HK1072618A - Self processing plants and plant parts - Google Patents

Description

Self-processing plants and plant parts

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RELATED APPLICATIONS

This application claims priority from application No.60/315,281, filed on 8/27/2001, which is incorporated herein by reference.

Technical Field

The present invention relates generally to the field of plant molecular biology and, more particularly, to the production of plants capable of expressing processing enzymes (enzymes) that impart desirable characteristics to the plant or product thereof.

Background

Enzymes are commonly used to process a wide variety of agricultural products such as wood, fruits and vegetables, starch, fruit juices, and the like. Generally, the enzymes used in the process are produced and recovered on an industrial scale from different sources, for example from microbial fermentation (bacillus alpha-amylase) or from plant isolation (coffee beta-galactosidase or papain from plant parts). By mixing the enzyme and substrate under appropriate water content, temperature, time and mechanical mixing conditions to allow the enzymatic reaction to be carried out in a commercially viable manner, the enzyme preparation is used in different processing applications. These methods involve separate steps of production of the enzyme, preparation of the enzyme preparation, mixing of the enzyme with the substrate, and subjecting the mixture to appropriate conditions to facilitate the enzymatic reaction, etc. Methods that reduce or eliminate time, energy, mixing, capital investment, and/or enzyme production costs, or produce improved or new products, are useful and advantageous. One example of the need for such an aspect is in the field of corn milling.

Currently, corn is ground to obtain corn starch and other corn grinding byproducts, such as corn gluten feed, corn gluten meal, and corn oil. The starch obtained from this process usually needs to be further processed into other products, such as derivatized starches and sugars, or by fermentation to produce a range of products including alcohols or lactic acid. The treatment of corn starch often involves the use of various enzymes, particularly enzymes capable of hydrolyzing and converting starch to fermentable sugars or fructose (alpha-and glucose-amylases, alpha-glucosidases, glucose isomerases, etc.). The processing procedures currently in commercial use are capital intensive because very large mills must be built to process the corn on a large scale as needed for reasonable cost efficiency. In addition, this process requires the preparation of enzymes that hydrolyze or modify starch separately and then machinery to mix the enzymes and substrates to produce the hydrolyzed starch product.

The process of recovering starch from corn kernels is well known and involves a wet milling process. The corn wet milling method comprises the following steps: steeping corn kernels, grinding the corn kernels and separating the components of the corn kernels. The kernels are steeped in a steeping vessel having a convection current of about 120 ° F for about 24 to 48 hours. The steepwater typically contains about 0.2% by weight of sulphur dioxide. Sulfur dioxide is used in the treatment to reduce microbial growth and also to reduce disulfide bonds in endosperm proteins to facilitate efficient starch-protein separation. Typically, about 0.59 gallons of steepwater per bushel of corn is used. The steepwater is considered a waste liquor and typically contains undesirable levels of residual sulphur dioxide.

The steeped kernels are then dewatered and subjected to a series of attrition type mills. The first set of attrition mills are capable of breaking the kernels and releasing the germ from the remainder of the kernels. A commercial mill of the friction type suitable for wet milling is sold under the Bauer brand. The germ is separated from the remainder of the kernel by centrifugation. One common commercial centrifugal separator is the Merco centrifugal separator. Both attrition mills and centrifugal separators are large, expensive machines that require energy to operate.

The next step in the process is to continue the remaining corn kernel components, including starch, husk, fiber and gluten, through another set of attrition milling processes and passing through a washing screen to separate the fiber components from the starch and gluten (endosperm protein). Starch and gluten will pass through this screen while fiber will not. Starch is separated from the endosperm protein by centrifugation or a third comminution after centrifugation. The starch slurry was formed by centrifugation, dewatered, washed with fresh water and dried to about 12% moisture. This substantially pure starch is usually further treated with enzymes.

Starch is separated from other components of the kernel because removal of seed coat, embryo and endosperm proteins effectively exposes the starch to enzymes, which results in a hydrolysate that is relatively free of contamination with other kernel components. The separation also ensures that other components of the kernel are effectively recovered and subsequently sold as by-products to increase the profitability of the plant.

After wet milling to obtain starch, it is usually subjected to gelatinization, liquefaction and dextrinization to obtain a maltodextrin product, followed by saccharification, isomerization and refining steps to produce glucose, maltose and fructose.

Since the currently available enzymes cannot hydrolyze crystalline starch rapidly, gelatinization occurs during the starch hydrolysis process. In order to make the starch available to the action of hydrolytic enzymes, the starch is usually slurried with water (20-40% dry matter) and then heated at a suitable gelatinization temperature. For corn starch, the temperature is between 105 ℃ and 110 ℃. Gelatinized starch is usually very viscous and is therefore thinned in the next step, called liquefaction. Liquefaction breaks down some of the bonds between the starch glucose molecules, which can be done enzymatically or using acid. Thermostable endo-alpha-amylase is used in this step and in the subsequent dextrinization step. The degree of hydrolysis is controlled during the dextrinisation step so that the desired percentage of glucose in the resulting hydrolysate is achieved.

Depending on the desired product, the dextrin product obtained from the liquefaction step can be further hydrolyzed by a number of different exoamylases and debranching enzymes. If the ultimate goal is fructose, then immobilized glucose isomerase is typically used to convert glucose to fructose.

The production of fermentable sugars (and then ethanol, for example) from corn starch by dry milling can facilitate the step of efficiently contacting the starch with exogenous amylase. These processes have lower capital investment than wet milling processes, but significant cost benefits are still desirable because the by-products from these processes are generally less valuable than those from wet milling processes. For example, in wet milled corn, the corn kernels are ground into a powder to facilitate efficient contact of the corn with degrading enzymes. Corn meal, after enzymatic hydrolysis, is also useful as a feed due to the residual solids containing protein and some other components. Eckhoff recently described the potential for improvement of wet milling and related problems in a paper entitled "Fermentation and costs of fuel ethanol from sugar with quick-germ processes" (appl. biochem. Biotechnol., 94: 41 (2001)). This "quick-germ" process enables oil-rich germ to be separated from starch using a reduced soaking time.

One such example where the regulation and/or levels of endogenous processing enzymes in a plant can form a product of interest is sweet corn. Typical sweet corn varieties differ from feed corn (field corn) varieties in that sweet corn is unable to biosynthesize normal levels of starch. Genetic mutations in genes encoding enzymes involved in starch biosynthesis are commonly used in sweet corn varieties to limit starch biosynthesis. Such mutations (e.g., sweet and supersweet mutations) are located in both the starch synthase and ADP-glucose pyrophosphorylase encoding genes. The three monosaccharides, fructose, glucose and sucrose, required to produce a palatable sweet taste, which is appreciated by consumers of edible fresh corn, accumulate in the developing endosperm within these mutants. However, if the level of starch accumulation is too high such that the corn matures too long (late harvest) or the corn is stored too long before consumption, sweetness is lost and starch flavor and mouthfeel (mouthfeel) changes. The harvest period (hardest window) of sweet corn is therefore very narrow and has a limited shelf life.

Another difficulty for farmers of growing sweet corn is that the application of these varieties is limited to edible foods only. If a farmer wishes to abandon the harvest of sweet corn for use as a consumable food during the seed development stage, the harvest is lost considerably. Grain yield and quality of sweet corn can be low for two basic reasons. The first reason is that mutations in the starch biosynthesis pathway disrupt the starch biosynthesis mechanism and maize is not yet fully populated, resulting in yield and quality losses. Second, due to the high levels of sugars present in corn, which cannot be stored as starch, the overall absorption strength (sink strength) of these seeds is reduced, which exacerbates the reduction in stored nutrients in the corn kernel. The endosperm of the seeds of these sweet corn varieties shrinks and cracks without undergoing a proper drying process and is susceptible to disease. This low quality of sweet corn grain can have further agronomic impacts; for example, the survival rate of seeds is low, the germination rate is low, seedlings are susceptible to diseases, seedlings in early stage are not easy to survive and the like, which are caused by various factors caused by too low starch accumulation amount. Thus, problems with low quality sweet corn can affect consumers, farmers/growers, distributors and seed producers.

Thus, for dry milling, a method that improves the process and/or increases the value of the by-products is desired. For wet milling, there is a need for a starch processing method that does not require equipment to be provided for the long soaking, milling, grinding and/or separation of the corn kernel components. For example, it would be desirable to be able to modify or eliminate the steeping step in a wet milling process because this would reduce the amount of waste stream that needs to be disposed of, thereby saving energy and time, and increasing the milling capacity (which would shorten the time of the corn kernels in the steeping vessel). There is also a need for methods of eliminating or improving the separation of starchy endosperm from the embryo.

Summary of The Invention

The present invention relates to self-processing plants and plant parts and methods of using the same. The self-processing plants and plant parts of the invention are capable of expressing and activating (mesophilic, thermophilic) and hyperthermophilic) enzymes. After activation of the (mesophilic, thermophilic and hyperthermophilic) enzyme, the plant and plant parts are able to self-process the substrate, thus obtaining the desired product.

The present invention provides an isolated polynucleotide a) comprising the nucleotide sequence of SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59 or a complement thereof, or a sequence capable of hybridizing to SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59, and encodes a polypeptide having α -amylase, pullulanase, α -glucosidase, glucose isomerase or glucoamylase activity, or b) encodes a polypeptide comprising the amino acid 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 an enzymatically active fragment thereof. Preferably, the isolated polynucleotide encodes a fusion polypeptide comprising a first polypeptide and a second peptide, wherein the first polypeptide has alpha-amylase, pullulanase, alpha-glucosidase, glucose isomerase, or glucoamylase activity. Most preferably, the second peptide comprises a signal sequence peptide capable of targeting the first polypeptide to the vacuole, endoplasmic reticulum, chloroplast, starch granule, seed or cell wall of the plant. For example, the signal sequence can be the N-terminal signal sequence of waxy, the N-terminal signal sequence of γ -zein, the starch binding domain, or the C-terminal starch binding domain. Still further included are the following polynucleotides: is capable of hybridizing to SEQ ID NO: 2, 9, or 52 and encodes a polypeptide having alpha-amylase activity, capable of hybridizing to the polypeptide of any one of SEQ ID NOs: 4 or 25 and encodes a polypeptide having pullulanase activity, capable of hybridizing to the polynucleotide of SEQ ID NO: 6 and encodes a polypeptide having alpha-glucosidase activity; is capable of hybridizing to SEQ ID NO: 19, 21, 37, 39, 41 or 43 and encodes a polypeptide having glucose isomerase activity; is capable of hybridizing to SEQ ID NO: 46, 48, 50 or 59 and encodes a polypeptide having glucoamylase activity.

In addition, expression cassettes are provided comprising a polynucleotide a) having the sequence of seq id NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 or a complement thereof, or a sequence capable of hybridizing to SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 and encodes a polypeptide having α -amylase, pullulanase, α -glucosidase, glucose isomerase, or glucoamylase activity, or b) a polynucleotide encoding a polypeptide comprising the amino acid 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 an enzymatically active fragment thereof. Preferably, the expression cassette further comprises a promoter operably linked to the polynucleotide, such as an inducible promoter, a tissue-specific promoter or preferably an endosperm-specific promoter. Preferably, the endosperm-specific promoter is a maize gamma-zein promoter or a maize ADP-gpp promoter. In a preferred embodiment, the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12. furthermore, in another preferred embodiment the polynucleotide is positioned in sense orientation relative to the promoter. The expression cassettes of the invention may further encode a signal sequence operably linked to the polynucleotide-encoding polypeptide. The signal sequence preferably targets the polypeptide to which it is operably linked to the vacuole, endoplasmic reticulum, chloroplast, starch granule (starch grain), seed or cell wall of a plant. Preferred signal sequences include the N-terminal signal sequence of waxy, the N-terminal signal sequence of gamma-zein, or the starch binding domain.

The invention also relates to vectors or cells containing the expression cassettes of the invention. The cells may be selected from: agrobacterium, a monocot plant cell, a dicot plant cell, a Liliopsida cell, a Panicoideae cell, a maize cell, and a cereal cell. Preferably the cell is a maize cell.

The invention also includes plants stably transformed with a vector of the invention. The present invention provides plants stably transformed with a vector comprising an alpha-amylase having the amino acid sequence of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33, or 35, or a polypeptide consisting of an amino acid sequence comprising SEQ id no: 2 or 9. Preferably, the alpha-amylase is resistant to hyperthermophiles.

In another embodiment, there is also provided a plant stably transformed with a vector comprising a pullulanase having the amino acid sequence of SEQ ID NO: 24 or 34, or a polypeptide consisting of a sequence comprising any one of SEQ ID NOs: 4 or 25. Also provided are plants stably transformed with a vector comprising an alpha-glucosidase having the amino acid sequence of SEQ ID NO: 26 or 27, or a polypeptide consisting of a sequence comprising any one of SEQ ID NOs: 6. Preferably, the alpha-glucosidase is resistant to hyperthermophiles. Also described are plants stably transformed with a vector containing a glucose isomerase enzyme having the amino acid sequence of SEQ ID NO: 18, 20, 28, 29, 30, 38, 40, 42, or 44, or a polypeptide consisting of an amino acid sequence comprising SEQ id no: 19, 21, 37, 39, 41 or 43. Preferably, the glucose isomerase is resistant to ultra-high temperatures. Also described in another embodiment is a plant stably transformed with a vector comprising a glucoamylase having the amino acid sequence of SEQ ID NO: 45, 47 or 49, or a polypeptide consisting of a sequence comprising SEQ ID NO: 46, 48, 50, or 59. Preferably, the glucoamylase is hyperthermophilic.

Also provided are plant products, such as seeds, fruits or grains, obtained from the stably transformed plants of the invention.

In another embodiment, the invention also relates to a transformed plant having added to its genome a recombinant polynucleotide operably linked to a promoter sequence and encoding at least one processing enzyme, the sequence of which has been optimized for expression in said plant. The plant may be a monocot, such as maize, or a dicot. Preferably the plant is a cereal or economic plant. The processing enzyme is selected from: alpha-amylase, glucoamylase, glucose isomerase, glucanase, beta-amylase, alpha-glucosidase, isoamylase, pullulanase, neopullulanase (neo-pullulanase), iso-pullulanase (iso-pullulanase), amylopullulanase (amylomullululanase), cellulase, exo-1, 4-beta-cellobiohydrolase, exo-1, 3-beta-D-glucanase, beta-glucosidase, endoglucanase, L-arabinase, alpha-arabinosidase, galactanase (galactanase), galactosidase, mannanase, mannosidase, xylanase, xylosidase, protease, glucanase, xylanase, esterase, phytase and lipase. Preferably the processing enzyme is a starch processing enzyme selected from the group consisting of: alpha-amylase, glucoamylase, glucose isomerase, beta-amylase, alpha-glucosidase, isoamylase, pullulanase, neopullulanase, isoamylase and amylopullulanase (amylomululanase). More preferably the enzyme is selected from: alpha-amylase, glucoamylase, glucose isomerase, alpha-glucosidase, and pullulanase. Further preferred are processing enzymes which are resistant to ultra-high temperatures. According to the invention, the enzyme may be a non-starch degrading enzyme selected from the group consisting of: proteases, glucanases, xylanases, esterases, phytases and lipases. These enzymes may also be hyperthermophilic. In a preferred embodiment, the enzyme is capable of accumulating in the vacuole, endoplasmic reticulum, chloroplast, starch granule, seed or cell wall of a plant. Moreover, in another embodiment, a second recombinant polynucleotide comprising a non-hyperthermophilic enzyme may be added to the genome of the plant.

In another aspect, the present invention provides a transformed plant having increased in its genome a recombinant polynucleotide encoding at least one processing enzyme selected from the group consisting of: an alpha-amylase, a glucoamylase, a glucose isomerase, an alpha-glucosidase and a pullulanase, said polynucleotide being operably linked to a promoter sequence, the sequence of the polynucleotide having been optimized for expression in the plant. Preferably, the processing enzyme is a hyperthermophilic enzyme and corn.

Another embodiment provides a transformed maize plant having increased in its genome a recombinant polynucleotide encoding at least one processing enzyme selected from the group consisting of: an alpha-amylase, a glucoamylase, a glucose isomerase, an alpha-glucosidase and a pullulanase, said polynucleotide being operably linked to a promoter sequence, the sequence of the polynucleotide having been optimized for expression in a maize plant. Preferably the processing enzyme is a hyperthermophilic enzyme.

Also provided are transformed plants whose genome has increased expression of a polypeptide having the sequence of SEQ ID NO: 2, 9 or 52, operably linked to a promoter and a signal sequence. In addition, transformed plants are described whose genome is augmented with a plant having the amino acid sequence of SEQ ID NO: 4 or 25, operably linked to a promoter and a signal sequence. In another embodiment, transformed plants are also described whose genome has increased expression of a polypeptide having the sequence of SEQ ID NO: 6, operably linked to a promoter and a signal sequence. Additionally, transformed plants are provided whose genome is increased by a plant having seq id NO: 19, 21, 37, 39, 41 or 43. A transformed plant whose genome has increased expression of a polypeptide having the sequence of SEQ ID NO: 46, 48, 50 or 59.

Products obtained from the transformed plants are further contemplated herein. Products include, for example: seeds, fruits, or grains. The product may also be a processing enzyme, starch or sugar.

Plants obtained from the stably transformed plants of the invention are further described. In this aspect, the plant may be a hybrid plant or an inbred (bred) plant.

In another embodiment of the invention, a starch composition is provided comprising at least one processing enzyme that is a protease, a glucanase, or an esterase. Preferably the enzyme is resistant to ultra high temperatures.

Another embodiment of the invention provides grain (grain) comprising at least one processing enzyme that is an alpha-amylase, pullulanase, alpha-glucosidase, glucoamylase or glucose isomerase. Preferably the enzyme is resistant to ultra high temperatures.

Another embodiment provides a method of making starch granules comprising: treating a grain comprising at least one non-starch processing enzyme under conditions effective to activate the at least one enzyme to produce a mixture comprising starch granules and non-starch degradation products, wherein the grain is obtained from a transformed plant having an increased genome of an expression cassette encoding the at least one enzyme; and separating the starch granules from the mixture. Wherein, the preferred enzyme is protease, glucanase, xylanase, phytase or esterase. Furthermore, the enzyme is preferably resistant to ultra-high temperatures. The grain may be cracked grain and/or may be treated under conditions of low or high moisture content. Alternatively, the grain may be treated with sulphur dioxide. Preferably the invention further comprises separating the non-starch product from the mixture. Starch products and non-starch products obtained by the process are further described.

In another embodiment, there is also provided a method of producing super sweet corn (hypersweet corn) comprising treating transformed corn or a portion thereof, the genome of which has been augmented with an expression cassette encoding at least one starch degrading enzyme or starch isomerase, under conditions capable of activating the at least one enzyme to convert polysaccharides in the corn to sugars (sugar) to obtain the super sweet corn, and expressing the expression cassette in the endosperm. The expression cassette preferably further comprises a promoter operably linked to the enzyme-encoding polynucleotide. The promoter may be, for example, a constitutive promoter, a seed-specific promoter or an endosperm-specific promoter. Preferably the enzyme is resistant to ultra high temperatures. More preferably the enzyme is an alpha-amylase. The expression cassette used herein may further comprise a polynucleotide encoding a signal sequence operably linked to the at least one enzyme. The signal sequence may direct the hyperthermophilic enzyme to, for example, the apoplast or the endoplasmic reticulum. Preferably, the enzyme comprises SEQ id no: 13, 14, 15, 16, 33 or 35.

In a most preferred embodiment, a method of producing super-sweet corn is described comprising treating transformed corn or a portion thereof, the genome of which has been augmented with an expression cassette encoding an alpha-amylase and expressing the expression cassette in the endosperm stream, under conditions that activate the at least one enzyme to convert polysaccharides in the corn to sugars, producing super-sweet corn. Preferably the enzyme is hyperthermophilic, the hyperthermophilic alpha-amylase comprising the amino acid sequence of SEQ ID NO: 10. 13, 14, 15, 16, 33, or 35, or an enzymatically active fragment thereof having α -amylase activity.

Also described herein is a method of making a solution of a hydrolyzed starch product comprising treating a plant part comprising starch granules and at least one processing enzyme under conditions effective to activate the at least one enzyme, thereby allowing the enzyme to process the starch granules to form an aqueous solution comprising a hydrolyzed starch product, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one starch processing enzyme; and collecting the aqueous solution containing the hydrolyzed starch product. The hydrolyzed starch product may contain dextrins, maltooligosaccharides, glucose and/or mixtures thereof. Preferably, the enzyme is an alpha-amylase, an alpha-glucosidase, a glucoamylase, a pullulanase, a starch pullulanase, a glucose isomerase, or a combination thereof. In addition, the enzyme is preferably resistant to ultra-high temperatures. On the other hand, the genome of the plant part can be further increased by an expression cassette encoding a non-hyperthermophilic starch processing enzyme. The non-hyperthermophilic starch processing enzyme may be selected from: an amylase, a glucoamylase, an alpha-glucosidase, a pullulanase, a glucose isomerase, or a combination thereof. On the other hand, the processing enzyme is preferably expressed in the endosperm. Preferably, the plant part is a grain, derived from maize, wheat, barley, rye, oats, sugar cane or rice. Preferably, the at least one processing enzyme is operably linked to a promoter and a signal sequence, said signal peptide being capable of directing the enzyme to the starch granule or to the endoplasmic reticulum or to the cell wall. The process may further comprise the step of isolating the hydrolysed starch product and/or fermenting the hydrolysed starch product.

In another aspect of the invention, a method of preparing a hydrolyzed starch product is described, comprising treating a plant part comprising starch granules and at least one starch processing enzyme under conditions effective to activate the at least one enzyme, thereby allowing the enzyme to process the starch granules to form an aqueous solution comprising the hydrolyzed starch product, wherein the plant part is obtained from a transformed plant, the genome of which has been augmented by an expression cassette encoding at least one alpha-amylase; and collecting the aqueous solution containing the hydrolyzed starch product. Preferably, the alpha-amylase is resistant to hyperthermophilic temperatures, more preferably the hyperthermophilic alpha-amylase comprises the amino acid sequence of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33 or 35, or a fragment thereof having alpha-amylase activity. Preferably, the expression cassette comprises a polynucleotide selected from the group consisting of: SEQ ID NO: 2, 9, 46 or 52 or a complement thereof, or a sequence capable of hybridizing to SEQ ID NO: 2, 9, 46 or 52 and encodes a polypeptide having alpha-amylase activity. Also, the present invention includes the genome of the transformed plant further comprising a polynucleotide encoding a non-hyperthermophilic starch processing enzyme. Alternatively, parts of the plant may be treated with non-hyperthermophilic starch processing enzymes.

The invention also provides a transformed plant part comprising at least one starch processing enzyme present in a plant cell, wherein the plant part is obtained from a transformed plant whose genome has been augmented with an expression cassette encoding at least one starch processing enzyme. Preferably, the enzyme is a starch processing enzyme selected from the group consisting of: alpha-amylase, glucoamylase, glucose isomerase, beta-amylase, alpha-glucosidase, isoamylase, pullulanase, neopullulanase, isoamylase and amylopullulanase. Also, the enzyme is preferably resistant to ultra-high temperatures. The plant may be any plant, but is preferably maize.

Another embodiment of the invention is a transformed plant part comprising at least one non-starch processing enzyme present within a cell or cell wall of a plant, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one non-starch processing enzyme or the at least one non-starch polysaccharide processing enzyme. Preferably, the enzyme is resistant to ultra-high temperatures. Also, the non-starch processing enzyme is preferably selected from: proteases, glucanases, xylanases, esterases, phytases and lipases. The plant part may be any plant part, but is preferably an ear, seed, fruit, grain, straw (stover), chaff or slag (bagass).

The invention also provides transformed plant parts. For example, transformed plant parts are described that contain an alpha-amylase having the amino acid sequence of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33 or 35, or a sequence consisting of an amino acid sequence comprising any one of SEQ ID NOs: 2, 9, 46 or 52; a transformed plant part comprising an alpha-glucosidase having the amino acid sequence of SEQ ID NO: 5, 26 or 27, or a sequence consisting of the amino acid sequence of any one of SEQ ID NOs: 6; a transformed plant part comprising a glucose isomerase enzyme having the amino acid sequence of SEQ ID NO: 28, 29, 30, 38, 40, 42 or 44, or a sequence consisting of the amino acid sequence of any one of SEQ ID NOs: 19, 21, 37, 39, 41 or 43; a transformed plant part comprising a glucoamylase having the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49 or by an amino acid sequence comprising SEQ ID NO: 46, 48, 50 or 59; and a transformed plant part comprising a pullulanase consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or 25, or a pharmaceutically acceptable salt thereof.

Another embodiment provides a transformation method for transforming starch in a plant part, comprising activating a starch processing enzyme contained therein. Also provided is a starch, dextrin, maltooligosaccharide or saccharide obtained according to the method.

The present invention also describes a method of using a transformed plant part containing at least one non-starch processing enzyme in the cell wall or cells of the plant part, comprising treating a transformed plant part containing at least one non-starch polysaccharide processing enzyme under conditions to activate the at least one enzyme to digest non-starch polysaccharides to form an aqueous solution containing oligosaccharides and/or sugars (sugar), wherein the plant part is obtained from a transformed plant whose genome has been augmented with an expression cassette encoding the at least one non-starch polysaccharide processing enzyme; and collecting the aqueous solution containing the oligosaccharides and/or sugars. Preferably, the non-starch polysaccharide processing enzyme is resistant to ultra high temperatures.

Also provided is a method of using transformed seed containing at least one processing enzyme, comprising treating transformed seed containing at least one protease or lipase under conditions capable of activating the at least one enzyme, thereby obtaining an aqueous mixture containing amino acids and fatty acids, wherein the seed is obtained from a transformed plant whose genome has been augmented with an expression cassette encoding the at least one enzyme; and collecting the aqueous mixture. Preferably the amino acid or the fatty acid or both are isolated. Preferably, the at least one protease or lipase is resistant to ultra-high temperatures.

A method of producing ethanol comprising treating a plant part comprising at least one polysaccharide processing enzyme under conditions capable of activating said at least one enzyme, thereby degrading a polysaccharide to form an oligosaccharide or a fermentable sugar, wherein said plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding said at least one polysaccharide processing enzyme; and incubating the fermentable sugar under conditions that promote conversion of the fermentable sugar or oligosaccharide to ethanol. Preferably the plant part is a grain, fruit, seed, stem, wood, vegetable or root. Preferably, the plant part is obtained from a plant selected from the group consisting of: oats, barley, wheat, berries, grapes, rye, corn, rice, potatoes, sugar beets, sugar cane, pineapple, grasses and trees. In another preferred embodiment, the polysaccharide processing enzyme is: an alpha-amylase, a glucoamylase, an alpha-glucosidase, a glucose isomerase, a pullulanase, or a combination thereof.

Also provided is a method of producing ethanol comprising heat treating a plant part comprising at least one enzyme selected from the group consisting of: an alpha-amylase, glucoamylase, alpha-glucosidase, glucose isomerase, pullulanase, or a combination thereof, wherein the plant part is obtained from a transformed plant, the genome of which has been augmented with an expression cassette encoding the at least one enzyme; and incubating the fermentable sugar under conditions that promote conversion of the fermentable sugar to ethanol. Preferably, the at least one enzyme is resistant to hyperthermia or mesophilic.

In another embodiment, a method of producing ethanol is described comprising treating a plant part comprising at least one non-starch processing enzyme to digest non-starch polysaccharides into oligosaccharides and fermentable sugars under conditions capable of activating the at least one enzyme, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one enzyme; and incubating the fermentable sugar under conditions that promote conversion of the fermentable sugar to ethanol. Preferably, the non-starch processing enzyme is a glucanase, a xylanase or a cellulase.

Also provided is a method of producing ethanol comprising treating a plant part comprising at least one enzyme selected from the group consisting of: an alpha-amylase, glucoamylase, alpha-glucosidase, glucose isomerase, pullulanase, or a combination thereof, wherein the plant part is obtained from a transformed plant, the genome of which has been augmented with an expression cassette encoding the at least one enzyme; and incubating the fermentable sugar under conditions that promote conversion of the fermentable sugar to ethanol. Preferably the enzyme is resistant to ultra high temperatures.

Also provided is a method of producing a sweetened farinaceous food product (farinaceous food product) without the need for additional sweeteners, comprising treating a plant part containing at least one starch processing enzyme under conditions capable of activating said at least one enzyme to process starch granules in the plant part to sugars to form a sweetened product, wherein the plant part is obtained from a transformed plant whose genome has been augmented with an expression cassette encoding said at least one enzyme; and processing the sweet product into a farinaceous food. The farinaceous food product may be made from a sweet product and water. In addition, the farinaceous food product may contain malt, flavoring agents, vitamins, minerals, coloring agents, or combinations thereof. Preferably, the at least one enzyme is resistant to ultra-high temperatures. The enzyme may be selected from: an alpha-amylase, an alpha-glucosidase, a glucoamylase, a pullulanase, a glucose isomerase, or any combination thereof. The plant may also be selected from: soybean, rye, oat, barley, wheat, corn, rice and sugarcane. Preferably, the farinaceous food product is a cereal, a breakfast food, a ready-to-eat food or a bakery product. The method may further comprise baking, boiling, heating, steam processing, electrical discharge (or combinations thereof).

The present invention also provides a method for sweetening a starch-containing product without the addition of a sweetener, comprising treating a starch containing at least one starch processing enzyme under conditions effective to activate the at least one enzyme to digest the starch to form a sugar to form a sweet starch, wherein the starch is obtained from a transformed plant whose genome has been augmented with an expression cassette encoding the at least one enzyme; and adding the sweet starch to the product to produce a sweet starch-containing product. The transformed plant is preferably selected from: corn, soybean, rye, oat, barley, wheat, rice and sugarcane. Preferably, the at least one enzyme is resistant to ultra-high temperatures. More preferably, the at least one enzyme is an alpha-amylase, an alpha-glucosidase, a glucoamylase, a pullulanase, a glucose isomerase, or a combination thereof.

The invention also provides farinaceous and sweet farinaceous products.

The present invention also provides a method for sweetening a fruit or vegetable containing polysaccharides, comprising treating a fruit or vegetable containing at least one polysaccharide processing enzyme under conditions capable of activating said at least one enzyme, whereby the polysaccharides in the fruit or vegetable are processed to form sugars (sugar), to obtain a sweet fruit or vegetable, wherein the fruit or vegetable is obtained from a transformed plant, the genome of which has been augmented with an expression cassette encoding said at least one polysaccharide processing enzyme. The fruit or vegetable is selected from potato, tomato, banana, pumpkin, pea and bean. Preferably, the at least one enzyme is resistant to ultra-high temperatures.

The present invention still further provides a process for preparing an aqueous solution comprising sugars, comprising treating starch granules obtained from plant parts under conditions capable of activating said at least one enzyme, thereby obtaining an aqueous solution comprising sugars.

Another embodiment provides a method of preparing a starch-derived product from grain without involving wet milling (wet milling) or dry milling (dry milling) of the grain prior to recovery of the starch-derived product, comprising treating a plant part comprising starch granules and at least one starch processing enzyme to process the starch granules to form an aqueous solution comprising a dextrin or sugar, wherein the plant part is obtained from a transformed plant whose genome has been augmented with an expression cassette encoding the at least one starch processing enzyme, under conditions capable of activating the at least one enzyme; and collecting the aqueous solution containing the starch-derived product. Preferably, the at least one starch processing enzyme is resistant to ultra high temperatures.

Also provided are methods of isolating alpha-amylase, glucoamylase, glucose isomerase, alpha-glucosidase and pullulanase comprising growing the transformed plant and isolating alpha-amylase, glucoamylase, glucose isomerase, alpha-glucosidase and pullulanase therefrom. Preferably the enzyme is resistant to ultra high temperatures.

Also provided is a method of making maltodextrin comprising mixing transgenic grain with water, heating the mixture, separating solids from the formed dextrin syrup, and collecting maltodextrin. Preferably the transgenic grain contains at least one starch processing enzyme. Preferably, the starch processing enzymes are alpha-amylases, glucoamylases, alpha-glucosidases, and glucose isomerases. Also provided are maltodextrins and compositions formed by the method.

The present invention provides a method of preparing dextrins or sugars from grain without involving mechanical disruption of the grain prior to recovery of a starch-derived product, comprising treating a plant part comprising starch granules and at least one starch processing enzyme under conditions capable of activating the at least one enzyme to process the starch granules to form an aqueous solution comprising dextrins or sugars, wherein the plant part is obtained from a transformed plant, the genome of which has been augmented with an expression cassette encoding the at least one processing enzyme; and collecting the aqueous solution containing the sugar and/or dextrin.

The present invention also provides a method of producing fermentable sugars comprising treating a plant part comprising starch granules and at least one starch processing enzyme under conditions capable of activating the at least one enzyme to process the starch granules to form an aqueous solution comprising dextrins or sugars, wherein the plant part is obtained from a transformed plant, the genome of which has been augmented with an expression cassette encoding the at least one processing enzyme; and collecting the aqueous solution containing fermentable sugars.

The invention also includes a corn plant stably transformed with a vector comprising a hyperthermophilic alpha-amylase. Preferably, for example, a maize plant stably transformed with a vector comprising a polynucleotide sequence encoding an alpha-amylase that hybridizes to SEQ ID NO: 1 or SEQ ID NO: 51 had a consistency of greater than 60%.

Drawings

FIGS. 1A and 1B show alpha-amylase activity expressed in isolated T1 grain from the pNOV6201 plant and in corn grain from six pNOV6200 lines and in the endosperm.

FIG. 2 shows alpha-amylase activity in isolated T1 grain from pNOV6201 line.

FIG. 3 shows the amount of ethanol formed after fermentation of transgenic corn mash containing thermostable 797GL3 alpha-amylase, which mash has undergone liquefaction at 85 ℃ and 95 ℃ for no more than 60 minutes. The figure illustrates that the ethanol yield obtained by fermentation for 72 hours hardly changed between 15 and 60 minutes of liquefaction. It also shows that the paste produced by liquefaction at 95 ℃ formed a higher yield of ethanol at each time point than the paste produced by liquefaction at 85 ℃.

FIG. 4 shows the amount (%) of starch remaining after liquefaction and then re-fermentation of a transgenic corn mash containing thermostable α -amylase at 85 ℃ and 95 ℃ for no more than 60 minutes. The graph shows that the ethanol yield at 72 hours of fermentation hardly changed between 15 minutes and 60 minutes of liquefaction. It also shows that liquefaction at 95 ℃ produced a paste with a higher yield of ethanol formed at various time points than liquefaction at 85 ℃.

FIG. 5 shows ethanol yields for mashes prepared at 85 ℃ and 95 ℃ for transgenic corn versus control corn and various mixtures thereof. The figure shows that: transgenic corn containing alpha-amylase has a significant improvement in making starch fermentatively usable due to the reduced residual amount of starch after fermentation.

FIG. 6 shows the residual starch amounts measured in the dry still (stillage) after fermentation of the mash prepared from transgenic grain, control corn and various mixtures thereof at 85 ℃ and 95 ℃.

FIG. 7 shows ethanol production as a function of fermentation time at various pH ranges of 5.2-6.4 for samples containing 3% transgenic corn over a period of 20-80 hours. The figure illustrates that fermentation at lower pH conditions proceeds faster than at pH6.0 or higher.

FIG. 8 shows ethanol production during fermentation of mash containing 0-12 wt% of different weight percentages of transgenic corn at different pH ranges of 5.2-6.4. The graph shows that ethanol production is independent of transgenic grain content in the sample.

Figure 9 shows the results of T2 seed analysis from different events transformed by pNOV 7005. High expression of pullulanase activity can be detected in a number of events compared to non-transgenic controls.

Fig. 10A and 10B show the results of HPLC analysis of starch hydrolysate produced by expressed pullulanase in transgenic corn meal. The flour of corn expressing pullulanase was incubated in reaction buffer at 75 ℃ for 30 minutes to form medium chain oligosaccharides (degree of polymerization (DP) of about 10-30) and short amylose (DP of about 100-200) from corn starch. FIGS. 10A and 10B show the effect of added calcium ions on the activity of pullulanase.

FIGS. 11A and 11B show data for the formation of starch hydrolysates from two reaction mixtures after HPLC analysis. The first reaction, designated "amylase", contained a sample mixture of corn meal containing transgenic corn expressing alpha-amylase and non-transgenic corn a188 [ 1: 1(w/w) ]; while the second reaction mixture of "amylase + pullulanase" contained a corn meal sample mixture of transgenic corn expressing alpha-amylase and transgenic corn expressing pullulanase [ 1: 1(w/w) ].

FIG. 12 shows the μ g amount of sugar product per 25 μ l reaction mixture for both mixtures. The first reaction, designated "amylase", contained a sample mixture of corn meal containing transgenic corn expressing alpha-amylase and non-transgenic corn a188 [ 1: 1(w/w) ]; while the second reaction mixture of "amylase + pullulanase" contained a sample mixture of corn meal from transgenic corn expressing alpha-amylase and transgenic corn expressing pullulanase [ 1: 1(w/w) ].

FIGS. 13A and 13B show the starch hydrolysates of both sets of reaction mixtures at the end of the 30 minute incubation at 85 ℃ and 95 ℃. Two reaction mixtures were present in each group; the first reaction, called "amylase X pullulanase", contains the corn meal of a transgenic corn (produced by cross-pollination) expressing both alpha-amylase and pullulanase, and the second reaction, an "amylase", is a sample mixture of corn meal of a transgenic corn expressing alpha-amylase and non-transgenic corn A188, mixed in a ratio that achieves the same amount of alpha-amylase activity observed for the hybrid corn (amylase X pullulanase).

FIG. 14 shows the degradation of starch to glucose using non-transgenic corn seeds (control), transgenic corn seeds containing 797GL3 alpha-amylase, and a combination of 797GL3 transgenic corn seeds and Mal A alpha-glucosidase.

