Autoprocessing plants and parts of plant

FIELD: molecular biology, genetic engineering, biochemistry.

SUBSTANCE: invention relates to polynucleotides optimized for expression in plants and encoding processing enzymes. Polynucleotides encode mesophilic, thermophilic or hyperthermophilic enzymes that are activated under conditions suitable for interaction with a necessary substrate. Incorporation of these polynucleotides into the plant genome results to preparing "self-processing" transgenic plants wherein their parts, for example, grain, fruit or seed express one or more indicated enzymes and have the varied composition. Autoprocessing plants can be used, for example, for preparing foodstuffs eliciting improved taste.

EFFECT: improved and valuable biological properties of plants.

29 cl, 23 dwg, 6 tbl, 41 ex

 

Related applications

In the present application claims the priority of application no. No. 60/215281, filed August 27, 2001, which introduced into the present description by reference.

The scope to which the invention relates.

In General terms, the present invention relates to molecular biology of plants, and more specifically to the creation of plants that Express processrule enzyme, which imparts the desired properties to the plant or its products.

Prior art

Enzymes are used for processing of some agricultural products, such as timber, fruits and vegetables, starches, juices etc. Usually processorsa enzymes produce and produce from a variety of sources on an industrial scale, for example, by microbial fermentation (α-amylase Bacillus) or selection of plants (using β-galactosidase coffee or papain from parts of the plant). Enzyme preparations used in various machining techniques by mixing enzyme and substrate at a corresponding humidity, temperature and in a certain period of time and mechanical agitation in order to implement the enzymatic reaction on an industrial scale. Methods include a separate stage of production of the enzyme, the production of enzyme preparation, mixing of enzyme and substrate and placing the mixture received in suitable conditions, stimulating the passage of the enzymatic reaction. Thus a suitable and preferred is a method, which allows you to save time and energy, to reduce the stage of mixing, reduce financial costs and/or cost of production of enzymes or to improve the results or to obtain new products. One example of a procedure that needs improvement is the grinding of corn.

Currently, corn grind with obtaining corn starch and other by-products generated during the grinding of corn, such as corn gluten product, corn gluten meal or corn oil. The starch obtained in this way is often subjected to further processing with other products, such as derivationally starches and sugar, or fermentation to produce different products, including alcohol or lactic acid. Treatment of corn starch often involves the use of enzymes, in particular enzymes that break down starch and turn it into formatiruem sugar or fructose (αand glucoamylase, α-glucosidase, glucose and the like). Modern industrial methods require large finansowych costs since the implementation of the processing of corn on an industrial scale it is necessary to design is the formation of very large mills, that does not meet acceptable economic terms of "cost - effectiveness". Furthermore, the method requires a separate production gidroliznaya or modifying starch enzymes with subsequent use of the equipment for mixing this enzyme and substrate in order to produce products of hydrolyzed starch.

The method of producing starch from corn grain is well known and includes the process of wet grinding. Wet corn milling stage includes soaking corn grain, grinding it corn grain and separating components of the grain. The grain is soaked in the tub for soaking with a reverse flow of water at about 120°F, and the grain left in the tub to soak for 24-48 hours. The water in the tub for soaking typically contains sulfur dioxide at a concentration of about 0.2 wt.%. In this method, sulfur dioxide is used to reduce microbial growth, and to restore the disulfide bonds in the proteins of the endosperm in order to better separation of the starch from the protein. Usually one bushel of corn used approximately 0.59 gallons of water for soaking. Water for soaking goes to waste and often contain undesirable levels of residual sulfur dioxide.

Then soaked the grain dehydrated and subjected to grinding in a series of fine grinding mills. In PE the howl of a series of mills, fine grinding is the grinding of grain, resulting from this grain is released germ. Industrial mill fine grinding, suitable for large-scale wet grinding, available commercially under the trade mark Bauer. To separate the germ from the rest of the grain using centrifugation. Widely used industrial centrifuge separator is a centrifugal separator Merco. Mill fine grinding and centrifugal separators are expensive equipment, which consumes a lot of energy.

In the next stage of this process, the remaining components of the grain, including starch, husk, fiber, and gluten, upload to another series of fine grinding mills and passed through a series of wash sieves for separating fibrous components from the starch and gluten (a protein of the endosperm). The starch and gluten to pass through sieves, and the fiber does not pass through the sieve. Centrifugation or third stage crushing followed by centrifugation is used to separate the starch from the protein of the endosperm. In the centrifugation is produced by a starch suspension, which dehydrate, and then washed with fresh water and dried before reaching about 12% moisture. Mostly pure starch is usually subjected to further treatment using enzymes.

Separation of starch from other components the grain is carried out in order to remove the peel of seeds and proteins of the embryo and endosperm, what contributes to effective communication starch with processormask enzymes, and so the resulting products of hydrolysis, generally, do not contain impurities other components of the grain. This separation allows you to effectively allocate other components of the grain, which can then be marketed as an auxiliary products to increase income from the use of the mill.

After the separation of the starch in the wet grinding the starch is usually subjected to such stages of processing, as gelatinization, liquefaction and dextrinization, in order to produce maltodextrin, and subsequent saccharification stages, isomerization and purification in order to produce glucose, maltose and fructose.

The gelatinization is used in the hydrolysis of starch, because currently available enzymes are not able to quickly hydrolyze crystalline starch. In order to make the starch accessible to hydrolytic enzymes, the starch is usually prepared in the form of an aqueous suspension (20-40% solids) and heated at a suitable temperature gelation. For corn starch temperature is 105-110°C. Gelatinizing starch is usually very viscous, and therefore it is diluted in the next stage, called liquefaction. When liquefaction some connection between lucassie starch molecules are broken and this is either under the action of the enzyme, either under the action of acid. In this stage and in subsequent stages of dextrinization used thermostable enzymes endo-α-amylase. The degree of hydrolysis is regulated under dextrinization, resulting receive the products of hydrolysis to the desired percentage of dextrose.

Subsequent hydrolysis dekstrinovym products after stage liquefaction carried out using different ectasias and deletada enzymes, depending on the desired product. And finally, if you want to get fructose, usually used immobilized enzyme glucose isomerase to convert glucose into fructose.

Implementation of dry grinding to obtain formatiruem sugars (for example, with the subsequent receipt of ethanol from corn starch promotes effective communication exogenous enzymes with starch. This method requires a lower financial cost than wet grinding method, however, it would be desirable to obtain greater economic benefits, because, in most cases, the by-products formed in these processes often are not as valuable as the products obtained by wet grinding. For example, during dry milling of corn grain is ground into powder, which facilitates effective contact of the starch with rasdal the sponding enzymes. After hydrolysis of corn flour enzymes residual solids have a definite food value, because they contain proteins and some other components. Recently in the work Eckhoff, entitled "Fermentation and costs of fuel ethanol from corn with quick germ process" (Appl. Biochem. Biotechnol., 94:41 (2001)), were described improvement opportunities and problems associated with dry grinding. The method of "rapid separation of the embryo allows us to distinguish the rich oil germ from the starch using a smaller time soaking.

One example of a plant where regulation and/or the level of endogenous processorbased enzymes can lead to obtaining the desired product is sweet corn. Typical varieties of sweet corn differ from field corn that sweet corn is not capable of the biosynthesis of normal levels of starch. Usually, to limit the biosynthesis of starch varieties of sweet corn are subjected to genetic mutations in genes encoding enzymes involved in the biosynthesis of starch. These mutations are in genes encoding cromulent and ADP-glucocerebrosidase (such as mutations in the sugar and supersearch maize varieties). Fructose, glucose and sucrose, which are simple sugars that are necessary for producing a pleasant sweet taste, which of predpochtitel the main consumer of fresh corn, accumulated in the developing endosperm of such mutants. However, if the level of accumulation of starch is too high, i.e. if the corn is left on too long term to maturity (late harvest), or if the corn before it is used, is stored for a very long period of time, this product loses its sweetness and acquires a starchy taste and undesirable organoleptic properties. So the window of harvest for sweet corn is too narrow, and its shelf life is limited.

Another serious problem faced by the farmers growing sweet corn stems from the fact that the value of these varieties of maize solely limited to their use in food products. If the farmer wants in advance, during the development of seeds, to harvest sweet corn for use as a food product, it will suffer significant losses in yield. Low yield and poor grain quality sweet corn can be explained by two fundamental reasons. The first reason is that mutations in the pathway of biosynthesis of starch have an inhibitory effect on the mechanisms of the biosynthesis of starch, and the grain is poured completely, resulting in reduced yield and quality of grain. Secondly, due to the high content of sugar is in the grain and the inability of these sugars to sequesterants as starches moisture of seeds decreases, which leads to further reduction of the retention of nutrients in the grain. Endosperm seed varieties of sweet corn shrink and plushevaya, but are not inherent desiccation and become susceptible to disease. In addition, the low quality of seeds sweet corn is associated with other agronomic problems such as low seed viability, poor germination, the susceptibility of seedlings to disease and poor early germination, due to a combination of factors caused by inadequate accumulation of starch. Thus, poor quality products sweet corn cost impact on consumers, farmers/producers, distributors products and seed.

Thus, as for dry grinding, it is necessary to develop a method that would allow to increase the efficiency of the process and/or to increase the value of by-products. As for wet grinding, it is necessary to develop a method of processing starch, which would avoid the use of equipment required for prolonged soaking, grinding, milling and/or separating components of the grain. For example, you need to modify or exclude the stage soaking wet grinding, which will reduce the amount of wastewater requiring is the pressure, and, thus, save energy and time and to increase the productivity of grinding (the time spent by grain in tubs for soaking should decrease). It is also necessary to exclude or to improve the way the Department of starch-containing endosperm from the embryo.

Brief description of the invention

The present invention relates to sameprinciples plants and parts of such plants, and to methods of their use. Sameprinciples plants and parts of plants of the present invention is able to Express and activate the enzyme(s) (mesophilic, thermophilic, or hyperthermophilic enzymes). After activation of the enzyme(s) (mesophilic, thermophilic, or hyperthermophilic enzymes) of plant or part of plant has the ability to autoprocessing substrate, which allows to achieve the desired result.

The present invention relates to selected polynucleotide, (a) contains the sequence SEQ ID NO:2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59, or a complementary sequence, or polynucleotide that hybridizes with a sequence complementary to any of sequences of SEQ ID NO:2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59, in conditions of low stringency and which encodes a polypeptide having α-amylase, pullulanase, α-glucosidases, glucose is isomerases or glucoamylase activity or (b) codereuse polypeptide containing the sequence of 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 enzymatically active fragment. Preferably, the selected polynucleotide encodes a hybrid polypeptide containing the first polypeptide and second polypeptide, where the first polypeptide has α-amylase, pullulanase, α-glucosidase, glucose isomerase, or glucoamylase activity. More preferably, the second peptide contains a signal peptide sequence that can be targeted first polypeptide to vacuole, endoplasmic reticulum, chloroplast, starch grains, seeds or the cell wall of plants. For example, the signal sequence may be N-terminal signal sequence, derived from waxy corn (waxy), N-terminal signal sequence, derived from γ-Zein, krokhmalskii domain or C-terminal krokhmalskii domain. In addition, the present invention encompasses polynucleotide that hybridizes with a sequence complementary to any of sequences of SEQ ID NO:2, 9 or 52, in conditions of low stringency, and encodes a polypeptide having α-amylase activity; polynucleotide that hybridizes with a sequence complementary to any of the C sequence SEQ ID NO:4 or 25, in conditions of low stringency, and encodes a polypeptide having pullulanase activity; polynucleotide that hybridizes with a sequence complementary to the sequence of SEQ ID NO:6 and encodes a polypeptide having α-glucosidase activity; polynucleotide that hybridizes with a sequence complementary to any of sequences of SEQ ID NO:19, 21, 37, 39, 41 or 43, in conditions of low stringency, and encodes a polypeptide having glucose isomerase activity; polynucleotide that hybridizes with a sequence complementary to any of sequences of SEQ ID NO:46, 48, 50, or 59, in conditions of low stringency, and encodes a polypeptide having glucoamylase activity.

In addition, the present invention relates to expressing the cassette containing polynucleotide, (and) having the sequence SEQ ID NO:2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59, or a complementary sequence, or polynucleotide that hybridizes with a sequence complementary to any of sequences of SEQ ID NO:2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59, or a complementary sequence under conditions of low stringency, and encodes a polypeptide having α-amylase, pullulanase, α-glucosidase, glucose isomerase, or glucoamylases what aktivnosti, or (b) encoding a polypeptide containing the sequence of 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 enzymatically active fragment. Preferably, expressing cassette further comprises a promoter functionally attached to polynucleotide, such as inducible promoter, a tissue-specific promoter or, preferably, an endosperm-specific promoter. Preferred endosperm-specific promoter is the promoter γ-Zein of corn or promoter ADP-gpp corn. In a preferred embodiment of the invention, the promoter contains a sequence of SEQ ID NO:11 or SEQ ID NO:12. In addition, in another preferred embodiment of the invention the specified polynucleotide is in sense orientation relative to this promoter. Expressing cassette according to the invention may optionally encode a signal sequence which is functionally attached to the polypeptide encoded by the specified polynucleotide. The signal sequence is preferably directs functionally attached to the polypeptide at the vacuole, endoplasmic reticulum, chloroplast, starch grains, seeds or on the cell wall of plants. The preferred signal sequences are N-terminal signal is supplemented flax sequence, derived from waxy corn (waxy), N-terminal signal sequence, derived from γ-Zein, or krokhmalskii domain.

In addition, the present invention relates to a vector or a cell expressing containing cassette according to the invention. The cell can be selected from the group consisting of Agrobacterium, the cells of monocotyledonous plants, the cells of dicotyledonous plants, the cells of a plant of the family Liliaceae (Liliopsida), the cells of plants of the family presovyh (Panicoideae), cells of maize cells and cereals. Preferred is a cell of corn.

In addition, the present invention encompasses a plant stably transformed with vectors of the present invention. The present invention relates to a plant stably transformed with the vector containing α-amylase, having any of the amino acid sequence SEQ ID NO:1, 10, 13, 14, 15, 16, 33 or 35 or encoded by polynucleotides, having any of the sequences SEQ ID NO:2 or 9. Preferred α-amylase is a hyperthermophilic α-amylase.

In another embodiment, its implementation of the present invention relates to a plant stably transformed with the vector containing pullulanase, having any of the amino acid sequence SEQ ID NO:24 or 34 or encoded by polynucleotides containing sequence is alnost SEQ ID NO:4 or 25. In addition, the present invention relates to a plant stably transformed with the vector containing α-glucosidase, having any of the amino acid sequence SEQ ID NO:26 or 27 or encoded by polynucleotide containing the sequence of SEQ ID NO:6. Preferred α-glucosidase is a hyperthermophilic α-glucosidase. In addition, the present invention relates to a plant stably transformed with the vector containing the glucose isomerase, having any of the amino acid sequence SEQ ID NO:18, 20, 28, 29, 30, 38, 40, 42 or 44 or encoded by polynucleotides containing any of the sequences SEQ ID NO:19, 21, 37, 39, 41 or 43. The preferred glucosinolate is hyperthermophilic glucose isomerase. In another embodiment, its implementation of the present invention relates to a plant stably transformed with the vector containing glucosamines, having any of the amino acid sequence SEQ ID NO:45, 47 or 49 or encoded by polynucleotides containing any of the sequences SEQ ID NO:46, 48, 50, or 59. Preferably glucosamines is hyperthermophilic.

In addition, the present invention relates to plant products such as seeds, fruits or grains, derived from stably transformed plants of the present invention.

In another embodiment, its implementation of the present invention relates to a transformed plant, the genome of which is increased at the expense of recombinant polynucleotide encoding at least one processrule the enzyme is functionally attached to a promoter sequence, where the sequence specified polynucleotide optimized for expression in the plant. The plant may be a monocotyledonous plant, such as corn, or dicotyledonous plant. The preferred plant is a cereal plant or a plant cultivated for commercial purposes. Processrule enzyme selected from the group consisting of α-amylase, glucoamylase, glucose, glucanase, β-amylase, α-glucosidase, isoamylase, pullulanase, neopolitans, soullans, aminophylline, cellulase, Exo-1,4-β-cellobiohydrolase, Exo-1,3-β-D-glucanase, β-glucosidase, endoglucanase, L-arabinose, α-arabinosides, galactans, galactosidase, mannanase, mannosidase, xylanase, xyloside, protease, glucanase, xylanase, esterase, phytase and lipase. Preferred processormask enzyme is cromartyshire enzyme selected from the group consisting of α-amylase, glucoamylase, glucose, β-amylase, α-glucosidase, isoamylase, pullulanase, neopolitans, soullans and aminophylline. More preferably, the specified enzyme selected from αand ilazi, glucoamylase, glucose, α-glucosidase and pullulanase. Preferably, processrule the enzyme was further hyperthermophilic. In accordance with this aspect of the present invention the enzyme can be necromantress enzyme selected from the group consisting of protease, glucanase, xylanase, esterase, phytase and lipase. In addition, these enzymes can be hyperthermophilic. In a preferred embodiment of the invention the specified enzyme accumulates in the vacuoles, endoplasmic reticulum, chloroplast, starch grains, seeds, or in the cell walls of plants. In addition, in another embodiment of the invention the genome of the plant can be further augmented second recombinant polynucleotide containing regimentally enzyme.

In another aspect the present invention relates to a transformed plant, the genome of which is increased at the expense of recombinant polynucleotide encoding at least one processrule enzyme selected from the group consisting of α-amylase, glucoamylase, glucose, α-glucosidase and pullulanase, and functionally attached to a promoter sequence, where the sequence specified polynucleotide optimized for expression in the plant. Preferred is entrusted processrule enzyme is hyperthermophilic and comes from corn.

In another embodiment, the present invention relates to a transformed plant corn, the genome of which is increased at the expense of recombinant polynucleotide encoding at least one processrule enzyme selected from the group consisting of α-amylase, glucoamylase, glucose, α-glucosidase and pullulanase, and functionally attached to a promoter sequence, where the sequence specified polynucleotide optimized for expression in a plant of maize. Preferred processormask enzyme is hyperthermophilic enzyme.

The present invention relates to a transformed plant, the genome of which is increased at the expense of recombinant polynucleotide having the sequence of SEQ ID NO:2, 9 or 52, and functionally attached to the promoter and signal sequence. It also describes a transformed plant, the genome of which is increased at the expense of recombinant polynucleotide having the sequence of SEQ ID NO:4 or 25, and functionally attached to the promoter and signal sequence. In another embodiment, the present invention relates to a transformed plant, the genome of which is increased at the expense of recombinant polynucleotide having the sequence of SEQ ID NO:6, and functional connec nomu to the promoter and signal sequence. It also describes a transformed plant, the genome of which is increased at the expense of recombinant polynucleotide having the sequence SEQ ID NO:19, 21, 37, 39, 41 or 43. Describes a transformed plant, the genome of which is increased at the expense of recombinant polynucleotide having the sequence of SEQ ID NO:46, 48, 50, or 59.

In the present application is also considered products of the transformed plants. Such products are, for example, seed, fruit or grain. Alternatively, this product may be processrule enzyme, starch or sugar.

It also describes the plant, obtained from stably transformed plants of the present invention. In this aspect of the present invention specified by the plant may be a hybrid plant or inbred plant.

In another embodiment of the invention the starch composition comprises at least one processrule enzyme, which is a protease, glucanase or esterase. The preferred enzyme is hyperthermophilic enzyme.

In another embodiment of the invention, the grain contains at least one processrule enzyme, which is α-amylase, pullulanase, α-glucosidase, glucoamylase or glucose isomerase. The preferred enzyme is hyperthermophilic enzyme.

In his other is ariante the present invention relates to a method for producing starch grains, including processing of grain, which contains at least one non-processrule starch enzyme, under conditions that promote the activation of at least one enzyme; obtaining a mixture containing starch grains and non-starch decomposition products, where the specified seed derived from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one enzyme; and the allocation of the starch grains of the mixture. This enzyme is preferably a protease, glucanase, xylanase, phytase or esterase. Furthermore, the enzyme preferably is hyperthermophilic. Grain can be crushed and/or processed in low or high humidity. Alternatively, the grain may be treated with sulfur dioxide. In addition, the present invention may, preferably, include the allocation of non-starch products of this mixture. Hereinafter described starch and Necromastery, obtained by this method.

In yet another embodiment, the present invention relates to a method for producing sverhslojnoe corn, which includes the processing of the transformed plant or part of a gene which is expressed in the endosperm and enriched by expressing cassette that encodes at least one krahmalistye the speaker or krahmalsoderzhaschih enzyme, in the conditions, contributes to the activation of at least one enzyme and, thus, the conversion of polysaccharides into sugar in the corn, and getting sverhslojnoe corn. Expressing cassette preferably further comprises a promoter functionally attached to polynucleotide, codereuse enzyme. The promoter can be, for example, a constitutive promoter, semispecific promoter or endoparasiticides promoter. The preferred enzyme is hyperthermophilic enzyme. The preferred enzyme is α-amylase. Used here expressing cassette may additionally contain polynucleotide, which encodes the signal sequence is functionally attached to polynucleotide, codereuse at least one enzyme. The signal sequence may send hyperthermophilic enzyme, for example, in the apoplast or in the endoplasmic reticulum. The enzyme preferably includes any of the sequences SEQ ID NO:13, 14, 15, 16, 33 or 35.

In its most preferred embodiment, the present invention relates to a method for producing sverhslojnoe corn, which includes the processing of the transformed plant corn or part of a gene which increased at the expense of expressing cassette, and indicated the cassette, coding α-amylase, is expressed in the endosperm under conditions that promote the activation of at least one enzyme and, thus, the conversion of polysaccharides into sugar in the corn; and receiving sverhslojnoe corn. The preferred enzyme is hyperthermophilic enzyme, and this hyperthermophilic α-amylase contains the amino acid sequence of SEQ ID NO:10, 13, 14, 15, 16, 33 or 35 or its enzymatically active fragment possessing α-amylase activity.

Describes a method for the solution of hydrolyzed krahmaloprodukt, which includes the processing parts of the plant, containing starch grains and at least one processrule enzyme under conditions that promote the activation of at least one enzyme thereby processing the starch granules, with formation of an aqueous solution containing hydrolyzed krahmaloprodukt where a specified part of the plant is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one cromartyshire enzyme; and collecting the aqueous solution containing gidralizovanny krahmaloprodukt. Gidralizovanny krahmaloprodukt may contain dextrin, maltooligosaccharide, glucose and/or mixtures thereof. The preferred enzyme is α-amylase, α-glucosidase, g is ulamila, pullulanase, aminophylline, glucose isomerase, or any combination thereof. Moreover, the preferred enzyme is hyperthermophilic enzyme. In another aspect of the present invention genome of the plant can be further increased at the expense of expressing cassette, the coding regimentally cromartyshire enzyme. Regimentally cromartyshire the enzyme may be selected from the group consisting of amylase, glucoamylase, α-glucosidase, pullulanase, glucose, or combinations thereof. In another aspect of the present invention processrule enzyme, preferably, is expressed in the endosperm. The preferred part of the plant is a grain, and that grain is corn, wheat, barley, rye, oats, sugar cane or rice. While it is preferable that at least one processrule enzyme was functionally attached to the promoter and signal sequence, which directs the specified enzyme on starch grains or on the endoplasmic reticulum or in the cell wall. This method may further include allocating hydrolyzed krahmaloprodukt and/or fermentation gidrolizovannogo of krahmaloprodukt.

In another aspect of the present invention describes a method for hydrolyzed is rahmanipour, including the processing of parts of plants, containing starch grains and at least one processrule enzyme under conditions that promote the activation of at least one enzyme thereby processing the starch grains with formation of an aqueous solution containing hydrolyzed krahmaloprodukt where a specified part of the plant is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one α-amylase; and collecting the aqueous solution containing the product of hydrolyzed starch. Preferably hyperthermophilic α-amylase, and more preferably hyperthermophilic α-amylase contains any of the amino acid sequence SEQ ID NO:1, 10, 13, 14, 15, 16, 33 or 35 or its active fragment, with α-amylase activity. While it is preferable that expressing cassette contained polynucleotide selected from the sequences SEQ ID NO:2, 9, 46, or 52, or complementary sequences, or polynucleotide that hybridizes with any of the sequences SEQ ID NO:2, 9, 46 or 52 under conditions of low stringency and which encodes a polypeptide having α-amylase activity. In addition, the present invention also relates to the genome of transformed plants, which further comprises Poliny eatig, encoding heterophily cromartyshire enzyme. Alternatively, the part of the plant can be processed nereparampil cromartyshire enzyme.

The present invention also relates to part of the transformed plants containing at least one cromartyshire enzyme present in the cells of this plant, where the plant part is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one cromartyshire enzyme. The preferred enzyme is cromartyshire enzyme selected from the group consisting of α-amylase, glucoamylase, glucose, β-amylase, α-glucosidase, isoamylase, pullulanase, neopolitans, soullans and aminophylline. Moreover, the enzyme is preferably hyperthermophilic. The plant can be any plant, preferably corn.

In another embodiment, the present invention also relates to part of the transformed plants containing at least one cromartyshire enzyme that is present in cell walls or in the cells of plants, where the plant part is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette, the coding on m is Nisha least one non-processrule starch enzyme or at least one enzyme, processrule polysaccharide, which is not a starch. This enzyme, preferably, is hyperthermophilic. Moreover, it is not processrule starch enzyme is preferably selected from the group consisting of protease, glucanase, xylanase, esterase, phytase and lipase. The plant part may be any part of a plant, and preferably, spike, seed, fruit, grain, corn straw, scales cereals or bagasse.

The present invention also relates to parts of the transformed plants. So, for example, described part of the transgenic plants containing α-amylase, having any of the amino acid sequence SEQ ID NO:1, 10, 13, 14, 15, 16, 33 or 35, or encoded by polynucleotides containing any of the sequences SEQ ID NO: 2, 9, 46, or 52; part transgenic plants containing α-glucosidase, having any of the amino acid sequence SEQ ID NO:5, 26 or 27 or encoded by polynucleotide containing the sequence of SEQ ID NO:6; part transgenic plants containing glucose isomerase, having any of the amino acid sequence SEQ ID NO:28, 29, 30, 38, 40, 42 or 44 or encoded by polynucleotides containing any of the sequences SEQ ID NO:19, 21, 37, 39, 41 or 43; part transgenic plants containing the glucoamylase having the amino acid sequence of SEQ ID NO:45 or EQ ID NO:47 or SEQ ID NO:49 or encoded by polynucleotides, containing any of the sequences SEQ ID NO:46, 48, 50, or 59; part transgenic plants containing pullulanase encoded by polynucleotides containing any of the sequences SEQ ID NO:4 or 25.

In another embodiment, the present invention relates to a method of converting starch in part of transgenic plants, including the activation of the contained cromartyshire enzyme. Described as starch, dextrin, maltooligosaccharide or sugar produced in accordance with this method.

The present invention also relates to a method of use part of the transgenic plants containing at least one non-processrule starch enzyme in the cell wall or in the cells of the plant, including the processing part of the transgenic plants containing at least one enzyme, processrule polysaccharide, non-starch under conditions that stimulate the activation of at least one enzyme, thereby contributing to the breakdown of the polysaccharide, non-starch, with formation of an aqueous solution containing oligosaccharide and/or sugar, where a specified part of the plant is obtained from a transformed plant, the genome of which is credited for the expense of expressing cassette that encodes at least one enzyme pretsessiruyushchei, non-starch; and collecting the aqueous solution containing oligosaccharides and/or sugar. The preferred enzyme processrule polysaccharide, non-starch, is a hyperthermophilic enzyme.

The method of application of transformed seeds containing at least one processrule enzyme includes processing the transformed seeds containing at least one protease or lipase under conditions that stimulate the activation of at least one enzyme with the formation of aqueous mixtures containing amino acids and fatty acids, where the seeds obtained from the transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one enzyme; and collecting water mixture. Thus preferably produce amino acids, fatty acids, or both. Preferably, at least one protease or lipase was hyperthermophilic.

The present invention relates to a method for producing ethanol, comprising the processing parts of the plant containing at least one polisaharidnyi enzyme under conditions that stimulate the activation of at least one enzyme, thereby contributing to the breakdown of the polysaccharide, with the formation of the oligosaccharide or fermentive sugar, where part of the plant is obtained from the t is informirovannii plants, the genome of which is increased at the expense of expressing cassette that encodes at least one polisaharidnyi enzyme; and incubating fermentive sugar under conditions that stimulate the transformation fermentive sugar or oligosaccharide in ethanol. The preferred part of the plant is corn, fruit, seed, stems, wood, vegetable or root. Preferably, the part of the plant is obtained from plants selected from the group consisting of oats, barley, wheat, berries, grapes, rye, corn, rice, potato, sugar beet, sugar cane, pineapple, herbaceous plants and trees. In another preferred embodiment of the invention polisaharidnyi enzyme is α-amylase, glucoamylase, α-glucosidase, glucose isomerase, pullulanase or combinations thereof.

