 93/07278. The 35S promoter of this vector contains two ATG 5 'sequences from the start site. These sites are mutated using normal PCR techniques in such a way as to remove the ATG and generate the 5 SspI and Pvull restriction sites. The new restriction sites are 96 and 37 bp away from the single Salí site and 42 bp away from the actual start site. The resulting derivative of pCIB246 is designated pCIB3025. The GUS gene then cleaves from pCIB3035 by digestion with Sail and SacI, the term becoming blunt and being re-ligated to generate the plasmid pCIB3060. The plasmid. pJIT82 can be obtained from John Innes Center, Norwich and a 400 bp Saml fragment containing the bar gene from streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al., EMBO J, 6: "" 2519 (1987)). This pCIB3064, which comprises the bar gene under the control of the CaMV 35S promoter and thermometer for herbicide selection, a gene for ampicillin resistance (for selection in E. coli) and a polylinker with unique sites Sphl, PstI, HindIII, and BamHI. This vector is suitable for the cloning of plant expression cartridges containing "their own regulatory signals. pS0G19 and pSOG35: The plasmid pSOG35 is a transformation vector using the E. coli gel of dihydrofolate reductase (DHFR) as a selectable marker that confers resistance to methotrexate. PCR is used to amplify the 35S promoter (-800 bp), intron 6 of the Adhl maize gene (-550 bp) and 18 bp of the GUS untranslated leader sequence of pSOGlO. A 250 bp fragment encoding the type II dihydrofolate reductase gene from E. coli is also amplified by PCR and these two PCR fragments are mounted on a SacI-PstI fragment of pB1221 (Clontech) comprising the structure of the pUC19 vector and the nopaline-synthase terminator. The assembly of these fragments generates pSOG19 containing the 35S promoter in fusion with the intron 6 sequence. The GUS guide, the DHFR gene and the nopaline synthase terminator. The replacement of the GUS guide in pSOG19 with the guiding sequence of the Clo-róticas Maize Virus Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35: carry the pUC gene for ampicillin resistance and have the HindIII, Sphl, PstI and EcoRI sites available for the cloning of foreign substances. c. Vector Suitable for Chloroplast Transformation For the expression of a nucleotide sequence of the present invention in plant plastids, the plastid transformation vector pPH143 is used (WO 97/32011, example 36). The nucleotide sequence is inserted into pPH143, thereby replacing the PROTOX coding sequence. This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance. Alternatively, the nucleotide sequence is inserted into pPH143 so that it replaces the aadH gene. In this case, the transformants are selected for resistance to PROTOX inhibitors.

Plant Hosts Subjected to Methods of Transformation Any plant tissue capable of subsequent clonal propagation, either by organogenesis or embryogenesis, can be transformed with a construction of the present invention. The term organogenesis means a process by which buds and roots develop sequentially of the meristematic centers while the term embryogenesis means a process by which shoots and roots develop together in a concerted manner (not in a way sequential), either from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and most suited to, the particular species that is transformed. Exemplary tissue targets include differentiated and undifferentiated tissues or plants, including but not limited to, leaf discs, roots, stems, shoots, leaves, pollen, seeds, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue , existing meristematic tissue (e.g., apical meristems, axillary shoots, and root meristems), and induced meristem tissue (e.g., cotyledon meristems and hypocotyl meristem), tumor tissue, and various cell forms and culture such as individual cells, protoplasts, embryos and callus tissue. The plant tissue can be in plants or in organ, tissue or cell culture. The plants of the present invention can take a variety of forms. Plants can be chimeras of transformed cells and untransformed cells; the plants can be clonal transformants (e.g., all cells transformed to contain the expression cassette); the plants may comprise grafts from transformed and untransformed tissues, (for example, a transformed root line inserted into a shoot not transformed into citrus species). Transformed plants can be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, -, first generation (or TI) transformed plants can be self-reproduced to give a second homozygous (or T2) generation of transformed plants, and T2 plants are further propagated through classical breeding techniques. A dominant selectable marker (such as npt II) can be associated with the expression cassette to aid in reproduction. The present invention can be used for the transformation of any plant species, including monocots or dicots, including but not limited to corn (Zea mays), Brassica sp. (for example, B. napus, B. rapa, B. júncea), particularly those Brassica species useful as sources of seed oil, alfalfa, (Medicago sativa), rice (Oryza sativa), rye (Sécale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (for example, pearl millet, (Pennisetum glaucum), millet proso (Panicum miliaceum), foxtail millet (Italic serria), finger millet (Eleusine coracana), sunflower (Helianthus annuus) ) safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), yucca (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucífera), pineapple (Ananas com sus), citrus trees (Citrus spp.) , cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), • cashew (Anacardium occidentale), macadam (Macadamia integrifolia), almond (Pronus amygdalus), sugar beet (Beta vulgaris), cane sugar (Saccharum spp.), oats, barley, vegetables, ornamental plants, timber plants such as conifers and deciduous trees, chayote, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, soy, sorghum, sugarcane , rapeseed, clover, carrot and Arabi dopsi s thal i ana. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (for example, Lactuca sativa), green beans (Phaseolus vulgaris), beans (Phaseolus limentis, pea (Lathyrus spp.), Cauliflower, broccoli, turnip, radish, spinach, asparagus, onion , garlic, pepper, celery, and members of the genus Cucumis such as cucumber (C. sativus), melon (C. cantalupensis), and musk melon (C. meló) .Ornamental plants such as azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), Tulips (Tulipa spp.), Daffodils (Narcissus spp.), Petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. The conifers that can be used in the - practice of the present invention include, for example, pine trees such as incense pine (Pinus taeda), felling pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), conifer pine (Pinus contorta), and Monterey pine (Pinus radiata), Douglas fir (Pseidotsuga menziesii); fir-tree "-Occidental (Tsuga canadensis), fir Sitka (Picea glauca), redwood (Sequioia sempervirens), royal firs such as silver fir (Abies amabilis) and balsamer fir (Abies balsamea), and cedars such as Western red cedar ( Thuja plicata) and Alaska yellow cedar (Chamaecyparis nootkatensis) .The legume plants include beans and peas.The beans include guar, white acacia, frenegreco, soybean, garden bean, cowpea, mango, bean, fava bean, lentil, chickpea, etc. The legumes include, without limitation, peanuts, for example, peanuts, Vicia, for example, crowned carob, hairy carob, adzuki beans, mung beans, and chickpeas, Lupinus, for example, lupine, trifolio, Phaseolus, for example, common bean and bean, Pisum, for example, garden bean, Melilotus, for example Medicago clover, for example alfalfa, Lotus, for example clover, Lens, for example, lentil, and false indigo. for the use in the methods of the invention include alfalfa, orchard grass, cañuela, perennial ryegrass, climbing grass and agrostis alba. Preferably, the plants of the present invention include crop plants, for example, corn, alfalfa, sunflower, Brassica, soy, cotton, safflower, peanut, sorghum, wheat, wheat, tobacco, barley, rice, tomato, potato, chayó, melons, vegetable crops, etc. Other preferred plants include Liliopsida and Panicoideae. Once a DNA sequence has been transformed into a particular plant species, it can be propagated in this species or it can be moved to other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques. Below are descriptions of representative techniques to transform both dicotyledonous and monocotyledonous plants, as well as a representative technique of plastid transformation. to. Dicotyledonous Transformation Dicotyledonous transformation techniques are well known in the art and include techniques based on Agrobacterium and techniques that do not require Agrobacterium. The techniques that do not require Agrobacterium include the uptake of exogenous genetic material directly by protoplasts or cells. This can be achieved by PEG or uptake mediated by electroporation, distribution mediated by particle bombardment, or microinjection. Examples of these techniques are described in Paszkowski et al., EMBO J, 3: 2717 (1984), Potrykus et al., Mol. Gen. Genet .. 199: 169 (1985), Reich et al., Biotechnology,: 1001 (1986), and Klein et al., Nature, 327: 70 (1987). In each case, the transformed cells are regenerated to whole plants using known normal techniques. The transformation mediated by Agrobacterium is a preferred technique for dicotyledonous transformation due to its high transformation efficiency and its wide utility with many different species. Transformation with Agrobacterium typically comprises transferring the binary vector having the foreign DNA of interest (eg pCIB200 or pCIB2001) to an appropriate strain of Agrobacterium which may depend on the complement of vir genes carried by the host strain of Agrobacterium already in a Resident or chromosomal form of Ti plasmid (for example, strain CIB542 for pCIB200 and pCIB2001 (Uknes et al., Plant Cell, 5 ..- 159 (1993)). The transfer of the recombinant binary vector to Agrobacterium is achieved by processing of triparenteral mating using E. coli having the recombinant binary vector, an auxiliary E. coli strain having a plasmid such as pRK2013 and which is capable of mobilizing the recombinant binary vector to the target Agrobacterium strain. recombinant binary can be transferred to Agrobacterium by DNA transformation (Hófgen & amp;; Willmitzer, Nucí. Acids Res. 16: 9877 (1988)). The transformation of the target plant species by recombinant Agrobacterium usually comprises the Agrobacterium cocuitive with plant explants and following well-known protocols. The transformed tissue generates a selectable medium of the antibiotic or herbicide resistance marker, present between the limits of T-DNA of binary plasmid. The vectors can be introduced into the plant cells in known ways. Preferred cells for transformation include Agrobacterium, monocotyledonous cells, including Liliopsida cells and Panicoideae cells. The preferred monocotyledone cells are cereal cells; for example, cells of corn (maize), barley and wheat and dicotyledonous cells that accumulate starch, for example, potato. Another approach to transforming a plant cell with a gene comprises driving inert or biologically active particles into tissues and plant cells. This technique is described in U.S. Patent Nos. 4,945,050, 5,036,006 and 5,100,792. In general, this method comprises impelling inert or biologically active particles in the cells under effective conditions to penetrate the outer surface of the cell and to give incorporation into the inside thereof. When inert particles are used, the vector can be introduced into the cell by coating the particles with the vector 'containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried in the cell by the wake of the particle. Biologically active particles (eg, dried yeast cells, bacteria or dried bacteriophages, each containing the DNA to be introduced) can also be propelled into the tissue or plant cell. b. Monocotyledon Transformation The transformation of most monocotyledonous species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using electroporation or polyethylene glycol (PEG) techniques and bombardment of particles in callus tissue. . Transformations can be undertaken with an individual species of DNA or multiple species of DNA (ie, co-transformation) and both of these techniques are suitable for use with this invention. The co-transformation may have the advantage of avoiding complete construction of vectors and generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, allowing the removal of the selectable marker in subsequent generations, if this should be considered desirable. However, a disadvantage of the use of co-transformation is less than 100% frequency with which separate species of DNA are integrated into the genome (Schocher et al., Biotechnology, 4: 1093 1986)). Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts of an elite corn breeding line, transformation of protoplasts using PEG or electroporation, and regeneration of corn plants from the transformed protoplasts. Gordon-Kamm et al. (Plant Cell, 2: 603 (1990)) and Fromm et al. (Biotechnology, 8: 833 (1990) -) have published techniques for transformation of the corn line derived from A188 using particle bombardment. Additionally WO 93/07278 and Koziel et al. (Biotechnology, 11: 194 (1993)) describe techniques for the transformation of elite corn breeding lines by particle bombardment. This technique uses immature maize embryos of 1.5-2.5 mm length excised from corn ears 14-15 days after pollination and a PDS-100OO Biolistics device for bombardment. Rice transformation can also be undertaken by direct gene transfer techniques using protoplasts or particle bombardment. Transformation mediated by protoplasts and the types of Japan and Indica have been described (Zhang et al., Plant Cell Rep, X. 379 (1988), Shimamoto et al., Nature, 338: 274 (1989), Datta et al., Biotechnology. ,, 8 736 (1990)). Both types are also transformable routinely using particle bombardment (Christou et al., Biotechnology, 9: 957 (1991)). Additionally, WO 93/21335 describes techniques for the transformation of rice by electroporation. Patent application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Additionally, the transformation of wheat by Vasil et al. (Biotechnology 10: 667 (1992)) using bombardment of particles in long-term regenerable callus cells of type C, and also by Vasil et al. (Biotechnology, 11: 1553 (1993)) and Weeks et al.

(Plant Physiol., 102: 1077 (1993) using bombardment of immature embryo particles and callus derived from immature embryos.) A preferred technique for wheat transformation, however, comprises the transformation of wheat as by bombardment of immature embryo particles and includes either a saccharine high pass or high maltose content before the gene distribution Before the bombardment, any number of embryos (0.75-1 mm in length) are plated in MS medium with 3% sucrose (Murashiga &Skoog, Physiologia Plantarum, - 15: 473 (1962)) and 3 mg / 1 of 2,4-D for the induction of somatic embryos, which is allowed to proceed in the dark.On the chosen day of the bombardment, the embryos they are removed from the induction medium and placed in the osmotricum (ie induction medium with sucrose or maltose added at the desired concentration, typically 15%) .The embryos are allowed to plasmolyze for 2-3 hours and then They bombard.Three embryos per target plate are typical but not critical. A suitable plasmid having a gene (such as pCIB3064 or pSG35) is precipitated in the gold particles of micrometer size using normal procedures. Each embryo plate is fired with the DuPont Biolistics ™ 1 helium device using a burst pressure of approximately 1000 lbs./in.2 using a normal 80 mesh screen. After the bombing, the embryos are placed back in the dark for recovery for approximately 24 hours (even in the osmoticum). After 24 hours, the embryos are removed from the osmoticum and placed back. in the induction medium where they rest for about a month before regeneration. Approximately one month later, the embryo explants with embryogenic callus in development are transferred to the regeneration medium (MS + 1 mg / liter NAA, mg / liter GA), which also contains the appropriate selection agent (10 mg / 1 coarse in the case of pCIB3064 and 2 mg / 1 methotrexate in the case of pS0G35). After about a month, the developed shoots are transferred to large sterile containers known as "GA7" containing MS, of medium concentration, 2% sucrose, and the same _concentration of the selection agent. . The "transformation of monocotyledons using Agrobacterium has also been described," see WO "94/00977 and the U.S. Patent No. 5,591,616, both of which are incorporated herein by reference. c. Transformation of Plastids The seed of Nicotiana tabacum c. v. ? Xanthi nc 'are germinated seven per plate in a 1-inch circular array on T agar medium and are bombarded 12-14 days after sowing with 1 μm tungsten particles (MIO, Biorad, Hercules, CA) coated with DNA from the plasmids pPH143 and pPH145 essentially as described (Svab and Maliga, PNAS, 9_0: 913 (1993)). The bombarded seedlings are incubated in medium T for two days after which the leaves are cut and placed abaxial side up in bright light (350-500 μmol photons / m / s) on RMOP medium plates (Svab, Hajdukiewicz and Maliga, PNAS, 87.:8526 (1990)) containing 500 μg / ml of spectinomycin dihydrochloride (Sigma, St.

Louis, MO). The resistant shoots that appear below the bleached leaves three to eight weeks after the bombardment are subcloned in the same selective medium, the callus is allowed to form and the secondary shoots are isolated and subcloned. Complete segregation of copies of the plastid-transformed genome (homoplasmicity) into independent subclones is assessed by normal Southern Blot techniques (Sambrook et al., Molecular Cloning: A Laborpatory Manual, Cold Spring Harbo'r "" Laboratqry, Cold Spring Harbor ( 1989)). BamHI / EcoRI-digested total. cellular DNA (Mettler, IJ Plant Mol Biol Reporter, 5. 346 (1987)) is separated on agarose gels of Tris-borate (TBE), transferred to nylon membranes (Amersham) and probed with primed DNA sequences randomly labeled with 32P corresponding to a 0.7 kb BamHI / HindIII DNA fragment of pC8 containing a portion of the plastic target selection sequence rps 7/12. The homoplastic shoots are aseptically rooted in the MS / IBA medium containing spectinomycin (McBride et al., PNAS, .91: 7301 (1994)) and transferred to the greenhouse.

Production and Characterization of Stably Transformed Plants The transformed plant cells are then placed in an appropriate selective medium for selection of transgenic cells, which are then grown to callus. Outbreaks of callus and seedlings generated from the shoot are cultivated in the middle of rooting. The various constructs will normally be attached to a marker for selection in plant cells. Conveniently, the label can make resistance to a biocide (particularly an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like). The particular marker used will allow the selection of transformed cells in comparison to cells lacking the DNA that has been introduced. Components of DNA constructs, including transcription / expression cartridges of this invention, can be prepared from sequences, which are native (endogenous) or foreign (exogenous) to the host. By "foreign" it is meant that the sequence is not is found in the wild-type host into which the construct is introduced The heterologous constructs will contain at least one region, which is not native to the gene from which the transcription initiation region is derived, to confirm the presence of transgenes in cells and transgenic plants, a Southern blot analysis can be performed using methods known to those skilled in the art.The integration of a polynucleic acid segment into the genome can be detected and quantified by Southern blotting., since they can easily be distinguished from constructions that contain the segments through light or appropriate restriction enzymes. Expression products of the transgenes can be detected in any of a variety of ways, depending on the nature of the product, and include Western blot and enzyme assay. A particularly useful way to quantify protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they can be grown to produce plant weeds or parts that have the desired phenotype. The plant tissue or plant parts can be collected and / or the seeds are collected. The seed can serve as a source to grow additional plants with tissues or parts that have the desired characteristics. The invention thus provides "a transformed plant or part of a plant, such as a spike, seed, fruit, grain, fodder, shredded straw or loaf comprising at least one polynucleotide, expression cassette and vector of the invention, methods for elaborate this plant and methods to use this plant or a part thereof The transformed plant or part of the plant expresses a processing enzyme, optionally located in a particular cellular or subcellular compartment of a certain tissue or in a developing grain. , the invention provides a transformed plant part comprising at least one starch processing enzyme present in the cells of the plant, wherein the plant part is obtained from a transformed plant, the genome of which is augmented with a Cassette expression that codes for at least one starch processing enzyme.The processing enzyme does not act on the target substrate unless it is activated by s such as heating, milling or other methods, which allow the enzyme to make contact with the substrate under conditions where the enzyme is activated.

Example Methods of the Present Invention The self-processing plants and plant parts of the present invention can be used in various methods employing the processing enzymes (mesophilic, thermophilic, or hyperthermophilic) expressed and activated therein. According to the present invention, a transgenic plant part obtained from a transgenic plant, the genome of which is increased with at least one processing enzyme, is placed under conditions in which the processing enzyme is expressed and activated. In activation, the processing enzyme is activated and functions to act on the substrate in which it normally acts to obtain the desired result. For example, starch processing enzymes act on starch to degrade, hydrolyze, isomerize, or otherwise modify to obtain the desired result in activation. Starch non-processing enzymes can be used to destabilize or break down the cell membrane of the plant in order to facilitate the extraction of starch, lipids, amino acids or other plant products. Still further, hyperthermophilic and non-hyperthermophilic enzymes can be used in combination in the self-processing plant or plant parts of the present invention. For example, a non-degrading starch enzyme, mesophilic, can be activated to break down the cell membrane of the plant for the extraction of starch, and subsequently a starch degrading enzyme can be activated in the self-processing plant. to degrade the starch. The enzymes expressed in the grain can be placed on the plant or part of the plant that contains them under conditions to which its activity is promoted. For example, one or more of the following techniques may be used. The plant part can be contacted with water, which provides a substrate for a hydrolytic enzyme and will thus activate the enzyme. The plant part can be put in contact with water that will allow the enzyme to migrate from the compartment from which it was deposited during the development of the plant part and in this way, be associated with its substrate. The movement of the enzyme is possible because division into compartments is ramified during ripening, grain drying and rehydration. The "intact or crushed grain can be put in contact with water which will allow the enzyme to migrate from the compartment in which it was deposited during the development of the part of the plant and in this way be associated with its substrate. activating enzymes by addition of an activating compound For example, a calcium-dependent enzyme can be activated by the addition of calcium Other activating compounds can be determined by those skilled in the art Enzymes can be activated by removal of an activator. , there are known inhibitors of amylase enzyme peptides, the amylase can be co-expressed with an amylase inhibitor and then activated by the addition of a protease.The enzymes can be activated by activating the pH to one of which is more active. The enzyme can also be activated by increasing the temperature, an enzyme generally increasing its activity up to the maximum temperature for that enzyme. mesophyll enzyme will increase its activity from the activity level at room temperature to the temperature at which it loses activity that is typically less than or equal to 70 ° C. Similarly, thermophilic and hyperthermophilic enzymes can also be activated by increasing the temperature. Thermophilic enzymes can be activated by heating at temperatures up to the maximum temperature of activity or stability. For a thermophilic enzyme, the maximum temperatures of stability and activity will be generally between 70 and 85 ° C. Hypertermophilic enzymes will have relative activation even greater than mesophilic or thermophilic enzymes due to the greater potential change in temperature from 25 ° C to 85 ° C to 95 ° C or even 100 ° C. The increased temperature can be achieved by any method, for example when heating such as by baking, boiling, heating, steam heating, electric discharge or any combination thereof. In addition, in plants that express mesophilic or thermophilic enzymes, the activation of the enzyme can be achieved by grinding, thereby allowing the enzyme to make contact with the substrate. Optimal conditions, for example, temperature, hydration, pH, etc., can be determined by one skilled in the art and can depend on the individual enzyme that is employed and the desired application of the enzyme.