FIG. 15 illustrates the conversion of crude starch at room temperature or 30 ℃. In this figure, reaction mixtures 1 and 2 are mixtures of water and starch at room temperature or 30 ℃, respectively. Reaction mixtures 3 and 4 are mixtures of barley alpha-amylase and starch, respectively, at room temperature or 30 ℃. Reaction mixtures 5 and 6 are mixtures of thermoanaerobacter (Thermoanaerobacterium) glucoamylase and starch at room temperature or 30 ℃. Reaction mixtures 7 and 8 are mixtures of barley alpha-amylase (sigma) and thermoanaerobacter glucoamylase and starch, respectively, at room temperature or 30 ℃. Reaction mixtures 9 and 10 are mixtures of barley alpha-amylase (sigma) control and starch at room temperature or 30 ℃, respectively. The Degree of Polymerization (DP) of the Thermoanaerobacterium glucoamylase product has been indicated.

FIG. 16 shows the production of fructose from amylase transgenic corn flour using a combination of alpha-amylase, alpha-glucosidase and glucose isomerase as described in example 19. The amylase corn flour and enzyme solution are mixed together with water or buffer. All reactions contained 60mg of amylase corn flour in a total volume of 600. mu.l and were incubated at 90 ℃ for 2 hours.

FIG. 17 shows the peak area of the reaction product as a function of moisture retention time at 90 ℃ for 0-1200 minutes for 100% amylase corn flour from self-processed grain.

FIG. 18 shows the peak area of the reaction product as a function of incubation time at 90 ℃ for 0-1200 minutes for 10% amylase corn flour from self-processed grain and 90% control corn flour.

FIG. 19 provides HPLC analysis of transgenic amylase corn flour incubated at 70 deg., 80 deg., 90 deg. or 100 deg.C for no more than 90 minutes to assess the effect of temperature on starch hydrolysis.

FIG. 20 shows the ELSD peak areas of samples of mixtures containing 60mg of transgenic amylase corn flour with enzyme solution and water or buffer under various reaction conditions. One set of reactions was buffered with 50mM MOPS, pH7.0, at room temperature plus 10mM MgSO₄And 1mMCoCl₂(ii) a In another set of reactions, the metal-containing buffer was replaced with water. All reactions were incubated at 90 ℃ for 2 hours.

Detailed Description

In the present invention, an isolated polynucleotide encoding a processing enzyme capable of being processed in a plant, e.g., modified, starch, polysaccharide, lipid, protein, etc., has been introduced into a "self-processing" plant or plant part, wherein the processing enzyme is resistant to moderate, high or ultra-high temperatures and can be activated by crushing, adding water, heating or otherwise providing the conditions necessary for the enzyme to function. The isolated polynucleotide encoding a processing enzyme is integrated into a plant or plant part for expression therein. Upon expression and activation of the enzyme, the plants or plant parts of the invention process the substrate for the action of the processing enzyme. Thus, the plants or plant parts of the invention are capable of self-processing the substrates of the processing enzyme after the enzyme is activated in the absence or presence of only reduced amounts of external factors normally necessary for processing such substrates. Thus, the transformed plants, transformed plant cells, and transformed plant parts of the present invention have "intrinsic" processing capabilities that enable processing of a desired substrate by the enzyme incorporated therein. Preferably, the polynucleotide encoding the processing enzyme is "genetically stable", i.e., the polynucleotide is capable of being stably maintained in the transformed plant or plant part of the invention and can be stably inherited by progeny through successive generations.

Methods of using these plants and plant parts according to the present invention can eliminate the need to pulverize or otherwise physically break the plant parts prior to recovery of the starch-derived product. For example, the present invention provides improved methods of treating corn and other grains to recover starch-derived products. The present invention also provides a method that allows for the recovery of starch granules that contain a level of a starch degrading enzyme in or on the starch granules sufficient to hydrolyze specific bonds within the starch without the addition of exogenous starch hydrolyzing enzymes. The present invention also provides improved products from the self-processing plants or plant parts obtained by the methods of the invention.

In addition, transformed plant parts such as grains, which are "self-processing", and transformed plants avoid the major problems of the prior art, namely that the processing enzymes are usually produced by microbial fermentation, which requires isolation of the enzymes from the culture supernatant, which is capital intensive; isolated enzymes need to be treated for specific applications and processes and machinery must be developed to add, mix and react the enzyme with its substrate. The transformed plants or parts thereof of the invention may also be sources of the processing enzymes themselves as well as substrates and products of the enzymes, such as sugars, amino acids, fatty acids and starch and non-starch polysaccharides. The plants of the invention may also be used to prepare progeny plants, such as hybrids and inbreds.

Processing enzymes and polynucleotides encoding same

Polynucleotides encoding processing enzymes (mesophilic, thermophilic or hyperthermophilic) are introduced into plants or parts thereof. The processing enzyme is selected based on the desired substrate for its action found in the plant or transgenic plant and/or the desired end product. For example, the enzyme may be a starch processing enzyme, such as a starch degrading or starch isomerizing enzyme, or a non-starch processing enzyme. Suitable processing enzymes include, but are not limited to, starch degrading or starch isomerizing enzymes, including, for example, alpha-amylase, endo-or exo-1, 4 or 1, 6-alpha-D, glucoamylase, glucose isomerase, beta-amylase, alpha-glucosidase and other exoamylases; and starch debranching enzymes such as isoamylase, pullulanase, neopullulanase, isoamylase, amylopullulanase and the like, glycosyltransferases such as cyclodextrin glycosyltransferase and the like, cellulases such as exo-1, 4- β -cellobiohydrolase, exo-1, 3- β -D-glucanase, hemicellulase, β -glucosidase and the like, endoglucanases such as endo-1, 3- β -glucanase and endo-1, 4- β -glucanase and the like; l-arabinases (L-arabinases) such as endo-1, 5-alpha-L-arabinases, beta-arabinosidases, etc., galactanases (galactanases) such as endo-1, 4-beta-D-galactanase, endo-1, 3-beta-D-galactanase, beta-galactosidase, alpha-galactosidase, etc.; mannanases such as endo-1, 4- β -D-mannanase, β -mannosidase, α -mannosidase, and the like; xylanases, such as endo-1, 4-beta-xylanase, beta-D-xylosidase, 1, 3-beta-D-xylanase, and the like; and a pectinase; and non-starch processing enzymes including proteases, glucanases, xylanases, thioredoxin/thioredoxin reductases, esterases, phytases and lipases.

In one embodiment, the processing enzyme is a starch degrading enzyme selected from the group consisting of: alpha-amylase, pullulanase, alpha-glucosidase, glucoamylase, amylopullulanase, glucose isomerase, or a combination thereof. As will be further described in the present invention: according to this embodiment, the starch degrading enzyme is capable of causing the self-processing plant or plant part to degrade starch upon activation of the enzyme comprised in the plant or plant part. The starch degrading enzyme may be selected according to the desired end product. For example, glucose isomerase may be selected to convert glucose (hexose) to fructose. Alternatively, the enzyme may be selected according to the desired starch-derived end product having different chain lengths or having various desired branching patterns based on, for example, a function of the degree of processing. For example, alpha-amylase, glucoamylase or amylopullulanase may be used to produce dextrin products at shorter incubation times, and short chain products or sugars at longer incubation times. Pullulanase can be used to specifically hydrolyze branch points of starch to form high amylose starch, or new pullulanase can be used to produce starch interspersed with 1, 6 linkage alpha-1, 4 linkage chains. Glucosidase can be used to produce limit dextrins, or different combinations can be used to make other starch derivatives.

In another embodiment, the processing enzyme is a non-starch processing enzyme selected from the group consisting of: proteases, glucanases, xylanases and esterases. These non-starch degrading enzymes allow the self-processing plants or plant parts of the invention to be incorporated into a target area of a plant and when activated destroy the plant while leaving the starch granules intact. For example, in a preferred embodiment, the non-starch degrading enzyme targets the endosperm matrix of a plant cell and, upon activation, disrupts the endosperm matrix while maintaining the starch granules within the endosperm matrix intact, and is more easily recovered from the resulting material.

The invention further provides various combinations of processing enzymes. For example, a combination of starch processing enzymes and non-starch processing enzymes may be used. Combinations of processing enzymes can be obtained using multigene constructs encoding various enzymes. Alternatively, plants containing multiple enzymes can be obtained by crossing a single transgenic plant that has been stably transformed with each enzyme by known methods. Another method involves the use of exogenous enzymes on transgenic plants.

The processing enzyme may be isolated or prepared from any source, and its corresponding polynucleotide may be determined by one of skill in the art. For example, the processing enzyme, preferably an alpha-amylase, may be derived from Pyrococcus (Pyrococcus) (e.g.Pyrococcus furiosus), Thermus (Thermus), Thermococcus (Thermococcus) (e.g.Thermococcus serohydrothermalis), Sulfolobus (Sulfolobus) (e.g.Thielavia sulphureus (Sulfolobus), Thermotoga (Thermotoga) (e.g.Thermoanaerobacter marinus (T.maritima), Thermoanaerobacterium (Thermoanaerobacterium) (e.g.Thermoanaerobacter), Aspergillus (Aspergillus) (e.g.Aspergillus shikonii and Thermococcus nigricans) (e), Thermococcus (Thermoascus), Thermomyces (Thermomyces), Thermomyces (e), Thermomyces (Thermomyces), Thermomyces (e) (, aeropyrum perinx and plants such as corn, barley and rice.

The processing enzyme of the present invention can be activated after being introduced into the genome of a plant and expressed. The conditions under which each individual enzyme is activated can be determined and can include various conditions such as temperature, pH, hydration, presence of metals, activating compounds, inactivating compounds, and the like. For example, temperature-dependent enzymes may include mesophilic, thermophilic and hyperthermophilic enzymes. Mesophilic enzymes generally have a maximum activity at 20-65 ℃ and are inactivated at temperatures above 70 ℃. Mesophilic enzymes have a significant activity at 30-37 ℃, preferably an activity at 30 ℃ of at least 10% of the maximum activity, more preferably at least 20% of the maximum activity.

Thermostable enzymes have a maximum activity at 50-80 ℃ and are inactivated at temperatures above 80 ℃. Preferably, the activity of the thermostable enzyme at 30 ℃ is less than 20% of the maximum activity, more preferably less than 10% of the maximum activity.

Hyperthermophilic enzymes are active at higher temperatures. Hyperthermophilic enzymes have a maximum activity at temperatures above 80 ℃ while retaining activity at temperatures of at least 80 ℃, more preferably at temperatures of at least 90 ℃, most preferably at temperatures of at least 95 ℃. The enzyme with ultrahigh temperature resistance still has reduced activity under the condition of low temperature. The activity of the hyperthermophilic enzyme at 30 ℃ may be less than 10% of the maximum activity, more preferably less than 5% of the maximum activity.

The polynucleotide encoding the processing enzyme is preferably modified to include codons optimized for expression in a selected organism, 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 Nos.5,096,825, 5,625,136, 5,670,356 and 5,874,304). Codon-optimized sequences are synthetic sequences, i.e., they are not naturally occurring and preferably encode the same polypeptide (or an enzymatically active fragment of a full-length polypeptide, and which fragment has substantially the same activity as the full-length polypeptide) encoded by a non-codon-optimized parent polynucleotide encoding a processing enzyme. Preferably, the polypeptide is biochemically unique or improved, for example, by recursive mutation (recursive mutagenesis) of DNA encoding a particular processing enzyme from a parent polypeptide, thereby improving its performance in processing applications. Preferably the polynucleotide is optimized for expression in the target host plant and it encodes a processing enzyme. Methods for making such enzymes include mutagenesis, such as recursive mutagenesis, and screening. Methods for mutations and nucleotide sequence changes 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. Pat. No.4,873,192; walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein and Arnold et al, chem.eng.sci., 51: 5091(1996)). Methods for optimizing the expression of nucleic acid fragments in a target plant or organism are well known in the art. Briefly, a codon usage table is obtained that indicates the optimal codons for use in the target organism, the optimal codons are selected for substitution of codons in the target polynucleotide, and then the optimized sequence is chemically synthesized. Maize preferred codons are described in US Patent No.5,625,136.

Nucleic acids complementary to the polynucleotides of the invention are further envisioned. An example of low stringency conditions for hybridization of complementary nucleic acids with more than 100 complementary residues in Southern or Northern blots performed on filters is 50% formamide, for example in 50% formamide, 1M NaCl, 1% SDS at 37 ℃ and washing with 0.1 XSSC at 60-65 ℃. Exemplary low stringency conditions include hybridization with 30-35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) in buffer at 37 ℃ and washing with 1X-2X SSC (20X SSC ═ 3.0M NaCl/0.3M trisodium citrate) at 50-55 ℃. Exemplary moderate stringency conditions include hybridization in 40-45% formamide, 1.0M NaCl, 1% SDS at 37 ℃ and washing with 0.5X-1X SSC at 55-60 ℃.

In addition, polynucleotides encoding fragments of the processive enzyme "enzymatic activity" are also included. Herein, "enzymatic activity" refers to a polypeptide fragment of the processing enzyme and which has substantially the same biological activity as the processing enzyme that modifies, under appropriate conditions, a substrate on which the processing enzyme normally acts.

In a preferred embodiment, the polynucleotide of the invention is a maize-optimized polynucleotide encoding an alpha-amylase, such as the polynucleotide of SEQ ID NO: 2, 9, 46 and 52. In another preferred embodiment, the polypeptide is a corn-optimized polynucleotide encoding a pullulanase, such as the nucleotide sequence set forth in SEQ ID NO: 4 or 25. In another preferred embodiment, the polynucleotide is a maize-optimized polynucleotide encoding an alpha-glucosidase, as set forth in SEQ ID NO: and 6. In another preferred embodiment, the polypeptide is a maize-optimized polynucleotide encoding a glucose isomerase enzyme, as set forth in SEQ ID NO: 19, 21, 37, 39, 41 or 43. In another preferred embodiment, a maize-optimized polynucleotide encoding a glucoamylase is preferred, as set forth in SEQ ID NO: 46, 48 or 50. In addition, SEQ ID NO: 57 also provides maize-optimized polynucleotides encoding the glucanase/mannanase fusion polypeptides. The invention further provides the complement of such polynucleotides that hybridize under moderate or, preferably, low stringency hybridization conditions and, as the case may be, encode a polypeptide having alpha-amylase, pullulanase, alpha-glucosidase, glucose isomerase, glucoamylase, glucanase, or mannanase activity.

Polynucleotides are used interchangeably with "nucleic acids" or "polynucleic acids" and refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form, composed of monomers (nucleotides) containing a sugar, a phosphate and a base, either a purine or pyrimidine. Unless otherwise indicated, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to control nucleotides and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, in addition to the sequence explicitly set forth. Specifically, substitution of degenerate codons may be obtained by generating sequences in which one or more of the third positions of selected (or all) codons are substituted with mixed base and/or deoxyinosine residues.

Also included are "variants" or substantially similar sequences. With respect to nucleotide sequences, variants include those sequences that, due to the degeneracy of the genetic code, encode the same amino acid sequence as the native protein. Naturally occurring allelic variants, e.g., these variants, can be identified using known molecular biology techniques, such as Polymerase Chain Reaction (PCR), hybridization techniques, and Ligation reassembly (Ligation reassembly) techniques. Variant nucleotide sequences also include nucleotide sequences of synthetic origin, such as those formed, for example, using site-directed mutagenesis to encode a native protein, as well as nucleotide sequences encoding polypeptides having amino acid substitutions. In general, the nucleotide sequence variants of the invention have at least 40%, 50%, 60%, preferably 70%, more preferably 80%, especially preferably 90%, most preferably 99% and percent identity based on these ranks of individual units to the native nucleotide sequence. E.g., 71%, 72%, 73%, etc., up to at least a 90% rating. Variants may also include the full-length gene corresponding to the identified gene fragment.

Regulatory sequences: promoter/Signal sequence/selectable marker

The polynucleotide encoding a processive enzyme of the invention may be operably linked to a polynucleotide sequence encoding a localization signal or signal sequence (at the N-or C-terminus of the polypeptide) to, for example, target the hyperthermophilic enzyme to a particular compartment within a plant. Examples of such targets include, but are not limited to, vacuoles, endoplasmic reticulum, chloroplasts, amyloplasts, starch granules or cell walls, or targeting to specific tissues, such as seeds. Expression in plants of polynucleotides encoding processing enzymes having signal sequences, particularly in combination with tissue-specific or inducible promoters, can result in high levels of localized processing enzymes in plants. Many signal sequences are known to affect the expression of a polynucleotide or to affect the targeting of a polynucleotide to a particular compartment or to a site other than a particular compartment. Suitable signal sequences and targeted promoters known in the art include, but are not limited to, those described herein.

For example, tissue-specific promoters may be used when expression in a particular tissue or organ is desired. Conversely, when gene expression in response to a stimulus is desired, an inducible promoter may be the regulatory element of choice. When sustained expression is desired throughout the cells of a plant, constitutive promoters may be used. Additional regulatory sequences upstream and/or downstream of the central promoter may also be included in the expression construct of the transformation vector to allow expression of the heterologous nucleotide sequence at various levels within the transgenic plant.

A number of plant promoters with diverse expression characteristics have been described. Some constitutive promoters that have been disclosed include rice actin 1(Wang et al, mol.cell.biol., 12: 3399 (1992); U.S. patent No.5,641,876), CaMV 35S (Odell et al, Nature, 313: 810(1985)), CaMV 19S (Lawton et al, 1987), nos (Ebert et al, 1987), Adh (Walker et al, 1987), sucrose synthase (Yang & Russell 1990), and ubiquitin promoters.

Vectors for tissue-specific targeting of genes in transgenic plants typically include a tissue-specific promoter, and may also include other tissue-specific regulatory elements, such as enhancer sequences. Those promoters capable of directing specific expression or enhancing expression in certain plant tissues will be apparent to those skilled in the art in light of the teachings of this disclosure. For example, including, the rbcS promoter, green tissue specific; the ocs, nos and mas promoters, which are more active in root or wounded leaf tissue; a truncated (-90 to +8)35S promoter capable of directing enhanced expression in roots, an alpha-tubulin gene which directs expression in roots, and a promoter from a zein storage protein gene which is capable of directing expression in endosperm.

Tissue-specific expression can be functionally accomplished by: a constitutively expressed gene (all tissues) is introduced together with an antisense gene which is expressed only in a specific tissue, that is, a tissue in which the gene product is not desired. For example, a gene encoding lipase can be introduced using 35S promoter from cauliflower mosaic virus, and thus can be expressed in all tissues. Expression of antisense transcripts of lipase genes in corn grain using, for example, a zein promoter can prevent accumulation of lipase proteins in seeds. Thus, the protein encoded by the introduced gene can be present in all tissues except the grain.

In addition, several tissue-specific regulatory genes and/or promoters have been reported within plants. Some reported tissue-specific genes include genes encoding Seed reserve proteins (e.g., napin, cruciferin, β -conglycinin, and dolichin) zein or oil body proteins (e.g., oleosin), or genes associated with fatty acid biosynthesis (including acyl carrier proteins, stearoyl-ACP desaturase, and fatty acid desaturase (fad 2-1)), as well as other genes expressed during embryonic development (e.g., Bce 4, see EP 255378 and Kridl et al, Seed Science Research, 1: 209 (1991)). Tissue-specific promoters which have been disclosed include lectin (Vodkin, prog. Clin. biol. Res., 138; 87 (1983); Lindstrom et al, der. Genet., 11: 160(1990)), maize alcohol dehydrogenase 1(Vogel et al, 1989; Dennis et al, Nucleic Acids Res., 12: 3983(1984)), maize light harvesting complex (Simpson, 1986; Bansal et al, Proc. Natl. Acad. Sci.USA, 89: 3654(1992)), maize heat shock protein (Odell et al, 1985, Rochester et al, 1986), pea small subunit Rucarboxylase (Poulsen et al, 1986; Cashmore et al, 1983), Ti mannopine synthase (Langridge et al, 1989), Ti plasmid synthase (Lagri et al, 1989), Langri et al, Devyd. 1989; phenyl Tolon isomerase J., 1987, Catalzhen et al, Ile et al, Biokum. J., 1259, Kleine et al, 1986), truncated Ti mannopine synthase (Langrime J., 1989), and Kleine et al, 1989, Kleine et al, Kleine, Shinekalliki, 1989, Shinek et al, Shih, 13: 347(1989)), root cells (Yamamoto et al, Nucleic Acids res, 18: 7449(1990)), zein of corn (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., 18: 7449(1990)), globulin-1 (Belanger et al, Genetics, 129: 863(1991)), alpha-tubulin, cab (Sullivan et al, mol.Gen.Genet., 215: 431(1989)), PEPCase (Hudspeth & Grula, 1989), R gene complex-associated promoter (Chandler et al, Plant Cell, 1: 1175(1989)), and the phenylketene synthase promoter (Franken et al, EMBO j., 10: 2605(1991)). Particularly suitable for seed-specific expression are the pea vicilin promoters (Czako et al, mol. Gen. Genet., 235: 33(1992), see also US5,625,136, incorporated herein by reference.) other promoters that can be used for expression in mature leaves are those that turn on upon initiation of senescence, such as the SAG promoter from Abu (Gan et al, Science, 270: 1986 (1995)).

A class of fruit-specific promoters expressed from flowering to the time or process of fruit development (at least until the onset of ripening) is disclosed in US4,943,674, which is incorporated herein by reference in its entirety. cDNA clones preferentially expressed in cotton fibers have been isolated (John et al, Proc. Natl. Acad. Sci. USA, 89: 5769 (1992)). Tomato cDNA clones that show differential expression during fruit development have been isolated and characterized (Mansson et al, Gen. Genet., 200: 356 (1985)), Slater et al, Plant mol. biol., 5: 137 (1985)). The promoter of the polygalacturonase gene is active at the stage when the fruit begins to mature. U.S. Pat. No. 4,535,060, U.S. Pat. No. 4,769,061, U.S. Pat. No. 4,801,590 and U.S. Pat. No. 5,107,065, which are incorporated herein by reference, describe polygalacturonase genes.

Other examples of tissue-specific promoters include those that are capable of directing expression in leaf cells after leaf damage (e.g., feeding by an insect), in tubers (e.g., patatin promoter), and in fiber cells (an example of a developmental related fiber cell protein is E6(John et al, Proc. Natl. Acad. Sci. USA 89: 5769 (1992)). The E6 gene is most active in fibers, although transcribed at low levels in leaves, ovules and flowers.

The tissue specificity of some "tissue-specific" promoters may not be absolute and can be tested by those skilled in the art using diphtheria toxin sequences. Tissue-specific expression can also be obtained by "leaky" expression using various combinations of different tissue-specific promoters (Bells et al, Plant Cell, 9: 1527 (1997)). Other tissue-specific promoters can also be isolated by those skilled in the art (see US 5,589,379).

In one embodiment, the orientation of the polysaccharide hydrolysis gene product (e.g., alpha-amylase) may be targeted to a specific organelle (e.g., apoplast) rather than the cytoplasm. This can be demonstrated using the maize gamma zein N-terminal signal sequence (SEQ ID NO: 17), which allows specific targeting of the protein to the apoplast. Targeting a protein or enzyme to a particular compartment allows the enzyme to be localized in a manner that does not contact the substrate. In this way, the enzyme will not start the enzymatic reaction until it contacts its substrate. The integrity of the plant cell or organ containing the enzyme can be disrupted by grinding (physically disrupting the cell integrity) or heating the cell or plant tissue to bring the enzyme into contact with its substrate. For example, mesophilic starch hydrolases may be targeted to the apoplast or endoplasmic reticulum, thereby rendering it inaccessible to the starch granules in the amyloplast. Grinding the grain disrupts the integrity of the grain so that the starch hydrolyzing enzymes contact the starch granules. In this way, the potential negative effects of co-localization of the enzyme and its substrate can be avoided.

In another embodiment, the tissue specific promoter includes an endosperm specific promoter, such as the maize gamma zein promoter (shown in SEQ ID NO: 12) or the maize ADP-gpp promoter (shown in SEQ ID NO: 11, which includes the 5' untranslated region and the intron sequence). Thus, the invention includes an isolated polynucleotide comprising a promoter comprising the sequence of SEQ ID NO: 11 or 12, a polynucleotide capable of hybridizing to its complement under low stringency hybridization conditions, or a fragment thereof, having promoter activity as set forth in SEQ ID NO: 11 or 12, preferably 50% of the activity of the promoter.

In another embodiment of the art, the polynucleotide encoding the hyperthermostable processing enzyme is operably linked to a chloroplast (amyloplast) transit peptide (CTP) and a starch binding domain from, for example, a waxy gene. Exemplary polynucleotides in this embodiment encode the nucleic acid sequence of SEQ ID NO: 10 (alpha-amylase linked to the starch binding domain of waxy). The other polynucleotide encodes a hyperthermostable enzyme linked to a signal sequence capable of targeting and secreting the enzyme to the endoplasmic reticulum into the apoplast (as exemplified by the polynucleotide encoding SEQ ID NO: 13, 27 or 30, which contains the N-terminal sequence of maize gamma-zein operably linked to alpha-amylase, alpha-glucosidase, glucose isomerase, respectively), a hyperthermostable enzyme linked to a signal sequence capable of retaining the enzyme in the endoplasmic reticulum (as exemplified by the polynucleotide encoding SEQ ID NO: 14, 26, 28, 29, 33, 34, 35 or 36, which contains the N-terminal sequence of maize gamma-zein operably linked to a hyperthermostable enzyme operably linked to SEKDEL, wherein the hyperthermostable enzyme is alpha-amylase, malA alpha-glucosidase, maritima glucose isomerase, t.neapolitan glucose isomerase), a hyperthermostable processing enzyme operably linked to an N-terminal sequence capable of targeting the enzyme to the amyloplast (e.g., a nucleic acid encoding SEQ ID NO: 15 comprising the N-terminal amyloplast targeting sequence of proxy operably linked to an alpha-amylase, an hyperthermophilic fusion polypeptide targeting the enzyme to starch granules (e.g., a polynucleotide encoding the amino acid sequence of SEQ ID NO: 16, comprising a waxy N-terminal sequence operably linked to an alpha-amylase/waxy fusion polypeptide comprising a waxy starch binding domain, an ER retention signal linked to a hyperthermostable processing enzyme (e.g., a polynucleotide encoding SEQ ID NO: 38 and 39). In addition, the hyperthermostable processing enzyme may be linked to a raw starch (raw starch) binding site having an amino acid sequence (SEQ ID NO: 53), wherein the polynucleotide encoding the processing enzyme is linked to a maize optimized nucleic acid sequence (SEQ ID NO: 54) encoding the binding site.

Several inducible promoters have been reported. Gatz in Current Opinion in Biotechnology, 7: 168(1996) and Gatz C in Annu.Rev.plant Physiol.plant mol.biol.48: 89(1997) already describe a number. Examples include the tetracycline repressor system, the Lac repressor system, the copper inducible system, the salicylic acid inducible system (e.g., the PR1a system), the glucocorticoid inducible system (Aoyama T et al, N-H Plant Journal, 11: 605(1997)) and the ecdysone inducible system. Other inducible promoters include the ABA-and swollenin-inducible promoter, the auxin-binding protein gene promoter (Schwob et al, Plant J.4: 423(1993)), the UDP glucose yellow ketotransferase gene promoter (Ralston et al, Genetics, 119: 185(1988)), the MPI protease inhibitor promoter (Cordero et al, Plant J.6: 141(1994)), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (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: 511 (1989)). Also included are benzenesulfonamide-inducible (U.S. Pat. No. 5,364,780) and ethanol-inducible (WO97/06269 and WO97/06268) systems as well as the glutathione S transferase promoter.

Other studies have focused on genes that respond to environmental stress or external stimuli by inducible regulation, such as increased salinity, drought, pathogens, and injury (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)). Accumulation of metallocarboxypeptidase inhibitor protein has been reported in leaves of damaged potato plants (Graham et al, biochem. Biophys. Res. Comm., 101: 1164 (1981)). Other plant genes have also been reported which can be induced by methyl jasmonate, an inducer, heat shock, anaerobic stress or a pesticide safener.

Controlled expression of the chimeric trans-acting (transducing) viral replication proteins may be further regulated by other genetic methods, such as Cre-mediated gene activation (Odell et al, mol.Gen.Genet., 113: 369 (1990)). Thus, a DNA fragment containing a 3' regulatory sequence bound by a lox site between the promoter and replicator protein coding sequence, which regulatory sequence is capable of blocking expression of the chimeric replicator gene from the promoter, may be removed by Cre-mediated excision, allowing expression of the trans-acting replicative gene. In this case, the chimeric Cre gene, the chimeric trans-acting replicative gene, or both, can be regulated by a tissue and development specific or inducible promoter. Another genetic approach is the use of tRNA suppressor genes. For example, controlled expression of tRNA suppressor genes conditionally regulates the expression of trans-acting replication protein coding sequences that contain appropriate stop codons (Ullmasov et al, Plant mol. biol., 35: 417 (1997)). Likewise, a chimeric tRNA suppressor gene, a chimeric trans-acting replicator gene, or both, can be regulated by a tissue-and development-specific or inducible promoter.

Preferably, for multicellular organisms, the promoter may also be specific to a particular tissue, organ or developmental stage. Examples of such promoters include, but are not limited to: maize (Zeamyys) ADP-gpp and maize gamma-zein promoters and maize globulin promoters.

It may be desirable to express a gene in a transgenic plant only for a certain period of time during plant development. Timing of development is often associated with the expression of tissue-specific genes. For example, zein store protein expression typically begins in the endosperm about 15 days after pollination.

In addition, vectors can be constructed and used to target specific gene products to intracellular components within transgenic plant cells, or to direct proteins to the extracellular environment. This is typically accomplished by linking a DNA sequence encoding a transit or signal peptide sequence to a specific gene coding sequence. The resulting transport or signal peptide transports the protein to a specific intracellular or extracellular destination, respectively. And then can be removed after translation. Transport or signal peptides act by facilitating the passage of proteins across various membranes (e.g., vacuolar, vesicle, plastid and mitochondrial membranes) within cells, while the signal peptide directs the passage of proteins across the outer membrane of cells.

Such a signal peptide sequence, such as the N-terminal signal sequence of maize gamma zein, which can be directed to the endoplasmic reticulum and secreted into the apoplast, can be operably linked to a polynucleotide of the present invention encoding a hyperthermophilic processing enzyme (Torrent et al, 1997). For example, SEQ ID NO: 13, 27 and 30 provide polynucleotides encoding hyperthermophilic enzymes operably linked to a maize gamma zein N-terminal sequence. Another signal sequence is the SEKDEL amino acid sequence used to retain the polypeptide within the endoplasmic reticulum (Munro and Pelham, 1987). For example, the nucleic acid sequence encoding seq id NO: 14, 26, 28, 29, 33, 34, 35, or 36, comprising a zea mays gamma zein N-terminal sequence operably linked to a processing enzyme, said enzyme being operably linked to a SEKDEL. Polypeptides may also be targeted to amyloplasts (Klosgen et al, 1986) or starch granules by fusion to a wax amyloplast targeting peptide. For example, a polynucleotide encoding an hyperthermostable processing enzyme may be operably linked to a chloroplast (amyloplast) transit peptide (CTP) and a starch binding domain from, for example, waxy. SEQ ID NO: 10 demonstrates α -amylase linked to the starch binding domain of waxy. SEQ ID NO: 15 demonstrates an alpha-amylase operably linked to the N-terminal sequence of the amyloplast targeting sequence of waxy. In addition, the polynucleotide encoding the processing enzyme may also be fused to a starch granule using a waxy starch binding domain. For example, SEQ ID NO: 16 demonstrates a fusion polypeptide comprising a wax N-terminal amyloplast targeting sequence operably linked to an alpha-amylase/wax fusion polypeptide comprising a wax starch binding domain.

As is well known in the art, the polynucleotides of the present invention may further comprise other regulatory sequences in addition to the processing signal. "regulatory sequence" and "appropriate regulatory sequence" both refer to a nucleotide sequence located upstream (5 'non-coding sequence), within or downstream (3' non-coding sequence) of a coding sequence, which sequence can affect the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include both natural and synthetic sequences as well as combinations of synthetic and natural sequences.

As is well known in the art, the present invention may also employ selectable markers to allow for the screening out of transformed plants or plant tissues. The skilled artisan may desire to use a selectable or screenable marker gene as, or in addition to, an expressible gene of interest. A "marker gene" is a gene that confers a unique phenotype on cells expressing the gene, thereby allowing transformed cells to be distinguished from cells that do not possess the marker. Such a gene may encode a selectable or screenable marker, depending on whether the marker confers a selectable property that can be selected chemically, i.e., with a selection agent (e.g., herbicide, antibiotic, etc.), or whether it is simply a property that can be observed or detected, i.e., identified by screening (e.g., an R locus property). Many examples of suitable marker genes are known in the art and can be used in the practice of the present invention.

The term selectable or screenable marker also includes within its scope a gene encoding a "secretable marker" whose secretion can be detected as a means of identifying or selecting transformed cells. Examples include a marker encoding a secretable antigen which can be identified by antibody interaction, or even a secretable enzyme which can be detected by its catalytic activity. The variety of secretable proteins is numerous, including small diffusible proteins that can be detected, for example, by ELISA; small active enzymes detectable in extracellular fluids (e.g., alpha-amylase, beta-lactamase, phosphinothricin transacetylase); and proteins inserted or embedded within the cell wall (such as proteins containing leader sequences, e.g., as observed in extensin or expression units of tobacco PR-S).

In the case of a selectable secretory marker, it is considered particularly advantageous to use a gene encoding a protein which contains a unique epitope and is sequestered in the cell wall. Such secretory antigen markers ideally use epitope sequences that have low background in plant tissues, enable efficient expression and targeting of the promoter-leader sequence across the plasma membrane, and form cell wall-associated proteins that make antibody access. Such a need can be met by a normally secreted cell wall protein that is modified to contain unique epitopes.

One protein suitable for modification in this way is extensin, or hydroxyproline-rich glycoprotein (HPRG). For example, The maize HPRG (Steifel et al, The Plant Cell, 2: 785(1990)) molecule has been characterized in detail in molecular biology, expression and protein structure. However, any extensin and/or glycine-rich cell wall protein can be modified by adding antigenic sites (Keller et al, EMBO Journal, 8: 1309 (1989)) to form a screenable marker.

a. Selectable markers

Selectable markers useful in the present invention include, but are not limited to: neo or nptII genes (Potrykus et al, mol.Gen.Genet., 199: 183(1985)) which encode kanamycin resistance, which can be selected using kanamycin, and G418 et al; a bar gene capable of conferring resistance to the herbicide phosphinothricin; the gene encoding the altered EPSP synthase protein (Hinche et al, Biotech, 6: 915(1988)) is thus capable of conferring glyphosate resistance; nitrilases, such as bxn from Klebsiella ozaenae, which confer resistance to bromoxynil (Stalker et al, Science, 242: 419 (1988)); mutant acetolactate synthase (ALS) which is capable of conferring resistance to imidazolinone, sulfonylurea or other ALS inhibiting compounds (european patent application 154, 204, 1985); methotrexate resistant DHFR gene (Thillet et al, J.biol.chem., 263: 12500 (1988)); a dalapon dehalogenase gene capable of conferring resistance to the herbicide dalapon; a phosphomannose isomerase (PMI) gene; a mutant anthranilate synthase gene capable of conferring 5-methyltryptophan resistance; a hph gene capable of conferring resistance to the antibiotic hygromycin; or the mannose-6-phosphate isomerase gene (also referred to herein as phosphomannose isomerase gene), which provides the ability to metabolize mannose (US 5,767,378 and 5,994,629). One skilled in the art will be able to select an appropriate selectable marker gene to practice the present invention. Additional benefits may also be obtained when using a mutated EPSP synthase, incorporating the appropriate chloroplast transit peptide CTP (EP0,218,571, 1987).

An illustrative example of a selectable marker gene that can be used to select transformants is a gene encoding phosphinothricin deacetylase, such as the bar gene of Streptomyces hygroscopicus (Streptomyces hygroscopicus) or the pat gene of Streptomyces viridochromogenes (Streptomyces viridochromogenes). Phosphinothricin deacetylase (PAT) is capable of inactivating phosphinothricin (PPT), an active ingredient in the herbicide bialaphos. PPT is able to inhibit glutamine synthase (Mrakami et al, mol. Gen. Genet., 205: 42 (1986); Twell et al, Plant physiol.91: 1270(1989)), leading to rapid accumulation of ammonia, which causes cell death. The success of using this selection system with monocotyledonous plants is surprising, since great difficulties in cereal transformation have been reported (Potrykus, Trends Biotehch., 7: 269 (1989)).

When it is desired to practice the invention using the bialaphos resistance gene, a very useful gene is the bar or pat gene available from Streptomyces species (e.g., ATCC No.21,705). Cloning of the bar gene has been disclosed (Murakami et al, mol. Gen. Genet., 205: 42 (1986); Thompson et al, EMBO Journal, 6: 2519(1987)) and the use of the bar gene in plants other than monocotyledonous plants (De Block et al, EMBO Journal, 6: 2513 (1987); De Block et al, Plant Physiol.91: 694 (1989)).

b. Screenable markers

Screenable markers that can be used include, but are not limited to, the β -glucuronidase or uidA Gene (GUS), various chromogenic substrates for the enzyme for which it is known to encode; an R-locus gene encoding a product capable of modulating the production of anthocyanin pigments (red) in plant tissues (Dellaporta et al, Chromosome texture and Function, 263-plus 282 (1988)); the beta-lactamase gene (Sutcliffe, PNAS USA 75: 3737(1978)), various chromogenic substrates for the enzymes known to be encoded by it (e.g., PADAC, a chromogenic cephalosporin); xylE gene (Zukowsky et al, PNAS USA, 80: 1101(1983)) which encodes a catechol dioxygenase enzyme which converts to produce chromocatechol; the alpha-amylase gene (Ikuta et al, Biotech., 8: 241 (1990)); tyrosinase gene (Katz et al, J.Gen.Microbiol., 129: 2703(1983)) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn are capable of condensing to form the easily detectable compound melanin; a beta-galactosidase gene encoding an enzyme having a plurality of chromogenic substrates; the luciferase (lux) gene (Ow et al, Science, 234: 856(1986)), which may allow bioluminescent detection; or aequorin gene (Prasher et al, biochem. Biophys. Res. Comm., 126: 1259(1985)) which can be used for calcium sensitive bioluminescence detection, or green fluorescent protein gene (Niedz et al, Plant Cell Reports, 14: 403 (1995)).