The present invention relates to a method for producing ethanol, comprising the processing parts of the plant containing at least one enzyme selected from the group consisting of α-amylase, glucoamylase, α-glucosidase, glucose, pullulanase or combinations thereof, when heated over a certain period of time and under conditions that stimulate the activation of at least one enzyme, thereby contributing to the breakdown of the polysaccharide with the formation of fermentive sugar, where part of the fatigue produced from transgenic plants, the genome of which is increased at the expense of expressing cassette that encodes at least one enzyme; and incubating the specified fermentive sugar under conditions that stimulate the transformation fermentive sugar into ethanol. Preferably, at least one enzyme was hyperthermophilic or mesophilic.

In another embodiment, the present invention relates to a method for producing ethanol, comprising the processing parts of the plant containing at least one non-processrule starch enzyme under conditions that stimulate the activation of at least one enzyme, thereby contributing to the breakdown of non-starch polysaccharide to oligosaccharide and fermentary sugar, where part of the plant is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one enzyme; and incubating fermentive sugar under conditions that stimulate the transformation fermentive sugar into ethanol. Preferred not processormask starch enzyme is glucanase, xylanase or cellulase.

The present invention relates to a method for producing ethanol, comprising the processing parts of the plant containing at least one enzyme selected from the group consisting of α-amylase, glucoamylase, α-glucosidase, gluco is isomerase, pullulanase or combinations thereof, under conditions that stimulate the activation of at least one enzyme, thereby contributing to the breakdown of the polysaccharide with the formation of fermentive sugar, where part of the plant is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one enzyme; and incubating the specified fermentive sugar under conditions that stimulate the transformation fermentive sugar into ethanol. Preferably, the specified enzyme was hyperthermophilic.

In addition, the described method of obtaining a sweetened pastry food product without adding additional sweeteners, including processing parts of the plant containing at least one cromartyshire enzyme, under conditions that stimulate the activation of at least one enzyme, thereby facilitating the processing of starch grains in the specified parts of the plant to sugars with the formation of sweetened product, where part of the plant is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one enzyme; processing sweetened product, obtaining farinaceous food product. Farinaceous food product can be obtained from the sweetened product and water. In addition to the CSO, specified farinaceous food product may contain malt, fragrances, vitamins, minerals, colorants, or combinations thereof. Preferably, at least one enzyme was hyperthermophilic. The enzyme may be selected from α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose, or any combination thereof. The plant can be optionally selected from the group consisting of soybean, rye, oats, barley, wheat, corn, rice and sugar cane. Preferred farinaceous food product is a cereal food product, a food product for Breakfast, the food product is ready to use, and pastries. The processing may include baking, boiling, heating, cooking for a couple, electrical discharge, or any combination thereof.

In addition, the present invention relates to a method of sweetening starch-containing product without adding additional sweeteners, including processing of starch containing at least one cromartyshire enzyme, under conditions that stimulate the activation of at least one enzyme, thereby contributing to the breakdown of starch with the formation of sweetened starch, where the specified starch is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one shall erment; and add sweetened starch in the product with the receipt of a food product containing sweetened starch. Preferably, the transformed plant may be selected from the group consisting of corn, soybeans, rye, oats, barley, wheat, rice and sugar cane.

Preferably, at least one enzyme was hyperthermophilic. More preferably, at least one enzyme was a α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof.

The present invention relates to a farinaceous food product and to the product containing sweetened starch.

The present invention also relates to a method for sweetening polisakharidami fruits or vegetables, including processed fruits or vegetables, containing at least one polysaccharidesilica enzyme under conditions that stimulate the activation of at least one enzyme, thereby facilitating the processing of polysaccharide in fruit and vegetables with the formation of sugar and obtaining sweetened fruit or vegetables where the fruits or vegetables are obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one polisaharidnyi enzyme. Fruits or vegetables are selected from the group, with the standing of potatoes, tomatoes, bananas, pumpkin, peas and beans. Preferably, at least one enzyme was hyperthermophilic.

The present invention also relates to a method for charcterised aqueous solution, which includes the processing of starch grains obtained from a plant part under conditions that stimulate the activation of at least one enzyme, thereby obtaining an aqueous solution containing sugar.

In another embodiment, the present invention relates to a method for producing derivatives of starch from grain, which is not subjected to wet or dry grinding prior to extraction of starch products, including the processing of parts of plants, containing starch grains, and at least one cromartyshire enzyme under conditions that stimulate the activation of at least one enzyme, thereby facilitating the processing of starch grains with formation of an aqueous solution containing dextrins or sugars, where the plant part is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one cromartyshire enzyme; and collecting the aqueous solution containing the derived krahmaloprodukt. Preferably, at least one cromartyshire enzyme was hyperthermophily is.

The present invention also relates to a method for allocating α-amylase, glucoamylase, glucose, α-glucosidase and pullulanase, which includes the cultivation of transgenic plants, and selection of it α-amylase, glucoamylase, glucose, α-glucosidase and pullulanase. Preferably, the specified enzyme was hyperthermophilic.

Described is a method of obtaining maltodextrin, which includes the mixing of transgenic grain with water, heating this mixture, separating the solids from the resulting dekstrinovym syrup and collection of maltodextrin. Preferably, the transgenic grains contained in at least one cromartyshire enzyme. Preferred cromartyshire enzyme is α-amylase, glucoamylase, α-glucosidase and glucose isomerase. In addition, the present invention relates to a maltodextrin obtained by the above method, and also to compositions obtained by this method.

The present invention relates to a method for producing dextrins or sugars from the grain, which is not subjected to mechanical failure prior to extraction of starch-containing product, including the treatment of parts of plants, containing starch grains and at least one cromartyshire enzyme in the conditions, stimuliruyushchie, at least one enzyme, thereby facilitating the processing of starch grains with formation of an aqueous solution containing dextrins or sugars, where the plant part is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one processrule enzyme; and collecting the aqueous solution containing sugar and/or dextrins.

The present invention relates to a method for fermentive sugar, which includes the processing parts of the plant, containing starch grains and at least one cromartyshire enzyme under conditions that stimulate the activation of at least one enzyme, thereby facilitating the processing of starch grains, with formation of an aqueous solution containing dextrins or sugars, where the plant part is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one processrule enzyme; and collecting the specified aqueous solution containing fermentary sugar.

In addition, the present invention relates to plant corn, stably transformed with the vector containing the hyperthermophilic α-amylase. For example, the present invention comprises, preferably, maize, stable Tran is formed by vector containing a polynucleotide sequence that encodes α-amylase and which is 60% identical to the sequence of SEQ ID NO:1 or SEQ ID NO:51.

Brief description of the graphical material

In figures 1A and 1B illustrate the activity α-amylase expressed in corn grain and endosperm as a result of segregation of the grains from T1 plants transformed NOV6201 and six NOV6200-lines.

The figure 2 illustrates the activity α-amylase in egregiously beans T1 line NOV6201.

The figure 3 shows the amount of ethanol produced during the fermentation of biomass of transgenic corn containing a thermostable alpha-amylase 797GL3 and subjected to liquefaction during the period of time up to 60 minutes at 85°and 95°C. this figure illustrates that the yield of ethanol after 72 hours of fermentation remained almost unchanged during liquefaction, comprising from 15 minutes to 60 minutes. In addition, it was shown that the biomass obtained by dilution with 95°With, has produced more ethanol at any point in time, than the mass obtained by liquefaction at 85°C.

The figure 4 shows the amount of residual starch (%) after fermentation biomass of transgenic corn containing a thermostable alpha-amylase and subjected to liquefaction during the period of time up to 60 mi the ut at 85° With 95°C. this figure illustrates that the yield of ethanol after 72 hours of fermentation remained almost unchanged during the period of time dilution of 15 minutes to 60 minutes. In addition, it was shown that the biomass obtained by dilution with 95°With, has produced more ethanol at any point in time, than the mass obtained by liquefaction at 85°C.

The figure 5 illustrates the output of ethanol to biomass of transgenic corn and control corn and their various mixtures obtained at 85°and 95°C. this figure shows that transgenic corn containing α-amylase gives a significant increase in the production of starch available for fermentation, because after fermentation, the decrease of the residual starch.

The figure 6 shows the amount of residual starch, measured in drained distillation Cuba after fermentation biomass of transgenic corn, the control of grain and their various mixtures obtained at 85°and 95°C.

The figure 7 shows the outputs of ethanol depending on the fermentation time of the sample containing 3% transgenic corn within 20-80 hours at different pH in the range from 5.2 to 6.4. The figure illustrates that the fermentation is carried out at a lower pH, proceeds faster than at pH 6.0 or above.

On the figure 8 shows the outputs of ethanol by fermentation of biomass, containing various weight percents of transgenic maize from 0 to 12 wt.% at different pH in the range from 5.2 to 6.4. This figure shows that the yield of ethanol depends on the number of transgenic grain contained in this sample.

The figure 9 illustrates the analysis of T2 seeds obtained in different experiments, transformed NOV7005. In some cases, can be detected high expression of pullulanase activity compared to non-transgenic control.

In figures 10A and 10B shows the results of HPLC analysis of the hydrolysis products produced downregulation of pullulanase of the starch in the flour transgenic corn. Incubation of flour from corn expressing pullulanase, in the reaction buffer at 75°C for 30 minutes resulted in the production of oligosaccharides with an average chain (degree of polymerization (DP) ˜10-30) and short amylose chains (DP ˜100-200) of corn starch. In figures 10A and 10B also shows the effect of adding calcium ions on the activity of pullulanase.

In figures 11A and 11B presents data HPLC analysis of the hydrolysis product of starch from two reaction mixtures. The first reaction mixture indicated as "Amylase", contains a mixture [1:1 (wt./wt.)] samples of corn flour obtained from transgenic corn expressing α-amylase, and not-tranche the Noah corn A; and the second reaction mixture Amylase + Pullulanase" contains a mixture [1:1 (wt./wt.)] samples of corn flour obtained from transgenic corn expressing α-amylase, and from transgenic corn expressing pullulanase.

The figure 12 presents the number of sugar product (μg) in 25 µl of reaction mixture for the two reaction mixtures. The first reaction mixture indicated as "Amylase", contains a mixture [1:1 (wt./wt.)] samples of corn flour obtained from transgenic corn expressing α-amylase, and non-transgenic maize A; and the second reaction mixture Amylase + Pullulanase" contains [1:1 (wt./wt.)] samples of corn flour obtained from transgenic corn expressing α-amylase, and from transgenic corn expressing pullulanase.

In figures 13A and 13B shows the hydrolysis product of starch obtained from two series of reaction mixtures after 30-minute incubation at 85°and 95°C. For each series were used two of the reaction mixture, where the first reaction mixture indicated as "Amylase × pullulanase", contains flour from transgenic maize (obtained by cross-pollination)expressing α-amylase and pullulanase, and the second reaction mixture is specified as "Amylase", contains a mixture of samples of corn flour, obtained from the tra is Chennai corn, expressing α-amylase, and from non-transgenic corn A in such relation that gives the same level α-amylase activity, and the level of activity observed in cross-pollination (Amylase × Pullulanase").

The figure 14 shows the decomposition of starch to glucose using seeds of non-transgenic maize (control), seeds of transgenic corn containing α-amylase 797GL3, and the combination of seeds of transgenic maize with 797GL3 and seeds with α-glucosidase MalA.

The figure 15 shows the transformation of the original starch at room temperature or at 30°C. In this figure, the reaction mixtures 1 and 2 are a combination of water and starch at room temperature and at 30°s, respectively. The reaction mixture 3 and 4 are a combination of α-amylase and starch and barley at room temperature and at 30°s, respectively. The reaction mixture 5 and 6 are a combination of glucoamylase Thermoanaerobacterium and starch at room temperature and at 30°s, respectively. The reaction mixture 7 and 8 are a combination of α-amylase barley (Sigma), glucoamylase Thermoanaerobacterium and starch at room temperature and at 30°s, respectively. The reaction mixture 9 and 10 are a combination of control α-amylase barley (Sigma), and starch at room temp is the temperature and at 30° S, respectively. Shows the degree of polymerization (DP) products glucoamylase Thermoanaerobacterium.

The figure 16 shows the production of fructose from flour amylase transgenic corn using a combination of alpha-amylase, alpha-glucosidase and glucose as described in example 19. Corn flour, contains amylase, was mixed with the enzyme solution + water or buffer. All of the reaction mixture contained 60 mg of flour with amylase and just 600 ál of liquid and were incubated for 2 hours at 90°C.

The figure 17 shows the peak areas for the reaction products produced using 100% amylase flour obtained from sameprinciples grain depending on the time of incubation for 0-1200 minutes at 90°C.

The figure 18 shows the peak areas for the reaction products produced using a mixture containing 10% transgenic flour with amylase derived from sameprinciples grain, and 90% control corn flour, depending on the time of incubation for 0-1200 minutes at 90°C.

Figure 19 presents the results of HPLC analysis of transgenic flour with amylase, incubated at 70°S, 80°S, 90°or 100°during the period of time up to 90 minutes, conducted to assess the effect of temperature on the hydrolysis of starch.

The figure 20 shows the area of PI is and ELSD sample, containing 60 mg amylase transgenic flour mixed with enzyme solution plus water or buffer at different reaction conditions. One series of reaction mixtures was buffered with 50 mm MOPS, pH 7.0 at room temperature, plus 10 mm MgSO4and 1 mm CoCl2as of the second series of reaction mixtures containing buffer solution was replaced by water. All reaction mixtures were incubated for 2 hours at 90°C.

Detailed description of the invention

In accordance with the present invention sameprinciples plant or plant part contains entered into him (or her) selected polynucleotide encoding processrule enzyme capable of processing such as modification of starches, polysaccharides, lipids, proteins, etc. in plants, where processrule enzyme may be mesophilic, thermophilic, or hyperthermophilic, and it can be activated by grinding, adding water, heating or any other conditions favourable for the functioning of this enzyme. Selected polynucleotide encoding processrule enzyme integrate into a plant or plant part for expression. After expression and activation processimage enzyme plant or plant part of the present invention is capable of autoprocessing substrate, after which the valid percent is serouse enzyme. Therefore, the plant or plant part of the present invention have the ability to autoprocessing substrate of the enzyme after activation contained in them processimage enzyme in the absence of external sources or with a reduced number of such sources, which are usually required for the processing of these substrates. Such transformed plant cells, transgenic plants and parts of transgenic plants possess "inherent" processormask abilities that allow you to processional desired substrate under the action entered in these enzymes in accordance with the present invention. While it is preferable that polynucleotide encoding processrule enzyme, was "genetically stable", it means that this polynucleotide stably preserved in the transformed plant or parts of such plants of the present invention and stably transmitted to offspring and subsequent generations.

In accordance with the present invention, the methods that use these plants and parts of plants, to avoid the necessity of grinding or any other physical damage to the integrity of the parts of the plant prior to extraction of starch. For example, the present invention relates to improved methods of processing the Kukura the s and other grains for selection of starch. The present invention also relates to a method which allows to separate the starch grains containing levels krahmalsoderzhaschih enzymes that are inside these grains or on these grains, which are adequate for the hydrolysis of specific linkages within the starch and does not require the addition of exogenous produced krahmalsoderzhaschih enzymes. The present invention also relates to improved products sameprinciples plants or parts of plants obtained by the methods of the present invention.

In addition, the use of part sameprinciples" transgenic plants, such as grain, and transgenic plants avoids the main problems associated with current technology, namely, that processorsa enzymes, which are usually produced during the fermentation of microbes, requiring expensive selection of enzymes from supernatant culture, with selected enzymes should be prepared for specific purposes, and should be developed methods and equipment for adding, mixing and performing the reaction of the enzyme with its substrate. Transformed plant of the present invention or part thereof also serve as a source of processimage enzyme, and the sub is tretow and products of this enzyme, such as sugars, amino acids, fatty acids and polysaccharides that are and are not starch. The plant of the present invention can also be used to produce progeny plants, such as hybrid and inbred plants.

Processorsa enzymes and encoding their polynucleotide

Polynucleotide encoding processrule enzyme (mesophilic, thermophilic, or hyperthermophilic), is introduced into a plant or plant part. Processrule enzyme is selected based on the desired substrate, on which, as it was discovered, he acts in plants or transgenic plants, and/or based on the desired final product. For example, the specified processormask enzyme can be cromartyshire enzyme, such as brahmarishi enzyme or chromolithography enzyme, or not processrule starch enzyme. Suitable processormask enzymes include, but are not limited to, krahmalosushilnye or krahmalsoderzhaschih enzymes, including, for example, α-amylase, endo - or Exo-1,4 - or 1,6-α-D-glucoamylase, glucose isomerase, β-amylase, α-glucosidase and other Exo-amylases, and krishnadevaraya enzymes, such as isoamylase, pullulanase, neo-pullulanase, soullans, aminophylline etc., glycosyltransferases, such as cyclodextrines sheraz etc., cellulase, such as Exo-1,4-β-cellobiohydrolase, Exo-1,3-β-D-glucanase, hemicellulase, β-glucosidase, etc., endoglucanase, such as endo-1,3-β-glucanase and endo-1,4-β-glucanase, etc., L-arabinose, such as endo-1,5-α-L-arabinose, α-arabinosides etc., galactans, such as endo-1,4-β-D-galactans, endo-1,3-β-D-galactans, β-galactosidase, α-galactosidase and the like, mannanase, such as endo-1,4-β-D-mannanase, β-mannosidase, α-mannosidase, etc., xylanase, such as endo-1,4-β-xylanase, β-D-xyloside, 1,3-β-D-xylanase, and the like, and pectinase, and not processorsa starch enzymes, including protease, glucanase, xylanase, thioredoxin/thioredoxin-reductase, esterase, phytase and lipase.

In one of the embodiments of the invention processormask enzyme is brahmarishi enzyme selected from the group consisting of α-amylase, pullulanase, α-glucosidase, glucoamylase, aminophylline, glucose, or combinations thereof. In accordance with this variant of the present invention brahmarishi enzyme allows sameprinciples the plant or its parts to break down the starch in the activation of the enzyme contained in the plant or plant part, as will be described below. Brahmarishi(s) enzyme(s) selected based on the need of the target products. For example, for the conversion of glucose (hexose) in fructose can be selected glucose isomerase. Alternatively, the enzyme may be selected based on the desired final krahmaloprodukt having chains with different lengths, depending on, for example, on the degree of processing, or having various desired types of branching. For example, α-amylase, glucoamylase or aminophylline can be used for a small period of time of incubation for producing dekstrinovym products, and a longer time of incubation for producing products or sugars with a shorter chain. Pullulanase can be used, in particular, for hydrolysis at the points of branching of the starch with the formation of vysokoimpulsnogo starch, and neopolitans can be used for the production of starch fragments with α-1,4-linkages interspersed with α-1,6-linkages. Glucosidase can be used for producing limit dextrins, and for other starch derivatives can be used in combination of enzymes.

In another embodiment of the invention, processormask enzyme is not processrule starch enzyme selected from a protease, glucanase, xylanase and esterase. These are not processorsa starch enzymes can penetrate into the target about the art sameprinciples plants or in part, and when activated to destroy the plant, leaving, however, the contained starch grains intact. For example, in the preferred embodiment, the invention is not Deplete starch enzymes act in the matrix of the endosperm of the plant cells and, when activated, destroy the matrix of the endosperm, leaving, however, the contained starch grains intact and more easily excreted from the received material.

In addition, in the present invention deals with the combination processorbased enzymes. For example, cromartyshire enzymes and not processorsa starch enzymes can be used in combination. Combination processorbased enzymes can be obtained using multiple gene constructs encoding each of these enzymes. Alternatively, the individual transgenic plant stably transformed by enzymes, can be crossed with known methods of obtaining plants containing both the enzyme. Another method involves the use of exogenous enzyme(s) and transgenic plants.

Processorsa enzymes can be isolated or obtained from any source, and its corresponding polynucleotide can be identified by a specialist. For example, processrule enzyme, preferably α-amylases obtained from Pyrococcu (for example, Pyrococcus furiosus), Thermus, Thermococcus (e.g., Thermococcus hydrothermalis), Sulfolobus (for example, Sulfolobus solfataricus), Thermotoga (e.g., Thermotoga maritima and Thermotoga neapolitana), Thermoanaerobacterium (e.g., Thermoanaerobacter tengcongensis), Aspergillus (e.g., 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.

Processorsa enzymes of the present invention can be activated after their introduction and expression in the plant genome. Conditions for activation of the enzyme is determined for each specific enzyme and can be used in a variety of conditions, such as appropriate temperature and pH, hydration, presence of metals, activating connections, inactivating compound and the like, for example, dependent on the temperature of the enzymes can be mesophilic, thermophilic and hyperthermophilic enzymes. Mesophilic enzymes typically have a maximum activity at a temperature of 20-65°and are inactivated at temperatures above 70°C. Mesophilic enzymes have significant activity at 30-37aboutWith, and their activity at 30°C is at least 10% of the maximum activity, and more preferably at least 20% of the maximum activity./p>

Thermophilic enzymes have a maximum activity at a temperature of from 50°C to 80°and are inactivated at temperatures above 80°C. Thermophilic enzyme at 30°preferably has less than 20% of the maximum activity, and more preferably less than 10% of the maximum activity.

"Hyperthermophilic" the enzyme has activity even at higher temperatures. Hyperthermophilic enzymes have a maximum activity at temperatures above 80°and retain activity at a temperature of at least 80°and more preferably, if they keep activity at a temperature of at least 90°and most preferably, if they keep activity at a temperature of at least 95°C. Hyperthermophilic enzymes also have low activity at low temperatures. Hyperthermophilic enzyme may possess activity at 30°S, i.e. less than 10% of the maximum activity, and preferably less than 5% of the maximum activity.

Polynucleotide encoding processrule enzyme, preferably modified so that it includes the codons optimized for expression in a selected microorganism, such as a plant (see, e.g., Wada et al., Nucl. Acids. Res., 18:2367 (1990), Murray et al., Nucl. Acids. Res., 17:477 (1989), U.S. patent No. is 5096825, 5625136, 5670356 and 5874304). Optimized codon sequences are synthetic sequences, i.e. they do not occur in nature and, preferably, encode the same polypeptide (or an enzymatically active fragment of full-length polypeptide, which has basically the same activity as the full-sized polypeptide)encoded not optimized codons source polynucleotide that encodes processrule enzyme. While it is preferable that the polypeptide differed in their biochemical properties or had improved biochemical properties (which can be achieved, for example, by a recursive mutagenesis of DNA that encodes a specific processrule enzyme), compared to the parent polypeptide source, so that it had a higher efficiency in this method of application. Preferred polynucleotide optimized for expression in the desired plant host and encode processrule enzyme. Methods of obtaining these enzymes include mutagenesis, for example recursive mutagenesis and selection. Methods mutagenesis and modification of the nucleotide sequence are well known in the art. See, for example, Kunkel, Proc.Natl. Acad. Sci., USA 82:488 (1985); Kunkel et al., Methods in Enzymol., 154:367 (1987); U.S. patent No. 4873192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) is quoted there and Arnold et al., Chem. Eng. Sci. 51:5091 (1996)). Optimization methods the expression of the nucleic acid segment in the desired plant or microorganism. To do this, get a table of occurrence of codons illustrating optimal codons that are used in the body of the target, and choose an optimal codons to replace them in the right polynucleotide, and then carry out chemical synthesis of optimized sequence. The preferred codons of corn is described in U.S. patent No. 5625136.

In addition, are considered complementary nucleic acid polynucleotide of the present invention. Examples of low stringency for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a southern or Northern-band, are the use of 50% formamide, for example carrying out hybridization in 50% formamide, 1M NaCl, 1% LTOs at 37°and washing in 0.1 x SSC at 60°-65°C. an Example of hybridization conditions with a low degree of hardness is hybridization with a buffer solution of 30 to 35% formamide, 1M NaCl, 1% LTOs (sodium dodecyl sulphate) at 37°and washing in 1X-2X SSC (20X SSC=3.0 m NaCl/0.3 m trinatriytsitrat) at 50-55°C. Examples of hybridization conditions of moderate stringency include hybridization in 40 to 45% formamide, 1.0m NaCl, 1% LTOs at 37°and washing in 0.5x-1X SSC 55-60° C.

In addition, it then examines polynucleotide encoding "enzymatically active fragment processimage enzyme. Used herein, the term "enzymatically active fragment of a polypeptide means a fragment processimage enzyme, which has basically the same biological activity as processrule enzyme, and is able to modify the substrate, after which processrule enzyme normally functions normally in suitable conditions.

In a preferred embodiment, polynucleotide of the present invention is polynucleotide, optimized codons corn and encoding α-amylase, such as SEQ ID NO:2, 9, 46, and 52. In another preferred embodiment, the invention polynucleotides is polynucleotide, optimized codons corn and encoding pullulanase, such as SEQ ID NO:4 and 25. In yet another preferred embodiment of the invention the specified polynucleotide is polynucleotide, optimized codons corn and encoding α-glucosidase, such as SEQ ID NO:6. Other preferred polynucleotides is polynucleotide, optimized codons corn and encoding the glucose isomerase, such as SEQ ID NO:19, 21, 37, 39, 41 or 43. In another embodiment of the invention, it is preferable poly is ucleotide, optimized codons corn and encoding a glucoamylase, such as SEQ ID NO:46, 48 or 50. In addition, optimized codons corn polynucleotide coding for a hybrid polypeptide glucanase/mannanase is polynucleotide SEQ ID NO:57. The present invention also relates to sequences complementary to such polynucleotides that hybridize under moderate, or preferably low stringency and which encode a polypeptide having α-amylase, pullulanase, α-glucosidase, glucose isomerase, glucoamylase, glucanases or mannanase activity where it is needed.

The term "polynucleotide" can be used along with the terms "nucleic acid" or "poliolefinovoy acid" means deoxyribonucleotides or ribonucleotides and polymers in single-stranded or double-stranded form, composed of monomers (nucleotides)containing a sugar, phosphate and base, which is either a purine or a pyrimidine. If it is not specifically mentioned, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the original nucleic acid and are metabolized in accordance with the same mechanism as natural nucleotides. If this is not the taxes the Reno particularly, the specific sequence of nucleic acids are also, of course, covers its reversible modified variants (for example, substitution of degenerate codons) and complementary sequences, as well as a specified sequence. In particular, substitution of degenerate codons can be achieved by generating sequences in which one or more selected (or all) codons in the third position replaced with mixed bases and/or deoxyinosine remains.

The present invention also encompasses the "options" or, basically, the same sequence. For variants of the nucleotide sequences are sequences that, because of the degeneracy of the genetic code, encode an identical amino acid sequence of a native protein. Natural allelic variants, such as those described here, can be identified using well known molecular biology techniques such as polymerase chain reaction (PCR), hybridization techniques and the secondary Assembly by ligating. Variant nucleotide sequences also are synthetic nucleotide sequences, such as sequences, obtained, for example, by site-directed mutagenesis and encoding natural white is to, as well as the sequence encoding the polypeptide having the amino acid replacement. In General, variants of the nucleotide sequence of the present invention, at least 40%, 50%, 60%, preferably 70%, more preferably 80%, even more preferably 90%, and most preferably 99% identical to the native nucleotide sequence of the same class or differ from it by only one nucleotide. For example, this similarity may be 71%, 72%, 73%, etc. at least 90%. Such variants can be also full-length gene corresponding to the identified gene fragment.

Regulatory sequences include promoter/signal sequence/selective markers.

Polynucleotide sequences encoding processrule the enzyme of the present invention can be functionally attached to the polynucleotide sequences coding for the signal localization signal or sequence (the N - or C-end of the polypeptide, for example, to target a hyperthermophilic enzyme to a specific compartment in the plant. Examples of such targets include, but are not limited to, vacuole, endoplasmic reticulum, chloroplast, amyloplast, starch grains or cell wall, or a specific tissue, such as the seed. The Pauline expression is of cleotide, coding processrule enzyme having the signal sequence in the plant, and in particular, in combination with tissue-specific or inducible promoter can produce high levels of localized processimage enzyme in the plant. It is known that different signal sequences affect the expression or targeting polynucleotide on a specific compartment or outside the specific compartment. Suitable signal sequences and target promoters known in the art, and such sequences include, but are not limited to, the sequence described here.

For example, if it is desirable that the expression occurred in specific tissues or organs, it can be used for tissue-specific promoters. In contrast, if it is desirable that the gene expressively in response to the stimulator, the regulatory elements necessary to choose inducible promoters. If desired continuous expression in all plant cells, the use of constitutive promoters. In the expression constructs of the transforming vectors may be additional regulatory sequences that are located above and/or below from the cow to the promoter sequence, and these sequences will provide the tested different levels of expression of heterologous nucleotide sequences in a transgenic plant.