The present invention also provides the use of exogenous enzymes, which aid in a particular process. For example, the use of a self-processing plant, or part of the plant of the present invention, can be used in combination with an exogenously provided enzyme to facilitate the reaction. As an example, transgenic a-amylase starch can be used in combination with other starch processing enzymes, such as pullulanase, α-glucosidase, glucose-isomerase, mannases, hermicellulases, etc., by hydrolyzing starch or producing ethanol. Indeed, it has been found that combinations of transgenic starch of α-amylase with these enzymes, has unexpectedly provided higher degrees of conversion of starch relative to the use of transgenic α-amylase starch, alone. Examples of suitable methods contemplated herein are provided. to. Extraction of Plant Starch The invention provides a method for facilitating the extraction of starch from plants. In particular, at least one polynucleotide encoding a processing enzyme that breaks the physically inhibitory matrix of the endosperm (cell walls, non-starch polysaccharide and protein matrix) is introduced to a plant so that the enzyme is preferably in close physical proximity to the starch granules in the plant. In this embodiment of the invention, the transformed plants express one or more of protease, glucanase, xylanase, thioredoxin / thioredoxin-reductase, cellulase, phytase, lipase, beta-glucosidase, esterase and the like, but the enzymes having any degrading activity of starch, to maintain the integrity of the starch granules. The expression of these enzymes in a plant part such as the grain thus improves the characteristics of the grain process. The processing enzyme can be mesophilic, thermophilic or hyperthermophilic. In one aspect, the grain of a transformed plant of the invention is heat-dried, probably inactivating non-thermophilic processing enzymes and improving the integrity of the seed. Grain (or crushed grain) is impregnated at low temperatures or high temperatures (where time is the essence) with high or low moisture content or conditions (see Primary Cereal Processing, Gordon and Willm, eds., Pp. 319- 337 (1994), the description of which is incorporated herein), with or without sulfur dioxide. Upon reaching high temperatures, optionally at certain moisture conditions, the integrity of the endosperm matrix is broken by activating enzymes, for example, proteases, xylanase, phytase or glucanases that degrade proteins and non-starch polysaccharides present in the endosperm leaving the starch granule intact therein and more easily recoverable from the resulting material. Additionally, the non-starch proteins and polysaccharides in the effluent are at least partially degraded and highly concentrated, so that they can be used for improved animal feed, feed or as a means for the fermentation of microorganisms. The effluent is considered a corn impregnation liquor with improved composition. In this way, the invention provides a method for preparing starch granules. The method comprises treating, for example, "fractionated grain, comprising, at least one non-starch processing enzyme under conditions that activate at least one enzyme, producing a mixture comprising the starch granules" and non-starch degradation products, eg, products of digested endosperm matrix.The non-starch processing enzyme can be mesophilic, thermophilic, hyperthermophilic.After activation of the enzyme, the starch granules are separated from the mixture. The grain is obtained, from a transformed plant, the genome of which comprises (is increased on) an expression cassette that codes for at least one processing enzyme For example, the processing enzyme can be a protease, glucanase, xylanase, phytase, thioredoxin / thioredoxin-reductase, esterase, cellulose, lipase or a beta-glucosidase. The processing enzyme can be hyperthermophilic. The grain can be treated under conditions of high or low humidity, in the presence or absence of sulfur dioxide. Depending on the activity and level of expression of the processing enzyme, in the grain of the transgenic plant, the transgenic grain can be mixed with commercial grain before or during processing. Also provided are products obtained by the method such as starch, non-starch products and improved fresh water comprising at least one additional component. b. Starch Processing Methods The transformed plants or plant parts of the present invention may comprise starch degrading enzymes "as described herein that degrade starch granules to dextrins, other modified starches or hexoses (eg," α-amylase "). , pullulanase, α-gluGosidase, glucoamylase, amylopululanase) or convert glucose to fructose (eg glucose isomerase). Preferably, the starch degrading enzyme is selected from α-amylase, α-glucosidase, glucoamylase, pullulanase, neopululanase. amilopululanase, glucose isomerase and combinations thereof is used to transform the grain. In addition, preferably, the enzyme is operably linked to a promoter and a signal sequence that directs the enzyme to the starch granule, an amyloplast, the apoplast, or the endoplasmic reticulum. More preferably, the enzyme is expressed in the endosperm, and particularly, corn endosperm, and is located in one or more cell compartments, or within the starch granule itself. The preferred plant part is the grain. The preferred plant parts are those of corn, wheat, barley, rye, oats, sugar cane or rice. According to a degrading method of the present invention, the transformed grain accumulates the starch degrading enzyme in the starch granules, is impregnated at conventional temperatures and 50 ° C-60 ° C, and wet milled as is known in the art. technique. Preferably, the starch degrading enzyme is hyperthermophilic. Due to the sub-cellular targeting of the enzyme to the starch granule, or by virtue of the association of the enzyme with starch granules, by contacting the enzyme and the starch granule during the wet milling process to conventional temperatures, the processing enzyme is co-purified with starch granules to obtain the mixture of starch / enzyme granules. Subsequent to the recovery of the starch / enzyme granule mixture, the enzyme is then activated by providing favorable conditions for the activity of the enzyme. For example, the processing may be carried out under various conditions of humidity and / or temperature to facilitate partial hydrolysis (in order to make derivatized starches or dextrins) or complete hydrolysis of the starch into hexoses. Syrups containing high dextrose or fructose equivalents are thus obtained. This method effectively reduces the time, energy and costs of enzymes and the efficiency with which the starch is converted to the corresponding hexose, and the efficiency of product production is increased, such as fresh water with higher sugar content and equivalent syrups with high dextrose content. In another embodiment, a plant or a plant product such as a fruit or grain, or flour made from the grain expressing the enzyme is treated to activate the enzyme and converts the polysaccharides expressed and contained within the plant into sugars. Preferably, the enzyme is fused to a signal sequence that directs the enzyme to a starch granule, an amyloplast, the apoplast or the endoplasmic reticulum as described herein. The sugar produced can then be isolated or recovered from the plant or the plant product. In another modality, it is placed. a processing enzyme capable of converting polysaccharides into sugars, under the control of an inducible promoter according to methods known in the art and described herein. The processing enzyme can be mesophilic, thermophilic or hyperthermophilic. The plant is curtíva a desired stage and the promoter is induced causing expression of the enzyme and conversion of the polysaccharide, within the plant or product of the plant, to sugars. Preferably, the enzyme is operably linked to a signal sequence that directs the enzyme to a starch granule, an amyloplast, an apoplast or the endoplasmic reticulum. In another modality, a transformed plant is produced that expresses a processing enzyme capable of converting starch into sugar. The enzyme is fused to a signal sequence that directs the enzyme to a starch granule within the plant. The starch is then isolated from the transformed plant containing the enzyme expressed by the transformed plant. The enzyme contained in the isolated starch can then be activated to convert the starch into sugar. The enzyme can be mesophilic, thermophilic or hyperthermophilic. Examples of hyperthermophilic enzymes capable of converting starch to sugar are provided herein. Methods can be used with any plant that produces a polysaccharide and that can express an enzyme capable of converting a polysaccharide to sugars or starch hydrolyzate product such as dextrin, maltooligosaccharide, glucose and / or mixtures thereof. The invention provides a method for producing dextrins and altered starches from a plant, or a product of a plant, that has been transformed with a processing enzyme that hydrolyzes certain covalent bonds of a polysaccharide to form a polysaccharide derivative. In one embodiment, a plant, or a product of the plant such as a fruit or grain, or flour made from the grain expressing the enzyme, is placed under sufficient conditions to activate the enzyme and convert the polysaccharides contained within the plant into polysaccharides of reduced molecular weight. Preferably, the enzyme is fused to a signal sequence that directs the enzyme to a starch granule, an amyloplast, the apoplast or the endoplasmic reticulum as described herein. The dextrin or the derived starch produced then can be isolated or recovered from the plant or the plant product. In another embodiment, a processing enzyme capable of converting polysaccharides to dextrins or altered starches is placed under the control of an inducible promoter according to methods known in the art and described herein. The plant is cultured at a desired stage and the promoter is induced by causing expression of the enzyme and conversion of the polysaccharides, within the plant or product of the plant, to dextrins or altered starches. Preferably, the enzyme is α-amylase, pullulanase, iso or neo-pullulanase and is operably linked to the signal sequence which directs the enzyme to a starch granule, an amyloplast, the apoplast or the endoplasmic reticulum. In one embodiment, the enzyme is directed to the apoplast or the endorreticle, In yet another embodiment, a transformed plant is produced that expresses an enzyme capable of converting starch to dextrins or altered starches.The enzyme is fused to a signal sequence that directs the enzyme to a starch granule within the plant.The starch is then isolated from the transformed plant containing the enzyme expressed by the transformed plant.The enzyme contained in the isolated starch can then be activated under conditions sufficient for activation to convert the starch in dextrins or altered starches Examples of hyperthermophilic enzymes, for example, capable of converting starch to starch hydrolysates, are provided in the present. The methods can be used with any plant that produces a polysaccharide and that can express an enzyme capable of converting a polysaccharide into sugar. In another embodiment, the grain of transformed plants of the invention that accumulates starch degrading enzymes that degrade the bonds in the granules of starch to dextrins, modified starches or hexose (for example, α-amylase, pullulanase, α-glucosidase, glucoamylase, amilopululanase) are impregnated under conditions that favor the activity of the starch degrading enzyme for various periods of time. The resulting mixture may contain high levels of the starch derivative product. The use of that grain: 1) eliminates the need to grind the grain, or otherwise process the grain to obtain first corn granules, 2) makes starch more accessible to enzymes by virtue of placing the enzymes directly within the grain endosperm tissue, and 3) eliminates the need for microbially produced starch hydrolyzing enzymes. In this way, the complete wet milling process before hexose recovery is eliminated by simply heating the grain, preferably corn kernel, in the presence of water to allow the enzymes to act on the starch. This process can also be used for the production of ethanol, syrups with high fructose content, fermentation media containing hexose (glucose), or any other use of starch that does not require the refinement of the grain components. The invention further provides a method for preparing dextrin, maltooligosaccharides, and / or sugar comprising treating a plant part comprising starch granules and at least one starch processing enzyme under conditions to activate at least one enzyme, thereby digesting the starch granules to form an aqueous solution comprising sugars. The plant part is obtained from a transformed plant, the genome of which is increased with an expression cassette that codes for at least one processing enzyme. The aqueous solution comprising dextrins, maltooligosaccharides, and / or sugar is then harvested. In one embodiment, the processing enzyme is α-amylase, α-glucosidase, pullulanase, glucoamylase, amylopululanase, gluco-iso erase, or any combination thereof. Preferably, the enzyme is hyperthermophilic. In another embodiment, the method further comprises isolating the dextrins, maltooligosaccharides and / or sugar. c. Improved Maize Varieties The invention also provides for the production of improved maize varieties (and varieties of other crops) that have normal levels of starch accumulation, and accumulate sufficient levels of amylolytic enzymes in their endosperm, or starch accumulation organ, such that in the activation of the enzyme contained therein, such as by boiling or heating the plant or a part of it in the case of a hyperthermophilic enzyme, the enzymes are activated and facilitate the rapid conversion of starch into simple sugars. These simple sugars (mainly glucose) will provide sweetness to the treated corn. The resulting corn plant is an improved variety for dual use as a grain that produces hybrid and as tender or sweet corn. In this manner, the invention provides a method for producing hyperdulce corn, which comprises treating the transformed corn or a part thereof, the genome of which has been augmented with, and expressing 'in the' endosperm, an expression cassette comprising a promoter operably linked to a first polynucleotide encoding at least one amylolytic enzyme, conditions that activate at least one enzyme to convert the polysaccharides in maize into sugar, producing hyperdulceous corn.The promoter can be a constitutive promoter, a seed-specific promoter , or a specific endosperm promoter, which is linked to a polynucleotide sequence encoding a processing enzyme such as α-amylase, for example, one comprising SEQ ID NO: 13, 14 or 16. Preferably, the enzyme is hyperthermophilic In one embodiment, the expression cassette further comprises a second polynucleotide that codes for a linked signal sequence. in the enzyme encoded for the first polynucleotide. The exemplary signal sequence in this embodiment of the invention directs the enzyme to the apoplast, in endoplasmic reticulum, a starch granule, or an amiloplast. The corn plant is grown such that the ears with the grains are formed and then the promoter is induced to cause the enzyme to be expressed and converted to the polysaccharide contained within the plant in sugar. d. Self-Fermenting Plants In another embodiment of the invention, plants such as corn, rice, wheat or sugarcane are handled to accumulate large quantities of processing enzymes in their cell walls, for example, xylanase, cellulases, hemicellulases, glucanases, pectinases, lipases, esterases, beta-glucosidases, phytases, proteases and the like (non-degrading enzymes of starch polysaccharides). After harvesting the grain component (or sugar in the case of sugarcane), the forage, straw or bagasse is used as a source of the enzyme, which was targeted as an expression and accumulation in cell walls, and as a source of biomass. Forage (or other leftover tissue) is used as a raw material in a process to recover fermentable sugars. The process of obtaining the fermentable sugars consists of activating the non-degrading enzyme of starch polysaccharides. For example, the activation may comprise heating the plant tissue in the presence of water for periods of time suitable for hydrolysis of the non-starch polysaccharide in the resulting sugars. In this way, this self-processing forage produces the enzymes required for the conversion of polysaccharides into monosaccharides, essentially without increasing the cost since they are a component of the raw material. Additionally, the temperature-dependent enzymes have no detrimental effects on the growth of the plant or on the development of the plant, and selection as an objective of the cell wall, or even targeting in microfibrils of polysaccharides by virtue of the domains of cellulose / xylose binding fused to the protein, improve the accessibility of the substrate to the enzyme In this way, the invention also provides a method for using a transformed plant part comprising at least one non-starch polysaccharide processing enzyme in the cell wall of the cells of the plant part The method comprises treating a transformed plant part comprising at least one non-starch polysaccharide processing enzyme under conditions that activate at least one enzyme, thereby digesting the starch granules to form an aqueous solution comprising sugars, wherein the plant part is obtained from a transformed plant, the genome of which is augmented with an expression cassette encoding at least one enzyme processing of polysaccharide not of starch; and collecting the aqueous solution comprising the sugars. The invention also includes a transformed plant or plant part comprising at least one non-starch polysaccharide processing enzyme present in the cell or cell wall of the cells of the plant or part of the plant. The plant part is obtained from a transformed plant, the genome of which is increased with an expression cassette that codes for at least one non-starch processing enzyme, for example, a xylanase, cellulase, glucanase, pectinase, lipase, esterase , beta-glucosidase, phytase, protease or any combination thereof.and. Aqueous Phase with High Protein and Sugar Content In yet another embodiment, proteases and lipases are managed to accumulate in seeds, for example, soybeans. After activation of the protease or lipase, such as, for example, by heating, these enzymes in the seeds hydrolyze the liquid and storage proteins present in the soybean during processing. In this way soluble products can be obtained which comprise amino acids, which can be used as feed, food or fermentation medium, and fatty acids. Typically, polysaccharides are found in the insoluble fraction of processed grain. However, by combining the expression of the polysaccharide degrading enzyme and the accumulation in seeds, proteins and polysaccharides can be hydrolyzed and found in the aqueous phase. For example, corn zeins and storage protein and polysaccharides not from soy starch can be solubilized in this way. The components of the aqueous and hydrophobic phases can be easily separated by extraction with organic solvent or supercritical carbon dioxide. In this way, what is provided is a method to produce an aqueous extract of grain that contains higher levels of protein, amino acids, sugars or saccharides. f. Self-Processing Fermentation The invention provides a method for producing ethanol, a fermented beverage, or other products derived from fermentation. The method comprises obtaining a plant, or the product or part of a plant, or a plant derivative such as grain meal, wherein a processing enzyme is expressed which converts polysaccharides into sugar. The plant, or product thereof, is treated such that the sugar is produced by polysaccharide conversion as described above. The sugars and other components of the plant are then fermented to form ethanol or a fermented beverage, or other products derived from the fermentation, according to methods known in the art. See, for example, United States Patent Number 4,929,452. Concisely, the sugar produced by conversion of polysaccharides is incubated with yeast under conditions that promote the conversion of sugar into ethanol. A suitable yeast includes yeast strains tolerant to high alcohol content and tolerant to high sugar content, i or X such as for example the yeast S. cerevis to ATCC No. - 20867. This strain was deposited in American Type Culture Collection, Rockville, MD, on September 17, 1987 and assigned ATCC No. 20867. Fermented product or fermented beverage can then be distilled to isolate ethanol or a distilled beverage, or the fermentation product recovered in another way. The plant used in this method can be any plant that contains a polysaccharide and is capable of expressing an enzyme of the invention. Many plants are described herein. Preferably, the plant is one that is grown commercially. More preferably, the plant is one that is normally used to produce ethanol or fermented beverages, or fermented products, such as, for example, wheat, barley, corn, rye, potato, grapes or rice. The method comprises treating a plant part comprising at least one polysaccharide processing enzyme under conditions to activate at least one enzyme, thereby digesting the polysaccharide in the plant part to form fermentable sugar. The "polysaccharide" processing enzyme can be mesophilic, thermophilic or hyperthermophilic, the plant part is obtained from a transformed plant, the genome of which is increased with an expression cassette that codes for at least one polysaccharide processing enzyme. plant of this embodiment of the invention include, without limitation, grain, fruit, seed, stem, plant or root.The plants include, without limitation, oats, barley, wheat, fence, grapes, rye, corn, rice, potato, "sugar beet, sugar cane, pineapple, grass and trees. The plant part can be combined with commercial grain or other commercially available substrates; the source of the substrate for processing may be a different source of the self-processing plant. The fermentable sugar is then incubated under conditions that promote the conversion of the fermentable sugar into ethanol, for example, with yeast and / or other microbes. In one embodiment, the plant is derived from maize transformed with α-amylase, which has been found to reduce the amount of time and cost of fermentation. It has been found that the amount of residual starch is reduced when the transgenic corn made according to the present invention which expresses a thermostable α-amylase, for example, is used in fermentation. This indicates that more starch is solubilized during fermentation. The reduced amount of residual starch results in distiller grains having higher protein content by weight and high value. In addition, the fermentation of the transgenic corn of the present invention allows the liquefaction process to be performed at a lower pH, resulting in savings in the cost of chemicals used to adjust the pH, at a higher temperature, for example more than 85 ° C, preferably, more than 90 ° C, more preferably, 95 ° C or higher, resulting in shorter times of liquefaction and more complete solubilization of starch, and reduction of liquefaction times, all giving for results in efficient fermentation reactions with high ethanol yields. On the other hand, it has been found that contacting the conventional plant parts with even a small portion of the transgenic plant made according to the present invention can reduce the fermentation time and the costs associated with it. As such, the present invention relates to the reduction in fermentation time for plants which comprises subjecting a transgenic plant part of a plant comprising a polysaccharide processing enzyme that converts polysaccharides to sugar in relation to the use of a part of plant that does not include the polysaccharide processing enzyme. g. Enzyme Processing of Natural Starch and Polysaccharides that Encode them A polynucleotide that encodes a mesophilic processing enzyme is introduced into a plant or part of a plant. In one embodiment, the polynucleotide of the present invention is a polynucleotide optimized for maize as provided in SEQ ID NOs: 48, 50 and 59, which codes for a glucoamylase, as provided in SEQ ID NOs: 47 and 49. In another embodiment, the polynucleotide of the present invention is a polynucleotide optimized for maize as provided in SEQ ID NO: 52, which codes for an alpha-amylase, as provided in SEQ ID NO: 51. On the other hand , fusion products of processing enzymes are also contemplated. In one embodiment, the polynucleotide of the present invention is a polynucleotide optimized for maize as provided in SEQ ID NO: 46, which codes for a fusion of alpha-amylase and glucoamylase, as provided in SEQ ID NO: 45. Combinations of processing enzymes are also contemplated by the present invention. For example, a combination of starch processing enzymes and non-starch processing enzymes is contemplated herein. These combinations of processing enzymes can be obtained by employing the use of multiple gene constructs that code for each of the enzymes. Alternatively, the individual transgenic plants stably transformed with the enzymes can be crossed by known methods to obtain a plant containing both enzymes. Another method includes the use of exogenous enzymes with the transgenic plant. The source of the starch processing and non-processing enzymes of "starch" can be isolated or derived from any source and the polynucleotides corresponding thereto can be determined by one skilled in the art.Ay-amylase can be derived from Aspergillus (by example, Aspergillus shirousami and Aspergillus niger), Rhizopus (for example Rhizopus oryzae) and plants such as corn, barley and rice.Glucoamylase can be derived from Aspergillus (for example, Aspergillus shirousami and Aspergillus Niger), Rhizopus) (for example, Rhizopus oryzae), and Thermoanaerobacter (for example, Thermoanaerobacter thermosaccharolyticum). In another embodiment of the invention, the polynucleotide encodes a mesophilic starch processing enzyme that is operably linked to a corn optimized polynucleotide as provided in SEQ ID NO: 54, which codes for a starch binding domain to the natural, as provided in SEQ ID NO: 53.

In another embodiment, a specific tissue promoter includes endosperm-specific promoters such as the corn? -zein promoter (exemplified by SEQ ID NO: 12) or the corn ADP-gpp promoter (exemplified by SEQ ID NO: 11) , which includes an intron and untranslated 5 'sequence) or a Q protein promoter (exemplified by SEQ ID NO: 98) -a_ a rice glutelin promoter (exemplified by SEQ.ID' NO: 67). Thus, the present invention includes an isolated polynucleotide comprising a promoter comprising SEQ ID NO: 11, 12, 67 or 98, a polynucleotide that hybridizes the complement thereof under conditions of low stringency hybridization, or a fragment thereof having promoter activity, for example, at least 10%, and preferably at least 50%, the activity of a promoter having SEQ ID NO: 11, 12, 67 or 98. In one embodiment , the product of a starch hydrolysis gene, such as α-amylase, glucoamylase or a-amylase / glucoamylase fusion can be directed to an organelle or particular location such as the endoplasmic reticulum or apoplast, instead of the cytoplasm. This is exemplified by the use of the N-terminal signal sequence of corn? -zein (SEQ ID NO: 17), which confers selection as a protein apoplast specific target, and the use of the N-signal sequence? ε-zein terminal (SEQ ID NO: 17) which is operably linked to the processing enzyme that is operably linked to the sequence SEKDE1 for retention in the endoplasmic reticulum. Addressing the enzyme or protein to a specific compartment will allow the enzyme to be located in a way that will not be in contact with the substrate. In this way, the enzymatic action of the enzyme will not occur until the enzyme makes contact with its substrate. The enzyme can be put in contact with its substrate by the process of grinding (physical breakdown of cell integrity) and hydration. For example, "a mesophilic starch hydrolyzing enzyme can be directed to the endoplasmic reticulum or apoplast and therefore will not be in contact with starch granules in the amyloplast.The grinding of the grain will break the integrity of the grain and the hydrolyzing enzyme of starch will then make contact with the starch granules.Thus, the potential negative effects of the co-localization of an enzyme and its substrates can be evaded. h. Food Products without Added Sweetener A method is also provided for producing a food product, farinaceous, sweetened without adding additional sweetener. Examples of farinaceous products include, but are not limited to, breakfast food, ready-to-eat food, baked goods, pasta products, and cereal such as breakfast cereal. The method comprises treating a plant part comprising at least one starch processing enzyme under conditions that activate the starch processing enzyme, thereby processing the starch granules in the plant part to sugars to form a sweetened product, for example, in relation to the product produced by processing starch granules from a part of the plant that does not comprise the hyperthermophilic enzyme. Preferably, the starch processing enzyme is hyperthermophilic and is activated by heating, such as by baking, boiling, heating, steam cooking, electric discharge or any combination of. the same. The plant part is obtained from a transformed plant, for example soy, rye, oats, barley, wheat, corn, rice or processed sugar cane, the genome of which is increased with an expression cassette that codes for at least one starch-processing hyperthermophilic enzyme, for example, α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose-isomerase, or any combination thereof. The sweetened product is then processed into a farinaceous food product. The invention also provides a farinaceous food product, for example, a cereal food, a breakfast food, a ready-to-eat food, or baked food, produced by the method. The farinaceous food product may be formed from the sweetened product and water, and may contain malt, flavors, vitamins, minerals, coloring agents or any combination thereof. The enzyme can be activated to convert the polysaccharides contained within the plant material into sugar prior to the inclusion of the plant material in the cereal product or during the processing of the cereal product. Accordingly, the polysaccharides contained within the plant material can be converted into sugar by activating the material, such as by heating in the chaos of a hyperthermophilic enzyme, before inclusion in the farinaceous product. The plant material containing sugar produced by conversion of the polysaccharides is then added to the product to produce a sweetened product. Alternatively, the polysaccharides can be converted into sugars by the enzyme during processing of the farinaceous product. Examples of processes used to make cereal products are well known in the art and include heating, baking, boiling and the like as described in U.S. Patent Nos. 6,183,788; 6,159,530; 6,149,965; 4,988,521 and 5,368,870. Concisely, a dough can be prepared by mixing several dry ingredients together with water and cooking to gelatinize the starchy components and develop a cooked flavor. The cooked material can then be mechanically worked to form a cooked dough, such as cereal dough. The dry ingredients may include various additives such as sugars, starch, salt, vitamins, minerals, dyes, flavors, salt and the like. In addition to water, various liquid ingredients such as corn or malt syrup can be added. The farinaceous material may include cereal grains, grains: cut, semolina or wheat, rice, corn, oats, barley, rye or other cereal grains and mixtures thereof of a transformed plant of the invention. The dough can then be processed to the desired shape through a process such as extrusion or stamping and further cooking using a medium such as a James cooker, an oven or an electric discharge device. A method for sweetening a product containing starch is additionally provided, without adding sweetener. The method comprises treating the starch comprising at least one starch processing enzyme under conditions to activate at least one enzyme which thereby digests the starch to form a sugar, thereby forming a treated (sweetened) starch for example in relation to the product produced by treating starch that does not comprise the hyperthermophilic enzyme. The starch of the invention is obtained from a transformed plant, the genome of which is increased with an expression cassette that codes for at least one processing enzyme. Enzymes include α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose-isomerase, or any combination thereof. The enzyme can be hyperthermophilic and heat activated. Preferred transformed plants include corn, soybeans, rye, oats, barley, wheat, rice and cane sugar. The starch then treated is added to a product to produce a sweetened starch-containing product, for example, a "farinaceous food product." A product containing sweetened starch produced by the method is also provided. invention further provides a method for sweetening a fruit or vegetable containing polysaccharide; comprising: treating a fruit or vegetable comprising at least one polysaccharide processing enzyme under conditions that activate at least one enzyme, thereby processing the polysaccharide in the fruit or vegetable to form sugar, producing a sweetened fruit or vegetable, for example , in relation to a fruit or vegetable of a plant that does not include the polysaccharide processing enzyme. The fruit or vegetable of the invention is obtained from a transformed plant, the genome of which is increased with an expression cassette that codes for at least one polysaccharide processing enzyme. Fruits and vegetables include potato, tomato, banana, chayote, pea and beans. Examples include α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose-isomerase, or any combination thereof. The enzyme can be hyperthermophilous. i. Sweetening a Plant or Plant Product Containing Polysaccharide, The method comprises obtaining a plant that expresses a polysaccharide processing enzyme that converts a polysaccharide into a sugar as described above. Accordingly, the enzyme is expressed in the. plant and in the products of the plant, such as in a fruit or vegetable. In one embodiment, the enzyme is placed under the control of an inducible promoter such that it can be induced "by an external stimulus of expression of the enzyme.These inducible promoters and constructs are well known in the art and are described herein. Expression of the enzyme within the plant or product thereof causes the polysaccharide contained within the plant product thereof to become sugar and sweeten the plant or product thereof In another embodiment, the polysaccharide processing enzyme is In this way, the plant or product thereof can be activated under sufficient conditions to activate the enzyme to convert the polysaccharides into sugar through the action of the enzyme to sweeten the plant or product thereof. As a result, this self-processing of the polysaccharide in the fruit or vegetable to form sugar produces a sweetened fruit or vegetable, for example in relation to a fr uta or plant of a plant that does not include the polysaccharide processing enzyme. The fruit or vegetable of the invention is obtained from a transformed plant, the genome of which is augmented with a pressure cartridge that codes for at least one polysaccharide processing enzyme. Fruits and vegetables include potato, tomato, banana, chayote, pea and beans. Enzymes include α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose-isomerase, or any combination thereof. The polysaccharide processing enzyme can be hyperthermophilic. j. Isolation of Transformed Grain Starch Containing an Enzyme that Breaks the Endosperm Matrix The invention provides a method for isolating starch from a transformed bean where an enzyme is expressed that destabilizes or breaks the endosperm matrix. The method comprises obtaining a plant that expresses an enzyme that breaks the endosperm matrix by modification of for example cell walls, non-starch polysaccharides and / or proteins. Examples of these enzymes include, without limitation, proteases, glucanases, thioredoxins, thioredoxin-reductase, phytases, lipases, cellulases, beta-glucosidases, xylanases, and esterases. These enzymes do not include any enzyme that exhibits starch degrading activity to maintain the integrity of the starch granules. The enzyme can be fused to a signal sequence that directs the enzyme to the starch granule. In one embodiment, the grain is dried with heat to activate the enzyme and activate the endogenous enzymes contained within the grain. The heat treatment causes the activation of the enzyme, which acts to break the endosperm matrix which then easily separates from the starch granules.In another embodiment, the grain is impregnated at low or high temperature, with high or low content of moisture, with or without sulfur dioxide, the grain is then thermally treated to break the endosperm matrix and allow easy separation of the starch granules, in another embodiment, appropriate conditions of temperature and humidity are created to allow the proteases to enter in the starch granules and degrades the proteins contained within the granules.This treatment will produce starch granules with high yield and little contaminating protein. k. Syrup that has a High Sugar Equivalent and the Use of Syrup to Produce Ethanol or a Fermented Drink. The method comprises obtaining a plant that expresses a polysaccharide processing enzyme that converts a polysaccharide to a sugar as described above. The plant, or product thereof, is impregnated in an aqueous stream under conditions where the expressed enzyme converts the polysaccharide contained within the plant, or product thereof, into dextrin, maltooligosaccharide, and / or sugar. The aqueous stream containing the dextrin, maltooligosaccharide and / or sugar produced through the conversion of the polysaccharide is then separated to produce a syrup having a high sugar equivalent. The method may or may not include an additional step of wet milling the plant or product thereof to obtain starch granules. Examples of enzymes that can be used within the method include, without limitation, α-amylase, glucoamylase, pullulanase, and α-glucosidase. The enzyme can be hyperthermophilous. The sugars produced according to the method include, without limitation, hexose, glucose and fructose. Examples of plants that can be used with the method include, without limitation, corn, wheat or barley. Examples of products of a plant that may be used include, without limitation, fruit, grain and vegetables. In one embodiment, the polysaccharide processing enzyme is placed under the control of an inducible promoter. Accordingly, before or during the impregnation step, the promoter is induced to cause the expression of the enzyme, which then provides for the conversion of the polysaccharide to sugar. Examples of inducible promoters and constructs containing them are well known in the art and are provided herein. In this way, where the processing of the polysaccharide is hyperthermophilic, the impregnation is carried out at high temperature to activate the hyperthermophilic enzyme and inactivate the endogenous enzymes found inside the plant or product thereof In another embodiment, a constituent is expressed hyperthermophilic enzyme capable of converting polysaccharide to sugar This enzyme can be directed or not to a compartment inside the plant through the use of a signal sequence.The plant, or product thereof, is impregnated under high temperature conditions to cause the conversion of polysaccharides contained within the plant or sugar.A method for producing ethanol or a fermented syrup beverage having a high sugar equivalent is also provided.The method comprises incubating the syrup with yeast under conditions that allow the conversion of sugar contained within the syrup into ethanol or a fermented beverage. Fermented beverages include, without limitation, beer and wine. Fermentation conditions are well known in the art and are described in U.S. Patent No. 4,929,452 and herein. Preferably, the yeast is a strain highly tolerant to alcohol and highly tolerant to yeast sugar such as S. cerevisiae ATCC No. 20867. The fermented product or fermented beverage can be distilled to isolate ethanol or a distilled beverage. 1. Accumulation of Hyperthermophilic Enzyme in the Cell Wall of a Plant The invention provides a method for accumulating a hyperthermophilic enzyme in the cell wall of a plant. The method comprises expressing within a plant a hyperthermophilic enzyme that is fused to a cell wall target selection signal such that the enzyme sought accumulates in the cell wall. Preferably, the enzyme is capable of converting polysaccharides to monosaccharides. Examples of target selection sequences include, but are not limited to, a cellulose or xylose binding domain. Examples of hyperthermophilic enzymes include those listed in SEQ ID NO: 1, 3, 5, 10, 13, 14, 15 or 16. Plant material containing cell walls can be added as a source of desired enzymes in a process for recover sugars from the raw material or as a source of enzymes for the conversion of polysaccharides that originate from other sources to monosaccharides. Additionally, cell walls can serve as a source from which enzymes can be purified. Methods for purifying enzymes are well known in the art and include, but are not limited to, gel filtration, ion exchange chromatography, chromatofocusing, isoelectric focusing, affinity chromatography, FPLC, HPLC, salt precipitation, dialysis, and Accordingly, the invention also provides purified enzymes isolated from the cell walls of plants.

. Method for Preparing and Isolating Processing Enzymes In accordance with the present invention, recombinantly produced processing enzymes of the present invention can be prepared by transforming plant tissue or plant cell comprising the processing enzyme of the present invention capable of being activated in the plant, selected for the tissue or cell of transformed plant, cultivating the transformed tissue or plant cell in a transformed plant, and isolating the processing enzyme from the transformed plant or part thereof. The recombinantly produced enzyme may be an α-amylase, glucoamylase, glucose isomerase, α-glucosidase, pullulanase, xylanase, protease, glucanase, beta-glucosidase, esterase, lipase, or phytase. The enzyme can be encoded by the polynucleotide selected from any of SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, 59, 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97 or 99. The invention will be further described by the following examples, which are not intended to limit the scope of the invention. invention in no way.

EXAMPLES Example 1 Construction of Optimized Genes for Processing / Starch Isomerization Genetics, Hyperthermophilic The enzymes α-amylase, pullulanase, α-glucosidase, and glucose-isomerase, included in the starch deliberation or glucose εomerization were selected by their desired profiles of activity. These include, for example, minimal activity at room temperature, activity / stability at high temperature, and activity at low pH. The corresponding genes are then designed using the preferred corn codons as described in U.S. Patent No. 5,625,136 and synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Α-Amylase 797GL3, having the amino acid sequence SEQ ID NO: 1, was selected for its hyperthermophilic activity. The nucleic acid sequence of this enzyme was deduced and optimized for maize as shown in SEQ ID NO: 2. Similarly, 6gp3 pullulanase having the amino acid sequence set forth in SEQ ID NO: 3 was selected. nucleic acid for pullulanase 6gp3 was deduced and optimized for corn as shown in SEQ ID NO: 4. The amino acid sequence for a-glucosidase malA from Sulfolobus solfataricus was obtained from the literature J. Bact. 177: 482-485- (1995); J. Bact. 180: 1287-1295 (1998). In -base to the published amino acid sequence of the protein (SEQ ID NO: 5), 'the corn optimized synthetic gene (SEQ ID NO: 6) which codes for a-glucosidase malA was designed. Several glucose-isomerase enzymes were selected. The amino acid sequence (SEQ ID NO: 18) for glucose-isomerase derived from maritime Thermotoga was predicted based on the published sequence of DNA having accession number NC_000853 and a synthetic gene optimized for maize was designed (SEQ ID NO: 19 ). Similarly, the amino acid sequence (SEQ ID NO: 20) for glucose-isomerase derived from Thermotoga neapolitana was predicted based on the published DNA sequence of Appl. Envir. Microbiol. 61 (5): 1867-1875 (1995), Accession No. L38994. A synthetic sequence optimized for corn coding for glucose-isomerase from Thermotoga neapolitana was designed (SEQ ID NO: 21).