Genes from the maize R gene complex are considered to be particularly effective screenable markers. The R gene complex in maize encodes a protein capable of modulating anthocyanin pigment production in most seeds and plant tissues. The gene from the R gene complex is suitable for transforming maize because expression of the gene in transformed cells does not harm the cells. Thus, the R gene introduced into such cells causes the expression of red pigment, and if stably introduced, the red part can be visually classified. If a maize line carries dominant alleles of genes encoding enzyme intermediates in the anthocyanin biosynthetic pathway (C2, a1, a2, Bz1 and Bz2), but carries recessive alleles at the R locus, transformation of any cell from that line with R will result in the production of red pigment. Exemplary lines include Wisconsin22, which contains the rg-Stadler allele and TR112, a derivative of K55, r-g, b, P1. Or any genotype of maize may be used if the C1 and R alleles are co-introduced. Another selectable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene. The presence of the lux gene in the transformed cells can be detected using, for example, an X-ray film, scintillation counting, fluorescence spectrophotometry, low-light camera, photon counting camera or multiwell luminometry (multiwell luminometry). It is also envisaged that the system may be developed for bioluminescent population screening, such as on tissue culture dishes, or even for whole plant screening.

Polynucleotides used to transform plants may include, but are not limited to, plant genetic and non-plant genetic DNA, such as DNA from bacteria, yeast, animals, or viruses. The introduced DNA may include modified genes, portions of genes, chimeric genes, including genes from the same or different maize genotypes. The term "chimeric gene" or "chimeric DNA" refers to a gene or DNA sequence or segment that contains at least two DNA sequences or segments from a species that does not incorporate the DNA in its natural environment, or in which the DNA sequences or segments are placed or linked in a manner that would not normally occur in the natural genome of an untransformed plant.

Also provided are expression cassettes comprising polynucleotides encoding hyperthermophile processing enzymes, preferably codon optimized polynucleotides. Preferably, the polynucleotide (first polynucleotide) in the expression cassette is operably linked to regulatory sequences, such as promoters, enhancers, termination sequences or combinations thereof, and may optionally be operably linked to a second polynucleotide encoding a signal sequence (N-or C-terminal) that directs the enzyme encoded by the first polynucleotide to a particular cellular or subcellular site. In this way, the promoter and one or more signal sequences enable high levels of expression of the enzyme at a particular location within the plant, plant tissue or plant cell. The promoter may be a constitutive promoter, an inducible (conditional) promoter or a tissue specific promoter, such as an endosperm specific promoter, for example the maize gamma zein promoter (e.g.SEQ ID NO: 12) or the maize ADP-gpp promoter (e.g.SEQ ID NO: 11, which contains 5' untranslated and intron sequences). The invention also provides an isolated polynucleotide comprising a promoter comprising the nucleotide sequence of SEQ ID NO: 11 or 12, a polynucleotide that hybridizes to its complement under low stringency hybridization conditions, or a fragment thereof having promoter activity, e.g., the promoter activity is a promoter having the sequence set forth in SEQ ID NO: 11 or 12, preferably at least 50% of the activity of the promoter. Also provided are vectors comprising the expression cassettes or polynucleotides of the invention and transformed cells comprising the polynucleotides, expression cassettes or vectors of the invention. The vector of the invention may contain a polynucleotide sequence encoding more than one hyperthermostable processing enzyme of the invention, which may be in sense or antisense orientation, and the transformed cell may contain one or more of the vectors of the invention. Vectors capable of introducing nucleic acids into plant cells are preferred.

Transformation of

The expression cassette or a vector construct containing the expression cassette can be inserted into a cell. The expression cassette or vector construct can be episomally carried or integrated into the genome of the cell. The transformed cells can then be cultured as transgenic plants. Accordingly, the present invention provides products of the transgenic plants. Such products may include, but are not limited to, products of seeds, fruits, progeny and progeny of transgenic plants.

A variety of techniques for introducing the constructs into host cells are available to those skilled in the art, and many such techniques are known. Transformation of bacteria and many eukaryotic cells can be performed using polyethylene glycol, calcium chloride, viral infection, phage infection, electroporation, and other methods known in the art. Methods for transforming plant cells or tissues include DNA transformation using agrobacterium tumefaciens (a. tumefaciens) or agrobacterium rhizogenes (a. rhizogenes) as transformation tools, electroporation, DNA injection, particle bombardment, particle acceleration, etc. (see e.g., EP 295959 and EP 138341).

In one embodiment, Ti and Ri plasmid binary vectors derived from Agrobacterium Ti-derived vectors are used to transform a variety of higher plants, including monocots and dicots, such as soybean, cotton, canola, 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.Genet., 199: 183 (1985); Park et al, J.plant biol.38: 365 (1985); Hiei et al, Plant J.6: 1994: 271). Transformation of Plant cells with T-DNA has been extensively studied and is disclosed in detail (EP 120516; Hoekema, The Binary Plant Vector System. offset-drukkerij Kanters B.V.; Alblaserdam (1985), Chapter V; Knauf et al, Genetic Analysis of Host Range Expression byAgeratorium, see Molecular Genetics of The Bacteria-Plant interaction, Puhler, eds., Springer-Verlag, New York, 1983, p.245; and An. et al, EMBO J., 4: 277 (1985)).

Other transformation methods are available to the person skilled in the art, such as direct uptake of the exogenous DNA construct (see EP295959), electroporation techniques (Fromm et al, Nature (London), 319: 791(1986)), or high-speed particle bombardment with metal particles covered with nucleic acid constructs (Kline et al, Nature (London) 327: 70(1987) and U.S. Pat. No. 4,945,050). After transformation, the cells can be regenerated by those skilled in the art. Of particular relevance are the recently published methods for transforming foreign genes into important commercial crops such as rapeseed rape (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); Hinche 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)) EP301749), rice (Hiei et al, Plant J.6: 271(1994)) and maize (Plant Kamm et al, Plant Cell 2: 603; Fromm et al, Biotechnology, 1990)).

Expression vectors containing genomic or synthetic fragments can be introduced into protoplasts or into intact tissues or isolated cells. Preferably, the expression vector is introduced into intact tissue. Commonly used Methods for culturing Plant tissue are known, see, e.g., Maki et al, "Procedures for introducing Foreign DNA into Plants" in Methods in Plant molecular biology &Biotechnology, Glich et al, (eds.) pp.67-68, CRC Press (1993); and Phillips et al, "Cell-Tissue Culture and In-Vitro Manipulation" inCorn& Corn Improvement，3^rdEdition10, Sprague et al, (eds.) pp.345-387, American Society of Agronomy Inc. (1988).

In one embodiment, the expression vector may be introduced into maize or other plant tissues using direct gene transfer methods, such as microprojectile-mediated delivery, DNA injection, electroporation, and the like. The expression vector can be introduced into plant tissue by microprojectile-mediated delivery using a biolistic device. See, for example, Tomes et al, "Direct DNA transfer inter integrate cell microprojectie bombardment" in Gamborg and Phillips (eds.) plant cell, Tissue and Organ Culture: fundamental Methods Springer Verlag, Berlin (1995). However, the present invention contemplates transforming plants with hyperthermophilic enzymes according to known transformation methods. See also, Weissinger et al, Annual rev, genet, 22: 421 (1988); sanford et al, Particulate Science and Techmology, 5: 27(1987) (onions); 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, aci.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 Ceu, 2, 603(1990) (maize); svab et al, proc.natl.acad.sci.usa, 87: 8526(1990) (tobacco chloroplasts); koziel et al, Biotechnology, 11: 194(1993) (corn); shimamoto et al, Nature, 338: 274(1989) (rice); christou et al, Biotechnology, 9: 957(1991) (rice); european patent application EP0332581 (dactylus glomerata and other Pooideae); vasil et al, Biotechnology, 11: 1553(1993) (wheat); weeks et al, Plant Physiol, 102: 1077(1993) (wheat). Method in Molecular Biology, 83.Arabidopsis Protocols, Martinez-Zaper and Salinas, 1998 Humana Press (Abu.).

Transformation of plants can be achieved with a single DNA molecule or with multiple DNA molecules (i.e., co-transformation), and these techniques are applicable to the expression cassettes and constructs of the invention. Many transformation vectors can be used for plant transformation, and the expression cassette of the present invention can be used with any such vector. The choice of vector will depend on the preferred transformation technique and the species of interest to be transformed.

Finally, the most desirable DNA fragment for introduction into the genome of a monocot plant may be a homologous gene or family of genes encoding a desired trait (e.g., hydrolysis of proteins, lipids or polysaccharides) and introduced under the control of a novel promoter or enhancer or the like, or even a promoter or regulatory element that may be homologous or tissue specific (e.g., root-, nucellar/leaf sheath-, wheel-, stem-, ear stem-, grain-or leaf-specific). It is also contemplated that particular applications of the present invention are gene targeting in a constitutive or inducible manner.

Examples of suitable transformation vectors

Many transformation vectors for plant transformation are known in the field of plant transformation, and the gene of the present invention can be used together with any such vector known in the art. The choice of vector depends on the preferred transformation technique and the target species to be transformed.

a. Vectors suitable for Agrobacterium transformation

A number of vectors can be used for transformation with Agrobacterium tumefaciens (Agrobacterium tumefaciens). These typically carry at least one T-DNA border (border) sequence, including, for example, pBIN19(Bevan, nucleic. The construction of two typical vectors suitable for Agrobacterium transformation is described below.

pCIB200 and pCIB2001

The binary vectors pCIB200 and pCIB2001, which can be used for constructing recombinant vectors for transformation using Agrobacterium, are as follows. pTJS75(Schmidhauser & Helinski, J.Bacteriol., 164: 446(1985)) was digested with NarI, the tetracycline resistance Gene was excised, and an AccI fragment from pUC4K, which carries NPTII (Messing & Vierra, Gene, 19: 259 (1982): Bevan et al, Nature, 304: 184 (1983): McBride et al, Plant Molecular Biology, 14: 266(1990)) was inserted to give pTJS75 kan. The XhoI linker was ligated to the EvoRV fragment of PCIB7, which contains the left and right T-DNA borders, the plant selectable nos/nptII chimeric Gene, and the pUC polylinker (Rothstein et al, Gene, 53: 153 (1987)). The XhoI-digested fragment was cloned into SalI-digested pTJS75kan to give pCIB200 (see EP0332104, example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, SstI, KpnI, BglII, XbaI and SalI. pCIB2001 is a derivative of pCIB200, and is obtained by inserting additional restriction sites into the polylinker. The unique restriction sites in the polylinker in pCIB2001 are: EcoRI, SstI, KpnI, BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI, and StuI. In addition to containing these unique restriction sites, pCIB2001 also contains plant and bacterial kanamycin selectable markers, left and right T-DNA borders for Agrobacterium-mediated transformation, RK 2-derived trfA energy for transfer between E.coli and other hosts, and OriT and OriV functions also from RK 2. The pCIB2001 polylinker is suitable for cloning plant expression cassettes containing self-regulatory signals.

pCIR10 and hygromycin selective derivatives thereof;

the binary vector pCIB10 contains sequences encoding the kanamycin resistance gene for selection in plants and the right and left border sequences of the T-DNA, inserted from a wide host range plasmid pRK252, enabling replication in E.coli (E.coli) and Agrobacterium. Rothstein et al describe the construction (Gene, 53: 153 (1987)). Various derivatives of pCIB10 were constructed that incorporated the Gene for hygromycin B phosphotransferase described by Gritz et al (Gene, 25: 179 (1983)). These derivatives enable selection of transgenic plants on hygromycin only (pCIB743) or hygromycin and kanamycin (pCIB715, pCIB 717).

b. Vectors suitable for non-agrobacterium transformation

Transformation without the use of Agrobacterium tumefaciens avoids the necessity to use T-DNA sequences in the transformation vector of choice, so that vectors lacking these sequences can be used in addition to, for example, the vectors containing T-DNA sequences described above. Agrobacterium-independent transformation techniques include transformation by particle bombardment, protoplast uptake (e.g., PEG and electroporation), and microinjection. The choice of vector will depend to a large extent on the preferred choice of the species to be transformed. Non-limiting examples of typical vector construction procedures suitable for non-agrobacterium transformation will be further described.

pCIB3064

pCIB3064 is a pUC-derived vector suitable for direct gene transfer in conjunction with selection using the herbicide basta (or phosphinothricin). Plasmid pCIB246 contains the CaMV35S promoter operably fused to the E.coli GUS gene and the CaMV35S transcriptional terminator. This plasmid is described in PCT published application WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5' to the initiation sites. These sequences have been mutated using standard PCR techniques, the mutation removing the ATG and creating SspI and PvuII restriction sites. The new restriction sites were 96 and 37bp from the unique SalI and 101 and 42bp from the actual start site. The obtained pCIB246 derivative was named pCIB 3025. The GUS gene was then excised from pCIB3025 by SalI and SacI digestion, blunt-ended and religated to form the pCIB3060 plasmid. This plasmid pJIT82 was obtained from John Innes centre, Norwich by excising a 400bp SmaI fragment containing the bar gene of Streptomyces viridochromogens and inserting it into the HpaI site of pCIB3060 (Thompson et al, EMBO J., 6: 2519 (1987)). This resulted in pCIB3064, which contains the bar gene under the control of the CaMV35S promoter and terminator for herbicide selection, an ampicillin resistance gene (for selection in E.coli) and a polylinker with unique SphI, PstI, HindIII and BamHI sites. The vector is suitable for cloning plant expression cassettes containing autoregulatory control signals.

pSOG19 and pSOG35

Plasmid pSOG35 is a transformation vector using the E.coli gene dihydrofolate reductase (DHFR) capable of conferring methotrexate resistance as a selectable marker. PCR was used to amplify the 35S promoter (-800bp), intron (-550bp) of the maize Adh1 gene and 18bp of the GUS untranslated leader from pSOG 10. A250 bp fragment encoding the E.coli dihydrofolate reductase type II gene was also amplified by PCR and assembled with the SacI-PstI fragment of pB1221(Clontech), the pB1221 fragment containing the backbone of the pUC19 vector and the nopaline synthase terminator. These fragments assembled to form pSOG19, which contains the 35S promoter fused to the intron 6 sequence, GUS leader sequence, DHFR gene and nopaline synthase terminator. Vector pSOG35 was formed by replacing the GUS leader sequence in pSOG19 with the leader sequence of Maize Chlorotic Mottle Virus (MCMV). pSOG19 and pSOG35 carried the pUC gene for ampicillin resistance and had HindIII, SphI, PstI and EcoRI sites which could be used for cloning of foreign material.

c. Vector for chloroplast transformation

In order to express the nucleotide sequence of the present invention in a plant plastid, the plastid transformation vector pPH143(WO 97/32011, example 36) may be used. This nucleotide sequence was inserted into pPH143 in place of the PROTOX coding sequence. This sequence was then used for plastid transformation and transformants were selected for spectinomycin resistance. Alternatively, the nucleotide sequence was inserted into pPH143 so as to replace the aadH gene. In this case, transformants were selected on the basis of resistance to the PROTOX inhibitor.

Plant hosts treated by transformation methods

Any plant tissue capable of subsequent vegetative propagation (clonal propagation), whether by organogenesis or embryogenesis, may be transformed with the constructs of the present invention. The term organogenesis refers to the process of sequentially developing shoots or roots from the center of a meristem, whereas embryogenesis refers to the process of co-development of shoots and roots in a consistent manner (rather than sequentially), whether starting from somatic cells or gametes. The selection of a particular tissue will depend on the clonal propagation system that is available and best suited to the particular species to be transformed. Exemplary target tissues include differentiated and undifferentiated tissues or plants, including but not limited to leaf discs, roots, stems, shoots, leaves, pollen, seeds, embryos, cotyledons, hypocotyls, macrogametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristem), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem), tumor tissue, and various forms of cells and cultures, such as single cells, protoplasts, embryos, and callus tissue. The plant tissue may be within a plant or organ, tissue or cell culture.

The plants of the invention may be in various forms. The plant may be a chimera of transformed and non-transformed cells; the plant may be a clonal transformant (e.g., all cells transformed to contain the expression cassette); the plant may contain grafts of transformed and non-transformed tissues (e.g., a transformed root stock grafted onto an untransformed scion in a citrus species). The transformed plants can be propagated in various ways, such as by asexual propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be self-pollinated to form a homozygous second generation (or T2) transformed plant, while a T2 plant may be further propagated by classical breeding techniques. A dominant selectable marker (e.g., nptII) can be associated with the expression cassette to aid in breeding.

The invention may also be used to transform any plant species, including monocots or dicots, including but not limited to, maize (maize), Brassica species (Brassica sp.) (such as Brassica napus (b. napus), turnip (b. rapa), mustard (b. jucare)), in particular those Brassica species as seed oil source, alfalfa (medical sativa), rice (oryza sativa), rye (Secale cereale), Sorghum (Sorghum biocolor), milo (Sorghum vulgare), millet (Sorghum vulgare)), millet (such as yugo (Pennisetum glaucum), wild millet (Panicum millecium), millet (Setaria italica), millaria italica), sunflower (Helianthus annuus), safflower (carthamus), wheat (Triticum aestivum), soybean (Glycine max), cotton (Solanum barbarum), cotton (sweet potato), cassava (Manihostesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananascomosus), Citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus Carica), guava (Psidium guajava), mango (Mangiferandica), olive (Olea europaea), papaya (Carica papaya), papaya (Anacardium occidentale), Macadamia (Macamiadilla integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugarcane (Saccharum saccharum spp.), Macadamia (Macamianthus integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), strawberry, olive, strawberry, grape vine, strawberry, grape vine, carrot and Arabidopsis thaliana (Arabidopsis thaliana).

Vegetables include tomato (Lycopersicon esculentum), lettuce (e.g., Lactussitiva), kidney bean (Phaseolus vulgaris), lima bean (Phaseolus limensis), Mucuna (Lathyrus spp.), cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, and members of the Cucumis genus (Cucumis), such as cucumber (C.sativus), cantaloupe (C.sativus), and cantaloupe (C.melo). Ornamental plants include Rhododendron (Rhododendron spp.), hydrangea (macrophylanangea), Hibiscus (Hibiscus rosanensis), Rosa (Rosa spp.), Tulipa (Tulipa spp.), Narcissus spp.), Solanum petunia (Petuniahybrid), Carcinia moschata (Dianthus caryophyllus), Rhododendron (Euphorbia pulcherrima) and Chrysanthemum. Conifers that may be used in the practice of the present invention include, for example, pine trees such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), yellow pine (Pinus poronida), Pinus parviflora (Pinus continenta) and radiata pine (Pinus radiata), Douglas fir (Pseudotsuga menziesii); firotus californicus (Tsuga canadens); white spruce (Picea glauca); sequoia sempervirens (Sequoia sempervirens); cool pines, such as pacific cool pine (Abies amabilis) and collybia albuminosa (Abies balasala); and cedars (cedars), such as arborvitae (Thuja plicata) and cypress (chamaetyparis noiseotkatenis). Leguminous plants include beans (beans) and peas. The vegetable and bean products include guar, locust bean, bitter bean (fenugreek), soybean, kidney bean, cowpea, mung bean, lima bean, broad bean, lentil, chickpea, etc. Leguminous plants include, but are not limited to, arachis, such as peanuts, Vicia, such as garden peas, vetch, adzuki beans, mung beans and chickpeas, lupine, such as lupine, clover, phaseolus, such as kidney beans, and lima beans, Pisum (Pisum), such as clover, melilotum (Medicago), such as clover, Medicago, such as ageratum, Lotus, such as clover, lentil, such as lentil, and pseudo-magnolia. Preferred feedstuffs and turf grasses for use in the method of the present invention include alfalfa, dactylus glomerulus, muscus niveus, rye grass, red top grass and chaff grass.

Preferably, the plants of the present invention include crops such as corn, alfalfa, sunflower, brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, barley, rice, tomato, potato, squash, melons, legume crops and the like. Other preferred plants include Liliopsida and Panicoideae.

When a DNA sequence of interest is transformed into a particular plant species, it can be transmitted through that species, or transferred to other entities of the same species, including particularly commercial entities, using conventional breeding techniques.

Representative methods for transforming dicotyledonous and monocotyledonous plants, as well as representative plastid transformation techniques, are described below.

a. Transformation of dicotyledonous plants

Techniques for transforming dicotyledonous plants are well known in the art and include Agrobacterium-based techniques as well as those that do not require Agrobacterium. Non-agrobacterium techniques involve the direct uptake of exogenous genetic material by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment mediated transport or microinjection. Paszkowski et al, EMBO j., 3: 2717(1984), Potrykus et al, mol.gen.genet, 1999: 169(1985), Reich et al, Biotechnology, 4: 1001(1986) and Klein et al, Nature, 327: 70(1987) all describe these techniques. In any event, the transformed cells can be regenerated into whole plants using standard techniques well known in the art.

Agrobacterium-mediated transformation is the preferred technique for transformation of dicotyledonous plants because of its high transformation efficiency and its wide application to many different species. Agrobacterium transformation typically involves transfer of a binary vector carrying the exogenous DNA of interest (e.g., pCIB200 or pCIB2001) into an appropriate Agrobacterium strain, possibly relying on the complementation of the vir genes carried by the host Agrobacterium strain either on a co-resident (co-residual) Ti plasmid or on the chromosome (e.g., strain CIB542 for pCIB200 and pCIB2001(Uknes et al, Plant Cell, 5: 159 (1993)). Transfer of the recombinant binary vector to agrobacterium is accomplished by a triparental mating process using escherichia coli carrying the recombinant binary vector, a helper escherichia coli strain carrying a plasmid such as pRK2013, which is capable of transferring the recombinant binary vector to a target agrobacterium strain. Alternatively, recombinant binary vectors can be transferred into Agrobacterium by DNA transformation (H * fgen & Willmitzer, Nucl. acids Res., 16: 9877 (1988)).

Transformation of a target plant species with recombinant agrobacterium typically involves co-culturing agrobacterium with an explant of the plant and using techniques well known in the art. Transformed tissues are regenerated on selection medium carrying antibiotic or herbicide resistance markers located between the binary plasmid T-DNA borders.

The vector may be introduced in a known manner. Preferred cells for transformation include Agrobacterium, monocot and dicot cells, including Liliopsida and Panicoideae cells. Preferred monocotyledonous cells are cereal cells, such as maize (maize), barley and wheat, and dicotyledonous cells which accumulate starch, such as potato.

Another method of transforming plant cells with genes involves the propulsion of inert or bioactive particles into plant tissues and cells. This technique is described in U.S. Pat. nos. 4,945,050, 5,036,006, and 5,100,792. Generally, the process involves propelling inert or bioactive particles into plant tissues and cells under conditions effective to penetrate the outer surface of the cells and incorporate them therein. When inert particles are used, the vector can be introduced into the cells by coating the particles with the vector containing the gene of interest. Alternatively, the target cell may be surrounded by a vector so that the vector can be brought into the cell by following the particle. Bioactive particles (such as stem yeast cells, stem bacteria or phages, all containing the DNA to be introduced) can also be propelled into plant cell tissue.

b. Transformation of monocots

Transformation of most monocot species has become a routine practice today. Preferred techniques include direct gene transfer to protoplasts using ethylene glycol (PEG) or electroporation techniques and particle bombardment into callus. Transformation can be performed with a single DNA species or with multiple DNA species (i.e., co-transformation), and such techniques are suitable for use in the present invention. Co-transformation may have the following advantages: avoiding the complete vector construction and preparing transgenic plants with unlinked genes of interest and selectable marker loci, enables the removal of the selectable marker in subsequent processes if deemed necessary. However, a drawback of co-transformation is that the frequency of integration of separate DNA species into the genome is less than 100% (Schocher et al, Biotechnolyy, 4: 1093 (1986)).

Patent applications EP0292435, EP0392225 and WO93/07278 describe methods for the preparation of callus and protoplasts from inbred lines of maize elite (elite), transformation of protoplasts using PEG or electroporation, and regeneration of maize plants from the transformed protoplasts. Gordon-kamm et al (Plant Cell, 2: 603(1990)) and Fromm et al (Biotechnolyy, 8: 833(1990)) both disclose methods of transforming A188-derived maize lines using particle bombardment. In addition, WO03/07278 and Koziel et al (Biotechnology, 11: 194(1993)) both describe the use of particle bombardment to transform elite inbred maize lines. The technique uses immature corn embryos 1.5-2.5mm long, cut from the ear of corn 14-15 days after pollination, and PDS-1000He Biolistics equipment.

Transformation of rice can also be achieved using protoplast or particle bombardment direct gene transfer. Protoplast-mediated transformation of the Japonica and Indica types has been disclosed (Zhang et al, plant cell Rep., 7: 379 (1988); Shimamoto et al, Nature, 338: 274 (1989); Datta et al, Biotechnology, 8: 736 (1990)). Both types can also be transformed conventionally using particle bombardment (Christou et al, Biotechnology, 9: 957 (1991.) additionally, WO93/21335 discloses methods for electroporating transformed rice. patent application EP0332581 discloses methods for preparing, transforming and regenerating Pooideae protoplasts, which can transform Dactylis (Dactylis) and wheat in addition Vasil et al (Biotechnology, 10: 667(1992)) discloses methods for transforming wheat using particle bombardment into type C long-term regenerable callus cells, Vasil et al (Biotechnology, 11: 1553(1993)) and Weeks et al (Plantpysol., 102: 1077(1993)) also discloses methods for particle bombardment transformation using immature embryos and immature embryo-derived callus, but preferred methods for transformation involve bombardment with immature embryo particles of wheat and include the delivery of sucrose or sucrose prior to the step of high-sucrose transport, any number of embryos (0.75-1mm long) were planted on MS medium (Murashiga & Skoog, physiologia plantarum, 15: 473(1962)) with 3% sucrose and 3mg/l 2, 4-D for induction of somatic embryos, and the process was performed in the dark. On the day of bombardment, embryos are removed from the induction medium and placed on osmoticum (i.e., induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryonic plasmodium is allowed to separate for 2-3 hours and then bombarded. Typically, there are 20 embryos per target plate, but this is not critical. The appropriate gene-carrying plasmid (e.g., pCIB3064 or pSG35) is precipitated onto micron-sized gold particles using standard methods. Each slab was bombarded with a DuPont Biolistics * helium apparatus, standard 80 mesh screen, at a burst pressure of about 1000 psi. After bombardment, the embryos are returned to the dark for about 24 hours (still on osmoticum). After 24 hours the embryos are removed from the osmoticum and placed on induction medium for about 1 month to regenerate. Approximately 1 month later, the embryogenic explants with developing embryogenic callus were transferred to regeneration medium (MS +1 mg/liter NAA, 5 mg/liter GA) which further contained the appropriate selection agent (pCIB3064 at 10mg/l basta, pSOG35 at 2mg/l methotrexate). After approximately 1 month, the emerging shoots were transferred to a larger sterile container designated "GA 7 s" containing half strength MS, 2% sucrose and the same concentration of selection agent.

Methods for transforming monocots using Agrobacterium have been disclosed, see WO94/00977 and US5,591,616, both incorporated herein by reference.

c. Plastid transformation

Seeds of Nicotiana tabacum c.v. ' Xanthi nc ' were germinated on a 1 ' circular matrix on T agar medium 7 per plate, bombarded with 1 μ M tungsten particles (M10, Biorad, Hercules, Calif.) coated with pPH143 and pPH145 plasmid DNA, approximately as described in the literature (Svab and Malega, PNAS, 90: 913 (1993)). The bombarded seedlings were grown for 2 days on T medium, then the leaves were cut and placed in bright light (350-²S) (Svab, Hajdukiewicz and Maliga, PNAS, 87: 8526(1990)), the medium containing 500. mu.g/ml spectinomycin dihydrochloride (Sigma St. Louis, MO). Tolerant shoots appearing below etiolated leaves were subcloned into the same selection medium 3-8 weeks after bombardment, allowed to callus, and a second shoot was isolated for subcloning. Complete isolation of genomic copies of transformants in independent subclones (bioplasmity) was assessed by standard techniques of Southern blotting (Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold spring Harbor (1989)). BamHI/EcoRI digested total cellular DNA was isolated on a 1% Tris-borate (TBE) agar gel (Mettler, I.J. plant mol. biol. Reporter, 5: 346(1987)), transferred to a nylon membrane (Amersham) and hybridized with a 32P-labelled random priming DNA sequence corresponding to the 0.7kb BamHI/HindIII DNA fragment of pC8 containing a portion of the rps7/12 plastid targeting sequence. The homoshoots were rooted under sterile conditions on MS/IBA medium containing spectinomycin (McBride et al, PNAS, 91: 7301(1994)) and transferred to the greenhouse.

Preparation and characterization of stably transformed plants

The transformed plant cells are then cultured to form callus in a selection medium suitable for selection of transgenic cells. Shoots (shoots) are formed from the callus and seedlings are formed from the shoots by growing in rooting medium. Typically, each construct will be linked to a marker to facilitate selection of plant cells. For convenience, the marker may be resistance to a biocide (particularly an antibiotic such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, a herbicide, etc.). The specific marker used will allow selection of transformed cells as compared to cells lacking the introduced DNA. The components of the DNA construct, including the transcription/expression cassette of the invention, can be prepared from sequences that are native (endogenous) or foreign (exogenous) to the host. By "foreign" is meant that the sequence is not present in the wild-type host into which the construct is introduced. The heterologous construct will contain at least one region that is not native to the gene from which the transcription initiation region is derived.

To confirm the presence of the transgene in the transgenic cells and plants, Southern blot analysis can be performed using methods well known in the art. Since polynucleotide segments can be readily distinguished from constructs containing the segments using appropriate restriction enzymes, integration of the polynucleotide segments into the genome can be detected and quantified by Southern blotting. Expression products of the transgene can be detected in a variety of ways, depending on the nature of the product, including Western blotting and enzymatic assays. One very efficient method for quantifying protein expression and detecting replication in different plant tissues is the use of reporter genes, such as GUS. Once the transgenic plant is obtained, it can be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant part may be collected and/or seeds harvested. The seeds may be used as a source for growing additional plants containing plant tissues or parts having the desired characteristics.

Thus the invention provides transformed plants or plant parts, such as ears, seeds, fruits, grains, stalks (stover), chaffs (chaff) or shavings (bagass), containing at least one polynucleotide, expression cassette or vector of the invention, methods of using such plants or parts thereof, and methods of making such plants. The transformed plant or part thereof will express a processing enzyme, optionally located in a particular cell or subcellular location of a tissue or developing grain. For example, the present invention provides a transformed plant part comprising at least one starch processing enzyme present in a plant cell, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one starch processing enzyme. The processing enzyme does not act on the target substrate unless activated by a process such as heating, milling or other means that allows the enzyme to contact the substrate under conditions in which the enzyme is active.

Preferred method of the invention

The self-processing plants and plant parts of the invention may be used in various methods utilizing processing enzymes (mesophilic, thermophilic or hyperthermophilic) expressed and activated therein. According to the invention, the transgenic plant part obtained from the transgenic plant, which has increased in its genome at least one starch processing enzyme, is subjected to conditions which enable the expression and activation of the processing enzyme. Upon activation, the processing enzyme is activated and is able to act on its normally acting substrate to obtain the desired product. For example, after activation the starch processing enzymes act on the starch to degrade, hydrolyze, isomerize, or otherwise modify it to achieve the desired result. Non-starch processing enzymes may be used to disrupt plant cell membranes to facilitate extraction of plant starch, lipids, amino acids or other products. In addition, non-hyperthermophilic and hyperthermophilic enzymes can be used in conjunction with the self-processing plants or plant parts of the present invention. For example, mesophilic non-starch degrading enzymes may be activated to disrupt plant cell membranes to facilitate starch extraction, and then hyperthermophilic starch degrading enzymes in the self-processing plant may be activated to degrade starch.

Enzymes expressed in cereals plants or parts thereof containing the enzyme may be activated by subjecting them to conditions which increase the enzymatic activity. For example, one or more of the following techniques may be used: the plant parts may be contacted with water, which provides a substrate for the hydrolase to activate the enzyme. The plant part may be contacted with water such that the enzyme migrates out of the area where it is deposited during development of the plant part to contact the substrate. Since regionalization is disrupted during ripening, grain drying and rehydration, enzyme migration is possible. Whole or broken grain may be contacted with water, which causes the enzyme to migrate away from its location of deposition during development of the plant part and thus contact the substrate. Activating compounds may also be added to activate the enzyme. For example, calcium-dependent enzymes can be activated by the addition of calcium. Other activating compounds can be determined by one skilled in the art. The enzyme may be activated by removing the inactivating agent. For example, some peptide inhibitors are known to inhibit amylases, which can be co-expressed with amylase inhibitors and then activated by the addition of proteases. The enzyme can be activated by adjusting the pH to a state of maximum enzymatic activity. The temperature may also be increased to activate the enzyme. Increasing the temperature without exceeding the maximum temperature of the enzyme generally increases the enzyme activity. The activity of the enzyme increases from the ambient temperature activity level up to a temperature at which the mesophilic enzyme loses activity, typically at a temperature of less than or equal to 70 ℃. Likewise, the temperature may be increased to activate both thermostable and hyperthermophilic enzymes. Thermostable enzymes can be activated by heating the temperature to the highest temperature of activity or stability. The maximum temperature for stability or activity is generally 70-85 ℃ for thermostable enzymes. Hyperthermal enzymes have a higher relative activation than mesophilic or thermophilic enzymes, because of their greater potential temperature variation, from 25 ℃ up to 85 ℃ or 95 ℃ or even 100 ℃. The temperature can be increased by any method, for example by heating such as by baking, boiling, heating or steaming, electrical discharge or a combination thereof. In addition, enzymes in plants expressing mesophilic or thermophilic enzymes may be activated by milling to bring the enzymes into contact with the substrate.

Optimal conditions, such as temperature, hydration, pH, etc., can be determined by one skilled in the art and will depend on the particular enzyme used and the desired application for that enzyme.

The invention also provides the use of exogenous enzymes that may assist in a particular action. For example, the self-processing plants or plant parts of the invention can be used in conjunction with exogenous enzymes to facilitate the reaction. For example, the transgenic alpha-amylase corn can be used in combination with other starch processing enzymes such as pullulanase, alpha-glucosidase, glucose isomerase, mannanase, hemicellulase, and the like, to hydrolyze starch or form ethanol. In fact, it has been found that the use of transgenic alpha-amylase corn in combination with such an enzyme unexpectedly provides superior starch conversion levels as compared to corn using only the transgenic alpha-amylase.

Examples of suitable methods contemplated herein are provided.

a. Extraction of starch from plants

The present invention provides methods for facilitating the extraction of starch from plants. In particular, at least one polynucleotide encoding a processing enzyme capable of disrupting the physically restricted matrix of the endosperm (cell wall, non-starch polysaccharide and protein matrix) is introduced into a plant so that the enzyme is in close physical proximity to starch granules within the plant. Preferably, in this embodiment of the invention the transformed plant expresses one or more proteases, glucanases, xylanases, thioredoxin/thioredoxin reductases, esterases etc. but does not express an enzyme with any starch degrading activity, so that the integrity of the starch granules can be maintained. Expression of these enzymes in plant parts such as grain will improve the processing characteristics of the grain. The processing enzyme may be resistant to moderate, high or ultra high temperatures. In one example, grain from a transformed plant of the invention is heat dried, likely to inactivate non-hyperthermophile processing enzymes and improve seed integrity. The grain (or cracked grain) is steeped at low or high temperatures (time is very important) with or without sulphur dioxide under high or low moisture content or conditions (see Primary Cereal Processing, Gordon and Willim, eds, pp.319-337(1994), incorporated herein by reference). After reaching the elevated temperature, the integrity of the endosperm matrix is disrupted, optionally under certain moisture content conditions, by activating enzymes such as proteases, xylanases, phytases or glucanases which degrade the protein and non-starch polysaccharides in the endosperm, leaving the starch granules intact and more easily recovered from the product material obtained. In addition, the protein and non-starch polysaccharides in the effluent (elluent) are at least partially degraded and highly concentrated and can therefore be used in modified animal feed, food products or as an intermediate component in microbial fermentation. The effluent is considered to be corn steep liquor of improved composition.