Was described by a number of promoters plants with different expression properties. Examples of some constitutive promoters that have been described in the literature, are actin 1 rice (Wang et al., Mol. Cell. Biol. 12:3399 (1992); U.S. patent No. 5641876), 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 the promoters ubicacin.

The vectors used for tissue-specific targeting of genes in transgenic plants, typically include tissue-specific promoters, and they may also include other tissue-specific regulatory elements such as enhancer sequences. Promoters that regulate specific or enhanced expression in some tissues of plants, known in the art from the description of the present invention. Such promoters are, for example, the rbcS promoter, specific for green tissue; the promoters of the ocs, nos and mas, which have higher activity in roots or in the tissue of the damaged leaves; the truncated 35S promoter (-90-(+8), which provides enhanced expression in roots, gene α-tubulin, which provides the expression in the roots, and promoters derived from the genes of the protein reserve of Zein and ensuring expression in the endosperm.

Tissue-specific expression may be functionally implemented by introducing the Constitution is utive expressed gene (in all tissues) in combination with antimuslim genome which is expressed only in those tissues in which the production of a given gene product is undesirable. For example, the gene encoding the lipase, can be introduced together with the 35S promoter of cauliflower mosaic virus so that it expressively in all tissues. Expression of the antisense transcript lipase gene in corn using, for example, Zein promoter will prevent the accumulation of lipase protein in the seeds. Therefore, the protein encoded by the specified introduced gene will be present in all tissues, except the beans.

In addition, as previously reported, in plants there are several tissue-specific regulatory genes and/or promoters. Some of these are described tissue-specific genes are the genes encoding the spare proteins of seeds (such as napin, cruciferin, beta-conglycinin and phaseolin), Zein or oil proteins cells (such as oleosin) or genes involved in the biosynthesis of fatty acids (including acyl-carrying protein, stearoyl-ASR-desaturase and Desaturate fatty acids (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)). The examples described tissue-specific promoters are the promoters of the lectin (Vodkin, Prog. Clin. Biol. Res. 138:87 (1983); Lindstrom et al., Der. Genet., 11:160 (1990)), alcohol dehydrogenase 1 corn (Voge et al., 1989, Dennis et al., Nucleic Acids. Res., 12:3983 (1984)), light-harvesting complex, corn (Simpson, 1986; Bansal et al., Proc. Natl. Acad. Sci. USA, 89:3654 (1992)), heat shock protein maize (Odell et al., 1985; Rochester et al., 1986), carboxylase small subunit RuBP peas (now et al., 1986; Cashmore et al., 1983), manopen synthase of the Ti plasmid (Langridge et al., 1989), nopalin synthase of the Ti plasmid (Langridge et al., 1989), balconyterrace petunias (vanTunen et al., EMBO J. 7:1257 (1988)), rich in glycine protein 1 soybean (Keller et al., Genes Dev. 3:1639 (1989)), truncated CaMV 35s (Odell et al., Nature, 313:810 (1985)), patatin potatoes (Wenzler et al., Plant Mol. Biol. 13:347 (1989)), root cells (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, 34:1015 (1983); Reina et al., Nucleic Acids Res., 17:7449 (1990)), globulin-1 (Belanger et al., Genetics, 129:863 (1991)), α-tubulin, cab (Sullivan et al., Mol. Gen. Genet. 215:431 (1989)), Rerise (Hudspeth &Grula, 1989), complex-associated promoters of the gene R (Chandler et al., Plant Cell 1:1175 (1989)and promoters malcontents (Franken et al., EMBO J. 10:2605 (1991)). Particularly suitable for somespecific expression is the promoter vicilin peas (Czako et al., Mol. Gen. Genet., 235:33 (1992)). (Cm. also U.S. patent No. 5625136, which is introduced into the present description by reference). Other suitable promoters for expression in the budding leaves are promoters that are switched at the beginning of the aging process, such as the SAG promoter of Arabidopsis plants (Gan et al., Science, 270:1986 (1995)).

The promoters of the fruit-specific class, expressed during flowering or during the period of time from beginning of flowering to fetal development, at least prior to its maturity, are discussed in U.S. patent No. 4943674, which is introduced into the present description by reference. Were isolated cDNA clones that are expressed predominantly in the fiber of cotton (John et al., Proc. Natl. Acad. Sci. USA, 89:5769 (1992)). There were also isolated and characterized cDNA clones of tomato, in which there is differential expression during fetal development (Mansson et al., Gen. Genet., 200:356 (1985), Staler et al., Plant. Mol. Biol., 5:137 (1985)). The promoter of the gene polygalacturonase is active on the stage of gestation. Gene polygalacturonase described in U.S. patent No. 4535060, in U.S. patent No. 4769061, in U.S. patent No. 4801590 and in U.S. patent No. 5107065 that are entered into the present description by reference.

Other examples of tissue-specific promoters are promoters that regulate expression in leaf cells following their injury (for example, after eating insects), in tubers (for example, the promoter of the gene patatina) and in the cells of the fiber (for example, regulated at the stage of development of the cells of the fiber protein is a protein E6 (John et al., Proc. Natl. Acad. Sci. USA, 89:5769 (1992)). Gene E6 has the greatest activity in the fiber, although small levels of transcripts detected is in the leaves, the ovules and flowers.

The tissue specificity of some "tissue-specific" promoters may not be absolute and may be tested by a technician using the sequence of diphtheria toxin. Tissue-specific expression may also be achieved by peak expression by combining different tissue-specific promoters. (Beals et al., Plant Cell 9:1527 (1997)). Other tissue-specific promoters can be selected by a person skilled in the art (see U.S. patent No. 5589379).

In one of the embodiments of the invention, the gene product of the hydrolysis of the polysaccharide, such as α-amylase, may be targeted to a specific organelle, such as the apoplast, and not in the cytoplasm. This can be achieved, for example, using N-terminal signal sequence γ-Zein of maize (SEQ ID NO:17), which provides the apoplast-specific targeting of proteins. The direction of the protein or enzyme in a specific compartment allows the enzyme to be localized so that it does not come in contact with the substrate. Thus, the enzymatic activity of the enzyme will not come until the enzyme is not precontractual with its substrate. The contact of the enzyme with the substrate can be carried out by milling (physical destruction of the integrity of the cells) or heating the cells or the tissue is her plants with the violation of the physical integrity of cells or organs of a plant, containing the enzyme. For example, mesophilic brahmarishi enzyme can be targeted to the apoplast or to the endoplasmic reticulum, and therefore he will not come in contact with starch grains in amyloplasts. After grinding grain integrity of this grain would be violated, then chromalveolates the enzyme will be in contact with starch grains. This way you can avoid possible negative effects of joint localization of the enzyme and its substrate.

In another embodiment of the invention tissue-specific promoter is an endosperm-specific promoters such as the promoter γ-Zein of maize (represented by SEQ ID NO:12) or the promoter ADP-gpp corn (represented by SEQ ID NO:11 and including 5'-noncoding sequence and intron sequence). Thus, the present invention relates to selected polynucleotide containing a promoter comprising the sequence of SEQ ID NO:11 or 12, polynucleotide that hybridizes with its complementary sequence under conditions of low stringency, or its fragment, the promoter activity which is at least 10%and preferably at least 50% of the activity of the promoter having the sequence of SEQ ID NO:11 or 12.

In another embodiment, izopet is of polynucleotide encodes hyperthermophilic processrule enzyme, which is functionally attached to the chloroplast (ameloblastoma) transport peptide (P) and krahmalevym domain, for example encoded gene waxy. Typical polynucleotide in this embodiment of the present invention encodes the sequence of SEQ ID NO:10 (α-amylase attached to krahmalevym domain waxy maize). Other characteristic polynucleotide encode hyperthermophilic processrule enzyme attached to a signal sequence that provides targeting the specified enzyme in the endoplasmic reticulum and secretion into the apoplast (for example, polynucleotide encoding the sequence of SEQ ID NO:13, 27 or 30, which contains the N-terminal sequence γ-Zein corn, functionally associated with αamylase, α-glucosidase, glucosinolate respectively); hyperthermophilic processrule the enzyme attached to the signal sequence, which ensures the retention of the enzyme in the endoplasmic reticulum (for example, polynucleotide encoding SEQ ID NO:14, 26, 28, 29, 33, 34, 35 or 36, which contain the N-terminal sequence γ-Zein corn, functionally attached to the hyperthermophilic enzyme, which is functionally attached to SEKDEL, where the enzyme is α-amylase, α-glucosidase malA, glucoses is merasa T.maritima, glucose isomerase T.neapolitana); hyperthermophilic processrule the enzyme attached to the N-terminal sequence, which provides the targeting of the enzyme to amyloplast (for example, polynucleotide encoding the sequence of SEQ ID NO:15, which contains the N-terminal sequence of providing the targeting of the enzyme to amyloplast waxy maize, and functionally attached to the sequence α-amylase); hyperthermophilic hybrid polypeptide that provides targeting the specified enzyme on starch grains (for example, polynucleotide encoding the sequence of SEQ ID NO:16, which contains the N-terminal sequence of providing the targeting of the enzyme to amyloplast sequence waxy maize, and functionally attached to the hybrid polypeptide α-amylase/waxy containing krokhmalskii domain waxy maize); hyperthermophilic processrule enzyme associated with a signal sequence that provides retention in the ER (for example, polynucleotide encoding SEQ ID NO:38 and 39). In addition, hyperthermophilic processrule the enzyme may be attached to the binding site with the original starch having the amino acid sequence (SEQ ID NO:53), where the specified polynucleotide encoding processrule enzyme, process is Dinan to a nucleic acid sequence, optimized codons maize (SEQ ID NO:54) and the coding specified binding site.

It was also reported on several inducible-promoters. Many of them described Gatz in the work of Current Opinion in Biotechnology, 7:168 (1996) and Gatz, C., Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 48:89 (1997). Examples of such promoters are the system suppression tetracycline, system suppression Lac, copper-inducible systems, salicylate-inducible systems (such as the PR1a system), glucocorticoid-inducible system (Aoyama T. et al., N-H Plant Journal, 11:605 (1997)) and ecdyson-inducible system. Other inducible promoters are ABA - and turgor-inducible promoters, the promoter of the gene of the protein, bind to auxin (Schwob et al., Plant J. 4:423 (1993)), the promoter of the gene UDP-glucanotransferase (Ralston et al., Genetics, 119:185 (1988)), the promoter of the proteinase inhibitor MPI (Cordero et al., Plant. J., 6:141 (1994)), the promoter of the gene of glyceraldehyde-3-phosphate dehydrogenase (Kohler et al., Plant. Mol. Biol., 29:1293 (1995); Quigley et al., J. Mol. Evol. 29:412 (1989); Martinez et al., J. Mol. Biol. 208:551 (1989)). These promoters also include benzosulfimide-inducible system (U.S. patent No. 5364780) and alcohol-inducible system (WO 97/06269 and WO 97/06268) and promoters glutathione-S-transferase.

Was also conducted other studies of genes inducible genes regulated in response to stress conditions generated by the environment, or stimulants, such as increasing the degree is Olanesti, drought, presence of pathogens and damage (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 protein inhibitor metallocarboxypeptidase was observed in the damaged leaves of potato plants (Graham et al., Biochem. Biophys. Res. Comm. 101:1164 (1981)). As previously reported, other plant genes induced by methylammonium, stimulants, heat shock proteins, anaerobic stress or herbicide preparations.

Regulated expression of a chimeric protein with TRANS-activity against replication of the virus, can additionally be regulated by other methods genetic strategies, for example by Cre-mediated gene activation (Odell et al., Mol. Gen. Genet., 113:369 (1990)). Thus, a DNA fragment containing a 3'-regulatory sequence associated with lox sites between the promoter and the sequence encoding the protein replication and blocking the expression of a chimeric gene replication, since the promoter may be deleted by Cre-mediated cut, which leads to the expression of TRANS-active gene replication. In this case, the chimeric Cre gene, chimeric TRANS-active gene replication or both of the gene can be under the control of tissue-specific and statespecific or inducible promoters. Alternative gene strategy involves the use of gene-PMPs who quarrel tRNA. So, for example, regulated expression of the suppressor tRNA may, depending on conditions, to regulate the expression of a sequence that encodes a TRANS-active protein replication and contains the appropriate termination codon (Ulmasov et al. Plant. Mol. Biol., 35:417 (1997)). And again, the chimeric gene is a suppressor tRNA, chimeric TRANS-active gene replication or both of the gene can be under the control of tissue-specific and statespecific or inducible promoters.

In the case of a multicellular organism, the promoter may also preferably be specific to a particular tissue, organ and developmental stage. Examples of such promoters include, but are not limited to, the promoter ADP-gpp Zea mays, promoter γZea mays Zein and the promoter of the globulin Zea mays.

Gene expression in transgenic plants may be desirable only in a certain period of time of plant development. The time development is often correlates with tissue-specific expression of the gene. For example, the expression of spare proteins seenow is initiated in the endosperm after about 15 days after pollination.

In addition, can be constructed vectors that can be used for intracellular targeting of a specific gene product in the cells of transgenic plants or to target the protein to the extracellular space. This usually is achieved by joining a DNA sequence, coding transport or signal peptide sequence to the coding sequence of a particular gene. Received or the signal peptide will transfer the protein to a particular intracellular or extracellular phase respectively, and then he will excision removed. Transport or signal peptides act via transport across intracellular membranes, such as vacuole, vesicles, plastid and mitochondrial membranes, whereas the signal peptides direct proteins through the extracellular membrane.

Signal sequence, such as the N-terminal signal sequence γ-Zein corn, providing targeting to the endoplasmic reticulum and secretion into the apoplast, may be functionally attached to polynucleotide, codereuse hyperthermophilic processrule the enzyme of the present invention (Torrent et al., 1997). For example, the sequence of SEQ ID NO:13, 27 and 30 are polynucleotide encoding hyperthermophilic enzyme, functionally attached to the N-terminal sequence of protein γ-Zein of corn. Another signal sequence is the amino acid sequence SEKDEL, ensuring the retention of polypeptides in the endoplasmic reticulum (Munro &Pelham, 1987). So, e.g. the measures polynucleotide encoding sequence SEQ ID NO:14, 26, 28, 29, 33, 34, 35 or 36, which contain the N-terminal sequence γ-Zein corn, functionally attached to processarea the enzyme that is functionally associated with SEKDEL. The polypeptide may also be aimed at amyloplast by joining peptide, providing targeting amyloplast waxy maize (Klosgen et al., 1986) or the starch grain. For example, polynucleotide encoding hyperthermophilic processrule the enzyme may be functionally attached to the chloroplast (ameloblastoma) transport peptide (P) and krahmalevym domain, for example, encoded by the gene waxy. Sequence of SEQ ID NO:10 represents α-amylase attached to krahmalevym domain waxy maize. Sequence of SEQ ID NO:15 represents the N-terminal sequence, providing targeting amyloplast waxy maize and functionally attached to α-amylase. In addition, polynucleotide encoding processrule the enzyme can be attached to the targeting starch grains using krahmalsoderzhashchego domain waxy maize. For example, the sequence of SEQ ID NO:16 is a hybrid polypeptide containing the N-terminal amyloplast nalivaemo the sequence, providing targeting amyloplast waxy maize and functionally attached to the hybrid polypeptide α-amylase/waxy containing krokhmalskii domain waxy maize.

Polynucleotide of the present invention, in addition to signal processing, may also include other regulatory sequences known in the art. The term "regulatory sequence" and "suitable regulatory sequence" means a nucleotide sequence that is localized above (5'-non-coding sequences), within, or below (3'-non-coding sequences) the coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, leader sequences broadcast, introns and the signal polyadenylation sequence. Such sequences are natural and synthetic sequences as well as sequences that can be a combination of synthetic and natural sequences.

In accordance with the present invention can be also used selective markers which allow for selection of transformed the s plants and plant tissues, as is well known in the art. As expressing the desired gene or in addition to this gene may be desirable to use a selective or skrinichenko marker gene. "Marker genes" are genes that give the cells expressing this marker gene, distinctive phenotypic trait and, thus, make it possible to identify such transformed cells from cells that do not have this marker. These genes may encode either a selective or scenerey token in the matter attaches if the specified token is a sign that can be carried out selection by chemical methods, i.e. using the selective agent (e.g., a herbicide, antibiotic, or the like), or it is simply a trait that one can identify through observation or testing, that is, by screening (for example, the characteristic of R-locus). Needless to say, there are many examples of suitable marker genes that can be used to implement the present invention.

The terms "selective" or "scrinium" marker genes also means the genes encoding the "secretory marker, the secretion of which can be detected by identifying or selecting for transformed cells. Examples of this stamp is s, encoding the secretory antigen that can be identified through interaction with the antibody, or even secreted enzymes that can be detected by their catalytic activity. Secreted proteins belong to several classes, including small diffundere proteins, detected, for example using ELISA; small active enzymes detected in the extracellular solution (for example, α-amylase, β-lactamase, phosphinotricin-acetyltransferase); and proteins, which are involved in cell wall or immobilized on the cell walls (e.g., proteins that include a leader sequence such as the sequence found in the expression element of extensin or PR-S tobacco).

With regard to selective secreted markers, it is particularly preferable to use a gene that encodes a protein that becomes sequestered in the cell wall and which includes a unique epitope. Such secretory antigenic marker is ideal for use sequence of the epitope, which should provide a low background level in the tissue of plants; promoter-leader sequence, which must ensure the efficient expression and passing through the plasma membrane and must produce the protein to the th associated with the cell wall and is also accessible to antibodies. Usually the secretory protein of the cell wall modified to include a unique epitope must meet all specified requirements.

One example of a protein suitable for such modification is extensin or hydroxyprolin-rich glycoprotein (HPRG). For example, the molecule HPRG corn (Steifel et al., The Plant Cell, 2:785 (1990)) is well characterized from the point of view of molecular biology, expression and protein structure. However, to create skrinichenko token any protein from a number of extension and/or glycine-rich cell wall proteins (Keller et al., EMBO Journal, 8:1309 (1989)) can be modified by adding the area of determinants.

A. Selective markers

Possible selective markers used in the present invention include, but are not limited to, a neo gene or nptII (Potrykus et al., Mol. Gen. Genet. 199:183 (1985)), which give resistance to kanamycin and can be selected for using kanamycin, G418, etc.; bar gene, which imparts resistance to the herbicide phosphinotricin; a gene that encodes a modified protein EPSP synthase (Hinchee et al., Biotech., 6:915 (1988)) and, thus, imparts resistance to glyphosate; gene nitrilase, such as bxn from Klebsiella ozaenae which gives resistance to bromoxynil (Stalker et al., Science, 242:419 (1988); a mutant gene acetolactate (ALS), which gives resis intesti to imidazolinone, the sulfonylurea or other ALS-inhibiting chemicals (European patent application 154204, 1985); a gene of resistance to methotrexate DHFR (Thillet et al., J. Biol. Chem. 263:12500 (1988)); a gene of dependableness, which imparts resistance to the herbicide dalapon; gene phosphomonoesterase (PMI); the mutated gene anthranilates, which gives resistance to 5-methyltryptophan; hph gene, which imparts resistance to the antibiotic hygromycin; or gene mannose-6-fortismere (also referred to here as the genome phosphomonoesterase), which gives the ability to metabolize mannose (U.S. patent No. 5767378 and 5994629). Every person is able to choose suitable selective marker gene for use in the present invention. When using the mutated gene, EPSP synthase, you can get additional advantage from the introduction of a suitable chloroplast transport peptide P (European patent application 0218571, 1987).

Illustrative option selective marker gene used in the selection of transformants, are genes encoding the enzyme phosphinotricin-acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes. The enzyme phosphinotricin-acetyltransferase (RAT) inactivates the active ingredient in the phosphinotricin (PPT) herbicide bialaphos. PPT inhibits glutam is synthetase (Murakami et al., Mol. Gen. Genet. 205:42 (1986); Twell et al., Plant. Physiol., 91:1270 (1989)), which leads to the rapid accumulation of ammonia and to cell death. Taking into account the fact that the main challenges, as it has been reported that occur during transformation of cereals, the successful use of this selective system in monocotyledonous plants was particularly unexpected (Potrykus, Trends Biotech., 7:269 (1989)).

If for implementing the present invention it is necessary to use a gene of resistance to bialaphos, the most suitable for these purposes are the genes of the bar or pat, which can be obtained from microorganisms of the species of Streptomyces (e.g., ATSC No. 21705). Cloning of the gene bar described in Murakami et al., Mol. Gen. Genet., 205:42 (1986) and Thompson et al., EMBO Journal, 6:2519 (1987), and has also been described using the bar gene for neededonline plants (De Block et al., EMBO Journal, 6:2513 (1987); De Block et al., Plant Physiol., 91:694 (1989)).

b. Scrinium markers

Senireemi markers that can be used are, but are not limited to, gene β-glucuronidase or uidA gene (GUS)that encodes an enzyme for which various chromogenic substrates; R gene-locus that encodes a product that regulates the production of anthocyanines pigments (red color) in plant tissues (Dellaporta et al., Chromosome Structure and Function, pp.263-282 (1988)); gene β-lactamase (Sutcliffe, PNAS USA, 75:3737 (1978))that encodes an enzyme for which various known the chromogenic substrates (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., PNAS USA, 80:1101 (1983)), encoding catechol-dioxygenase that can convert chromagene category; gene α-amylase (Ikuta et al., Biotech, 8:241 (1990)); a tyrosinase gene (Katz et al., J. Gen. Environ., 129:2703 (1983)), encoding an enzyme capable of oxidizing tyrosine to DOPA and dauphinee, which, in turn, is condensed with education legkodeformiruemyh compound melanin; gene β-galactosidase encoding an enzyme for which there are chromogenic substrates; a luciferase gene (lux) (Ow et al., Science 234:856 (1986)), which provides bioluminescent detection; gene aquarina (Prasher et al., Biochem. Biophys. Res. Comm., 126:1259 (1985)), which can be used for sensitive calcium bioluminescent detection, or gene protein, fluorescent in the green range of the spectrum (Niedz et al., Plant Cell Reports, 14:403 (1995)).

It is believed that the genes of the R-gene complex of corn are especially valuable as scrinium markers. R-gene complex in maize encodes a protein that acts as a regulator of production anthocyanines pigments in most seeds and plant tissues. R gene-gene complex is suitable for transformation of corn, because the expression of this gene in transformed cells has no negative effects on these cells. Thus, R-gene, introduced into these cells, will bring the e to the expression of red pigment, and, when stable embedding in the red sector, can provide a visual assessment. If the line corn carries the dominant alleles of genes encoding enzyme intermediate in the biosynthesis pathway of anthocyanin (C2, A1, A2, Bz1 and Bz2), but also carries a recessive allele at the R locus, then this R-transformation of any cell of this line will lead to the formation of the red pigment. Examples of such lines are line Wisconsin 22, which contains the allele rg-Stedler and R112, and derived GOST types K55, which is r-g, b, P1. Alternatively, it may be used any genotype of corn, if C1 and R alleles are introduced together. Other scrinium marker suitable for use in the present invention, is the liquidation luciferase encoded by the genome of lux. The presence of the lux gene in transformed cells can be detected using, for example, x-ray film, scintillation counter, fluorescent spectrophotometry, cameras in low light, counters photons or multivalency luminometry. It is also believed that there may be developed a system for screening populations for bioluminescence, such as tablets for the cultivation of tissues or even for screening of whole plant.

Polynucleotide used for transformation of plants can be but are not limited to, DNA consisting of plant genes and practically genes, such as genes in bacteria, yeast, animals or viruses. Integrated DNA may include modified genes, parts of genes or chimeric genes, including genes from the same or from different genotypes of maize. The terms "chimeric gene" or "chimeric DNA" means a gene or DNA sequence or segment that contains at least two DNA sequences or segments from species that are not merged into a single DNA under natural conditions, either DNA sequences or segments located or attached as this does not usually occur in the native genome of the untransformed plants.

The present invention also relates to expressing the cassettes containing polynucleotide encoding hyperthermophilic processrule enzyme, preferably optimized codons polynucleotide. While it is preferable that polynucleotide specified in expressing cassette (the first polynucleotide) was functionally joined to regulatory sequences such as promoter, enhancer, intron, the sequence of termination or any combination, and, optionally, the second polynucleotide, codereuse signal sequence (N - or C-terminal), which directs the enzyme encoded by the first palynol what Atidim, in specific cellular or subcellular site. Thus, the promoter and one or more signal sequences can provide high levels of expression of the enzyme in specific areas of the plant, plant tissue or plant cells. Such promoters may be constitutive promoters, inducible conditional promoters or tissue-specific promoters, such as the endosperm-specific promoters such as the promoter γ-Zein of maize (represented by SEQ ID NO:12) or the promoter ADP-gpp corn (represented by SEQ ID NO:11, which includes 5′-and noncoding intron sequence). The present invention also relates to the selected polynucleotide containing a promoter comprising the sequence of SEQ ID NO:11 or 12, polynucleotide that hybridizes with its complement under low stringency, or its fragment, the promoter activity which is at least 10%and preferably at least 50% of the activity of the promoter having the sequence of SEQ ID NO:11 or 12. The present invention also relates to vectors that include expressing cassette or polynucleotide of the present invention, and the transformed cells containing polynucleotide expressing cassette or vector according to the invention. The vector according to the image is the shadow may contain polynucleotide sequence, encoding more than one hyperthermophilic processrule the enzyme of the present invention, where the sequence can be in sense or in antisense orientation, and a transformed cell can contain one or more vectors of the present invention. Preferred vectors are vectors that are suitable for injection of nucleic acid into cells of a plant.

Transformation

Expressing cassette or vector construct that contains expressing cassette which can be introduced into the cell. Expressing cassette or vector construct may be present in the formation of the episome or they can be integrated into the host cell genome. Then the transformed cell can be cultured, obtaining transgenic plants. In accordance with this present invention relates to products of transgenic plants. Such products can be, but are not limited to, seeds, fruit, offspring and progeny of transgenic plants.

To introduce the construct into the host cell has a number of known and available specialists methods. Transformation of bacteria and many eukaryotic cells can be carried out using polyethylene glycol, calcium chloride, viral infection, fagboy infection, electroporation and other methods known work, istam. Methods of transformation of plant cells or tissues are transformation using DNA as the transforming means is used A.tumefaciens or A.rhizogenes, electroporation, injection of DNA, bombardment by particles, particle acceleration, and the like (see, for example, EP and the EP 295959 138341).

In one of the embodiments of the invention the binary plasmid vectors Ti and Ri Agrobacterium spp., that is, derived Ti-vectors are used to transform a wide range of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape, 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 T-DNA for transformation of plant cells has received wide dissemination in scientific research and is described in detail in the literature (EP 120516; Hoekema, In: The Binary Plant Vector_System. Offset-drukkerij Kanters B.V.; 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)).

Specialists and other known methods of transformation, such as direct capture of foreign DNA constructs (see EP 295959), electroporation (Fromm et al., Nature (London)319:791 (1986) or high-speed ballistic bombardment metallicheskaya, covered by the structures of nucleic acids (Kline et al., Nature (London)327:70 (1987) and U.S. patent No. 4945050). After the transformation, the cells can be regenerated by methods known in the art. Particularly suitable are the recently described methods transform foreign genes into commercially valuable crops such as rapeseed (De Block et al., Plant Physiol. 91:694-701(1989)), sunflower (Everett et al., Bio/Technology. 5:1201(1987)), soybean (McCabe et al., Bio/Technology. 6:923 (1988); Hinchee et al., Bio/Technology. 6:915 (1988); Chee et al., Plant Physiol., 91:1212 (1989); Christou et al., Proc. Natl. Acad. Sci USA, 86:7500 (1989) EP 301749), rice (Hiei 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)).

Expressing the vectors containing genomic or synthetic fragments can be introduced into protoplasts or in intact tissue, or in the selected cells. Preferably expressing vectors injected into intact tissue. General methods of culturing plant tissues are described, 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 & Com Improvement, 3rd Edition 10, Sprague et al. (Eds.) pp. 345-387, American Society of Agronomy Inc. (1988).

In one of the embodiments of the invention expressing vectors can be introduced into corn or other plant tissue by the methods of direct gene transfer, such as mediated by microcast the AMI targeting, the DNA injection, electroporation and the like, Expressing the vectors are introduced into plant tissue by microparticle bombardment using booballistics 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 (1995). However, in the present invention considers the transformation of plants using hyperthermophilic processimage enzyme in accordance with known methods of transformation. Cm. 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. Natl. Acad. Sci. USA, 85:4305 (1988) (maize); Klein et al., Bio/Technology, 6:559 (1988) (maize); Klein et al., Plant Physiol., 91:440 (1988) (maize); Fromm et al., Bio/Technology, 8:833 (1990) (maize); and Gordon-Kamm et al., Plant Cell. 2, 603 (1990) (maize); Svab et al., Proc. Natl. Acad. Sci. USA, 87:8526 (1990) (tobacco chloroplast); Koziel et al., Biotechnology, 11:194 (1993) (maize); Shimamoto et al., Nature. 338:274 (1989) (rice); Christou et al., Biotechnology, 9:957 (1991) (rice); European patent application EP 0332581 (cocksfoot and other Grasses); Vasil et al., Biotechnology, 11:1553 (1993) (wheat). Methods in Molecular Biology, 82. Arabidopsis Protocols Ed. Martinez-Zapater and Salinas 1998 Humana Press (Arabidopsis).