Example 2 Expression of Fusion of a-amylase 797GL3 and Regions of Encapsulation of Starch in E. coli A construct encoding for hyperthermophilic α-amylase 797GL3 fused to the starch encapsulation region (SER) of granulated starch-synthase (waxy) was introduced and expressed in E. coli. The cDNA (SEQ ID NO: 7) of corn granule-bound starch synthase encoding the amino acid sequence (SEQ ID NO: 8) (Klosgen RB, et al. 1986) was cloned as a source of a starch binding domain, or starch encapsulation region (SER). The full-length cDNA was amplified by ... RT-PCR of the RNA prepared from corn seed using the SV57 primers (5'AGCGAATTCATGGCGGCTCTGGCCACGT 3 ') (SEQ ID NO: 22) and SV58 (5'AGCTAAGCTTCAGGGCGCGGCCACGTTCT 3 ') (SEQ ID NO: 23) designed from GenBank Accession No. X03935. The complete cDNA was cloned into pBluescript as an EcoRI / HindIII fragment and the plasmid was designated pNOV4022. The C-terminal portion (encoded by bp 919-1818) of the cDNA, which includes the starch binding domain, was amplified by pNOV4022 and fused in frame to the 3 'end of the full length corn optimized gene 797GL3 (SEQ ID NO. NO: 2). The fused gene product, 797GL3 / Waxy, having the nucleic acid sequence SEQ ID NO: 9 and coding for the amino acid sequence, SEQ ID NO: 10, was cloned as a Ncol / Xbal fragment in pET28b (NOVAGEN, Madison , Wl) that was cut with Ncol / Nhel. The 797GL3 gene alone was also cloned into the vector pET28b as an NcOl / Xbal fragment. The pET28 / 797GL3 and pET28 / 797GL3 / Waxy vectors were transformed into E.coli BL21 / DE3 cells (NOVAGEN) and cultured and induced according to the manufacturer's instruction. Analysis by PAGE / tinsion- with Coomassie revealed an induced protein in both extracts corresponding to the predicted sizes of fused and unfused amylase, respectively. The total cell extracts were analyzed for hyperthermophilic amylase activity as follows: 5 mg of starch was suspended in 20 μl of water then diluted with μl of ethanol. The positive control of normal amylase or the sample that was tested for amylase activity was added to the mixture and water was added to a final reaction volume of 500 μl. The reaction was carried out at 80 ° C for 15-45 minutes. The reaction was then cooled to room temperature, and 500 μl of a mixture of o-dianisidine and glucose oxidase / peroxidase (Sigma) was added. The mixture was incubated at 37 ° C for 30 minutes. 500 μl of 12 N sulfuric acid was added to retain the reaction. The absorbance at 540 nm was measured to quantify the amount of glucose released by the amylase / sample. The assay of both the fused and unfused amylase extracts gave similar levels of hyperthermophilic amylase activity, while the control extracts were negative. This indicated that amylase 797GL3 was still active (at high temperatures) when it was fused to the C-terminal portion of the waxy protein.

Example 3 Isolation of promoter fragments for endosperm specific expression in YM corn The promoter and the 5 'non-coding region I (which includes the first control) of the large subunit of ADP-gpp (ADP-glucose-pyrophosphorylase) from Zea mays was amplified as a fragment of 1515 basess pairs (SEQ ID NO: 11) from corn genomic AN using the primers designated from Genbank accession M81603. The ADP-gpp promoter has been shown to be of specific endosperm (Shaw and Hannah, 1992). * The promoter of the? -zein gene of Zea mays was amplified as a 673 bp fragment (SEQ ID NO: 12) of the plasmid pGZ27.3 (obtained from Dr. Brian Larkins). The? -zein promoter has been shown to be of specific endosperm (Torrent et al., 1997).

Example 4 Construction of transformation vectors for hyperthermophilic a-amylase 797GL3 Expression cartridges were constructed to express the hyperthermofila 797GL3 amylase in maize endosperm with several target selection signals, as follows: pNOV6200 (SEQ ID NO: 13) comprises the N-terminal signal sequence of? -zaina de maíz (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to the synthetic amylase 797GL3 as described above - in Example 1 for targeting the endoplasmic reticulum and secretion in the apoplast (Torrent et al., 1997). The fusion was cloned behind the ADP-gpp corn promoter for expression specifically in the endosperm. . - pNOV6201 (SEQ ID NO: 14) comprises the N-terminal signal sequence of? -zein fused to synthetic amylase 797GL3 with a C-terminal addition of the SEKDEL sequence for target selection and retention in the endoplasmic reticulum (ER) (Munro and Pelham, 1987). The fusion was cloned behind the corn ADP-gpp promoter for expression specifically in the endosperm. pNOV7013 comprises the N-terminal signal sequence of? -zein fused to the synthetic amylase 797GL3 with a C-terminal addition of the SEKDEL sequence for target selection and retention in the endoplasmic reticulum (ER). PNOV7013 is the same as pNOV6201, except that the corn? -zein promoter (SEQ ID NO: 12) was used in place of the corn ADP-spp promoter in order to express the fusion in the endosperm. pNOV4029 (SEQ ID NO: 15) comprises the waxy amyloplast target selection peptide (Klosgen et al., 1986) fused to the synthetic amylase 797GL3 for targeting the amyloplast. The fusion was cloned behind the corn ADP-gpp promoter for expression specifically in the endosperm. pNOV4031 (SEQ ID NO: 16) comprises the waxy amyloplast target selection peptide fused to the synthetic 797GL3 / waxy fusion protein for target selection to starch granules. The fusion was cloned behind the ADP-gpp promoter of corn for the expression - specifically of the endosperm. Additional constructs were elaborated, with these fusions -cloned behind the β-zein promoter to obtain higher levels of enzyme expression. All expression cartridges were moved to a binary vector for transformation into corn by Agrobacterium infection. The binary vector contained the phosphomannanase-isomerase gene (PMI) that allows the selection of transgenic cells with mannose. The transformed corn plants either self-pollinated or crossed and the seed was collected for analysis. Additional constructs were prepared with target selection signals described above fused to either the 6gp3 pullulase or the 340g a-glucosidase 12 in precisely the same manner as described for α-amylase. These fusions were cloned behind the corn ADP-gpp promoter and / or the? -zein promoter transformed into corn as described above. The transformed corn plants either self-pollinated or crossed and the seed was collected by analysis. Combinations of the enzymes can be produced either by crossing plants expressing the individual enzymes or by cloning several expression cartridges in the same binary vector to allow co-transformation.

Example 5 Construction of plant transformation vectors for thermophilic pullulanase 6GP3. An expression cassette was constructed to express the thermophilic pullulanase 6GP3 in the endosplasmic corn endosperm reticulum as follows: pNOV7005 (SEQ ID NOs: 24 and 25) "comprises the N-terminal signal sequence of corn? -zein fused to synthetic 6GP3 pullulanase with a C-terminal addition of the SEKDEL sequence for targeting and retention in the ER.The SEKDEL amino acid peptide was fused to the C-terminal end of the enzymes using PCR with primers designed to amplify the synthetic gene and simultaneously add the 6 amino acids at the C-terminal end of the protein.The fusion was cloned behind the corn? -zein promoter for expression specifically in the endosperm.

Example 6 Construction of plant transformation vectors for hyperthermophilic a-glucosidase malA Expression cartridges were constructed to express hyperthermophilic α-glucosidase Sulfolobus solfataricus malA in maize endosperm with several target selection signals as follows. PNOV4831 (SEQ ID NO: 26) comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ.

ID NO: 17) fused to α-glucosidase with a C-terminal addition of the SEKDEL sequences for target selection and retention in the endoplasmic reticulum (ER) (Munro and Pelham, 1987). The fusion was cloned behind the corn? -zein "promoter for expression specifically in the endosperm pNOV4839 (SEQ ID NO: 27) comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ.

ID NO: 17) fused to synthetic α-glucosidase malA for direction to the endoplasmic reticulum and secretion in the apoplast (Torrent et al., 1997). The fusion was cloned behind the corn? -zein promoter for expression specifically in the endosperm. pNOV4837 comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to synthetic malA a-glucosidase with a C-terminal addition of the SEKDEL sequence for a-direction and retention in the ER. The fusion was cloned behind the corn ADP-gpp promoter for expression specifically in the endosperm. The amino acid sequence for this clone is identical to that of pNOV4831 (SEQ ID NO: 26).

Example 7 Construction of plant transformation vectors for glucose-isomerases from Thermotoga maritima and Thermotoga neapolitana hyperthermophilics Se'- constructed expression cartridges to express hyperthermophilic glucose-isomerases from Thermotoga maritima and Thermotoga neapolitana in maize endosperm with various direction signals to target as follows: pNOV4832 (SEQ ID NO: 28) comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to the synthetic glucose-isomerase of Thermotoga maritima with a C-terminal addition of the SEKDEL sequence for targeting and retention in ER. The fusion was cloned behind the promotor.de? -zein corn for expression specifically in the endosperm. pNOV4833 (SEQ ID NO: 29) comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to synthetic Glucose-isomerase from Thermotoga neapolitana with a C-terminal addition of SEKDEL sequence for a-direction and ER retention. The fusion was cloned behind the corn? -zein promoter for expression specifically in the endosperm. pNOV4840 (SEQ ID NO: 30) comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ.

ID NO: 17) fused to synthetic glucose-isomerase of Thermotoga neapolitana for direction to the endoplasmic reticulum and secretion in the apoplast (Torrent et al., 1997). The fusion was cloned behind the corn? -zein promoter for expression specifically in the endosperm. pNOV4838 comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to the synthetic glucose-isomerase of Thermotoga neapolitana with a C-terminal addition of the SEKDEL sequence for targeting and retention in the ER The fusion was cloned behind the corn ADPgpp promoter for expression specifically in the endosperm. The amino acid sequence for this clone is identical to that of pNOV4833 (SEQ ID NO: 29).

Example 8 Construction of plant transformation vectors for the expression of glucanase EglA hyperthermophilus pNOV4800 (SEQ ID NO: 58) comprises the AMY32b alpha-amylase signal sequence of barley (MGKNGNLCCFSLLLLLLAGLASGHQ) (SEQ ID NO: 31) fused to the mature protein sequence of EglA for localization to the apoplast. The fusion was cloned behind the corn? -zein promoter for expression specifically in the endosperm.

Example 9 Construction of plant transformation vectors for the expression of multiple hyperthermophilic eszymes pNOV4841 comprises a double gene construct of an a-amylase 797GL3 fusion and a fusion of pullulanase 6GP3. Both the 797GL3 fusion (SEQ ID NO: 33) and the 6GP3 fusion (SEQ ID NO: 34) possess the N-terminal signal sequence of corn? -zein and the SEKDEL sequences for direction a and retention in the ER. Each fusion was cloned behind a separate? -zein corn promoter for expression specifically in the endosperm. pNOV 842 comprises a double gene construct of a fusion of α-amylase 797GL3 and a fusion of α-glucosidase or malA. Both the polypeptide of the 797GL3 fusion (SEQ ID NO: ) as the α-glucosidase fusion polypeptide malA (SEQ.

. ID NO: 36) possess the N-terminal signal sequence of? -zein from corn and SEKDEL sequences for a-direction and retention in the ER. Each fusion was cloned behind a promoter of? -5 separate corn zein for expression specifically in the endosperm. pNOV4843 comprises a double gene construct of a? -amylase 797GL3 fusion and a malA a-glucosidase fusion. Both the 797GL3 fusion and the a-glucosidase or malA fusion possess the N-terminal signal sequence of corn? -zein and the sequence SEKDEL for direction and retention in ER. The 797GL3 fusion was cloned behind the corn? -zein promoter and the malA fusion was cloned behind the corn ADP-gpp promoter for expression specifically of the endosperm. The amino acid sequence of the 797GL3 fusion and the malA fusion are identical to those of pNOV4842 (SEQ ID Nos: 35 and 36, respectively). pNOV4844 comprises a triple gene construct of an a-amylase 797GL3 fusion, a fusion of pullulanase 6GP3, and a fusion of a-glucosidase malA. 797GL3, malA, and 6GP3 possess all of the N-terminal signal sequences of corn? -cein and SEKDEL sequence for a-direction and retention in the ER. The 797GL3 and malA fusions were cloned behind 2 separate promoters of corn? -zein, and the 6GP3 fusion was cloned behind the ADP-gpp corn promoter for expression specifically in the endosperm. The amino acid sequences for the 797GL3 and malA fusions are identical to those of pNOV4842 (SEQ ID NOs: 35 and 36, respectively). The amino acid sequence for the 6GP3 fusion is identical to that of the 6GP3 fusion in pNOV4841 (SEQ ID NO: 34). All the expression cartridges exposed in this Example as well as in the following Examples were moved in the binary vector pNOV2117 for transformation into corn by infection with Agrobacterium. pNOV2117 contains the phosphomannanase-isomerase (PMI) gene that allows the selection of transgenic cells with mannose. pNOV2117 is a binary vector with both pVSl and ColEl origins of replication. This vector contains the constitutive VirG gene of pAD1289 (Hansen, G., et al., PNAS USA 91: 7603-7607 (1994), incorporated herein by reference) and a spectinomycin resistance gene of Tn7. Cloned in the polylinker between the right and left boundaries is the corn ubiquitin promoter, the PMI coding region and the nopaline-sitase terminator of pN0V117 (Negrotto, D., et al., Plant Cell Reports 19: 798-803 (2000), incorporated by reference herein). The transformed corn plants either self-pollinated or crossed and the seed was collected by analysis. The combinations of the different enzymes can be produced either by crossing plants that express the individual enzymes or by transforming a plant with one of the multi-gene cartridges.

Example 10 'Construction of bacterial and Pichia expression vectors Expression vectors were constructed to express a-glucosidase and glucose-isomerases hyperthermophilus either in Pichia or bacteria as follows: pNOV4829 (SEQ ID Nos: 37 and 38) comprises a fusion of synthetic glucose-isomerase from Thermotoga maritima with ER retention signal with the bacterial expression vector pET29a. The glucose-isomerase fusion gene was cloned into the Ncol and Sacl sites of pET29a, which resulted in the addition of an N-terminal S-label for protein purification. pNOV4830 (SEQ ID NOS: 39 and 40) comprises a synthetic glucose-isomerase fusion of Thermotoga neapolitana with ER retention signal in the bacterial expression vector pET29a. The glucose-isomerase fusion gene was cloned into the NcOI and Sacl sites of pET29a, which resulted in the addition of an N-terminal S-label for protein purification. pNOV4835 (SEQ ID NO: 41 and 42) comprises the synthetic glucose-isomerase gene from Thermotoga maritima cloned at the BamHI and EcoRI sites of the bacterial expression vector pET28C. This resulted in the fusion of a His-tag (for protein purification) to the N-terminal end of the glucose isomerase. ..AA pNOV4836 (SEQ ID NO: 43 and 44) comprises the synthetic glucose-isomerase gene from Thermotoga neapolitana cloned at the BamHI and EcoRI sites of the bacterial expression vector pET28C. This gave. resulting in the fusion of a His-tag (for protein purification) to the N-terminal end of glucose isomerase.

Example 11 Transformation of immature maize embryos was performed essentially as described in Negrotto et al. , Plant Cell Reports 19: 798-803. For this example, all constituents of the media are as described in Negrotto et al., Supra. However, several constituents of the media described in the literature can be substituted. A. Transformation plasmids and selectable marker The genes used for transformation were cloned into a vector suitable for corn transformation. The vectors used in this example contained the phosphomannanase isomerase (PMI) gene for selection of transgenic lines (Negrotto et al (2000) Plant Cell Reports 19: 798-803).

B. Preparation of Agrobacterium tumefaciens Agrobacterium strain LBA4404 (psBl) containing the plant transformation plasmid was cultured in YEP (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L) , 15 g / 1 agar, pH 6.8) solid medium, for 2-4 days at 28 ° C. Approximately 0.8 X 10s Agrobacterium were suspended in the LS-inf media supplemented with 100 μM of As (Negrotto et al., (2000) Plant Cell-Re 19: 798-803). The cells were pre-incubated in this medium for 30-60 minutes.

C. Inoculation Immature embryos of Al88 or other suitable genotype were excised from spikes of 8-12 days of age in LS - info + 100 μM of As. The embryos were rinsed once with fresh medium of infection. Then Agrobacterium solution was added and the embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos were then transferred by staking it upward to the medium LSAs and cultured in the dark for 2 to 3 days. Subsequently, between 20 and 25 embryos per petri dish were transferred to the LSDc medium supplemented with cetotaxime (250 mg / 1) and silver nitrate (1.6 mg / 1) and cultured in the dark at 28 ° C for 10 days .

D. Selection of transformed cells and regeneration of transformed plants Immature embryos producing embryogenic callus were transferred to the LSD1M0.5S medium. The cultures were selected in this medium for 6 weeks with a sub-cultivation step at 3 weeks. The surviving calli were transformed to Regí medium supplemented with mannose. After light culture (16 hours light / 8 hours dark), the green tissues were then transferred to Reg2 medium without growth regulators and incubated for 1-2 weeks. "The seedlings were transferred to the boxes of Magenta GA-7 (Magenta Corp, Chicago III) containing the Reg3 medium and cultured in light.After 2-3 weeks, the plants were tested for the presence of the PMI genes and other genes of interest by PCR. Positive plants from the PCR assay were transferred to the greenhouse.

Example 12 IT seed analysis of maize plants expressing apoplast-a-amylase or ER. IT seeds were obtained from self-pollinated maize plants transformed with either pNOV6200 or pNOV6201 as described in Example 4. The accumulation of starch in these grains appeared to be normal, based on the visual inspection and on the normal tinsion of starch. with an iodine solution before any high temperature exposure. The immature grains were cut and the purified endosperms were individually placed in microcentrifuge tubes and immersed in 200 μl of 50 mM NaP04 buffer. The tubes were placed in a water bath at 85 ° C for 20 minutes. Then they cooled on ice. Twenty microliters were added to the 1% iodine solution and mixed. Approximately 25% of the cereal grains. they were usually stained by starch. The remaining 75% failed to stain, indicating that the starch is degraded sugars of low molecular weight that do not stain with iodine. It was found that the TI grains of pNOV6200 and pNOV6201 were self-hydrolyzed from corn starch. There was no detectable reduction in starch after incubation at 37 ° C. The expression of amylase was further analyzed by isolation of the hyperthermophilic protein fraction from the endosperm followed by tinsion by PAGE / Coomassie. A segregating protein band of appropriate molecular weight (50 kD) was observed. These samples are subjected to an a-amylase assay using commercially available dry amylose (AMYLAZYME; from Megazyme, Ireland). The high levels of hyperthermophilus amylase activity correlated with the presence of the 50 kD protein. Additionally it was found that the starch in the grains of a majority of transgenic maize expressing hyperthermophilic α-amylase, directed to the amyloplast, was sufficiently active at room temperature to hydrolyze most of the starch if the enzyme was left in direct contact with granule of starch. Of the eighty lines that have hyperthermophilic α-amylase directed to the amyloplast, four lines identified that the starch accumulates in the grains. Three of these lines were analyzed for thermostable α-amylase activity using a colorimetric amylazyme assay (Megazyme). The amylase enzyme assay indicated that these three lines have low levels of thermostable amylase activity. When starch was purified from these three lines it was treated with appropriate conditions of. moisture and heat, the starch was hydrolysed indicating the presence of adequate levels of α-amylase to facilitate . self-hydrolysis of the starch prepared from these lines. '.- •' Multi-line IT seed was obtained -And independent of both transformants pNOV6200 and pNOV6201.

Individual granules were cut from each line and the purified endosperms were homogenized individually in 300 μl of 50 mM NaP04 buffer. The aliquots of the endosperm suspensions were analyzed for α-amylase activity at 85 ° C. Approximately 80% of the lines secreted hyperthermophilic activity (See Figures 1A, IB and 2). Grains of wild type plants or plants transformed with pNOV6201 were heated at 100 ° C for 1, 2, 3 or 6 hours and then stained for starch with an iodine solution. Little or no starch was detected in mature grains after 3 or 6 hours, respectively. In this manner, the starch in mature transgenic maize grains expressing hyperthermophilus amylase which is directed to the endoplasmic reticulum was hydrolyzed when iated at high temperature. In another experiment, partially purified starch of mature IT grains of pNOV6201 plants that were impregnated at 50 ° C for 16 hours was hydrolyzed after heating at 85 ° C for 5 minutes. This illustrates that the α-amylases directed to the endoplasmic reticulum binds to starch after grinding the grain, and is able to hydrolyze the starch on heating. Thinning with iodine indicated that the starch remained intact in mature seeds after impregnation for 16 hours at 50 ° C. In another experiment, they were heated at 95 ° C for 16 hours and then dried segregant mature grains, from plants transformed with pNOV6201. In seeds expressing hyperthermophilic α-amylase, the hydrolysis of starch and sugar resulted in a wrinkled appearance after drying.

EXAMPLE 13 IT seed analysis of maize plants expressing amyloplast-directed amylase IT seed was obtained from self-pollinated maize plants transformed with either pNOV 029 or pNOV4031 as described in Example 4. Corn accumulation in the grains of these lines was clearly not normal. All the lines segregated, with some variation of severity, very little or nothing of the starch phenotype. The purified endosperm of immature grains stained only weakly with iodine before exposure to high temperatures. After 20 minutes at 85 ° C, there was no tinsión. When the ears dried, the grains withered. This particular amylase clearly has sufficient activity at greenhouse temperatures to hydrolyze the starch if it is left in direct contact with the granule.

Example 14 Grain fermentation of corn plants expressing α-amylase 100% transgenic grains, 85 ° C, versus 95 ° C, variable liquefaction time. Transgenic corn (pNOV6201) containing a thermostable α-amylase performs well without the addition of exogenous α-amylase, requires much less time for liquefaction and results in more complete starch solubilization. Fermentations at laboratory scale were analyzed by a protocol with the following steps (detailed below): 1) grinding, 2) moisture analysis, 3) preparation of a thick suspension containing ground corn, water, countercurrent and α-amylase , 4) liquefaction and 5) simultaneous saccharification and fermentation (SSF). In this example, the temperature and time of the liquefaction step were varied as described below. In addition, the transgenic maize was liquefied with and without exogenous α-amylase and the performance in ethanol production was compared to control corn treated with commercially available α-amylase. The transgenic corn used in this example was made according to the procedures set forth in Example 4 using a vector comprising the a-amylase vector and the PMI selectable marker specifically pNOV6201. The transgenic corn was produced by pollinating a commercial hybrid (N3030BT) with pollen from a transgenic line that expresses a high level of thermostable α-amylase. The corn was dried at 11% humidity and stored at room temperature. The α-amylase content of the transgenic corn meal was 95 unit / g where one unit of enzyme generates 1 micromole reducing corn flour ends per minute at 85 ° C in buffer pH 6.0 The control corn used was a well-known yellow tooth corn that performs well in the production of ethanol 1) Grinding: Transgenic corn (1180 g) was milled in a Perten 3100 hammer mill equipped with a 2.0 mm screen thus generating transgenic corn flour Control corn was ground in the same mill after complete cleaning to prevent contamination by transgenic maize 2) Moisture analysis: Weighed samples (20 g) of control transgenic corn were weighed in aluminum canisters and heated at 100 ° C for 4 hours The samples were weighed again and the moisture content of the weight loss was calculated.The moisture content of the transgenic flour was 9.26% of that of the control flour was 12.54% 3) Preparation of thick suspensions: The composition of thick suspensions was designed to produce a mass with 36% solids at the beginning of the SFF. In the control samples, they were prepared in 100 ml plastic bottles and contained 21.50 g of corn flour control, 23 ml of deionized water, 6.0 ml of countercurrent (8% solids by weight), and 0.30 ml of a commercially available α-amylase diluted 1/50 with water. The dose of alpha-amylase was chosen as representative of industrial use. When, it was evaluated under the conditions described above for the transgenic α-amylase assay, the dose of control α-amylase was 2 U / g corn flour. The pH was adjusted to 6.0 by the addition of ammonium hydroxide. Transgenic samples were prepared in the same way but contained 20 g of corn flour due to the lower moisture content of the transgenic flour. Thick suspensions of transgenic flour were prepared either with a-amylase at the same dose as the control samples or without exogenous α-amylase. 4) Liquefaction: The bottles containing slurries of transgenic corn flour were submerged in water baths at either 85 ° C or 95 ° C for 5, 15, 30, 45 or 60 minutes. The control slurries were incubated for 60 minutes at 85 ° C. During the high temperature incubation, the thick suspensions were mixed vigorously by hand every 5 minutes. After the high temperature step, the thick suspensions were cooled in ice. 5) Simultaneous saccharification and fermentation: The mass produced by liquefaction was mixed with glucoamylase (0.65 ml of a 1/50 dilution of a commercially available glucoamylase L-400), protease (0.60 ml of a 1,000-fold dilution of a commercially available protease), 0.2 mg of Lactocide and urea (0.85 ml of a dilution of 10 times of Urea Liqueur 50%). A hole was cut in the lid of the 100 ml bottle containing the dough to allow the C02 to vent. The mass was then inoculated with yeast (1.44 ml) and incubated in a water bath set at 90 ° F. After 24 hours of fermentation, the temperature was lowered to 86 ° F; at 48 hours it was adjusted to. 82 ° F. Yeast was propagated for inoculation when preparing a mixture containing yeast (0.12 g) with 70 grams of maltodextrin, 230 ml of water, 100 ml of countercurrent, glucoamylase (0.88 ml of a 10-fold dilution of a commercially available glucoamylase), protease (1.76 ml of a 100-fold dilution of a commercially available enzyme), urea (1.07 grams), penicillin (0.67 mg) and zinc sulfate (0.13 g). The propagation culture was started the day before it was needed and incubated with mixing at 90 ° F. At 24, 48 and 72 hours samples were taken from each fermentation vessel, filtered through 0.2 μm filters, and analyzed by HPLC for ethanol and sugars. At 72 hours, samples for dissolved total solids and for residual starch were analyzed.

The HPLC analysis was performed in a binary gradient system equipped with refractive index detector, column heater and Bio-Rad Aminex HPX-87H column. The system was equilibrated with 0.005 M H2SO4 in water at 1 ml / min. The temperature of the column was 50 ° C. The injection volume of the sample was 5 μl; the avoidance was in the same solvent. The Rl response was calibrated by injection of known standards. Both etariol and glucose were measured in each injection. "~ ~ - Residual starch was measured as follows The samples and standards were dried at 50 ° C in a furnace, then ground to a powder in a sample mill The powder (0.2 g) was weighed into a tube 15 ml graduated centrifuge The powder was washed three times with 10 ml of aqueous ethanol (80% v / v) by vortexing followed by centrifugation and discarding supernatant DMSO (2.0 ml) was added to the sediment followed by 3.0 ml of a thermostable alpha-amylase (300 units) in MOPS buffer After vigorous mixing, the tubes were incubated in a water bath at 85 ° C for 60 minutes During the incubation, the tubes were mixed 4 times. and was added with 4.0 ml of sodium acetate buffer (200 mM, pH 4.5) followed by 0.1 ml of glucoamylase (20 U) The samples were incubated at 50 ° C for 2 hours, mixed, then centrifuged for 5 hours. minutes at 3,500 rpm. The supernatant was filtered through a 0.2 μm filter and set at was analyzed for glucose by the HPLC method described above. An injection size of 50 μl was used for samples with little residual starch (< 20% solids). Results: Transgenic corn performed well in fermentation without added α-amylase. The yield of ethanol at 72 hours was essentially the same with or without exogenous α-amylose as shown in Table 1. This Table also shows that a high ethanol yield is achieved when the liquefaction temperature is higher; the present enzyme expressed in the transgenic corn has activity at higher temperatures than other commercially used enzymes such as Bacillus liquefaciens α-amylase.