Thus, the present invention provides a method of preparing starch granules. The method includes treating grain, such as cracked grain, containing starch granules and a non-starch degradation product, such as a digested endosperm matrix product, under conditions effective to activate the at least one enzyme, wherein the grain contains at least one non-starch processing enzyme. The non-starch processing enzyme may be resistant to moderate, high or ultra high temperatures. After activation of the enzyme, the starch granules are separated from the mixture. The grain is obtained from a transformed plant, the genome of which contains (or is augmented by) an expression cassette encoding the at least one enzyme. For example, the processing enzyme may be a protease, a glucanase, a xylanase, a phytase, a thioredoxin/thioredoxin reductase or an esterase. Preferably the processing enzyme is resistant to ultra high temperatures. The grain may be treated under low or high moisture conditions, in the presence or absence of sulphur dioxide. Depending on the activity and expression level of processing enzymes in the grain of the transgenic plant, the transgenic grain may be mixed with commercial grain before or during processing. Products obtained by the process, such as starch, non-starch products and modified steepwater (corn steep liquor) containing at least one additional component are also provided.

b. Starch processing method

The transformed plants or plant parts of the invention may contain a starch processing enzyme as disclosed herein which is capable of degrading starch granules to dextrins, other modified starches or hexoses (e.g. alpha-amylase, pullulanase, alpha-glucosidase, glucoamylase, amylopullulanase) or converting glucose to fructose (e.g. glucose isomerase). Preferably, the starch degrading enzyme is selected from the group consisting of alpha-amylase, alpha-glucosidase, glucoamylase, pullulanase, neopullulanase, amylopullulanase, glucose isomerase, and combinations thereof, applied to the transformed grain. In addition, the enzyme is preferably operably linked to a promoter and a signal sequence that targets the enzyme to a starch granule, amyloplast, apoplast or endoplasmic reticulum. Most preferably, the enzyme is expressed in the endosperm, particularly the corn endosperm, and is localized in one or more cellular compartments, or in the starch granule itself. Preferred plant parts are grains. Preferred plant parts are from maize, wheat, barley, rye, oats, sugarcane or rice.

According to one starch degradation process of the present invention, the transformed grain accumulates starch degrading enzymes in the starch granules, steeps the grain at conventional temperatures of 50 ℃ to 60 ℃, and wet mills in a manner well known in the art. Preferably, the starch degrading enzyme is resistant to ultra high temperatures. Since the enzyme is subcellularly targeted to the starch granule, or the enzyme is linked to the starch granule by contacting the enzyme with the starch granule during the wet milling process at conventional temperatures, the process enzyme is co-purified with the starch granule to obtain a starch granule/enzyme mixture. After recovery of the starch granule/enzyme mixture, the enzyme is activated by conditions favoring the activity of the enzyme. For example, the enzyme can be processed under various moisture content and/or temperature conditions to promote partial (to make derivatized starches or dextrins) or complete hydrolysis to hexoses. In this way a syrup is obtained which contains a high concentration of glucose or fructose equivalents. The method can effectively save time, energy and enzyme consumption, and improve the efficiency of starch conversion to the corresponding hexose and the production efficiency of products such as high sugar steepwater (corn steep liquor) and syrup with higher glucose equivalent.

In another embodiment, the plant expressing the enzyme or a product of the plant, such as fruit or grain or flour prepared from the grain, is treated to activate the enzyme and convert polysaccharides expressed and contained within the plant to sugars (sugar). Preferably, the enzyme is fused to a signal sequence that targets the enzyme to a starch granule, amyloplast, apoplast or endoplasmic reticulum, as described herein. The formed sugars can then be isolated from the plant or plant product. In another embodiment, a processing enzyme capable of converting polysaccharides to sugars (sugar) is placed under the control of an inducible promoter according to methods well known in the art and disclosed herein. The processing enzyme may be resistant to moderate, high or ultra high temperatures. The plant is grown to the desired stage and then the promoter is induced to express the enzyme and convert the polysaccharide in the plant or plant product to a sugar. Preferably the enzyme is operably linked to a signal sequence capable of targeting it to a starch granule, amyloplast, apoplast or endoplasmic reticulum. In another embodiment, transformed plants are prepared that express a processing enzyme capable of converting starch to sugars. The enzyme is fused to a signal sequence which directs the enzyme to starch granules in plants. The starch is then isolated from the transformed plant containing the enzyme expressed by the transformed plant. The enzyme contained in the isolated starch is then activated to convert the starch into sugars. The enzyme may be mesophilic, thermophilic or hyperthermophilic. Examples of hyperthermophilic enzymes capable of converting starch to sugars are provided herein. The method can be used with any plant that is capable of producing polysaccharides and that is capable of expressing enzymes capable of converting the polysaccharides into sugars or hydrolyzed starch products, such as dextrins, maltooligosaccharides, glucoses and/or mixtures thereof.

The present invention provides methods for preparing dextrins and modified starches from plants or plant products that have been converted by processing enzymes capable of hydrolyzing certain covalent bonds of polysaccharides to form polysaccharide derivatives. In one embodiment, the plant or product thereof, such as fruit or grain, or flour made from the grain, expressing the enzyme is subjected to conditions sufficient to activate the enzyme and convert polysaccharides contained in the plant to polysaccharides of reduced molecular weight. Preferably, the enzyme is fused to a signal sequence that targets the enzyme to a starch granule, amyloplast, apoplast or endoplasmic reticulum, as described herein. The resulting dextrin or derivatized starch may then be isolated or recovered from the plant or plant product. In another embodiment, a processing enzyme capable of converting a polysaccharide to dextrin or modified starch is placed under the control of an inducible promoter according to methods well known in the art and disclosed herein. The plant is grown to the desired stage and then the promoter is induced to express the enzyme and convert the polysaccharide in the plant or plant product to dextrin or modified starch. Preferably the enzyme is an alpha-amylase, pullulanase, exo-or neopullulanase, and preferably the enzyme is operably linked to a signal sequence capable of targeting it to a starch granule, amyloplast, apoplast or endoplasmic reticulum. In one embodiment, the enzyme is targeted to the apoplast or the endoplasmic reticulum. In another embodiment, transformed plants are prepared that express an enzyme capable of converting starch to dextrin or modified starch. The enzyme is fused to a signal sequence capable of targeting it to starch granules in plants. The starch is then isolated from the transformed plant containing the enzyme expressed by the transformed plant. The enzyme contained in the starch is then activated under conditions sufficient to activate the enzyme, converting it to a dextrin or modified starch. Examples of hyperthermophilic enzymes capable of converting starch to a hydrolyzed starch product are provided herein. The method may be used with any plant that is capable of producing a polysaccharide and that is capable of expressing an enzyme capable of converting the polysaccharide to a sugar.

In another embodiment, the grain from the transformed plant of the invention is soaked for various periods of time and under conditions conducive to the activity of starch degrading enzymes capable of degrading bonds in the starch granules to form dextrins, modified starches or hexoses (e.g., alpha-amylase, pullulanase, alpha-glucosidase, glucoamylase, amylopullulanase). The resulting mixture may contain high levels of starch derived products. The application of the grain comprises: 1) the need to grind or otherwise process the grain to obtain starch granules in the first place is avoided, 2) the starch is made more accessible to enzymes by placing the enzymes directly within the endosperm tissue of the grain, and 3) no microbially produced starch hydrolyzing enzymes are required. In this way, the entire wet milling process prior to hexose recovery can be avoided by heating the grain, preferably corn grain, in the presence of water so that the enzyme acts on the substrate.

The process can also be used to prepare ethanol, high fructose syrups, hexose (glucose) -containing fermentation media or any other starch application that does not require refining of the grain component.

The invention also provides a process for the preparation of dextrins, malto-oligosaccharides and/or sugars (sugar), involving treating a plant part containing starch granules and at least one starch processing enzyme under conditions which activate the at least one enzyme, thereby causing enzymatic digestion of the starch granules to form a sugar-containing aqueous solution. The plant part is obtained from a transformed plant, to the genome of which an expression cassette encoding said at least one processing enzyme has been added. The aqueous solution containing dextrin, maltooligosaccharides and/or sugars is then collected. In one embodiment, the processing enzyme is an alpha-amylase, an alpha-glucosidase, a pullulanase, a glucoamylase, a starch pullulanase, a glucose isomerase, or any combination thereof. Preferably the enzyme is resistant to ultra high temperatures. In another embodiment, the method further comprises the step of isolating the dextrin, maltooligosaccharide and/or sugar.

c. Improved maize varieties

The present invention also provides a method for producing improved corn varieties (as well as other crop varieties) having normal levels of starch accumulation and accumulating sufficient levels of amylolytic enzymes in their endosperm or starch accumulating organs such that upon activation of the enzymes contained therein, for example, for ultra-high temperature tolerant enzymes, by boiling or heating the plant or portion thereof, the enzymes are activated and promote rapid conversion of starch to monosaccharides. These monosaccharides (primarily glucose) can impart sweetness to the treated corn. The obtained corn plant is an improved variety and has dual purposes of producing hybrid and sweet corn as grain. Thus, the present invention provides a method for producing super-sweet corn comprising treating transformed corn or a portion thereof having an increased expression cassette in its genome and expressing the expression cassette in the endosperm under conditions effective to activate the at least one enzyme, the expression cassette comprising a promoter operably linked to a first polynucleotide encoding at least one amylolytic enzyme, such that polysaccharides in corn are converted to sugars, resulting in super-sweet corn. The promoter may be a constitutive promoter, a seed-specific promoter or an endosperm-specific promoter linked to a polynucleotide sequence encoding a processing enzyme such as an alpha-amylase (e.g., an enzyme comprising SEQ ID NO: 13, 14 or 16). Preferably, the enzyme is resistant to ultra-high temperatures. In one embodiment, the expression cassette further comprises a second polynucleotide encoding a signal sequence operably linked to the enzyme encoded by the first polynucleotide. Exemplary signal sequences in this embodiment of the invention are capable of directing the enzyme to an apoplast, endoplasmic reticulum, starch granule or amyloplast. The corn plant is grown such that ears of corn with grains are formed, and then the promoter is induced to express the enzyme and convert the polysaccharides contained in the plant into sugars (sugar).

d. Self-fermenting plant

In another embodiment of the invention, a plant, such as corn, rice, wheat or sugarcane, is engineered to accumulate in its cell wall a large amount of processing enzymes, such as xylanases, cellulases, hemicellulases, glucanases, pectinases, etc. (non-starch polysaccharide degrading enzymes). After harvesting the grain components (or sugar if sugar cane), the straw, chaff or shavings are used as a source of enzymes that are directed to be expressed and accumulated within the cell walls and used as a source of biomass. Straw (or other residual tissue) is used as a feedstock for a process for recovering fermentable sugars. This process of obtaining fermentable sugars consists of activating non-starch polysaccharide degrading enzymes. For example, activation can include heating plant tissue in the presence of water for a time sufficient to hydrolyze the non-starch polysaccharides to final sugars. Thus, the self-processing straw produces the enzymes needed to convert polysaccharides to monosaccharides without substantial additional cost, as they are a component of the feedstock. In addition, temperature dependent enzymes have no detrimental effect on plant growth and development, whereas cell wall targeting via the cellulose/xylose binding domain fused to the protein, even into polysaccharide microfibrils, improves substrate accessibility to the enzyme.

Thus, the present invention also provides a method of using a transformed plant part comprising at least one non-starch polysaccharide processing enzyme within the cell wall of the cells of the plant part. The method comprises treating a transformed plant part comprising at least one non-starch polysaccharide processing enzyme, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one non-starch polysaccharide processing enzyme, under conditions which activate the at least one processing enzyme, thereby digesting starch granules to form an aqueous solution comprising sugars; and collecting the aqueous solution containing the sugar. The invention also includes a transformed plant or plant part comprising at least one non-starch polysaccharide processing enzyme in the cells of the plant or part thereof or in the cell walls of the cells. The plant part is obtained from a transformed plant, which has increased in its genome an expression cassette encoding said at least one non-starch processing enzyme, such as xylanase, cellulase, glucanase, pectinase or any combination thereof.

e. Aqueous phase with high protein and sugar content

In another embodiment, the protease and esterase are engineered to accumulate in seeds, such as soybean seeds. After activation of proteases or esterases, for example by heating, these enzymes in the seed hydrolyze lipids and stored proteins in the soybean during processing. In this way, a soluble product containing amino acids and fatty acids is obtained, which can be used as feed, food or fermentation medium. Polysaccharides are typically found in the insoluble components of the grain after processing. However, by combining the expression and accumulation of polysaccharide degrading enzymes in the seed, the protein and polysaccharide can be hydrolyzed and then found in the aqueous phase. For example, zein from corn and stock proteins and non-starch polysaccharides from soybeans can be solubilized in this manner. The components of such aqueous and hydrophobic phases can be easily separated by extraction with organic solvents or supercritical carbon dioxide. This provides a method for preparing an aqueous extract of a cereal grain which extract contains higher levels of proteins, amino acids, sugars (sugar) or carbohydrates (saccharoides).

f. Self-processing fermentation

The present invention provides a process for the preparation of ethanol, fermented beverages or other fermentation derived products. The process involves obtaining a plant or a product or part of a plant, or a plant derivative such as cereal flour, in which a processing enzyme capable of converting a polysaccharide to a sugar (sugar) is expressed. The plant or its product is treated to allow the production of sugars by polysaccharide transformation as described above. The sugars and other components of the plant are then fermented according to techniques well known in the art to form ethanol or fermented beverages, or other fermentation-derived products. See, for example, US 4,929,452. Briefly, sugars formed by polysaccharide conversion are incubated with yeast under conditions that promote the conversion of the sugars to ethanol. Suitable yeasts include high alcohol and sugar tolerant yeast strains, such as the yeast Saccharomyces cerevisiae (S.cerevisiae) ATCC No. 20867. The strain was deposited at 17.9.1987 with the American Type Culture Collection (American Type Culture Collection, Rockville, Md.) under the accession number ATCC No. 20867. The fermentation product or fermented beverage may then be subjected to distillation to separate the ethanol or distilled beverage, or the fermentation product may be otherwise recovered. The plant used in the method may be any plant which contains a polysaccharide and is capable of expressing the enzyme of the invention. Many such plants are disclosed herein. Preferably, the plant is a commercial crop. More preferably the plants are those typically used for the preparation of ethanol or fermented beverages, or fermented products, such as wheat, barley, maize, rye, potato, grape or rice. Most preferably, the plant is maize.

The method comprises treating a plant part containing at least one polysaccharide processing enzyme under conditions that activate the at least one enzyme, thereby digesting polysaccharides in the plant part to form fermentable sugars. The polysaccharide processing enzyme may be resistant to moderate, high or ultra high temperatures. Preferably the enzyme is resistant to ultra high temperatures. The plant part is obtained from a transformed plant, to the genome of which an expression cassette encoding said at least one polysaccharide processing enzyme has been added. Plant parts of this embodiment of the invention include, but are not limited to, grain, fruit, seed, stem, wood, vegetable or root. Preferred plants include, but are not limited to, oats, barley, wheat, berries, grapes, rye, corn, rice, potatoes, sugar beets, sugar cane, pineapples, grasses, and trees. Plant parts may be used in combination with commercial grains or other commercially available substrates; the source of the processing substrate may be a source other than the processed plant. The fermentable sugars are then incubated, for example, with yeast and/or other microorganisms, under conditions that promote the conversion of the fermentable sugars to ethanol. In a preferred embodiment, the plant part is from corn transformed with an alpha-amylase, which has been found to reduce fermentation time and cost.

It has been found that when fermentation is performed using transgenic corn expressing a thermostable alpha-amylase prepared according to the method of the present invention, the amount of residual starch is reduced. This indicates that more starch is dissolved during fermentation. This reduction in the amount of residual starch results in distiller grains with higher weight protein content and higher value. In addition, fermentation of the transgenic corn of the invention can be carried out at lower pH and at higher temperatures, e.g., greater than 85 ℃, preferably greater than 90 ℃, more preferably 95 ℃ or higher, with the liquefaction process being carried out using lower pH resulting in lower cost of chemicals used to adjust pH, and higher temperature resulting in shorter liquefaction time and more complete solubilization of starch, reducing liquefaction time, all of which results in higher yield and efficiency of ethanol for the fermentation reaction.

In addition, it has been found that contacting conventional plant parts with even small proportions of transgenic plants prepared according to the invention can reduce fermentation time and associated costs. Thus, the present invention relates to reducing the fermentation time of plants relative to the use of plant parts that do not contain a polysaccharide processing enzyme, including the use of transgenic plant parts from plants containing a polysaccharide processing enzyme that can convert polysaccharides to sugars.

g. Crude starch processing enzymes and polynucleotides encoding same

Polynucleotides encoding mesophilic processing enzymes are introduced into plants or plant parts. In a preferred embodiment, the polynucleotide of the invention is a maize-optimized polynucleotide, such as the polynucleotide of SEQ id no: 48, 50 and 59, which encodes a glucoamylase such as SEQ ID NO: 47 and 49. In another preferred embodiment, the polynucleotide of the invention is a maize-optimized polynucleotide, such as SEQ ID NO: 52, which encodes an alpha-amylase, such as the polypeptide of SEQ ID NO: shown at 51. In addition, the invention further includes processing the enzyme fusion product. In a preferred embodiment, the polynucleotide of the invention is a maize-optimized polynucleotide, such as SEQ ID NO: 46 encoding an alpha-amylase and glucoamylase fusion, such as SEQ ID NO: shown at 45. The invention further provides combinations of processing enzymes. For example, combinations of starch processing enzymes and non-starch processing enzymes are provided. Such combinations of processing enzymes can be obtained by using multigene constructs encoding various enzymes. Alternatively, each transgenic plant stably transformed with the enzyme may be crossed by a known method to obtain a plant containing both enzymes. Another method involves the use of exogenous enzymes with the transgenic plants.

The source of the starch processing and non-starch processing enzymes may be isolated or derived from any source, the corresponding polynucleotides of which may be determined by one skilled in the art. Preferably, the alpha-amylase is derived from the genera Aspergillus (e.g., Aspergillus shirousami and Aspergillus niger), Rhizopus (e.g., Rhizopus oryzae) and plants such as corn, barley and rice. Preferred glucoamylases are from Aspergillus (e.g., Aspergillus shirousami and Aspergillus niger), Rhizopus (e.g., Rhizopus oryzae) and Thermoanaerobacter (e.g., Thermoanaerobacter thermosaccharomyces saccharolyticus).

In another embodiment of the invention, the polynucleotide encodes a mesophilic starch processing enzyme operably linked to a maize-optimized polynucleotide, such as the nucleotide sequence set forth in SEQ ID NO: 54, the maize-optimized polynucleotide encodes a crude starch binding domain, such as SEQ ID NO: shown at 53.

In another embodiment, the tissue-specific promoter includes an endosperm-specific promoter such as a maize gamma-zein promoter (e.g., as shown in SEQ ID NO: 12) or a maize ADP-gpp promoter (e.g., as shown in SEQ ID NO: 11, which includes 5' untranslated sequences and intron sequences). Thus, the invention includes an isolated polynucleotide comprising a promoter comprising the sequence of SEQ ID NO: 11 or 12, or a polynucleotide that hybridizes to its complement under low stringency hybridization conditions, or a fragment thereof having promoter activity, e.g., the activity being a promoter having the sequence of seq id NO: 11 or 12 sequence, preferably at least 10%, preferably at least 50%.

In one embodiment, the product of a starch hydrolyzing gene, such as an alpha-amylase, glucoamylase or alpha-amylase/glucoamylase fusion, may be targeted to a specific organelle or site, such as the endoplasmic reticulum or apoplast, rather than the cytoplasm. Examples are the use of the maize gamma-zein N-terminal signal sequence (SEQ ID NO: 17) which confers protein specificity targeting to the apoplast, and the use of the gamma-zein N-terminal signal sequence (SEQ ID NO: 17) which is operably linked to a processing enzyme which is operably linked to the SEKDEL sequence for retention in the endoplasmic reticulum. Directing a protein or enzyme to a specific region can position the enzyme in such a way that the enzyme does not come into contact with the substrate. In this way, enzymatic action of the enzyme does not occur until the enzyme contacts the substrate. The enzyme may be contacted with its substrate by grinding (physical disruption of cell integrity) and hydration processes. For example, mesophilic starch hydrolyzing enzymes may be directed to the apoplast or endoplasmic reticulum so as not to contact the starch granules in the amyloplast. Grinding the grain disrupts the integrity of the grain and the starch hydrolyzing enzyme then contacts the starch granules. In this way, potential side effects of co-localization of the enzyme and its substrate can be avoided.

h. Food product without added sweetener

Also provided is a method of producing a sweetened farinaceous food product without the addition of a sweetener. Examples of farinaceous products include, but are not limited to, breakfast foods, ready-to-eat foods, baked goods, pasta, and cereal products such as breakfast cereals. The method comprises treating a plant part containing at least one starch processing enzyme under conditions that activate the starch processing enzyme to process starch granules in the plant part to sugars to form a sweet product, relative to, for example, a product made from starch granules from a plant part that does not contain the hyperthermophilic enzyme. Preferably, the starch processing enzyme is resistant to ultra high temperatures and is activated by heating, such as baking, boiling, heating, steaming, electrical discharge or combinations thereof. The plant is obtained from a transformed plant, such as transformed soybean, rye, oat, barley, wheat, maize, rice or sugarcane, which has increased in its genome an expression cassette encoding said at least one hyperthermophilic starch processing enzyme, such as an alpha-amylase, an alpha-glucosidase, a glucoamylase, a pullulanase, a glucose isomerase or any combination thereof. The sweet product is then processed into a farinaceous food product. The invention also provides a farinaceous food product prepared by the method, such as a cereal, a breakfast food, a ready-to-eat food, a baked food. The farinaceous food product can be made from a sweet product and water, and can contain malt, flavoring agents, vitamins, minerals, colorants, or any combination thereof.

The enzyme may be activated prior to introducing the plant material into the cereal product or during processing of the cereal product to convert polysaccharides in the plant material into sugars (sugar). Accordingly, the polysaccharides contained within the plant material may be converted into sugars by activating the material prior to addition to the farinaceous product, for example by heating for ultra-high temperature resistant enzymes. Plant material containing sugars converted from polysaccharides is then added to the product to produce a sweet product. Alternatively, the polysaccharide may be converted to sugar by an enzyme during processing of the farinaceous product. Examples of methods for preparing cereal products are well known in the art and include heating, baking, boiling, etc., as described in US 6,183,788; 6,159,530, respectively; 6,149,965, respectively; 4,988,521 and 5,368,870.

Briefly, a dough may be prepared by mixing various dry ingredients with water, and cooking to gelatinize the starch component and form a cooked flavor. The cooked material can then be mechanically processed to form a cooked dough, such as a cereal dough. The dry ingredients may include various additives such as sugars, starches, salts, vitamins, minerals, pigments, flavors, salts, and the like. In addition to water, various liquid ingredients such as corn (maize) or malt syrup may also be added. The farinaceous material may include cereal grains, cut grains (cut grains), grits (grits) or flours from wheat, rice, maize, oats, barley, rye or other grains and mixtures thereof, derived from the transgenic plants of the invention. The dough may then be processed into a desired shape by methods such as extrusion or stamping, and further cooked using devices such as James cookers, ovens, or electric discharge equipment.

Methods of sweetening starch-containing products without the addition of sweeteners are also provided. The method comprises treating a starch comprising at least one starch processing enzyme under conditions effective to activate the at least one enzyme, thereby digesting the starch to form a sugar to form a treated (sweetened) starch, e.g., the sweetness relative to a product made by treating the starch without the hyperthermophilic enzyme. The starch of the invention is obtained from a transformed plant, the genome of which has been augmented with an expression cassette encoding the at least one processing enzyme, preferably an enzyme comprising an alpha-amylase, an alpha-glucosidase, a glucoamylase, a pullulanase, a glucose isomerase or a combination thereof. Preferably the enzyme is resistant to ultra high temperatures and can be activated by heat. Preferred transformed plants include maize, soybean, rye, oats, wheat, barley, rice and sugarcane. The treated starch is then added to a product to form a sweetened starch-containing product, such as a farinaceous food product. Sweetened starch-containing products prepared by the method are also provided.

The present invention also provides a method for sweetening a fruit or vegetable containing polysaccharides, comprising: treating a fruit or vegetable containing at least one polysaccharide processing enzyme under conditions effective to activate the at least one enzyme, thereby processing the polysaccharide in the fruit or vegetable to form a sugar, resulting in a sweetened fruit or vegetable, e.g., relative to a vegetable or fruit from a plant not containing the polysaccharide processing enzyme. The fruit or vegetable of the invention is obtained from a transformed plant, the genome of which is increased by an expression cassette encoding said at least one polysaccharide processing enzyme.

Preferred fruits and vegetables include potatoes, tomatoes, bananas, pumpkins, peas and beans. Preferred enzymes include alpha-amylase, alpha-glucosidase, glucoamylase, pullulanase, glucose isomerase, or combinations thereof. Preferably the enzyme is resistant to ultra high temperatures.

i. Sweetening polysaccharide-containing plants or plant parts

The method involves obtaining a plant expressing a polysaccharide processing enzyme as described above, which enzyme converts a polysaccharide to a sugar (sugar). Accordingly, the enzyme is expressed in plants and plant products, such as fruits or vegetables. In one embodiment, the enzyme is placed under the control of an inducible promoter such that expression of the enzyme can be induced by an external stimulus. Such inducible promoters and constructs are well known in the art and are described herein. Expression of the enzyme in a plant or product thereof can convert polysaccharides contained in the plant or product thereof to sugars and sweeten the plant or product thereof. In another embodiment, the polysaccharide processing enzyme is constitutively expressed. Thus, the plant or product thereof can be activated under conditions sufficient to activate the enzyme, thereby converting the polysaccharide to a sugar by activating the enzyme, and sweetening the plant or plant product. Thus, the self-processing of the polysaccharide to sugar in the fruit or vegetable results in a sweetened fruit or vegetable, e.g., relative to a vegetable or fruit from a plant not containing the polysaccharide processing enzyme. The fruit or vegetable of the invention is obtained from a transformed plant, the genome of which is increased by an expression cassette encoding said at least one polysaccharide processing enzyme. Preferred fruits and vegetables include potatoes, tomatoes, bananas, pumpkins, peas and beans. Preferred enzymes include alpha-amylase, alpha-glucosidase, glucoamylase, pullulanase, glucose isomerase, or combinations thereof. Preferably the polysaccharide processing enzyme is resistant to ultra high temperatures.

i. Isolation of starch from transformed grain containing enzymes that disrupt endosperm matrix

The present invention provides a process for isolating starch from transformed grain wherein an enzyme capable of disrupting the endosperm matrix is expressed. The method involves obtaining plants expressing enzymes that disrupt the endosperm matrix by modifying, for example, the cell wall, non-starch polysaccharides and/or proteins. Examples of such enzymes include, but are not limited to: proteases, glucanases, thioredoxins, thioredoxin reductases and esterases. Such enzymes do not include any enzymes exhibiting starch degrading activity, such that the integrity of the starch granules is maintained. Preferably the enzyme is fused to a signal sequence capable of targeting the enzyme to the starch granule. In one embodiment the grain is heat dried to activate the processing enzymes and inactivate endogenous enzymes contained within the grain. The heat treatment results in activation of the enzyme, which acts to break down the endosperm matrix, and the starch granules can then be easily separated from the endosperm matrix. In another embodiment, the grain is steeped at low or high temperature with or without the use of sulphur dioxide at high or low moisture content. The grain is then heat treated to break the endosperm matrix and allow easy separation of starch granules. In another embodiment, suitable temperature and moisture conditions are created to allow the protease to enter the starch granules and degrade the proteins contained within the granules. Such treatment results in high yields of starch granules with little contaminating protein.

k. Syrup with high sugar equivalent and application of syrup in preparing ethanol or fermented beverage

The method involves obtaining a plant expressing a polysaccharide processing enzyme as described above, which enzyme can convert a polysaccharide to a sugar. The plant or its product is soaked in an aqueous stream (aqueous stream) under conditions such that the expressed enzyme converts the polysaccharides contained in the plant or its product into dextrins, maltooligosaccharides and/or sugars. The aqueous stream containing dextrins, malto-oligosaccharides and/or sugars formed by the conversion of polysaccharides is then separated to produce a syrup having a high sugar equivalent. The method may or may not include the additional step of wet milling the plant or its product to obtain starch granules. Examples of enzymes that may be used in the present process include, but are not limited to, alpha-amylase, glucoamylase, pullulanase, and alpha-glucosidase. Preferably the enzyme is resistant to ultra high temperatures. Sugars formed according to this process may include, but are not limited to, hexoses, glucose, and fructose. Examples of plants that can be used in the present method include, but are not limited to, maize, wheat or barley. Examples of plant products that can be used include, but are not limited to, fruits, grains, and vegetables. In another embodiment, the polysaccharide processing enzyme is under the control of an inducible promoter. Accordingly, the promoter is induced to express the enzyme before or during the soaking process, and then the enzyme will convert the polysaccharide into sugar. Examples of inducible promoters and constructs containing the same are well known in the art and are disclosed herein. Thus, if the polysaccharide processing enzyme is resistant to ultra-high temperatures, soaking may be performed at high temperatures to activate the ultra-high temperature resistant enzyme and inactivate endogenous enzymes within the plant or product thereof. In another embodiment, the hyperthermophilic enzyme capable of converting the polysaccharide into a sugar is constitutively expressed. The enzyme may not or may be targeted to a plant component by a signal sequence. The plant or its product is soaked under high temperature conditions to convert polysaccharides contained in the plant into sugars.

Also provided are methods of preparing an ethanol or fermented beverage from a syrup having a high dextrose equivalent. The method involves incubating the syrup with yeast under conditions that allow the sugars contained in the syrup to be converted into ethanol or fermented beverages. Examples of such fermented beverages include, but are not limited to, beer and wine. Fermentation conditions are well known in the art and are described in US 4,929,452 and herein. Preferred yeasts are high alcohol and sugar tolerant strains, such as Saccharomyces cerevisiae (S.cerevisiae) ATCC No. 20867. The fermented product or fermented beverage may be distilled to separate ethanol or distilled beverage.

l. Accumulation of hyperthermophilic enzymes in plant cell walls

The invention provides a method for accumulating ultra-high temperature resistant enzyme in plant cell walls. The method involves expressing in the plant a hyperthermophilic enzyme fused to a cell wall targeting signal such that the targeted enzyme accumulates at the cell wall. Preferably the enzyme is capable of converting polysaccharides to monosaccharides. Examples of targeting sequences include, but are not limited to, cellulose or xylose binding domains. Examples of hyperthermophilic enzymes include SEQ ID NO: 1, 3, 5, 10, 13, 14, 15 or 16. Plant material containing cell walls may be added to the process for recovering sugars from the feed as a source of the enzyme of interest, or the plant material may be added as a source of an enzyme that converts polysaccharides from other sources into monosaccharides. In addition, the cell wall can also be used as a source of purified enzyme. Methods for purifying enzymes are well known in the art and include, but are not limited to, gel filtration, ion exchange chromatography, chromatofocusing, isoelectric focusing, affinity chromatography, FPLC, HPLC, salt precipitation, dialysis, and the like. Accordingly, the present invention also provides a purified enzyme isolated from plant cell walls.

m. Process for preparing and isolating processing enzymes

According to the invention, the recombinantly produced processing enzyme of the invention may be prepared by: transforming plant tissue or plant cells containing a processing enzyme of the invention, which enzyme can be activated in plants, selecting transformed plant tissue or cells, culturing the transformed plant tissue or cells into transformed plants, and isolating the processing enzyme from the transformed plants or parts thereof. Preferably, the recombinantly produced enzymes are alpha-amylases, glucoamylases, glucose isomerases, alpha-glucosidases and pullulanases. Most preferably the enzyme is encoded by a polynucleotide selected from any of the following sequences: SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59.

The following examples further illustrate the invention but are not intended to limit the scope of the invention in any way.

Examples

Example 1

Construction of maize optimized hyperthermophilic starch processing enzyme/isomerase genes the various enzymes involved in starch degradation or glucose isomerization, i.e., alpha-amylase, pullulanase, alpha-glucosidase and glucose isomerase, were selected according to the desired activity profile. These activity profiles include, for example, minimal activity at ambient temperature, high temperature activity/stability, and activity at low pH. The corresponding gene was then designed using maize preferred codons as described in US patent 5,625,136 and synthesized by Integrated DNA Technologies, Inc.

The gene having the sequence of SEQ ID NO: 1 amino acid sequence 797GL3 alpha-amylase. The nucleic acid sequence of the enzyme was deduced and optimized in maize as shown in SEQ id no: 2, respectively. Similarly, the peptide having SEQ ID NO: 3 amino acid sequence 6gp3 pullulanase. The nucleic acid sequence of the enzyme was deduced and optimized in maize as shown in SEQ ID NO: 4, respectively.

According to document j.bact.177: 482-; J.Bact.180: 1287-1295(1998) obtained the malA. alpha. -glucosidase amino acid sequence of Sulfolobus solfataricus. Based on the disclosed amino acid sequence of this protein (SEQ ID NO: 5), a maize optimized synthetic gene (SEQ ID NO: 6) was designed that encodes malA α -glucosidase.

Several glucose isomerases were selected. The glucose isomerase amino acid sequence of Thermotoga maritima (Thermotoga maritima)) was predicted from the published DNA sequence with accession number NC-000853 (SEQ ID NO: 18) and designing the optimized corn synthetic gene (SEQ ID NO: 19). Similarly, according to appl.envir.microbiol.61 (5): 1867-1875(1995), the DNA sequence of accession number L38994 predicts the glucose isomerase amino acid sequence (SEQ ID NO: 20) of Thermotoga neracustricola (T.neapolitana), and designs a maize-optimized synthetic gene (SEQ ID NO: 21) encoding the glucose isomerase of Thermotoga nerusicola.

Example 2

797GL3 alpha-amylase and starch encapsulation region Expression of the fusions in E.coli

Constructs encoding fusions of the hyperthermophilic 797GL3 alpha-amylase and a starch-encapsulating region (SER) from corn grain-bound starch synthase (wax) were introduced into E.coli and expressed. The maize granule-bound starch synthase cDNA (SEQ ID NO: 7) (Klosgen RB et al, 1986) encoding the amino acid sequence (SEQ ID NO: 8) was cloned and used as the source of the starch-binding or starch-encapsulating region (SER). RT-PCR amplification of full-length cDNA was performed from RNA prepared from maize seeds using primers SV 57(5 'AGCGAATTCATGGCGGCTCTGGCCACGT 3') (SEQ ID NO: 22) and SV 58(5 'AGCTAAGCTTCAGGGCGCGGCCACGTTCT 3') (SEQ ID NO: 23) designed according to GenBank accession number X03935. The full-length cDNA was cloned into pBluescript using the EcoRI/HindIII fragment and the plasmid was named pNOV 4022.

The C-terminal portion of the waxy cDNA (encoded by 919-1818 bp) was amplified from pNOV4022, including the starch-binding domain, and fused in reading frame to the 3' end of the full-length maize optimized 797GL3 gene (SEQ ID NO: 2). The fusion gene product, 797GL3/wax, has the sequence of SEQ ID NO: 9 and encodes the nucleic acid sequence of SEQ ID NO: 10, cloned as an NcoI/XbaI fragment into pET28b (NOVAGEN, Madison, Wis.) digested with NcoI/NheI. In addition, the 797GL3 gene was cloned individually into pET28b vector as NcoI/XbaI fragment.

pET28/797GL3 and pET28/797GL3/Waxy vectors were transformed into BL21/DE3 E.coli cells (NOVAGEN) and cultured and induced according to the manufacturer's instructions. PAGE/coomassie staining analysis showed induced proteins in both extracts, corresponding to the predicted sizes of the fused and non-fused amylases, respectively.

Hyperthermophilic amylase activity in total cell extracts was analyzed as follows: 5mg of starch was suspended in 20. mu.l of water and then diluted with 25. mu.l of ethanol. Samples of the amylase activity to be tested or standard amylase positive controls were added to the mixture and water was added to a final reaction volume of 500. mu.l. The reaction is carried out at 80 ℃ for 15-45 minutes. The reaction was then cooled to room temperature and 500. mu.l o-dianisidine and glucose oxidase/peroxidase mixture (Sigma) were added. The mixture was incubated at 37 ℃ for 30 minutes. The reaction was terminated by adding 500. mu.l of 12N sulfuric acid. The absorbance at 540nm was measured to quantify the amount of glucose released by the amylase/sample. The experimental results of the fused and unfused amylase extracts show that: the level of hyperthermophilic amylase activity was similar, whereas the control extract was negative. This indicates that 797GL3 amylase is still active when fused to the C-terminal portion of the waxy protein (at high temperatures).

Example 3

Isolation of promoter fragments for embryo milk specific expression in maize

The promoter and 5' non-coding region I (including the first intron) of the maize (Zea mays) ADP-gpp (ADP-glucose pyrophosphorylase) large subunit of the 1515 base pair fragment (SEQ ID NO: 11) were amplified from maize genomic DNA using primers designed according to GenBank accession number M81603. The ADP-gpp promoter has been shown to be endosperm specific (Shaw and Hannah, 1992).

The maize (Zea mays) gamma-zein gene promoter of the 673bp fragment was amplified from plasmid pgz27.3 (obtained from dr. brian Larkins). The gamma-zein gene promoter has been shown to be endosperm specific (Torrent et al, 1997).

Example 4

797GL3 construction of superhigh temperature resistant alpha-amylase transformation vector

Expression cassettes with various targeting signals were constructed to express 797GL3 hyperthermophilic alpha-amylase in maize embryo milk as follows:

pNOV6200(SEQ ID NO: 13) contains a fusion of the maize γ -zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) for targeting and secretion into the apoplast of the endoplasmic reticulum and 797GL3 amylase synthesized as described in example 1 (Torrent et al, 1997). The fusion product is cloned into corn ADP-gpp promoter for specific expression in endosperm.

pNOV6201(sEQ ID NO: 14) contains a fusion of the gamma-zein N-terminal signal sequence with synthetic 797GL3 amylase with the C-terminal additional sequence SEKDEL for targeting and retention in the Endoplasmic Reticulum (ER) (Munro and Pelham, 1987). The fusion product is cloned into corn ADP-gpp promoter for specific expression in endosperm.

pNOV7013 contains a fusion of the N-terminal signal sequence of gamma-zein with a synthetic 797GL3 amylase with the C-terminal additional sequence SEKDEL for targeting and retention into the Endoplasmic Reticulum (ER). pNOV7013 is identical to pNOV6021, except that the maize gamma-zein promoter (SEQ ID NO: 12) in pNOV 7103 is used in place of the maize ADP-gpp promoter to express the fusion product in the endosperm.

pNOV4029(SEQ ID NO: 15) contains a fusion of the wax amyloplast targeting peptide (Klosgen et al, 1986) with synthetic 797GL3 amylase for targeting to amyloplasts. The fusion product is cloned into corn ADP-gpp promoter for specific expression in endosperm.

pNOV4031(SEQ ID NO: 16) contains a fusion of the wax amyloplast targeting peptide with synthetic 797GL3/wax fusion protein for targeting to starch granules. The fusion product is cloned into corn ADP-gpp promoter for specific expression in endosperm.