The transformation can be carried out one DNA molecule or multiple DNA molecules (i.e. co-transformation), and both of these methods are suitable for the clients for use in conjunction with expressing cassettes and structures according to the invention. For plant transformation, there are many suitable transformation vectors, and expressing cassette according to the invention can be used in conjunction with any such vectors. The choice of vector depends on the preferred method of transformation and as targets for transformation.

And finally, the most preferred DNA segments for introduction into the genome of monocotyledonous plants can be a homologous genes or family of genes that encode a desired trait (e.g., hydrolysis of proteins, lipids or polysaccharides), which is administered under the control of new promoters or enhancers, etc. or maybe even under the control of homologous or tissue-specific (e.g., conspecifics, Sheiko/legalisations, motokosmetika, telesperience, postcompetition, perspectivistic or listspecific) promoters or regulatory elements. Indeed, provided that the specific use of the present invention will allow for directed gene targeting constitutive or inducible manner.

Examples of suitable transformation vectors

Numerous transformation vectors suitable for transformation of plants, known in the art for transformation of plants, and genes of the present invention can b shall be used in conjunction with any of the vectors, well-known specialists. The choice of vector will depend on the preferred method of transformation and the type of target used for the transformation.

A. Vectors suitable for Agrobacterium transformation

For transformation using Agrobacterium tumefaciens there are many vectors. These vectors usually carry at least one T-DNA border sequence, and such vectors are BIN19 (Bevan, Nucl. Acids. Res. (1984)). Below is the design of two types of vectors suitable for Agrobacterium-mediated transformation.

RSV and RSV

To construct recombinant vectors using Agrobacterium were used binary vectors RSV and RSV, which were constructed as follows. TJS75kan designed by splitting NarI TJS75 (Schmidhauser &Helinski, J. Bacteriol., 164:446 (1985)), that allowed us to cut out the gene of resistance to tetracycline, followed by embedding Ass fragment from plasmid pUC4 carrying NPTII (Messing & Essen, Gene, 19:259 (1982): Bevan et al., Nature, 304:184 (1983): McBride et al., Plant Molecular Biology, 14:266 (1990)). XhoI-linkers ligated with EcoRV fragment RSV, which contains the left and right border of T-DNA sequences, selective chimeric gene nos/nptII plants and polylinker pUC (Rothstein et al., Gene, 53:153 (1987)), a XhoI-cleaved fragment was cloned into SalI-cleaved pTJS75kan obtaining RSV (see also EP 0332104, example 19). RSV contain what it the following unique restriction sites polylinker: EcoRI, SstI, Kpnl, BglII, Xbal and Sail. pCIB2001 is a derivative of RSV constructed by embedding in polylinker additional restriction sites. Unique restriction sites in polylinker pCIB2001 are EcoRI, SstI, Kpnl, BglII, Xbal, Sall, Mlul, Bc1l, AvrII, Apa1, Hpa1 and StuI. Polylinker pCIB2001, in addition to these unique restriction sites also contains the gene for resistance to kanamycin for selection in plants and bacteria, the left and right border of T-DNA sequences for Agrohacterium-mediated transformation, functional trfA gene, derived from RK2, for activation in E. coli and other hosts, and functional genes OriT and OriV, also derived from RK2. Polylinker pCIB2001 is suitable for the cloning of plant expressing cassettes containing their own regulatory signals.

pCIB10 and its derivatives for selection on hygromycin

The binary vector pCIB10 contains the gene for resistance to kanamycin for selection in plants and the left and right border of T-DNA sequence and introduces a sequence derived from plasmid pRK252 broad host range, allowing the vector to replicate in E. coli and in Agrobacterium. Its construction is described Rothstein et al. (Gene., 53:153 (1987)). Were designed into various derivative RSV that impose gene hygromycin In-phosphotransferase described by Gritz et al. (Gene, 25179 (1983)). These derivatives allow for the selection of transgenic plant cells only hygromycin (RSV) or hygromycin and kanamycin (RSV, RSV).

b. Vectors suitable for transformation, not mediated by Agrobacterium

Transformation without the use of Agrobacterium tumefaciens allows you to "bypass" the requirement of the presence of T-DNA sequences when choosing a transforming vector, and therefore, addition of vectors, such as described above vectors containing T-DNA sequences, can be used vectors that do not have these sequences. The transformation functions that do not depend on Agrobacterium, provide for the implementation of the transformation by microparticle bombardment, the capture of protoplasts (e.g., PEG and electroporation) and microinjection. The choice of vector, mainly depends on the preferred selection of transformed plants. The following are non-limiting examples of design typical vectors suitable for transformation without the use of Agrobacterium.

RSV

RSV is a vector originating from the pUC and suitable for implementing the methods of direct gene transfer in combination with the method of selection by the herbicide basta (or phosphinothricin). Plasmid RSV contains the promoter CaMV 35S, functionally attached to gene GUS gene and the terminator of transcription of the 35S CaMV and described in the published PCT application WO 93/07278. The 35S promoter of this vector contains two sequence ATG from 5′-end from the site of initiation. These sites were subjected to mutation using standard PCR techniques in order to remove the ATG and generation of restriction sites Sspl and Pvul. New restriction sites were located at a distance of 96 and 37 BP from the unique SalI site and at a distance of 101 and 42 BP from the existing site of initiation. Derived RSV meant RSV. Then GUS gene was cut out from RSV by splitting enzymes SalI and Sad, the end was a small mistake and again ligated to obtain plasmid RSV. Plasmid JI82 can be taken from the Centre John Innes (Norway), and 400 BP-SmaI fragment containing the bar gene from Streptomyces viridochromogenes, carved and built into the HpaI site RSV (Thompson et al., EMBO J. 6:2519 (1987)). This generated vector RSV contains the bar gene under the control of the promoter of CaMV 35S and terminator for selection for herbicide resistance gene for ampicillin (for selection in E. coli) and polylinker with a unique SphI sites, PST, HindIII and BamHI. This vector is suitable for the cloning of plant expressing cassettes containing their own regulatory signals.

PSOG19 and pSOG35

Plasmid pSOG35 is a transforming vector that contains a gene digidrofolatreduktazy E.coli (DHFR) as a selective marker, giving resistentes is to methotrexate. To amplify the 35S promoter (-800 BP), intron 6 of the gene of maize Adh1 (-550 BP) and 18 BP untranslated leader sequence from GUS SOG10 used PCR. 250-BP fragment encoding the gene hydropolitics type II gene is also amplified by PCR and these two PCR fragments were assembled using SacI- > PST fragment from the vector RV (Clontech), which contains the skeleton of the vector pUC19 and the terminator nopaline-synthase. The Assembly of these fragments leads to the generation of vector SOG19, which contains the 35S promoter attached to the sequence of intron 6, a leader sequence GUS, DHFR gene and the terminator nopaline-synthase. Replacement leader sequence GUS in SOG19 on leader sequence derived from virus chlorotic leaf spot of maize (MCMV), leads to the generation of vector SOG35. Vectors SOG19 and SOG35 carry the gene Fig resistance to ampicillin and have HindIII-, SphI -, PST and EcoRI sites available for the cloning of foreign material.

C. a Vector suitable for transformation into chloroplasts

For expression of the nucleotide sequence of the present invention in plastids of plants used vector for transformation in plastids RRN (WO 97/32011, example 36). The nucleotide sequence was built in rn to replace the coding sequence of PROTOX. Then this vector was used for transformation is rmacie in plastids and for selection of transformants for resistance to spectinomycin. Alternatively, the nucleotide sequence was built in rn to replace aadH gene. In this case, the transformants were selected for resistance to PROTOX inhibitors.

Host-plants subjected to transformation

Any plant tissue capable of clonal reproduction, regardless of organogenesis or embryogenesis, may be transformed with constructs of the present invention. The term "organogenesis" means the process by which shoots and roots consistently develop from meristematic centers, and the term "development" means the process by which shoots and roots are developed simultaneously (not sequentially) or from somatic cells or gametes. The choice of a particular tissue can vary depending on systems clonal reproduction available and most appropriate for specific transformable species. Examples of target tissues are differentiated and undifferentiated tissues or plants, including but not limited to, disks, leaves, roots, stems, shoots, leaves, pollen, seeds, embryos, cotyledons, hypocotyl, megagametophytes, callus tissue, present meristematic tissue (e.g., apical meristem, axillary buds, and root meristem), and induced meristem tissue (for example, the meristem of the cotyledons and the hypocotyl meristem), tumors of ewww fabric and various forms of cells and culture such as single cells, protoplasts, embryos, and callus tissue. This plant tissue may be present in the plant or in an organ or tissue or cell culture.

Plants of the present invention can take various forms. Such plants may be chimeras of transformed cells and normal cells; plants can be clonal transformants (e.g., all cells transformed to contain the expressing cassette); plant may contain grafts of transformed or untransformed tissues (e.g., transformed root escape, grafted to the untransformed escape citrus plants). Transgenic plants can be propagated in various ways, such as clonal propagation or the classic way of natural breeding. For example, the first generation (or T1) transformed plants may be subject to self-pollination to obtain a second generation (or T2) of the transformed plants and plant T2 can then be multiplied by classical methods of plant breeding. To facilitate the cultivation of a dominant selective marker (such as npt II) can be associated with expressing cassette.

The present invention can be used for transformation of any kind is in plants, namely monocotyledonous or dicotyledonous plants, including, but not limited to, corn (Zea mays), Brassica sp.(for example, B.napus, B.rapa, B.juncea), and in particular, species of Brassica plants used as a source for oil seed, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., millet American (Pennisetum glaucum), pearl millet (Panicum miliaceum), Italian millet (Setaria italica), Elefsina (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctoris), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), figs (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.), oats, barley, vegetable plants, ornamental plants, woody plants such as coniferous and deciduous trees, pumpkin large ordinary pumpkin, hemp, zucchini, Apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, BlackBerry, soybean, sorghum, sugar cane, rapeseed, clover, carrots, resusci tal (Arabidopsis thalina).

Vegetable plants are tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), Lima beans (Phaseolus limensis), peas (Lathyrus spp.), cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery and members of the genus Cucumis such as cucumber (C.sativus), cantalupa melon (C.cantalupensis) and cantaloupe (.melo). Ornamental plants are azaleas (Rhododendron spp.), hydrangea (hydrangea Macrophylla), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum. Coniferous trees, which can be used to implement the present invention are, for example, pines such as pine incense (Pinus taeda)pine Elliott (Pinus elliotii), yellow pine (Pinus ponderosa), pine twisted sirakorola (Pinus contorta), and Monterey pine (Pinus radiata), getsuga tesselata (Pseudotsuga menziesii); Western Hemlock (Tsuga canadensis); Sitka sichinskaja (Picea glauca); Sequoia sempervirens (Sequoia sempervirens); fir real, such as white fir (Abies amabilis) and balsam fir (Abies balsamea), and cedars such as arborvitae (Thuja plicata) and yellow cypress Alaska (Chamaecyparis nootkatensis). Legume plants are beans and peas. Beans are guar, carob, fenugreek Sennoy, soy, beans crop, cowpea Chinese, mung bean, Lima beans, Fava beans, lentils, peas, baranyi etc. Legume plants are, but are not limited to, Arachis, for example, peanuts, Vicia, for example, Basel, hairy vetch, beans radiant, mung bean and pea lamb, Lupinus, for example, lupine, clover, Phaseolus, e.g., kidney beans and Lima beans, pea, for example, field beans, Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil, Lens, for example, lentils, and Sophora Australian. Preferred forage and tarnobrzeska plants, suitable for use in the methods of the present invention are Lucerne, cocksfoot, ovsyannitsa high, perennial ryegrass, bentgrass and white bentgrass small.

Preferred plants of the present invention are crop plants such as corn, alfalfa, sunflower, cabbage Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, barley, rice, tomato, potato, pumpkin garden, melons, legumes, etc. in Other preferred plants are Liliopsida and Panicoideae.

After the transformation, the desired DNA sequence in a plant of a specific form, it may be amplified in the plant of the same species or introduced in another plant variety of the same species, and in particular, industrial grade, used in traditional methods of cultivation.

The following describes the transformation of dicotyledonous and monocotyledonous plants, and representative way of transformation in plastids.

A. Transformation of dicotyledonous plants

Methods for transformation of dicotyledonous plants is well known in the art, and such methods are methods using Agrobacterium and methods without the use of Agrobacterium. Methods without the use of Agrobacterium include the capture of exogenous genetic material directly protoplasts or cells. This can be done by PEG-mediated introduction, introduction by electroporation, delivery by microparticle bombardment or by microinjection. Examples of such methods are described in the literature (Paszkowski et al., EMBO J. 3:2717 (1984), Potrykus et al. Mol. Gen. Genet., 199:169 (1985), Reich et al., Biotechnology, 4:1001 (1986), and Klein et al., Nature, 327:70 (1987)). In each case, the transformed cells regenerate with getting the whole plant by standard methods known to experts.

Agrobacterium-mediated transformation is the preferred method of transformation of dicotyledonous plants because of its high efficiency of transformation and availability for many species of plants. Agrobacterium transformation typically involves the transfer of the binary vector carrying interest of foreign DNA (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain, and may depend on the complement of vir genes, which are present is in the strain-host Agrobacterium, or present together on the Ti-plasmid or on the chromosome (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 carried out by crossing the three parental lines using recombinant binary vector and the helper strain E. coli carrying a plasmid, such as RK2013, and which is able to mobilize this recombinant binary vector to the strain-target Agrobacterium. Alternatively, the indicated recombinant binary vector can be transferred to Agrobacterium by DNA transformation sequence (Höfgen & Willmitzer, Nucl. Acids. Res., 16:9877 (1988)).

Transformation of plants desired recombinant agrobacteria usually provides for joint cultivation of Agrobacterium with Explant taken from a plant, in accordance with the following protocols well known in the art. Transformed tissue regenerate on the selective medium bearing a token resistance to the antibiotic or herbicide found between T-DNA flanking sequences in the binary plasmid.

Vectors can be introduced into plant cells by the known methods. Cells are preferred for transformation, Agrobacterium are, monocotyledonous cells and cells of dicotyledonous plants, including cells (Liliopsida) and Panicoideae. Preference is sustained fashion by cells of monocotyledonous plants are the cells from cereals, for example, maize (corn), barley, and wheat, and kamalabadi dicotyledonous plants, such as potatoes.

Another way of transforming cells of a plant provides a "run" of particles, which is inert or biologically active in the cells and tissues of plants. This method is described in U.S. patent No. 4945050, 5036006 and 5100792. In General, this procedure involves the putting in motion of particles, which is inert or biologically active cells, under conditions effective to pass through the outer surface of the cell and activate inside cells. If you are using inert particle, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, cell-target may be surrounded by a vector to this vector was introduced into the cell using a given particle. Biologically active particles (e.g., dried yeast cells, drained bacteria or bacteriophage, which contain DNA, which is designed for injection) may also be introduced into cells and plant tissue.

b. The transformation of monocotyledonous plants

Currently, the transformation of most species of monocotyledonous plants has become a routine procedure. Preferred methods is a direct capture of the gene protoplasts using polyethylene glycol (PEG), or the technique of switching-mode power the radio, as well as the bombardment of callus tissue particles. The transformation can be carried out using one type of DNA or multiple types of DNA (i.e. co-transformation), and both of these methods can be used in the present invention. Co-transformation may have the advantage that it avoids the construction of the full vector and to generate transgenic plants with non-linked loci for the desired gene and a selective marker and, thus, allows to delete this selective marker in subsequent generations, which should be considered as a positive factor. However, the disadvantage of using co-transformation is that in this case, certain types of DNA integrated into the genome with a frequency of less than 100% (Schocher et al., Biotechnology, 4:1093, 1986).

In patent applications EP 0292453, ER and WO 93/07278 described methods of obtaining callus and protoplasts of elite imbrogno lines of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. In the work of Gordon-Kamm et al. (Plant Cell, 2:603 (1990)) and Fromm et al. (Biotechnology, 8:833 (1990)described the methods of transformation lines of maize originating from A through bombardment by particles. In addition, WO 93/07278 and work Koziel et al. (Biotechnology, 11:194 (1993)) describes the methods of transformation of elite inbred the lines of maize by particle bombardment. This method was used immature embryos of maize in length, 1.5-2.5 mm, cut from the cob of corn after 14-15 days after pollination, and booballistics device PDS-ne for the bombing.

Transformation of rice can also be carried out by methods of direct gene transfer using protoplasts or by particle bombardment. Mediated by protoplasts transformation has been described for species Japonica and Indica (Zhang et al., Plant. Cell. Rep., 7:379 (1988); Shimamoto et al., Nature 338: 274 (1989); Datta et al., Biotechnology, 8:736 (1990)). Both of these types can be routinely transformed by particle bombardment (Christou et al., Biotechnology, 9:957 (1991)). In addition, in WO 93/21335 described methods for the transformation of rice via electroporation. In patent application EP 0332581 described methods of generation, transformation and regeneration of protoplasts of plants of the family of Grasses (Pooideae). These methods allow for a transformation of Dactylis and wheat. In addition, transformation of wheat was described by Vasil et al. (Biotechnology, 10:667 (1992)) by particle bombardment of long-lived cells of the regenerated callus type, and Vasil et al. (Biotechnology 11:1553 (1993)) and Weeks et al. (Plant Physiol., 102:1077 (1993)), by particle bombardment of immature embryos and calli derived from immature embryo. However, the preferred method of transformation of wheat involved Tran the formation of wheat by particle bombardment of immature embryos and includes a step of cultivation with a high content of sucrose or high content of maltose before delivery of the gene. Before the bombing any number of embryos (length of 0.75-1 mm) were sown in the MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum, 15:473 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, which was left in the dark. On the day selected for bombardment, the embryos were removed from the medium to induce and placed in conditions osmosis (i.e. in the environment to induce containing sucrose or maltose in the desired concentration, usually 15%). Embryos were left for 2-3 hours for plasmolysis, and then was bombarded. Number twenty embryos at this tablet is typical, but not critical. The plasmid that carries the appropriate gene (such as RSV or SG35) flocked to the gold particle size of several microns using standard procedures. Each tablet embryos were shot using helium booballistics device DuPont Biolistics® when the burst pressure of approximately 1000 psi using a standard sieve of 80 mesh. After bombardment, the embryos were placed back into the darkness for about 24 hours (still in terms of osmosis). After 24 hours, the embryos were removed from the environment in terms of osmosis and put back on Wednesday for induction, where they were about one month before regeneration. About a month later the embryo explants with developing embryogenic callus was transferred to medium DL is regeneration (MS+1 mg/liter NAA, 5 mg/liter GA), which additionally contains the appropriate selective agent (10 mg/l basta if RSV and 2 mg/l of methotrexate in the case of SOG35). After about one month developed shoots was transferred into a larger sterile containers, known as "GA7s", which contained policecontributing MS, 2% sucrose and the same concentration of selective agent.

Was also described transformation of monocotyledonous plants using Agrobacterium. Cm. WO 94/00977 and U.S. patent No. 5591616 that are entered into the present description by reference.

C. Transformation of plastids

Seeds of Nicotiana tabacum, cultivar Xanthi nc ' are germinated in the number seven seed in the tablet in 1-inch circular array on T agar medium and 12-14 days after sowing was bombarded with tungsten particles with a size of 1 μm (M10, Biorad, Hercules, CA)coated with DNA derived from the plasmid rn and rn, basically as described in the literature (Svab &Maliga, PNAS, 90:913 (1993)). Bombed seedlings are incubated on T medium for two days after which leaves are cut and placed with its back side in bright light (350-500 µmol·Foton/m2/s) on tablets with RMOP medium (Svab, Hajdukiewicz & Maliga, PNAS, 87:8526 (1990))containing 500 μg/ml spectinomycin dihydrochloride (Sigma, St Louis, MO). After three to eight weeks after the bombing resistant shoots are poobustroennej leaves, was subjected to subclavian on the same selective medium, then left for the formation of callus and secondary shoots were separated and subclinically. Full segregation copies of the transformed plastid genome (homoplasies) in independent subclones were analyzed by standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1989)). Full cellular DNA cleaved BamHI/EcoRI (Mettler I.J. Plant. Mol. Biol. Reporter, 5:346 (1987)), was separated on an agarose gel with 1% Tris-borate (TBE), transferred to nylon membranes (Amersham) and probed32P-labeled random praimirovanie DNA sequences corresponding to a 0.7 TPN-BamHI/HindIII-DNA fragment from plasmid RS containing part of the plastid targeting sequence rps7/12. Homoplasmic shoots aseptically were rooted on MS medium/IBA containing spectinomycin (McBride et al., PNAS, 91:7301 (1994)), and transferred into the greenhouse.

Production and characterization of stably transformed plants

Cells transformed plants were placed in the appropriate selective medium for selection of transgenic cells, which are then grown to callus. From callus was grown shoots, and from these shoots were generated small plants by culturing in the medium for rooting. Various designs were usually attached to the marker for selection in ledah plants. Typically, the marker is a gene for resistance to a biocide (and in particular, to the antibiotic, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide or the like). Specifically used the marker allows to differentiate transformed cells from cells that do not contain introduced DNA.

Components of DNA structures, including transcription/expressing cassette according to the invention, can be obtained from the sequences which are native (endogenous) or alien (exogenous) to the host. The term "alien" means that the sequence is not found in the host wild-type, in which introduced this design. Heterologous constructs will contain at least one region that is not native to the gene originates from the region of transcription initiation.

To confirm the presence of transgenes in transgenic cells and plants can be carried out southern blot analysis by methods known in the art. Integration segments poliolefinovoy acid into the genome can be detected and quantified using southern blot analysis, as they can be easily differentiated from structures containing these segments, using suitable restrictively enzymes. The products of expression of tra is of shenow can be detected by any means depending on the nature of this product, and those methods are Western blot analysis and enzyme analysis. One of the specific methods used for quantitative assessment of protein expression and detection of replication in different tissues of plants is the use of gene-reporter, such as GUS. After obtaining transgenic plants, they can be cultured to generate tissues or parts of plants that have the desired phenotype. Can be obtained from plant tissues or plant part and/or collected seeds. Seeds can serve as a source for the cultivation of other plants with tissues or parts having the desired characteristics.

Thus, the present invention relates to a transformed plant or plant part, such as a spike, seeds, fruit, grain, straw, scales cereals or bagasse containing at least one polynucleotide expressing cassette or vector according to the invention, the method of production of such plants and to methods of using such plant or part thereof. Specified the transformed plant or part of plant expresses processrule enzyme, optionally present in a particular cell or subcellular compartment of some tissues or in germinating grain. For example, the present invention relates to a transformed plant containing at least one is rahmenpresse enzyme, present in the cells of specified plants, where the plant part is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one cromartyshire enzyme. Processrule the enzyme does not act on the substrate-target, unless it is activated by methods such as heating, grinding, or other methods that ensure the engagement of the specified enzyme with the substrate under conditions where the specified enzyme is active.

Preferred methods of the present invention

Sameprinciples plants and parts of plants of the present invention can be used in various methods using processorbased enzymes (mesophilic, thermophilic, or hyperthermophilic), expressed and activated in these plants. In accordance with the present invention a portion of a transgenic plant derived from the transgenic plant, the genome of which is replenished at least one processrule the enzyme is placed in conditions in which the antigen is expressed and activated this processrule enzyme. After activation processrule the enzyme is activated and interacts with the substrate to which it normally operates, with the production of the desired product. For example, cromartyshire enzymes de is available in the starch, followed by its decomposition, hydrolysis, isomerization, or any other modification, and with the formation of the desired product during activation. Enzymes, not processorsa starch, can be used to destroy the cell membrane of a plant in order to stimulate the extraction of starch, lipids, amino acids or other products of plants. In addition, neperturbativnye and hyperthermophilic enzymes can be used in combination with sameprinciples plants or parts of plants of the present invention. For example, mesophilic enzyme that does not destroy starch, can be activated for the destruction of the cell membrane of a plant for the extraction of starch, and then in this sameprinciples the plant can be activated hyperthermophilic brahmarishi enzyme to break down starch.

The enzymes expressed in the grain, can be activated by placing plants or parts of plants containing these enzymes, the conditions under which stimulates their activity. So, for example, can be used one or more of the following methods. Part of the plant may come in contact with water, which is a substrate for hydrolytic enzyme, and thereby activate the enzyme. Part of the plant may come in contact with water, which contributes to the migration of the enzyme from compartment, to who m it settles in the process of development of the plant part and, thus, is associated with its substrate. Migration of the enzyme is due to the fact that during maturation, drying and rehydration occurs violation of compartmentalization. Whole or broken grain may come in contact with water, allowing the enzyme to migrate from compartment, in which he deposited in the process of developing parts of the plant, and thus be associated with its substrate. Enzymes can be activated by adding an activating compound. For example, Kalnyshevsky the enzyme can be activated by the addition of calcium. Other activating compounds can be determined by methods known in the art. Enzymes can be activated by removing inactivator. For example, such inactivators are known peptide inhibitors of amylase enzymes, where amylase can be co-expressed with amylase inhibitor, and then activated by the addition of protease. Enzymes can be activated by changing the pH to a value at which a specified enzyme is most active. Enzymes can be activated by increasing the temperature. The enzyme activity usually increases when reaching the maximum temperature for this enzyme. The activity of the mesophilic enzyme will increase, since the level of the I activity at room temperature and allowed to reach temperature, in which his activity will be reduced, i.e. usually at a temperature of less than 70°or at temperatures equal to 70°C. Similarly, thermophilic and hyperthermophilic enzymes can be activated by increasing the temperature. Thermophilic enzymes can be activated by raising the temperature to the maximum temperature of their activity or stability. For thermophilic enzyme maximum temperature stability and activity are usually in the range from 70 to 85°C. Hyperthermophilic enzymes will have even higher relative activity than mesophilic or thermophilic enzymes, due to their stronger change of potential at a temperature of from 25°C to 85°C-95°or even up to 100°C. the Elevated temperature may be achieved by any method, such as heating, such as baking, boiling, heat treatment with hot steam, electric discharge or any combination thereof. In addition, plants expressing mesophilic or thermophilic enzyme(s), activates a specific enzyme can be carried out by crushing plants, thereby, facilitates the contacting of the specified enzyme with the substrate.

Optimal conditions, such as temperature, hydration, pH, etc. can be determined using any of the JV is the expert and can depend on the specific of enzyme used and purpose of use of the enzyme.

In addition, the present invention relates to the use of exogenous enzymes that can stimulate a specific process. For example, sameprinciples plant or plant part of the present invention can be used in combination with exogenous enzyme to accelerate the reaction. For example, transgenic maize containing α-amylase may be used in combination with other cromartyshire enzymes, such as pullulanase, α-glucosidase, glucose isomerase, mannanase, hemicellulase etc. for the hydrolysis of starch or the production of ethanol. Indeed, it was found that the combination of transgenic corn containing α-amylase, such enzymes resulted in unexpectedly high levels of conversion of the starch in comparison with the use of transgenic corn containing only α-amylase.

Below is an example of suitable methods discussed in this application.

A. Extraction of starch from plants

The invention relates to a method of facilitating the extraction of starch from plants. In particular, at least one polynucleotide encoding processrule the enzyme that destroys physically limiting matrix of the endosperm (the cell wall of non-starch polysaccharide and the protein matrix is injected into rasteniya, to this enzyme, it is preferable that were in close physical proximity to the starch grains in plant. In this embodiment of the invention transgenic plants, preferably, Express one or more enzymes, such as protease, glucanase, xylanase, thioredoxin/thioredoxin, esterase and the like, but not enzymes that have any brahmarakshasa activity that contributes to maintaining the integrity of the starch grains. Thus, the expression of these enzymes in plants, such as grains, leads to improvement of the functional properties of the grain. Processrule enzyme may be mesophilic, thermophilic, or hyperthermophilic. In one example of the grain of transgenic plants of the present invention is dried by heating, which probably leads to inactivation neperturbatiwnyh processorbased enzymes and to better preserve the integrity of the seed. The grain (or crushed grain) soaked at low temperatures or at high temperatures (where time plays an important role in conditions of high or low humidity (see manual Primary Cereal Processing, Gordon & Willm, eds., pp.319-337 (1994), the content of which is introduced into the present description by reference), in the presence or in the absence of sulfur dioxide. When increasing the temperature, optionally in the conditions is x specific humidity, the integrity of the matrix of the endosperm is broken through the activation of enzymes such as protease, xylanase, phytase or a glucanase which break down proteins and polysaccharides, non-starch present in the endosperm, resulting starch grains remain intact and can be more easily separated from the resulting material. In addition, facing proteins and polysaccharides, non-starch, become at least partially cleaved and highly concentrated, and therefore they can be used for improved animal feed, food products or as components of media used for fermentation of microorganisms. It is assumed that leaving the substance is a liquid corn extract with an improved structure.