Table 1 When the liquefaction time was varied, it was found that the liquefaction time required for the efficient production of ethanol was much lower than the hour required by the conventional process. Figure 3 shows that the yield of ethanol at 72 hours of fermentation was almost without liquefaction change from 15 minutes to 60 minutes. In addition, liquefaction at 95 ° C gave more ethanol at each point of time than liquefaction at 85 ° C. This observation demonstrates the process improvement achieved by the use of the hyperthermophilic enzyme. Control corn yields a higher final ethanol yield than transgenic corn, but the control was chosen because it performs very well in fermentation. In contrast, the transgenic maize has a genetic background chosen to facilitate the transformation. The introduction of the α-amylase trait in the elite corn plasma germ by well-known reproductive techniques should eliminate this difference. - Examination of the residual levels of starch in the beer produced at 72 hours (Figure 4) shows that the transgenic α-amylase results in a significant improvement in the preparation of starch available for fermentation; much less starch was left after fermentation. Using both ethanol levels and residual starch levels, the optimal liquefaction times were 15 minutes at 95 ° C and 30 minutes at 85 ° C. In the present experiments, these times were the total time that the fermentation vessels were in the water bath and thus include a period of time during which the temperature of the samples increased from room temperature to 85 ° C or 95 ° C. ° C. Optimal shorter liquefaction times may be optimal in large-scale industrial processes that quickly heat the mass by the use of equipment such as burners. Conventional industrial liquefaction processes require holding tanks to allow the dough to incubate at a high temperature for one or more hours. The present invention eliminates the need for these retention tanks and increases the productivity of the liquefaction equipment. An important function of o-amylase in fermentation processes is to reduce the viscosity of the dough. At all time points, the samples containing transgenic maize meal were markedly less viscous than the control sample. In addition, the transgenic samples do not seem to experience the gelatinous phase observed with all the control samples; Gelatization usually occurs when thick suspensions of corn are cooked. In this way, having the α-amylase distributed throughout the length of the endosperm fragments gives the advantageous physical properties of the dough during cooking by preventing the formation of large gels that slow down diffusion and increase the energy costs of mixed and pumping the dough. The high dose of α-amylase in transgenic corn may also contribute to the favorable properties of the transgenic mass. At 85 ° C, the activity of transgenic corn a-amylase was many times greater than that of the exogenous α-amylase dose used in controls. The latter was chosen as representative of commercial speeds of use.

EXAMPLE 15 Effective Function of Transgenic Corn When Shown with Control Corn Tragenic corn flour was mixed with control corn flour at various levels from 5% to 100% transgenic maize meal. These were treated as described in Example 14. The masses containing trangically expressed α-amylase were liquefied at 85 ° C for 30 minutes or at 95 ° C for 15 minutes; Control masses were prepared as described in Example 14 and liquefied at 85 ° C for 30 or 60 minutes (one for each) or at 95 ° C for 15 or 60 minutes (one for each). The data for ethanol at 48 and 72 hours and for the residual starch are given in Table 2. The levels of ethanol at 48 hours are plotted in Figure 5; Residual starch determinations are shown in Figure 6. These data show that transgenic, thermostable a-amylase, give very good performance in ethanol production even when the transgenic grain is only a small portion (as little as 5% of Total grain in the dough The data also show that the residual starch is markedly lower than in the control mass when the transgenic grain comprises at least 40% of the total grain.

Table 2 * Control samples. Values of the average of determinations.

Example 16 Production of ethanol as a function of liquefaction, pH using tragenic corn, at a ratio of 1.5 to 12% of total corn. Because transgenic corn performed well at a level of 5-10% of total corn in a fermentation , an additional series of fermentations were carried out in which the tragenic corn comprised 1.5 to 12% of the total corn. The pH was varied from 6.4 to 5.2 and the a-amylase enzyme expressed in the transgenic corn was optimized for the activity at a lower pH than what conventionally used in industrial form. The experiments were performed as described in Example 15, with the following exceptions: 1) Transgenic flour was mixed with control flour with a percent of total dry weight at levels ranging from 1.5% to 12.0%. 2) The control maize was N3030BT which is more similar to the transgenic corn than the control used in Examples 14 and 15. 3) No exogenous α-amylase was added to the samples containing transgenic flour 4) The samples were adjusted at pH 5.2, 5.6, 6.0, or 6.4 before liquefaction At least 5 samples ranging from "0% transgenic maize to 12% transgenic maize meal were prepared for each pH. 5) Liquefaction for all samples was carried out at 85 ° C for 60 minutes. The change in ethanol content as a function of fermentation time is shown in Figure 7. This figure shows the data obtained from samples that contained 3% transgenic corn. At the lower pH, the fermentation proceeds more quickly than at pH 6.0 and above; Similar behavior was observed in samples with other doses of transgenic maize. The pH profile of the transgenic enzyme activity combined with high levels of expression will allow pH liquefactions that result in faster fermentations and thus higher yields than is possible in the conventional process at pH 6.0. The ethanol yields at 72 hours are shown in Figure 8. As can be seen, based on the ethanol yield ,. the results showed little dependence on the amount of transgenic grain included in the sample. In this way, the grain contains abundant amylase to facilitate the fermentative production of ethanol. It also shows that the "'pH less than liquefaction results in higher yield of' ethanol. 0 The viscosity of the samples after the __ liquefaction was monitored and it was observed that at pH 6.0, 6% of tragenic grain is sufficient for adequate reduction of viscosity. At pH 5.2 and 5.6, the viscosity is equivalent to that of the control at 12% of transgenic grain, but not at lower percentages of transgenic grain. Example 17 Production of fructose from corn flour using thermophilic enzymes Corn expressing hyperthermophilic α-amylase, C) 797GL3, was shown to facilitate the production of fructose when mixed with an α-amylase (MalA) and a xyloseisomerase (XylA). The seeds of transgenic plants pNOV6201 expressing 797GL3 were ground to a flour in a Kleco cell thus creating amyla meal. Non-trangic corn grains were milled in the same way to generate control flour. The α-glucosidase, MalA (from S. solfataricus), was expressed in E. coli. The collected bacteria were suspended in 50 mM potassium phosphate buffer, pH 7. 0 containing 4- (2-aminoethyl) benzenesulfonyl fluoride 1 mM, then used in a French press cell. The lysate was centrifuged at 23,000 x g for 15 minutes at 4 ° C. The supernatant solution was stirred, heated at 70 ° C for 10 minutes, cooled on ice for 10 minutes, then centrifuged at 34,000 x g for 30 minutes at 4 ° C. The supernatant solution was removed and the MalA was concentrated twice in centricon 10 devices. The filtrate from the centricon 10 step was retained for use as a negative control for MalA. Xylose (glucose) isomerase was prepared by expressing the xylA gene of T. neapoli tana in E. coli. The bacteria were suspended in 100 mM sodium phosphate pH 7.0 and used by passage through a French press cell. After precipitation of cell debris, the extract was heated at 80 ° C for 10 minutes, then centrifuged. The supernatant solution contained the enzymatic activity XylA. An empty vector control extract was prepared in parallel with the XylA extract. Corn meal (60 mg per sample) was mixed as a buffer and extracts of E. coli. As indicated in Table 3, the samples contained amylase corn flour (amylase) or control corn flour (control), 50 μl of each extract (+) or filtrate (-) of MalA, and 20 μl of either abstract (+) or control (-) empty vector of XylA. All samples also contained 230 μl of 50 mM MOPS, 10 mM MgSO4, 1 mM CoCl2; and pH of the buffer was 7.0 at room temperature. Samples were incubated at 85 ° C for 18 hours. At the end of the incubation period, the samples were diluted with 0.9 ml of water at 85 ° C and centrifuged to remove insoluble material. The supernatant fraction was then filtered through a Centricon 3 ultrafiltration device and analyzed by HPLC with ELSD detection. The gradient HPLC system was equipped with an ASTEC Polymer Amino Column, with a particle size of 5 microns, of 250 x 4.6 mM and an Altech ELSD 2000 detector. The system was pre-equilibrated with a 15:85 water mixture: acetonitrile. The flow rate was 1 ml / min. The initial conditions were maintained for 5 minutes after injection followed by a 20 minute gradient to water: 50:50 acetonitrile followed by 10 minutes of the same solvent. The system was washed with 20 minutes of water: acetonitrile 80:20 and then re-equilibrated with the starting solvent. The fructose was diluted at 5.8 minutes and the glucose at 8.7 minutes. Table 3 The HPLC results also indicated the presence of major maltooligosaccharides in all samples containing the α-amylase. These results show that the three thermophilic enzymes can work together to produce fructose from corn flour at a high temperature.

EXAMPLE 18 Amylase Flour with Isomerase In another example, amylase flour was mixed with MalA and each of the two bacterial xylose isomerases: XylA from T. maritima, and an enzyme designated BD8037 obtained from Diversa. Amylase flour was prepared as described in Example 18. MalA from S. solfataricus with a purification label. His was expressed in E. coli, cell lysate was prepared as described in Example 18, then purified to apparent homogeneity using a nickel affinity resin (Probond, Invitrogen) and following the manufacturer's instructions for purification of native protein.

XylA of T. maritina with the addition of a S-tag and a retention signal of ER was expressed in E. coli and was prepared in the same manner as the XylA of T. neapoli tana described in Example 18. The xylose-isomerase BD8037 was obtained as a lyophilized powder and resuspended in 0.4x of the original volume of water. Amylase corn flour was mixed with enzyme solutions plus water or buffer. All reactions contained 60 mg of amylase flour and a total of 600 μl of liquid. One set of reactions was buffered with 50 mM MOPS, pH 7.0 at room temperature, plus 10 mM MgSO4 and 1 mM CoCl 2.; in a second set of reactions, the buffer solution containing metal was replaced by water. The amounts of isomerase enzyme were varied as indicated in Table 4. All reactions were incubated for 2 hours at 90 ° C. The reaction supernatant fractions were prepared by centrifugation. The pellets were washed with an additional 600 μl of H20 and recentrifuged. The supernatant fractions from each reaction were combined, filtered through a Centricon 10, and analyzed by HPLC with ELSD detection as described in Example 17. The observed amounts of glucose and fructose are plotted in Figure 15.

Table 4 With each of the isomerases, corn flour fructose was produced in a dose-dependent manner when the α-amylase and α-glucosidase were present in the reaction. The results demonstrate that the 797GL3 amylase expressed in grain can function with MalA and a variety of different thermophilic isomerases, with or without added metal ions, to produce corn flour fructose at a high temperature. In the presence of added divalent metal ions, the isomerases can achieve the expected balance of fructose: glucose at 90 ° C of about 55% fructose. This will be an improvement over the current process using mesophilic isomerases, which requires a chromatographic separation to increase the fructose concentration.

Example 19 Expression of a pullulanase in maize. Transgenic plants that were homozygous for either pNOV7013 or pNOV7005 were crossed to generate transgenic maize seeds expressing both a-amylase 797GL3 and pullulanase 6GP3. TI or T2 seeds were obtained from self-pollinated maize plants transformed with either pNOV 7005 or pNOV 4093. pNOV4093 is a fusion of a synthetic gene optimized for maize for 6GP3 (SEQ-ID No: 3, 4) with the sequence of direction of amyloplast (SEQ ID No: 7, 8) for placement of the amylloplast fusion protein. This fusion protein is under the control of the ADPgpp promoter (SEQ ID No: 11) for the expression specifically of the endosperm. The pNOV7005 construct directs the expression of pullulanase in the endoplasmic reticulum of the endosperm. The placement of this enzyme in the ER allows the normal accumulation of the starch less grains. Normal staining was also observed for starch with iodine solution, before any high temperature exposure. As described in the case of α-amylase, the expression of pullulanase directed to the amyloplast (pNOV 4093) resulted in abnormal accumulation of starch in the grains. When the ears of corn were dried, the grains withered. Apparently, this thermophilic pullulanase is sufficiently active at low temperatures and hydrolyzes I 177 starch if it is left in direct contact with the starch granules in the endosperm of the seed.

Enzyme preparation or enzyme extraction from corn flour: The pullulanase enzyme was extracted from the transgenic seeds by grinding them in a Kleco grinder, followed by incubation of the flour in 50 mM NaOAc, buffer pH 5.5 for 1 hour at room temperature, with agitation continues. The incubated mixture was then centrifuged for 15 minutes at 14,000 rpm. The supernatant was used as an enzyme source. Pullulanase assay: The assay reaction was carried out in 96-well plates. The enzyme extracted from corn flour (100 μl) was diluted 10 times with 900 μl of 50 mM NaOAc, buffer pH 5.5, containing 40 mM CaCl2. The mixture was vortexed, a Limit-Dextricima tablet (bluish-reticulate-pullulan, from Megazyme) was added to each reaction mixture and incubated at 75 ° C for 30 minutes (or as mentioned). At the end of the incubation, the reaction mixtures were centrifuged at 3500 rpm for 15 minutes. The supernatants were diluted 5 times and transferred into a 96-well flat bottom plate for absorbance measurement at 590 nm. Hydrolysis of the bluein-reticulate-pullulan substrate by pullulanase produces fragments of water-soluble dyes and the release rate of these (measured as the increase in absorbance at 590 nm) is directly related to the activity of the enzyme.

Figure 9 shows the analysis of T2 seeds of different events transformed with pNOV7005. The high expression of pullulanase activity can be detected in several events, in comparison to the non-transgenic control. To a measured amount (approximately 100 μg) of dried corn flour of transgenic (expressing pullulanase, or amylase, or both enzymes) and / or control (non-transgenic), 1000 μl of pH 5.5 buffer of 50 mM NaOAc was added. contains 40 mM CaCl2. The reaction mixtures were vortexed and incubated on a shaker-for 1 hour. The enzymatic reaction was initiated by transferring the incubation mixtures at high temperature "(75 ° C, the optimum reaction temperature for pullulanase or as mentioned in the figures) during a period of time as indicated in the figures. The reactions stopped when cooled with ice. The reaction mixtures were then centrifuged for 10 minutes, at 14,000 rpm. An aliquot (100 μl) of the supernatant was diluted three times, filtered through a 0.2 micron filter by HPLC analysis. The samples were analyzed by HPLC using the following conditions: Column: Alltech Prevail Carbohydrate ES, 5 microns, 250 X 4.6 mm. Detector: Alltech ELSD 2000 Pump: Gilson 322 Injector: Gilson 215 Injector / Diluent Solvents: HPLC grade Acetonitrile (Fisher Scientific) and water (purified by Waters Millipore System).

Gradient used for oligosaccharides under polymerization degree (DP 1-15) Time% water% acetonitrile 0 15 85 5 15 85 25 50 50 35 50 50 -.- 36 - 80 20 -55 80 20 56 15 85 76 15 85 Gradient used for high degree polymerization saccharides (DP 20-100 and above) Time "% water% acetonitrile 0 35 65 60 85 15 70 85 15 85 35 65 100 35 65 System used for data analysis: Wilson Unipoint Software Version 3.2 Figures 10A and 10B show the HPLC analysis of the hydrolytic products generated by expressed pullulanase of starch in the tragenic cornmeal. Incubation of corn flour expressing pullulanase in reaction buffer at 75 ° C for 30 minutes results in production of medium chain oligosaccharides (DP about 10-30) and short amylase chains (DP about 10-200) of cornstarch. This figure also shows the dependence of pullulanase activity in the presence of calcium ions. Transgenic maize expressing pullulanase can be used to produce modified starch / dextrin which is branched (split al-6 bonds) and will therefore have high level of straight chain amylose / dextrin. Also depending on the kind of starch (eg, waxy, high amylose, etc.) used, the chain length distribution of the amylose / dextrin generated by the pullulanase will vary, and thus the property of the modified starch /dextrin. Hydrolysis of the al-6 bond was also demonstrated using pullulan as the substrate. Pullulanase isolated from corn flour efficiently hydrolyzed pullulan. HPLC analysis (as described) of the product generated at the end of the incubation showed maltotriose production, as expected, due to the hydrolysis of the al-6 bonds in the pullulan molecules by the corn enzyme.

Example 20 Expression of pullulanase in corn The expression of pullulanase 6gp3 was further analyzed by extraction of corn flour followed by PAGE and Coomassie staining. Corn flour was prepared by grinding seeds for 30 seconds in a Kleco grinder. The enzyme was extracted from approximately 150 mg of flour with 1 ml of buffer 5.5 of 50 mM NaOAc. The mixture was vortexed and incubated on a shaker at room temperature for 1 hour, followed by another 15 minute incubation at 70 ° C. The mixture was then pelleted (14000 rpm for 15 minutes at room temperature) and the supernatant was used as SDS-PAGE analysis. The protein band of appropriate molecular weight (95 kDal) was observed. These samples were subjected to a commercially available "conjugated dye-conjugated pullulanase-dextrin assay" (LIMIT-DEXTRIZYME, from Megazyme, Ireland). High levels of thermophilic pullulanase activity correlated with the presence of the.95 kD protein. Western blotting and ELISA analysis of the transgenic maize seed also demonstrated the expression of approximately 95 kD protein that reacted with antibody produced against pullulanase (expressed in E. coli).

Example 21 Increase in starch hydrolysis rate and improved yield of small chain oligosaccharides (fermentable) by the addition of pullulanase-expressing corn The data shown in Figures HA and 11B were generated from HPLC analysis, as described above, of the starch hydrolysis products of two reaction mixtures. The first reaction indicated as "Amylase" contains a mixture [1: 1 (w / w)] of corn flour samples of transgenic maize expressing α-amylase prepared according to the method described in Example 4, for example, and starch non-transgenic A188; and the second reaction mixture "Amylase + Pullulanase" contains a mixture [1: 1 (w / w)] of corn flour samples of transgenic maize expressing α-amylase and transgenic maize expressing pullulanase prepared according to the method described in Example 19. The results obtained support the benefit of the use of pullulanase in combination with α-amylase during the processes of starch hydrolysis. The benefits are of the increased rate of starch hydrolysis (Figure HA) and increases the yield of fermentable oligosaccharides with low DP (Figure 11B). It was found that α-amylase. alone or α-amylase and pullulanase (or any other combination of starch hydrolytic enzymes) expressed in corn can be used to produce maltodextrin (straight or branched oligosaccharides) (Figures HA, 11B, 12 and 13A). Depending on the reaction conditions, the type of hydrolytic enzymes and their combinations will vary, and the type of starch used, the composition of the maltodextrins produced, and therefore their properties. Figure 12 represents the results of an experiment carried out in a similar manner as described for Figure 11. The different temperature and time schedules followed during the incubation of the reactions are indicated in the figure. The optimal reaction temperature for pullulanase is 75 ° C and for a-amylase it is >; 95 ° C. Therefore, the indicated schemes were followed to provide scope to carry out catalysis by pullulanase and / or α-amylase at their respective optimum reaction temperature. It can be clearly deduced from the result shown that the combination of α-amylase and pullulanase performed better in the hydrolysis of cornstarch at the end of the 60 minute incubation period.The HPLC analysis, as described above (except that used approximately 150 mg of corn flour in these reactions), the starch hydrolysis product of two "- sets of reaction mixtures at the end of 30 minutes of incubation is shown in Figures 13A and 13B. The first set of reactions was incubated at 85 ° C and the second was incubated at 95 ° C. For each set, there are two reaction mixtures, the first reaction indicated as "Amylase X Pullulanase" contains tragenic corn flour (generated by cross-pollination) that expresses both a-amylase and pullulanase, and the second reaction indicated as a mixture of "Amylase" from corn flour sample of transgenic maize expressing α-amylase and non-trangic corn A188 in a ratio to obtain the same amount of a-amylase activity as observed at the cross (Amylase X Pullulanase). The total yield of low DP oligosaccharides was more in the case of a-amylase and cross pullulanase compared to corn expressing α-amylase alone, when the corn flour samples were incubated at 85 ° C. The incubation temperature of 95 ° C inactivates (at least partially) the pullulanase enzyme, therefore little difference can be observed between "Amylanase X Pullulanase" and "Amylase". However, the data for both incubation temperatures show significant improvement in the amount of glucose produced (Figure 13B), at the end of the incubation period, when maize flour from the a-amylase and pullulanase cross was used in comparison to corn that expresses α-amylase alone. Therefore, the use of starch expressing both α-amylase and pullulanase for purposes when full hydrolysis of starch to glucose is important may be especially beneficial. The above examples provide broad support for pullulanase expressed in corn seeds, when used in combination with α-amylase, improves the starch hydrolysis process. The pullulanase enzyme activity, which is specific to the al-6 bond, de-branches the starch more efficiently than α-amylase (an enzyme specific for the al-4 bond), thereby reducing the amount of branched oligosaccharides (e.g. , limit-dextrin, panosa, these usually are not fermentable) increasing the amount of straight chain short oligosaccharides (easily fermentable to ethanol, etc.). Second, the fragmentation of starch molecules by pullulanase-catalyzed debranching increases the accessibility to the substrate for α-amylase, thereby resulting in a reduction in the efficiency of the reaction catalyzed by α-amylase.

Example 22 To determine whether alpha-amylase 797GL3 and alpha-glucosidase MalA could function under similar conditions of pH and temperature to generate an increased amount of glucose over that produced by any enzyme alone, approximately 0.35 μg of the enzyme alpha-glucosidase was added. malA (produced in bacteria) to a solution containing 1% starch and purified starch from either non-transgenic maize seed (control) or transgenic maize seed 797GL3 (in 979GL3 maize seed, alpha-amylase is co-purified with starch). In addition, purified starch from transgenic 797GL3 and non-transgenic corn seed was added to 1% corn starch in the absence of any malA enzyme. The mixtures were incubated at 90 ° C, pH 6.0 for 1 hour, centrifuged to remove any insoluble material, and the soluble fraction was analyzed by HPLC for glucose levels. As shown in Figure 14, alpha-amylase 797GL3 and alpha-glucosidase malA function at a similar temperature and pH to decompose starch into glucose. The amount of glucose generated is significantly greater than that produced by any single enzyme.

Example 23 The utility of glucoamylase of Thermoanaerobacterium for hydrolysis of natural starch was determined. As shown in Figure 15, the conversion by hydrolysis of starch to the native was tested with water, barley α-amylase (commercial preparation of Sigma), glucoamylase of Thermoanaerobacterium, and combinations thereof were determined at room temperature and at 30 °. C. As shown, the combination of barley α-amylase with glucoamylase from Thermoanaerobacterium was able to hydrolyze starch naturally in glucose. In addition, the amount of glucose produced by barley amylase and GA of Thermoanaerobacterium is significantly greater than that -produced by any single enzyme.

Example 24 Genes and sequences optimized for maize for hydrolysis of native starch and vectors for plant transformation Enzymes were selected on the basis of their ability to hydrolyze starch naturally at temperatures ranging from about 20 ° -50 ° C. The corresponding genes or gene fragments were then designed by using codons preferred for maize for the construction of synthetic genes as set forth in Example 1. A fusion polypeptide of α-amylase-glucoamylase from Aspergillus shirousami (without signal sequence) was selected and has the amino acid sequence as set forth in SEQ ID No-. 45 as identified in Biosci. Biotech Biochem., 56: 884-889 (1992); Agrie. Biol. Chem. 545: 1905-14 (1990); Biosci. Biotechnol. Biochem. 56: 174-79 (1992). The corn-optimized nucleic acid was designed and depicted in SEQ ID NO: 46. Similarly, glucoamylase from Thermoanaerobacterium thermosaccharolyticu, having the amino acid sequence of SEQ ID No: 47 as published in Biosci. Biotech Biochem., 62: 302-308 (1998). The nucleic acid optimized for corn was designated (SEQ ID Nos: 48). Rhizopus oryzae glucoamylase having the amino acid sequence (without signal sequence) was selected (SEQ ID NO: 50), as described in the literature (Agrie Biol. Chem. (1986) 50-, - pg 857 -964) The corn-optimized nucleic acid was "" designed and is depicted in SEQ ID NO: 51. In addition, the corn α-amylase was selected and the amino acid sequence (SEQ ID NO: 51) was obtained. and the nucleic acid sequence (SEQ ID NO: 52) from the literature See, for example, Plant Physiol., 105: 759-760 (1994) .Expression cartridges were constructed to express the α-amylase / glucoamylase fusion polypeptide. Aspergillus shirousami of the corn-optimized nucleic acid was designed as depicted in SEQ ID NO: 46, the Thermoanaerobacterium thermosaccharolyticum glucoamylase of the corn-optimized nucleic acid was designed as depicted in SEQ ID NO: 48, Rhizopus oryzae glucoamylase was selected to have the amino acid sequence idos (without signal sequence) (SEQ ID NO: 49 of the nucleic acid optimized for corn, it was designed and represented in SEQ ID NO: 50, and the maize α-amylase. A plasmid comprising the N-terminal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ ID NO: 17) is fused to the synthetic gene encoding the enzyme. Optionally, the SEKDEL sequence is fused to the C-terminus of the synthetic gene for targeting and retention in the ER. The fusion is cloned behind the corn? -zein promoter for expression significantly in the endosperm in a plant transformation plasmid. The fusion is distributed to the corn tissue by transfection with Agrobacterium.

EXAMPLE 25 Expression cartridges are constructed comprising the enzymes selected to express the enzymes. A plasmid comprising the sequence for a naturally occurring starch binding site is fused to the synthetic gene encoding the enzyme. The natural starch binding site allows the fusion of the enzyme to bind to the non-gelatinized starch. The amino acid sequence (SEQ ID NO: 53) of the wild-type starch binding site was determined based on the literature, and the nucleic acid sequence was optimized for maize to give SEQ ID NO: 54. The nucleic acid sequence optimized for corn is fused to the synthetic gene encoding the enzyme in a plasmid for expression in a plant.

EXAMPLE 26 Construction of Genes and Vectors Optimized for Corn for Plant Transformation Genes or gene fragments were designed by using codons preferred for maize for the construction of synthetic genes as set forth in Example 1. The amino acid sequence of hyperthermophilic endoglucanase was selected. , EGLA, from Pyrococcus furiosus (without signal sequence) and has the amino acid sequence as set forth in SEQ ID NO: 55, as identified in Journal of Bacteriplogy (1999) 181, pg 284-290). The corn-optimized nucleic acid is designed and depicted in SEQ ID NO: 56. Thermus flavus xylose-isomerase was selected and has the amino acid sequence as set forth in SEQ ID NO: 57, - as described in Applied Biochemistry and Biotechnology 62: 15-27 (1997) Expression cartridges are constructed to express the EGLA (endoglucanase) of Pyrococcus furiosus of corn-optimized nucleic acid (SEQ ID NO: 56) and xylose-isomerase of Thermus flavus of an amino acid sequence encoding nucleic acid optimized for corn SEQ ID NO: 57. A plasmid containing an N-terminal signal sequence of? -zein (MRVLLVALALLALAASATS) (SEQ ID NO: 17) is fused to the gene optimized for maize, synthetic coding for the enzyme. Optionally, the SEKDEL sequence is fused to the C-terminus of the synthetic gene for the a-direction and retention in the ER. The fusion is cloned behind the corn? -zein promoter for expression specifically in the endosperm in a plant transformation plasmid. The fusion is distributed to the corn tissue by transfection with Agrobacterium.

Example 27 Production of glucose from corn flour using thermophilic enzymes expressed in corn. It is shown that the expression of hyperthermophilic α-amylase, 797GL3 and α-glucosidase (MalA) results in the production of glucose when mixed with an aqueous solution and incubated at 90 ° C. A transgenic maize line (line 168A10B, pNOV4831) expressing the MalA enzyme was identified by measuring a-glucosidase activity as indicated by hydrolysis of p-nitrophenyl-a-glucoside. Corn kernels from transgenic plants expressing 797GL3 were ground to a flour in a Kleco cell, thereby creating amylase flour. Corn kernels were ground from transgenic plants that express MalA to a flour in a Kleco cell thus creating MalA flour. The non-transgenic corn grains were milled in a similar manner to generate control flour. The buffer was absorber MES 50 mM, pH 6.0. Corn flour hydrolysis reactions: Samples were prepared as indicated in Table 5 below. Corn meal (approximately 60 mg per sample) was mixed with 40 ml of 50 ml MES buffer, pH 6.0. The samples were incubated in a water bath set at 90 ° C for 2.5 and 14 hours. At the indicated incubation times the samples were removed and analyzed for glucose content.