Additional constructs were made to allow higher levels of enzyme expression by cloning these fusions into the maize gamma-zein promoter followed by preparation of additional constructs. All expression cassettes were transferred into binary vectors for transformation of maize by Agrobacterium infection. Binary vectors containing the phosphomannose isomerase (PMI) gene may allow for the selection of transgenic cells with mannose. Transformed maize plants were either self-pollinated or outcrossed and seeds were collected for analysis.

Other constructs were made by fusing the above-described targeting signal sequence to either 6gp3 pullulanase or 340g12 α -glucosidase in exactly the same manner as for the above-described α -amylase. These fusion products were cloned behind the maize ADP-gpp promoter and/or gamma-zein promoter and transformed into maize as described above. Transformed maize plants were either self-pollinated or outcrossed and seeds were collected for analysis.

Combinations of enzymes can be obtained by crossing plants expressing a single enzyme or by co-transformation by cloning several expression cassettes into the same binary vector.

Example 5

Construction of 6GP3 high temperature resistant pullulanase plant transformation vector

The expression cassette was constructed as follows to express 6GP3 thermostable pullulanase in the endoplasmic reticulum of maize endosperm.

pNOV7005(SEQ ID NOS: 24 and 25) contains a fusion of the N-terminal signal sequence of maize gamma-zein with a synthetic 6GP3 pullulanase with the C-terminal additional sequence SEKDEL, targeted to and retained in the ER. The amino acid peptide SEKDEL was fused to the C-terminus of the enzyme by PCR using primers designed to allow amplification of the synthetic gene and simultaneous addition of 6 amino acids at the C-terminus of the protein. The fusion product was cloned behind the maize gamma-zein promoter for specific expression in the endosperm.

Example 6

Construction of malA hyperthermophilic alpha-glucosidase-resistant plant transformation vector

Expression cassettes with various targeting signals were constructed as follows to express sulfolobus solfataricus malA hyperthermophilic alpha-glucosidase in maize embryo milk.

pNOV4831(SEQ ID NO: 26) contains a fusion of the maize gamma-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) with a synthetic malA alpha-glucosidase with the C-terminal additional sequence SEKDEL for targeting and retention into the endoplasmic reticulum (Munro and Pelham, 1987). The fusion product was cloned behind the maize gamma-zein promoter for specific expression in the endosperm.

pNOV4839(SEQ ID NO: 27) contains a fusion of the maize gamma-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) with synthetic malA alpha-glucosidase, allowing targeting to the endoplasmic reticulum and secretion into the apoplast (Torrent et al, 1997). The fusion product was cloned behind the maize gamma-zein promoter for specific expression in the endosperm.

pNOV4837 contains a fusion of the maize gamma-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) with a synthetic malA alpha-glucosidase with the C-terminal additional sequence SEKDEL, allowing targeting and retention into the ER. This fusion product was cloned behind the maize ADP-gpp promoter for specific expression in endosperm. The amino acid sequence of this clone was identical to that of pNOV4831(SEQ ID NO: 26).

Example 7

Construction of a plant transformation vector for hyperthermophilic Thermotoga maritima and Thermotoga neapolitana glucose isomerase

Expression cassettes with various targeting signals were constructed as follows to express hyperthermophilic glucose isomerase of Thermotoga maritima and Thermotoga neapolitana in maize embryo milk.

pNOV4832(SEQ ID NO: 28) contains the maize gamma-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to the synthetic Thermotoga maritima glucose isomerase with the C-terminal additional sequence SEKDEL to allow targeting and retention into the ER. The fusion product was cloned behind the maize gamma-zein promoter for specific expression in the endosperm.

pNOV4833(SEQ ID NO: 29) will contain the maize gamma-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to the synthetic Thermotoga nardus glucose isomerase with the C-terminal additional sequence SEKDEL to allow targeting and retention into the ER. The fusion product was cloned behind the maize gamma-zein promoter for specific expression in the endosperm.

pNOV4840(SEQ ID NO: 30) contains the maize gamma-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to synthetic Thermotoga neapolitana glucose isomerase to target the ER and secrete into the apoplast (Torrent et al, 1997). The fusion product was cloned behind the maize gamma-zein promoter for specific expression in the endosperm.

pNOV4838 contains the gamma-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to the synthetic Thermotoga neapolitana glucose isomerase with the C-terminal additional sequence SEKDEL to allow targeting and retention into the ER. This fusion product was cloned behind the maize ADP-gpp promoter for specific expression in endosperm. The amino acid sequence of this clone was identical to that of pNOV4833(SEQ ID NO: 29).

Example 8

Construction of plant transformation vector for expressing hyperthermophilic dextranase EglA

pNOV4800(SEQ ID NO: 58) contains a fusion of the barley alpha-amylase AMY32b signal sequence (MGKNGNLCCFSLLLLLLAGLASGHQ) (SEQ ID NO: 31) with the mature protein sequence of EglA for localization to apoplasts. The fusion product was cloned behind the maize gamma-zein promoter for specific expression in the endosperm.

Example 9

Construction of plant transformation vector for expressing multiple hyperthermophilic enzymes

pNOV4841 contains a two-gene construct, 797GL3 alpha-amylase fusion and 6GP3 pullulanase fusion. 797GL3 fusion (SEQ ID NO: 33) and 6GP3 fusion (SEQ ID NO: 34) both possess the maize gamma-zein N-terminal signal sequence and the SEKDEL sequence for targeting and retention in the ER. Each fusion was cloned behind a separate gamma-zein promoter for specific expression in endosperm.

pNOV4842 contains a two gene construct, 797GL3 alpha-amylase fusion and malA alpha-glucosidase fusion. 797GL3 fusion polypeptide (SEQ ID NO: 35) and malA α -glucosidase fusion polypeptide (SEQ ID NO: 36) both have a maize γ -zein N-terminal signal sequence and a SEKDEL sequence for targeting to and retention in the ER. Each fusion polypeptide was cloned behind a separate gamma-zein promoter for specific expression in the endosperm.

pNOV4843 contains a two-gene construct, 797GL3 alpha-amylase fusion and malA alpha-glucosidase fusion. 797GL3 and malA α -glucosidase fusion polypeptides both have a maize γ -zein N-terminal signal sequence and a SEKDEL sequence for targeting to and retention in the ER. 797GL3 fusion polypeptide was cloned behind the gamma-zein promoter, while the malA fusion polypeptide was cloned behind the maize ADP-gpp promoter for specific expression in the endosperm. 797GL3 and malA fusions have the same amino acid sequences as those of pNOV4842 (SEQ ID NOS: 35 and 36, respectively).

pNOV4844 contains the three gene construct 797GL3 alpha-amylase fusion, 6GP3 pullulanase fusion and malA alpha-glucosidase fusion. 797GL3, malA and 6GP3 all have a maize gamma-zein N-terminal signal sequence and a SEKDEL sequence for targeting and retention in the ER. 797GL3 and malA fusions were cloned behind two separate maize gamma-zein promoters, while 6GP3 fusions were cloned behind a maize ADP-gpp promoter for specific expression in endosperm. 797GL3 and the amino acid sequence of the malA fusion were identical to that of pNOV4842 (SEQ ID NOS: 35 and 36, respectively). The amino acid sequence of the 6GP3 fusion was identical to that of the 6GP3 fusion in pNOV4841 (SEQ ID NO: 34).

All expression cassettes were transferred into the binary vector pNOV2117 for transformation of maize by agrobacterium infection. pNOV2117 contains the phosphomannose isomerase (PMI) gene, allowing the use of mannose for screening of transgenic cells. pNOV2117 is a binary vector with the origins of replication of pVS1 and ColE 1. The vector contained the constitutive VirG gene from pAD1289 (Hansen, G., et al, PNAS USA 91: 7603-7607(1994)) and the actinospectinomycin resistance gene from Tn 7. Cloned into the polylinker between the left and right borders is the maize ubiquitin promoter, the PMI coding sequence and the nopaline synthase terminator of pNOV117 (Negrotto, D et al, Plant Cell Reports 19: 798-. Transformed maize plants can be self-pollinated or outcrossed and seeds collected for analysis. Combinations of different enzymes can be obtained by crossing plants expressing a single enzyme or by transforming plants with a multiple gene expression cassette.

Example 10

Construction of bacterial and Pichia expression vectors

The expression cassette was constructed for expression of hyperthermophilic alpha-glucosidase and glucose isomerase in pichia or bacteria as follows:

pNOV4829(SEQ ID NOS: 37 and 38) contains a fusion of the synthetic Thermotoga maritima glucose isomerase with the ER retention signal in the bacterial expression vector pET29 a. The glucose isomerase fusion gene was cloned into the NcoI and SacI sites of pET29a, thus adding the N-terminal S-tag required for protein purification.

pNOV4830(SEQ ID NOS: 39 and 40) contained a fusion of the synthetic Thermotoga nardus glucose isomerase with the ER retention signal in the bacterial expression vector pET29 a. The glucose isomerase fusion gene was cloned into the NcoI and SacI sites of pET29a, thus adding the N-terminal S-tag required for protein purification.

pNOV4835(SEQ ID NOS: 41 and 42) contained a synthetic Thermotoga maritima glucose isomerase gene cloned into the BamHI and EcoRI sites of the bacterial expression vector pET 28C. This fused the His tag (required for protein purification) to the N-terminus of glucose isomerase.

pNOV4836(SEQ ID NOS: 43 and 44) contained a synthetic Thermotoga neapolitana glucose isomerase gene that was cloned into the BamHI and EcoRI sites of the bacterial expression vector pET 28C. This fused the His tag (required for protein purification) to the N-terminus of glucose isomerase.

Example 11

Essentially as in Negrotto et al, Plant Cell Reports 19: 798-803 the transformation of immature maize embryos is carried out. For this example, all media components are as described in Negrotto et al, supra, however, each of the media components described in this document may be substituted.

A. Transformation plasmids and selectable markers

The genes used for transformation were cloned into vectors suitable for transforming maize. The vector used in this example contains the phosphomannose isomerase (PMI) gene to screen for transgenic lines (Negrotto et al, (2000) Plant Cell Reports 19: 798-803).

B. Preparation of Agrobacterium tumefaciens

Agrobacterium strain LBA4404(pSB1) containing the plant transformation plasmid was cultured on YEP (yeast extract (5g/L), peptone (10g/L), NaCl (5g/L), 15g/L agar, pH6.8) solid medium at 28 ℃ for 2-4 days. Then about 0.8X 10⁹Agrobacterium is suspended in LS-inf medium supplemented with 100. mu.M As (Negrotto et al, (2000) Plant Cell Rep 19: 798-803). The bacteria were pre-induced in this medium for 30-60 minutes.

C. Inoculation of

Immature embryos cut from 8-12 day old ears of corn from A188 or other appropriate genotype are placed in liquid LS-inf + 100. mu.M As. Embryos were rinsed once with fresh infection medium. The agrobacterium solution was then added, the embryos shaken on a vortex machine for 30 seconds, and allowed to stand with the bacteria for 5 minutes. The embryo-retaining scutellum was then transferred in an upward orientation into LSAs medium and cultured in the dark for 2-3 days. Then, 20-25 embryos from each dish were transferred to LSDc medium supplemented with cefotaxime (250mg/l) and silver nitrate (1.6mg/l) and cultured in the dark at 28 ℃ for 10 days.

D. Selection of transformed cells and regeneration of transformed plants

Immature embryos forming embryogenic callus were transferred to lsd1m0.5s medium. Cultures were screened on this medium for 6 weeks and a subculture step was performed at week 3. Surviving calli were transferred to Reg1 medium supplemented with mannose. After incubation under light (16 hours light/8 hours dark), the green tissue was transferred to Reg2 medium without growth regulators and incubated for 1-2 weeks. The plantlets (plantalets) were transferred to a Magenta GA-7 box (Magenta Corp, Chicago Ill) containing Reg3 medium and grown under light. After 2-3 weeks, the plants were PCR tested for the presence of the PMI gene and other genes of interest. Positive plants tested by PCR were transferred to the greenhouse.

Example 12

Analysis of expression targeting apoplast or ERT1 seed of corn plants with alpha-amylase

T1 seed from self-pollinated maize plants transformed with pNOV6200 or pNOV6201 as described in example 4 was obtained. The accumulation of starch within these corn kernels appeared to be normal upon visual inspection and normal staining of the starch with iodine solution prior to exposure to elevated temperatures. Dissecting the immature corn kernels, placing the purified endosperm separately in a microcentrifuge tube and soaking in 200. mu.l 50mM NaPO ₄In a buffer solution. The tube was placed in a water bath at 85 ℃ for 20 minutes and then cooled on ice. Add 20 μ l of 1% iodine solution to each tube and mix. Approximately 25% of the isolated kernels showed normal starch staining. The remaining 75% failed to stain, indicating that the starch had been degraded to low molecular weight sugars that could not be stained with iodine. The T1 corn kernels of pNOV6200 or pNOV6201 were found to be capable of autohydrolyzing corn starch. No starch reduction was detected after incubation at 37 ℃.

The expression of amylase was further analyzed by separating the hyperthermophilic protein fraction from the endosperm followed by PAGE/coomassie staining. An isolated protein band of appropriate molecular weight (50kD) was observed. These samples were tested for alpha-amylase testing using commercially available colored amylose (AmYLAZYME, from Megazyme, Ireland). High levels of hyperthermophilic amylase activity are associated with the presence of this 50kD protein.

It was further found that corn kernel starch from most transgenic corn expressing hyperthermophilic alpha-amylase (the enzyme is directed to the amyloplasts) is sufficiently active at ambient temperature to hydrolyze most of the starch if the enzyme is allowed to come into direct contact with the starch granules. Of the 80 lines with hyperthermophilic alpha-amylase (the enzyme directed to the amyloplasts), 4 were identified as capable of accumulating starch within the corn kernel. 3 of these lines were analyzed for thermostable alpha-amylase activity using the colorimetric amylazyme assay. The amylase test showed a very low level of thermostable amylase activity in these 3 lines. When the starch purified from these 3 lines was treated at the appropriate moisture content (moisture) and heating conditions, the starch was hydrolyzed, indicating that sufficient levels of alpha-amylase were present to promote autohydrolysis of the starch prepared from these lines.

T1 seeds from multiple independent lines of pNOV6200 or pNOV6201 transformants were obtained. Individual kernels of each line were dissected at 300. mu.l 50mM NaPO₄Each purified endosperm was homogenized separately in buffer. Samples of endosperm suspension were analyzed for alpha-amylase activity at 85 ℃. About 80% of the lines underwent segregation for hyperthermal activity (see FIGS. 1A, 1B and 2).

Corn kernels from wild-type plants or pNOV 6201-transformed plants were heated at 100 ℃ for 1, 2, 3, or 6 hours and then starch stained with iodine solution. After 3 or 6 hours, little or no starch was detected in the mature corn kernels, respectively. Thus, when incubated at high temperature, starch within the mature corn kernels of transgenic corn expressing hyperthermophilic amylase, which is directed to the endoplasmic reticulum, is hydrolyzed.

In another experiment, mature T1 kernels from the pNOV6201 plant were steeped at 50 ℃ for 16 hours and partially purified starch from the kernels was hydrolyzed after heating at 85 ℃ for 5 minutes. This demonstrates that after milling of the corn kernels, an alpha-amylase targeted to the endoplasmic reticulum binds to the starch and is able to hydrolyze the starch upon heating. Iodine staining indicated that the starch remained intact in the mature seeds after 16 hours of soaking at 50 ℃.

In another experiment, mature corn kernels isolated from pNOV6201 transformed plants were heated at 95 ℃ for 16 hours and then dried. In seeds expressing hyperthermophilic alpha-amylase, hydrolysis of starch to sugars results in a wrinkled appearance after drying.

Example 13

Analysis of T1 seeds from maize plants expressing amyloplast-directed alpha-amylase

T1 seeds of an autologous pollinated maize plant transformed with pNOV4029 or pNOV4031 as described in example 4 were obtained. Starch accumulation in the corn kernels of these lines was clearly abnormal. All lines isolated either very low or no starch phenotype, although varying in severity. Iodine only weakly stains endosperm purified from immature kernels before exposure to high temperatures. After 20 minutes at 85 ℃ no staining occurred. When the ear is dried, the kernels shrink. Such amylases are apparently active enough to hydrolyze starch at greenhouse temperatures if allowed to come into direct contact with starch granules.

Example 14

Grain of corn plants fermented to express alpha-amylase

100% transgenic grain, 85 ℃ vs 95 ℃ with different liquefaction times

Transgenic corn containing thermostable alpha-amylase (pNOV6201) performs well in fermentations without the addition of exogenous alpha-amylase, with a greatly reduced time required for liquefaction and more complete solubilization of starch. The laboratory fermentation was carried out using the following procedure (detailed below): 1) milling, 2) moisture content (moisture) analysis, 3) preparation of a slurry containing milled corn, water, backsset and alpha-amylase, 4) liquefaction and 5) Simultaneous Saccharification and Fermentation (SSF). In this example, the temperature and time of the liquefaction step were varied as described below. In addition, the transgenic corn was liquefied with or without exogenous alpha-amylase and ethanol production performance was compared to control corn treated with commercially available alpha-amylase.

The transgenic maize used in this example was prepared according to the method described in example 4 using a vector containing the α -amylase gene and a PMI selectable marker, pNOV 6201. Pollen from a transgenic line expressing high levels of thermostable alpha-amylase was pollinated to a commercial hybrid (N3030BT) to obtain transgenic maize. The corn was dried to 11% moisture and stored at room temperature. The transgenic corn meal has an alpha-amylase content of 95 units/g, while 1 unit of enzyme can produce 1 micromole reducing ends per minute from corn flour in pH6.0 MES buffer at 85 ℃. The control corn used was yellow dent corn known to produce good ethanol.

1) Grinding: transgenic corn (1180g) was ground in a Perten 3100 hammer mill equipped with a 2.0mm screen, thus producing transgenic corn meal. After thorough washing, control corn was ground in the same mill to avoid contamination by transgenic corn.

2) And (3) analyzing the water content: transgenic and control maize samples (20g) were weighed on an aluminum scale (weighboat) and heated at 100 ℃ for 4 hours. Reweighed and the water content calculated from the weight loss. The moisture content of the transgenic flour was 9.26%, and the control flour was 12.54%.

3) Preparing slurry: the slurry composition was designed to form a slurry with 36% solids at the start of SSF. A control sample was prepared in a 100ml plastic bottle and contained 21.50g of control corn flour, 23ml of deionized water, 6.0ml of backsset (8% solids by weight), and 0.30ml of commercially available alpha-amylase diluted 50-fold with water. The alpha-amylase dosage is selected to represent industrial applications. When the experiment was performed under the conditions of the transgenic alpha-amylase test described above, the dose of control alpha-amylase was 2U/g corn meal. Ammonium hydroxide was added to adjust the pH to 6.0. Transgenic samples were prepared in the same manner, but with 20g of corn flour because of the lower moisture content of the transgenic corn flour. Transgenic flour slurries were prepared with the same dosage of alpha-amylase as the control samples or without exogenous alpha-amylase.

4) Liquefaction: bottles containing the transgenic corn flour slurry were soaked in a water bath at 85 ℃ or 95 ℃ for 5, 15, 30, 45 or 60 minutes. The control slurry was incubated at 85 ℃ for 60 minutes. During the high temperature incubation, the slurry was mixed vigorously by hand every 5 minutes. The slurry was cooled on ice after the high temperature step.

5) Simultaneous saccharification and fermentation: the resulting slurry was then liquefied and mixed with glucoamylase (0.65ml1/50 diluted commercially available L-400 glucoamylase), protease (0.60ml 1000 fold diluted commercially available protease), 0.2mg Lactocide &urea (0.85ml of 50% urealyquor diluted 10-fold) was mixed. A hole was made in the cap of a 100ml bottle containing the syrup as CO₂A discharge passage. The slurry was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; the time was set to 82F at 48 hours.

The yeast used for inoculation was propagated by preparing a mixture containing yeast (0.12g) and 70 grams maltodextrin, 230ml water, 100mlbackset, glucoamylase (0.88ml 10 fold diluted commercially available glucoamylase), protease (1.76ml 100 fold diluted commercially available enzyme), urea (1.07 grams), penicillin (0.67mg) and zinc sulfate (0.13 g). The propagation culture was prepared and incubated with mixing at 90 ° F starting the day before the propagation culture was needed.

At 24, 48 and 72 hours, samples were removed from each fermentor, filtered through a 0.2 μm filter and analyzed by HPLC for ethanol and sugars. The samples were analyzed for total dissolved solids and residual starch over 72 hours.

HPCL analysisIn a binary gradient system equipped with a refractive index detector, column heater and Bio-Rad AminexHPX-87H column. With 0.005M H₂SO₄The system was equilibrated at a rate of 1 ml/min. The column temperature was 50 ℃. The sample loading volume is 5 mul; the same solvent was used for elution. RI responses were calibrated by injection of known standards. Ethanol and glucose were measured in the injected samples.

Residual starch was determined as follows. The sample and standards were dried in an oven at 50 ℃ and then ground to a powder in a sample grinder. The powder (0.2g) was weighed into a 15ml graduated centrifuge tube. The powder was washed 3 times with 10ml aqueous ethanol (80% v/v) by centrifugation after vortexing and removal of the supernatant. DMSO (2.0ml) was added to the pellet, followed by 3.0ml of thermostable a-amylase (300 units) in MOPS buffer. After vigorous mixing, the tube was incubated in a water bath at 85 ℃ for 60 minutes. During incubation, tubes were mixed 4 times. The sample was cooled and 4.0ml of sodium acetate buffer (200mM, pH4.5) was added, followed by 0.1ml of glucoamylase (20U). The samples were incubated at 50 ℃ for 2 hours, mixed and then centrifuged at 3500rpm for 5 minutes. The supernatant was filtered through a 0.2 μ M filter and analyzed for glucose by HPLC as described above. The 50 μ l injection was used as the loading of samples with low residual starch (< 20% solids).

ResultsTransgenosisCorn performs well in fermentations without the addition of alpha-amylase. The ethanol yield at 72 hours was essentially the same with or without the addition of exogenous alpha-amylase, as shown in table 1. These data also indicate that greater ethanol yields are obtained when the liquefaction temperature is higher; the enzymes of the invention expressed in transgenic maize are active at higher temperatures than other commercially used enzymes such as Bacillus liquefaciens (Bacillus liquefaciens) alpha-amylase.

TABLE 1

Liquefaction temperature C	Liquefaction time min	Exogenous alpha-amylase	Number of repetitions	Average ethanol% v/v	% standard deviation v/v
						85	60	Is provided with	4	17.53	0.18
85	60	Is free of	4	17.78	0.27
						95	60	Is provided with	2	18.22	ND
95	60	Is free of	2	18.25	ND

When the liquefaction time was varied, it was found that the liquefaction time required for efficient production of ethanol was much shorter than that required by the conventional process. Figure 3 shows that liquefaction from 15 minutes to 60 minutes hardly changed the ethanol yield of the 72 hour fermentation. In addition, liquefaction at 95 ℃ resulted in a greater amount of ethanol at each time point than liquefaction at 85 ℃. These results indicate that the use of hyperthermophilic enzymes improves the process.

The control maize showed a higher final ethanol yield than the transgenic maize, but the control was chosen because of its superior fermentation performance. In contrast, transgenic maize has a genetic background selected to facilitate transformation. Introduction of this alpha-amylase trait into elite (elite) maize germplasm by well-known breeding techniques should eliminate this difference.

Examination of residual starch levels in 72 hour produced beer (fig. 4) showed that transgenic alpha-amylase significantly increased the amount of starch available for fermentation; the amount of residual starch after fermentation is much reduced.

The optimum liquefaction times were 95 ℃ for 15 minutes and 85 ℃ for 30 minutes using ethanol levels and residual starch levels. These times are the total time of the fermenter water bath in this experiment, and thus include the time taken for the sample to rise from room temperature to 85 ℃ or 95 ℃. Shorter liquefaction times may be optimal for large-scale industrial processes where the slurry may be rapidly heated by using steam pressure cookers and the like. Conventional industrial liquefaction processes require a holding tank to incubate the slurry at elevated temperatures for 1 or more hours. The present invention avoids the need for such a holding tank and increases the capacity of the liquefaction plant.

An important function of alpha-amylase in fermentation is to reduce the viscosity of the pulp. At each time point, the samples containing the transgenic corn meal were significantly less viscous than the control samples. Additionally, the transgenic samples did not appear to undergo the gel phase that would occur in all control samples; gelatinization typically occurs when corn steep liquor is heated and processed. Thus, having the alpha-amylase distributed throughout the endosperm fragment will impart beneficial physical properties to the slurry during the heat processing by preventing the formation of large gels that slow diffusion and increase the energy input required to mix and pump the slurry.

High doses of alpha-amylase in transgenic corn can also increase the beneficial properties of the transgenic pulp. At 85 ℃, the alpha-amylase activity of the transgenic corn is many times higher than the activity of the dosage of exogenous alpha-amylase used in the control. The latter is chosen as representative of the commercial grades.

Example 15

Efficient action of transgenic maize when mixed with control maize

The transgenic corn flour was blended with the control corn flour at various levels ranging from 5% to 100% transgenic corn flour. The process was as described in example 14. Liquefying the pulp containing the transgenically expressed alpha-amylase at 85 ℃ for 30 minutes or at 95 ℃ for 15 minutes; control slurries were prepared as described in example 14 and were liquefied at 85 ℃ for 30 minutes or 60 minutes (one each) or 95 ℃ for 15 or 60 minutes (one each).

The ethanol yields at 48 and 72 hours and data for residual starch are shown in table 2. Ethanol levels at 48 hours are shown in figure 5; the measured values for residual starch are shown in figure 6. These data show that when the transgenic grain represents only a small fraction (as low as 5%) of the total grain in the slurry, the transgenically expressed thermostable a-amylase still performs very well in ethanol production. The data also show that when at least 40% transgenic grains were contained in the total grain, the residual starch was significantly lower than the control slurry.

TABLE 2

	Liquefaction at 85 DEG C			Liquefaction at 95 DEG C
							Transgenic grain wt%	Residual starch	Ethanol for 48h	Ethanol% v/v72h	Residual starch	Ethanol for 48h	Ethanol% v/v72h
100	3.58	16.71	18.32	4.19	17.72	21.14
							80	4.06	17.04	19.2	3.15	17.42	19.45
60	3.86	17.16	19.67	4.81	17.58	19.57
							40	5.14	17.28	19.83	8.69	17.56	19.51
20	8.77	17.11	19.5	11.05	17.71	19.36
							10	10.03	18.05	19.76	10.8	17.83	19.28
50	10.67	18.08	19.41	12.44	17.61	19.38
							0*	7.79	17.64	20.11	11.23	17.88	19.87

^*Control samples. All were mean values of 2 determinations.

Example 16

Ethanol production with liquefaction pH using 1.5-12% transgenic corn in total corn Change of

Since transgenic maize performs well in fermentations at a rate of 5-10% of the total maize, an additional series of fermentations is followed in which the total maize contains 1.5-12% of the transgenic maize. The pH varies from 6.4 to 5.2, and the optimum pH for activity of the alpha-amylase expressed in transgenic maize is lower than that conventionally used in the industry.

The experiment was carried out as described in example 15, with the following exceptions:

1) the transgenic corn flour is blended with a control corn flour at a level of 1.5% to 12.0% of total dry weight.

2) The control maize was N3030BT, which is closer to the transgenic maize than the controls used in examples 14 and 15.

3) Exogenous alpha-amylase was not added to the samples containing the transgenic powder.

4) The sample pH was adjusted to 5.2, 5.6, 6.0 or 6.4 prior to liquefaction. At least 5 samples were prepared at each pH, covering the range of 0% transgenic corn meal to 12% transgenic corn meal.

5) All samples were liquefied at 85 ℃ for 60 minutes.

The change in ethanol content as a function of fermentation time is shown in FIG. 7. The figure shows data obtained from samples containing 3% transgenic corn. Fermentation at lower pH proceeds faster than at pH6.0 or higher. Similar behavior was observed in samples with other doses of transgenic maize. The pH profile of the activity of the transgenic enzyme combined with high levels of expression may allow liquefaction at lower pH, resulting in faster fermentation, which results in higher productivity than the conventional fermentation process at pH 6.0.

The 72 hour ethanol yield is shown in FIG. 8. It can be seen that these results show little dependence on the amount of transgenic grain contained within the sample, in terms of ethanol yield. Thus, the grain is rich in amylase to facilitate fermentative production of ethanol. It also shows that lower pH liquefaction can result in higher ethanol yields.

The viscosity of the sample after liquefaction was monitored and it was found that 6% transgenic grain was sufficient to reduce viscosity sufficiently at ph 6.0. At pH5.2 and 5.6, the viscosity was equal to that of the 12% transgenic grain control, but not for the lower percentage of transgenic grain.

Example 17

Production of fructose from corn flour using thermostable enzymes

It was demonstrated that maize expressing hyperthermophilic alpha-amylase 797GL3, when mixed with alpha-glucosidase (MalA) and xylose isomerase (XylA), promoted fructose production.

Seeds of pNOV6201 transgenic plants expressing 797GL3 were ground in a Kleco trough to flour, which formed amylase flour. Control flours were obtained by milling non-transgenic corn grain in the same manner.

Alpha-glucosidase, MalA (from sulfolobus solfataricus) was expressed in E.coli. The harvested bacteria were suspended in 50mM potassium phosphate buffer containing 1mM 4- (2-aminoethyl) benzenesulfonyl fluoride, pH7.0, and then lysed in a French cell crusher. Lysates were centrifuged at 23000Xg for 15 min at 4 ℃. The supernatant was taken out, heated to 70 ℃ for 10 minutes, cooled on ice for 10 minutes, and then centrifuged at 34000Xg for 30 minutes at 4 ℃. The supernatant was removed and MalA was concentrated 2-fold in a centricon10 apparatus. The filtrate from the centricon10 step was retained as a negative control for MalA.

Xylose (glucose) isomerase was prepared by expressing the xylA gene of Thermotoga nardus in E.coli. The bacteria were suspended in 100mM sodium phosphate pH7.0 and lysed by French cell crusher. After precipitating the cell debris, the extract was heated to 80 ℃ for 10 minutes and then centrifuged. The supernatant contained the XylA enzyme activity. Control extracts of empty vehicle were prepared in parallel with the XylA extract.

Corn meal (60mg per sample) was mixed with buffer and extract from e. As shown in Table 3, the samples contained either amylase corn flour (amylase) or control corn flour (control), 50. mu.l MalA extract (+) or filtrate (-), and 20. mu.l XylA extract (+) or empty vector control (-). All samples contained 230. mu.l of 50mM MOPS, 10mM MgSO₄And 1mM CoCl₂(ii) a The buffer pH was 7.0 at room temperature.

The samples were incubated at 85 ℃ for 18 hours. At the end of the incubation period, the sample was diluted with 0.9ml of water at 85 ℃ and centrifuged to remove undissolved material. The supernatant fraction was then filtered through a Centricon3 ultrafiltration unit and analyzed by HPLC and ELSD detection.

The gradient HPLC system was assembled with an Astec Polymer Amin column, 5 micron particle size, 250X 4.6mm, and an Alltech hELSD 2000 detector. The system was pre-equilibrated with a 15: 85 mixture of water and acetonitrile. The flow rate was 1 ml/min. Initial conditions were maintained for 5 minutes after injection loading, followed by a 20 minute gradient to 50: 50 water to acetonitrile and 10 minutes with the same solvent. The system was washed with 80: 20 water acetonitrile for 20 minutes and then re-equilibrated with the starting solvent. Fructose eluted at 5.8min, glucose eluted at 8.7 min.

TABLE 3

Sample (I)	Corn flour	MalA	XylA	Fructose Peak area X10-6	Area of glucose peak X10-6
						1	Amylase	+	+	25.9	110.3
2	Amylase	-	+	7.0	12.4
						3	Amylase	+	-	0.1	147.5
4	Amylase	-	-	0	25.9
						5	Control	+	+	0.8	0.5
6	Control	-	+	0.3	0.2
						7	Control	+	-	1.3	1.7
8	Control	-	-	0.2	0.3

The HPLC results also indicated that more malto-oligosaccharides were present in all samples containing alpha-amylase. These results demonstrate that three thermostable enzymes can co-act at high temperatures to produce fructose from corn flour.

Example 18

Amylase flour with isomerase

In another example, the amylase flour was mixed with purified MalA and one of two bacterial xylose isomerases, XylA of Thermotoga maritima and an enzyme known as BD8037 from Diversa. Amylase flour was prepared as described in example 18.

The sulfolobus solfataricus MalA with 6His purification label is expressed in Escherichia coli. Cell lysates were prepared as described in example 18 and then purified to a homogeneous appearance using nickel affinity resin (Probond, Invitrogen) according to the manufacturer's instructions for purification of native proteins.

Thermotoga maritima XylA carrying an S-tag and an ER retention signal was expressed in E.coli and prepared in the same manner as Thermotoga maritima XylA described in example 18.

The xylose isomerase BD8037 was obtained as a lyophilized powder, which was resuspended in 0.4x the initial volume of water.

The amylase corn meal is mixed with an enzyme solution and water or buffer. All reactions contained 60mg of amylase flour and a total of 600. mu.l of liquid. One group of reactions is added with 10mM MgSO₄，1mM CoCl₂50mM MOPS pH7.0 at room temperature as a buffer; the second set of reactions replaced the metal-containing buffer with water. The amount of isomerase varied as shown in Table 4. All reactions were incubated at 90 ℃ for 2 hours. The reaction supernatant was prepared by centrifugation. The pellet was washed with an additional 600. mu.l of water and recentrifuged. The supernatant fractions from each reaction were combined, filtered through Centrico 10 and analyzed by HPLC with ELSD detection as described in example 17. The amounts of glucose and fructose observed are shown in the figureShown at 15.

TABLE 4

Sample (I)	Amylase flour	MalA	Isomerase enzyme
				1	60mg	+	Is free of
2	60mg	+	Thermotoga maritima, 100. mu.l
				3	60mg	+	Thermotoga maritima, 10. mu.l
4	60mg	+	Thermotoga maritima, 2. mu.l
				5	60mg	+	BD8037，100μl
7	60mg	+	BD8037，2μl
				C	60mg	Is free of	Is free of

For each isomerase, fructose was produced from corn flour in a dose-dependent manner when alpha-amylase and alpha-glucosidase were present in the reaction. These results demonstrate that cereals expressing amylase 797GL3 can work with MalA and various thermostable isomerases with or without metal ions to produce fructose from corn meal at high temperature. When added with divalent metal ions, the isomerase can achieve the desired fructose of about 55% fructose at 90 ℃: and (4) glucose balancing. This is an improvement over the prior art methods using mesophilic isomerases, as the prior art requires chromatographic separation to increase fructose concentration.

Example 19

Expression of pullulanase in maize

Transgenic plants homozygous for pNOV7013 or pNOV7005 were crossed to produce transgenic corn seeds co-expressing 797GL3 a-amylase and 6GP3 pullulanase.

T1 or T2 seeds of pNOV7005 or pNOV4093 transformed autologous maize pollen-receptive plants were obtained. pNOV4093 is a fusion protein of the maize optimized synthetic 6GP3 gene (SEQ ID NO: 3, 4) with an amyloplast targeting sequence (SEQ ID NO: 7, 8) for localization of the fusion protein to an amyloplast. The fusion protein is under the control of the ADPgpp promoter (SEQ ID NO: 11) for specific expression in endosperm. The pNOV7005 construct targets the expression of pullulanase to the endosperm endoplasmic reticulum. The localization of the enzyme in the ER allows for the normal accumulation of starch within the corn kernel. Normal staining of the starch with iodine solution prior to high temperature treatment was also observed.

As with alpha-amylase, expression of amylopectinase (pNOV4093) targeted to amyloplasts causes an abnormal accumulation of starch within the corn kernel. When the ear of corn is dried, the kernels shrink. It is clear that the high temperature resistant pullulanase has sufficient activity at low temperatures and is capable of hydrolyzing starch if in direct contact with starch granules within the endosperm of seeds.

Preparation of enzymes or enzyme extracts from corn flour: the transgenic seeds were ground on a Kleco grinder and the powder was incubated in 50mM NaOAc pH5.5 buffer for 1 hour at room temperature with constant shaking to extract pullulanase therefrom. The incubated mixture was then centrifuged at 14000rpm for 15 minutes. The supernatant was used as the source of the enzyme.

Pullulanase assay: the experimental reactions were performed in 96-well plates. Pullulanase (100. mu.l) extracted from corn meal was used in 900. mu.l containing 40mM CaCl₂Diluted 10-fold in 50mM pH5.5NaOAc buffer. The mixture was vortexed and a sheet of Limit-Dextrizyme (azurin cross-linked amylopectin, from Megazyme) was added to each reaction mixture and incubated at 75 ℃ for 30 min (or as mentioned). At the end of the incubation period, the reaction mixture was centrifuged at 3500rpm for 15 minutes. Supernatants were diluted 5-fold and transferred to 96-well flat-bottom plates for measurement of 590nm absorbance. Hydrolysis of azurin-crosslinked amylopectin by pullulanase forms a water-soluble stained fragment, the release rate of which (measured as an increase in absorbance at 590 nm) is directly related to the enzymatic activity.

Figure 9 shows analysis of T2 seeds from different events of pNOV7005 transformation. Expression of pullulanase activity was detected in many events as higher than in non-transgenic controls.