Thus, the present invention relates to a method for producing starch grains. The method includes processing the grain, such as crushed grain, which includes at least one non-processrule starch enzyme under conditions which activate the at least one enzyme, to obtain a mixture containing starch grains and not containing starch decomposition products, such as split products matrix of the endosperm. Not processrule starch enzyme may be mesophilic, thermophilic, or hyperte Mogilny. After activation of the enzyme to the starch granules are separated from the mixture. The grain is obtained from a transformed plant, the genome of which contains (refilled) expressing cassette encoding at least one processrule enzyme. For example, the specified processormask enzyme may be a protease, glucanase, xylanase, phytase, thioredoxin/thioredoxin or esterase. Specified processrule enzyme preferably is hyperthermophilic. Grain can be processed in low or high humidity, in the presence or in the absence of sulfur dioxide. Depending on the activity and expression level processimage enzyme in the seed of transgenic plants transgenic seed can be mixed with commodity grain, before or during processing. Also discusses the products obtained by the above method, such as starch, food, containing no starch; and water for soaking, containing at least one accessory component.

b. Methods of processing starch

Transgenic plants or parts of plants of the present invention may contain described here krahmalosushilnye enzymes that break down starch grains to dextrins and other modified starches or hexoses (e.g., α-amylase, pullulanase, α-glucosidase, glucoamylase, is mylopoulos) or convert glucose into fructose (for example, glucose isomerase). Preferably, the specified brahmarishi enzyme selected from α-amylase, α-glucosidase, glucoamylase, pullulanase, neopolitans, aminophylline, glucose and combinations thereof, and its use for transformation of grain. In addition, it is preferable that the specified enzyme was functionally attached to the promoter or signal sequence that targets the enzyme to the starch grain, amyloplast, the apoplast or to the endoplasmic reticulum. More preferably, if the enzyme is expressed in the endosperm and in particular, in the endosperm of corn, and is localized in one or more cell compartments or within the starch grains. The preferred part of the plant is corn. The preferred parts of the plant are plant parts of maize, wheat, barley, rye, oats, sugar cane or rice.

In accordance with one method of the decomposition of the starch of the present invention, the transformed grain accumulating brahmarishi enzyme in starch grains, soaked, usually at 50°-60°and subjected to wet-milling method known in the art. Preferably, the specified brahmarishi enzyme was hyperthermophilic. Thanks subcellular targeting of the enzyme to krahmann the e grain or due to the Association of the specified enzyme with starch grains by contact of the enzyme and starch grains in the process of wet grinding at standard temperature processrule the enzyme is subjected to joint treatment with starch grains with obtaining a mixture of starch grains/enzyme". After receiving a mixture of starch grains/enzyme, the enzyme is activated in conditions conducive to the activation of this enzyme. So, for example, processing may be carried out in different conditions of humidity and/or temperature to facilitate partial (for derivatizing of starches or dextrins or complete hydrolysis of starch in hexose. In the same way also get the syrups with a high content of equivalents of dextrose or fructose. This method allows you to effectively save time, energy, and the cost of the enzyme and increase the efficiency of the conversion of starch in the corresponding hexose and efficiency of production of products, such as water for steeping high in sugar and syrups with a high content of dextrose equivalents.

In another embodiment of the invention, the plant or product of plants, such as fruit, or grain, or flour made from grain, which expresses the specified enzyme process for activation of the enzyme and transformation expressed and contained in the plant polysaccharides in sugar. This enzyme, preferably, attached to a signal sequence that targets the enzyme to the one described descramble grain, amyloplast, the apoplast or the endoplasmic reticulum. Produced sugar can then be selected or extracted from a plant or product of plants. In another embodiment of the invention processrule enzyme capable of converting polysaccharides in sugar, placed under the control of the inducible promoter by methods known in the art, and the methods described in this application. Processrule enzyme may be mesophilic, thermophilic, or hyperthermophilic. The plant is grown to the desired stage, and at this stage is induced by the promoter driving the expression of the enzyme and the conversion of polysaccharides in the plant or product plants in sugar. The enzyme is preferably functionally attached to a signal sequence that targets the enzyme described herein starch grains, amyloplast, the apoplast or to the endoplasmic reticulum. In another embodiment of the invention produce a transformed plant that expresses processrule enzyme capable of converting starch into sugar. The enzyme attached to the signal sequence, the target of this enzyme on starch grain plants. After that, the starch isolated from the transgenic plants, which contains the enzyme expressed by the transformed plant is receiving. Then the enzyme contained in the selected starch, can be activated for the conversion of starch into sugar. This enzyme can be mesophilic, thermophilic, or hyperthermophilic. In this application describes examples of hyperthermophilic enzymes capable of converting starch into sugar. The methods described can be applied to any plant that produces a polysaccharide and which can Express the enzyme, capable of converting the polysaccharide in sugar or hydrolyzed krahmaloprodukt, such as dextrin, maltooligosaccharide, glucose and/or mixtures thereof.

The present invention relates to a method for producing dextrins and modified starches from plants or product of such plants, which were transformed processormask enzyme gidrolizuut some covalent bonds of the polysaccharide with the formation of the polysaccharide derivative. In one of the embodiments of the invention, the plant or the product of this plant, such as fruit, or grain, or flour made from corn expressing the indicated enzyme is placed in conditions favorable for activation of the enzyme and the conversion of polysaccharides contained in this plant, in the polysaccharides of lower molecular weight. The enzyme is preferably added to the signal sequence, which Nazeli the AET of this enzyme described herein starch grains, amyloplast, the apoplast or the endoplasmic reticulum. Produced dextrin or derivationally starch can then be selected or extracted from plants or from plants. In another embodiment of the invention processrule an enzyme able to convert polysaccharides into dextrins and modified starches, placed under the control of the inducible promoter by methods known in the art and described in this application. The plant is grown to the desired stage, and then is induced promoter that initiates the expression of the enzyme and the conversion of polysaccharides in the plant or in the product of this plant in dextrins or modified starches. The preferred enzyme is α-amylase, pullulanase, ISO - or neo-pullulanase, and this enzyme is functionally connected to a signal sequence that targets the enzyme described herein starch grains, amyloplast, the apoplast or the endoplasmic reticulum. In one of the embodiments of the invention the specified enzyme targeting to the apoplast or to the endoplasmic reticulum. In yet another embodiment of the invention receive a transformed plant that expresses an enzyme capable of converting starch into dextrin or modified starches. The enzyme PR is connected to the signal sequence, which directs the enzyme to the starch grains of the plant. Then the starch isolated from the transgenic plants, which contains the enzyme expressed by the transformed plant. The enzyme contained in the selected starch may then be activated under conditions conducive to the transformation of starch into dextrins or modified starches. In this application describes examples of hyperthermophilic enzymes capable of converting starch to split the starch. The methods described can be applied to any plant that produces a polysaccharide and which can Express the enzyme, capable of converting the polysaccharide in sugar.

In another embodiment of the present invention, the grain obtained from the transformed plants of the present invention, which accumulate amylolytic enzymes that disrupt the communication of starch grains with the formation of dextrins modifitsirovannyh starches or hexose (for example, α-amylase, pullulanase, α-glucosidase, glucoamylase, aminophylline), soaked in conditions conducive to the activation krahmalsoderzhashchego enzyme within a certain period of time. The mixture may contain high levels of krahmaloprodukt. The use of such grain (1) avoids the need to grind grain or to the some or other processing of grain, usually used to obtain starch grains, (2) makes the starch more accessible to enzymes due to the presence of enzymes directly into the tissue of the endosperm of the grain, and (3) avoids the need for microbiological production krahmalsoderzhaschih enzymes. Thus, the whole process of wet grinding to highlight hexose can be replaced by simple heating of the grain, preferably corn, in the presence of water, which stimulates the action of enzymes on starch.

This method can also be used to produce ethanol, syrups, high fructose fermentation medium containing hexose (glucose), or for any other transformation of starch, which does not require purification of the components of the grain.

The present invention also relates to a method for producing dextrin, maltooligosaccharides and/or sugar, including the processing of parts of plants, containing starch grains or at least one cromartyshire enzyme under conditions that stimulate the activation of at least one enzyme with subsequent enzymatic breakdown of starch grains and obtaining an aqueous solution containing sugar. Plant part is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes a p is at least one processrule enzyme. Then collect the aqueous solution containing dextrins, maltooligosaccharide and/or sugar. In one of the embodiments of the invention processormask enzyme is α-amylase, α-glucosidase, pullulanase, glucoamylase, aminophylline, glucose isomerase, or any combination thereof. Preferably, the specified enzyme was hyperthermophilic. In another embodiment of the invention the method additionally includes the allocation of dextrins, maltooligosaccharides and/or sugar.

C. Improved varieties of maize

The present invention also relates to the production of improved varieties of maize (and varieties of other crops), which have normal levels of accumulation of starch and accumulate sufficient levels of amylolytic enzyme(s) in its endosperm, or nakaplivaya starch body, such that after activation contained enzyme, for example by boiling or heating of the specified plant or part thereof, in the case of a hyperthermophilic enzyme specified(e) the enzyme(s) was activated and stimulated rapid conversion of starch into simple sugars. These simple sugars (mainly glucose) will give a processed corn sweetness. The resulting plant corn will be an improved variety, designed for dual-use spacecraft is as thereproductive hybrid, and as sweet corn. For example, the present invention relates to a method for producing sverhslojnoe corn, including the processing of transformed corn or part thereof, which the gene is credited with the introduction of expressing cassette and expresses this expressing cassette in the endosperm, where this cassette contains a promoter functionally attached to the first polynucleotide, codereuse at least one brahmarishi the enzyme under conditions which activate at least one enzyme, so that it turned polysaccharides corn sugar, with the formation of sverhslojnoe corn. The promoter may be a constitutive promoter, semispecific promoter or endoparasiticides promoter that is joined to a polynucleotide sequence that encodes processrule enzyme, such as α-amylase, for example, contains the sequence of SEQ ID NO:13, 14, or 16. The preferred enzyme is hyperthermophilic. In one of the embodiments of the invention expressing cassette further comprises a second polynucleotide, which encodes the signal sequence is functionally attached to the enzyme encoded by the first polynucleotide. The signal sequence characteristic of this variant implementation from the retene, target the specified enzyme in the apoplast, endoplasmic reticulum, starch grains or amyloplast. Plant corn is grown prior to the formation of cobs with grain, after the induction of the promoter, resulting in expression of the enzyme and the conversion of polysaccharide contained in this plant, sugar.

d. Samoformatiranja plants

In another embodiment of the invention are plants, such as corn, rice, wheat or sugar cane, are manufactured so that their cell walls have accumulated large quantities of processorbased enzymes, such as xylanase, cellulase, hemicellulase, glucanase, pectinase, etc. (enzymes that destroy polysaccharides, non-starch). After collecting the components of the grain (or sugar if sugar cane) as the source of enzyme, which goes for expression and accumulation in the cell walls, and as a source of biomass use straw, scaly grains or bagasse. Straw (or other unwanted tissue) used as starting material in the method of selection of ferment sugars. The method of obtaining such formatiruem sugars is activation of the enzyme that destroys polysaccharides, non-starch. For example, activation may include heating plant tissues in Pris the accordance of water for a period of time, sufficient to break down non-starch polysaccharide with the formation of sugars. Thus, sameprinciples straw produces enzymes that are necessary for the conversion of polysaccharides into monosaccharides, and who, in General, does not require additional costs, because they are components of the source material. In addition, heat-dependent enzymes do not have any adverse effect on the growth and development of plants, and on targeting the cell wall, and even on targeting polysaccharide microfiber thanks binding cellulose/xylose domains attached to a protein that increases the availability of substrate for this enzyme.

Thus, the invention relates to a method of use part of the transgenic plants, including targeting, at least one enzyme, gidrolizuemye polysaccharide, non-starch in the cell wall of the cells of this part of the plant. The method includes processing part transgenic plants containing at least one enzyme, processrule polysaccharide, non-starch under conditions that promote the activation of at least one enzyme with subsequent splitting of the starch grains and the formation of an aqueous solution containing sugar, where part of the plant is obtained from Tr is sformirovannogo plants, the genome of which is increased at the expense of expressing cassette that encodes at least one enzyme, processrule polysaccharide, non-starch; and collecting the aqueous solution containing sugar. The present invention also relates to transformed plant or plant part containing at least one enzyme that processes polysaccharide, non-starch, and is present in the cell or in the cell wall of plant cells or plant part. Plant part is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one non-processrule starch enzyme, such as a xylanase, a cellulase, glucanase, pectinase, or any combination of them.

that is, the High content of the aqueous phase in the protein and sugar

In yet another embodiment of the invention the protease and lipase are manufactured so that they accumulated in seeds, such as soybean seeds. After activation of proteases or lipases, for example, by heating these enzymes in the seeds hydrolyzing lipids and spare proteins present in soybean during processing. Thus, it can be obtained soluble products containing amino acids that can be used as a nutrient, food or a fermentation medium, and fatty acids. Polish the IDA, usually present in the insoluble fraction processioning grain. However, due to the expression of polysaccharide-destroying enzyme and its accumulation in the seeds, can be hydrolyzed proteins and polysaccharides present in the aqueous phase. For example, this method can be solubilisation Seine corn and spare protein and non-starch polysaccharides of soybean. The components of the aqueous and hydrophobic phases can be easily separated by extraction with an organic solvent or supercritical carbon dioxide. Thus, the present invention relates to a method for producing an aqueous extract of the grain, which contains high levels of protein, amino acids, sugars or saccharides.

f. Fermentation sameprinciples plants

The present invention relates to a method for producing ethanol, beverages or other products obtained during fermentation. This method provides for plants, or product, or plant part or derivative of a plant, such as flour from grain, which is expressed processrule enzyme, converting polysaccharides in sugar. A plant or its product is treated so that when the conversion of the polysaccharide formed sugar as described above. Then sugar and other plant components subjected to fermentation with obtaining ethanol, written in the Cove or other products, produced by fermentation methods known in the art. See, for example, U.S. patent No. 4929452. Briefly, sugar, produced by conversion of polysaccharides, incubated with yeast under conditions that stimulate the conversion of sugar to ethanol. Suitable yeast are yeast strains tolerant to high alcohol and sugar content, for example, the yeast S.cerevisiae ATSS No. 20867. This strain was deposited in the American type culture collection, Rockville, MD, 17 Sept., 1987 and registered under the number of ATSS No. 20867. Then the product or a beverage obtained by fermentation, can be subjected to distillation with the separation of ethanol or beverage obtained by distillation, or the fermentation product may be selected in any other way. The plant used in this way, can be a plant containing a polysaccharide and is able to Express the enzyme of the present invention. In the present application describes many such plants. Preferred is a plant cultivated for commercial purposes. More preferred is a plant that is commonly used for ethanol production or food or beverage obtained by fermentation, for example, such plants as wheat, barley, corn, rye, potatoes, grapes or rice. The most preferred plant is corn.

The method includes the processing parts of the plant, containing at least one polisaharidnyi enzyme under conditions that stimulate the activation of at least one enzyme, thereby contributing to the breakdown of the polysaccharide in the specified parts of the plant with obtaining fermentable sugar. Polisaharidnyi enzyme may be mesophilic, thermophilic, or hyperthermophilic. Preferred is a hyperthermophilic enzyme. Plant part is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one polisaharidnyi enzyme. In this embodiment of the invention the parts of the plant are, but are not limited to, grain, fruits, seeds, stems, wood, vegetables or roots. Preferred plants include, but are not limited to, oats, barley, wheat, berries, grapes, rye, corn, rice, potato, sugar beet, sugar cane, pineapple, grass and trees. Part of the plant can be combined with commodity grain or with other commercially available products, as a source of substrate for processing can be a source of non sameprinciples plant. Then fermentary sugar incubated in conditions that promote the conversion of fermentable sugar into ethanol, for example, yeast and/or other microorganism is mi. In a preferred embodiment of the invention the part of the plant is derived from corn, transformed αamylase, which has been found, saves the fermentation time and the cost of implementation of this fermentation.

It was found that the amount of residual starch is reduced in the case when transgenic corn produced in accordance with the present invention, expresses thermostable α-amylase, for example, when used in fermentation. This indicates that during the fermentation solubilizated more starch. At low content of residual starch in the distiller get grain with a higher content (by weight) protein and higher values. Furthermore, fermentation of transgenic maize allows liquefaction at lower pH, which enables to reduce the cost of chemicals used for pH correction at a higher temperature, for example at more than 85°and preferably more than 90°S, more preferably at 95°With or higher, which reduces the time of dissolution, and to more fully solubilize the starch and reduces the time of dilution, resulting in can be made effective response fermentation with higher outputs of ethanol.

To the ome, it was found that the engagement of the respective parts of the plant, even with a small part of the transgenic plants produced in accordance with the present invention may result in reduced fermentation time and the cost of its implementation. Thus, the present invention relates to a method of reducing fermentation time for plants, including the use of part of the transgenic plants obtained from plants containing polisaharidnyi the enzyme that converts polysaccharides in sugar, instead of using a plant that does not contain polysaccharidesilica enzyme.

g. Enzymes, processorsa the original starch, and polynucleotide, encoding

Polynucleotide encoding mesophilic processrule enzyme(s), is introduced into a plant or plant part. In a preferred embodiment, the invention polynucleotides the present invention is to polynucleotide, optimized codons corn, such as polynucleotide having the sequence SEQ ID NO:48, 50 and 59 and encoding a glucoamylase having the sequence of SEQ ID NO:47 and 49. In another preferred embodiment, the invention polynucleotides the present invention is to polynucleotide, optimized codons corn, such as polynucleotide with the settlement of egovernance SEQ ID NO:52, encoding α-amylase having the sequence of SEQ ID NO:51. It also deals with hybrid products processorbased enzymes. In one of the preferred embodiments of the invention polynucleotides the present invention is to polynucleotide, optimized codons corn, such as polynucleotide having the sequence of SEQ ID NO:46 encoding a hybrid alpha-amylase and glucoamylase having the sequence of SEQ ID NO:45. In addition, in the present invention deals with the combination processorbased enzymes. So, for example, is considered a combination krahmalsoderzhaschih enzymes and not processorbased starch enzymes. Such combinations processorbased enzymes can be obtained using multiple gene constructs encoding each of the enzymes. Alternatively, the individual transgenic plant stably transformed by enzymes, can be subjected to crossing with known methods of obtaining plants containing both the enzyme. Another method involves the use of exogenous enzyme(s) in obtaining transgenic plants.

Source cromartyshire enzyme and not processimage starch enzyme can be isolated or obtained from any material, and its polynucleotide can be determined using any specialistkompetens, α-amylase derived from Aspergillus (e.g., Aspergillus shirousami and Aspergillus niger), Rhisopus (for example, Rhizopus oryzae) and plants such as corn, barley and rice. Preferably, the glucoamylase derived from Aspergillus (e.g., Aspergillus shirousami and Aspergillus niger), Rhisopus (for example, Rhizopus oryzae) and Thermoanaerobacter (e.g., Thermoanaerobacter thermosaccharolyticum).

In another embodiment, the invention polynucleotide encodes mesophilic cromartyshire enzyme and functionally attached to the optimized codons of corn to polynucleotide, such as SEQ ID NO:54, codereuse linking the original starch domain, such as SEQ ID NO:53.

In another embodiment of the invention tissue-specific promoters are antispermalnye promoters such as the promoter γ-Zein of maize (represented by SEQ ID NO:12) or the promoter ADP-gpp corn (represented by SEQ ID NO:11, which includes the 5'-noncoding and intron sequence). Thus, the present invention relates to selected polynucleotide containing a promoter comprising SEQ ID NO:11 or 12; for polynucleotide that hybridizes sequence, the complementary sequence under conditions of low stringency, or its fragment, the promoter activity of which is, for example, at least 10%and preferably at least 50% of the promotional activity is ora, having the sequence of SEQ ID NO:11 or 12.

In one of the embodiments of the invention, the gene product of the hydrolysis of starch, such as gene α-amylase, glucoamylase or hybrid α-amylase/glucoamylase, can be targeted to a specific organelle or on a particular area, such as the endoplasmic reticulum or the apoplast, but not in the cytoplasm. This can be, for example, implemented using a N-terminal signal sequence γ-Zein of maize (SEQ ID NO:17), which provides apoplastically targeting proteins, and the use of N-terminal signal sequence γ-Zein (SEQ ID NO:17), functionally attached to processarea the enzyme, which, in turn, functionally attached to the sequence SEKDEL for retention in the endoplasmic reticulum. Targeting protein or enzyme in a specific compartment provides such a localization of the specified enzyme, where it cannot come in contact with the substrate. Thus, the enzymatic action of the specified enzyme will not be carried out prior to contact of the enzyme with the substrate. The enzyme may come in contact with its substrate in the grinding process (physical destruction of the integrity of the cells) and hydration. For example, mesophilic cromartyshire enzyme can be targeted n is the apoplast or to the endoplasmic reticulum, and so it will not come in contact with starch grains in amyloplasts. Grinding grain will destroy the integrity of the grain, and brahmarishi the enzyme will be in contact with starch grains. This way you can avoid the potentially negative effects of joint localization of the enzyme and its substrate.

h. Foods without added sweetener.

The present invention also relates to a method for producing sweetened baked food product without adding additional sweeteners. Examples of farinaceous food products include, but are not limited to, food for Breakfast, the food product is ready to use, pastries, pasta and cereal foods, such as cereal Breakfast. The method includes processing parts of the plant containing at least one cromartyshire enzyme, under conditions that stimulate the activation of cromartyshire enzyme, thereby facilitating the processing of starch grains in the specified parts of the plant to sugars with the formation of sweetened product, for example product produced during the processing of starch granules from parts of the plant, which does not contain a hyperthermophilic enzyme. Preferred cromartyshire enzyme is hyperthermophilic enzyme, and riverwest when heated, for example when baking, boiling, heating, cooking for a couple, electrical discharge, or any combination thereof. Part of the plant is obtained from the transformed plants, for example of transformed plants of soybean, rye, oats, barley, wheat, corn, rice or sugar cane, the genome of which is increased at the expense of expressing cassette that encodes at least one hyperthermophilic cromartyshire enzyme, for example α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. Then sweetened product processed with getting farinaceous food product. The present invention also relates to a farinaceous food product, such as grain food product, food product for Breakfast food, ready to eat, or for baking, prepared by this method. Specified farinaceous food product can be obtained from the sweetened product and water and may contain malt, fragrances, vitamins, minerals, colorants, or combinations thereof.

The enzyme can be activated so that it turned the polysaccharides contained in the specified plant material, in sugar before turning plant material in the grain product, or when the processing of the grain product. In accordance with these polysaccharides, provided the given plant material, can be converted into sugar by activating material, for example by heating in the case of a hyperthermophilic enzyme before turning in flour product. Then the plant material containing sugar produced by transformation of polysaccharides, add in the specified product from obtaining a sweetened product. Alternatively, the polysaccharides can be converted into sugar by the enzyme during processing flour product. Examples of methods of obtaining grain products are well known in the art, and such methods are heating, baking, boiling, etc. as described in U.S. patent No. 6183788, 6159530, 6149965, 4988521 and 5368870.

Briefly, the dough can be prepared by mixing various dry ingredients together with water and exposure to heat treatment for gelatinization of starch components and flavour development preposterously cream. This heat-treated material may then be subjected to mechanical processing with getting ready test, such as grain dough. The dry ingredients can include various additives, such as sugar, starch, salt, vitamins, minerals, colorants, odorants, salts, etc. in Addition to water, may be added various liquid ingredients, such as corn or malt syrup. Flour product may include grains, split is the second grain, the coarse grain or flour from wheat, rice, corn, oats, barley, rye or other cereals and mixtures thereof derived from a transformed plant according to the invention. Then the dough can be processed to obtain the desired shape, such as extrusion or stamping, with subsequent heat treatment using tools such as cooking apparatus James, furnace or device for the supply of electric discharge.

In addition, the present invention relates to a method of sweetening starch-containing product without added sweetener. The method includes processing a starch containing at least one cromartyshire enzyme, under conditions that stimulate the activation of at least one enzyme, thereby contributing to the breakdown of starch to sugars with the formation of the processed (sweetened) starch, for example, for the product produced during the processing of starch, not containing hyperthermophilic enzyme. The starch of the present invention is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one processrule enzyme. The preferred enzymes are α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. Before occhialino, to the specified enzyme was hyperthermophilic and activated by heating. Preferred transformed plants are corn, soy, rye, oats, barley, wheat, rice and sugar cane. Then the processed starch is added to the product, obtaining sweetened starch-containing product, such as flour-based food product. The present invention also relates to Podkamennaya starch-containing product obtained by this method.

In addition, the present invention relates to a method of sweetening polisakharidami fruits or vegetables, including processed fruits or vegetables, containing at least one polisaharidnyi the enzyme under conditions which activate at least one enzyme, and subsequent conversion of the specified polysaccharide fruits or vegetables in sugar and more fruits and vegetables, for example, compared with fruits and vegetables plants that do not contain polisaharidnyi enzyme. The fruits or vegetables of the present invention is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one polisaharidnyi enzyme. Preferred fruits and vegetables are potatoes, tomatoes, banana, pumpkin, peas and beans. The preferred enzymes are the camping α -amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. The enzyme preferably is hyperthermophilic.

i. The sweetening polysacharides plant or plant product.

This method involves obtaining a plant that expresses polisaharidnyi enzyme, converting the polysaccharide in sugar, as described above. In accordance with this enzyme is expressed in the plant and in the products of plants, such as fruits or vegetables. In one of the embodiments of the invention the enzyme is placed under the control of the inducible promoter so that expression of this enzyme was induced under the influence of external factors. Such inducible promoters and designs are well known in the art and described in this application. The expression of the enzyme in the plant or product of plants leads to the conversion of the polysaccharide contained in the plant or its product, sugar and sweetening of this plant or product. In another embodiment of the invention polisaharidnyi enzyme is expressed constitutively. Thus, the plant or its derived product can be activated under conditions sufficient to activate the enzyme for the conversion of polysaccharides into sugar by the action of the enzyme and subsequent sweetening plants or derived from the product. As a result, autoprocessing polysaccharide in fruit and vegetables with the formation of sugar leads to production of more fruits or vegetables than fruits or vegetables plants not containing the specified polisaharidnyi enzyme. The fruits or vegetables of the present invention is obtained from a transformed plant, the genome of which is increased at the expense of expressing cassette that encodes at least one polisaharidnyi enzyme. Preferred fruits and vegetables are potatoes, tomatoes, banana, pumpkin, peas and beans. The preferred enzymes are α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. Preferably, polisaharidnyi enzyme was hyperthermophilic.

j. Selection of starch from transgenic grain, which contains an enzyme that destroys the matrix of the endosperm.

The present invention relates to a method of extraction of starch from transgenic grain, where is expressed enzyme that destroys the matrix of the endosperm. The method comprises obtaining a plant that expresses an enzyme that destroys the matrix of the endosperm, by modifying, for example, cell wall polysaccharides, non-starch and/or proteins. Examples of such enzymes are, n is not limited to, protease, glucanase, thioredoxin, thioredoxin and esterase. These enzymes do not include enzymes having brahmarishi activity, since it is necessary to preserve the integrity of the starch grains. While it is preferable that this enzyme was attached to the signal sequence, the target of this enzyme on starch grains. In one of the embodiments of the invention, the grain is subjected to thermal drying for activation of the enzyme to inactivate endogenous enzymes in the grain. Thermal treatment leads to the activation of an enzyme that destroys the matrix of the endosperm, which can then be easily separated from the starch grains. In another embodiment of the invention, the grain is soaked at low or high temperatures, high or low humidity and in the presence or in the absence of sulfur dioxide. Then the grain is subjected to heat treatment for the destruction of the matrix of the endosperm, which makes it easy to extract the starch grains. In another embodiment of the invention, the conditions with the appropriate temperature and humidity, which provide the penetration of proteases in starch grains and decomposition of proteins found in grains. Such treatment leads to the production of starch grains with high output and small with the holding of protein impurities.

k. A syrup having a high content of sugar equivalent, and use this syrup for the production of ethanol or beverage obtained by fermentation.