The samples were evaluated for glucose by a glucose-oxidase / horseradish peroxidase-based assay. The GOPOD reagent contained: 0.2 mg / ml of o-dianisidine, 100 mM Tris, pH 7.5, 100 U / ml of glucose oxidase and 10 U / ml of horseradish peroxidase, 20 μl of sample or diluted sample was fixed in a 96-well plate along with glucose standards (ranging from 0 to 0.22 mg / ml). 100 μl of GOPOD was added to each cavity with mixing and the plate was incubated at 37 ° C for 30 minutes. 100 μl of sulfuric acid (9M) was added and the absorbance was read at 540 nm.

The glucose concentration of the sample was determined by reference to the normal curve. The amount of glucose observed in each sample is shown in Table 5.

Table 5 Hyperhermophilic α-amylase and α-glucosidase in corn results in a corn product that will generate glucose when hydrated and heated under appropriate conditions.

Example 28 Production of Maltodextrins Grain expressing thermophilic α-amylase was used to prepare maltodextrins. The exemplified process does not require previous isolation of starch nor does it require the addition of exogenous enzymes. Corn grains from transgenic plants expressing 797GL3 were ground to a flour in a Kleco cell to create "-amylase meal". A mixture of 10% transgenic grains /; 90% non-transgenic grains was ground in the same way to create "10% amylase flour". Amylase flour and 10% amylase flour (approximately 60 mg / samples) were mixed with water at a ratio of 5 μl of water per mg of flour. The resulting slurry was incubated at 90 ° C for up to 20 hours as indicated in Table 6. The reactions were stopped by the addition of 0.9 ml of 50 mM EDTA at 85 ° C and mixed by pipette. Samples of 0.2 ml of slurry were removed, centrifuged to remove insoluble material and diluted 3x in water. Samples were analyzed by HPLC with detection of ELSD with sugars and maltodextrins. The gradient HPLC system was equipped with an Astee Polymer Amino Column, particle size of 5 microns, 250 x 4.6 mm and an Alltech ELSD 2000 detector. The system was pre-equilibrated with a 15:85 water: acetonitrile mixture. The flow rate "was 1 ml / min.The initial conditions were maintained for 5 minutes after the injection followed by a 20 minute gradient to water: 50:50 acetonitrile followed by 10 minutes of the same solvent. with 20 min of water: acetonitrile 89:20 and then re-equilibrated with the starting solvent.The resulting peak areas were normalized for flour volume and weight.The ELSD response factor per μg of carbohydrate decreases with increase of DP Thus, maltodextrins higher than DP represent a greater percentage of the total than indicated by the peak area.The relative peak areas of the reaction products with 100% amylase flour are shown in Figure 17. Peak areas Relative "of the reaction products with 10% amylase flour are shown in Figure 18. These data demonstrate that a variety of maltodextrin mixtures can be produced by varying the heating time. The level of a-amylase activity can be varied by mixing transgenic corn expressing α-amylase with wild type corn to alter the profile of maltodextrins. The products of the hydrolysis reactions described in this example can be concentrated and purified for food and other applications by using a variety of well-defined methods including: centrifugation, filtration, ion exchange, gel permeation, ultrafiltration, nanofiltration, osmosis inverted, discoloration with carbon particles, spray drying and other known normal techniques.

EXAMPLE 29 Time and Temperature Effector in Maltodextrin Production The composition of the maltodextrin products of the grain self-hydrolysis containing thermophilic α-amylase can be altered by varying the reaction time and temperature. In another experiment, amylase flour was produced as described in Example 28 above and mixed with water at a ratio of 300 μl of water per 60 mg of flour. These samples were incubated at 70 °, 80 °, 90 ° at 100 ° C for up to 90 minutes. The reactions were stopped by adding 900 ml of 50 mM EDTA at 90 ° C, centrifuged to remove insoluble material and filtered through 0.45 μm nylon filters. The filtrates were analyzed by HPLC as described in Example 28. The result of this analysis is presented in Figure 9. The nomenclature of DP numbers refers to the degree of polymerization. DP2 is maltose; DP3 is maltotriose, etc. The larger DP maltodextrins eluted at an individual peak near the end of the elution and marked "> D012". This aggregate includes dextrins that pass through 0.45 μm filters and through the protection column and does not include any very large starch fragments trapped with the filter or protection column. This experiment demonstrates that the maltodextrin composition of the product can be altered by varying both the temperature and the incubation time to obtain the desired maltooligosaccharide or maltodextrin product.

EXAMPLE 30"Production of maltodextrins The composition of transgenic maize maltodextrins products containing thermophilic α-amylase can also be altered by the addition of other enzymes such as α-glucosidase and xylose isomer as well as by" including salts in the aqueous mixture ". of flour before heat treatment In another amylase meal, prepared as described above, mixed with purified MalA and / or a bacterial xylose-isomerose, designated BD8037. MaLA from S. sulfotaricus was expressed with a 6His purification label. in E. coli Cell lysate was prepared as described in Example 28, and then purified to apparent homogeneity using nickel affinity resin (Probond, Invitrogen) and following the manufacturer's instructions for purification of native proteins. xylose-isomerase BD8037 as a lyophilized powder from Diversa and re-suspended in 0.4x the original volume of the water. Enzyme solutions plus water or buffer. All reactions contained 60 mg of amylase flour and a total of 600 μl of liquid. One set of reactions was buffered with MOPS, 50 mM, pH 7.0 at room temperature, plus 10 mM MgSO 4, 1 mM C0C12; in a second set of reactions, the buffer solution containing metal was replaced by water. All reactions were incubated for 2 hours at 90 ° C. The supernatant reaction fractions were prepared by centrifugation. The pellets were "washed with an additional 600 μl of H20 and re-centrifuged.The supernatant fractions" of each reaction were combined, filtered through: Centricon 10, and analyzed by HPLC with ELSD detection as described. described above. The results are plotted in Figure 20. They show that 797GL3 amylase expressed in grain can work with other thermophilic enzymes, with or without additional metal ions, to produce a variety of corn flour maltodextrin mixtures at a high temperature. In particular, the inclusion of a glucoamylase or a-glucosidase can result in a product with more glucose and other products of low DP. The inclusion of an enzyme with glucose isomerase activity results in a product having fructose and thus will be sweeter than that produced by amylase alone or α-glucosidase amylase. In addition, the data indicate that the proportion of maltooligosaccharides of DP5, DP6 and DP7 can be increased by including divalent cationic salts, such as CoCl2 and MgSO4. Other means for altering the maltodextrin composition produced by reaction such as that described herein include: variation of the pH of the reaction, variation of the type of starch in the transgenic or non-transgenic grain, variation of the solids ratio, or by addition of organic solvents.

EXAMPLE 31 Preparation of Dextrins, or Grain Sugars Without Mechanical Grain Breaking Anees of Recovery of Starch Derivatives Products Sugars and maltodextrins were prepared by contacting the transgenic maize expressing the aramylase, 797GL3, with water and heating at 90 ° C. during the night "(> 14 hours) .The liquid was separated from the grain by filtration.The liquid product was analyzed by HPLC by the method described in Example 15. Table 6 presents the profile of detected products.

Table 6 * Quantification of DP3 includes maltotriose and may include maltotriose isomers having a linkage to (1-6) in place of a (1-4) linkage. Similarly, the quantification of DP4 to DP7 includes the linear maltooligosaccharides of a given chain length as well as isomers having one or more bonds to (I 6) instead of one or more bonds to (I-> 4). These data demonstrate that sugars and maltodextrins can be prepared by contacting intact grain that expresses the amylase grain with water and heat. The products can then be separated from the intact grain by filtration or centrifugation or by gravitational settling.

EXAMPLE 32 Fermentation of wild-type starch in maize expressing glucoamylase from Rhizipus oryzae Transgenic maize grains are harvested from engineered transgenic plants as described in Example 29.

The grains are ground to a flour. The corn grains express a protein containing an active fragment of the glucoamylase from Rhizopus oryzae (SEQ ID NO: 49) directed to the endoplasmic reticulum. The corn kernels are ground to a flour as described in Example 15. A dough containing 20 g of corn flour, 23 ml of deionized water, 6.0 ml of countercurrent (8% by weight solids) is then prepared. The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the mass: protease (0.60 ml of a 1,000-fold dilution of a commercially available protease), 0.2 mg of Lactoside and urea (0.85 ml of a 10-fold dilution of 50% Urea Liqueur. A hole is cut in the lid of the 100 ml bottle containing the dough to allow the C02 to vent, the dough is then inoculated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is reduced to 86 ° C, at 48 hours it is adjusted to 82 ° C. Yeast is propagated by inoculation as described in Example 14. Samples are recovered as described in Example 14 and then are analyzed by the methods described in Example 14. 5 EXAMPLE 33 Transgenic maize grains are harvested from transgenic plants prepared as described in Example 28.

The grains are ground to a flour. Corn kernels n express a protein containing an active fragment of the glucoamylase from Rhizopus oryzae (SEQ ID NO: 49) directed to the endoplasmic reticulum. The corn kernels are ground to a flour as described in Example 15. A dough containing 20 g of corn flour, 23 ml of deionized water, 6.0 ml of countercurrent (8% by weight solids) is then prepared. The pH 6.0 is adjusted by the addition of ammonium hydroxide. The following components are added to the mass: protease (0.60 ml of a 1,000-fold dilution of a commercially available protease), 0.2 mg of Lactoside and urea (0.85 ml of a 10-fold dilution of 50% Urea Liqueur). A hole is cut in the lid of the 100 ml bottle containing the dough to allow the C02 to vent. The mass is then inoculated with the cam: hard (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C; at 48 hours it is adjusted to 82 ° C. Yeast is propagated by inoculation as described in Example 14. ~ - z_ Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 34 Example of fermentation of native starch in whole grains of corn expressing Rhizopus oryzae glucoamylase with addition of hexogenic α-amylase Transgenic maize grains are harvested from transgenic plants processed as described in Example 28. Corn grains express a protein containing an active fragment of glucoamylase from Rhizopus oryzae (SEQ ID NO: 49) directed to the endoplasmic reticulum. The corn kernels are contacted with 20 g of corn flour, 23 ml of deionized water, 6.0 ml of countercurrent (8% solids by weight). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added: barley α-amylase purchased from Sigma (2 mg), protease (0.60 ml of a 1,000-fold dilution of a commercially available protease), 0.2 mg of Lactoside and urea (0.85 ml of a 10-fold dilution) of Urea liqueur at 50%). A hole is cut from the lid of the 100 ml bottle containing the mixture to allow the C02 to vent. The mixture is then inoculated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C; at 48 hours it is adjusted to 82 ° C. ? - ~ - Yeast is propagated by inoculation as described in Example 14. Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

EXAMPLE 35 Fermentation of starch native to maize expressing Rhizopus oryzae glucoamylase and Zea mays amylase Transgenic maize grains were harvested from transgenic plants processed as described in Example 28. Corn kernels express a protein containing an active fragment of the glucoamylase of Rhizopus oryzae (SEQ ID NO: 49) directed to the endoplasmic reticulum. The grains also express corn amylase with the native starch binding domain as described in Example 28.

The corn grains "" are ground to a flour as described in Example 14. Then a dough containing 20 g of corn flour, 23 ml of deionized water is prepared, 6. 0 ml countercurrent (8% by weight solids). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the mass: protease (0.60 ml of a 1,000-fold dilution of a commercially available protease), 0.2 mg of Lactoside and urea (0.85 ml of a 10-fold dilution of 50% Urea Liqueur) . A hole is cut from the lid of the 100 ml bottle containing the dough to allow it to vent from the C02. The dough is then inoculated with yeast (1.44 ml) and incubated in a bath of -water adjusted to 90 ° F. After 24 hours of fermentation, the ---- Temperature is lowered to 86 ° F; at 48 hours it adjusts to 82 ° F. It propagates yeast. by inoculation as described in Example 14.. The samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

EXAMPLE 36 Example of fermentation of starch native to maize expressing glucoamylase from Thermoanaerobacter thermosaccharolyticum Transgenic maize grains are harvested from transgenic plants prepared as described in US Pat.

Example 28. Corn kernels express a protein containing an active fragment of the glucoamylase from Thermoanaerobacter thermosaccharolyticum (SEQ ID NO: 47) directed to the endoplasmic reticulum. The corn kernels are ground to a flour as described in Example 15. A dough containing 20 g of corn flour, 23 ml of deionized water, is then prepared. 6. 0 ml countercurrent (8% by weight solids). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the mass: protease (0.60 ml of a 1,000-fold dilution of a commercially available protease), 0.2 mg of Lactoside and urea (0.85 ml of a "10-fold dilution of 50% Urea Liqueur) A hole is cut in the lid of the 100 ml bottle containing the dough to allow the C02 to vent, then the dough is inoculated with yeast (1.44 ml) and incubated in a water bath set at 90 ° F. After 24 hours of fermentation, the temperature is lowered to 86 ° C, to 48 hours it is adjusted to 82 ° C. Yeast is propagated by inoculation as described in Example 14. Samples are removed as described in Example 14 and then it is analyzed by the methods described in the Example 14 EXAMPLE 37 Example of fermentation of starch native to maize expressing Aspergillus niger glucoamylase Transgenic maize grains are harvested from transgenic plants made as described in Example 28. Corn grains express a protein containing an active fragment of glucoamylase of Aspergillus niger (Fiil, NP "Glucoamylases Gl and G2 from Aspergillus niger are synthesized from two different but closely related RNAs" EMBO J. 3 (5), 1097-1102 (1984), accession number P04064). The corn-optimized nucleic acid encoding glucoamylase has SEQ ID NO: 59 and is directed to the endoplasmic reticulum. The corn kernels are ground to a flour as described in Example 14. A dough is then prepared containing 20 mg of corn flour, 23 ml of deionized water, 6.0 ml of countercurrent (8% by weight solids). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the mass: protease (0.60 ml of a dilution of 1, 000 times of a commercially available protease), 0.2 mg of Lactoside and urea (0.85 ml of a 10-fold dilution of 50% Urea Liqueur). A hole is cut in the lid of the 100 ml bottle containing the dough to allow the C02 to vent. The mass is then inoculated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is reduced to 86 ° C; at 48 hours it is adjusted to 82 ° C. Yeast is propagated by inoculation as described in Example 14. Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

EXAMPLE 38 Example of fermentation of starch native to corn that expresses glucoamylase from Aspergillus nicrer and Zea mays Transgenic corn kernels are harvested from plants. Transgenic plants prepared as described in Example 28. Corn grains express a protein containing an active fragment of Aspergillus niger glucoamylase (Fiil, NP "Glucoamylases Gl and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs" EMBO J. 3 (5), 1097-1102 (1984), accession number P04064) (SEQ ID NO: 59, nucleic acid optimized for maize) and targets the endoplasmic reticulum. The grains also express corn amylase with natural-starch binding domain as described in Example 28. The corn grains are ground to a flour as described in Example 14. A dough containing 20 g is then prepared. of corn flour, 23 ml of deionized water, 6.0 ml of countercurrent (8% by weight of solids). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the mass: protease (0.60 ml of a 1,000-fold dilution of a commercially available protease), 0.2 mg of Lactoside and urea (0.85 ml of a 10-fold dilution of 50% Urea Liqueur). A hole is cut in the lid of the 100 ml bottle that contains the dough to allow the C02 to vent. The mass is then inoculated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C; at 48 hours it is adjusted to 82 ° C. Yeast is propagated by inoculation as described in Example 14. Samples are removed as described in Example 14 and then they are analyzed by the methods described in Example 14 Example 39; "Example of fermentation of native starch in maize expressing Thermoanaerobacter glucoamylase" - thermosaccharolyticum and barley amylase Transgenic maize grains are harvested from transgenic plants prepared as described in Example .28. The corn grains express a protein containing an active fragment of the glucoamylase from Thermoanaerobacter thermosaccharolyticum (SEQ ID NO: 47) directed to the endoplasmic reticulum. Grains also express the barley amylase gene of pl (Rogers, JC and Milliman, C. "Isolation and sequence analysis of a barley alpha-amylase cDNA clone" J. Biol. Chem. 258 (13), 8169-8174 ( 1983) modified to direct the expression of the protein to the endoplasmic reticulum, The corn kernels are ground to a flour as described in Example 14. A dough containing 20 g of corn flour, 23 ml of deionized water is then prepared. , 6.0 ml of countercurrent (8% by weight solids) The pH is adjusted to 6.0 by addition of ammonium hydroxide The following components are added to the mass: protease (0.60 ml of a 1,000-fold dilution of a protease commercially available), 0.2 mg of Lactoside and urea (0.85 ml of a 10-fold dilution of 50% Urea Liqueur) A hole is cut in the lid of the 100 ml bottle containing the dough to allow the C02 to vent The mass is then inoculated with yeast (1.44 ml) and incubated in a bath of water set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C; at 48 hours it is adjusted to 82 ° C. Yeast is propagated by inoculation as described in Example 14. Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 40 Example of fermentation of starch in natural whole grains of corn expressing glucoamylase from Thermoanaeirobacter thermosaccharolyticum and barley amylase Transgenic maize grains are harvested from transgenic plants made as described in Example 28. Corn grains express a protein containing an active fragment of the glucoamylase from Thermoanaerobacter thermosaccharoliticum (SEQ ID NO: 47) to the endoplasmic reticulum. The grains also express the amyl gene of low pl barley amylase (Rogers, J.C. and Milliman, C.? solation and sequence analysis of a barley alpha-amylase cDNA clone "J. Biol. Chem. 258 (13), 8169-8174 (1983) modified to direct the expression of the protein to the endoplasmic reticulum. of maize are contacted with 20 g of corn flour, 23 ml of deionized water, 6.0 ml of countercurrent (8% by weight of solid) The pH is adjusted to 6.0 by the addition of ammonium hydroxide. add to the mixture: protease (0.60 ml of a 1,000-fold dilution of a commercially available protease), 0.2 mg of Lactocide and urea (0.85 ml of a 10-fold dilution of 50% urea liquor). A hole is cut "in the lid of the 100 ml bottle containing the dough to allow the C02 to vent, the mixture is then inoculated with yeast (1.44 ml) and incubated in a water bath set at 90 °. C. After 24 hours of fermentation, the temperature is lowered to 86 ° C; at 48 hours it is adjusted to 82 ° C. Yeast is propagated by inoculation as described in example 14. Samples are removed as described in example 14 and then analyzed by the methods described in Example 14.

EXAMPLE 41 Example of fermentation of native starch in maize expressing an alpha-amylase and glucoamylase function Transgenic maize grains are harvested from transgenic plants made as described in Example 28. Corn kernels express an optimized corn polynucleotide such as provided in SEQ ID NO: 46, which codes for an alpha-amylase and glucoamylase function, as provided in SEQ ID NO: 45, which are directed to the endoplasmic reticulum. The corn grains are ground to a ... arina as described in Example 14. A dough containing 20 g of corn flour, 23 ml of deionized water, 6.0 ml of countercurrent (8% by weight) is then prepared. of solids). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the mass: protease (0.60 ml of a 1,000-fold dilution of a commercially available protease), 0.2 mg of Lactocide and urea (0.85 ml of a 10-fold dilution of 50% urea liquor). A hole is cut in the lid of the 100 ml bottle containing the dough to allow the C02 to vent. The mass is then inoculated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C; and at 48 hours it is adjusted to 82 ° C.

Yeast is propagated by inoculation as described in example 14. Samples are removed as described in example 14 and then analyzed by the methods described in Example 14.

Example 42 Construction of transformation vectors Expression cartridges were constructed to express hyperthermophilic beta-glucanase EglA in corn as follows: pNOV4800 comprises the barley Amy32B signal peptide (MGKNGNLCCFSLLLLLLAGLASGHQ) fused to the synthetic gene for beta-glucanase EglA for the direction to the endoplasmic reticulum and secretion in the apoplast. The fusion was cloned behind the? -zein promoter of corn for expression especially in the endosperm. pNOV4803 comprises the signal peptide Amy32b from barley fused to the synthetic gene for beta-glucanase EglA for targeting the endoplasmic reticulum and secretion in the apoplast. The fusion was cloned behind the corn ubiquitin promoter for expression throughout the plant. Expression cartridges were constructed to express the thermophilic beta-glucanase / mannanase 6GP1 (SEQ ID NO: 85) in corn as follows: pNOV4819 comprises the signal peptide PRla of tobacco (MGFVLFSQLPSFLLVSTLLLFLVISHSCRA) fused to the gene "synthetic for beta-glucanase / mannanase 6GP1 for targeting the endoplasmic reticulum and secretion in the apoplast.The fusion was cloned behind the corn? -zein promoter for expression specifically in the endosperm pNOV4820 comprises the synthetic gene for 6GP1 cloned behind the? -zein promoter of corn for cytoplasmic location and specifically expression of the endosperm pNOV4823 comprises tobacco PRla signal peptide fused to the synthetic gene for beta-glucanase / mannanase 6GP1 with a C-terminal addition of the KDEL sequence for the direction to and retention of the endoplasmic reticulum. was cloned behind the corn? -zein promoter for expression specifically of endosperam. NOV 825 comprises the signal peptide PRla of tobacco fused to the synthetic gene for the beta-glucanase / mannanase 6GP1 with a C-terminal addition of the KDEL sequence for the a direction and retention of the endoplasmic reticulum. The fusion was cloned behind the corn ubiquitin promoter for expression throughout the plant. Expression cartridges were constructed to express alpha-amylase A and barley (SEQ ID NO: 87) in corn as follows: pNOV4867 comprises the N-terminal signal sequence of corn? -zein fused to Amyl alpha-amylase barley with a C-terminal addition of the SEKDEL sequence for the direction- and retention in the endoplasmic reticulum. The merger was cloned behind the developer of? -zeine corn for expression specifically in the endosperm. pNOV4879 comprises the N-terminal signal sequence of corn? -zein fused to barley Amyl alpha-amylase with a C-terminal addition of the SEKDEL sequence for the a direction and retention in the endoplasmic reticulum. The fusion was cloned behind the corn globulin promoter for expression specifically in the embryo. pNOV4897 comprises the N-terminal signal sequence of corn? -zein fused to the Amyl alpha-amylase of barley for targeting the endoplasmic reticulum and secretion in the apoplast. The fusion was cloned behind the corn globulin promoter for expression specifically in the embryo. pNOV4895 comprises the N-terminal signal sequence of corn? -zein fused to barley Amyl alpha-amylase for direction to the endoplasmic reticulum and secretion in the apoplast. The fusion was cloned behind the? -zein promoter of corn for expression specifically in the endosperm. pNOV4901 comprises the gene for Amyl alpha-amylase of barley cloned behind the corn globulin promoter for cytoplasmic localization and expression specifically in the embryo. Expression cartridges for expressing Rhizopus glucoamylase (SEQ ID NO: 50) were constructed in corn as follows: pNOV4872 comprises the N-terminal signal sequence of? -zein-corn fused to the synthetic gene for Rhizopus glucoamylase with an addition C -terminal of the SEKDEL sequence for the direction to and retention in the endoplasmic reticulum. The fusion was cloned behind the? -zein promoter of corn for expression specifically in the endosperm. pNOV4880 comprises the N-terminal signal sequence of corn? -zein fused to the synthetic gene for Rhizopus glucoamylase with a C-terminal addition of the SEKDEL sequence for the a direction and retention in the endoplasmic reticulum. The fusion was cloned behind the corn globulin promoter for expression specifically in the embryo. pNOV4889 comprises the N-terminal signal sequence of corn? -zein fused to the synthetic gene for Rhizopus glucoamylase for targeting the endoplasmic reticulum and secretion in the apoplast. The fusion was cloned behind the corn globulin promoter for expression specifically in the embryo. pNOV4890 comprises the N-terminal signal sequence of corn? -zein fused to the synthetic gene for Rhizopus glucoamylase for targeting the endoplasmic reticulum and secretion in the apoplast. The fusion was cloned behind the? -zein promoter of corn for expression specifically in the endosperm. pNOV48.91 comprises the. Synthetic gene for Rhizopus glucoamylase cloned "behind the? -zeine corn promoter for cytoplasmic location and expression specifically in the endosperm.

Example 43 Expression of Rhizopus mesophyll glucoamylase in maize A variety of constructs were generated for the expression of Rhizopus glucoamylase in corn. The globulin and corn? -zein promoters were used to express glucoamylase specifically in the endosperm or embryo, respectively. In addition, the corn? -zein signal sequence and a synthetic ER retention signal were used to regulate the subcellular location of the glucoamylase protein. The 5 constructs (pNOV4872, pNOV4880, pNOV4889, pNOV4890, and pNOV4891) produced transgenic plants with glucoamylase activity detected in the seed. Tables 7 and 8 show the results for individual transgenic seed (construction pNOV4872) and mixed seed (construction pNOV4889), respectively. No harmful phenotype was observed for any transgenic plant expressing this Rhizopus glucoamylase. Glucoamylase assay: seeds were ground to a flour and the flour was suspended in water. The samples were incubated at 30 ° C for 50 minutes to allow the glucoamylase to react with starch. The insoluble material was sedimented and determined at the glucose concentration for the supernatants. The amount of glucose released in each sample is taken as an indication of the level of glucoamylase present. The concentration of glucose was determined by incubating the samples with GOHOD reagent (Tris / Cl 300 mM, pH 7.5, glucose oxidase (20 U / ml, horseradish peroxidase (20 U / ml), o-dianisidine 0.1 mg / ml) for 30 minutes at 37 ° C, adding 0.5 volumes of H2SO, 12N measuring the OD540.

Table 7 shows glucoamylase activity of Rhizopus in individual transgenic corn seed (construction pNOV4872). Table 7 Flour Seed U / g Wild type # 1 0.07 Wild type # 2 0.55 Wild type # 3 0.25 Wild type # 4 0.33 Wild type # 5 0.30 Wild type # 6 0.42 Wild type # 7 -0.01 Wild type # 8 0.31 MD9L022156 # 1 5.17 MD9L022156 # 2 1.66 MD9L022156 # 3 7.66 MD9L022156 # 4 1.77 MD9L022156 # 5 7.08 MD9L022156 # 6 4.46 MD9L022156 # 7 2.20 MD9L022156 # 8 3.50 MD9L023377 # 1 9.23 MD9L023377 # 2 4.30 MD9L023377 # 3 6.72 MD9L023377 # 4 3.35 MD9L023377 # 5 0.56 MD9L023377 # 6 4.79 Flour Seed U / g MD9L023377 # 7 4.60 MD9L023377 # 8 6.01 MD9L023043 # 1 4.93 MD9L023043 # 2 8.74 MD9L023043 # 3 2.70 MD9L023043 # 4 0.72 MD9L023043 # 5 3.33 MD9L023043 # 6 3.53 MD9L023043 # 7 3.'94 MD9L023043 # 8 11.51 MD9L023334 # 1 4.28 MD9L023334 # 2 2.86 MD9L023334 # 3 0.56 MD9L023334 # 4 6.96 MD9L023334 # 5 3.29 MD9L023334 # 6 3.18 MD9L023334 # 7 4.57 MD9L023334 # 8 7.44 MD9L022039 # 1 6.25 MD9L022039 # 2 2.85 MD9L022039 # 3 4.32 MD9L022039 # 4 2.51 Flour Seed U / g MD9L022039 # 5 5.06 MD9L022039 # 6 5.03 MD9L022039 # 7 2.79 MD9L022039 # 8 2.98 Table 8 shows the activity of Rhizopus glucoamylase in mixed transgenic maize seed "1 ° (construction pNOV4889) Table 8 Flour Seed U / g _" Wild type 0.38 15 MD9L023347 2.14 MD9L023352 2.34 MD9L023369 1.66 MD9L023469 1.42 MD9L023477 1.33 20 MD9L023482 1.95 MD9L023484 1.32 MD9L024170 1.35 MD9L024177 1.48 MD9L024184 1.60 25 Flour Seed U / g MD9L024186 1.34 MD9L024196 1.38 MD9L024228 1.69 MD9L024263 1.70 MD9L024315 1.32 MD9L024325 1.73 MD9L024333 1.41 MD9L024339 1.84 All expression cartridges were inserted into the binary vector pNOV2117 for transformation of maize by infection with Agrobacterium. The binary vector contained the phosphomannanase-isomerase (PMI) gene that allows the selection of transgenic cells with mannose. The transformed corn plants either self-pollinated or crossed and seeds were collected for analysis.