To a measured amount (. about.100. mu.g) of dry corn meal from the transgene (expressing pullulanase, amylase or both) and/or control (non-transgenic) was added 1000. mu.l of dry corn meal containing 40mM CaCl₂50mM pH5.5 NaOAc buffer. The reaction mixture was vortexed and incubated for 1 hour on a shaker. As shown, the incubation mixture is transferred to a high temperature (75 ℃, the optimum reaction temperature for pullulanase or as shown) to start the enzymatic reaction for a period of time. The reaction was quenched by cooling on ice. The reaction mixture was then centrifuged at 14000rpm for 10 minutes. An aliquot of the supernatant (100 μ l) was diluted 3-fold and filtered through a 0.2 μm filter for HPLC analysis.

HPLC analysis of the samples was performed using the following conditions:

column: alltech Prevail ES 5 micron 250 x 4.6mm

A detector: alltech ELSD 2000

A pump: gilson 322

An injector: gilson 215 injector/diluter

Solvent: HPLC grade acetonitrile (Fisher Scientific) and water (purified by Waters Millipore System)

Gradient for oligosaccharide of Low degree of polymerization (DP1-15)

Time% water% acetonitrile

0 15 85

5 15 85

25 50 50

35 50 50

36 80 20

55 80 20

56 15 85

76 15 85

Gradient for high degree of polymerization (DP20-100 and above) saccharides

Time% water% acetonitrile

0 35 65

60 85 15

70 85 15

85 35 65

100 35 65

System for data analysis: gilson Unipoint software System version 3.2

FIGS. 10A and 10B show HPLC analysis of hydrolysates formed by the action of expressed pullulanase on transgenic corn starch. The pullulanase expressing maize meal was incubated in reaction buffer at 75 ℃ for 30 minutes resulting in the formation of medium chain oligosaccharides (DP 10-30) and short straight chain starch chains (DP 100-200) from maize starch. The figure also shows that the activity of pullulanase is dependent on calcium ions.

Transgenic corn expressing pullulanase can be used to produce debranched (broken α 1-6 bonds) modified starches and/or dextrins such that amylose/amylose levels are high. In addition, the modified starch/dextrin also has different characteristics because the chain length distribution of amylose/dextrin formed by pullulanase varies depending on the kind of starch used (e.g., waxy, high amylose starch, etc.).

The hydrolysis of the alpha 1-6 bond was also demonstrated using amylopectin as substrate. The pullulanase isolated from corn flour efficiently hydrolyzes amylopectin. HPLC analysis of the product formed after incubation (as described) indicated that maltotriose was formed from maize as expected due to enzymatic hydrolysis of the α 1-6 linkage within the amylopectin molecule.

Example 20

Expression of pullulanase in maize

Expression of 6gp3 pullulanase was further analyzed by PAGE and Coomassie staining after extraction from corn meal. The seeds were ground in a Kleco pulverizer for 30 seconds to obtain corn flour. The enzyme was extracted from approximately 150mg of flour with 50mM pH5.5 NaOAc buffer. The mixture was vortexed and incubated with shaker at room temperature for 1 hour, followed by incubation at 70 ℃ for an additional 15 minutes. The reaction mixture was centrifuged (14000 rpm for 15 minutes at room temperature) and the supernatant was used for SDS-PAGE analysis. Protein bands of appropriate molecular weight (95kDal) were observed. Pullulanase experiments were performed on samples using commercially available dye-conjugated LIMIT dextrin (LIMIT-DEXTRIZYME, from Megazyme, Ireland). High levels of thermostable pullulanase activity are associated with the presence of a 95kD protein.

Western blot and ELISA analysis of transgenic maize seeds also showed expression of the 95kD protein, which reacted with the prepared anti-pullulanase antibody (expressed in E.coli).

Example 21

Addition of pullulanase to express corn increases the rate of starch hydrolysis and improves short chain (fermentable) Yield of oligosaccharides

The data shown in FIGS. 11A and 11B are from HPLC analysis of starch hydrolysates from two reaction mixtures as described above. The first reaction, labeled "amylase", contained a sample mixture of α -amylase expressing transgenic corn prepared as described in example 4 and corn flour such as non-transgenic corn a188 [ 1: 1(w/w) ]; the second reaction mixture was "amylase + pullulanase" and contained a sample mixture of corn flour [ 1: 1(w/w) ] of α -amylase expressing transgenic corn and pullulanase expressing transgenic corn prepared as described in example 19. The results obtained support the beneficial effect of using pullulanase in combination with alpha-amylase in starch hydrolysis processes. These benefits result from an increase in the rate of starch hydrolysis (FIG. 11A) and an increase in the production of fermentable low DP oligosaccharides (FIG. 11B).

Alpha-amylase expressed in maize alone or together with pullulanase (or any other combination of starch hydrolyzing enzymes) was found to be useful for the preparation of maltodextrins (linear or branched oligosaccharides) (FIGS. 11A, 11B, 12 and 13A). The composition of the maltodextrin formed and thus its properties vary according to the different reaction conditions, the type of hydrolytic enzyme and its combination and the type of starch used.

Fig. 12 depicts the results of an experiment performed in a similar manner as described in fig. 11. The different temperature and time protocols adopted in the incubation reactions are indicated in the figure. The optimum reaction temperature for pullulanase is 75 ℃ and for alpha-amylase > 95 ℃. Thus, according to the scheme shown, ranges can be provided in which pullulanase and/or α -amylase, respectively, catalyze at their optimal reaction temperatures. From these results it is clearly deduced that the combination of alpha-amylase and pullulanase hydrolyses corn starch better at the end of the 60 min incubation period.

HPLC analysis of starch hydrolysates in both sets of reaction mixtures was performed as described above (except-150 mg corn flour was used in these reactions) at the end of 30 min incubation, and the results are shown in fig. 13A and 13B. The first set of reactions was incubated at 85 ℃ and the second at 95 ℃. Each group had two reaction mixtures; the first, called "amylase X pullulanase", contains the corn meal of transgenic corn expressing alpha-amylase and pullulanase (formed by cross pollination), while the second reaction, called "amylase" mixture, is formed by mixing samples of corn flour of transgenic corn expressing alpha-amylase and non-transgenic corn a188 in a ratio that allows it to obtain the same amount of activity as the alpha-amylase activity observed in the hybrid (amylase X pullulanase). When a sample of corn flour is incubated at 85 ℃, the total production of low DP oligosaccharides in the case of hybridization between alpha-amylase and pullulanase is higher than in corn expressing only alpha-amylase. Incubation at 95 ℃ will (at least partially) inactivate the pullulanase, so that little difference is observed between "amylase X pullulanase" and "amylase". However, when using amylase and pullulanase hybrid corn meal, at the end of the incubation period, the data at both incubation temperatures show: glucose production was significantly improved over that using corn expressing only alpha-amylase (fig. 13B). Thus, the use of corn expressing both alpha-amylase and pullulanase may be particularly beneficial where complete hydrolysis of starch to glucose is important.

The above examples provide sufficient evidence that pullulanase expressed in corn seeds improves the starch hydrolysis process when used in combination with alpha-amylase. Pullulanase specific for alpha 1-6 bonds is far more effective than alpha-amylase (alpha 1-4 bond specificity) in debranching starch, thereby reducing the amount of branched oligosaccharides (e.g., limit dextrins, panose, which are generally non-fermentable) and increasing the amount of linear short oligosaccharides (readily fermentable to ethanol, etc.). Second, fragmentation of the starch molecule by pullulanase catalyzed debranching increases the accessibility of the alpha-amylase to the substrate and thus the efficiency of the alpha-amylase catalyzed reaction.

Example 22

To determine whether 797GL3 alpha-amylase and mal a alpha-glucosidase were able to function under similar pH and temperature conditions to produce a yield of glucose that was higher than that produced by the individual enzymes, approximately 0.35ug of mal a alpha-glucosidase (produced in bacteria) was added to a solution containing 1% starch and purified starch obtained from either non-transgenic corn seed (control) or 797GL3 transgenic corn seed (797GL3 corn seed, co-purified with starch). In addition, starch purified from non-transgenic and 797GL3 transgenic corn seeds was added to 1% corn starch in the absence of any malA enzyme. The mixture was incubated at 90 ℃ pH6.0 for 1 hour, the insoluble material was removed by centrifugation, and the soluble fraction was analyzed by HPLC for glucose level. As shown in FIG. 14, 797GL3 α -amylase and malA α -glucosidase were operated under similar pH and temperature conditions to hydrolyze starch to glucose. The resulting glucose yields are significantly higher than those formed by a single enzyme.

Example 23

The use of Thermoanaerobacterium (Thermoanaerobacterium) glucoamylase in the hydrolysis of raw starch (raw-starch) was determined. As shown in FIG. 15, the hydrolytic conversion of crude starch was determined at room temperature and 30 ℃ using water, barley alpha-amylase (a commercial product from Sigma), Thermoanaerobacter glucoamylase and combinations thereof. As shown, the combination of barley alpha-amylase and Thermoanaerobacter glucoamylase can hydrolyze crude starch to glucose. In addition, the yield of glucose formed by barley amylase and thermoanaerobacterium GA was significantly higher than that formed by the single enzyme.

Example 24

Maize optimized genes and sequences for crude starch hydrolysis and plant transformation vectors

The enzyme is selected for its ability to hydrolyze the crude starch at a temperature of about 20 ℃ to 50 ℃. The maize preferred codons were then used to design the corresponding gene or gene fragment for the construction of synthetic genes as described in example 1.

Selecting an Aspergillus shirousami alpha-amylase/glucoamylase fusion polypeptide (without a signal sequence) having an amino acid sequence as set forth in SEQ ID NO: 45, by biosci.biotech.biochem, 56: 884 + 889 (1992); agric.biol.chem.545: 1905-14 (1990); biosci.biotechnol.biochem.56: 174-79 (1992). Designing a maize optimized nucleic acid sequence as set forth in SEQ ID NO: 46, respectively.

Similarly, Thermoanaerobacterium thermosaccharolyticum (Thermoanaerobacterium thermosaccharolyticum) glucoamylase was selected, the amino acid sequence of which is shown in SEQ ID NO: 47, by biosci.biotech.biochem, 62: 302-308 (1998). A maize optimized nucleic acid sequence (SEQ ID NO: 48) was designed.

Rhizopus oryzae (Rhizopus oryzae) glucoamylase was selected, the amino acid sequence (without signal sequence) (SEQ ID NO: 50) of which is disclosed in the literature (Agric. biol. chem., (1986)50, pg 957-964). Designing a maize optimized nucleic acid sequence as set forth in SEQ ID NO: shown at 51.

In addition, a maize alpha-amylase was selected, the amino acid sequence (SEQ ID NO: 51) and the nucleic acid sequence (SEQ ID NO: 52) of which are from the literature. See, for example, Plant physiol.105: 759-760(1994).

The expression cassette was constructed to express the sequence of the desired SEQ ID NO: 46 maize optimized nucleic acid expression Aspergillus shirousami alpha-amylase/glucoamylase fusion polypeptide, encoded by a sequence of SEQ ID NO: 48 optimized nucleic acid expression in maize thermoanaerobacterium thermosaccharolyticum glucoamylase, engineered from SEQ ID NO: 50 expresses a Rhizopus oryzae glucoamylase with amino acids (without signal sequence) (SEQ ID NO: 49) and expresses a maize alpha-amylase.

The maize gamma-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) contained in the plasmid was fused to a synthetic gene encoding the enzyme. Optionally, a SEKDEL sequence can be fused to the C-terminus of the synthetic gene to target and retain to the ER. The fusion product was cloned behind the maize gamma-zein promoter in a plant transformation plasmid for specific expression in the endosperm. The fusion product is transported to maize tissue by agrobacterium infection.

Example 25

Expression cassettes containing the selected enzyme were constructed to express the enzyme. A plasmid containing the crude starch binding site sequence is fused to a synthetic gene encoding an enzyme. The coarse starch binding site allows the fused enzyme to bind to non-gelatinized starch. The amino acid sequence of the crude starch binding site (SEQ ID NO: 53), which has been optimized for maize, is determined from the literature as shown in SEQ ID NO: as shown at 54. The maize optimized nucleic acid sequence is fused in a plasmid to a synthetic gene encoding an enzyme for expression in plants.

Example 26

Construction of maize optimized genes and vectors for usePlant transformation

Genes or gene fragments were designed using maize preferred codons to construct synthetic genes as described in example 1.

Selecting Pyrococcus furiosus (Pyrococcus furiosus) EGLA ultra-high temperature resistant endoglucanase, the amino acid sequence (without signal sequence) of which is as shown in SEQ ID NO: 55, identified by Journal of Bacteriology (1991)181, pg 284-290. Designing a maize optimized nucleic acid sequence as set forth in SEQ ID NO: as shown at 56.

Selecting Thermus flavus (Thermus flavus) xylose isomerase with an amino acid sequence shown as SEQ ID NO: 57, by Applied Biochemistry and Biotechnology 62: 15-27 (1997).

The expression cassette was constructed to express Pyrococcus furiosus EGLA (endoglucanase) from a maize optimized nucleic acid (SEQ ID NO: 56), and to express the polypeptide sequence from SEQ ID NO: the maize optimized coding nucleic acid of the 57 amino acid sequence expresses the Thermus flavus xylose isomerase.

A plasmid containing the maize gamma-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) was fused to a synthetic maize optimized gene encoding the enzyme. Optionally, a SEKDEL sequence can be fused to the C-terminus of the synthetic gene to target and retain to the ER. The fusion product was cloned into a plant transformation plasmid behind the maize gamma-zein promoter for specific expression in the endosperm. The fusion product is transported to maize tissue by agrobacterium infection.

Example 27

Production of glucose from corn flour using thermostable enzymes expressed in corn

Expression of hyperthermophilic alpha-amylase 797GL3 and alpha-glucosidase (MalA) has been shown to result in glucose production when mixed with aqueous solutions and incubated at 90 ℃.

Transgenic maize lines expressing MalA enzyme (168a10B line, pNOV4831) were identified by measuring alpha-glucosidase activity, which is indicated by hydrolysis of p-nitrophenyl-alpha-glucoside.

Transgenic plant corn kernels expressing 797GL3 were ground in a Kleco tank to flour, thereby forming amylase flour. Transgenic plant maize kernels expressing MalA are ground to flour in a Kleco tank to form MalA flour. Non-transgenic plant corn kernels were ground in the same manner to form control flours.

The buffer was 50mM MES buffer pH6.0

Hydrolysis reaction of corn flour: samples were prepared as shown in table 5 below. Corn meal (approximately 60mg per sample) was mixed with 40ml 50mM MES buffer, pH 6.0. The samples were incubated in a 90 ℃ water bath for 2.5 hours and 14 hours. Samples were taken at the indicated incubation time points and analyzed for glucose content.

The glucose in the sample was analyzed by the glucose oxidase/horseradish peroxidase assay. The GOPOD agent contains: 0.2mg/ml o-dianisidine, 100mM Tris pH7.5, 100U/ml glucose oxidase and 10U/ml horseradish peroxidase. Mu.l of sample or diluted sample and glucose standards (varying from 0-0.22 mg/ml) were arrayed on 96-well plates. Add 100. mu.l GOPOD reagent to each well and mix, then incubate the plate for 30 min at 37 ℃. Absorbance was read at 540nm with the addition of 100. mu.l sulfuric acid (9M). The glucose concentration of the sample was determined with reference to a standard curve. The amounts of glucose observed in each sample are shown in table 5.

TABLE 5

Sample (I)	WT flour mg	Amylase flour mg	MalA flour mg	Buffer solution ml	Glucose 2.5hmg	Glucose 14h mg
							1	66	0	0	40	0	0
2	31	30	0	40	026	050
							3	30	0	31.5	40	0	0.09
4	0	32.2	30.0	40	2.29	12.30
							5	0	6.1	56.2	40	1.16	8.52

These data demonstrate that when hyperthermophilic alpha-amylase and alpha-glucosidase are expressed in corn to form a corn product, glucose can be formed when the product is hydrolyzed and heated under appropriate conditions.

Example 28

Production of maltodextrin

Maltodextrins are prepared using grains expressing a thermostable alpha-amylase. This exemplary process does not require pre-separation of the starch, nor the addition of exogenous enzymes.

Transgenic plant corn kernels expressing 797GL3 were ground in a Kleco tank to a flour to form "amylase flour". The mixture of 10% transgenic/90% non-transgenic corn kernels was ground in the same manner in a Kleco tank to form "10% amylase flour".

The amylase flour and 10% amylase flour (approximately 60 mg/sample) were mixed with water at a rate of 5 μ l water per mg flour. As shown in Table 6, the obtained slurry was incubated at 90 ℃ for not more than 20 hours. The reaction was stopped by adding 0.9ml of 50mM EDTA at 85 ℃ and mixing by pipetting. A0.2 ml sample of the slurry was removed, centrifuged to remove insoluble material and diluted 3-fold with water.

The samples were analyzed for sugars and maltodextrins by HPLC with ELSD detection. The gradient HPLC system was assembled using an Astec Polymer Amino column, 5 micron particle size, 250X 4.6mm and Alltech ELSD2000 detector. The system was pre-equilibrated with a 15: 85 mixture of water and acetonitrile. The flow rate was 1 ml/min. Initial conditions were maintained for 5 minutes after injection loading, followed by a 20 minute gradient to 50: 50 water to acetonitrile and 10 minutes run with the same solvent. The system was washed with 80: 20 water acetonitrile for 20 minutes and then re-equilibrated with the starting solvent.

The peak areas obtained are normalized to the volume and weight of the flour. The ELSD response factor per μ g carbohydrate decreases with increasing DP, so that the higher DP maltodextrins represent a higher percentage of the population than indicated by the peak area.

The relative peak areas of the reaction products of 100% amylase flour are shown in figure 17. The relative peak areas of the reaction products of 10% amylase flour are shown in figure 18.

These data demonstrate that different maltodextrin blends can be produced by varying the heating time. The maltodextrin property profile can be altered by altering the level of alpha-amylase activity by mixing transgenic alpha-amylase expressing maize with wild type maize.

The hydrolysis reaction products described in this example can be concentrated and purified for food and other applications by a variety of established techniques, including: centrifugation, filtration, ion exchange, gel permeation, ultrafiltration, nanofiltration (nanofiltration), reverse osmosis, decolorization with carbon particles, spray drying, and other techniques known in the art.

Example 29

Effect of time and temperature on maltodextrin production

The composition of the maltodextrin product obtained from the autohydrolysis of grains containing thermostable alpha-amylase can be varied by varying the reaction time and temperature.

In another experiment, amylase flour was prepared as described above in example 28 and mixed with water at a ratio of 300 μ l water per 60mg flour. The samples were incubated at 70 ℃, 80 ℃, 90 ℃ or 100 ℃ for no more than 90 minutes. The reaction was stopped by adding 900ml of 50mM EDTA at 90 ℃. Insoluble matter was removed by centrifugation and filtered through a 0.45 μm nylon filter. The filtrate was analyzed by HPLC as described in example 28.

The analysis results are shown in FIG. 19. DP number designation refers to the degree of polymerization. DP2 is maltose; DP3 is maltotriose and the like. The larger DP maltodextrin eluted in a single peak near the end of the elution, labeled "> DP 12". The aggregates include dextrins that can pass through a 0.45 μm filter and through a guard column, and do not contain very large starch fragments that cannot pass through the filter or guard column.

This experiment demonstrates that the maltodextrin composition of the product can be varied by varying the temperature and incubation time to obtain the desired maltooligosaccharide or maltodextrin product.

Example 30

Production of maltodextrin

Other enzymes such as alpha-glucosidase and xylose isomerase may also be added to the water and flour mixture prior to heat treatment, or by including salts to alter the composition of the maltodextrin product formed from transgenic corn containing thermostable alpha-amylase.

In another experiment, the amylase flour prepared as above was mixed with purified MalA and/or bacterial xylose isomerase designated BD 8037. The sulfolobus solfataricus MalA with a 6His purification tag is expressed in Escherichia coli. Cell lysates were prepared as described in example 28 and then purified to a homogeneous appearance using nickel affinity resin (Probond, Invitrogen) according to the manufacturer's instructions for purification of native proteins. Xylose isomerase BD8037 obtained from Diversa was a lyophilized powder, which was resuspended in 0.4x the initial volume of water.

The amylase corn meal is mixed with an enzyme solution and water or buffer. All reactions contained 60mg of amylase flour and a total of 600. mu.l of liquid. One group of reactions is added with 10mM MgSO₄，1mM CoCl₂50mM MOPS pH7.0 at room temperature as a buffer; the second set of reactions replaced the metal-containing buffer with water. All reactions were incubated at 90 ℃ for 2 hours. The reaction supernatant was prepared by centrifugation. The pellet was washed with an additional 600. mu.l of water and recentrifuged. Supernatants from each reaction were pooled, filtered by Centrico 10, and analyzed by HPLC with ELSD detection as described above.

The results are shown in FIG. 20. The figure illustrates that cereals expressing amylase 797GL3 can be used with other thermostable enzymes, with or without the addition of metal ions, to produce various maltodextrin blends from corn meal at elevated temperatures. In particular, inclusion of a glucoamylase or alpha-glucosidase may result in more glucose products and other low DP products. The inclusion of glucose isomerase allows fructose to be present in the product, which may be sweeter than the product formed by amylase alone or amylase with alpha-glucosidase. In addition, the data also show that the ratio of DP5, DP6 and DP7 maltooligosaccharides can be adjusted by adding a divalent cation salt, such as CoCl ₂And MgSO₄To be improved.

Other methods of altering the composition of the maltodextrin formed by the reaction described herein include: change the reaction pH, change the starch type in transgenic or non-transgenic grain, change the solids ratio or add organic solvents.

Example 31

Preparation of dextrins or sugars from cereals without mechanical disruption prior to recovery of starch-derived products

Sugars and maltodextrins were prepared by contacting transgenic grain expressing alpha-amylase 797GL3 with water and heating at 90 ℃ overnight (> 14 hours). The liquid is then separated from the grain by filtration. The liquid product was analyzed by HPLC by the method described in example 15. Table 6 shows the results of the detection of the products.

TABLE 6

Molecular species	Product concentration μ g/25 μ l injection volume
		Fructose	0.4
Glucose	18.0
		Maltose	56.0
DP3*	26.0
		DP4*	15.9
DP5*	11.3
		DP6*	5.3
DP7*	1.5

^*The quantitative determination of DP3 includes that maltotriose may also include the maltotriose isomer having an α (1 → 6) bond in place of the α (1 → 4) bond. Similarly, quantification of DP4-DP7 includesLinear malto-oligosaccharides of a given chain length and isomers having one or more alpha (1 → 4) linkages replaced by one or more alpha (1 → 6) linkages.

These data indicate that sugars and maltodextrins can be made by contacting whole grain expressing an alpha-amylase with water and heating. The product can then be separated from the whole grain by filtration or centrifugation or gravity settling.

Example 32

Corn medium-coarse starch for fermenting and expressing rhizopus oryzae glucoamylase

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 29. The corn kernels are ground into flour. The kernel expresses a protein comprising an active fragment of Rhizopus oryzae glucoamylase (SEQ ID NO: 49), which is targeted to the endoplasmic reticulum.

The corn kernels were ground into a flour as described in example 15. A slurry was then prepared containing 20g of corn meal, 23ml of deionized water and 6.0ml of backsset (8% by weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following ingredients were added to the slurry: protease (0.60ml of a 1000-fold dilution of a commercially available protease), 0.2mg of Lactocide&Urea (0.85ml of 50% Urea Liquor diluted 10-fold). A hole was made in the cap of a 100ml bottle containing the syrup as CO₂A discharge passage. The slurry was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48h, 82F was set.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

Example 33

Example of fermentation of crude starch in corn expressing Rhizopus oryzae Glucoamylase

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 28. Grinding corn kernels into powder. The kernel expresses a protein comprising an active fragment of Rhizopus oryzae glucoamylase (SEQ ID NO: 49), which is targeted to the endoplasmic reticulum.

The corn kernels were ground into a flour as described in example 15. A slurry was then prepared containing 20g of corn meal, 23ml of deionized water and 6.0ml of backsset (8% by weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following ingredients were added to the slurry: protease (0.60ml of a 1000-fold dilution of a commercially available protease), 0.2mg of Lactocide&Urea (0.85ml of 50% Urea Liquor diluted 10-fold). A hole was made in the cap of a 100ml bottle containing the syrup as CO₂A discharge passage. The slurry was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48h, 82F was set.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

Example 34

Corn whole corn added with exogenous alpha-amylase for fermenting and expressing rhizopus oryzae glucoamylase Examples of raw starch in rice grains

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 28. The kernel expresses a protein comprising an active fragment of Rhizopus oryzae glucoamylase (SEQ ID NO: 49), which is targeted to the endoplasmic reticulum.

The corn kernels were contacted with 20g corn meal, 23ml deionized water and 6.0ml backsset (8% weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following ingredients were added: barley alpha-amylase from sigma (2mg), protease (0.60ml 1000 fold dilution of commercial protease), 0.2mg Lactocide&Urea (0.85ml of 50% Urea Liquor diluted 10-fold). A hole was made in the cap of a 100ml bottle containing the mixture as CO₂A channel. The mixture was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48hSet to 82F.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

Example 35

Fermentation of corn expressing Rhizopus oryzae Glucoamylase and corn (Zea mays) amylase Examples of crude starch

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 28. The kernel expresses a protein comprising an active fragment of Rhizopus oryzae glucoamylase (SEQ ID NO: 49), which is targeted to the endoplasmic reticulum. The kernel also expresses a maize amylase with a crude starch binding domain, as described in example 28.

The corn kernels were ground into a flour as described in example 14. A slurry was then prepared containing 20g of corn meal, 23ml of deionized water and 6.0ml of backsset (8% by weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following ingredients were added to the slurry: protease (0.60ml of a 1000-fold dilution of a commercially available protease), 0.2mg of Lactocide&Urea (0.85ml of 50% Urea Liquor diluted 10-fold). A hole was made in the cap of a 100ml bottle containing the syrup as CO₂A channel. The slurry was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48h, 82F was set.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

Example 36

Implementation of fermentation of raw starch in corn expressing Thermoanaerobacter thermosaccharolyticus glucoamylase Example (b)

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 28. The corn kernel expresses a protein comprising an active fragment of Thermoanaerobacterium thermosaccharolyticum glucoamylase (SEQ ID NO: 47) that is targeted to the endoplasmic reticulum.

The corn kernels were ground into a flour as described in example 15. A slurry was then prepared containing 20g of corn meal, 23ml of deionized water and 6.0ml of backsset (8% by weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following ingredients were added to the slurry: protease (0.60ml of a 1000-fold dilution of a commercially available protease), 0.2mg of Lactocide &Urea (0.85ml of 50% Urea Liquor diluted 10-fold). A hole was made in the cap of a 100ml bottle containing the syrup as CO₂A channel. The slurry was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48h, 82F was set.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

Example 37

Fermentation of crude starch in corn expressing Aspergillus niger glucoamylase Examples of powders

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 28. The corn grain expresses a protein containing an active fragment of Aspergillus niger glucoamylase (Fiil, N.P. "Aspergillus niger glucoamylase G1 and G2 synthesized from two different but closely related mRNAs" EMBO J.3(5), 1097-1102(1984) accession number P04064). The maize-optimized nucleic acid encoding the glucoamylase has the amino acid sequence of SEQ ID NO: 59, which protein targets the endoplasmic reticulum.

The corn kernels were ground into a flour as described in example 14. A mixture was then prepared containing 20g of corn meal, 23ml of deionized water and 6.0ml of backsset (8% weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following were added to the mixture: protease (0.60ml of a 1000-fold dilution of a commercially available protease), 0.2mg of Lactocide &Urea (0.85ml 50% Urea Liqu 10-fold diluted)or). A hole was made in the cap of a 100ml bottle containing the mixture as CO₂A channel. The slurry was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48h, 82F was set.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

Example 39

Fermentation of corn expressing Aspergillus niger glucoamylase and corn (Zea mays) amylase Examples of crude starch

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 28. The corn grain expresses a protein (SEQ ID NO: 59, maize-optimized nucleic acid) containing an active fragment of Aspergillus niger glucoamylase (Fiil, N.P. "Glucoamylases synthesize Aspergillus niger Glucoamylases G1 and G2" EMBO J.3(5), 1097-1102(1984) accession number P04064 from two different but closely related mRNAs), which is targeted to the endoplasmic reticulum. The kernel also expresses a maize amylase with a crude starch binding domain, as described in example 28.

The corn kernels were ground into a flour as described in example 14. A slurry was then prepared containing 20g of corn meal, 23ml of deionized water and 6.0ml of backsset (8% by weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following ingredients were added to the slurry: protease (0.60ml of a 1000-fold dilution of a commercially available protease), 0.2mg of Lactocide &Urea (0.85ml of 50% Urea Liquor diluted 10-fold). A hole was made in the cap of a 100ml bottle containing the syrup as CO₂A channel. The slurry was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48h, 82F was set.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

Example 39

In corn fermenting expressing Thermoanaerobacter thermosaccharolyticus glucoamylase and barley amylase Examples of raw starch

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 28. The corn kernel expresses a protein comprising an active fragment of Thermoanaerobacterium thermosaccharolyticum glucoamylase (SEQ ID NO: 47), which is targeted to the endoplasmic reticulum. The kernel also expressed the low pI barley amylase amy1 gene (Rogers, j.c. and Milliman, c. "isolated and sequenced barley alpha-amylase cDNA clone" j.biol. chem.258(13), 8169-.

The corn kernels were ground into a flour as described in example 14. A slurry was then prepared containing 20g of corn meal, 23ml of deionized water and 6.0ml of backsset (8% by weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following ingredients were added to the slurry: protease (0.60ml of a 1000-fold dilution of a commercially available protease), 0.2mg of Lactocide &Urea (0.85ml of 50% Urea Liquor diluted 10-fold). A hole was made in the cap of a 100ml bottle containing the syrup as CO₂A channel. The slurry was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48h, 82F was set.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

Example 40

Fermentation of corn expressing Thermoanaerobacter thermosaccharolyticus glucoamylase and barley amylase Examples of crude starch in whole corn kernels

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 28. The corn kernel expresses a protein comprising an active fragment of Thermoanaerobacterium thermosaccharolyticum glucoamylase (SEQ ID NO: 47), which is targeted to the endoplasmic reticulum. The kernel also expressed the low pI barley amylase amy1 gene (Rogers, j.c. and Milliman, c. "isolated and sequenced barley alpha-amylase cDNA clone" j.biol. chem.258(13), 8169-.

The corn kernels were contacted with 20g corn meal, 23ml deionized water and 6.0ml backsset (8% weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following ingredients were added to the mixture: protease (0.60ml of a 1000-fold dilution of a commercially available protease), 0.2mg of Lactocide &Urea (0.85ml of 50% Urea Liquor diluted 10-fold). A hole was made in the cap of a 100ml bottle containing the mixture as CO₂A channel. The mixture was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48h, 82F was set.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

EXAMPLE 41

Example of fermentation of raw starch in corn expressing alpha-amylase and glucoamylase fusions

Transgenic corn kernels were harvested from transgenic plants prepared as described in example 28. The corn grain expresses, for example, SEQ ID NO: 46, which encodes an alpha-amylase and glucoamylase fusion as set forth in SEQ ID NO: 45, which targets the endoplasmic reticulum.

The corn kernels were ground into a flour as described in example 14. A slurry was then prepared containing 20g of corn meal, 23ml of deionized water and 6.0ml of backsset (8% by weight solids). Ammonium hydroxide was added to adjust the pH to 6.0. The following ingredients were added to the slurry: protease (0.60ml of a 1000-fold dilution of a commercially available protease), 0.2mg of Lactocide&Urea (0.85ml of 50% Urea Liquor diluted 10-fold). In a slurry-containing 10 A hole is dug on the bottle cap of the 0ml bottle to be used as CO₂A channel. The slurry was then inoculated with yeast (1.44ml) and incubated in a 90F water bath. After fermenting for 24 hours, the temperature is reduced to 86F; at 48h, 82F was set.

The inoculated yeast was propagated as described in example 14.

Samples were removed as described in example 14 and then analyzed as described in example 14.

All publications, patents and patent applications are herein incorporated by reference. Having thus described the invention by reference to certain of its preferred embodiments, numerous details are set forth in order to illustrate the invention, and it will be understood by those skilled in the art that: the present invention is susceptible to other embodiments and its several details are capable of modifications in various obvious respects, all without departing from the basic principles of the invention.