This method provides for obtaining plants expressing polisaharidnyi the enzyme that converts the polysaccharide in sugar, as described below. A plant or its product is soaked in water flow in the conditions under which expressed the enzyme converts the polysaccharide contained in the plant or its product, dextrin, maltooligosaccharide and/or sugar. Then an aqueous stream containing dextrin, maltooligosaccharide and/or sugar produced through the conversion of polysaccharide, isolated to obtain a syrup having a high content of sugar equivalent. The method may include and not include the additional step of wet grinding plant or its product with the receipt of starch grains. Examples of enzymes that can be used in the method include, but are not limited to, the α-amylase, glucoamylase, pullulanase and α-glucosidase. Preferably, the enzyme is hyperthermophilic. Sugars produced in this way are, but are not limited to, hexose, glucose and fructose. Examples of plants that can be used in the method include, but are not limited to them is, corn, wheat or barley. Examples of products of plants that can be used are, but are not limited to, fruits, grains and vegetables. In one of the embodiments of the invention polisaharidnyi enzyme is placed under the control of the inducible promoter. In line with this, before soaking or steeping process, induced the promoter driving the expression of the indicated enzyme, which then converts the polysaccharide in sugar. Examples of inducible promoters and structures containing these promoters are well known in the art and described in this application. Thus, if polysaccharideprotein enzyme is hyperthermophilic enzyme, the soaking is carried out at room temperature to activate the hyperthermophilic enzyme for the inactivation of endogenous enzymes contained in the plant or its product. In another embodiment of the invention hyperthermophilic enzyme, capable of converting the polysaccharide in sugar, is expressed constitutively. The enzyme may be (or may not) be aimed at the compartment of the plant using the signal sequence. A plant or its product is soaked in conditions of high temperature to initiate the conversion of polysaccharides contained in this plant, sugar.

On toadie the invention also relates to the production of ethanol or preparation of the drink, obtained by fermentation, from a syrup having a high content of sugar equivalent. The method involves incubation syrup, yeast under conditions that ensure the transformation of the sugar contained in the syrup, ethanol, or in a beverage obtained by fermentation. Examples of such beverages obtained by fermentation, include, but are not limited to, beer and wine. The conditions of fermentation are well known in the art and described in U.S. patent No. 4929452 and in this application. Preferred yeast is a yeast strain that is tolerant to high alcohol content and high sugar content, such as S.cerevisiae, reg. ATSS No. 20867. Fermented product or a beverage obtained by fermentation, can be subjected to distillation for separation of ethanol or beverage obtained by distillation.

l. Accumulation of hyperthermophilic enzyme in the cell walls of plants.

The present invention relates to a method of accumulation hyperthermophilic enzyme in the cell wall of plants. The method provides for expression in the plant hyperthermophilic enzyme, which is associated with the signal, the target of this enzyme in the cell wall, so that the target enzyme accumulated in the cell wall. Preferably, the enzyme was able to convert polysaccharides into monosaccharides. Examples aim is to her sequences are, but not limited to, a domain that binds cellulose or xylose. Examples of hyperthermophilic enzymes are enzymes presented in SEQ ID NO:1, 3, 5,10, 13, 14, 15 or 16. In this method, the plant material containing the cell wall, may be added as a source of the necessary enzymes for separation of sugars from the original food material or as a source of enzymes for the conversion of polysaccharides originating from other sources, into monosaccharides. In addition, the cell wall can serve as a source from which can be selected enzymes. Methods of purification of enzymes are well known in the art, and such methods include, but are not limited to, gel filtration, ion exchange chromatography, chromatofocusing, isoelectric focusing, affinity chromatography, JAHB, HPLC, salt deposition, dialysis, etc. In accordance with this present invention also relates to purified enzymes isolated from cell walls of plants.

m. The method of obtaining and allocating processorbased enzymes.

In accordance with the present invention recombinante produced processorsa enzymes of the present invention can be obtained by transformation of plant tissue or plant cell containing processrule the enzyme of the present invention, capable of aktivirovat is in the plant, chosen to produce transformed tissue or cells; culturing the transformed tissue or cell to obtain a transformed plant, and selection processimage enzyme from the indicated transgenic plants or parts of it. Preferred recombinante produced by the enzyme is α-amylase, glucoamylase, glucose isomerase, α-glucosidase and pullulanase. Most preferably, the specified enzyme encoded by polynucleotides selected from the sequences SEQ ID NO:2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59.

The present invention is also described in the following examples, which should not be construed as limiting its scope.

Examples

Example 1

Design of optimized codons maize genes for hyperthermophilic krahmalsoderzhaschih/isomerizing enzymes

Enzymes, such as α-amylase, pullulanase, α-glucosidase and glucose isomerase, involved in the decomposition of starch or isomerization of glucose were selected for their desired activity. These profiles are, for example, minimal activity at room temperature, the activity/stability at high temperature and activity at low pH. Then designed the corresponding genes using the preferred code for the new corn, described in U.S. patent No. 5625136, and synthesized in accordance with the General technology of recombinant DNA (Integrated DNA Technologies, Inc. (Coralville, IA).

α-Amylase 797GL3 having the amino acid sequence of SEQ ID NO:1, was selected for its hyperthermophilic activity. Extracted nucleic acid sequence of the enzyme and its optimized for codons corn, as presented in SEQ ID NO:2. Similarly took pullulanase 6gp3 having the amino acid sequence represented in SEQ ID NO:3. The sequence of the nucleic acid to pullulanase 6gp3 taken out and optimized according to the codons of corn, as shown in SEQ ID NO:4.

Amino acid sequence of α-glucosidase malA Sulfolobus solfataricus received, as described in the literature, J.Bact. 177:482-485 (1995); J.Bact. 180:1287-1295 (1998). Based on the published amino acid sequences (SEQ ID NO:5) was designed synthetic gene optimized codons maize (SEQ ID NO:6) encoding α-glucosidase malA.

We selected several enzymes glucosinolates. Amino acid sequence (SEQ ID NO:18) glucose, derived from Thermotoga maritima, was predicted on the basis of published DNA sequences with reg. No. NC_000853, and was designed synthetic gene optimized codons maize (SEQ ID NO:19). Similarly, the amino acid the percentage sequence (SEQ ID NO:20) glucose, derived from Thermotoga neapolitana was predicted on the basis of DNA sequence, published in Appl. Envir. Environ. 61(5):1867-1875 (1995), reg no.L38994. Was designed synthetic gene optimized codons corn and encoding the glucose isomerase Thermotoga neapolitana (SEQ ID NO:21).

Example 2

Expression of hybrid α-amylase 797GL3 and the field of encapsulation of starch in E.coli

Design, coding hyperthermophilic α-amylase 797GL3 attached to the field of encapsulation of starch (SER), derived from starch-synthase, associated with dried corn (waxy), was integrated and expressed in E.coli. cDNA for associated with corn grain starch synthase (SEQ ID NO:7)encoding the amino acid sequence (SEQ ID NO:8) (Klosgen R.B., et al.,1986), cloned as a source krahmalsoderzhashchego domain or region encapsulating starch (SER). Full-size cDNA amplified using RT-PCR from RNA obtained from the seeds of corn, using primers SV57: (5'-AGCGAATTCATGGCGGCTCTGGCCACGT-3') (SEQ ID NO:22) and SV58 (5'-AGCTAAGCTTCAGGGCGCGGCCACGTTCT-3') (SEQ ID NO:23), available in Genbank, reg. No. h. Full-size cDNA cloned in pBluescript as EcoRI/HindIII fragment and the plasmid was designated NOV4022.

C-terminal part (encoded PN-1818) cDNA waxy maize, including krokhmalskii domain, amplified from NOV4022 and was joined in the same reading frame to the 3'-con is in full gene 797GL3, optimized codons maize (SEQ ID NO:2). The hybrid gene product, 797GL3/waxy, having the nucleic acid SEQ ID NO:9 and encodes the amino acid sequence of SEQ ID NO:10, cloned as NcoI/XbaI fragment into the vector 28b (NOVAGEN, Madison, WI), who cut NcoI/NheI. Gene 797GL3, taken separately, is also cloned into the vector 28b as NcoI/XbaI fragment.

Vectors RET/797GL3 and RET/797GL3/waxy transformed into BL21 cells/D3 E. coli (NOVAGEN), were cultured and induced in accordance with the manufacturer's instructions. Analysis by electrophoresis in SDS page/coloring Kumasi revealed the presence of induced protein in both extracts, corresponding to the predicted sizes of hybrid and non-hybrid amylase, respectively.

Total cell extracts were analyzed for the activity of hyperthermophilic amylase as follows: 5 mg of starch suspended in 20 μl of water, and then diluted with 25 μl of ethanol. To this mixture was added a standard smilasaponin control or sample being tested for amylase activity, then added water to a final reaction volume of 500 μl. The reaction was carried out at 80°C for 15-45 minutes. Then the reaction mixture was cooled to room temperature and was added 500 μl of o-dianisidine and a mixture of glucose oxidase/peroxidase (Sigma). The resulting mixture was incubated at 37the C for 30 minutes. To terminate the reaction, was added 500 μl of 12 N. sulfuric acid. To determine the amount of glucose released by amylase/sample, was measured by optical density at 540 nm. Analysis of extracts of hybrid and non-hybrid amylase showed similar levels of activity of hyperthermophilic amylase, whereas the control extracts were negative for this activity. This testified to the fact that when joining the C-terminal part of the protein waxy maize, amylase 797GL3 was still active (at high temperatures).

Example 3

The selection of fragments of the promoter for endoparasitism expression in maize

The promoter and 5'-non-coding region I (including the first intron), derived from the large subunit of ADP-gpp Zea mays (ADP-glucocerebrosidase), amplified in the form of a fragment from 1515 pairs of nucleotides (SEQ ID NO:11)derived from genomic DNA of maize, using primers available in Genbank, reg. No. m. It was shown that the promoter ADP-gpp is endoparasitism (Shaw & Hannah, 1992).

The promoter of the gene γZea mays Zein amplified in the form of a 673 BP fragment (SEQ ID NO:12)derived from the plasmid pGZ27.3 (obtained from Dr. Brian Larkins). It was shown that the promoter γ-Zein was the endosperm-specific (Torrent et al., 1997).

Example 4

Designing transforming vectors for the ISU is thermopiles α -amylase 797GL3

Expressiona cartridge designed for the expression of hyperthermophilic amylase 797GL3 in the endosperm of maize, designed using different signals for targeting, as follows.

pNOV6200 (SEQ ID NO:13) contains N-terminal signal sequence γ-Zein of maize (MRVLLVALALLALAASATS)(SEQ ID NO:17), attached to synthetic amylase 797GL3, obtained as described above in example 1, and used for targeting to the endoplasmic reticulum and secretion to the apoplast (Torrent et al., 1997). Hybrid cloned in the area not covered by the promoter ADP-gpp corn, for specific expression in the endosperm.

pNOV6201 (SEQ ID NO:14) contains N-terminal signal sequence γ-Zein attached to synthetic amylase 797GL3 adding in the end of the sequence SEKDEL for targeting to and retention in the endoplasmic reticulum (ER) (Munro &Pelham, 1987). Hybrid cloned in the area not covered by the promoter ADP-gpp corn, for specific expression in the endosperm.

pNOV7013 contains N-terminal signal sequence γ-Zein attached to synthetic amylase 797GL3 adding in the end of the sequence SEKDEL for targeting to and retention in the endoplasmic reticulum (ER). pNOV7013 was similar pNOV6201, except that for the expression of ecologized in the endosperm, instead promoter ADP-gpp corn, used moter γ-Zein of maize (SEQ ID NO:12).

pNOV4029 (SEQ ID NO:15) contains meloplasty target peptide waxy maize (Klosgen et al., 1986)attached to synthetic amylase 797GL3 to target a specified amyloplast. This hybrid was cloned in the area not covered by the promoter ADP-gpp corn, for specific expression in the endosperm.

pNOV4031 (SEQ ID NO:16) contains meloplasty target peptide waxy maize attached to synthetic hybrid protein 797GL3/waxy for targeting starch grains. Hybrid cloned in the area not covered by the promoter ADP-gpp corn, for specific expression in the endosperm.

Were created more designs with the use of these hybrids, cloned behind the promoter γ-Zein of corn, in order to obtain higher levels of expression of the enzyme. All expressing cassette was transferred into a binary vector for transformation into maize by Agrobacterium infection. The binary vector contains a gene phosphomonoesterase (PMI), which allows for the selection of transgenic cells using mannose. Transgenic corn plants were either subjected to self-pollination, or crossed, and the resulting seeds were collected for analysis.

Were created additional design is the use of the above described signals for targeting attached either to pullulanase Dr or α-glucosidase 340g12, similarly as described for α-amylase. These hybrids were cloned in the area not covered by the promoter ADP-gpp corn and/or promoter γ-Zein of corn, and transformed into maize, as described above. Transgenic corn plants were either subjected to self-pollination, or crossed, and the resulting seeds were collected for analysis.

Combinations of enzymes can be produced either by crossing plants expressing individual enzymes or by cloning, expressing several cassettes in the same binary vector for co-transformation.

Example 5

Construction of vectors designed for plant transformation for the introduction of thermophilic pullulanase 6GP3

Expressing cassette was designed for the expression of thermophilic pullulanase 6GP3 in the endoplasmic reticulum of the maize endosperm as follows.

pNOV7005 (SEQ ID NO:24 and 25) contains N-terminal signal sequence γ-Zein of corn attached to synthetic pullulanase 6GP3, adding in the end of the sequence SEKDEL for targeting to and retention in the ER. Peptide SEKDEL was attached to the C-end of these enzymes by PCR using primers designed for amplif the requirements of the synthetic gene with the simultaneous addition of 6 amino acids at the C-end of the protein. Hybrid cloned in the area not covered by the promoter γ-Zein of corn, for specific expression in the endosperm.

Example 6

Construction of vectors designed for plant transformation, for hyperthermophilic α-glucosidase malA

Expressing cassette was designed for the expression of hyperthermophilic α-glucosidase malA Sulfolobus solfataricus in the endosperm of maize using different signals for targeting, as follows.

pNOV4831 (SEQ ID NO:26) contains N-terminal signal sequence γ-Zein (MRVLLVALALLALAASATS) (SEQ ID NO:17), attached to synthetic α-glucosidase malA adding in the end of the sequence SEKDEL for targeting to and retention in the endoplasmic reticulum (ER) (Munro &Pelham, 1987). Hybrid cloned in the area not covered by the promoter γ-Zein of corn, for specific expression in the endosperm.

pNOV4839 (SEQ ID NO:27) contains N-terminal signal sequence γ-Zein of maize (MRVLLVALALLALAASATS) (SEQ ID NO:17), attached to synthetic α-glucosidase malA for targeting to the endoplasmic reticulum and secretion into the apoplast (Torrent et al., 1997). Hybrid cloned in the area not covered by the promoter γ-Zein of corn, for specific expression in the endosperm.

pNV4837 contains N-terminal signal sequence γ -Zein of maize (MRVLLVALALLALAASATS) (SEQ ID N0:17)attached to the synthetic α-glucosidase malA, adding in the end of the sequence SEKDEL for targeting to and retention in the ER. Hybrid cloned in the area not covered by the promoter ADP-gpp corn, for specific expression in the endosperm. Amino acid sequence of the clone was identical to the sequence pNOV4831 (SEQ ID NO:26).

Example 7

Construction of vectors intended for transformation of plants, in order to introduce the hyperthermophilic glucosinolates Thermotoga maritima and Thermotoga neapolitana

Below is described the design of expressing cassettes for the expression of hyperthermophilic glucosamines Thermotoga maritima and Thermotoga neapolitana in the endosperm of maize using different targeting signals.

pNOV4832 (SEQ ID NO:28) contains N-terminal signal sequence γ-Zein of maize (MRVLLVALALLALAASATS) (SEQ ID NO:17), attached to a synthetic glucose isomerase Thermotoga maritima, adding in the end of the sequence SEKDEL for targeting to and retention in the ER. Hybrid cloned in the area after promoter γ-Zein of corn, for specific expression in the endosperm.

NOV4833 (SEQ ID NO:29) contains N-terminal signal sequence γ-Zein of maize (MRVLLVALALLALAASATS)(SEQ ID NO:17), attached to a synthetic glucose isomerase Thermotoga neapoitana adding in the end of the sequence SEKDEL for targeting to and retention in the ER. Hybrid cloned in the area not covered by the promoter γ-Zein of corn, for specific expression in the endosperm.

NOV4840 (SEQ ID NO:30) contains N-terminal signal sequence γ-Zein of maize (MRVLLVALALLALAASATS)(SEQ ID NO:17), attached to a synthetic glucose isomerase Thermotoga neapolitana for targeting to the endoplasmic reticulum and secretion into the apoplast (Torrent et al., 1997). Hybrid cloned in the area not covered by the promoter γ-Zein of corn, for specific expression in the endosperm.

NOV4838 contains N-terminal signal sequence γ-Zein of maize (MRVLLVALALLALAASATS)(SEQ ID NO:17), attached to a synthetic glucose isomerase Thermotoga neapolitana with the addition of in-the end of the sequence SEKDEL for targeting to and retention in the ER. Hybrid cloned in the area not covered by the promoter ADP-gpp corn, for specific expression in the endosperm. Amino acid sequence of the clone was identical to the sequence NOV4833 (SEQ ID NO:29).

Example 8

Construction of vectors intended for transformation of plants for the expression of hyperthermophilic glucanase EglA

NOV4800 (SEQ ID NO:58) contains the signal sequence of the alpha-amylase barley AMY32b (MGKNGNLCCFSLLLLLLAGLASGHQ) (SEQ ID NO:31)attached to the sequence of the Mature protein EglA for localization in the apoplast. Hybrid kleinova and in the region, covered by promoter γ-Zein of corn, for specific expression in the endosperm.

Example 9

Construction of vectors intended for transformation of plants for the expression of many hyperthermophilic enzymes

NOV4841 contains a double gene construct consisting of hybrid α-amylase 797GL3 and hybrid pullulanase 6GP3. Hybrid 797GL3 (SEQ ID NO:33) and hybrid 6GP3 (SEQ ID NO:34) had a N-terminal signal sequence γ-Zein of corn and the sequence SEKDEL for targeting to and retention in the ER. Each hybrid was cloned in an area not covered by separate promoters γ-Zein of corn, for specific expression in the endosperm.

NOV4842 contains a double gene construct consisting of hybrid α-amylase 797GL3 and hybrid α-glucosidase malA. Hybrid polypeptide 797GL3 (SEQ ID NO:35) and the hybrid polypeptide α-glucosidase malA (SEQ ID NO:36) had a N-terminal signal sequence γ-Zein of corn and the sequence SEKDEL for targeting to and retention in the ER. Each hybrid was cloned in an area not covered by separate promoters γ-Zein of corn, for specific expression in the endosperm.

NOV4843 contains a double gene construct consisting of hybrid α-amylase 797GL3 and hybrid α-glucosidase malA. Both hybrid 797GL3 and α-glucosidase malA had the N-to the core of the signal sequence γ -Zein of corn and the sequence SEKDEL for targeting to and retention in the ER. Hybrid 797GL3 was cloned in the area not covered by the promoter γ-Zein of maize and hybrid malA was cloned in the area not covered by the promoter ADP-gpp corn, for specific expression in the endosperm. Amino acid sequence of the hybrid 797GL3 and hybrid malA were identical to sequences NOV4842 (SEQ ID No:35 and 36, respectively).

NOV4844 contains triple gene structure consisting of hybrid α-amylase 797GL3, hybrid pullulanase 6GP3 and hybrid α-glucosidase malA. All hybrids 797GL3, malA, and 6GP3 had N-terminal signal sequence γ-Zein of corn and the sequence SEKDEL for targeting to and retention in the ER. Hybrids 797GL3 and malA were cloned in the area for 2 separate promoters γ-Zein of maize and hybrid 6GP3 was cloned in the area not covered by the promoter ADP-gpp corn for specific expression in the endosperm. Amino acid sequence of hybrids 797GL3 and malA were identical to sequences NOV4842 (SEQ ID No:35 and 36, respectively). Amino acid sequence of the hybrid 6GP3 was identical to the sequence of the hybrid 6GP3 in NOV4841 (SEQ ID NO:34).

All expressing cassette was transferred into the binary vector pNOV2117 for transformation into maize by Agrobacterium infection. pNOV2117 gene phosphomonoesterase (PMI) for selection of transgenic cells using mannose. pNOV2117 is a binary vector with sites of replication initiation pVS1 and ColE1. This vector contains a constitutive VirG gene, originating from pAD1289 (G. Hansen et al., PNAS USA 91:7603-7607 (1994)), and the gene of resistance to spectinomycin, originating from Tn7. This vector, cloned in polylinker between its right and left borders, contained the promoter ubicacin corn, the coding region PMI and terminator nepalensis pNOV117 (D. Negrotto et al., Plant Cell Report 19:798-803 (2000)). Transgenic corn plants were either subjected to self-pollination, or were crossed and the resulting seeds were collected for analysis. A combination of different enzymes can be obtained by crossing plants expressing individual enzymes, or by transformation of plants cassettes containing one gene, or multigene cassettes.

Example 10

Construction of bacterial and expressing Pichia vectors

Below is described the design of expressing cassettes for the expression of hyperthermophilic α-glucosidase and glucosamines or Pichia, or in bacteria.

pNOV4829 (SEQ ID NO:37 and 38) contains a synthetic hybrid glucose Thermotoga maritima signal retention in the ER in bacterial expressing vector rate. Hybrid gene glucose cloned into the NcoI and SacI sites rate, which led to the accession with the N-terminal S-tag used for the cleaning of the protein.

PNOV4830 (SEQ ID NO:39 and 40) contains a synthetic hybrid glucose Thermotoga neapolitana signal retention in the ER in bacterial expressing vector rate. Hybrid gene glucose cloned into the NcoI and SacI sites pET29a, which led to the accession with the N-terminal S-tag used for protein purification.

NOV4835 (SEQ ID NO:41 and 42) contains a synthetic gene for the glucose Thermotoga maritima cloned into BamHI - and EcoRI sites of bacterial expressing vector rats. The result has been a hybrid of the His-tag (for protein purification) N-end glucose.

NOV4836 (SEQ ID NO:43 and 44) contains a synthetic gene for the glucose Thermotoga neapolitana cloned into BamHI - and EcoRI sites of bacterial expressing vector rats. The result has been a hybrid of the His-tag (for protein purification) N-end glucose.

Example 11

Transformation of immature embryos of maize was carried out basically as described by D. Negrotto et al., Plant Cell Reports 19:798-803. In this example, all components of the environments are as they were described by D. Negrotto et al., see above. However, the various components of the environments described in the literature, can be replaced.

A. Transforming plasmids and selective marker

The genes used for transformation, cloned in a vector suitable for transformation of maize. The vectors used in this example contained the gene F. somanathapura (PMI) for selection of transgenic lines (Negrotto et al. (2000), Plant Cell Reports 19:798-803).

C. Obtaining Agrobacterium tumefaciens

Strain LBA4404 Agrobacterium (pSBI)containing plasmid for transformation of plants cultivated on solid medium YEP (yeast extract (5 g/l), peptone (10 g/l), NaCl (5 g/l), 15 g/l agar, pH 6.8) for 2-4 days at 28°C. Approximately 0.8×109Agrobacterium suspended in the environment LS-inf, to which was added 100 μm As (Negrotto et al. (2000), Plant Cell Report 19:798-803). Bacteria previously induced in the environment within 30-60 minutes.

C. Inoculation

Immature embryos from A or other appropriate genotype were cut from 8-12-day cob and put in a liquid LS-inf+100 μm As. The embryos once washed fresh infectious environment. Then the solution was added with agrobacteria, embryos were shaken for 30 seconds and left with bacteria for 5 minutes to precipitate. Then these embryos are the side where the flap of the embryo was transferred on Wednesday LSA and cultured in the dark for two to three days. After about 20-25 embryos per Petri dish were transferred to the environment LSDs that were added Cefotaxime (250 mg/l) and silver nitrate (1.6 mg/l), 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, moved on Wednesday LSD10.5S. Kul is URS was selected in this medium for 6 weeks, at this stage subculturing was carried out for 3 weeks. Viable calli was transferred on Wednesday Reg1, to which was added mannose. After culturing in the light (day/night=16 hours/8 hours) green tissue was transferred on Wednesday Reg2 in the absence of growth regulators and incubated for 1-2 weeks. Seedlings transferred in pots Magenta GA-7 (Magenta Corp., Chicago III)containing environment Reg3, and cultured in the light. After 2-3 weeks the plants were tested for the presence of genes PMI and other target genes by PCR. Positive plants identified by PCR analysis, was transferred to a greenhouse.

Example 12

Analysis of T1 seeds obtained from maize plants expressing α-amylase targeted to the apoplast or at AYR

Received T1 seeds from self-pollinated maize plants transformed or NOV6200 or NOV6201 as described in example 4. It was found that the accumulation of starch in these grains was normal, as was recognized by visual assessment and normal staining iodine solution to test for starch, conducted before the heat treatment. Immature grain was dissected and cleaned endosperm separately placed in microcentrifuge tubes and immersed in 200 μl of 50 mm NaPO4-buffer. The tubes were placed in a water bath at 85°for 20 minutes, and then cooled on ice. the each tube was added twenty microlitres 1% iodine solution and mixed. Approximately 25% egregiously grains gave a positive color on the starch. The remaining 75% gave no color, indicating that the starch was degraded to low molecular weight sugars that are not stained with iodine. It was found that grain T1 NOV6200 and NOV6201 was a grain with autohydrolysis corn starch. Any noticeable reduction in the level of starch after incubation at 37aboutSince it was not observed.

Then analyzed the expression of amylase by allocating a fraction of the hyperthermophilic protein from the endosperm followed by electrophoresis in SDS page/coloring Kumasi. Observed band egregiousness protein with the corresponding molecular weight (50 kDa). These samples were analyzed by α-amylase using commercially available dyed amylose (AMYLAZYME, from Megazyme, Ireland). High levels of activity of hyperthermophilic amylase correlated with the presence of a 50 kDa protein.

In addition, it was found that the starch in the grains obtained from most plants transgene corn, which is expressed hyperthermophilic α-amylase transported in amyloplast, quite active hydrolizable at room temperature, if the specified enzyme was able to engage directly with starch grains. Of eighty lines, the content is related hyperthermophilic α -amylase aimed at amyloplast were identified four lines, which have accumulated starch in the grain. Three of these lines were analyzed for stable α-amylase activity using colorimetric analysis on milazim (Megazyme). Analysis of amylase enzyme showed that these three lines had low levels of thermostable amylase activity. When processing of the pure starch obtained from these three lines, in suitable conditions of moisture and heat, the starch was obtained that indicated the presence of adequate levels α-amylase required to ensure autohydrolysis starch derived from these lines.

Were obtained from the seeds of T1 from multiple independent lines of both transformants NOV6200 and NOV6201. Separate the wheat from each line was cut and peeled endosperm separately homogenized in 300 μl of 50 mm NaPO4-buffer. Aliquots of the suspensions of the endosperm were analyzed on a α-amylase activity at 85°C. Approximately 80% of the lines were segregationalist by hyperthermophilic activity (see figures 1A, 1B and 2).

Grains from wild-type plants or plants transformed NOV6201, was heated at 100°C for 1, 2, 3, or 6 hours, and then stained for the presence of starch iodine solution. After 3 or 6 hours, respectively, in Mature grains nab who was udalos or negligible content of starch, or starch absent. Thus, the starch in Mature seeds of transgenic corn, which is expressed hyperthermophilic amylase targeted to the endoplasmic reticulum, hydrolizable at incubation at high temperature.

In another experiment partially purified starch obtained from Mature seeds of T1 plants with NOV6201, which were soaked in 50°C for 16 hours, hydrolizable after heating at 85°C for 5 minutes. It was pointed out that α-amylase targeted to the endoplasmic reticulum, is associated with starch after grinding grain and is able to hydrolyze starch after heating. The iodine staining showed that the starch remains intact in Mature seeds after soaking for 16 hours at 50°C.

In another experiment egregiousness Mature grain, obtained from plants transformed NOV6201, was heated at 95°C for 16 hours and then dried. In seeds expressing the hyperthermophilic α-amylase, after drying, was the hydrolysis of starch into sugar, which was indicated view wrinkled grain.

Example 13

Analysis of T1 seeds obtained from maize plants expressing α-amylase aimed at amyloplast

Received T1 seeds from self-pollinated maize plants transformed or NOV4029, l is Bo NOV4031, as described in example 4. The accumulation of starch in the grains from these lines was obviously abnormal. All lines were segregationalist, with a certain degree of variability in the phenotype of a very low content of starch or no starch. Before processing at high temperatures, the endosperm, purified from immature grains, gave only a weak coloration with iodine. After incubation for 20 minutes at 85°color disappeared. After drying corn grain was wrinkled. This specific amylase had, apparently, sufficient activity at temperatures that are generated in the greenhouse for hydrolysis of starch, if this enzyme were in direct contact with starch grains.