Example 44 Expression of hyperthermophilic beta-glucanase EglA in corn For the expression of the hyperthermophilic beta-glucanase EglA in maize the ubiquitin promoter was used for expression in all parts of the plant and the promoter of? -zein for expression specifically in the endosperm of the corn seed. The Amy32B barley signal peptide was fused to EglA for location in the apoplast. The expression of the hyperthermophilic beta-glucanase EglA in transgenic maize seeds and leaves was analyzed using an enzymatic assay and western blot. Transgenic seeds secreted for construction were analyzed pNOV4800 or pNOV4803 using both western blot and an enzymatic assay for beta-glucanase. The endosperm was isolated from individual seeds after soaking in water for 48 hours. The protein was extracted by grinding the endosperm in 50 mM NaP04 buffer (pH 6.0). Heat-stable proteins were isolated by heating the extracts at 100 ° C for 15 minutes, followed by sedimentation of the insoluble material. The supernatant containing the heat stable proteins was analyzed for beta-glucanase activity using the barley azo-glucan method (Megazyme). The samples were pre-incubated at 100 ° C for 10 minutes and analyzed for 10 minutes at 100 ° C using the barley azo-glucan substrate. After incubation, 3 volumes of precipitation solution were added to each sample, the samples were centrifuged for 1 minute, and the OD590 of each supernatant was determined. In addition, 5 ug of protein was separated by SDS-PAGE and transferred to nitrocellulose for western blot analysis using antibodies against EglA protein. Western blot analysis detected specific heat-stable proteins in EglA-positive endosperm extracts, and not in negative extracts. The western transfer signal correlates with the activity level of EglA detected enzymatically. The activity of EglA in leaves and seeds of plants containing the transgenic constructs pNOV4803 and pNOV4800, - respectively, was analyzed. Assays (carried out as described above) showed that heat-stable EglA beta-glucanase was expressed at several levels in the leaves (Table 9) and seeds (Table 10) of transgenic plants while no activity was detected in 'non-transgenic control plants. The expression of EglA in corn using the constructs pNOV4800 and pNOV4803 did not result in any detectable negative phenotype. Table 9 shows the activity of hyperthermophilic beta-glucanase EglA in leaves of transgenic corn plants. Enzymatic assays were carried out on the extracts of the leaves of transgenic plants pNOV4803 to detect hyperthermophilic beta-glucanase activity. The tests were carried out at 100 ° C using the barley azo-glucan method (Megazyme). The results indicate that the transgenic leaves have variable levels of hyperthermophilic beta-glucanase activity.

Table 9 Plant Abs 590 ipo wild 0 266A-17D 0.008 266A-18E 0.184 266A-13C 0.067 266A-15E 0.003 266A-11E 0 265C-1B 0.024 265C-1C 0.065 265C-2D 0.145 265C-5C-. 0.755 265C-5D 0.133 265C-3A 0.076 266A-4B 0.045 266A-12B 0.066 266A-11C 0.096 266A-14B 0.074 266A-4C 0.107 266A-4A 0.084 266A-12A 0.054 266A-15B 0.052 266A-11A 0.109 266A-20C 0.044 Abs Plant 590 266A-19D 0.02 266A-12C 0.098 266A-4E 0.248 266A-18B 0.367 265C-3D 0.066 266A-20E. 0.163 266A-13D 0.084 -265C-3B 0.065 10 26'6A-15A 0.131 266A-13A 0.169 265C-3E 0.116 266A-20A 0.365 266A-20B 0.521 15 266A-19C 0.641 266A-20D 0.561 266A-4D 0.363 266A-18A 0.676 265C-5E 0.339 20 266A-17E 0.221 266A-11B 0.251 265C-4E 0.138 265C-4D 0.242 Table 10 shows the activity of hyperthermophilic beta-glucanase EglA in seeds of transgenic maize plants. Enzymatic assays were carried out on individual segregative seed extracts of pNOV4800 transgenic plants to detect hyperthermophilic beta-glucanase activity. The tests were carried out at 100 ° C using the barley azo-glucan method (Megazyme). The results indicate that the transgenic seed has variable levels of hyperthermophilic beta-glucanase activity Table 10 Seed Abs 590 Wild type 0 1A 1.1 IB 0 1C 1.124 ID 1.323 2A 0 2B 1.354 2C 1.307 2D 0 3A 0.276 3B 0.089 3C 0.463 3D 0 Seed Abs 590 4A 0.026 4B 0.605 4C 0.599 4D 0.642 5A 1.152 5B 1.359 5C 1.035 5D 0 6A 0.006 6B 1,201 6C 0.034"6D 1,227 7A 0.465 7B 0 7C 0.366 7D 0.77 8A 1,494 8B 1,427 8C 0.003 8D 1.413 EglA endoglucanase transgene expression effect in cell wall composition and analysis of in vitro digestion capacity Five individual seeds from each of the two lines, # 263 and # 266, which do not express or express Egla (pNOV4803) respectively they grew in the greenhouse. Protein extracts made from small leaf samples of immature plants were used to verify that the transgenic endoglucanase activity was present in plants # 266 but not in plants # 263. At full maturity of the plant, approximately 30 days after pollination, the whole plants were collected above the ground, chopped in general terms, and dried in the oven for 72 hours. Each sample was divided "into 2 duplicate samples (labeled A and B, respectively), ~ -and subjected to in vitro digestion capacity analysis using cast herbal fluid using common procedures (forage, fiber, analysis, apparatus, reagents, procedures and some applications, by HK Goering and PJ Van Soest, Goering, H. Keith 1941 (Washington, DC): Agricultural Research Service, EU Dept. of Agriculture, 1970, iv, 20 p .: ill - Agriculture handbook, no. 379), except that the material was treated by a pre-incubation at either 40 ° C or 90 ° C before analysis of in vitro digestion capacity The analysis of in vitro digestibility was performed as follows: they were chopped to about 1 mm with a wiley mill, and then subdivided into 16 heavy aliquots for analysis.The material was suspended in buffer and incubated at either 40 ° C or 90 ° C for 2 hours, then cooled overnight They added mic ronutrients, trypticase and casein and sodium sulfide, followed by strained herbal fluid, and incubated for 30 hours at 37 ° C. Analyzes of neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (AD-L) were performed using normal g-ravimetric methods (Van Soest &Wine, Use of Detergents in the Analysis of Fibrous Feeds. IV. '' Determination of plant cell-wall constituents, PJ Van Suest &RH Wine (1967), Journal of the AOAC, 50: 50-55, see also Methods for dietry fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition (1991) PJ Van Soest, JB Robertson & amp;; B. A. Lewis J. Dairy Science, 74: 3583-3597). The data showed that the transgenic plants that express EglA (# 266) contain more NDF than the control plants (# 233), while ADF of lignin were relatively unchanged. The NDF fraction of the transgenic plants is more easily digested than that of the non-transgenic plants, and this is due to an increase in the cellulose digestion capacity (NDF-ADF-AD-L), consistent with the "self-digestion" of the cell wall cellulose by the transgenically expressed endoglucanase enzyme.

Example 45 _ - Expression of Thermophilic Beta-Glucanase / Mannanase (6GP1) in Corn Transgenic seeds were analyzed for pNOV4820 and . pNOV4823 for the 6GP1 beta-glucanase activity using the barley azo-glucan method (Megazyme). Enzymatic assays carried out at 50 ° C indicate that the transgenic seed has thermophilic beta-glucanase 6GP1 activity while no activity was detected in non-seed Transgenic (positive signal represents background noise associated with this assay). Table 11 shows 6GP1 thermophilic beta-glucanase / mannanase activity in transgenic maize seed. Transgenic seeds were analyzed for pNOV4820 0 (events 1-6) and pNOV4823 (events 7-9) for 6GP1 beta-glucanase activity using the barley azo-glucan method (Megazyme). Enzymatic assays were carried out at 50 ° C and the results indicate that the transgenic seed has thermophilic beta-glucanase 6GP1 activity while no activity is detected in non-transgenic seed.

Table 11 Seed Abs 590 Type silveí 3tre 0 - 1 0.21 2 0.31 3 0.36 4 0.23 5 0.16 6 0.14 7 0.52 8 0/54 9 0.49 EXAMPLE 46 Expression of amyl amylose Amyl from misophilic barley in corn A variety of constructs for the expression of Amyl alpha-amylase from barley in corn was generated. The globulin and? -zein corn promoters were used to express amylase specifically in the endosperm or embryo, respectively. In addition, the corn? -zein signal sequence and the synthetic ER retention signal were used to regulate the subcellular location of the amylase protein. The 5 constructs (pNOV4867, pNOV4879, pNOV4897, pNOV4895, and pNOV4901) produced transgenic plants with alpha-amylase activity detected in the seed. Table 12 shows the activity in individual seeds for 5 independent segregative events (constructs pNOV4879 and pNOV4897). All constructions produced some teransgenic events with a countersunk seed phenotype indicating that the barley Amyl amylase synthesis can affect the formation, accumulation or decomposition of starch. Table 12 shows the Amyl alpha-amylase activity of barley on individual maize seeds (constructs pNOV4879 and pNOV4897). Individual segregative seeds for constructs pNOV4879 (seed samples 1 and 2) and pNOV4897 (seed samples 3-5) were analyzed for alpha-amylase activity as described above. Table 12 Seed Corn meal U / g ÍA 19. 29 IB 1. 49 1C 18. 36 ID 1. 15 1E 1. 62 Seed Corn meal U / g 1F 14.99 1G 1.88 1H 1.83 2A 2.05 2B 36.79 2C 30.11 2D 2.25 2E 32.37 2F 1.92 2G 20.24 2H 35.76 3A 22.99 3B 1.72 3C 25.38 3D 18.41 3E 28.51 3F 2.11 20 3G 16.67 3H 1.89 4A 1.57 4B 36.14 4C 23.35 25 Seed Corn meal U / g 4D 1.70 4E 1.94 4F 14.38 4G 2.09 4H 1.83 5A 11.64 5B 18.20 5C 1.87 5D 2.07"5E 1.71 5F 1.92 5G 12.94 5H 15.25 Example 47 Preparation of xylanase constructs Table 13 lists 9 binary vectors each containing a single xylanase expression cartridge. The xylanase expression cartridges include a promoter, a synthetic xylanase gene (coding sequence), an intron (PEPC, inverted), and a terminator (35S). Two maize optimized endo-xylanase genes were cloned into the binary vector pNOV2117. These two xylanase genes were designated BD7436 (SEQ ID NO: 61) and BD6002A (SEQ ID NO: 63). Additional binary vectors containing a third optimized sequence can be prepared for corn, BD6002B (SEQ ID NO: 65). Two promoters were used: the corn glutelin-2 promoter (27 kD gamma-zein promoter) (SEQ ID NO: 12) and the rice glutelin-1 (Osgtl) promoter (SEQ ID NO: 67) . The first 6 vectors listed in Table 1 have been used to generate transgenic plants. The last 3 vectors can also be developed and used to generate transgenic plants. Vectors 11560 and 11562 encode the polypeptide shown "in SEQ ID NO: 62 (BD7436)." Constructs 11559 and 11561 encode a polypeptide consisting of SEQ ID NO: 17 fused to the N-terminus of SEQ ID NO: 62. SEQ ID NO: 17 is the signal sequence of 19 amino acids of the 27 kD gamma-zein protein Vector 12175 codes for the polypeptide shown in SEQ ID NO: 64 (BD6002A). fusion consisting of the gamma-zein signal sequence (SEQ ID NO: 17) fused to the N-terminus of SEQ ID NO: 64. Vectors pWIN062 and pWIN064 encode the polypeptide shown in SEQ ID NO: 66 (BD6002B). The vector pWIN058 codes for a fusion protein consisting of the corn chloroplast transit chloroplast transit peptide (SEQ ID NO: 68) fused to the N-terminus of SEQ ID NO: 66.

Table 13. Linear xylanase vectors All constructs include an expression cassette for PMI, allowing positive selection of regenerated transgenic tissue in medium containing mannose.

Example 48 Xylanase Activity Testing Results The data shown in Tables 14 and 15 demonstrate that the xylanase activity accumulates in the Ti generation seed harvested from regenerated corn plants (TO) stably transformed with binary vectors containing the xylanase genes BD7436 (SEQ ID NO: 61 in Example 47) and BD6002A (SEQ ID NO: 63 in Example 47). Using an Azo-WAXY (Megazyme) assay, activity was detected in extracts from both mixed (segregative) transgenic seeds and individual transgenic seeds. TI seeds were sprayed and soluble proteins extracted from four samples using citrate-phosphate buffer (pH 5.4). Flour suspensions were stirred at room temperature for 60 minutes, and the insoluble material was removed by centrifugation. The xylanase activity of the supernatant fraction was measured using an Azo-WAXY assay (McCleary, BV "Problems in the measurement of beta-xylanase, beta-glucanase and alpha-amylase in feed enzy es and animal feeds'X In proceedings of Second European Symposium on Feed Enzymes "(W. van Hartingsveldt, M. Hessing, JP van der Jugt, and WA C Somers Eds.), Noordwiij kerhout, The Netherlands, 25-27 October 1995). The extracts and substrate were pre-incubated at 37 ° C. To a volume of the supernatant of extract IX, 1 volume of substrate (1% wheat Azo-arabinoxylan S-AWAXP) was added and then incubated at 37 ° C for 5 minutes. Activity . "- xylanase in the extract of maize flour desiza eriza Azo-arabinoxylan of trigó-by an endo-mecanis oy produces dyed fragments of low molecular weight in the form of xylo-oligomers.After incubation for 5 minutes, the reaction it was terminated by the addition of 5 volumes of 95% EtOH.The alcohol addition causes the dry substrate not to depolymerize precipitate so that some of the low molecular weight xylo-oligomers remain in solution.The insoluble material is removed by centrifugation The absorbance of the supernatant fraction is measured at 590 nm, and the xylanase units per gram of flour are determined by comparison to the absorbance values of identical assays using a xylanase standard of known activity.The activity of this standard was determined by a BCA assay.The enzyme activity of the standard was determined using wheat arabinoxylan as a substrate and measuring the release of reducing ends by reaction. n reducing ends with 2,2'-bicinchoninic acid (BCA). The substrate was prepared as a 1.4% w / w solution of wheat arabinoxyla (Megazyme P-WAXYM) in 100 mM sodium acetate buffer pH 5.30 containing 0.02% sodium azide. The BCA reagent was prepared by combining 50 parts of reagent A with one part of reagent B (reagents A and B are from Pierce, product numbers 23223 and 23224, respectively). These reagents were combined no more than four hours before use. The assay was performed by combining 200 microliters of substrate to 80 microliters of enzyme sample. After incubation at the desired temperature for the desired duration of time, 2.80 milliliters of BCA reagent was added. The contents were mixed and placed at 80 ° C for 30-45 minutes. The contents were allowed to cool and then transferred to specimens and the absorbance at 560 nm was measured relative to the known concentrations of xylose. The choice of enzyme dilution, incubation time, incubation temperature can be varied by one skilled in the art. The experimental results shown in Table 14 demonstrate the presence of recombinant xylanase activity in flour prepared from the Ti generation corn seed. The seeds of 12 T0 plants (derived from independent integration events of T-DNA) were analyzed. The 12 transgenic events were derived from 6 different vectors as indicated (refer to Table 13 in Example 47 for description of vectors). Extracts of non-transgenic corn flour (negative control) do not contain mediated xylanase activity (see Table 15). The xylanase activity in these 12 samples varied from 10-87 units / grams of flour.

Table 14. Analysis of mixed Ti seeds presence of xylanase activity in corn flour derived from individual grains. Ti seeds from two TO plants containing vectors 11561 and 11559 were analyzed. These vectors are described in Example 47. Eight seeds from each of the two plants were sprayed and two samples were extracted for each seed. The table shows results of individual trials of each extract. No xylanase activity was found in the essays of extracts of seeds 1, 5 and 8 for both transgenic cases. These seeds represent null segregants. Seeds 2, 3, 4, 6 and 7 for both transgenic events accumulated measurable xylanase activity attributable to the expression of the recombinant BD7436 gene. The 10 seeds tested positive for xylanase activity (> 10 units / gram of flour) had an almond-shaped or wrinkled or shrunken appearance. In contrast, the 6 seeds that were tested negative for xylanase activity (= 1 unit / gram of flour) had a normal appearance. This result suggests that the recombinant xylanase depolymerized the endogenous (arabino) xylan substrate during development and / or maturation.

Table 15. Analysis of individual Ti seeds Example 49 Starch corn starch recovery using enzymes Wet milling of corn includes the steps of impregnation of the corn kernel, milling of the corn kernel, and separation of the grain components. A bench enzyme assay (the broken corn test) was developed to mimic the wet milling process of corn. The "broken corn test" was used to identify enzymes that improve the starch production of corn seeds which result in improved efficiency of the wet milling process of corn. The distribution of enzymes was either by exogenous addition, transgenic maize seeds, or a combination of both. In addition to the use of enzymes to facilitate separation of corn components, S02 removal from the process is also shown.

Broken Corn Test One gram of seeds was impregnated overnight at S02 to 4000, 2000, 1000, 500, "40-0, 40 or 0 ppm at 50 ° C or 37 ° C. The seeds were cut halfway and the germ was removed, each half seed was cut in half again, Soaking water from each soaked seed sample was retained and diluted to a final concentration ranging from 400 ppm to 0 ppm S02. of soaking water with or without enzymes to the degerminated seeds and the samples were placed at 50 ° C or 37 ° C for 2-3 hours.Each enzyme was added to 10 units per sample.All the samples were vortexed approximately every 15 minutes After 2-3 hours the samples were filtered through the scope in a 50 ml centrifuge tube, the seeds were washed with 2 ml of water and the samples were mixed with the first supernatant. 15 minutes at 3000 rpm.After centrifugation, the supernatant s and poured and the sediment was placed at 37 ° C to dry. All weights of the sediment were recorded. Determinations of starch and protein were also carried out in the samples to determine the ratio: protein released during the treatments (data not shown). The analysis of the Ti and T2 seeds of corn plants expressing 6GP1 endoglucanase in the broken corn test. Transgenic corn (pNOV4819 and pNOV4823) containing a thermostable endoglucanase performed well when performed in the broken corn test. The recovery of starch from the pNOV4819 line was found to be 2 times higher in seeds expressing the endoglucanase when soaked in 2000 ppm of S02. The addition of a protease and cellobiohydrolase to the endoglucanase seed increased the recovery of starch by approximately 7-fold over the control seeds. See Table 16.

Table 16 Results of Broken Corn Assay for Endoglucanase Expressed Cytosolic (pNOV4820). Control line, A188 / HÜ1, lines pNOV4819, 42C6A-1-21 and 27 Similar results were seen in transgenic seeds containing endoclucanase directed to endosperm ER (pNOV4823), which again result in a 2-7 fold increase in starch recovery when compared to control seeds. See Table 17.

Table 17 Results of Tests of Broken Maize for endoclucanase expressed in ER pNOV4823). Control Line, Al88 / Hill; line pNOV4823, 101D11A-1-28 These results that the expression of an endoglucanase improves the separation of starch and protein components of corn seed. Furthermore, it can be shown that the reduction or removal of S02 during the impregnation or soaking step resulted in recovery of starch that was comparable to or better than the control seeds normally impregnated or soaked. See Table 18. The removal of high levels of S02 from the wet milling process can provide added value benefits.

Table 18 Comparison of various concentrations of S02 in the recovery of transgenic 6GP1 seed starch Example 50 Construction of transformation vectors for bromelain optimized for corn. Expression cartridges were constructed to express bromelain optimized for maize in corn endosperm with various directional signals as follows: pSYNHOOO (SEQ ID No: 73) comprises the signal sequence of bromelain (MAWKVQWFLFLFLCVMWASPSAASA) (SEQ ID NO: 72) and the synthetic bromelain sequence fused to a C-terminal addition of the VFAEAIAANSTLVAE sequence for the direction to and retention in the PVS (Vítale and Raikhel Trends in Plant Science Vol. 4 No. 4 , pp. 149-155). The fusion was cloned behind the corn gamma-zein promoter for expression specifically of the endosperm. pSYN11587 (SEQ ID NO: 75) comprises the N-terminal signal sequence of bromelain (MAWKVQWFLFLFLCVMWASPSAASA) and the synthetic bromelain sequence with a C-terminal addition of the SEKDEL sequence for targeting and retention in the endoplasmic reticulum (ER) ( Munro and Pelma, 1987). The fusion was cloned after the corn gamma-zein promoter for expression specifically in the endosperm. pSYN11589 (SEQ ID NO: 74) comprises the bromelain signal sequence (MAWKVQWFLFLFLCVMWASPSAASA) (SEQ ID NO: 72) fused to the lytic vacuolar targeting sequences SSSSFADSNPIRVTDRAAST (Neuhaus and Rogers Plant Molecular Biology 38: 127-144, 1998) and Synthetic bromelain for the direction to the lytic vacuole. The fusion was cloned behind the corn gamma-zein promoter for expression specifically in the endosperm. pSYN12169 (SEQ ID NO: 76) comprises the N-terminal signal sequence of? -. corn zeina (MRVLLVALALLALAASATS) (SEQ ID No: 17) fused to synthetic bromelain for targeting the endoplasmic reticulum and secretion in the apoplast (Torrent et al., 1997). The fusion was cloned behind the maize gamma-zein promoter for expression specifically of the endosperm. pSYN12575 (SEQ ID NO: 77) comprises the sero-amyloplast targeting peptide (Klosgen et al., 1986) fused to the synthetic bromelain for targeting the amyloplast. The fusion was cloned behind a gamma-zein promoter for expression specifically of the endosperm. pSM270 (SEQ ID No: 78) comprises the N-terminal signal sequence of bromelain fused to the lytic vacuolar targeting sequence SSSSFADSNPIRVTDRAAST (Neuhaus and Rogers Plant Molecular Biology-_. 38 :: 127-144, 1998) and synthetic bromelain for the direction to the lytic vacuole. The fusion was cloned behind the aleurone specific promoter P19 (U.S. Patent No. 6,392,123) for expression specifically in aleurone.

Example 51 Expression of bromelain in corn Seeds of transgenic IT lines transformed with vectors containing the synthetic bromelain gene with targeting sequence for expression in several subcellular locations of the seeds were analyzed for protease activity. Corn flour was made by grinding seeds, for 30 seconds in a Kleco grinder. The enzyme was extracted from 100 mg of flour with 1 ml of 50 mM NaOAc buffer, pH 4.8 or 50 mM Tris pH 7.0 containing 1 mM EDTA and 5 mM DTT. The samples were vortexed, then placed at 4 ° C with continuous agitation for 30 minutes. Extracts from each transgenic line were assessed using resorufin-labeled casein (Roche, Cat. No. 1,080,733) as summarized in the product brochure. Flours of the T2 seeds were evaluated using a bromelain-specific assay as summarized in Methods in Enzymology Vol. 244: p 557-558 with the following modifications. 100 mg of corn seed flour were extracted with 1 ml of 50 mM Na2HP04 / 50 mM NaH2P04, pH7.0, 1 mM EDTA + 1 μM EDTA +/- leupeptin for 15 minutes at 4 ° C. The extracts were centrifuged for 5 minutes at 14,000 rpm at 4 ° C. The extracts were made in duplicates. The flour of transgenic lines T2 was evaluated for bromelain activity using Z-Arg-Arg-NHMee (Sigma) as a substrate. Four aliquots of 100 μl corn seed extracts were added to 96-well flat bottom plates (Corning) containing 50 μl of 100 mM Na2HP04 / 100 mM Na2HP04, pH 7.02, 2 mM EDTA, 8 mM DTT / well The reaction was initiated by the addition of 50 μl of 20 μM Z-Arg-Arg-NHMec The reaction rate was monitored using a SpectraFluorPlus (Tecan) equipped with a 360 nm excitation filter and emission from 465 nm to 40 nm. ° C at 2.5 minute intervals Table 19 shows the seed analysis of different bromelain TI events We found that bromelain expression is 2-7 times higher than control lines A188 and JHAF. replanted and T2 seeds were obtained The analysis of the T2 seeds showed bromelain expression Figure 21 shows bromelain activity assay using Z-Arg-Arg-NHMec_ in T2 seeds for bromelain directed to ER (11587) and directed to lytic vacuolar (11589).

Analysis of T2 seeds of corn plants expressing Bromelain Transgenic bromelain T2 seeds, 11587-2 seeds were analyzed in the broken corn test for improved recovery of starch. The above experiments - using exogenously added bromelain showed an increased recovery in starch when tested alone and in combination with other enzymes, particularly cellulases. The T2 seeds of line 11587-2 showed a 1.3-fold increase in the starch recovered on the control seeds when soaked at 37 ° C / 2000 ppm S02 overnight. More importantly, there was a 2-fold increase in the starch of the T2 bromelain line, 11587-2 when a cellulase (C8546) was used when the seeds were soaked or impregnated at 37 ° C / 2000 ppm S02. The transgenic line showed a similar tendency in increased starch on the control seed when the seeds were soaked at 37 ° C / 400 ppm of S02. A 1.6-fold increase was observed in the recovered starch with respect to the control in the transgenic seeds and a 2.5-fold increase in starch with the addition of a cellulase (C8546). See Table 20. These results are significant in showing that it is possible at reduced temperature and reduced levels of S02 while improving the recovery of starch during the wet milling process when using transgenic seed that expresses a bromelain. Table 19 Summary of Specific Expression in Bromelain Grain in IT Corn Table 20 Results of Broken Corn Tests for bromelain T2 seeds Example 52 Construction of transformation vectors for ferulic acid-esterase optimized for maize. Expression cartridges were constructed to express maize-optimized ferulic esterase acid in the maize endosperm with or without several directional signals as follows: Plasmid 13036 (SEQ. ID No: 101) comprises the ferulic acid esterase (FAE) sequence optimized for maize (SEQ ID No: 99). The sequence was cloned behind the maize gamma-zein promoter without any targeting sequence for expression specifically in the endosperm cytosol. Plasmid 13038 (SEQ ID NO: 103) comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to synthetic FAE for targeting the endoplasmic reticulum and secretion in the apoplast (Torrent et al., 1997). The fusion was cloned behind the corn gamma-zein promoter for expression specifically in the endosperm. Plasmid 13039 (SEQ ID NO: 105) comprises the waxy amylloplast targeting peptide (MLAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRHQHQ QARRGARFPSLWCASAGA) (Klosgen et al., 1986) fused to the synthetic FAE for targeting the amyloplast. The fusion was cloned behind the gamma-zein promoter for expression specifically in the endosperm. Plasmid 13347 (SEQ ID NO: 107) comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ ID No: 17) fused to the synthetic FAE sequence with a C-terminal addition of the sequence SEKDEL for the direction and retention in the endoplasmic reticulum (ER) (Munro and Pelham, 1987). The fusion was cloned behind the corn gamma-zein promoter for expression specifically in the endosperm. All expression cartridges were moved to a binary vector pNOV2117 for transformation into maize by Agrobacterium infection. The binary vector contained the phosphomannanase-isomerase (PMI) gene that allowed the selection of transgenic cells with mannose. Transformed corn plants either self-pollinated or crossed and seeds were collected for analysis. Combinations of the enzymes can be produced either by crossing plants expressing the individual Menzymes or by cloning several expression cartridges in the same binary vector to allow co-transformation.

Acid Sequence Ferulic-Synthetic Esterase (SEO ID No: 99) atggccgcctccctcccgaccatgccpccgtccggctaceaccaggtgcpcaacggceteccgcgcggccaggrgg? Gaaca? C? Cctacpctccat: c? .ccat. ctccacccgcccggcccgcgtgtacctcccgccgggctactccaaggacaagaagtactccgtgctctacctcctccacggcatcggcggctccgagaacgactggtt aa cgagggcggcggccgcgccaacgtgatcgccgacaacctcatcgccgagggcaagatcaagccgctcatcatcgtgaccccgaacaccaacgccgccggcccgg gcatcgccgacggctacgaeaacttcaccaaggacctcctcaactccctcatcccgtacatcgagtccaactactccgtgtacaccgaccgcgagcaccgcgccatcgc cggcctctctatgggcggcggccaetccttcaacatcggcctaaccaacctcgacaagttcgcctacatcggcccgatctccgccgccccgaacacctacccgaacga gcgcctcttcccggacggcggcaaggccgcccgcgagaagctcaagctcctcttcatcgcctgcggcaccaacgactccctcatcggcttcggccagcgcgtgcacg agtactgcgtggccaacaacatcaaccacgtgtactggctcatccagggcggcggccacgacttcaacgtgtggaagccgggcctctggaacttcctccagatggccg acgaggccggcctcacccgcgacggcaacaccccggtgccgaccccgtccccgaagccggccaacacccgcatcgaggccgaggactacgacggcatcaactcc tcctccatcgagatcatcggcgtgccgccggagggcggccgcggcatcggctacatcacctccggcgactacctcgtgtacaagtccatcgacttcggcaacggcgcc acctccttcaaggccaaggtggccaacgccaacacctccaacatcgagcttcgcctcaacggcccgaacggcaccctcatcggcaccctctccgtgaagtccaccggc gactggaacacctacgaggagcag acctgctccatctccaaggtgaccggcatcaacgacctctacctcgtgttcaagggcccggtgaacatcgactggttcaccttcg gcgtgtag Amino acid sequence ferulic acid esterase Sintética (SEO ID No: 100) maaslptmppsgydqv ngvprgqvv usvfstatns roarvylppgvskdk v5ylyllhgiggsend fegggtanviadrüiaegk giadgyenftkdllnsHpyiesnvsv lf ^ dreru-aiaglsmgggqsfrugltnldkfavigpisaapntvpnerlfodggkaareklkpfiac vaiminhvy liqggghdfiív kpgl nilqpadeagltrdgntpyptpspkpan rieaedvdginsssieiigvppeggrgi sfka??? anantsnieij] Dgpngtligtlsvkstgd ntveeqtcsiskvtgindlylvfkgpynidwftfgv * 13036 sequence (SEQ ID No: 101) atggccgcctccctcccgaccatgccgccgtccggctacgaccaggtgcgcaacggcgtgccgcgcggccaggtggtgaacatctcctacttctccaccgccaccaa ctccacccgcccggcccgcgtgtacctcccgccgggctactccaaggacaagaagtactccgtgctctacctcctccacggcatcggcggctccgagaacgactggtt cgagggcggcggccgcgccaacgtgatcgccgacaacctcatcgccgagggcaagatcaagccgctcatcatcgtgaccccgaacaccaacgccgccggcccgg gcatcgccgacgectacgagaacttcaccaaggacctcctcaactccctcatcccgtacatcgagtccaactactccgtgtacaccgaccgcgagcaccgcgccatcgc cggcctctctatgggcggcggccaetccttcaacatcggcctcaccaacctcgacaag tcgcctacatcggcccgatctccgccgccccgaacacctacccgaacga gcgcctcttcccggacggcggcaaggccgcccgcgagaagctcaagctcctcttcatcgcctgcggcaccaacgactccctcatcggcttcggccagcgcgtgcacg agtactgcgtggccaacaacatcaaccacg gtactggctcatccagggcggcggccacgacttcaacgtgtggaagccgggcctctggaacttcctccagatggccg acgaggccggcctcacccgcgacggcaacaccccggtgccgaccccgtccccaaagccggccaacacccgcatcgaggccgaggactacgacggcatcaactcc tcctccatcgagatcatcggcg gccgccggagggcggccgcggcatcggctacatcacctccggcgactacctcgtgtacaagtccatcgacttcggcaac ggcgcc acctccttcaaggccaaggtggccaacgccaacacctccaacatcgagcttcgcctcaacggcccgaacggcaccctcatcggcaccctctccgtgaagtccaccggc gactggaacacctacgaggagcagacctgctccatctccaaggtgaccggcatcaacgacctctacctcgtgttcaagggcccggtgaacatcgactgg tcaccttcg gcgtgtag AA sequence of 13036 (SEO ID No: 102 iraaslptmppsgydqvmgvprgqvvmsyfstatest arvylppgvskdklcy giadgyenftkdllnsHpyiesnvsvvtdrehraiaglsmgggqsÍTOgltnldkfa ^ ^^ ^ vaniiinhvywliqggghdfnvwlrogl nflqrnadeagltrdgntpyftpspkpantá sfkakvanantsnielrlngpngtligtlsvkstgdwntveeqtcsiskvfgindiylvfkgpynídwftfgv * 13038 sequence (SEQ ID No: 103) atgaggatgttgctcgttgccctcgctctcctagctctcgctgcgagcgccacctccatggccgcctccctcccgaccatgccgccgtccggctaegaccaggtgcgca acggcgtgccgcgcggccaggtggtgaacatctcctacttctccaccgccaccaactecacccgcccggcccgcgtgtacctcccgccgggctactccaaggacaag aagtactccgtgctctacctcctccacggcatcggcggctccgagaacgactggttcgagggcggcggccgcgccaacgtgatcgccgacaacctcatcgccgaggg caagatcaagccgctcatcatcgtgaccccgaacaccaacgccgccggcccgggcatcgccgacggctacgagaacttcaccaaggaectcctcaactccctcatccc gtacatcgagtccaactactccgtgtacaccgaccgcgagcaccgcgccatcgccggcctctctatgggcggcggccagtccttcaacatcggcctcaccaacctcgac aagttcgcctacatcggcccgatctccgccgccccgaacacctacccgaacgagcgcctc tcccggacggcggcaaggccgcccgcgagaagctcaagctcctctt catcgcctgcggcaccaacgactccctcatcggcttcggccagcgcgtgcacgagtactgcgtggccaacaacatcaaccacgtgtactggctcatccagggcggcgg ccacgacttcaacgtgtggaagccgggcctctggaacttcctccagatggccgacgaggccggcctcacccgcgacggcaacaccccggtgccgaccccgtccccg aagccggccaacacccgcatcgaggccgaggactacgacggcatcaactcctcctccatcgagatcatcggcgtgccgccggagggcggccgcggca tcggctac atcacctccggcgactacctcgtgtacaagtccatcgacttcggcaacggcgccacctccttcaaggccaaggtggccaacgccaacacctccaacatcgagcttcgcc tcaacggcccgaacggcaccctcatcggcaccctctccgtgaagtccaccggcgactggaacacctacgaggagcagacctgctccatctccaagg'tgaccggcatc aacgacctctacctcgtgttcaagggcccggtgaacatcgactgg tcaccttcggcgtgtag AA sequence of 13038 (SEQ ID NO: 104) mrvilva1allalaasats aaslpttnppsgydqvmgvprgqvvnisvfstatnstrparvylppgvskdkkysylyllhgiggsendwfegggranviadnliae gkikpliivtpntoaagpgiadgyenftkdlínslipyiesnysv ^ drehraiaglsm cgmdsligfgqrv 'eycvanninhvv liqggghdfnvwkpglwnflqmadeaglfr ^ tsgdylvyksidfgrigatsfkakvanantsnielrlngpngtligtlsvkstgd ntveeqtcsiskvtgindlylvílcgpyriidwftfgv * 13039 sequence (SEQ ID No: 105) atgctggcggctctggccacgtcgcagctcgtcgcaacgcgegccggcctgggcgtcccggacgcgtccacgttccgccgcggcgccgcgcagggcctgagggg ggcccgggcgtcggcggcggcggacacgctcagcatgcggaccagcgcgcgcgcggcgcccaggcaccagcaccagcaggcgcgccgcggggccaggttcc cgtcgctcgtcgtgtgcgccagcgccggcgccatggccgcctccctcccgaccatgccgccgtccggctacgaccaggtgcgcaacggcgtgccgcgcggccaggt ggtgaacatctcctacttctccaccgccaccaactccacccgcccggcccgcgtgtacctcccgccgggctactccaaggacaagaagtactccgtgctctacctcctcc acggcatcggcggctccgagaacgactggttcgagggcggcggccgcgccaacgtgatcgccgacaacctcatcgccgagggcaagatcaagccgctcatcatcgt gaccccgaacaccaacgccgccggcccgggcatcgccgacggctacgagaacttcaccaaggacctcctcaactccctcatcccgtacatcgagtccaactactccgt gtacaccgaccgcgagcaccgcgccatcgccggcctctctatgggcggcggccagtccttcaacatcggcctcaccaacctcgacaagttcgcctacatcggcccgat ctccgccgccccgaacacctacccgaacgagcgcctcttcccggacggcggcaaggccgcccgcgagaagctcaagctcctcttcatcgcctgcggcaccaacgact ccctcatcggcttcggccagcgcgtgcacgagtactgcgtggccaacaacatcaaccacgtgtactggctcatccagggcggcggccacgacttcaacgtgtggaagc cgggcctctggaacttcctccagatggccgacgaggccggcctcacccgcgacggcaacaccccggtgccgaccccgtccccgaagccggccaacacccgcatcg aggccgaggacacgacggcatcaactcctcctccatcgagatcatcggcgtgccgccggagggcggccgcggcatcggctacatcacctccggcgactacctcgtg tacaagtccatcgacttcggcaacggcgccacctccttcaaggccaaggtggccaacgccaacacctccaacatcgagcttcgcctcaacggcccgaacggcaccctc atcggcaccctctccgtgaagtccaccggcgactggaacacctacgaggagcagacctgctccatctccaaegtgaccggcatcaacgacctctacctcgtgttcaagg gcccggtgaacatcgactggttcacc ttcggcgtgtag AA sequence of 13039 (SEO ID No: 106) irJaalatsqlvatragigvpdas fp-gaaqgkgarasaaadtlsputsaraaprhqhqqanrga stvvca svfstamsftparvylppgvskdJdcysylytlhgiggsend yfegggranviadnliaegki ^^^ ^ v ^ hraiagls gggqsfiíigltnldkfavigpisaapntvpperlfdggkaareidkllfiacgtndsligfgqrvheycvannin nflqiradeagltrdgntpyptpsplfantrieaedvdginsssieiigvppeggrgigvitsgdylvvksidfgn ^^ stgdwntveeqtcsiskvtgindlvivfkgpynid ftfgv * Sequence "13347 (SEO ID No: 107) atgagggtgttgctcgttgccctcgctctcctggctctcgctgcgagcgccacctccatggccgcctccctcccgaccatgccgccgtccggctacgaccaggtgcgca acggcgtgccgcgcggccaggtggtgaacatctcctacttctccaccgccaccaactccacccgcccggcccgcgtgtacctcccgccgggctactccaaggacaag aagtactccgtgctctacctcctccacggcatcggcggctccgagaacgactggttcgagggcggcggccgcgccaacgtgatcgccgacaacctcatcgccgaggg caagatcaagccgctcatcatcgtgaccccgaacaccaacgccgccggcccgggcatcgccgacggctacgagaacttcaccaaggacctcctcaactccctcatccc gtacatcgagtccaac actccgtgtacaccgaccgcgagcaccgcgccatcgccggcctctctatgggcggcggccagtccttcaacatcggcctcaccaacctcgac aagttcgcctacatcggcccgatctccgccgccccgaacacctacecgaacgagcgcctcttcccggacggcggcaaggccgcccgcgagaagctcaagctcctctt catcgcctgcggcaccaacgactccctcatcggcttcggccagcgcgtgcacgagtactgcgtggccaacaacatcaaccacgtgtactggctcatccagggcggcgg ccacgacttcaacgtgtggaagccgggcctctggaact cctccagatggccgacgaggccggcctcacccgcgacggcaacaccccggtgccgaccccgtccccg aagccggccaacacccgcatcgaggccgaggactacgacggcatcaactcctcctccatcgagatcatcggcgtgccgccggagggcggccgcgg catcggctac atcacctccggcgactacctcgtgtacaagtccatcgacttcggcaacggcgccacctccttcaaggccaaggtggccaacgccaacacctccaacatcgagcttcgcc tcaacggcccgaacggcaccctcatcggcaccctctccgtgaagtccaccggcgactggaacacctacgaggagcagacctgctccatctccaaggtgaccggcatc aacgacctctacctcgtgttcaagggcccggtgaacatcgactggttcaccttcggcgtgtccgagaaggacgaactctag Sequence 13347 (SEO ID No: 108) piryllvalapalaasatsmaaslptappsgydqvmgvfrgqvvmsyfstatostfarvylppgysk ^ gkilflii \ ^ ntnaagpgiadgvenftkdllnslipyiesnvsvytdrehraiagb ^ cgtndsligfgqprhevcvanninhvwliqggghdfovwkpglwnflqmadeagta ^ tsgdvivyksidfgngatsfkakvanantstijelrlrigpngtligtlsvkstgdwntveeqtcsiskvtgindlylvfkgpynidwftfgvsekdél * Ex emplo 53 Hydrolytic degradation of corn fiber for ferulic acid esterase Corn fiber is a major by-product of the wet milling and dry corn . The fiber component is composed, of course, of fiber arising from the pericarp (shell) of the seed and aleurone, with a smaller fraction of fine fiber coming from the endosperm cell walls. - Ferulic acid, a hydroxycinnamic acid, is found in high concentrations in the cell walls of cereal grains resulting in a cross-linking of lignin, hemicellulose and cellulose components of the cell wall. The enzymatic degradation of the crosslinking of ferulató is an important step in the hydrolysis of corn fiber that can result in the accessibility of the additional enzymatic degradation by other hydrolytic enzymes.

Acid Ferulic-Esterase Activity Assay Ferulic acid esterase, FAE-1, (synthetic gene optimized for C. thermocellum maize) was expressed in E. coli. Cells were harvested and stored at -80 ° C overnight. The bacteria harvested were suspended in 50 mM Tris buffer, pH 7.5. Lysozyme was added to a final concentration of 200 μg / mL and the sample was incubated 10 minutes at room temperature with gentle shaking. The sample was centrifuged at 4 ° C for 15 minutes at 4000 rpm. After centrifugation, the supernatant was transferred to a 50 mL conical tube, and placed in a water bath at 70 degrees Celsius for 30 minutes. The sample was then centrifuged for 15 minutes at 4000 rpm of the clarified supernatant was transferred to a conical tube (Blum et al., J. Bacteriology, March 2000, pp. 1346-1351). Recombinant FAE-1 was tested for activity using 4-methylumbelliferyl ferulate as described in Mastihubova et al., (2002) Analytical Biochemistry 309 96-101. Recombinant protein FAE-1 (104-3) was diluted 10, 100 and 1000 times and evaluated. The results of the activity test are shown in Figure 22.

Preparation of Corn Seed Fiber The thick fiber of the corn pericarp was isolated by soaking # 2 grains of yellow tooth for 48 hours at 50 ° C in sodium metabisulfite 2000 ppm (Aldrich). The grains were mixed in equal parts and mixed in a heavy-duty mixer from the Waring laboratory with the knife in inverted orientation. The mixer was controlled with a variable self-transformer (Staco Energy) at a 50% voltage output for 2 minutes. The mixed material was washed with tap water on a # 7 standard test sieve (Fisher scientific) to separate the coarse fiber from the starch fractions. The thick fiber and embryos were separated by floating the fiber of the embryos with warm tap water in a 4 L laboratory beaker (Fisher scientific). The fiber was then soaked in ethanol before drying overnight in a vacuum oven (Accuracy) at 60 ° C. The coarse corn fiber derived from the corn grain pericarp was milled with a 3100 laboratory mill equipped with a 3170 mill feeder (Perten instrumente) at a particle size of 0.5 mM.

Corn Fiber Hydrolysis Assay The coarse fiber (CF) was suspended in 10 mM citrate-phosphate buffer, pH 5.2 at 30 mg / 5 ml of buffer. The CF material was vortexed and transferred to a 40 ml modular tank (Beckman, Cat. No. 322790). The solution was mixed well then 100 μl was transferred to a 96-well plate (Corning Inc., Cat. "No. 9017, polystyrene, flat bottom), enzyme was added at 1-10 μl / well and the final volume was adjusted at 110 μl with buffer The CF bottom controls contained 10 μl of buffer only The plates were sealed with aluminum foil and incubated at 37 ° C with constant agitation for 18 hours The plates were centrifuged for 15 minutes at 4000 rpm: 1-10 μl of CF supernatant was transferred to a 96-well plate pre-filled with 100 μl of BCA reagents (BCA reagents: Reagent A (Pierce, Prod. # 23223), Reagent B (Pierce, Prod # 23224). The final volume was adjusted to 100 μl The plate was sealed with aluminum foil and placed at 85 ° C for 30 minutes After incubation at 85 ° C, the plate was centrifuged for 5 minutes at 2500 rpm. Absorbance values at 562 nm (Molecular Devices, Spectramax Plus). s were quantified with calibration curves of D-glucose and D-xylose (Sigma). The test results are reported as total sugar released.

Measurement of total sugar released Ferulic acid-esterase in Corn Seed Fiber Hydrolysis Assay The results of the hydrolysis test of fiber of Recombinant FAE-1 showed no increase in total reducing sugars (data not shown). These results were not expected since it has been reported in the literature that an increase in total reducing sugars is detectable only when other hydrolytic enzymes are used in combination with FAE (Yu et al., J. Agrie. Food Chem. 2003, 51, 218-223) Figure 23 shows that the addition of FAE-2 to a frogal supernatant that has been cultured in corn fiber, shows and increases total reducing sugars.This suggests that FAE plays an important role in hydrolysis of corn fiber Figure 23 shows the results of the hydrolysis of corn fiber showing an increase in the release of total reducing sugars of corn fiber with the addition of FAE-2 to the supernatant fangal (FS9).

Analysis of Ferulic Acid Released from Corn Seed Fiber by FAE-1 FAE activity was tested on maize seed following the release of ferulic acid as described in Walfron and Parr (1996) (Waldron, KW, Parr AJ 1996 Vol. 7 pp. 305-312 Phytochem Anal) with slight modification. The coarse corn fiber derived from the corn kernel pericarp was milled with a 3100 laboratory mill equipped with a 3170 mill feeder (Perten instruments) at a particle size of 0.5 mm, and was used as a substrate at a concentration of 10. mg / ml. One ml assays were carried out in 24-well Becton Dickenson Multiwell1 ™. The substrate was incubated in 50 mM citrate-phosphate pH 5.4 at 50 ° C at 110 rpm for 18 hours in the presence and absence of recombinant FAE. After the incubation period, the samples were centrifuged for 10 minutes at 13,000 rpm before extraction with ethyl acetate. All solvents and acids used were from Fisher Scientific. 0.8 ml of supernatant was acidified with 0.5 ml of glacial acetic acid and extracted three times with equivalent volume of ethyl acetate. The organic fractions were combined and the speed vac apparatus was subjected to dryness (Savant) at 40 ° C. The samples were then suspended with 100 μl of methanol and used for HPLC analysis. HPLC chromatography was carried out as follows. Ferulic acid (ICN Biomedicals) was used as a standard in HPLC analysis (data not shown). The HPLC analysis was carried out with a HPLC 1100 series system from Hewlett Packard. The procedure employed a completely capped inverted phase C18 column (XterraRp18, 150 mM x 3.9 mM internal diameter, particle size 5 μm) operated at 1.0 ml min "1 at 40 ° C. Ferulic acid was eluted with a gradient of 25 to 70% B in 32 minutes (solvent A: H20, 0.01% TFA, solvent B, MeCN, 0.0075%) As shown in Figure 24, the FA release of the corn fiber was 2-3 times greater than the control when treated with 10 or 100 μl of FAE-1, These results clearly show that FAE-1 is able to hydrolyze corn fiber.

Example 54 Fermentation functionality of glucoamylase and amylase expressed in corn This example demonstrates that the enzymes expressed in corn will support fermentation of starch in a thick suspension of corn in the absence of added enzyme and without cooking the corn slurry. Corn kernels containing glucoamylase from Rhizopus ozyzae (ROGA) (SEQ ID NO: 49) were produced as described in Example 32. As described in Example 46, corn kernels containing the α-amylase from pl barley (AMYI) (SEQ ID No: 88). The following materials were used in this example: Aspergillus niger glucoamylase (ANGA) was purchased from Sigma. glucoamylase of Rhizopus species (RxGA) was purchased from Wako as a dry crystalline powder and constituted in 10 mM sodium acetate, pH 5.2, 5 mM CaCl2 at 10 mg / ml. MAMYI, microbially produced AMYI was prepared at approximately 0.25 mg / ml in 10 mM sodium acetate, pH 5.2, mM CaCl2. Yeast was Saccharomyces cerviceae YE was a 5% sterile solution of yeast extract in water. The yeast initiator contained 50 g of maltodextrin, 1.5 g of yeast extract, 0.2 mg of ZnSO4 in a total volume of 300 ml of water, the medium was sterilized by autoclaving after preparation. After cooling to room temperature, 1 ml of tetracycline (10 mg / ml in ethanol), 100 μl of glucoamylase AMG300 and 155 mg of active dry yeast were added. The mixture was then stirred at 30 ° C for 22 hours. The yeast culture during the night. it was diluted 1/10 with water and A600 was measured to determine the yeast index, as described in Current Protocols in Molecular Biology. ROGA flour. The beads of several T0- lines were mixed and shown to have active glucoamylase. The seeds were ground in the Kleco and all the flour was mixed. AMYI flour. T0 corn kernels expressing AMYI were mixed and ground as before. Control flour. Grains with a similar genetic background were ground in the same way as the corn that expresses ROGA. An inoculation mixture was prepared in a sterile tube; contained by 1.65 ml: yeast cells (1 x 107), yeast extract (8.6 mg), tetracycline (55 μg). 1.65 ml / g of flour was added to each fermentation tube. Fermentation preparation: the flour was weighed at 1.8 g / tube in sterile polypropylene of 17 x 100 mm, tared. 50 μl of 0.9 M H2SO4 was added to bring the final pH before fermentation to 5. The inoculation mixture (2.1 ml) was added / tube, together with RXGA, AMYI-P and amylase misalignment buffer as indicated ahead. The amount of buffering was adjusted based on the moisture content of each flour so that the total solids content was constant in each tube. The tubes were thoroughly mixed, weighed and placed in a plastic bag and incubated at 30 ° C.

Table 21 The fermentation tubes were weighed at intervals during the course of 67 hours.

The weight loss corresponds to the evolution of C02 during fermentation. The ethanol content of the samples was determined after 67 h of fermentation by the DCL ethanol assay method. The equipment (catalog # 229-29) was purchased from Diagnostic Chemicals Limited, Charlottetown, PE, Canada, DIE 1B0. Samples (10 μl) were taken in triplicate from each fermentation tube and diluted in 990 μl of water. 10 μl of the diluted samples were mixed with 1.25 ml of a 12.5 / 1 mixture of assay buffers / ADH-NAD reagent. The standards were diluted (0, 5, 10, 15 and 20% v / v of ETOH) and evaluated in parallel. The reactions were incubated at 37 ° C for 10 minutes, then A340 was read. The standards were prepared in duplicate, samples of each fermentation were prepared in triplicate (including initial dilution). The weight of the samples changed with time as detailed in the table below. The weight loss is expressed as a percentage of the initial sample weight at time 0.

Table 22 These data show that the ROGA enzyme expressed in corn increases the fermentation rate compared to the control without enzyme. It also confirms previous data indicating that the AMYI enzyme expressed in corn kernels is a potent activator of starch fermentation in corn. The ethanol contents are detailed below.

Table 23 These data also show that the expression of glucoamylase from Rhizopus oryzae is corn facilitates the increased fermentation of starch in corn. Similarly, the expression of barley amylase in corn makes corn starch more fermentable without adding exogenous enzymes.

Example 55 Cellobiohydrolase I The cellobiohydrolase I (CBH I) gene from Trichoderma reesei was amplified and cloned by RT-PCR based on a published database sequence (access # E00389). The cDNA sequence was analyzed for the presence of a signal sequence using the SignalP program, which predicted a signal sequence of 17 amino acids. The DNA sequence encoding the signal sequence was replaced with an ATG by PCR, as shown in the sequence (SEQ ID NO: 79). This sequence of. DNC was used to make subsequent constructions. Additional constructions were made by substituting a maize optimized version of the gene (SEQ ID NO: 93).

Example 56 Cellobiohydrolase II The cellobiohydrolase II (CBH II) gene from Trichoderma reesei was amplified and cloned by RT-PCR based on a published database sequence (accession # M55080). The cDNA sequence was analyzed for the presence of a signal sequence using the SignalP program, which predicted a signal sequence of 18 amino acids. The DNA sequence encoding the signal sequence was replaced with an ATG by PCR, as shown in the sequence (SEQ ID NO: 81). This cDNA sequence was used to make subsequent constructions. Additional constructions were made by substituting an optimized corn version (SEQ ID NO: 94) of the gene. "TO .

Example 57 - Construction of transformation vectors of cellobiohydrolase I and cellobiohydrolase II of Trichoderma reeseii The cloning of cellobiohydrolase I (cbhi) cDNA of Trichoderma reeseii without the native N-terminal signal sequence is described in Example 55. constructed expression cartridges to express the cellobiohydrolase I cDNA of Trichoderma reeseii in maize endosperm with various targeting signals as follows: Plasmid 12392 comprises the cbhi cDNA of Trichoderma reeseii cloned behind the? -zein promoter for expression specifically of the endosperm for expression in the cytoplasm Plasmid 12391 comprises the N-terminal signal sequence of corn? -zein (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to the cbhi cDNA of Trichoderma reeseii as described above in the Example 1 for targeting the endoplasmic reticulum and secretion in the apoplast (Torrent et al., 1997) The fusion was cloned back of the? -zein promoter for expression specifically in the endosperm. Plasmid 12392 comprises the N-terminal signal sequence of? -zein fused to the cbhi cDNA of Trichoderma reeseii with a C-terminal addition of the KDEL sequence for the a direction and retention in the endoplasmic reticulum (ER) (Munro and Pelham, 1987). The fusion was cloned behind the corn? -zein promoter for expression specifically of the endosperm. Plasmid 12656 comprises the waxy amylloplast targeting peptide (Klosgen et al., 1986) fused to the cbhi cDNA of Trichoderma reeseii for the a-1 amyloplast address. The fusion was cloned behind the corn? -zein promoter for expression specifically of the endosperm. All expression cartridges were moved to a binary vector (pNOV2117) for transformation into maize by Agrobacterium infection. The binary vector contained the phosphomannanase-isomerase (PMI) gene that allows the selection of transgenic cells with mannose. The transformed corn plants either self-pollinated or crossed and seeds were collected for analysis. Additional constructs (plasmids 12652, 12653, 12654 and 12655) with the address signals described above fused to the cellobiohydrolase II (cbhii) cDNA of Trichoderma reeseii were made in precisely the same manner as described for the cbhi cDNA of Trichoderma reeseii. These fusions were cloned after the corn Q protein promoter (50 kd? -zein) (SEQ ID NO: 98) for expression specifically in the endosperm and transformed into corn as described above. The transformed corn plants either self-pollinated or crossed and the seeds were collected for analysis. Enzyme combinations can be produced either by crossing plants expressing the individual enzymes or by cloning several expression cartridges in the same binary vector to allow co-transformation.

Example 58 Expression of a Cbhi in corn Self-pollinated maize plant seeds transformed either with the plasmid 12390, 12391 or 12932 were obtained. Construction 12390 directs the expression of the Cbhl in the endoplasmic reticulum of the endosperm, construction 12391 directs the expression of Cbhl in the endosperm apoplast and construction 12392 directs the expression of Cbhl in the cytoplasm of the endosperm.

Extraction and detection of corn flour Cbhl: polyclonal antibodies were produced to Cbhl and Cbhll in goat according to established protocols. The flour of the transgenic Cbhl seeds was obtained by grinding them in an Autogizer grinder. Approximately 50 mg of flour was suspended in 0.5 ml of 20 mM NaP04 buffer (pH 7.4), 150 mM NaCl followed by incubation for 15 minutes at room temperature with continuous agitation. The incubated mixture was then centrifuged for 10 minutes at 10,000 xg. The supernatant was used as an enzyme source. 30 μl of this extract was loaded on a 4-12% NuPAGE gel (invitrogen) and separated in the buffer of NuPAGE MES run. Proteins are transferred into nitrocellulose membranes and Western blot analysis was performed following established protocols using the specific antibodies described above followed by goat anti-goat (H + L) IgG conjugated to alkaline phosphatase. Alkaline phosphatase activity was detected by incubation of the membranes with BCIP / MBT (plus) ready-to-use substrate from Moss Inc. Western blot analysis was performed on the IT seeds of different events transformed with the plasmid 12390. The Cbhl protein expression to non-transgenic control, and was detected in several events. The broken corn test was performed essentially as described in Example 49, using transgenic seeds expressing Cbhl. The starch recovery of the transgenic seeds was measured and the results are shown in the Table, 24.