Example 14

Fermentation of grain of maize plants expressing α-amylase

100% transgenic corn, 85°and 95°With varying time dilution

Transgenic corn (NOV6201), which contained a thermostable α-amylase, well were subjected to fermentation without the addition of exogenous α-amylase, required less time to liquefy and gave a more complete solubilization of the starch. Fermentation in laboratory scale were carried out in accordance with the Protocol, including the following steps (described in detail below): 1) grinding, (2) analysis on humidity, 3) obtaining a suspension containing the crushed corn, water bard and α -amylase, 4) liquefaction and 5) simultaneous saccharification and fermentation (SSF). In this example, the temperature and time dilution was varied as described below. In addition, transgenic corn was subjected to dilution with and without the use of exogenous α-amylase and the efficiency of producing ethanol were compared with a control efficiency of corn processed commercially available α-amylase.

Transgenic corn used in this example was obtained in accordance with the procedures described in example 4, using the vector containing the gene α-amylase and selective marker PMI, namely NOV6201. Transgenic maize was produced by pollination of commercially available hybrid (N3030) pollen of transgenic lines expressing high levels of thermostable α-amylase. Corn was dried to state 11% moisture content and stored at room temperature. Content α-amylase in flour transgenic corn was 95 units/g, where 1 unit of enzyme generates 1 micromoles of reducing ends in one minute in corn flour at 85°in MES buffer, pH 6.0. As control was used yellow dent corn, which is known to be well produces ethanol.

1) Grinding: transgenic corn (1180 g) were crushed in a hammer mill Perten 3100, where is Noah a 2.0-mm sieve, and thus gained the flour transgenic corn. Control corn was ground in the same mill after a thorough cleaning to prevent contamination by transgenic corn.

2) Analysis of the moisture content: the samples (20 g) of transgenic and control corn was weighed in an aluminum boat for weighing and heated at 100°C for 4 h Then the samples were again weighed and the moisture content was calculated by the mass loss. The moisture content in transgenic flour was 9,26%, and in the control flour - 12,54%.

3) Obtaining suspensions: the composition of the suspensions were prepared so that the resulting biomass contained 36% solids in the early stages of SSF. Control samples were prepared in plastic containers with a capacity of 100 ml, and these samples contained 21.50 per g flour control maize, 23 ml of deionized water, 6.0 ml of bards (8% solids by weight) and 0.30 ml of a commercially available α-amylase, diluted 1/50 water. The selected dose α-amylase was a dose commonly used in industrial production. In the analysis under the conditions described above for the analysis of transgenic α-amylase dose control α-amylase was 2 u/g of corn flour. the pH is brought to 6.0 by addition of ammonium hydroxide. Transgenic samples were prepared similarly, but because of the lower content of vlahov transgenic flour they contained 20 g of corn flour. Suspension transgenic flour received either α-amylase in the same dose as control samples, with or without exogenous α-amylase.

4) Liquefaction: a vessel containing a suspension of flour transgenic corn, was immersed in a water bath at 85°or at 95°at 5, 15, 30, 45 or 60 minutes. Control suspensions were incubated for 60 minutes at 85°C. During the incubation at high temperature the suspension is intensively stirred manually every 5 minutes. After the stage of processing at high temperature the suspension was cooled on ice.

5) Simultaneous saccharification and fermentation of biomass, obtained by dilution, mixed with glucoamylase (0,65 ml of a 1/50 dilution of a commercially available glucoamylase L-400), protease (0,60 ml 1000-fold dilution of a commercially available protease) and 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the specified biomass, making the hole for the release of CO2for the purposes of ventilation. Then the specified biomass was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated by obtaining a mixture containing yeast (0.12 g), with 70 grams of maltodextrin, 230 ml of water and 100 ml of bards, glucoamylase (0,88 ml-10-fold dilution of a commercially available glucoamylase), protease (1,76 ml of 100-fold dilution of a commercially available enzyme), urea (1,07 g), penicillin (0,67 mg) and zinc sulfate (0,13 g). The reproduction of culture initiated one day before use, and incubated under stirring at 90°F.

After 24, 48 and 72 hours, the samples were removed from each fermenter were filtered through 0.2 μm filters and analyzed by HPLC for ethanol and sugar. After 72 hours, the samples were analyzed for complete dissolution of solids and residual starch.

HPLC analysis was performed using a dual gradient system equipped with a detector of the refractive index, the heating column and the column of Bio-Rad Aminex HPX-N. The system was balanced 0.005 M H2SO4in water at a concentration of 1 ml/min column Temperature was 50°C. the injection Amount of the sample was 5 μl; elution was performed in the same solvent. The response RI was calibrated by injection of known standards. The level of ethanol and glucose were measured for each injection.

The residual starch was measured as follows. Samples and standards were dried at 50°in the oven and then crushed to powder in the mill for samples. Powder (0.2 g) was weighed into a graduated centrifuge tube with a capacity of 15 ml Powder 3 times washed with 10 ml of aqueous ethanol (80% vol./about.) through intensive paramasivan is followed by centrifugation and removal of supernatant. To the precipitate was added DMSO (2.0 ml)and then added 3.0 ml of thermostable alpha-amylase (300 units) in MOPS buffer. After intensive mixing, the tubes were incubated in a water bath at 85°C for 60 min In the incubation process the contents of the tubes were mixed four times. The samples were cooled and added to 4.0 ml nitroacetate buffer (200 mm, pH 4.5), and then 0.1 ml of glucoamylase (20%). Samples were incubated at 50°C for 2 hours, stirred, and then centrifuged for 5 minutes at 3500 rpm./minutes the Supernatant was filtered through 0.2 μm filter and analyzed for glucose HPLC method described above. For samples with a low content of residual starch (<20% solids) volume of injection was 50 µl.

Results. Transgenic corn can be well managed fermentation without adding α-amylase. The outputs of ethanol in 72 hours were mostly identical, regardless of the presence or absence of exogenous α-amylase, as shown in table 1. These data also showed that higher yield of ethanol is achieved at a higher temperature liquefaction; the enzyme of the present invention expressed in transgenic maize, have activity at higher temperatures than other commercially available enzymes, such as α-amylase of Bacillus liquefaciens.

Table 1
The pace. liquefaction, °The liquefaction time, minExogenous α-amylase# repeatThe average yield of ethanol, vol.%/about.Stockl., % vol./about.
8560Yes417,530,18
8560No417,780,27
9560Yes218,22ND
9560No218,25ND

When you change the time of the dissolution it was found that the liquefaction time required for the efficient production of ethanol, was less than one hour is required for the implementation of the standard process. The figure 3 shows that the yield of ethanol after 72 hours of fermentation remained almost unchanged over a period of time thinning from 15 minutes to 60 minutes. In addition, the liquefaction at 95°gave more ethanol in each period of time than the dilution at 85°C. This observation demonstrated the improvement of performance when using hyperthermophilic enzyme.

Control maize gave higher the yield of ethanol, than transgenic corn, but was selected control corn because it was found highly efficient fermentation. In contrast, transgenic corn had a genetic background that is selected to stimulate transformation. Introduction α-amylase trait into elite germplasm of maize by well-known methods of crossing should resolve this difference.

Assessment of residual levels of starch in the beer, prepared after 72 hours (figure 4)showed that transgenic α-amylase gave a significant increase in the production of starch available for fermentation, after fermentation remained much less starch.

Using levels of ethanol and levels of residual starch optimal liquefaction time was 15 minutes at 95°C and 30 minutes at 85°C. In these experiments, this time was a total time during which the fermenters were in a water bath, and, thus, included the period for which the temperature of the samples was increased from room temperature to 85°or 95°C. When large-scale industrial processes, at which the rapid heating of biomass with the use of such equipment, as the digester inkjet type can be optimal even shorter time Rasi is possible. Standard industrial processes require dilution of the vessels-collections, which allow to incubate biomass at high temperatures for one or more hours. The present invention avoids the necessity of using such tanks-collections and to improve the performance of the equipment to liquefy.

One of the important functions α-amylase in fermentation processes is to reduce the viscosity of the biomass. At all times, the samples containing flour transgenic maize were significantly less viscous than the control sample. In addition, transgenic samples, obviously not passed the phase of gelatinization, which was observed for all control samples and which usually occurs when cooking corn suspensions. Thus, the presence of α-amylase, distributed in all the fragments of the endosperm, attached biomass in the process of cooking a preferential physical properties, thus preventing the formation of large quantities of gel, which slows down the diffusion and increases the energy consumption for mixing and applying the specified biomass.

High dose α-amylase in transgenic maize may also give the indicated transgenic biomass of preferred properties. At 85°activity α-amylase transgenic corn was many times greater than the activity of the th same dose of exogenous α -amylase used in the control. This control α-amylase was selected as representative α-amylase for industrial applications.

Example 15

Effective function of transgenic corn by mixing with a control corn

The transgenic corn flour mixed with flour control of maize at different levels of transgenic corn flour from 5% to 100%. This mixture was processed as described in example 14. Biomass containing transgenic downregulation α-amylase were subjected to liquefaction at 85°C for 30 minutes and at 95°C for 15 minutes, and the control amount of biomass was obtained as described in example 14, and subjected to liquefaction at 85°C for 30 or 60 minutes (once each) or at 95°C for 15 or 60 minutes (one time each).

The data obtained for ethanol, 48 and 72 hours, and the residual starch are shown in table 2. The levels of ethanol after 48 hours represented graphically in figure 5, and the determination of residual starch is shown in figure 6. These data showed that transgenic expressing thermostable α-amylase gave a very good level of production of ethanol, even when transgenic grain accounted for only a small proportion (at least 5%) of the total grain biomass. These data also showed that the level of residual starch was significantly lower, che is the level in the control biomass, even when transgenic corn was at least 40% of the total amount of grain.

Table 2
Liquefaction at 85°Dilution with 95°
Transgenic grain, wt.%Residual starchEthanol, 48 hoursEthanol % vol./about., 72 hoursResidual starchEthanol, 48 hoursEthanol % vol./about., 72 hours
100to 3.5816,7118,324,1917,7221,14
804,0617,0419,23,1517,4219,45
603,8617,1619,674,8117,5819,57
405,1417,2819,838,6917,5619,51
208,7717.11 per bbl19,511,0517,7119,36
10there is a 10.0318,0519,7610,817,8319,28
510,67 18,0819,41to 12.4417,6119,38
0*7,7917,6420,1111,2317,8819,87
* Control samples. Average for 2 definitions

Example 16

The production of ethanol, depending on the pH of the dilution with the use of transgenic corn at levels of 1.5% to 12% of the total maize

Because transgenic corn lends itself well to the fermentation at the level of 5-10% of the total maize was conducted an additional series of fermentati, in which transgenic corn was 1.5-12% of the total maize. the pH ranged from 6.4 to 5.2, and the enzyme α-amylase expressed in transgenic maize was optimized for activity at a lower pH than is typically used in the industry.

The experiments were carried out as described in example 15, with the following exceptions:

1. Transgenic flour mixed with the control flour, and the percentage of transgenic flour on the total dry weight was within 1.5%-12,0%.

2. Control corn was N3030, which had greater similarity with transgenic corn than the control corn used in examples 14 and 15.

3. In samples containing transgenic flour, without added exogenous α-amylase.

4. Re the dilution pH of the samples was brought to 5.2, 5,6, 6,0 6,4 or. For each pH received at least 5 samples in which the content of flour transgenic corn was 0%-12%.

5. Dilution for all samples was carried out at 85°C for 60 minutes.

Change the content of ethanol depending on the time of fermentation are shown in figure 7. This figure presents data obtained from samples containing 3% transgenic corn. At lower pH, the fermentation proceeds faster than at pH 6.0 and above; however, a similar dependence was observed for the samples with different content of transgenic grain. the pH-activity profile of transgenic enzyme, along with high levels of expression, allows liquefaction at lower pH, which will result in faster fermentation and thereby increase performance than was possible in the standard processes at pH 6.0.

The outputs of ethanol in 72 hours is shown in figure 8. As you can see on the outputs of ethanol, these results show little dependence on the number of transgenic grains contained in the sample. Thus, to improve the fermentative production of ethanol grain should contain excessive amounts of amylase. It also indicates that liquefaction at lower pH gives a higher yield of ethanol.

Spent monitor the ring viscosity of the samples after dilution, and it was observed that at pH 6.0 to adequately reduce the viscosity was enough for 6% of the transgenic grain. At pH of 5.2 and 5.6 viscosity was equivalent viscosity control 12% of the content of transgenic seed, but not at a lower content of transgenic grain.

Example 17

Production of fructose from corn flour using thermophilic enzymes

It was shown that corn that expresses hyperthermophilic α-amylase 797GL3, more efficiently produces fructose when mixed with α-glucosidase (MalA) and ksilozoizomerazy (XylA).

Seeds from NOV6201-transgenic plants expressing 797GL3, crushed into flour in the camera Kleco, and thus received amylase flour. Grain non-transgenic corn was ground in the same way and got the control flour.

α-Glucosidase, MalA (from S.solfataricus), expressed in E.coli. The collected bacteria suspended in 50 mm phosphate potassium buffer, pH 7.0, containing 1 mm 4-(2-amino-ethyl)benzolsulfonate, and then subjected to lysis in a French press. The lysate was centrifuged at 23000×g for 15 minutes at 4°C. the supernatant Solution was removed, heated to 70°C for 10 minutes, cooled on ice for 10 minutes and then centrifuged at 34000×g for 30 minutes at 4°C. the supernatant Solution was removed and MalA was concentrated until workrates decline in the centrifuge Method 10. The filtrate obtained in the stage of centrifugation on the Method 10, left for use as a negative control for MalA.

Xylose(glucose)isomerase was obtained by gene expression l T.neapolitana in E.coli. Bacteria suspended in 100 mm sodium phosphate, pH 7.0, and were subjected to lysis by passing through a French press. After precipitation of cell debris extract was heated at 80°C for 10 minutes and then centrifuged. The supernatant solution had l-enzymatic activity. Simultaneously with the extract l got an extract empty control vector.

Corn flour (60 mg per sample) was mixed with buffer and extracts from E. coli. As shown in table 3, the samples contained corn flour with amylase (amylase) or control corn flour (control), 50 μl of either extract (+) MalA or filtrate (-), and 20 μl of either extract l (+)or control empty vector (-). All samples also contained 230 μl of 50 mm MOPS, 10 mm MgSO4and 1 mm CoCl2; the pH of the buffer was 7.0 at room temperature.

Samples were incubated at 85°C for 18 hours. At the end of incubation time, the samples were diluted with 0.9 ml of water at 85°C and centrifuged to remove undissolved material. Then the supernatant fraction was filtered by passing through the device for ultrafiltration entricon 3 and analyzed using HPLC and ELSD detection.

Gradient HPLC system was equipped with a column Astec Amino Polymer Column, particle size 5 µm, 250×4.6 mm and the detector Alltech ELSD 2000. This system is pre-balanced mixture of water:acetonitrile =15:85. The flow rate was 1 ml/min Initial conditions were maintained for 5 minutes after injection, then for 20 minutes spent elution gradient of water:acetonitrile =50:50 and within 10 minutes the same solvent. The system was washed for 20 minutes with a mixture of water:acetonitrile =80:20, and then balanced the original solvent. Fructose was lirowaus 5.8 minutes, and glucose - 8.7 minutes.

Table 3
SampleCorn flourMalAlThe peak area of fructose×10-6The peak area of glucose×10-6
1Amylase++25,9110,3
2Amylase-+7,012,4
3Amylase+-0,1147,5
4Amylase--025,9
5Control++0,80,5
6Control-+0,30,2
7Control+-1,31,7
8Control--0,20,3

The results of the HPLC also showed the presence of higher level of maltooligosaccharides in all samples containing α-amylase. These results demonstrated that the three thermophilic enzyme, taken together, can produce fructose from corn flour at a high temperature.

Example 18

Amylase flour with isomerase

In another example, amylase flour was mixed with purified MalA, and with each of the two bacterial xylothamia: l T.maritima and enzyme labeled BD8037 and received from Diversa. Amylase flour was obtained as described in example 18.

MalA S.solfataricus with 6His-tag for purification of expressed in E.coli. Cell lysate was obtained as described in example 18, after which the lysate was purified to apparent homogeneity using a resin having affinity to Nickel (Probond, Invitrogen), in accordance with the manufacturer's instructions for cleaning a native protein.

l T.maritma with added S-tag and signal retention in the ER expressed in E.coli and received in the same way, as l T.neapolitana described in example 18.

Xylothamia D8037 received in the form of liofilizirovannogo powder and resuspendable in water in the amount of 0.4 from the original.

Amylase corn flour mixed with enzyme solutions containing water or buffer. All of the reaction mixture contained 60 mg amylase flour and 600 μl of the total quantity of liquid. One series of reaction mixtures sauterelle 50 mm MOPS, pH 7.0, at room temperature, with the addition of 10 mm MgSO4and 1 mm CoCl2and in the second series of reaction mixtures containing buffer solution was replaced with water. The number isomerase enzyme was varied, as shown as table 4. All reaction mixtures were incubated for 2 hours at 90°C. Fraction of the reaction supernatant was obtained by centrifugation. The precipitate again washed with 600 μl of H2O and centrifuged. Fractions of supernatant from each reaction were combined, filtered through the Method 10 and analyzed using HPLC with ELSD-detection, as described in example 17. The amount of glucose and fructose are graphically presented in figure 15.

Table 4
SampleAmylase flourMalAThe isomerase
160 mg+/td> no
260 mg+T.maritima, 100 µl
360 mg+T.maritima, 10 ál
460 mg+T.maritima, 2 ál
560 mg+BD8037, 100 µl
760 mg+BD8037, 2 ál
860 mgnono

In the presence of each isomerase fructose was producirovanie of corn flour depending on the dose in the case when the reaction was attended by α-amylase and α-glucosidase. These results demonstrated that expressed corn amylase 797GL3 can function with MalA and with a variety of thermophilic isomers, in the presence or in the absence of metal ions, with the production of fructose from corn flour at high temperature. In the presence of added divalent metal ions these isomerases can give balanced predicted levels of fructose:glucose at 90°With approximately 55% glucose. The process can be seen as an improved compared with the conventional process that uses mesophilic isomerases and which requires chromatographic the division to increase the concentration of fructose.

Example 19

Expression of pullulanase in maize

Transgenic plants that are homozygous or NOV7013 or NOV7005, were crossed to obtain seeds of transgenic corn expressing how α-amylase 797GL3 and pullulan 6GP3.

Received the seeds of T1 or T2 from self-pollinated maize plants transformed or NOV7005 or NOV4093. NOV4093 is a hybrid synthetic gene 6GP3, optimized codons maize (SEQ ID NO:3, 4), with amyloplasts target sequence (SEQ ID NO:7, 8), which provides localization of the hybrid protein on amyloplast. The hybrid protein is under the control of the promoter ADP-gpp (SEQ ID NO:11) for specific expression in the endosperm. Design NOV7005 provides targeted expression of pullulanase in the endoplasmic reticulum of the endosperm. The localization of this enzyme in the ER ensures the normal accumulation of starch in the grains. In addition, we observed normal staining starch iodine solution before any processing at high temperatures.

As described in the case α-amylase expression of pullulanase aimed at amyloplast (NOV4093), leading to abnormal accumulation of starch in the grains. When drying corn grain was wrinkled. It is obvious that thermophilic of pullulanase is rises is active at low temperatures and hydrolyzes starch in that case, if there is direct contact with the starch granules in the endosperm of the seed.

Receiving enzyme or extraction of the enzyme from maize flour.

Enzyme pullulanase was extracted from transgenic seeds by grinding in a mill Kleco, followed by incubation flour in 50 mm NaOAc buffer, pH 5.5, for 1 hour at room temperature and under continuous shaking. Then, after incubation, the mixture was centrifuged for 15 minutes at 14000 rpm, the Supernatant was used as source of enzyme.

Analysis on pullulanase: Analytical reaction was carried out in 96-well pad. The enzyme extracted from corn flour (100 μl), 10-fold diluted with 900 μl of 50 mm NaOAc buffer, pH 5.5, containing 40 mm CaCl2. The mixture was intensively stirred, and then to each reaction mixture were added 1 tablet Limit-Dextrizyme (structured Surinam pullulan from Megazyme) and incubated at 75°C for 30 minutes (or as mentioned above). At the end of the incubation the reaction mixture was centrifuged for 15 minutes at 3500 rpm the Supernatant 5-fold diluted and transferred to flat-bottomed 96-well plate to measure the optical density at 590 nm. Hydrolysis structured Surinam pullulanase substrate by pullulanase led to the production of water-soluble dyed fragments, and the speed of the x release (measured by optical density at 590 nm) was directly correlated with enzymatic activity.

The figure 9 illustrates the analysis of T2 seeds at different times of transformation NOV7005. In some cases it was possible to detect high expression pullulanase activity compared to non-transgenic control.

To the measured quantity (˜100 μg) of dry corn flour obtained from transgenic plants (expressing pullulanase or amylase or the other enzyme) and/or from the control (non-transgenic) plants, was added to 1000 μl of 50 mm NaOAc buffer, pH 5.5, containing 40 mm CaCl2. The reaction mixture was intensively mixed and incubated on a shaker for 1 hour. The enzymatic reaction was initiated by transferring mixtures for incubation in conditions of high temperature (75°S, the optimal reaction temperature for pullulanase or as shown in the figures), and incubated for a period of time indicated on the figures. The reaction was stopped by cooling on ice. Then the reaction mixture was centrifuged for 10 minutes at 14000 rpm Aliquot (100 μl) of the supernatant three times diluted and filtered through an 0.2 micron filter for HPLC analysis.

The samples were analyzed by HPLC using the following conditions:

Column: Alltech Prevail Carbohydrate ES, 5 micron, 250×4.6 mm

Detector: Alltech ELSD 2000

Pump: Gilson 322

Injector: injector/diluent Gilson 215

Solvents: In the LC, acetonitrile high quality (Fisher Scientific) and water (purified on a system of Waters Millipore System)

The gradient used for oligosaccharides with a low degree of polymerization (DP 1-15).

Time% Water% Acetonitrile
01585
51585
255050
355050
368020
558020
561585
761585

The gradient used for saccharides with a high degree of polymerization (DP 20-100 and above).

Time% Water% Acetonitrile
03565
608515
708515
853565
1003565

The system used for data analysis: Gilson Unipoint Software System Version 3.2.

In figures 10A and 10B shows the HPLC analysis of the products hydro is the study of the starch in the flour transgenic corn, generated expressed by pullulanase. Incubation of flour from expressing pullulanase corn in the reaction buffer at 75°C for 30 minutes resulted in the production of oligosaccharides with an average size of chain (DP ˜10-30) and amylose with short chains (DP ˜100-200) of corn starch. This figure also shows the dependence pullulanase activity from the presence of calcium ions.

Transgenic corn expressing pullulanase, can be used for producing modified starch/dextrin, which is unbranched (split α-1-6-relations), and therefore will have a high level of amylose/dextrin with a straight chain. In addition, depending on the type of starch (for example, waxy, vysokoimpulsnogo and the like), the distribution on the chain length of amylose/dextrin generated by pullulanase will vary, and therefore, will vary and properties of the modified starch/dextrin.

It was also demonstrated processing α-1-6-ties using pullulan as substrate. Pullulanase isolated from corn flour, effectively hydrolyzed in pullulan. HPLC-analysis (described) of the product generated at the end of incubation, indicated, as expected, producing maltotriose that would be what about the due processing α -1-6-bonds in molecules pullulan enzyme corn.

Example 20

Expression of pullulanase in maize

The expression of pullulanase 6gp3 additionally analyzed by extraction of corn flour with subsequent electrophoresis in SDS page and staining Kumasi blue. Corn flour was obtained by grinding the seeds for 30 seconds at the mill Kleco. This enzyme was extracted from about 150 mg of flour with 1 ml of 50 mm NaOAc buffer, pH 5.5. The mixture was intensively mixed and incubated on a shaker at room temperature for 1 hour, and then incubated for another 15 minutes at 70°C. the mixture is Then centrifuged (14000 rpm for 15 minutes at room temperature) and the supernatant was used for analysis by electrophoresis in LTO-PAG. When this was observed, the protein band corresponding to a molecular weight of 95 kDa). These samples were analyzed on pullulanase using commercially available conjugated with dye-limit dextrins (LIMIT-DEXTRIZYME, from Megazyme, Ireland). High levels of thermophilic pullulanase activity correlated with the presence of 95 kDa protein.

Western blot analysis and ELISA analysis of transgenic maize has also demonstrated the expression of ˜95 kDa protein that reacts with the antibody against pullulanase (expressed in E.coli).

Example 21

The expansion of the rate of hydrolysis of starch and increase the yield of oligosaccharides from short circuit (formatiruem) adding corn, expressing pullulanase

The data presented in figures 11A and 11B were obtained based on the above HPLC analysis of the cleavage products of starch from two reaction mixtures. The first reaction mixture indicated as "amylase", contained a mixture [1:1 (wt./wt.)] samples of corn flour for transgenic corn expressing α-amylase and cooked by way of, for example, described in example 4, and for non-transgenic corn A; and the second reaction mixture is specified as "amylase + pullulanase", contained a mixture [1:1 (wt./wt.)] samples of corn flour for transgenic corn expressing α-amylase, and transgenic corn expressing pullulanase, and prepared by the method described in example 19. The obtained results confirmed the advantage of using pullulanase in combination with α-amylase during hydrolysis of starch. This advantage is in increasing the rate of hydrolysis of starch (figure 11A) and the increase in the output of formatiruem oligosaccharides with a low DP (figure 11B).

It was found that for the production of maltodextrin (oligosaccharides with a straight or branched chain) can be used either only α-amylase, or α-amylase and pullulanase (or any other combination krahmalsoderzhaschih enzymes), expressed in corn (igure 11A, 11, 12 and 13A). Depending on the reaction conditions, the type of hydrolytic enzymes and their combinations and the type of starch, the composition of the produced maltodextrins, and hence their properties will vary.

The figure 12 presents the results of an experiment carried out in a manner analogous to the method described in figure 11. This figure shows the different temperature and time regimes used in the process of incubation of the reaction mixtures. The optimal reaction temperature for pullulanase is 75°, and α-amylase she is >95°C. Consequently, these modes correspond to the implementation of catalysis pullulanase and/or α-amylase at their respective optimal temperatures of the reaction. From these results we can conclude that the combination of α-amylase and pullulanase gave more effective in the hydrolysis of corn starch at the end of the 60-minute incubation period.

HPLC analysis described above (except that in these reactions used ˜150 mg of corn flour) and assessment products of the hydrolysis of starch from two series of reaction mixtures after 30-minute incubation, illustrated in figure 13A and 13B. The reaction mixture in the first series of reactions were incubated at 85°and the reaction mixture is in the second series were incubated at 95° C. For each series were obtained from two of the reaction mixture; the first reaction mixture indicated as "amylase x pullulanase", contains flour from transgenic corn (produced by cross-pollination), expressing how α-amylase and pullulanase, and the second reaction mixture is specified as "amylase", is a mixture of samples of corn flour from expressing α-amylase transgenic maize and non-transgenic maize A in this regard, where α-amylase activity present in the same number as this occurs when cross-pollination (amylase × pullulanase). The total yield of oligosaccharides with a low DP was higher in the case of crosses "α-amylase × pullulanase" compared with corn expressing only α-amylase, at incubation samples of corn flour at 85°C. Incubation at a temperature of 95°led to inactivation (at least partially) of the enzyme pullulanase, and therefore between samples amylase × pullulanase" and "amylase" it was possible to observe small differences. However, the data obtained for both incubation temperatures, showed that the use of corn flour obtained from crosses "amylase × pullulanase", there was a significant increase in the number of the produced glucose (figure 13B) p is the end of the incubation period, than using corn flour, expressing only α-amylase. Therefore, the use of corn expressing how α-amylase and pullulanase, may be particularly preferred in processes where the desired complete hydrolysis of starch into glucose.

The above examples provide additional evidence that pullulanase expressed in corn seeds and used in combination with α-amylase that improves the process of hydrolysis of starch. The activity of the enzyme pullulanase, which is specific to α-1-6-relations, hydrolyzes the point of branching of the starch is much more efficient than α-amylase (an enzyme specific to α-1-4-PR), which leads to reduction in the number of branched oligosaccharides (e.g., limit dextrin, Panoz, which usually does not get fermented) and increase the number of oligosaccharides with a short, straight chain (easy formatiruem to ethanol, and the like). Secondly, the fragmentation of the molecules of starch catalyzed by pullulanase hydrolysis of branching points starch leads to increased availability of substrate for α-amylase, and hence to increase the efficiency of the reactions catalyzed α-amylase.