Table 24 Conditions Control that does not express Starch expresses line 3 CBHI of line 4 (mg) 400ppm of S02-No 40.2 78.1 Bromelain - _- 400ppm of S02-Plus 48.1 118.7 Bromelain 2000ppm of S02-No 47.5 73.1 Bromelain 2000ppm of S02-Plus 49.2 109 Bromelain Example 59 Preparation of Endoglucanase Constructs I The endoglucanase I gene from Trichoderma reesei (EGLI) was amplified and cloned by PCR based on a published database sequence (access # M15665; Penttila et al., 1986). Because only genomic sequences can be obtained, the cDNA was generated from the genomic sequence by removing 2 introns using overlap PCR. The resulting cDNA sequence was analyzed for the presence of a signal sequence using the SignalP program, which predicted a signal sequence of 22 amino acids. The DNA sequence encoding the signal sequence was replaced with an ATG by PCR, as shown in the sequence (SEQ ID NO: 83). This cDNA sequence was used to make subsequent constructions as discussed below.

Overlap PCR Overlap PCR is a technique (Ho et al., 1989) used to fuse complementary ends of two or more PCR products, and can be used to make basess pair (bp) changes, to add bp, "or delete bp. At the site of the proposed change in bp, forward and inverted mutagenic primers (Mut-F and Mut-R) containing the proposed change and 15 bp of the sequence are made on either side of the change. to remove an intron, the primers will consist of the final 15 bp of exon 1 fused to the first 15 bp of exon 2. Primers are also prepared which are fixed to the ends of the sequence to be amplified, for example, primers of codon ATG and STOP PCR amplification of the products proceeds with the pair of ATG / Mut-R primers and the pair of Mut-F / STOP primers in separate reactions.The products are gel purified and fused together in a PCR without additional primers The fus reaction The ion is separated in a gel, and the band of the correct size is gel purified and cloned. Multiple changes can be achieved simultaneously through the addition of additional mutagenic primer pairs.

Expression Constructions in EGLI Plant Expression cartridges were made to express EGLI cDNA from Trichoderma resei in maize endosperm as follows: 13025 comprises the T. reesei EGLI gene cloned behind the corn? -zein promoter for cytoplasmic localization and expression specifically in the endosperm. 13026 comprises the N-terminal signal peptide of corn? -zein (MRVLLVALALLALAASATS) fused to the EGLI gene of T. reesei for targeting the endoplasmic reticulum and secretion in the apoplast. The fusion was cloned behind the? -zein promoter of corn for expression specifically in the endosperm. 13027 comprises the N-terminal signal peptide of corn? -zein fused to the E. Glygene of T. reesei with a C-terminal addition of the KDEL sequence for targeting and retention in the endoplasmic reticulum. The fusion was cloned behind the corn? -zein promoter for specifically expression of the endosperm. "13028 comprises the N-terminal signal peptide of Granule-bound Starch-I Synthase (GBSSI) (N-terminal 77 amino acids) fused to T. reesei EGLI gene for targeting the amyloplast lumen The fusion was cloned behind the corn? -zein promoter for specifically expression of the endosperm 13029 comprises the N-terminal signal peptide of maize GBSSI fused to the gene of T. reesei EGLI with a C-terminal addition of the starch binding domain (C-terminal 301. amino acids) of the maize GBSSI gene for targeting the starch granule.The fusion was cloned behind the? -zein promoter of corn for expression specifically in the endosperm Additional expression cartridges were generated using an optimized corn version of EGLI (SEQ ID NO: 95) EGLI Enzyme Assays The activity of the EGLI enzyme in transgenic maize is measured using the Beta-Glucanase Assay Kit from Malta (Cat # K-MBGL) (Megazyme International Ireland Ltd). The enzymatic activity of the EGLI expressors is tested in the corn fiber hydrolysis assay as described in Example 53.

Example 60 β-Glucosidase 2 The β-glucosidase 2 (BGL2) gene from Trichoderma reesei was amplified and cloned by RT-PCR based on the sequence with accession number AB003110 (Takashima et al., 1999).

Expression Constructs of BGL2 Plant Expression cartridges were made to express the BGL2 cDNA of Trichoderma reesei (SEQ ID '"' NO: 89) in maize endosperm as follows: 13030 comprises the T. reesei gene BGL2 cloned behind the corn? -zein promoter for cytoplasmic location and expression specifically in the endosperm 13031 comprises the N-terminal signal peptide of corn? -zein (MRVLLVALALLALAASATS) fused to the BGL2 gene of T. reesei for targeting the endoplasmic reticulum and secretion in the apoplast The fusion was cloned behind the corn? -zein promoter for expression specifically in the endosperm 13032 comprises the N-terminal signal peptide of? -zein fused to the BGL2 gene of T. reesei with an addition C-terminal sequence of the KDEL for targeting and retention in the endoplasmic reticulum The fusion was cloned behind the corn? -zein promoter for expression specifically in the endosper M. 13033 comprises the N-terminal signal peptide of Granule-bound Starch-Synthase I (GBSSI) (N-terminus, 77 amino acids) fused to the T. reesei BGL2 gene for direction to the aminoplast lumen. The fusion was cloned behind the? -zein promoter of corn for expression specifically in the endosperm. 13034 comprises the N-terminal signal peptide of GBSSI maize fused to the BGL2 gene of T. reesei with a C-terminal addition of the starch binding domain (C-terminal, 301 amino acids) of the maize GBSSI gene for the address to the starch granule. The fusion was cloned behind the? -zein promoter of corn for expression specifically in the endosperm. Additional expression cartridges were generated by substituting a corn-optimized version of BGL2 (SEQ ID NO: 96). All expression cartridges are inserted into the binary vector pNOV2117 for transformation into maize by Agrobacterium infection. The binary vector contained the phosphomannanase-isomerase (PMI) gene that allows the selection of transgenic cells with mannose. The transformed corn plants either self-pollinated or crossed and the seeds were collected for analysis.

BGL2 Enzyme Assays BGL2 enzyme activity was measured in transgenic maize using a modified Bauer and Kelly protocol (Bauer, MW and Kelly, RM 1998. The family 1 ß-glucosidases from Pyrococcus - furiosus and Agrobacterium faecalis share a co mon catalytic mechanism, Biochemistry 37: 17170-17178). The protocol can be modified to incubate samples at 37 ° C instead of 100 ° C. The enzymatic activity of the BGL2 expressors is tested in the fiber hydrolysis assay.

Example 61 ß-Glucosidase D The g of ß-Glucosidase D (CEL3D) from Trichoderma reesei was amplified and cloned by PCR based on the published database sequence (access # AY281378: Foreman et al., 2003). Because only genomic sequences can be obtained, the cDNA was generated from the genomic sequence by removing an intron using Traslape PCR, as described in Example 58. The resulting ADnc (SEQ ID NO: 91) was used for subsequent constructions . An optimized version for maize (SEQ ID NO: 97) of the resulting cDNA can also be used for constructions.

Plant constructs can be generated and ß-glucosidase assays can be performed as described for BGL2 in Example 60, replacing BGL2 with CEL3D.

EXAMPLE 62 Lipases cDNAs encoding lipases were generated using Access Number-sequences D85895, AF04488: AF04489 (Tsuchiya et al., 1996; Yu et al., 2003) and the methodology set forth in Examples 59-60. Measure the activity of the lipase enzyme in transgenic maize using the Fluorescent Lipase Test Kit (Cat # M0612) (Marker Gene Technologies, Inc.). Lipase activity can also be measured in vivo using the fluorescent substrate 1,2-dioleoyl-3- (piren-1-yl) decanoyl-rac-glycerol (M0258), also from Marker Gene Technologies, Inc.

Example 63 Expression of Phytase in Rice Vectors 11267 and 11268 comprise binary vectors encoding Nov9x phytase. The expression of the phytase gene Nov9x in both vectors is under the control of the rice glutelin-1 promoter (SEQ ID NO: 67). Vectors 11267 and 11268 are derived from pN0V2117. The Nov9x phytase expression cassette in vector 11267 comprises the rice glutelin-1 promoter, the Nov9x phytase gene with the apoplast targeting signal, a PEPC intron, and the 35S terminator. The product of the Nov9x phytase coding sequence in vector 11267 is shown in SEQ ID NO: 110. The Nov9x phytase expression cassette in vector 11268 comprises the rice glutelin-1 promoter, the Nov9x phytase gene with retention in ER (SEQ ID NO: 111), an intron of PEPC and the 35S terminator. The product of the phytase coding sequence Nov9x in vector 11268 shows a SEQ ID NO: 112.

Phytase Nov9x 11267 with apoplasto-targeting DNA sequence (SEQ ID NO: 109). The translation start and finisher codons are underlined. The sequence coding for the signal sequence of the gamma-zein protein of 27-kD-is in bold. atgagggtgttgctcgttgccctcgctctcctggctctcgctgcgagcgccaccagcgctgcgcagtccgagccggagctgaagctgg agtccgtggtgatcgtgtcccgccacggcgígcgcgccccgaccaaggccacccagctcatgcaggacgtgaccccggacgcctggcc gacctggccggtgaagctcggcgagctgaccccgcgcggcggcgagctgatcgcctacctcggccactactggcgccagcgcctcgtg gccgacggcctcctcccgaagtgcggctgcccgcagtccggccaggtggccatcatcgccgacgtggacgagcgcacccgcaagacc ggcgaggccttcgccgccggcctcgccccggactgcgccatcaccgtgcacacccaggccgacacctcctccccggacccgctcttcaa cccgctcaagaccggcgtgtgccagctcgacaacgccaacgtgaccgacgccatcctggagcgcgccggcggctccatcgccgacttc accggccactaccagaccgccttccgcgagctggagcgcgtgctcaacttcccgcagtccaaccíctgcctcaagcgcgagaagcagga cgagtcctgctccctcacccaggccctcccgtccgagctgaaggtgíccgccgactgcgtgtccctcaccggcgccgtgtccctcgcctcc atgctcaccgaaatcttcctcctccagcaggcccagggcatgccggagccgggctggggccgcatcaccgactcccaccagtggaacac cctcctctccctccacaacgcccagttcgacctcctccagcgcaccccggaggtggcccgctcccgcgccaccccgctcctcgacctcaíc aagaccgccctcaccccgcacccgccgcagaagcaggcctacggcgtgaccctcccgacctccgtgctcttcatcgccggccacgacac caacctcg ccaacctcggcggcgccctggagctgaactggaccctcccgggccagccggacaacaccccgccgggcggcgagctggt gttcgagcgctggcgccgcctctccgacaactcccagtggattcaggtgtccctcgtgttccagaccctccagcagatgcgcgacaagacc ccgctctccctcaacaccccgccgggcgaggtgaagctcaccctcgccggctgcgaggagcgcaacgcccagggcatgtgctccctcg ccggcttcacccagatcgtgaacgaggcccgcatcccggcctgcíccctctaa Fitaa Nov9x 11267 with apoplasto targeting gene product (SEQ ID NO: 110). The sequence of the gamma -zei.na protein of 27 kD is in bold. mrvllvalallalaasatsaaqsepelkleswivsrhgvraptkatqlmqdvtpdawptwpvklgeltprggeliaylghy rqrlva dgllpkcgcpqsgqvaiiadvdertrktgeafaaglapdcaitvhtqadtsspdplfhplktgvcqldnanvtdaileraggsiadftghy qtafrelervin qsnlclkrekqdescsltqalpselkvsadcvsltgavslasmlteifllqqaqgmpepg gritds qwntllslhn aqfdllqrtpevarsratplldliktakphppqkqaygvtlptsvlfíaghdtnlanlggalelnwtlpgqpdntppggelvferwrrlsdn sqwiqvslvfqtlqqmrdktplslntppgevkltlagceernaqgmcslagftqivnearipacsl Phytase Nov9x 11268 with DNA sequence with retention in ER (SEQ ID NO: 111). The sequence coding for the signal sequence of the 27-kD gamma-zein protein is in bold. The sequence encoding the ER retention signal of the SEKDEL hexapeptide is underlined. at8aggSt8tt8ctcgttgccctcgctctcctggctctcgctgcgagcgccaccagcgctgcgcagtccgagccggagctgaagctgg agtccgtggtgatcgtgtcccgccacggcgtgcgcgccccgaccaaggccacccagctcaígcaggacgtgaccccggacgcctggcc gacctggccggtgaagctcggcgagctgaccccgcgcggcggcgagctgatcgcctacctcggccactactggcgccagcgcctcgtg gccgacggcctcctcccgaagtgcggctgcccgcagtccggccaggtggccatcatcgccgacgtggacgagcgcacccgcaagacc ggcgaggccttcgccgccggcctcgccccggactgcgccatcaccgtgcacacccaggccgacacctcctccccggacccgctcttcaa cccgctcaagaccggcgtgtgccagctcgacaacgccaacgtgaccgacgccatcctggagcgcgccggcggctccatcgccgacttc accggccactaccagaccgcctíccgcgagctggagcgcgtgctcaacttcccgcagtccaacctctgcctcaagcgcgagaagcagga cgagtcctgctccctcacccaggccctcccgtccgagctgaaggtgtccgccgactgcgtgtccctcaccggcgccgtgtccctcgcctcc atgctcaccgaaatcttcctcctccagcaggcccagggcatgccggagccgggctggggccgcatcaccgactcccaccagtggaacac cctcctctccctccacaacgcccagttcgacctcctccagcgcaccccggaggtggcccgctcccgcgccaccccgctcctcgacctcatc aagaccgccctcaccccgcacccgccgcagaagcaggcctacggcgtgaccctcccgacctccgtgctcttcatcgccggccacgacac caacctcgcc aacctcggcggcgccctggagctgaactggaccctcccgggccagccggacaacaccccgccgggcggcgagctggt gttcgagcgctggcgccgcctctccgacaactcccagtggattcaggtgtccctcgtgttccagaccctccagcagatgcgcgacaagacc ccgctctccctcaacaccccgccgggcgaggtgaagctcaccctcgccggctgcgaggagcgcaacgcccagggcatgtgctccctcg ccggcttcacccagatcgtgaacgaggcccgcatcccggcctgctccctctccgagaaggacgagctgtaa Fitaa Nov9x 11268 with ER retention gene product, (SEQ ID NO: 112). The signal sequence of the 27-kD gamma-zein protein is in bold. The retention signal in ER is underlined. mrvllvalallalaasatsaaqsepelkleswivsrhgvraptkatqlmqdvtpdawptwpvklgeltprggeliaylghywrqrlva dgllpkcgcpqsgqvaiiadvdertrktgeafaaglapdcaitvhtqadtsspdplf plktgvcqldnanvtdaileraggsiadftghy qtafrelervln qsnlclkjekqdescsltqalpselkvsadcvsltgavslasmlteifllqqaqgmpepgwgritdshqwntllslhn aqfdllqrtpevarsratplldliktaltphppqkqaygvtlptsvlfiaghdtnlanlggalelnwtlpgqpdntppggelvfenvrrlsdn dei sqwiqvslvfqtlqqmrdktplslntppgevkltlagceernaqgmcslagftqivnearipacslsd Generation of transgenic rice plants Rice (Oryza sativa) is used to generate transgenic plants. Several varieties of rice can be used (Hiei et al., 1994, Plant Journal 6: 271-282, Dong et al., 1996, Molecular "Breeding 2: 267-276, Hiei et al., 1997, Plant Molecular Biology, 35: 205-218.) Also, the various constituents of the media described below can either be varied in concentration or can be substituted.Embryogenic responses are initiated and / or cultures are established from mature embryos when culturing in the medium MS-CIM (basal salts MS, 4.3 g / liter, vitamins B5 (200 x), 5 ml / liter, Sucrose, 30 g / liter, proline, 50 mg / liter, glutamine, 500 mg / liter, casein hydrolyzate, 300 mg / liter, 2,4-D (1 mg / ml), 2 ml / liter, adjust pH to 5 pH with 1 N KOH, Phytagel, 3 g / liter.) Either mature embryos in the early stages of the culture response or established cultivation lines are inoculated and co-cultivated with the Agrobacterium strain LBA4404 containing the desired vector construct.Agrobacterium is grown from glycerol materials in the YPC solid medium (100 mg / L spectinomycin and any other appropriate antibiotic) for approximately 2 days at 28 ° C. The Agrobacterium is re-dispersed in the liquid MS-CIM medium. The Agrobacterium culture is diluted to an OD600 of 0.2-0.3 and the acetosyringone is added to a final concentration of 200 uM. Agrobacterium is induced with acetosyringone before mixing the solution with the rice cultures. For inoculation, the cultures are immersed in the bacterial suspension. The liquid bacterial suspension is removed and the inoculated cultures are placed in the co-culture medium and incubated at 22 ° C for 2 days. The cultures are then transferred to the MS-CIM medium with Ticarcillin (400 mg / liter) to inhibit the growth of Agrobacterium. For constructs using the PMI selectable marker gene (Reed et al., In Vitro Cell, Dev. Biol. -Plant 37: 127-132), the cultures are transferred to the Mannosa-containing selection medium as a carbohydrate source ( MS with 2% Mannose, 300 mg / liter of Ticarcillin) after 7 days, and are grown for 3-4 weeks in the dark. Then the resistant colonies are transferred for regeneration of the induction medium (MS without 2,4-D, 0.5 mg / liter of IAA, 1 mg / liter of zeatin, 200 mg / liter of Ticarcillin, 2% of Mannose and 3% of Sorbitol) and are grown in the dark for 14 days. Then proliferating colonies are transferred another round of regeneration induction medium and moved to the light growing room. Regenerated shoots are transferred to medium GA7-1 (MS without hormones and 2% Sorbitol) for 2 weeks and then moved to the greenhouse where they are sufficiently large and have adequate roots. The plants are transplanted to the soil in the greenhouse and grown to maturity.

Example 64 Analysis of Transgenic Rice Seeds Expressing Fitasa Nov9x ELISA for the Quantification of Fitasa Nov9x Seeds Rice The quantification of phytase expressed in seeds of transgenic rice was evaluated by ELISA. A rice seed (1 g) was milled for flour in a Kleco seed mill. 50 mg of flour was suspended in the sodium acetate buffer described in the example - for the Nov9X phytase activity assay and diluted as required for the immunoassay. The Nov9X immunoassay is a quantitative intercalation assay for the detection of phytase - which employs two polyclonal antibodies. The rabbit antibody was purified using protein A, and the goat antibody was purified by immunoaffinity against recombinant phytase protein (Nov9x) produced in E. coli inclusion bodies. Using these highly specific antibodies, the assay can measure picogram levels of phytase in transgenic plants. There are three basic parts to the essay. The phytase protein in the example is captured in the solid phase microtiter cavity using the rabbit antibody. Then the solid-phase antibody, the phytase protein, and the secondary antibody that has been added to the cavity are interspersed. After a washing step, where unbound secondary antibody has been removed, the bound antibody is detected using an alkaline phosphatase-labeled antibody. The substrate for the enzyme is added and the color development is measured by reading the absorbance of each cavity. The normal curve uses an adjustment to the four parameter curve to plot the concentrations versus absorbance.

Phytase Activity Assay The determination of phytase activity, based on the estimation of inorganic phosphate released in the hydrolysis of phytic acid, can be carried out at 37 ° C following the method of Engelen, AJ et al., J. AOAC , inter. , 8, 629 (2001). One unit of enzymatic activity is referred to as the amount of enzyme that releases 1 μmol of inorganic phosphate per minute under test conditions. For example, the activity of phytase can be measured by incubating 2.0 ml of the enzyme preparation with 4.0 ml of 9.1 mM sodium phytase in 250 mM sodium acetate buffer, pH . 5, supplemented with 1 mM CaC12 for 60 minutes at 37 ° C.

After incubation, the reaction stops when adding 4. 0 ml of a color stopping reagent consisting of equal parts of 10% ammonium molybdate (w / w) and a concentrated solution of 0.235% ammonium vanadate (w / w). The precipitate is removed by centrifugation, and the released phosphate is measured against a set of phosphate standards spectrophotometrically at 415 nm. The phytase activity is calculated by interpolating the absorbance values of A415 nm obtained for samples containing phytase using the normal phosphate curve generated. This procedure can be scaled down to adjust smaller volumes and adapt to preferred containers. Preferred containers include glass test tubes and plastic microplates. The partial immersion of the reaction vessels in a water bath is essential to keep the temperature constant during the reaction of the enzymes.

Table 24 * μg of phytase was assessed by an intercalation ELISA ** Phytase activity was assessed by 'phytase activity assay as described above.

Inorganic Phosphate Release Assay During Firing of Transgenic Rice Expressing Phytase Two samples of μg seed of selected transgenic rice lines and a wild type control line were dehusked using a Kett TR200 bank automatic rice shelling machine. Then a sample was ground for 30 seconds in a Kett Rice burnisher. Two volumes of H20 were added to each sample and the rice was cooked by submerging the tubes in a laboratory beaker with water. The water was boiled and kept in full boil for 10 minutes. The "cooked" rice seed was then ground to a paste with water bringing the total volume of the slurry to 6 ml. The slurry was centrifuged at 15, 000 xg for 10 minutes and the clear supernatant was evaluated for the endogenous inorganic phosphate released. The released phosphate test was based on the color form as a result of molybdate and vanadate ions returned complexed with inorganic phosphate and measured spectrophotometrically at 415 mm as described in the example, for phytase enzyme activity. The results are given in Table 2. All publications, patents and patent applications are incorporated herein by reference. While in the above specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is amenable to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention. It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention.

Claims (31)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. Isolated polynucleotide, characterized in that a) comprises SEQ ID Nos: 61, 63, 65, 69, 73, 74, 75, 76, 77 , 78, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 101, 103, 105, 107, 109 or 111, or the complement thereof, or "" a polynucleotide that hybridizes to the complement of any of "SEQ ID NO: 61, 63, 65, 73, 74, 75, 76, 77, 78, 79, 81, 10 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 101, 103, 105, 107, 109 or 111, under conditions of low stringency hybridization and encodes a polypeptide having cellulose activity, hemicellulase, exo-1, 4- β-cellobiohydrolase, exo- 1, 3-β-D-glucanase, β-glucosidase, endoglucanase, L-arabinase, -JC a-arabinosidase, galactanasa, galactosidase, mannanase, mannosidase, xylanase, xylosidase, protease, glucanase, or esterase, or b) which codes for a polypeptide comprising SEQ ID NO: 62, 64, 66, 70, 80, 82 , 84, 86, 88, 90, 92, 100, 102, 104, 106, 108, 110 or 112, or an enzymatically active fragment thereof.
2. 2. An isolated polynucleotide according to claim 1, characterized in that the polynucleotide encodes a fusion polypeptide comprising a first polypeptide and a second peptide, wherein the first polypeptide has cellulase, hemicellulase, exo-1,4 activity. β-cellobiohydrolase, exo-1,3-β-D-glucanase, β-glucosidase, endoglucanase, L-arabinase, α-arabinosidase, galactanase, galactosidase, mannanase, mannosidase, xylanase, xylosidase, protease, glucanase or esterase.
3. 3. An isolated polypeptide according to claim 2, characterized in that the second peptide comprises a peptide of a directing sequence.
4. 4. An isolated polypeptide according to claim 3, characterized in that the targeting peptide directs the first polypeptide to a vacuole, endoplasmic reticulum, chloroplast, starch granule or cell wall of a plant.
5. 5. Isolated polynucleotide according to claim 3, characterized in that the targeting sequence is a Waxy N-terminal signal sequence, a N-terminal signal sequence of? -zein, a starch binding domain, or a domain of binding to C-terminal starch.
6. 6. An expression cassette, characterized in that it comprises the isolated polynucleotide according to claim 1.
7. 7. An expression cassette according to claim 6, characterized in that it is operably linked to a promoter.
8. 8. Cassette of expression according to claim 7, characterized in that the promoter is an inducible, tissue-specific, constitutive promoter of specific endosperm.
9. 9. The expression cassette according to claim 8, characterized in that the specific endosperm promoter is a promoter of corn? -zein, rice glutenin-1, or maize ADP-gpp.
10. 10. An expression cassette according to claim 9, characterized in that the corn? -zein promoter is a? -zein promoter of 27 kD or 55 kD.
11. 11. Cassette of expression according to claim 9, characterized in that the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12.
12. 12. Cassette of expression according to claim 6, characterized in that the polynucleotide is oriented in the orientation of transcribed strand in relation to the promoter: - 13.
13. Expression cassette according to claim 6, characterized in that the - polynucleotide further encodes a targeting sequence that is operably linked to the polypeptide encoded by the polynucleotide.
14. An expression according to claim 13, characterized in that the polypeptide is operably linked to a vacuole, endoplasmic reticulum, chloroplast, starch granule or cell wall of a plant
15. 15. Expression cassette according to claim 1 , characterized in that the direction sequence is a signal sequence N-terminal Waxy, a N-terminal signal sequence of? -zein or a starch binding domain.
16. 16. Cell, characterized in that it comprises the expression cassette according to claim 6.
17. 17. Plant, or part thereof, characterized in that it comprises the cell according to claim 16.
18. 18. Cell according to claim 16, characterized in that it is selected from the group consisting of an Agrobact erium, a monocotyledonous cell, a dicotyledonous cell, a Liliopsida cell, a Panicoideae cell, a maize cell or a cereal cell.
19. 19. Seed, fruit, stem, leaf, stem, or grain, characterized in that it is of the plant according to claim 17.
20. 20. Plant according to claim 1, characterized in that it is sugar beet, cane sugar, oats, barley, wheat, rye, corn or rice.
21. 21. Method for preparing fermentable sugar, monosaccharide or oligosaccharide, characterized in that it comprises the steps of treating a part of transgenic monocot plant that comprises a cellulase enzyme, hemicellulase, exo-1, 4-b-cellobiohydrolase, exo-1, 3-bD -glucanase, b-glucosidase, endoglucanase, L-arabinase, α-arabinosidase, galactanase, galactosidase, mannanase, mannosidase, xylanase, xylosidase, protease, glucanase or stearase under conditions to activate the enzyme, thereby digesting the polysaccharide to form fermentable sugar or oligosaccharide, wherein the part of plant a transformed monocotyledonous plant is obtained, the genome of which is augmented with an expression cassette comprising a promoter operably linked to a polynucleotide encoding the enzyme and a targeting sequence.
22. 22. Method for preparing fermentable sugar, monosaccharide or oligosaccharide according to claim 21, characterized in that the promoter is an inducible promoter, of specific tissue, of specific or constitutive endosperm.
23. 23. Method for preparing fermentable sugar, monosaccharide or oligosaccharide according to claim 22, characterized in that the specific endosperm promoter is a promoter of corn? -zein, rice glutenin-1, or corn ADP-gpp.
24. 24. Cassette of expression according to claim 23, characterized in that the promoter of maize? -zain is a gamma-zein promoter of 27 kD or 55 kD.
25. 25. Method for preparing fermentable sugar, monosaccharide or oligosaccharide according to claim 23, characterized in that the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12.
26. 26. Method for preparing fermentable sugar, monosaccharide or oligosaccharide in accordance with claim 21, characterized in that the plant part is a seed, fruit, trunk, leaf, stem or grain.
27. 27. Method for preparing fermentable sugar, monosaccharide or oligosaccharide according to claim 21, characterized in that the plant part is obtained from sugar beet, sugar cane, oats, barley, wheat, rye, corn or rice.
28. 28. Method for preparing fermentable sugar, monosaccharide or oligosaccharide according to claim 21, characterized in that the treatment comprises heating the plant part for a quantity of time and under conditions to activate the enzyme, thereby digesting the polysaccharide to form monosaccharide , oligosaccharide or fermentable sugar.
29. 29. Method for preparing fermentable sugar, monosaccharide or oligosaccharide according to claim 21, characterized in that the enzyme is encoded by the polynucleotide sequence comprising SEQ ID NOs: 61, 63, 65, 69, 73, 74, 75, 76 , 77, 78, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 101, 103, 105, 107, 109 or 111.
30. 30. Method for preparing fermentable sugar , monosaccharide or oligosaccharide according to claim 21, characterized in that the polynucleotide sequence encodes a polypeptide comprising SEQ ID NO: 62, 64, 66, 70, 80, 82, 84, 86, 88, 90, 92, 100 , 102, 104, 106, 108, 110 or 112.
31. 31. The method according to claim 21, characterized in that it further comprises the step of incubating the monosaccharide, oligosaccharide, or fermentable sugar under conditions that promote the conversion of the oligosaccharide or sugar fermentable in ethanol.