Example 22

In order to determine whether alpha-amylase 797GL3 and alpha-glucosidase malA to function under the same condition is the conditions of pH, temperature and producing an increased amount of glucose compared to the amount of glucose, produced by any of these enzymes, taken separately, to a solution containing 1% starch and starch, selected either from the seeds of non-transgenic maize (control)or from seeds of transgenic corn 797GL3 (from corn seed 797GL3, where alpha-amylase is secreted together with starch), was added around 0.35 µg of the enzyme alpha-glucosidase malA produced in bacteria. In addition, the starch isolated from non-transgenic corn seed and seeds of transgenic corn 797GL3, was added to 1% corn starch in the absence of any enzyme malA. The mixture was incubated at 90°C, pH 6.0, for 1 hour, centrifuged to remove any nerastvorimogo material and the soluble fraction was analyzed by HPLC for glucose levels. As shown in figure 14, the alpha-amylase 797GL3 and alpha-glucosidase malA operate under similar pH and temperature, decomposing starch into glucose. In this case, the number of produced glucose was significantly higher than the amount of glucose produced by any one enzyme.

Example 23

Was determined by the efficiency of glucoamylase Thermoanaerobacterium for the initial hydrolysis of starch. As shown in figure 15, hydrolytic transformation of the original starch was tested with water, α-amylase barley (commercially available drug Sigma), glucoamylase Thermoaaerobacterium and their combinations at room temperature and at 30° C. As shown, the combination of α-amylase barley with glucoamylase Thermoanaerobacterium able to hydrolyze the original starch into glucose. Moreover, the amount of glucose produced by amylase barley and GA Thermoanaerobacter, significantly exceeded the amount of glucose produced by any one enzyme.

Example 24

Optimized codons maize genes and sequences for the initial hydrolysis of starch and vectors for plant transformation

The enzymes were selected for their ability to hydrolyze the original starch at temperatures of approximately 20-50°C. Then the corresponding genes or gene fragments were designed using codons corn, preferred to construct synthetic genes as described in example 1.

Selected hybrid polypeptide α-amylase/glucoamylase Aspergillus shirousami (without signal sequence), which has the amino acid sequence represented in SEQ ID NO:45 and identified in Biosci. Biotech. Biochem. 56:884-889 (1992); Agric. Biol. Chem. 545:1905-14 (1990); Biosci. Biotechnol. Biochem. 56:174-79 (1992). Designed also nucleic acid, optimized codons corn, and this nucleic acid represented in SEQ ID NO:46.

Similarly selected the glucoamylase Thermoanaerobacterium thermosaccharolyticum, having amino acid sequence SEQ ID NO:47, published in Biosci. iotech. Biochem. 62:302-308 (1998). Designed nucleic acid, optimized codons maize (SEQ ID NO:48).

Selected the glucoamylase Rhizopus oryzae, having the amino acid sequence (without signal sequence) (SEQ ID NO:49), described in the literature (Agric. Biol. Chem. (1986) 50, pg. 957-964). Designed nucleic acid, optimized codons corn, and this nucleic acid represented in SEQ ID NO:50.

In addition, selected α-amylase corn, and its amino acid sequence (SEQ ID NO:51) and nucleic acid sequence (SEQ ID NO:52) was obtained from the literature. See, for example, Plant Physiol. 105:759-760 (1994).

Designed expressing cassette for expression of the hybrid polypeptide α-amylase/glucoamylase Aspergillus shirousami designed and optimized codons corn nucleic acid represented in SEQ ID NO:46; glucoamylase Thermoanaerobacterium thermosaccharolyticum, designed and optimized codons corn nucleic acid represented in SEQ ID NO:48; glucoamylase Rhizopus oryzae, having the amino acid sequence (without signal sequence) (SEQ ID NO:49), designed and optimized codons corn nucleic acid represented in SEQ ID NO:50, and α-amylase corn.

The plasmids containing N-terminal signal sequence γ-Zein cook who rouses (MRVLLVALALLALAASATS) (SEQ ID NO:17), was attached to a synthetic gene, codereuse enzyme. Optional to-end of the synthetic gene was annexed sequence SEKDEL for targeting to and retention in the ER.

Hybrid cloned in the area not covered by the promoter γ-Zein of corn, for specific expression in the endosperm in the plasmids for plant transformation. Hybrid targets the tissue of maize via Agrobacterium-transfection.

Example 25

For the expression of enzymes designed expressing cassette containing the selected enzymes. A plasmid containing the sequence of the binding site with the original starch, was added to the synthetic gene, codereuse enzyme. The binding site with the original starch was provided by the binding of the enzyme with regulationsgoverning starch. Amino acid sequence of the binding site with the original starch (SEQ ID NO:53) was determined from the literature and sequence nucleic acids optimized for codons corn with obtaining SEQ ID NO:54. The sequence of the nucleic acid, optimized codons corn, was added to the synthetic gene, codereuse enzyme, plasmid for expression in the plant.

Example 26

Designing gene-optimized codons corn, and vectors for plant transformation

Genes or Genn is e fragments were designed using codons corn, preferred to create synthetic genes as described in example 1.

Selected EGLA, hyperthermophilic endoglucanase Pyrococcus furiosus (without signal sequence)having the amino acid sequence represented in SEQ ID NO:55 and identified in Journal of Bacteriology (1999) 181, pg.284-290). Designed nucleic acid, optimized codons corn and presented in SEQ ID NO:56.

Selected xylothamia Thermus flavus, having the amino acid sequence represented in SEQ ID NO:57, and is described in Applied Biochemistry and Biotechnology, 62:15-27 (1997).

Designed expressing cassettes for the expression of EGLA (endoglucanase) Pyrococcus furiosus of optimized codons corn nucleic acid (SEQ ID NO:56) and xylose isomerase Thermus flavus from optimized codons corn nucleic acid that encodes the amino acid sequence of SEQ ID NO:57. The plasmids containing N-terminal signal sequence γ-Zein of maize (MRVLLVALALLALAASATS) (SEQ ID NO:17), was added to synthetic and optimized codons maize gene, codereuse enzyme. Optional to-end of the synthetic gene was annexed sequence SEKDEL for targeting to and retention in the ER. Hybrid cloned in the area not covered by the promoter γ-Zein of corn, for specific expression in the endosperm in plasmas is de for the transformation of plants. Hybrid targets the tissue of maize via Agrobacterium-transfection.

Example 27

The production of glucose from corn flour using thermophilic enzymes expressed in maize

It was shown that the expression of hyperthermophilic α-amylase, 797GL3, and α-glucosidase (MalA) leads to the production of glucose when mixed with aqueous solution and incubation at 90°C.

A line of transgenic corn (line AV, pNOV4831)expressing the enzyme l identified by measuring α-glucosidase activity, indicating that the hydrolysis of p-nitrophenyl-α-glucoside.

Corn obtained from transgenic plants expressing 797GL3, crushed into flour in the camera Kleco and got flour containing amylase. Corn obtained from transgenic plants expressing MalA, was ground into flour in the camera Kleco and got flour containing MalA. Grain non-transgenic corn was ground in a similar way and got the control flour.

The buffer consisted of 50 mm MES buffer, pH 6.0.

Hydrolysis of corn flour: the Samples were received, as described below in table 5. Corn flour (about 60 mg per sample) was mixed with 40 ml of 50 mm MES buffer, pH 6.0. Samples were incubated in a water bath at 90°C for 2.5 and 14 hours. At the indicated time of incubation samples were taken and the analysis is whether glucose.

The samples were analyzed for glucose analysis using glucose oxidase/horseradish peroxidase. The GOPOD reagent contained: 0.2 mg/ml o-dianisidine, 100 mm Tris, pH 7.5, 100 units/ml glucose oxidase and 10 units/ml horseradish peroxidase. 20 μl of sample or diluted sample was placed in the wells of 96-hole tablet together with the standards of glucose (which ranged from 0 to 0.22 mg/ml). To each well was added, with stirring, 100 μl of GOPOD reagent and the plates were incubated at 37aboutC for 30 minutes. Then added 100 μl of sulfuric acid (9 M) and read the optical density at 540 nm. The glucose concentration in the samples was determined from the standard curve, constructed using the pattern. The amount of glucose observed in each sample are listed in table 5.

Table 5
SampleFlour dt, mgAmylase flour, mgMalA-flour, mgThe buffer mlGlucose, 2.5 h, mgGlucose, 14 h mg
166004000
231300400,260,50
330 31,54000,09
4032,230,040to 2.2912,30
406,156,2401,16charged 8.52

These data demonstrated that the expression of hyperthermophilic α-amylase and α-glucosidase in maize leads to the formation of a corn product, which will produce glucose during hydration and heated under appropriate conditions.

Example 28

The production of maltodextrins

To obtain the maltodextrins used grain expressing thermophilic α-amylase. Illustrated method does not require pre-allocation of starch and does not require the addition of exogenous enzymes.

Corn obtained from transgenic plants expressing 797GL3, crushed into flour in the camera Kleco and received "amylase flour". A mixture of 10% transgenic/90% non-transgenic grains were crushed in a similar way and got "10% amylase flour".

Amylase flour and 10% amylase flour (approximately 60 mg/sample) was mixed with water at a ratio of 5 μl of water per one mg of flour. The obtained suspensions were incubated at 90°during the period of time up to 20 hours, as indicated in table 6 Reaction was stopped by adding 0.9 ml of 50 mm EDTA at 85° C and the mixture stirred with a pipette. Took 0.2 ml samples of the suspension were centrifuged to remove nerastvorimogo material and bred 3 in water.

The samples were analyzed for sugars and maltodextrins using HPLC with ELSD-detection. Gradient HPLC system was equipped with a column Astec Amino Polymer Column, particle size 5 µm, 250×4.6 mm, and the detector Alltech ELSD 2000. This system is pre-balanced mixture of water:acetonitrile =15:85. The flow rate was 1 ml/min Initial conditions were maintained for 5 minutes after injection, after which elution was performed for 20 minutes with a gradient of water:acetonitrile =50:50, and then for 10 minutes with the same solvent. The system was washed for 20 minutes with a mixture of water:acetonitrile =80:20, and then balanced the original solvent.

The obtained peak areas were normalized to the volume and weight of the flour. The response factor ELSD one μg of carbohydrate decreased with increasing DP, and thus, a higher DP of maltodextrins corresponds to their higher percentage overall than was determined by peak area.

The relative area of peaks for the reaction products with a 100% amylase flour are shown in figure 17. The relative peak areas for the products of the reactions with 10% amylase flour are shown in figure 18.

These data demonstrated that different see the sea maltodextrins can be produced in various periods of heating. Level α-amylase activity may vary with the mixing of transgenic corn expressing α-amylase, with maize wild-type, which leads to a change of the profile of maltodextrin.

The products of hydrolysis reactions described in this example, concentrated and purified in order to cook the food product and for other purposes, using a number of well known methods, including centrifugation, filtration, ion exchange, gel filtration, ultrafiltration, nanofiltration, reverse osmosis, discoloration of the carbon particles, spray drying and other standard methods known to experts.

Example 29

The influence of time and temperature on the production of maltodextrin

The composition maltodextrine products when autohydrolysis grain containing thermophilic α-amylase, may be changed depending on time and temperature of reaction.

In another experiment amylase flour was obtained as described above in example 28, and was mixed with water at a ratio of 300 μl of water at 60 mg of flour. Samples were incubated at 70°S, 80°S, 90°or 100°during the period of time up to 90 minutes. The reaction was stopped by adding 900 μl of 50 mm EDTA at 90°C, centrifuged to remove nerastvorimogo material and filtered through 0.45 µm nylon filters. The filtrates EN who was literally using HPLC, as described in example 28.

The results of this analysis are presented in figure 19. The number of DP on the nomenclature indicates the degree of polymerization. DP2 refers to maltose, DP3 refers to maltotriose etc. Maltodextrins with a higher DP was loirevalley with one peak near the end of the elution and were marked ">DP12". This unit consisted of dextrins, which are passed through 0.45 µm filters and via the relief column, and did not include any very large fragments of starch retained on the filter or on the relief column.

This experiment demonstrated that the composition maltodextrine product may vary depending on temperature and time of incubation, which may be obtained the desired maltooligosaccharides or maltodextrine product.

Example 30

The production of maltodextrin

The composition maltodextrine products derived from transgenic corn containing thermophilic α-amylase, may be modified by adding other enzymes, such as α-glucosidase and xylothamia, as well as by the inclusion of salts in the water the flour mixture before thermal processing.

In another experiment amylase flour, obtained as described above was mixed with purified MalA and/or bacterial ksilozoizomerazy marked BD8037. MalA S. sulfotaricus with 6His-tagged for isdi expressed in E.coli. Cell lysate was obtained as described in example 28, and then the lysate was purified to apparent homogeneity using a resin having affinity to Nickel (Probond, Invitrogen), in accordance with the manufacturer's instructions for cleaning a native protein. Xylothamia D8037 received in the form of liofilizirovannogo powder from Diversa and resuspendable in water in the amount of 0.4 from the original.

Amylase corn flour mixed with enzyme solutions containing water or buffer. All of the reaction mixture contained 60 mg amylase flour and 600 μl of the total quantity of liquid. One series of reaction mixtures sauterelle 50 mm MOPS, pH 7.0, at room temperature, with the addition of 10 mm MgSO4and 1 mm CoCl2and in the second series of reaction mixtures containing buffer solution was replaced with water. All reaction mixtures were incubated for 2 hours at 90°C. the supernatant Fraction of the reaction was obtained by centrifugation. The precipitate was washed another 600 ál of H2O and centrifuged. Fractions of supernatant from each reaction were combined, filtered through the Method 10 and analyzed using HPLC with ELSD-detection, as described above.

The results are graphically presented in figure 20. This figure demonstrated that downregulation of grain amylase 797GL3 can operate together with other thermophilic the enzymes in the presence or in the absence of metal ions, resulting from corn flour at a high temperature is produced by a number of maltodextrin mixtures. In particular, the inclusion of glucoamylase or α-glucosidase may lead to the formation of product with more glucose and other products with a low DP. The inclusion of the enzyme with glucose isomerase activity results in a product that contains fructose, and therefore should be more sweet than the product produced only by amylase or amylase with α-glucosidase activity. In addition, these data showed that the content of maltooligosaccharides with DP5, DP6 and DP7 can be increased by incorporating divalent cationic salts, such as CoCl2and MgSO4.

Other ways to change the composition of maltodextrins produced by reactions described here are: the change of the pH value of the reaction, changing the type of starch in the transgenic or non-transgenic corn, the change in the ratio of solids or adding organic solvents.

Example 31

Getting dextrins or sugars from the grain, not subjected to mechanical destruction to release the starch

Sugars and maltodextrins received by contacting the transgenic grain, expressing α-amylase, 797GL3, water and heating to 90°during the night (>14 hours). For the eat 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 rectified products.

Table 6
Type moleculesThe concentration of the products

µg/25 µl injection
Fructose0,4
Glucose18,0
Maltose56,0
DP3*26,0
DP4*15,9
DP5*11,3
DP*65,3
DP*71,5
* Quantitative assessment DP3 includes evaluating maltotriose and may include an assessment of the isomers maltotriose that instead of α(1→4)communication has α(1→6)communication. Similarly, quantifying DP4-DP7 includes evaluating linear maltooligosaccharides with this chain length, as well as evaluating isomers, which instead of one or more α(1→4)relationships have one or more α(1→6)relationships.

These data demonstrated that sugars and maltodextrins can be obtained by contacting the intact grain, expressing α-amylase, water and heating. Then the obtained products can be separated from in the akt grain either by filtration or centrifugation, either by gravitational deposition.

Example 32

Fermentation source of starch in corn expressing the glucoamylase Rhizopus oryzae

The grain of transgenic maize were collected from transgenic plants obtained as described in example 29. The grain was ground into flour. Corn expresses a protein that contains an active fragment glucoamylase Rhizopus oryzae (sequence SEQ ID NO:49), targeted to the endoplasmic reticulum.

The corn was ground into flour, as described in example 15. Then received the biomass containing 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. To this biomass was added the following ingredients: protease (0,60 ml 1000-fold dilution of a commercially available protease), 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the specified biomass, making the hole for the release of CO2for the purposes of ventilation. Then the biomass was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described in example 14, and then analyzed by the methods described in example 14.

Example 33

An example of a fermentation source of starch in corn expressing the glucoamylase Rhizopus oryzae

The grain of transgenic maize were collected from transgenic plants obtained as described in example 28. The grain was ground into flour. Corn expresses a protein that contains an active fragment glucoamylase Rhizopus oryzae (sequence SEQ ID NO:49), targeted to the endoplasmic reticulum.

The corn was ground into flour, as described in example 15. Then received the biomass containing 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. To this biomass was added the following ingredients: protease (0,60 ml 1000-fold dilution of a commercially available protease), 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the specified biomass, making the hole for the release of CO2for the purposes of ventilation. Then the specified biomass was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described in example 14, and then analyzed by the methods described in example 1.

Example 34

Fermentation source of starch in whole grains maize expressing the glucoamylase Rhizopus oryzae with the addition of exogenous α-amylase

The grain of transgenic maize were collected from transgenic plants obtained as described in example 28. Corn expresses a protein that contains an active fragment glucoamylase Rhizopus oryzae (sequence SEQ ID NO:49), targeted to the endoplasmic reticulum.

Corn was subjected to contact with 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. Then added the following components: α-amylase barley purchased from Sigma (2 mg), protease (0,60 ml 1000-fold dilution of a commercially available protease), 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the specified biomass, making the hole for the release of CO2for the purposes of ventilation. Then the mixture was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation, the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described in example 14, and then analyzed by the methods described in example 14.

Example 35

armentizia source of starch in the corn, expressing the glucoamylase Rhizopus oryzae and amylase Zea mays

The grain of transgenic maize were collected from transgenic plants obtained as described in example 28. Corn expresses a protein that contains an active fragment glucoamylase Rhizopus oryzae (sequence SEQ ID NO:49), targeted to the endoplasmic reticulum. The grain also expresses amylase corn with domain linking the original starch as described in example 28.

The corn was ground into flour, as described in example 14. Then received the biomass containing 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. To this biomass was added the following ingredients: protease (0,60 ml 1000-fold dilution of a commercially available protease), 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the specified biomass, making the hole for the release of CO2for the purposes of ventilation. Then the biomass was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described in example 14, and then analyzed by the methods described in example 14.

Example 36

Fermentation source of starch in corn expressing the glucoamylase Thermoanaerobacter thermosaccharolyticum

The grain of transgenic maize were collected from transgenic plants obtained as described in example 28. Corn expresses a protein that contains an active fragment glucoamylase Thermoanaerobacter thermosaccharolyticum (sequence SEQ ID NO:47), targeted to the endoplasmic reticulum.

The corn was ground into flour, as described in example 15. Then received the biomass containing 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. The biomass was added the following ingredients: protease (0,60 ml 1000-fold dilution of a commercially available protease), 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the specified biomass, making the hole for the release of CO2for the purposes of ventilation. Then the biomass was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described in example 14, and then analyzed by the methods described in example 14.

Example 37

The farm is the level of initial starch in the corn, expressing the glucoamylase of Aspergillus niger

The grain of transgenic maize were collected from transgenic plants obtained as described in example 28. Corn expresses a protein that contains an active fragment glucoamylase Aspergillus niger (Fiil, N.P. "Glucoamylases G1 and G2 from Aspergillus niger are synthesized unit from two different but closely related mRNAs" EMBO J. 3(5), 1097-1102 (1984), reg. No. R). Nucleic acid-optimized codons corn and encoding the glucoamylase has the sequence of SEQ ID NO:59 and targets the endoplasmic reticulum.

The corn was ground into flour, as described in example 14. Then received the biomass containing 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. The biomass was added the following ingredients: protease (0,60 ml 1000-fold dilution of a commercially available protease), 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the biomass, making the hole for the release of CO2for the purposes of ventilation. Then the biomass was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described is about in example 14, and then analysed using the methods described in example 14.

Example 38

An example of a fermentation source of starch in corn expressing the Aspergillus niger glucoamylase and amylase Zea mays

The grain of transgenic maize were collected from transgenic plants obtained as described in example 28. Corn expresses a protein that contains an active fragment glucoamylase Aspergillus niger (Fiil, N.P. "Glucoamylases G1 and G2 from Aspergillus niger are synthesized unit from two different but closely related mRNAs" EMBO J. 3(5), 1097-1102 (1984), reg. No. R) (SEQ ID NO:59, nucleic acid, optimized codons corn) and targets the endoplasmic reticulum. The grain also expresses amylase corn with domain linking the original starch as described in example 28.

The corn was ground into flour, as described in example 14. Then received the biomass containing 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. The biomass was added the following ingredients: protease (0,60 ml 1000-fold dilution of a commercially available protease), 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the specified biomass, making the hole for the release of CO2for the purposes of ventilation. Then the biomass was inoculable yeast (1,44 ml) and incubated in a water is ane if 90° F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described in example 14, and then analyzed by the methods described in example 14.

Example 39

Fermentation source of starch in corn expressing the glucoamylase Thermoanaerobacter thermosaccharolyticum and amylase barley

The grain of transgenic maize were collected from transgenic plants obtained as described in example 28. Corn expresses a protein that contains an active fragment glucoamylase Thermoanaerobacter thermosaccharolyticum (sequence SEQ ID NO:47), targeted to the endoplasmic reticulum. The grain also expresses the gene amyl amylase barley with low pI (Rogers, J.C. & Milliman, C. "Isolation and sequence analysis of a barley alpha-amylase cDNA clone" J. Biol. Chem. 258(13), 8169-8174 (1983)), modified for directed expression of the indicated protein in the endoplasmic reticulum.

The corn was ground into flour, as described in example 14. Then received the biomass containing 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. The biomass was added the following ingredients: protease (0,60 ml 1000-fold dilution of a commercially available protease), 0.2 mg lactoside and urea (0,85 ml of 10-fold is svedeniya 50% solution of urea). In the cover 100 ml vessel containing the specified biomass, making the hole for the release of CO2for the purposes of ventilation. Then the biomass was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described in example 14, and then analyzed by the methods described in example 14.

Example 40

Fermentation source of starch in whole grains of corn expressing the glucoamylase Thermoanaerobacter thermosaccharolyticum and amylase barley

The grain of transgenic maize were collected from transgenic plants obtained as described in example 28. Corn expresses a protein that contains an active fragment glucoamylase Thermoanaerobacter thermosaccharolyticum (sequence SEQ ID NO:47), targeted to the endoplasmic reticulum. The grain also expresses the gene amyl amylase barley with low pI (Rogers, J.C. & Milliman, C. "Isolation and sequence analysis of a barley alpha-amylase cDNA clone" J. Biol. Chem. 258(13), 8169-8174 (1983)), modified for directed expression of the protein in the endoplasmic reticulum.

Corn was subjected to contacting with 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. To the mixture is added the following ingredients: protease (0,60 ml 1000-fold dilution of a commercially available protease), and 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the biomass, making the hole for the release of CO2for the purposes of ventilation. Then the mixture was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described in example 14, and then analyzed by the methods described in example 14.

Example 41

Fermentation source of starch in corn expressing the hybrid alpha-amylase and glucoamylase

The grain of transgenic maize were collected from transgenic plants obtained as described in example 28. Corn expresses optimized codons corn polynucleotide presented in SEQ ID NO:46 encoding a hybrid alpha-amylase and glucoamylase presented in SEQ ID NO:45, which is aimed at the endoplasmic reticulum.

The corn was ground into flour, as described in example 14. Then received the biomass containing 20 g of corn flour, 23 ml of deionized water and 6.0 ml of bards (8% solids by weight). the pH is brought to 6.0 by addition of ammonium hydroxide. The biomass was added the following ingredients: protease (0,60 ml 1000-fold dilution of a commercially available is Phnom protease), 0.2 mg lactoside and urea (0,85 ml of 10-fold dilution of a 50% solution of urea). In the cover 100 ml vessel containing the biomass, making the hole for the release of CO2for the purposes of ventilation. Then the specified biomass was inoculable yeast (1,44 ml) and incubated in a water bath at 90°F. After 24 hours of fermentation the temperature was lowered to 86°F, and after 48 hours it was brought up to 82°F.

Yeast for inoculation were propagated as described in example 14.

Samples were taken as described in example 14, and then analyzed by the methods described in example 14.

All publications, patents and patent applications are introduced in the present description by reference. Although in the above description of the present invention described some preferred variations in its implementation and presents many specific variants, with illustrative, however, it is obvious that the present invention can be made more variations in its implementation, and that some specific options can be modified, unless it is beyond the scope of the basic idea of the present invention.

Amino acid sequence of α-amylase 797GL3 (SEQ ID NO:1)

1. Selected polynucleotide, (a) contains the sequence of SEQ ID NO: 2, 9, or 50, or a complementary sequence, or polynucleotide that hybridizes with a sequence complementary to any of sequences of SEQ ID NO: 2, 9 or 50, in conditions of low hybridization stringency, and encodes a polypeptide having α-amylase activity, or (b) encoding a polypeptide containing the sequence of SEQ ID NO: 1, 10, 49, or 51, or enzymatically active fragment.

2. Selected polynucleotide according to claim 1, characterized in that the specified polynucleotide encodes a hybrid polypeptide containing the first polypeptide and second polypeptide, with said first polypeptide has α-amylase activity.

3. Selected polynucleotide according to claim 2, characterized in that the second peptide contains a signal peptide sequence.

4. Selected polynucleotide according to claim 3, characterized in that this peptide is the AE signal sequence directs the first polypeptide to vacuole, the endoplasmic reticulum, chloroplast, starch grains, seeds or cell wall of a plant.

5. Selected polynucleotide according to claim 3, characterized in that the signal sequence is an N-terminal signal sequence, derived from waxy, N-terminal signal sequence, derived from γ-Zein, krokhmalskii domain or C-terminal krokhmalskii domain.

6. Chimeric gene containing a promoter sequence operatively associated with polynucleotide according to claim 1.

7. Expressing cassette comprising a chimeric gene according to claim 6.

8. Expressing cassette according to claim 7, where the promoter is an inducible promoter, a tissue-specific promoter or endoparasiticides promoter.

9. Expressing cassette of claim 8, where endoparasitism promoter is the promoter γ-Zein of corn or promoter ADP-gpp corn.

10. Expressing cassette according to claim 9, where the promoter contains a sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

11. Expressing cassette according to claim 7, where polynucleotide is in sense orientation relative to the promoter.

12. Expressing cassette according to claim 7, where polynucleotide in accordance with (a) further encodes the signal sequence is functionally attached to the polypeptide encoded by Pauline what cleotides.

13. Expressing cassette according to item 12, where the signal sequence directs functionally attached to the polypeptide at the vacuole, endoplasmic reticulum, chloroplast, starch grains, seeds or cell wall of a plant.

14. Expressing cassette according to item 13, where the signal sequence is N-terminal signal sequence, derived from waxy, or N-terminal signal sequence, derived from γ-Zein.

15. Expressing cassette according to claim 7, where polynucleotide in accordance with (b) functionally attached to the tissue-specific promoter.

16. Expressing cassette according to item 15, where tissue-specific promoter is the promoter γZea mays Zein or promoter ADP-gpp Zea mays.

17. A vector expressing the cartridge according to claim 7.

18. Transgenic cell expressing containing cassette according to claim 7.

19. Transgenic cell according p, characterized in that it is selected from the group consisting of Agrobacterium, the cells of monocotyledonous plants, the cells of dicotyledonous plants, the plant cells Liliopsida, the plant cells Panicoidae, cells of maize cells and cereals.

20. Transgenic cell according to claim 19, wherein the cell is a cell of corn.

21. Transgenic plant stably transformed with the vector according to 17, where the aforementioned transgenic plant expresses α-is milazo.

22. Transgenic plant stably transformed with the vector containing polynucleotide containing any of the sequences SEQ ID NO: 2 or 9, or polynucleotide encoding the enzyme α-amylase, having any of the amino acid sequence SEQ ID NO: 1, 10, 13, 14, 15, 16, 33 or 35, but such transgenic plant expresses the enzyme α-amylase.

23. Transgenic seed, fruit or grain, stably transformed polynucleotide according to any one of claims 1 to 5, where the specified seed, fruit or grain expresses the enzyme α-amylase.

24. Transformed plant, the genome of which is increased at the expense of recombinant polynucleotide having the sequence of SEQ ID NO: 2, 9 or 50, functionally attached to the promoter and signal sequence, but such transformed plant expresses the enzyme α-amylase.

25. Transgenic seed, fruit or grain, stably transformed expressing cassette according to any one of claims 7 to 16, where the specified seed, fruit or grain expresses the enzyme α-amylase.

26. The product obtained from a transformed plant according to paragraph 24, where the product is cromartyshire enzyme, starch or sugar.

27. Part transgenic plants containing α-amylase, having any of the amino acid sequence SEQ ID NO: 1, 10, 49 of the 51 whether or encoded by polynucleotides, containing any of the sequences SEQ ID NO: 2, 9 or 50.

28. The method of converting starch in part of transgenic plants according to item 27, which includes the processing parts of the plant in conditions of humidity and/or temperature to activate the contained cromartyshire enzyme.

29. Plant corn, stably transformed with the vector containing a polynucleotide sequence that encodes α-amylase and which more than 60% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 50, but such transformed plant expresses the enzyme α-amylase.



 

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