797GL3 alpha-amylase amino acid sequence (SEQ ID NO: 1)

MAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSM

GYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVLADIVINHRAGGDLEWNPF

VGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQES

YAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSG

AKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAY

AFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPG

LITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPANGQ

YGYSVWSYCGVG

797GL3 alpha-amylase maize optimized nucleic acid sequence (SEQ ID NO: 2)

ATGGCCAAGTACCTGGAGCTGGAGGAGGGCGGCGTGATCATGCAGGCGTTCTACTGGGA

CGTCCCGAGCGGAGGCATCTGGTGGGACACCATCCGCCAGAAGATCCCCGAGTGGTACG

ACGCCGGCATCTCCGCGATCTGGATACCGCCAGCTTCCAAGGGCATGTCCGGGGGCTACT

CGATGGGCTACGACCCGTACGACTACTTCGACCTCGGCGAGTACTACCAGAAGGGCACG

GTGGAGACGCGCTTCGGGTCCAAGCAGGAGCTCATCAACATGATCAACACGGCGCACGC

CTACGGCATCAAGGTCATCGCGGACATCGTGATCAACCACAGGGCCGGCGGCGACCTGG

AGTGGAACCCGTTCGTCGGCGACTACACCTGGACGGACTTCTCCAAGGTCGCCTCCGGCA

AGTACACCGCCAACTACCTCGACTTCCACCCCAACGAGCTGCACGCGGGCGACTCCGGC

ACGTTCGGCGGCTACCCGGACATCTGCCACGACAAGTCCTGGGACCAGTACTGGCTCTGG

GCCTCGCAGGAGTCCTACGCGGCCTACCTGCGCTCCATCGGCATCGACGCGTGGCGCTTC

GACTACGTCAAGGGCTACGGGGCCTGGGTGGTCAAGGACTGGCTCAACTGGTGGGGCGG

CTGGGCGGTGGGCGAGTACTGGGACACCAACGTCGACGCGCTGCTCAACTGGGCCTACT

CCTCCGGCGCCAAGGTGTTCGACTTCCCCCTGTACTACAAGATGGACGCGGCCTTCGACA

ACAAGAACATCCCGGCGCTCGTCGAGGCCCTGAAGAACGGCGGCACGGTGGTCTCCCGC

GACCCGTTCAAGGCCGTGACCTTCGTCGCCAACCACGACACGGACATCATCTGGAACAA

GTACCCGGCGTACGCCTTCATCCTCACCTACGAGGGCCAGCCCACGATCTTCTACCGCGA

CTACGAGGAGTGGCTGAACAAGGACAAGCTCAAGAACCTGATCTGGATTCACGACAACC

TCGCGGGCGGCTCCACTAGTATCGTGTACTACGACTCCGACGAGATGATCTTCGTCCGCA

ACGGCTACGGCTCCAAGCCCGGCCTGATCACGTACATCAACCTGGGCTCCTCCAAGGTGG

GCCGCTGGGTGTACGTCCCGAAGTTCGCCGGCGCGTGCATCCACGAGTACACCGGCAAC

CTCGGCGGCTGGGTGGACAAGTACGTGTACTCCTCCGGCTGGGTCTACCTGGAGGCCCCG

GCCTACGACCCCGCCAACGGCCAGTACGGCTACTCCGTGTGGTCCTACTGCGGCGTCGGC

6gp3 pullulanase amino acid sequence (SEQ ID NO: 3)

MGHWYKHQRAYQFTGEDDFGKVAVVKLPMDLTKVGIIVRLNEWQAKDVAKDRFIEIKDGK

AEVWILQGVEEIFYEKPDTSPRIFFAQARSNKVIEAFLTNPVDTKKKELFKVTVDGKEIPVSRV

EKADPTDIDVTNYVRIVLSESLKEEDLRKDVELIIEGYKPARVIMMEILDDYYYDGELGAVYS

PEKTIFRVWSPVSKWVKVLLFKNGEDTEPYQVVNMEYKGNGVWEAVVEGDLDGVFYLYQL

ENYGKIRTTVDPYSKAVYANNQESAVVNLARTNPEGWENDRGPKIEGYEDAIIYEIHIADITG

LENSGVKNKGLYLGLTEENTKGPGGVTTGLSHLVELGVTHVHILPFFDFYTGDELDKDFEKY

YNWGYDPYLFMVPEGRYSTDPKNPHTRIREVKEMVKALHKHGIGVIMDMVFPHTYGIGELS

AFDQTVPYYFYRIDKTGAYLNESGCGNVIASERPMMRKFIVDTVTYWVKEYHIDGFRFDQM

GLIDKKTMLEVERALHKIDPTIILYGEPWGGWGAPIRFGKSDVAGTHVAAFNDEFRDAIRGSV

FNPSVKGFVMGGYGKETKIKRGVVGSINYDGKLIKSFALDPEETINYAACHDNHTLWDKNY

LAAKADKKKEWTEEELKNAQKLAGAILLTSQGVPFLHGGQDFCRTTNFNDNSYNAPISINGF

DYERKLQFIDVFNYHKGLIKLRKEHPAFRLKNAEEIKKHLEFLPGGRRIVAFMLKDHAGGDP

WKDIVVIYNGNLEKTTYKLPEGKWNVVVNSQKAGTEVIETVEGTIELDPLSAYVLYRE

6gp3 pullulanase maize optimized nucleic acid sequence (SEQ ID NO: 4)

ATGGGCCACTGGTACAAGCACCAGCGCGCCTACCAGTTCACCGGCGAGGACGACTTCGG

GAAGGTGGCCGTGGTGAAGCTCCCGATGGACCTCACCAAGGTGGGCATCATCGTGCGCC

TCAACGAGTGGCAGGCGAAGGACGTGGCCAAGGACCGCTTCATCGAGATCAAGGACGGC

AAGGCCGAGGTGTGGATACTCCAGGGCGTGGAGGAGATCTTCTACGAGAAGCCGGACAC

CTCCCCGCGCATCTTCTTCGCCCAGGCCCGCTCCAACAAGGTGATCGAGGCCTTCCTCAC

CAACCCGGTGGACACCAAGAAGAAGGAGCTGTTCAAGGTGACCGTCGACGGCAAGGAG

ATCCCGGTGTCCCGCGTGGAGAAGGCCGACCCGACCGACATCGACGTGACCAACTACGT

GCGCATCGTGCTCTCCGAGTCCCTCAAGGAGGAGGACCTCCGCAAGGACGTGGAGCTGA

TCATCGAGGGCTACAAGCCGGCCCGCGTGATCATGATGGAGATCCTCGACGACTACTACT

ACGACGGCGAGCTGGGGGCGGTGTACTCCCCGGAGAAGACCATCTTCCGCGTGTGGTCC

CCGGTGTCCAAGTGGGTGAAGGTGCTCCTCTTCAAGAACGGCGAGGACACCGAGCCGTA

CCAGGTGGTGAACATGGAGTACAAGGGCAACGGCGTGTGGGAGGCCGTGGTGGAGGGC

GACCTCGACGGCGTGTTCTACCTCTACCAGCTGGAGAACTACGGCAAGATCCGCACCACC

GTGGACCCGTACTCCAAGGCCGTGTACGCCAACAACCAGGAGTCTGCAGTGGTGAACCT

CGCCCGCACCAACCCGGAGGGCTGGGAGAACGACCGCGGCCCGAAGATCGAGGGCTAC

GAGGACGCCATCATCTACGAGATCCACATCGCCGACATCACCGGCCTGGAGAACTCCGG

CGTGAAGAACAAGGGCCTCTACCTCGGCCTCACCGAGGAGAACACCAAGGCCCCGGGCG

GCGTGACCACCGGCCTCTCCCACCTCGTGGAGCTGGGCGTGACCCACGTGCACATCCTCC

CGTTCTTCGACTTCTACACCGGCGACGAGCTGGACAAGGACTTCGAGAAGTACTACAACT

GGGGCTACGACCCGTACCTCTTCATGGTGCCGGAGGGCCGCTACTCCACCGACCCGAAG

AACCCGCACACCCGAATTCGCGAGGTGAAGGAGATGGTGAAGGCCCTCCACAAGCACGG

CATCGGCGTGATCATGGACATGGTGTTCCCGCACACCTACGGCATCGGCGAGCTGTCCGC

CTTCGACCAGACCGTGCCGTACTACTTCTACCGCATCGACAAGACCGGCGCCTACCTCAA

CGAGTCCGGCTGCGGCAACGTGATCGCCTCCGAGCGCCCGATGATGCGCAAGTTCATCGT

GGACACCGTGACCTACTGGGTGAAGGAGTACCACATCGACGGCTTCCGCTTCGACCAGA

TGGGCCTCATCGACAAGAAGACCATGCTGGAGGTGGAGCGCGCCCTCCACAAGATCGAC

CCGACCATCATCCTCTACGGCGAGCCGTGGGGCGGCTGGGGGGCCCCGATCCGCTTCGG

CAAGTCCGACGTGGCCGGCACCCACGTGGCCGCCTTCAACGACGAGTTCCGCGACGCCA

TCCGCGGCTCCGTGTTCAACCCGTCCGTGAAGGGCTTCGTGATGGGCGGCTACGGCAAGG

AGACCAAGATCAAGCGCGGCGTGGTGGGCTCCATCAACTACGACGGCAAGCTCATCAAG

TCCTTCGCCCTCGACCCGGAGGAGACCATCAACTACGCCGCCTGCCACGACAACCACACC

CTCTGGGACAAGAACTACCTCGCCGCCAAGGCCGACAAGAAGAAGGAGTGGACCGAGG

AGGAGCTGAAGAACGCCCAGAAGCTCGCCGGCGCCATCCTCCTCACTAGTCAGGGCGTG

CCGTTCCTCCACGGCGGCCAGGACTTCTGCCGCACCACCAACTTCAACGACAACTCCTAC

AACGCCCCGATCTCCATCAACGGCTTCGACTACGAGCGCAAGCTCCAGTTCATCGACGTG

TTCAACTACCACAAGGGCCTCATCAAGCTCCGCAAGGAGCACCCGGCCTTCCGCCTCAAG

AACGCCGAGGAGATCAAGAAGCACCTGGAGTTCCTCCCGGGCGGGCGCCGCATCGTGGC

CTTCATGCTCAAGGACCACGCCGGCGGCGACCCGTGGAAGGACATCGTGGTGATCTACA

ACGGCAACCTGGAGAAGACCACCTACAAGCTCCCGGAGGGCAAGTGGAACGTGGTGGTG

AACTCCCAGAAGGCCGGCACCGAGGTGATCGAGACCGTGGAGGGCACCATCGAGCTGGA

CCCGCTCTCCGCCTACGTGCTCTACCGCGAG

Sulfolobus solfataricus malA alpha-glucosidase amino acid sequence (SEQ ID NO: 5)

METIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIVQQGNKVIVEKSLDLKEHIIG

LGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISVKDGVATGYFFNSASKVIFDV

GLEEYDKVIVTTPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPPMWAFGYMISRYSYYPQDK

VVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPKKLIDELHKRNVKLITIVDHGI

RVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFREDTREWWAGLISEWLSQGVD

GIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVVHYLRGKRVKHEKVRNAYPL

YEAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPSWDDLKLQLQLVLGLSISGVP

FVGCDIGCFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKATDGIDTEPVFLPDYYKEKVK

EIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRIEDEYMVGKYLLYAPIVSKEE

SRLVTLPRGKWYNYWNGEIINGKSVVKSTHELPIYLREGSIIPLEGDELIVYGETSFKRYDNAE

ITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQNTYVAKINQKIRGKINLE

Sulfolobus solfataricus malA alpha-glucosidase maize optimized nucleic acid sequence (SEQ ID NO: 6)

ATGGAGACCATCAAGATCTACGAGAACAAGGGCGTGTACAAGGTGGTGATCGGCGAGCC

GTTCCCGCCGATCGAGTTCCCGCTCGAGCAGAAGATCTCCTCCAACAAGTCCCTCTCCGA

GCTGGGCCTCACCATCGTGCAGCAGGGCAACAAGGTGATCGTGGAGAAGTCCCTCGACC

TCAAGGAGCACATCATCGGCCTCGGCGAGAAGGCCTTCGAGCTGGACCGCAAGCGCAAG

CGCTACGTGATGTACAACGTGGACGCCGGCGCCTACAAGAAGTACCAGGACCCGCTCTA

CGTGTCCATCCCGCTCTTCATCTCCGTGAAGGACGGCGTGGCCACCGGCTACTTCTTCAA

CTCCGCCTCCAAGGTGATCTTCGACGTGGGCCTCGAGGAGTACGACAAGGTGATCGTGA

CCATCCCGGAGGACTCCGTGGAGTTCTACGTGATCGAGGGCCCGCGCATCGAGGACGTG

CTCGAGAAGTACACCGAGCTGACCGGCAAGCCGTTCCTCCCGCCGATGTGGGCCTTCGGC

TACATGATCTCCCGCTACTCCTACTACCCGCAGGACAAGGTGGTGGAGCTGGTGGACATC

ATGCAGAAGGAGGGCTTCCGCGTGGCCGGCGTGTTCCTCGACATCCACTACATGGACTCC

TACAAGCTCTTCACCTGGCACCCGTACCGCTTCCCGGAGCCGAAGAAGCTCATCGACGAG

CTGCACAAGCGCAACGTGAAGCTCATCACCATCGTGGACCACGGCATCCGCGTGGACCA

GAACTACTCCCCGTTCCTCTCCGGCATGGGCAAGTTCTGCGAGATCGAGTCCGGCGAGCT

GTTCGTGGGCAAGATGTGGCCGGGCACCACCGTGTACCCGGACTTCTTCCGCGAGGACA

CCCGCGAGTGGTGGGCCGGCCTCATCTCCGAGTGGCTCTCCCAGGGCGTGGACGGCATCT

GGCTCGACATGAACGAGCCGACCGACTTCTCCCGCGCCATCGAGATCCGCGACGTGCTCT

CCTCCCTCCCGGTGCAGTTCCGCGACGACCGCCTCGTGACCACCTTCCCGGACAACGTGG

TGCACTACCTCCGCGGCAAGCGCGTGAAGCACGAGAAGGTGCGCAACGCCTACCCGCTC

TACGAGGCGATGGCCACCTTCAAGGGCTTCCGCACCTCCCACCGCAACGAGATCTTCATC

CTCTCCCGCGCCGGCTACGCCGGCATCCAGCGCTACGCCTTCATCTGGACCGGCGACAAC

ACCCCGTCCTGGGACGACCTCAAGCTCCAGCTCCAGCTCGTGCTCGGCCTCTCCATCTCC

GGCGTGCCGTTCGTGGGCTGCGACATCGGCGGCTTCCAGGGCCGCAACTTCGCCGAGATC

GACAACTCGATGGACCTCCTCGTGAAGTACTACGCCCTCGCCCTCTTCTTCCCGTTCTACC

GCTCCCACAAGGCCACCGACGGCATCGACACCGAGCCGGTGTTCCTCCCGGACTACTAC

AAGGAGAAGGTGAAGGAGATCGTGGAGCTGCGCTACAAGTTCCTCCCGTACATCTACTC

CCTCGCCCTCGAGGCCTCCGAGAAGGGCCACCCGGTGATCCGCCCGCTCTTCTACGAGTT

CCAGGACGACGACGACATGTACCGCATCGAGGACGAGTACATGGTGGGCAAGTACCTCC

TCTACGCCCCGATCGTGTCCAAGGAGGAGTCCCGCCTCGTGACCCTCCCGCGCGGCAAGT

GGTACAACTACTGGAACGGCGAGATCATCAACGGCAAGTCCGTGGTGAAGTCCACCCAC

GAGCTGCCGATCTACCTCCGCGAGGGCTCCATCATCCCGCTCGAGGGCGACGAGCTGATC

GTGTACGGCGAGACCTCCTTCAAGCGCTACGACAACGCCGAGATCACCTCCTCCTCCAAC

GAGATCAAGTTCTCCCGCGAGATCTACGTGTCCAAGCTCACCATCACCTCCGAGAAGCCG

GTGTCCAAGATCATCGTGGACGACTCCAAGGAGATCCAGGTGGAGAAGACCATGCAGAA

CACCTACGTGGCCAAGATCAACCAGAAGATCCGCGGCAAGATCAACCTCGAGTGA

Waxy cDNA sequence (SEQ ID NO: 7)

ATGGCGGCTCTGGCCACGTCGCAGCTCGTCGCAACGCGCGCCGGCCTGGGCGTCCCGGA

CGCGTCCACGTTCCGCCGCGGCGCCGCGCAGGGCCTGAGGGGGGCCCGGGCGTCGGCGG

CGGCGGACACGCTCAGCATGCGGACCAGCGCGCGCGCGGCGCCCAGGCACCAGCACCAG

CAGGCGCGCCGCGGGGCCAGGTTCCCGTCGCTCGTCGTGTGCGCCAGCGCCGGCATGAA

CGTCGTCTTCGTCGGCGCCGAGATGGCGCCGTGGAGCAAGACCGGAGGCCTCGGCGACG

TCCTCGGCGGCCTGCCGCCGGCCATGGCCGCGAACGGGCACCGTGTCATGGTCGTCTCTC

CCCGCTACGACCAGTACAAGGACGCCTGGGACACCAGCGTCGTGTCCGAGATCAAGATG

GGAGACGGGTACGAGACGGTCAGGTTCTTCCACTGCTACAAGCGCGGAGTGGACCGCGT

GTTCGTTGACCACCCACTGTTCCTGGAGAGGGTTTGGGGAAAGACCGAGGAGAAGATCT

ACGGGCCTGTCGCTGGAACGGACTACAGGGACAACCAGCTGCGGTTCAGCCTGCTATGC

CAGGCAGCACTTGAAGCTCCAAGGATCCTGAGCCTCAACAACAACCCATACTTCTCCGG

ACCATACGGGGAGGACGTCGTGTTCGTCTGCAACGACTGGCACACCGGCCCTCTCTCGTG

CTACCTCAAGAGCAACTACCAGTCCCACGGCATCTACAGGGACGCAAAGACCGCTTTCT

GCATCCACAACATCTCCTACCAGGGCCGGTTCGCCTTCTCCGACTACCCGGAGCTGAACC

TCCCCGAGAGATTCAAGTCGTCCTTCGATTTCATCGACGGCTACGAGAAGCCCGTGGAAG

GCCGGAAGATCAACTGGATGAAGGCCGGGATCCTCGAGGCCGACAGGGTCCTCACCGTC

AGCCCCTACTACGCCGAGGAGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTCGACAA

CATCATGCGCCTCACCGGCATCACCGGCATCGTCAACGGCATGGACGTCAGCGAGTGGG

ACCCCAGCAGGGACAAGTACATCGCCGTGAAGTACGACGTGTCGACGGCCGTGGAGGCC

AAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACA

TCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGCCCCGACGTCATGGCG

GCCGCCATCCCGCAGCTCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCAC

GGGCAAGAAGAAGTTCGAGCGCATGCTCATGAGCGCCGAGGAGAAGTTCCCAGGCAAG

GTGCGCGCCGTGGTCAAGTTCAACGCGGCGCTGGCGCACCACATCATGGCCGGCGCCGA

CGTGCTCGCCGTCACCAGCCGCTTCGAGCCCTGCGGCCTCATCCAGCTGCAGGGGATGCG

ATACGGAACGCCCTGCGCCTGCGCGTCCACCGGTGGACTCGTCGACACCATCATCGAAG

GCAAGACCGGGTTCCACATGGGCCGCCTCAGCGTCGACTGCAACGTCGTGGAGCCGGCG

GACGTCAAGAAGGTGGCCACCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGC

GTACGAGGAGATGGTGAGGAACTGCATGATCCAGGATCTCTCCTGGAAGGGCCCTGCCA

AGAACTGGGAGAACGTGCTGCTCAGCCTCGGGGTCGCCGGCGGCGAGCCAGGGGTTGAA

GGCGAGGAGATCGCGCCGCTCGCCAAGGAGAACGTGGCCGCGCCC

Waxy amino acid sequence (SEQ ID NO: 8)

MAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRHQHQQ

ARRGARFPSLVVCASAGMNVVFVGAEMAPWSKTGGLGDVLGGLPPAMAANGHRVMVVSP

RYDQYKDAWDTSVVSEIKMGDGYETVRFFHCYKRGVDRVFVDHPLFLERVWGKTEEKIYG

PVAGTDYRDNQLRFSLLCQAALEAPRILSLNNNPYFSGPYGEDVVFVCNDWHTGPLSCYLKS

NYQSHGIYRDAKTAFCIHNISYQGRFAFSDYPELNLPERFKSSFDFIDGYEKPVEGRKINWMK

AGILEADRVLTVSPYYAEELISGIARGCELDNIMRLTGITGIVNGMDVSEWDPSRDKYIAVKY

DVSTAVEAKALNKEALQAEVGLPVDRNIPLVAFIGRLEEQKGPDVMAAAIPQLMEMVEDVQ

IVLLGTGKKKFERMLMSAEEKFPGKVRAVVKFNAALAHHIMAGADVLAVTSRFEPCGLIQL

QGMRYGTPCACASTGGLVDTIIEGKTGFHMGRLSVDCNVVEPADVKKVATTLQRAIKVVGT

PAYEEMVRNCMIQDLSWKGPAKNWENVLLSLGVAGGEPGVEGEELAPLAKEMVAAP

797 GL3/wax nucleic acid sequence (SEQ ID NO: 9)

ATGGCCAAGTACCTGGAGCTGGAGGAGGGCGGCGTGATCATGCAGGCGTTCTACTGGGA

CGTCCCGAGCGGAGGCATCTGGTGGGACACCATCCGCCAGAAGATCCCCGAGTGGTACG

ACGCCGGCATCTCCGCGATCTGGATACCGCCAGCTTCCAAGGGCATGTCCGGGGGCTACT

CGATGGGCTACGACCCGTACGACTACTTCGACCTCGGCGAGTACTACCAGAAGGGCACG

GTGGAGACGCGCTTCGGGTCCAAGCAGGAGCTCATCAACATGATCAACACGGCGCACGC

CTACGGCATCAAGGTCATCGCGGACATCGTGATCAACCACAGGGCCGGCGGCGACCTGG

AGTGGAACCCGTTCGTCGGCGACTACACCTGGACGGACTTCTCCAAGGTCGCCTCCGGCA

AGTACACCGCCAACTACCTCGACTTCCACCCCAACGAGCTGCACGCGGGCGACTCCGGC

ACGTTCGGCGGCTACCCGGACATCTGCCACGACAAGTCCTGGGACCAGTACTGGCTCTGG

GCCTCGCAGGAGTCCTACGCGGCCTACCTGCGCTCCATCGGCATCGACGCGTGGCGCTTC

GACTACGTCAAGGGCTACGGGGCCTGGGTGGTCAAGGACTGGCTCAACTGGTGGGGCGG

CTGGGCGGTGGGCGAGTACTGGGACACCAACGTCGACGCGCTGCTCAACTGGGCCTACT

CCTCCGGCGCCAAGGTGTTCGACTTCCCCCFGTACTACAAGATGGACGCGGCCTTCGACA

ACAAGAACATCCCGGCGCTCGTCGAGGCCCTGAAGAACGGCGGCACGGTGGTCTCCCGC

GACCCGTTCAAGGCCGTGACCTTCGTCGCCAACCACGACACGGACATCATCTGGAACAA

GTACCCGGCGTACGCCTTCATCCTCACCTACGAGGGCCAGCCCACGATCTTCTACCGCGA

CTACGAGGAGTGGCTGAACAAGGACAAGCTCAAGAACCTGATCTGGATTCACGACAACC

TCGCGGGCGGCTCCACTAGTATCGTGTACTACGACTCCGACGAGATGATCTTCGTCCGCA

ACGGCTACGGCTCCAAGCCCGGCCTGATCACGTACATCAACCTGGGCTCCTCCAAGGTGG

GCCGCTGGGTGTACGTCCCGAAGTTCGCCGGCGCGTGCATCCACGAGTACACCGGCAAC

CTCGGCGGCTGGGTGGACAAGTACGTGTACTCCTCCGGCTGGGTCTACCTGGAGGCCCCG

GCCTACGACCCCGCCAACGGCCAGTACGGCTACTCCGTGTGGTCCTACTGCGGCGTCGGC

ACATCGATTGCTGGCATCCTCGAGGCCGACAGGGTCCTCACCGTCAGCCCCTACTACGCC

GAGGAGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCPCGACAACATCATGCGCCTCAC

CGGCATCACCGGCATCGTCAACGGCATGGACGTCAGCGAGTGGGACCCCAGCAGGGACA

AGTACATCGCCGTGAAGTACGACGTGTCGACGGCCGTGGAGGCCAAGGCGCTGAACAAG

GAGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACATCCCGCTGGTGGCGTT

CATCGGCAGGCTGGAAGAGCAGAAGGGCCCCGACGTCATGGCGGCCGCCATCCCGCAGC

TCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAAGAAGAAGTTC

GAGCGCATGCTCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCGTGGTCAA

GTTCAACGCGGCGCTGGCGCACCACATCATGGCCGGCGCCGACGTGCTCGCCGTCACCA

GCCGCTTCGAGCCCTGCGGCCTCATCCAGCTGCAGGGGATGCGATACGGAACGCCCTGC

GCCTGCGCGTCCACCGGTGGACTCGTCGACACCATCATCGAAGGCAAGACCGGGTTCCA

CATGGGCCGCCTCAGCGTCGACTGCAACGTCGTGGAGCCGGCGGACGTCAAGAAGGTGG

CCACCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGAGGAGATGGTG

AGGAACTGCATGATCCAGGATCTCTCCTGGAAGGGCCCTGCCAAGAACTGGGAGAACGT

GCTGCTCAGCCTCGGGGTCGCCGGCGGCGAGCCAGGGGTTGAAGGCGAGGAGATCGCGC

CGCTCGCCAAGGAGAACGTGGCCGCGCCC

797 GL3/wax amino acid sequence (SEQ ID NO: 10)

MAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSM

GYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVIADIVINHRAGGDLEWNPF

VGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQES

YAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGEYWDTNVDALLNWAYSSG

AKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAY

AFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVYYDSDEMIFVRNGYGSKPG

LITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYSSGWVYLEAPAYDPANGQ

YGYSVWSYCGVGTSIAGILEADRVLTVSPYYAEELISGLARGCELDNIMRLTGITGIVNGMDV

SEWDPSRDKYIAVKYDVSTAVEAKALNKEALQAEVGLPVDRNIPLVAFIGRLEEQKGPDVM

AAAIPQLMEMVEDVQIVLLGTGKKKFERMLMSAEEKFPGKVRAVVKFNAALAHHIMAGAD

VLAVTSRFEPCGLIQLQGMRYGTPCACASTGGLVDTIIEGKTGFHMGRLSVDCNVVEPADVK

KVATTLQRAIKVVGTPAYEEMVRNCMIQDLSWKGPAKNWENVLLSLGVAGGEPGVEGEEIA

PLAKENVAAP

Maize ADP-gpp promoter nucleic acid sequence (SEQ ID NO: 11)

GGAGAGCTATGAGACGTATGTCCTCAAAGCCACTTTGCATTGTGTGAAACCAATATCGAT

CTTTGTTACTTCATCATGCATGAACATTTGTGGAAACTACTAGCTTACAAGCATTAGTGA

CAGCTCAGAAAAAAGTTATCTATGAAAGGTTTCATGTGTACCGTGGGAAATGAGAAATG

TTGCCAACTCAAACACCTTCAATATGTTGTTTGCAGGCAAACTCTTCTGGAAGAAAGGTG

TCTAAAACTATGAACGGGTTACAGAAAGGTATAAACCACGGCTGTGCATTTTGGAAGTA

TCATCTATAGATGTCTGTTGAGGGGAAAGCCGTACGCCAACGTTATTTACTCAGAAACAG

CTTCAACACACAGTTGTCTGCTTTATGATGGCATCTCCACCCAGGCACCCACCATCACCT

ATCTCTCGTGCCTGTTTATTTTCTTGCCCTTTCTGATCATAAAAAAACATTAAGAGTTTGC

AAACATGCATAGGCATATCAATATGCTCATTTATTAATTTGCTAGCAGATCATCTTCCTAC

TCTTTACTTTATTTATTGTTTGAAAAATATGTCCTGCACCTAGGGAGCTCGTATACAGTAC

CAATGCATCTTCATTAAATGTGAATTTCAGAAAGGAAGTAGGAACCTATGAGAGTATTT

TCAAAATTAATTAGCGGCTTCTATTATGTTTATAGCAAAGGCCAAGGGCAAAATTGGAAC

ACTAATGATGGTTGGTTGCATGAGTCTGTCGATTACTTGCAAGAAATGTGAACCTTTGTT

TCTGTGCGTGGGCATAAAACAAACAGCTTCTAGCCTCTTTTACGGTACTTGCACTTGCAA

GAAATGTGAACTCCTTTTCATTTCTGTATGTGGACATAATGCCAAAGCATCCAGGCTTTTT

CATGGTTGTTGATGTCTTTACACAGTTCATCTCCACCAGTATGCCCTCCTCATACTCTATA

TAAACACATCAACAGCATCGCAATTAGCCACAAGATCACTTCGGGAGGCAAGTGCGATT

TCGATCTCGCAGCCACCTTTTTTTGTTCTGTTGTAAGTATACCTTCCCTTACCATCTTTATC

TGTTAGTTTAATTTGTAATTGGGAAGTATTAGTGGAAAGAGGATGAGATGCTATCATCTA

TGTACTCTGCAAATGCATCTGACGTTATATGGGCTGCTTCATATAATTTGAATTGCTCCAT

TCTTGCCGACAATATATTGCAAGGTATATGCCTAGTTCCATCAAAAGTTCTGTTTTTTCAT

TCTAAAAGCATTTTAGTGGCACACAATTTTTGTCCATGAGGGAAAGGAAATCTGTTTTGG

TTACTTTGCTTGAGGTGCATTCTTCATATGTCCAGTTTTATGGAAGTAATAAACTTCAGTT

TGGTCATAAGATGTCATATTAAAGGGCAAACATATATTCAATGTTCAATTCATCGTAAAT

GTTCCCTTTTTGTAAAAGATTGCATACTCATTTATTTGAGTTGCAGGTGTATCTAGTAGTT

GGAGGAG

Maize gamma-zein nucleic acid sequence (SEQ ID NO: 12)

GATCATCCAGGTGCAACCGTATAAGTCCTAAAGTGGTGAGGAACACGAAACAACCATGC

ATTGGCATGTAAAGCTCCAAGAATTTGTTGTATCCTTAACAACTCACAGAACATCAACCA

AAATTGCACGTCAAGGGTATTGGGTAAGAAACAATCAAACAAATCCTCTCTGTGTGCAA

AGAAACACGGTGAGTCATGCCGAGATCATACTCATCTGATATACATGCTTACAGCTCACA

AGACATTACAAACAACTCATATTGCATTACAAAGATCGTTTCATGAAAAATAAAATAGG

CCGGACAGGACAAAAATCCTTGACGTGTAAAGTAAATTTACAACAAAAAAAAAGCCATA

TGTCAAGCTAAATCTAATTCGTTTTACGTAGATCAACAACCTGTAGAAGGCAACAAAACT

GAGCCACGCAGAAGTACAGAATGATTCCAGATGAACCATCGACGTGCTACGTAAAGAGA

GTGACGAGTCATATACATTTGGCAAGAAACCATGAAGCTGCCTACAGCCGTCTCGGTGG

CATAAGAACACAAGAAATTGTGTTAATTAATCAAAGCTATAAATAACGCTCGCATGCCT

GTGCACTTCTCCATCACCACCACTGGGTCTTCAGACCATTAGCTTTATCTACTCCAGAGCG

CAGAAGAACCCGATCGACA

pNOV6200 Amylase fusion amino acid sequence (SEQ ID NO: 13)

MRVLLVALALLALAASATSAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGI

SAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVI

ADIVINNHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDI

CHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGE

YWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAV

TFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVY

YDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYS

SGWVYLEAPAYDPANGQYGYSVWSYCGVG

pNOV6201 Amylase fusion amino acid sequence (SEQ IE NO: 14)

MRVLLVALALLALAASATSAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGI

SAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVI

ADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDI

CHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGE

YWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAV

TFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVY

YDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYS

SGWVYLEAPAYDPANGQYGYSVWSYCGVGSEKDEL

pNOV4029 Amylase fusion amino acid sequence (SEQ ID NO: 15)

MLAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRHQHQ

QARRGARFPSLVVCASAGAMAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDA

GISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIK

VIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYP

DICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAV

GEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFK

AVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSI

VYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYV

YSSGWVYLEAPAYDPANGQYGYSVWSYCGVGTSI

pNOV4031 amylase fusion amino acid sequence (SEQ ID NO: 16)

MLAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRHQHQ

QARRGARFPSLVVCASAGAMAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDA

GISAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIK

VIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYP

DICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAV

GEYWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFK

AVTFVANHDTDIIWNKYAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSI

VYYDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYV

YSSGWVYLEAPAYDPANGQYGYSVWSYCGVGTSIAGILEADRVLTVSPYYAEELISGIARGC

ELDNIMRLTGITGIVNGMDVSEWDPSRDKYIAVKYDVSTAVEAKALNKEALQAEVGLPVDR

NIPLVAFIGRLEEQKGPDVMAAAIPQLMEMVEDVQIVLLGTGKKKFERMLMSAEEKFPGKVR

AVVKFNAALAHHIMAGADVLAVTSRFEPCGLIQLQGMRYGTPCACASTGGLVDTIIEGKTGF

HMGRLSVDCNVVEPADVKKVATTLQRAIKVVGTPAYEEMVRNCMIQDLSWKGPAKNWEN

VLLSLGVAGGEPGVEGEEI

APLAKENVAAP

Maize gamma-maize zymolytic protein N-terminal signal sequence (SEQ ID NO: 17)

(MRVLLVALALLALAASATS)

Thermotoga maritima (Thermotoga maritima) glucose isomerase amino acid sequence (SEQ ID NO: 18)

MAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPT

AERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIK

ERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVF

WGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIGFTGQFLIEPKPKEPTKHQYDFDVAT

AYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQ

FPTNIYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKIAYK

LAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKEDIELPSGKQEYLESLLNSYIV

KTIAELR

Thermotoga maritima glucose isomerase-maize optimized nucleic acid sequence (SEQ ID NO: 19)

ATGGCCGAGTTCTTCCCGGAGATCCCGAAGATCCAGTTCGAGGGCAAGGAGTCCACCAA

CCCGCTCGCCTTCCGCTTCTACGACCCGAACGAGGTGATCGACGGCAAGCCGCTCAAGG

ACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGT

TCGGCGACCCGACCGCCGAGCGCCCGTGGAACCGCTTCTCCGACCCGATGGACAAGGCC

TTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGC

TTCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCT

CGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCT

GGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCT

CCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAG

GAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCT

CAACACCGACCTCGGCCTGGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGA

GTACGCCAAGAAGATCGGCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGC

CGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGAACCAC

GGCCTCGAC

GAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAG

CACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGG

CGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACATCTACGACACCACCCT

CGCCATGTACGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACG

CCAAGGTGCGCCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGC

ATGGACACCTTCGCCCTCGGCTTCAAGATCGCCTACAAGCTCGCCAAGGACGGCGTGTTC

GACAAGTTCATCGAGGAGAAGTACCGCTCCTTCAAGGAGGGCATCGGCAAGGAGATCGT

GGAGGGCAAGACCGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGGACATC

GAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCCTCAACTCCTACATCGTGAAG

ACCATCGCCGAGCTGCGCTGA

Thermotoga neapoliana (Thermotoga neocolitana) glucose isomerase amino acid sequence (SEQ ID NO: 20)

MAEFFPEIPKVQFEGKFSTNPLAFKFYDPEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPT

ADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERI

KERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYV

FWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQFLIEPKPKEPTKHQYDFDVA

TAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTD

QFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKVA

YKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFEKLEEYIIDKETIELPSGKQEYLESLINSY

IVKTILELR

Nonelles Thermotoga glucose isomerase maize optimized nucleic acid sequence (SEQ ID NO: 21)

ATGGCCGAGTTCTTCCCGGAGATCCCGAAGGTGCAGTTCGAGGGCAAGGAGTCCACCAA

CCCGCTCGCCTTCAAGTTCTACGACCCGGAGGAGATCATCGACGGCAAGCCGCTCAAGG

ACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGT

TCGGCGACCCGACCGCCGACCGCCCGTGGAACCGCTACACCGACCCGATGGACAAGGCC

TTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGC

TTCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCT

CGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCT

GGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCT

CCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAG

GAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCT

CAACACCGACCTCGGCTTCGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGA

CTACGCCAAGCGCATCGGCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGC

CGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGTCCCACG

GCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCAC

ACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCC

AACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACGTGTACGA

CACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCA

ACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACA

TCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGGTGGCCTACAAGCTCGTGAAGGACG

GCGTGCTCGACAAGTTCATCGAGGAGAAGTACCGCTCCTTCCGCGAGGGCATCGGCCGC

GACATCGTGGAGGGCAAGGTGGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGG

AGACCATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCATCAACTCCTAC

ATCGTGAAGACCATCCTGGAGCTGCGCTGA

SV57(SEQ ID NO：22)(5’AGCGAATTCATGGCGGCTCTGGCCACGT 3’)

SV58(SEQ ID NO：23)(5’AGCTAAGCTTCAGGGCGCGGCCACGTTCT 3’)

pNOV 70056 GP3 fusion amino acid sequence (SEQ ID NO: 24)

MRVLLVALALLALAASATSAGHWYKHQRAYQFTGEDDFGKVAVVKLPMDLTKVGIIVRLN

EWQAKDVAKDRFIFIKDGKAEVWILQGVEEIFYEKPDTSPRIFFAQARSNKVIEAFLTNPVDT

KKKELFKVTVDGKEIPVSRVEKADPTDIDVTNYVRIVLSESLKEEDLRKDVELIIEGYKPARVI

MMEILDDYYYDGELGAVYSPEKTIFRVWSPVSKWVKVLLFKNGEDTEPYQVVNMEYKGNG

VWEAVVEGDLDGVFYLYQLENYGKUIRTTVDPYSKAVYANNQESAVVNLARTNPEGWENDR

GPKIEGYEDAIIYEIHIADITGLENSGVKNKGLYLGLTEENTKAPGGVTTGLSHLVELGVTHVH

ILPFFDFYTGDELDKDFEKYYNWGYDPYLFMVPEGRYSTDPKNPHTRIREVKEMVKALHKH

GIGVIMDMVFPHTYGIGELSAFDQTVPYYFYRIDKTGAYLNESGCGNVIASERPMMRKFIVDT

VTYWVKEYHIDGFRFDQMGLIDKKTMLEVERALHKIDPTIILYGEPWGGWGAPIRFGKSDVA

GTHVAAFNDEFRDAIRGSVFNPSVKGFVMGGYGKETKIKRGVVGSINYDGKLIKSFALDPEE

TINYAACHDNHTLWDKNYLAAKADKKKEWTEEELKNAQKLAGAILLTSQGVPFLHGGQDF

CRTTNFNDNSYNAPISINGFDYERKLQFIDVFNYHKGLIKLRKEHPAFRLKNAEEIKKHLEFLP

GGRRIVAFMLKDHAGGDPWKDIVVIYNGNLEKTTYKLPEGKWNVVVNSQKAGTEVIETVEG

TIELDPLSAYVLYRESEKDEL

pNOV 70056 GP3 fusion maize optimized nucleic acid sequence (SEQ ID NO: 25)

ATGAGGGTGTTGCTCGTTGCCCTCGCTCTCCTGGCTCTCGCTGCGAGCGCCACCAGCGCT

GGCCACTGGTACAAGCACCAGCGCGCCTACCAGTTCACCGGCGAGGACGACTTCGGGAA

GGTGGCCGTGGTGAAGCTCCCGATGGACCTCACCAAGGTGGGCATCATCGTGCGCCTCA

ACGAGTGGCAGGCGAAGGACGTGGCCAAGGACCGCTTCATCGAGATCAAGGACGGCAA

GGCCGAGGTGTGGATACTCCAGGGCGTGGAGGAGATCTTCTACGAGAAGCCGGACACCT

CCCCGCGCATCTTCTTCGCCCAGGCCCGCTCCAACAAGGTGATCGAGGCCTTCCTCACCA

ACCCGGTGGACACCAAGAAGAAGGAGCTGTTCAAGGTGACCGTCGACGGCAAGGAGAT

CCCGGTGTCCCGCGTGGAGAAGGCCGACCCGACCGACATCGACGTGACCAACTACGTGC

GCATCGTGCTCTCCGAGTCCCTCAAGGAGGAGGACCTCCGCAAGGACGTGGAGCTGATC

ATCGAGGGCTACAAGCCGGCCCGCGTGATCATGATGGAGATCCTCGACGACTACTACTA

CGACGGCGAGCTGGGGGCGGTGTACTCCCCGGAGAAGACCATCTTCCGCGTGTGGTCCC

CGGTGTCCAAGTGGGTGAAGGTGCTCCTCTTCAAGAACGGCGAGGACACCGAGCCGTAC

CAGGTGGTGAACATGGAGTACAAGGGCAACGGCGTGTGGGAGGCCGTGGTGGAGGGCG

ACCTCGACGGCGTGTTCTACCTCTACCAGCTGGAGAACTACGGCAAGATCCGCACCACCG

TGGACCCGTACTCCAAGGCCGTGTACGCCAACAACCAGGAGTCTGCAGTGGTGAACCTC

GCCCGCACCAACCCGGAGGGCTGGGAGAACGACCGCGGCCCGAAGATCGAGGGCTACG

AGGACGCCATCATCTACGAGATCCACATCGCCGACATCACCGGCCTGGAGAACTCCGGC

GTGAAGAACAAGGGCCTCTACCTCGGCCTCACCGAGGAGAACACCAAGGCCCCGGGCGG

CGTGACCACCGGCCTCTCCCACCTCGTGGAGCTGGGCGTGACCCACGTGCACATCCTCCC

GTTCTTCGACTTCTACACCGGCGACGAGCTGGACAAGGACTTCGAGAAGTACTACAACTG

GGGCTACGACCCGTACCTCTTCATGGTGCCGGAGGGCCGCTACTCCACCGACCCGAAGA

ACCCGCACACCCGAATTCGCGAGGTGAAGGAGATGGTGAAGGCCCTCCACAAGCACGGC

ATCGGCGTGATCATGGACATGGTGTTCCCGCACACCTACGGCATCGGCGAGCTGTCCGCC

TTCGACCAGACCGTGCCGTACTACTTCTACCGCATCGACAAGACCGGCGCCTACCTCAAC

GAGTCCGGCTGCGGCAACGTGATCGCCTCCGAGCGCCCGATGATGCGCAAGTTCATCGT

GGACACCGTGACCTACTGGGTGAAGGAGTACCACATCGACGGCTTCCGCTTCGACCAGA

TGGGCCTCATCGACAAGAAGACCATGCTGGAGGTGGAGCGCGCCCTCCACAAGATCGAC

CCGACCATCATCCTCTACGGCGAGCCGTGGGGCGGCTGGGGGGCCCCGATCCGCTTCGG

CAAGTCCGACGTGGCCGGCACCCACGTGGCCGCCTTCAACGACGAGTTCCGCGACGCCA

TCCGCGGCTCCGTGTTCAACCCGTCCGTGAAGGGCTTCGTGATGGGCGGCTACGGCAAGG

AGACCAAGATCAAGCGCGGCGTGGTGGGCTCCATCAACTACGACGGCAAGCTCATCAAG

TCCTTCGCCCTCGACCCGGAGGAGACCATCAACTACGCCGCCTGCCACGACAACCACACC

CTCTGGGACAAGAACTACCTCGCCGCC

AAGGCCGACAAGAAGAAGGAGTGGACCGAGGAGGAGCTGAAGAACGCCCAGAAGCTCG

CCGGCGCCATCCTCCTCACTAGTCAGGGCGTGCCGTTCCTCCACGGCGGCCAGGACTTCT

GCCGCACCACCAACTTCAACGACAACTCCTACAACGCCCCGATCTCCATCAACGGCTTCG

ACTACGAGCGCAAGCTCCAGTTCATCGACGTGTTCAACTACCACAAGGGCCTCATCAAGC

TCCGCAAGGAGCACCCGGCCTTCCGCCTCAAGAACGCCGAGGAGATCAAGAAGCACCTG

GAGTTCCTCCCGGGCGGGCGCCGCATCGTGGCCTTCATGCTCAAGGACCACGCCGGCGG

CGACCCGTGGAAGGACATCGTGGTGATCTACAACGGCAACCTGGAGAAGACCACCTACA

AGCTCCCGGAGGGCAAGTGGAACGTGGTGGTGAACTCCCAGAAGGCCGGCACCGAGGTG

ATCGAGACCGTGGAGGGCACCATCGAGCTGGACCCGCTCTCCGCCTACGTGCTCTACCGC

GAGTCCGAGAAGGACGAGCTGTGA

pNOV4831 malA fusion amino acid sequence (SEQ ID NO: 26)

MRVLLVALALLALAASATSMEIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIV

QQGNKVIVEKSLDLKEHIIGLGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISV

KDGVATGYFFNSASKVIFDVGLEEYDKVIVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPP

MWAFGYMISRYSYYPQDKVVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPK

KLIDELHKNRVKLITIVDHGIRVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFRE

DTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVV

HYLRGKRVKHEKVRNAYPLYEAMATFKGFRRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPS

WDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKA

TDGIDTEPVFLPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRI

EDEYMVGKYLLYAPIVSKEESRLVTLPRGKWYNYWNGEIINGKSVVKSTHELPIYLREGSIIP

LEGDELIVYGETSFKRYDNAEITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQ

NTYVAKINQKIRGKINLESEKDEL

pNOV4839malA fusion amino acid sequence (SEQ ID NO: 27)

MRVLLVALALLALAASATSMETIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIV

QQGNKVIVEKSLDLKEHIIGLGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISV

KDGVATGYFFNSASKVIFDVGLEEYDKVIVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPP

MWAFGYMISRYSYYPQDKVVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPK

KLIDELHKRNVKLITIVDHGIRVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFRE

DTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVV

HYLRGKRVKHEKVRNAYPLYEAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPS

WDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKA

TDGIDTEPVFLPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRI

EDEYMVGKYLLYAPIVSKEESRLVTLPRGKWYNYWNGEIINGKSVVKSTHEELPIYLREGSIIP

LEGDELIVYGETSFKRYDNAEITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQ

NTYVAKINQKIRGKINLE

pNOV4832 glucose isomerase fusion amino acid sequence (SEQ ID NO: 28)

MRVLLVALALLALAASATSMAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFS

VAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAP

EGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAA

QVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIGFTGQF

LIEPKPKEPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGK

LGSIDANQGDLLLGWDTDQFPTIYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDL

FIGHLAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKE

DIELPSGKQEYLESLLNSYIVKTIAELRSEKDEL

pNOV4833 glucose isomerase fusion amino acid sequence (SEQ ID NO: 29)

MRVLLVALALLALAASATSMAEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGKPLKDHLKFS

VAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIA

PEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAA

AQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQ

FLIEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILG

KLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVE

DLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFEKLEEYIID

KETIELPSGKQEYLESLINSYIVKTILELRSEKDEL

pNOV4840 glucose isomerase fusion amino acid sequence (SEQ ID NO: 30)

MRVLLVALALLALAASATSMAEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGKPLKDHLKFS

VAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIA

PEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAA

AQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQ

FLIEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILG

KLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVE

DLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFEKLEEYIID

KETIELPSGKQEYLESLINSYIVKTILELR

Barley alpha-amylase AMY32b signal sequence (SEQ ID NO: 31)

(MGKNGNLCCFSLLLLLLAGLASGHQ)

PR1a signal sequence (SEQ ID NO: 32)

(MGFVLFSQLPSFLLVSTLLLFLVISHSCRA)

797GL3 fusion (SEQ ID NO: 33)

MRVLLVALALLALAASATSAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGI

SAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVI

ADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDI

CHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGE

YWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAV

TFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVY

YDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYS

SGWVYLEAPAYDPANGQYGYSVWSYCGVGSEKDEL

6GP3 fusion (SEQ ID NO: 34)

MRVLLVALALLALAASATSAGHWYKHQRAYQFTGEDDFGKVAVVKLPMDLTKVGIIVRLN

EWQAKDVAKDRFIEIKDGKAEVWILQGVEEIFYEKPDTSPRIFFAQARSNKVIEAFLTNPVDT

KKKELFKVTVDGKEIPVSRVEKADPTDIDVTNYVRIVLSESLKEEDLRKDVELIIEGYKPARVI

MMEILDDYYYDGELGAVYSPEKTIFRVWSPVSKWVKVLLFKNGEDTEPYQVVNMEYKGNG

VWEAVVEGDLDGVFYLYQLENYGKIRTTVDPYSKAVYANNQESAVVNLARTNPEGWENDR

GPKIEGYEDAIIYEIHIADITGLENSGVKNKGLYLGLTEENTKAPGGVTTGLSHLVELGVTHVH

ILPFFDFYTGDELDKDFEKYYNWGYDPYLFMVPEGRYSTDPKNPHTRIREVKEMVKALHKH

GIGVIMDMVFPHTYGIGELSAFDQTVPYYFYRIDKTGAYLNESGCGNVIASERPMMRKFIVDT

VTYWVKEYHIDGFRFDQMGLIDKKTMLEVERALHKIDPTIILYGEPWGGWGAPIPFGKSDVA

GTHVAAFNDEFRDAIRGSVFNPSVKGFVMGGYGKETKIKRGVVGSINYDGKLIKSFALDPEE

TINYAACHDNHTLWDKNYLAAKADKKKEWTEEELKNAQKLAGAILLTSQGVPFLHGGQDF

CRTTNFNDNSYNAPISINGFDYERKLQFIDVFNYHKGLIKLRKEHPAFRLKNAEEIKKHLEFLP

GGRRIVAFMLKDHAGGDPWKDIVVIYNGNLEKTTYKLPEGKWNVVVNSQKAGTEVIETVEG

TIELDPLSAYVLYRESEKDEL

797GL3 fusion (SEQ ID NO: 35)

MRVLLVALALLALAASATSAKYLELEEGGVIMQAFYWDVPSGGIWWDTIRQKIPEWYDAGI

SAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVI

ADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDI

CHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWVVKDWLNWWGGWAVGE

YWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAV

TFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIVY

YDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYS

SGWVYLEAPAYDPANGQYGYSVWSYCGVGSEKDEL

malA fusion (SEQ ID NO: 36)

MRVLLVALALLALAASATSMETIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIV

QQGNKVIVEKSLDLKEHIIGLGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISV

KDGVATGYFFNSASKVIFDVGLEEYDKVIVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPP

MWAFGYMISRYSYYPQDKVVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPK

KLIDELHKRNVKLITIVDHGIRVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFRE

DTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVV

HYLRGKRVKHEKVRNAYPLYEAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPS

WDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKA

TDGIDTEPVFLPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRI

EDEYMVGKYLLYAPIVSKEESRLVTLPRGKWYNYWNGEIINGKSVVKSTHELPIYLREGSIIP

LEGDELIVYGETSFKRYDNAEITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQ

NTYVAKINQKIRGKINLESEKDEL

pNOV4829 glucose isomerase fusion nucleotide sequence (SEQ ID NO: 37)

ATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGG

TACCCTGGTGCCACGCGGTTCCATGGCCGAGTTCTTCCCGGAGATCCCGAAGATCCAGTT

CGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCCGCTTCTACGACCCGAACGAGGTGA

TCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCG

TGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGAGCGCCCGTGGAACCGCTTC

TCCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAG

CTCAACATCGAGTACTTCTGCTTCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTC

CGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGG

ACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGC

ACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAG

AAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCG

CGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCCTGGAGCTGGAGAACCTCGCCC

GCTTCCTCCGCATGGCCGTGGAGTACGCCAAGAAGATCGGCTTCACCGGCCAGTTCCTCA

TCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCC

TACGCCTTCCTCAAGAACCACGGCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAAC

CACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGC

AAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCA

GTTCCCGACCAACATCTACGACACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCG

GCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTG

GAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGATC

GCCTACAAGCTCGCCAAGGACGGCGTGTTCGACAAGTTCATCGAGGAGAAGTACCGCTC

CTTCAAGGAGGGCATCGGCAAGGAGATCGTGGAGGGCAAGACCGACTTCGAGAAGCTG

GAGGAGTACATCATCGACAAGGAGGACATCGAGCTGCCGTCCGGCAAGCAGGAGTACCT

GGAGTCCCTCCTCAACTCCTACATCGTGAAGACCATCGCCGAGCTGCGCTCCGAGAAGG

ACGAGCTGTGA

pNOV4829 glucose isomerase fusion amino acid sequence (SEQ ID NO: 38)

MKETAAAKFERQHMDSPDLGTLVPRGSMAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGK

PLKDHLKFSVAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYF

CFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCS

ADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEY

AKKIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQH

ELRMARILGKLGSIDANQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAGGFTKGGLNFDAKV

RRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDF

EKLEEYIIDKEDIELPSGKQEYLESLLNSYIVKTIAELRSEKDEL

pNOV4830 glucose isomerase fusion nucleotide sequence (SEQ ID NO: 39)

ATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGG

TACCCTGGTGCCACGCGGTTCCATGGCCGAGTTCTTCCCGGAGATCCCGAAGGTGCAGTT

CGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCAAGTTCTACGACCCGGAGGAGATCA

TCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCG

TGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGACCGCCCGTGGAACCGCTAC

ACCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAA

GCTCAACATCGAGTACTTCTGCTTCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCT

CCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAG

GACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATG

CACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAG

AAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCG

CGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCTTCGAGCTGGAGAACCTCGCCC

GCTTCCTCCGCATGGCCGTGGACTACGCCAAGCGCATCGGCTTCACCGGCCAGTTCCTCA

TCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCC

TACGCCTTCCTCAAGTCCCACGGCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAAC

CACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGC

AAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCA

GTTCCCGACCAACGTGTACGACACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCG

GCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTG

GAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGGTG

GCCTACAAGCTCGTGAAGGACGGCGTGCTCGACAAGTTCATCGAGGAGAAGTACCGCTC

CTTCCGCGAGGGCATCGGCCGCGACATCGTGGAGGGCAAGGTGGACTTCGAGAAGCTGG

AGGAGTACATCATCGACAAGGAGACCATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTG

GAGTCCCTCATCAACTCCTACATCGTGAAGACCATCCTGGAGCTGCGCTCCGAGAAGGAC

GAGCTGTGA

pNOV4830 glucose isomerase fusion amino acid sequence (SEQ ID NO: 40)

MKETAAAKFERQHMDSPDLGTLVPRGSMAEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGK

PLKDHLKFSVAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCEKLNIEY

FCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCS

ADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAVDY

AKRIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHE

LRMARILGKLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVR

RASYKVEDLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFE

KLEEYIIDKETIELPSGKQEYLESLINSYIVKTILELRSEKDEL

pNOV4835 Thermotoga maritima glucose isomerase fusion nucleotide sequence (SEQ ID NO: 41)

ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCA

TATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGATCCCCATGGCCGAGTTCTTCCC

GGAGATCCCGAAGATCCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCCGCT

TCTACGACCCGAACGAGGTGATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCC

GTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCC

GAGCGCCCGTGGAACCGCTTCTCCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGC

CCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGCTTCCACGACCGCGACAT

CCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCG

CATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCT

TCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCT

ACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGG

CTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCCT

GGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGAGTACGCCAAGAAGATCG

GCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTAC

GCTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGAACCACGGCCTCGACGAGTACTTCA

AGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTG

CGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTC

CTCGGCTGGGACACCGACCAGTTCCCGACCAACATCTACGACACCACCCTCGCCATGTAC

GAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCG

CCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTT

CGCCCTCGGCTTCAAGATCGCCTACAAGCTCGCCAAGGACGGCGTGTTCGACAAGTTCAT

CGAGGAGAAGTACCGCTCCTTCAAGGAGGGCATCGGCAAGGAGATCGTGGAGGGCAAG

ACCGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGGACATCGAGCTGCCGTC

CGGCAAGCAGGAGTACCTGGAGTCCCTCCTCAACTCCTACATCGTGAAGACCATCGCCG

AGCTGCGCTGA

pNOV4835 Thermotoga maritima glucose isomerase fusion amino acid sequence (SEQ ID NO: 42)

MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIPMAEFFPEIPKIQFEGKESTNPLAFRFYD

PNEVIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEF

CEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYM

HGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARF

LRMAVEYAKKIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATL

AGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAGGFTKGG

LNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEI

VEGKTDFEKLEEYIIDKEDIELPSGKQEYLESLLNSYIVKTIAELR

pNOV4836 Napolis Thermotoga glucose isomerase fusion nucleotide sequence (SEQ ID NO: 43)

ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCA

TATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGATCCCCATGGCCGAGTTCTTCCC

GGAGATCCCGAAGGTGCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCAAGT

TCTACGACCCGGAGGAGATCATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCC

GTGGCCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCC

GACCGCCCGTGGAACCGCTACACCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGC

CCTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGCTTCCACGACCGCGACAT

CCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCG

CATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCT

TCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCT

ACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGG

CTACGTGTTCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCTT

CGAGCTGGAGAACCTCGCCCGCTTCCTCCGCATGGCCGTGGACTACGCCAAGCGCATCG

GCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTAC

GACTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGTCCCACGGCCTCGACGAGTACTTC

AAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCT

GCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCT

CCTCGGCTGGGACACCGACCAGTTCCCGACCAACGTGTACGACACCACCCTCGCCATGTA

CGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGC

GCCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCT

TCGCCCTCGGCTTCAAGGTGGCCTACAAGCTCGTGAAGGACGGCGTGCTCGACAAGTTCA

TCGAGGAGAAGTACCGCTCCTTCCGCGAGGGCATCGGCCGCGACATCGTGGAGGGCAAG

GTGGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGACCATCGAGCTGCCGTC

CGGCAAGCAGGAGTACCTGGAGTCCCTCATCAACTCCTACATCGTGAAGACCATCCTGG

AGCTGCGCTGA

pNOV4836 Napolis Thermotoga glucose isomerase fusion amino acid sequence (SEQ ID NO: 44)

MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIPMAEFFPEIPKVQFEGKESTNPLAFKFYD

PEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFC

EKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMH

GAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGFELENLARFL

RMAVDYAKRIGFTGQFLIEPKPKPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLA

GHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGL

NFDAKVRRASYKVEDLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDI

VEGKVDFEKLEEYIIDKETIELPSGKQEYLESLINSYIVKTILELR

Aspergillus shirousami alpha-amylase/glucoamylase fusion amino acid sequence (without signal sequence)

(SEQ ID NO：45)

ATPADWRSQSIYFLLTDRFARTDGSTTATCNTADQKYCGGTWQGIIDKLDYIQGMGFTAIWI

TPVTAQLPQTTAYGDAYHGYWQQDIYSLNENYGTADDLKALSSALHERGMYLMVDVVAN

HMGYDGAGSSVDYSVFKPFSSQDYFHPFCFIQNYEDQTQVEDCWLGDNTVSLPDLDTTKDV

VKNEWYDWVGSLVSNYSIDGLRIDTVKHVQKDFWPGYNKAAGVYCIGEVLDVDPAYTCPY

QNVMDGVLNYPIYYPLLNAFKSTSGSMDDLYNMINTVKSDCPDSTLLGTFVENHDNPRFASY

TNDIALAKNVAAFIILNDGIPIIYAGQEQHYAGGNDPANREATWLSGYPTDSELYKLIASANAI

RNYAISKDTGFVTYKNWPIYKDDTTIAMRKGTDGSQIVTILSNKGASGDSYTLSLSGAGYTA

GQQLTEVIGCTFVTVGSDGNVPVPMAGGLPRVLYPTEKLAGSKICSSSKPATLDSWLSNEAT

VARTAILNNIGADGAWVSGADSGIVVASPSTDNPDYFYTWTRDSGIVLKTLVDLFRNGDTDL

LSTIEHYISSQAIIQGVSNPSGDLSSGGLGEPKFNVDETAYAGSWGRPQRDGPALRATAMIGF

GQWLLDNGYTSAATEIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVE

GSAFATAVGSSCSWCDSQAPQILCYLQSFWTGSYILANFDSSRSGKDTNTLLGSIHTFDPEAG

CDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDSYYNGNPWFLCTLA

AAEQLYDALYQWDKQGSLEIIDVSLDFFKALYSGAATGTYSSSSSTYSSIVSAVKTFADGFVS

IVETHAASNGSLSEQFDKSDGDELSARDLTWSYAALLTANNRRNSVVPPSWGETSASSVPGT

CAATSASGTYSSVTVTSWPSIVATGGTTTTATTTGSGGVTSTSKTTTTASKTSTTTSSTSCTTP

TAVAVTFDLTATTTYGENTYLVGSISQLGDWETSDGIALSADKYTSSNPPWYVTVTLPAGESF

EYKFIRVESDDSVEWESDPNREYTVPQACGESTATVTDTWR

Optimized nucleic acid sequence (without signal sequence) from Aspergillus shirousami alpha-amylase/glucoamylase fusion maize (SEQ ID) NO：46)

GCCACCCCGGCCGACTGGCGCTCCCAGTCCATCTACTTCCTCCTCACCGACCGCTTCGCC

CGCACCGACGGCTCCACCACCGCCACCTGCAACACCGCCGACCAGAAGTACTGCGGCGG

CACCTGGCAGGGCATCATCGACAAGCTCGACTACATCCAGGGCATGGGCTTCACCGCCA

TCTGGATCACCCCGGTGACCGCCCAGCTCCCGCAGACCACCGCCTACGGCGACGCCTACC

ACGGCTACTGGCAGCAGGACATCTACTCCCTCAACGAGAACTACGGCACCGCCGACGAC

CTCAAGGCCCTCTCCTCCGCCCTCCACGAGCGCGGCATGTACCTCATGGTGGACGTGGTG

GCCAACCACATGGGCTACGACGGCGCCGGCTCCTCCGTGGACTACTCCGTGTTCAAGCCG

TTCTCCTCCCAGGACTACTTCCACCCGTTCTGCTTCATCCAGAACTACGAGGACCAGACC

CAGGTGGAGGACTGCTGGCTCGGCGACAACACCGTGTCCCTCCCGGACCTCGACACCAC

CAAGGACGTGGTGAAGAACGAGTGGTACGACTGGGTGGGCTCCCTCGTGTCCAACTACT

CCATCGACGGCCTCCGCATCGACACCGTGAAGCACGTGCAGAAGGACTTCTGGCCGGGC

TACAACAAGGCCGCCGGCGTGTACTGCATCGGCGAGGTGCTCGACGTGGACCCGGCCTA

CACCTGCCCGTACCAGAACGTGATGGACGGCGTGCTCAACTACCCGATCTACTACCCGCT

CCTCAACGCCTTCAAGTCCACCTCCGGCTCGATGGACGACCTCTACAACATGATCAACAC

CGTGAAGTCCGACTGCCCGGACTCCACCCTCCTCGGCACCTTCGTGGAGAACCACGACAA

CCCGCGCTTCGCCTCCTACACCAACGACATCGCCCTCGCCAAGAACGTGGCCGCCTTCAT

CATCCTCAACGACGGCATCCCGATCATCTACGCCGGCCAGGAGCAGCACTACGCCGGCG

GCAACGACCCGGCCAACCGCGAGGCCACCTGGCTCTCCGGCTACCCGACCGACTCCGAG

CTGTACAAGCTCATCGCCTCCGCCAACGCCATCCGCAACTACGCCATCTCCAAGGACACC

GGCTTCGTGACCTACAAGAACTGGCCGATCTACAAGGACGACACCACCATCGCCATGCG

CAAGGGCACCGACGGCTCCCAGATCGTGACCATCCTCTCCAACAAGGGCGCCTCCGGCG

ACTCCTACACCCTCTCCCTCTCCGGCGCCGGCTACACCGCCGGCCAGCAGCTCACCGAGG

TGATCGGCTGCACCACCGTGACCGTGGGCTCCGACGGCAACGTGCCGGTGCCGATGGCC

GGCGGCCTCCCGCGCGTGCTCTACCCGACCGAGAAGCTCGCCGGCTCCAAGATATGCTCC

TCCTCCAAGCCGGCCACCCTCGACTCCTGGCTCTCCAACGAGGCCACCGTGGCCCGCACC

GCCATCCTCAACAACATCGGCGCCGACGGCGCCTGGGTGTCCGGCGCCGACTCCGGCAT

CGTGGTGGCCTCCCCGTCCACCGACAACCCGGACTACTTCTACACCTGGACCCGCGACTC

CGGCATCGTGCTCAAGACCCTCGTGGACCTCTTCCGCAACGGCGACACCGACCTCCTCTC

CACCATCGAGCACTACATCTCCTCCCAGGCCATCATCCAGGGCGTGTCCAACCCGTCCGG

CGACCTCTCCTCCGGCGGCCTCGGCGAGCCGAAGTTCAACGTGGACGAGACCGCCTACG

CCGGCTCCTGGGGCCGCCCGCAGCGCGACGGCCCGGCCCTCCGCGCCACCGCCATGATC

GGCTTCGGCCAGTGGCTCCTCGACAACGGCTACACCTCCGCCGCCACCGAGATCGTGTGG

CCGCTCGTGCGCAACGACCTCTCCTACGTGGCCCAGTACTGGAACCAGACCGGCTACGAC

CTCTGGGAGGAGGTGAACGGCTCCTCCTTCTTCACCATCGCCGTGCAGCACCGCGCCCTC

GTGGAGGGCTCCGCCTTCGCCACCGCCGTGGGCTCCTCCTGCTCCTGGTGCGACTCCCAG

GCCCCGCAGATCCTCTGCTACCTCCAGTCCTTCTGGACCGGCTCCTACATCCTCGCCAACT

TCGACTCCTCCCGCTCCGGCAAGGACACCAACACCCTCCTCGGCTCCATCCACACCTTCG

ACCCGGAGGCCGGCTGCGACGACTCCACCTTCCAGCCGTGCTCCCCGCGCGCCCTCGCCA

ACCACAAGGAGGTGGTGGACTCCTTCCGCTCCATCTACACCCTCAACGACGGCCTCTCCG

ACTCCGAGGCCGTGGCCGTGGGCCGCTACCCGGAGGACTCCTACTACAACGGCAACCCG

TGGTTCCTCTGCACCCTCGCCGCCGCCGAGCAGCTCTACGACGCCCTCTACCAGTGGGAC

AAGCAGGGCTCCCTGGAGATCACCGACGTGTCCCTCGACTTCTTCAAGGCCCTCTACTCC

GGCGCCGCCACCGGCACCTACTCCTCCTCCTCCTCCACCTACTCCTCCATCGTGTCCGCCG

TGAAGACCTTCGCCGACGGCTTCGTGTCCATCGTGGAGACCCACGCCGCCTCCAACGGCT

CCCTCTCCGAGCAGTTCGACAAGTCCGACGGCGACGAGCTGTCCGCCCGCGACCTCACCT

GGTCCTACGCCGCCCTCCTCACCGCCAACAACCGCCGCAACTCCGTGGTGCCGCCGTCCT

GGGGCGAGACCTCCGCCTCCTCCGTGCCGGGCACCTGCGCCGCCACCTCCGCCTCCGGCA

CCTACTCCTCCGTGACCGTGACCTCCTGGCCGTCCATCGTGGCCACCGGCGGCACCACCA

CCACCGCCACCACCACCGGCTCCGGCGGCGTGACCTCCACCTCCAAGACCACCACCACC

GCCTCCAAGACCTCCACCACCACCTCCTCCACCTCCTGCACCACCCCGACCGCCGTGGCC

GTGACCTTCGACCTCACCGCCACCACCACCTACGGCGAGAACATCTACCTCGTGGGCTCC

ATCTCCCAGCTCGGCGACTGGGAGACCTCCGACGGCATCGCCCTCTCCGCCGACAAGTAC

ACCTCCTCCAACCCGCCGTGGTACGTGACCGTGACCCTCCCGGCCGGCGAGTCCTTCGAG

TACAAGTTCATCCGCGTGGAGTCCGACGACTCCGTGGAGTGGGAGTCCGACCCGAACCG

CGAGTAC

ACCGTGCCGCAGGCCTGCGGCGAGTCCACCGCCACCGTGACCGACACCTGGCGC

Thermoanaerobacterium thermosaccharolyticum (Thermoanaerobacterium thermosaccharolium) glucoamylase amino acid sequence (without signal sequence) (SEQ ID NO: 47)

VLSGCSNNVSSIKIDRFNNISAVNGPGEEDTWASAQKQGVGTANNYVSRVWFTLANGAISEV

YYPTIDTADVKEIKFIVTDGKSFVSDETKDAISKVEKFTDKSLGYKLVNTDKKGRYRITKEIFT

DVKRNSLIMKAKFEALEGSIHDYKLYLAYDPHIKNQGSYNEGYVIKANNNEMLMAKRDNV

YTALSSNIGWKGYSIGYYKVNDIMTDLDENKQMTKHYDSARGNIIEGAEIDLTKNSEFEIVLS

FGGSDSEAAKTALETLGEDYNNLKNNYIDEWTKYCNTLNNFNGKANSLYYNSMMILKASED

KTNKGAYIASLSIPWGDGQRDDNTGGYHLVWSRDLYHVANAFIAAGDVDSANRSLDYLAK

VVKDNGMIPQNTWISGKPYWTSIQLDEQADPIILSYRLKRYDLYDSLVKPLADFIIKIGPKTGQ

ERWEEIGGYSPATMAAEVAGLTCAAYIAEQNKDYESAQKYQEKADNWQKLIDNLTYTENG

PLGNGQYYIRIAGLSDPNADFMINIANGGGVYDQKEIVDPSFLELVRLGVKSADDPKILNTLK

VVDSTIKVDTPKGPSWYRYNHDGYGEPSKTELYHGAGKGRLWPLLTGERGMYEIAAGKDA

TPYVKAMEKFANEGGIISEQVWEDTGLPTDSASPLNWAHAEYVILFASNIEHKVLDMPDIVY

Thermoanaerobacterium thermosaccharolyticum (Thermoanaerobacterium thermosaccharolium) glucoamylase corn optimized cores Sequence (without signal sequence) (SEQ ID NO: 48)

GTGCTCTCCGGCTGCTCCAACAACGTGTCCTCCATCAAGATCGACCGCTTCAACAACATC

TCCGCCGTGAACGGCCCGGGCGAGGAGGACACCTGGGCCTCCGCCCAGAAGCAGGGCGT

GGGCACCGCCAACAACTACGTGTCCCGCGTGTGGTTCACCCTCGCCAACGGCGCCATCTC

CGAGGTGTACTACCCGACCATCGACACCGCCGACGTGAAGGAGATCAAGTTCATCGTGA

CCGACGGCAAGTCCTTCGTGTCCGACGAGACCAAGGACGCCATCTCCAAGGTGGAGAAG

TTCACCGACAAGTCCCTCGGCTACAAGCTCGTGAACACCGACAAGAAGGGCCGCTACCG

CATCACCAAGGAAATCTTCACCGACGTGAAGCGCAACTCCCTCATCATGAAGGCCAAGT

TCGAGGCCCTCGAGGGCTCCATCCACGACTACAAGCTCTACCTCGCCTACGACCCGCACA

TCAAGAACCAGGGCTCCTACAACGAGGGCTACGTGATCAAGGCCAACAACAACGAGATG

CTCATGGCCAAGCGCGACAACGTGTACACCGCCCTCTCCTCCAACATCGGCTGGAAGGG

CTACTCCATCGGCTACTACAAGGTGAACGACATCATGACCGACCTCGACGAGAACAAGC

AGATGACCAAGCACTACGACTCCGCCCGCGGCAACATCATCGAGGGCGCCGAGATCGAC

CTCACCAAGAACTCCGAGTTCGAGATCGTGCTCTCCTTCGGCGGCTCCGACTCCGAGGCC

GCCAAGACCGCCCTCGAGACCCTCGGCGAGGACTACAACAACCTCAAGAACAACTACAT

CGACGAGTGGACCAAGTACTGCAACACCCTCAACAACTTCAACGGCAAGGCCAACTCCC

TCTACTACAACTCCATGATGATCCTCAAGGCCTCCGAGGACAAGACCAACAAGGGCGCC

TACATCGCCTCCCTCTCCATCCCGTGGGGCGACGGCCAGCGCGACGACAACACCGGCGG

CTACCACCTCGTGTGGTCCCGCGACCTCTACCACGTGGCCAACGCCTTCATCGCCGCCGG

CGACGTGGACTCCGCCAACCGCTCCCTCGACTACCTCGCCAAGGTGGTGAAGGACAACG

GCATGATCCCGCAGAACACCTGGATCTCCGGCAAGCCGTACTGGACCTCCATCCAGCTCG

ACGAGCAGGCCGACCCGATCATCCTCTCCTACCGCCTCAAGCGCTACGACCTCTACGACT

CCCTCGTGAAGCCGCTCGCCGACTTCATCATCAAGATCGGCCCGAAGACCGGCCAGGAG

CGCTGGGAGGAGATCGGCGGCTACTCCCCGGCCACGATGGCCGCCGAGGTGGCCGGCCT

CACCTGCGCCGCCTACATCGCCGAGCAGAACAAGGACTACGAGTCCGCCCAGAAGTACC

AGGAGAAGGCCGACAACTGGCAGAAGCTCATCGACAACCTCACCTACACCGAGAACGGC

CCGCTCGGCAACGGCCAGTACTACATCCGCATCGCCGGCCTCTCCGACCCGAACGCCGAC

TTCATGATCAACATCGCCAACGGCGGCGGCGTGTACGACCAGAAGGAGATCGTGGACCC

GTCCTTCCTCGAGCTGGTGCGCCTCGGCGTGAAGTCCGCCGACGACCCGAAGATCCTCAA

CACCCTCAAGGTGGTGGACTCCACCATCAAGGTGGACACCCCGAAGGGCCCGTCCTGGT

ATCGCTACAACCACGACGGCTACGGCGAGCCGTCCAAGACCGAGCTGTACCACGGCGCC

GGCAAGGGCCGCCTCTGGCCGCTCCTCACCGGCGAGCGCGGCATGTACGAGATCGCCGC

CGGCAAGGACGCCACCCCGTACGTGAAGGCGATGGAGAAGTTCGCCAACGAGGGCGGC

ATCATCTCCGAGCAGGTGTGGGAGGACACCGGCCTCCCGACCGACTCCGCCTCCCCGCTC

AACTGGGCCCACGCCGAGTACGTGATCCTCTTCGCCTCCAACATCGAGCACAAGGTGCTC

GACATGCCGGACATCGTGTAC

Rhizopus oryzae (Rhizopus oryzae) glucoamylase amino acid sequence (without signal sequence) (SEQ ID NO: 49)

ASIPSSASVQLDSYNYDGSTFSGKIYVKNIAYSKKVTVTYADGSDNWNNNGNTIAASYSAPIS

GSNYEYWTFSASINGIKEFYIKYEVSGKTYYDNNNSANYQVSTSKPTTTTATATTTTAPSTST

TTPPSRSEPATFPTGNSTISSWIKKQEGISRFAMLRNINPPGSATGFIAASLSTAGPDYYYAWTR

DAALTSNVIVYEYNTTLSGNKTILNVLKDYVTFSVKTQSTSTVCNCLGEPKFNPDASGYTGA

WGRPQNDGPAERATTFILFADSYLTQTKDASYVTGTLKPAIFKDLDYVVNVWSNGCFDLWE

EVNGVHFYTLMVMRKGLLLGADFAKRNGDSTRASTYSSTASTIANKISSFWVSSNNWIQVSQ

SVTGGVSKKGLDVSTLLAANLGSVDDGFFTPGSEKILATAVAVEDSFASLYPINKNLPSYLGN

SIGRYPEDTYNGNGNSQGNSWFLAVTGYAELYYRAIKEWIGNGGVTVSSISLPFFKKFDSSAT

SGKKYTVGTSDFNNLAQNLALAADRFLSTVQLHAHNNGSLAEEFDRTTGLSTGARDLTWSH

ASLITASYAKAGAPAA

Rhizopus oryzae glucoamylase maize optimized nucleic acid sequence (without signal sequence) (SEQ ID NO: 50)

GCCTCCATCCCGTCCTCCGCCTCCGTGCAGCTCGACTCCTACAACTACGACGGCTCCACC

TTCTCCGGCAAAATCTACGTGAAGAACATCGCCTACTCCAAGAAGGTGACCGTGATCTAC

GCCGACGGCTCCGACAACTGGAACAACAACGGCAACACCATCGCCGCCTCCTACTCCGC

CCCGATCTCCGGCTCCAACTACGAGTACTGGACCTTCTCCGCCTCCATCAACGGCATCAA

GGAGTTCTACATCAAGTACGAGGTGTCCGGCAAGACCTACTACGACAACAACAACTCCG

CCAACTACCAGGTGTCCACCTCCAAGCCGACCACCACCACCGCCACCGCCACCACCACC

ACCGCCCCGTCCACCTCCACCACCACCCCGCCGTCCCGCTCCGAGCCGGCCACCTTCCCG

ACCGGCAACTCCACCATCTCCTCCTGGATCAAGAAGCAGGAGGGCATCTCCCGCTTCGCC

ATGCTCCGCAACATCAACCCGCCGGGCTCCGCCACCGGCTTCATCGCCGCCTCCCTCTCC

ACCGCCGGCCCGGACTACTACTACGCCTGGACCCGCGACGCCGCCCTCACCTCCAACGTG

ATCGTGTACGAGTACAACACCACCCTCTCCGGCAACAAGACCATCCTCAACGTGCTCAAG

GACTACGTGACCTTCTCCGTGAAGACCCAGTCCACCTCCACCGTGTGCAACTGCCTCGGC

GAGCCGAAGTTCAACCCGGACGCCTCCGGCTACACCGGCGCCTGGGGCCGCCCGCAGAA

CGACGGCCCGGCCGAGCGCGCCACCACCTTCATCCTCTTCGCCGACTCCTACCTCACCCA

GACCAAGGACGCCTCCTACGTGACCGGCACCCTCAAGCCGGCCATCTTCAAGGACCTCG

ACTACGTGGTGAACGTGTGGTCCAACGGCTGCTTCGACCTCTGGGAGGAGGTGAACGGC

GTGCACTTCTACACCCTCATGGTGATGCGCAAGGGCCTCCTCCTCGGCGCCGACTTCGCC

AAGCGCAACGGCGACTCCACCCGCGCCTCCACCTACTCCTCCACCGCCTCCACCATCGCC

AACAAAATCTCCTCCTTCTGGGTGTCCTCCAACAACTGGATACAGGTGTCCCAGTCCGTG

ACCGGCGGCGTGTCCAAGAAGGGCCTCGACGTGTCCACCCTCCTCGCCGCCAACCTCGGC

TCCGTGGACGACGGCTTCTTCACCCCGGGCTCCGAGAAGATCCTCGCCACCGCCGTGGCC

GTGGAGGACTCCTTCGCCTCCCTCTACCCGATCAACAAGAACCTCCCGTCCTACCTCGGC

AACTCCATCGGCCGCTACCCGGAGGACACCTACAACGGCAACGGCAACTCCCAGGGCAA

CTCCTGGTTCCTCGCCGTGACCGGCTACGCCGAGCTGTACTACCGCGCCATCAAGGAGTG

GATCGGCAACGGCGGCGTGACCGTGTCCTCCATCTCCCTCCCGTTCTTCAAGAAGTTCGA

CTCCTCCGCCACCTCCGGCAAGAAGTACACCGTGGGCACCTCCGACTTCAACAACCTCGC

CCAGAACATCGCCCTCGCCGCCGACCGCTTCCTCTCCACCGTGCAGCTCCACGCCCACAA

CAACGGCTCCCTCGCCGAGGAGTTCGACCGCACCACCGGCCTCTCCACCGGCGCCCGCG

ACCTCACCTGGTCCCACGCCTCCCTCATCACCGCCTCCTACGCCAAGGCCGGCGCCCCGG

CCGCC

Corn alpha-amylase amino acid sequence (SEQ ID NO: 51)

MAKHLAAMCWCSLLVLVLLCLGSQLAQSQVLFQGFNWESWKKQGGWYNYLLGRVDDIAA

TGATHVWLPQPSHSVAPQGYMPGRLYDLDASKYGTHAELKSLTAAFHAKGVQCVADVVIN

HRCADYKDGRGIYCVFEGGTPDSRLDWGPDMICSDDTQYSNGRGHRDTGADFAAAPDDIHL

NPRVQQELSDWLNWLKSDLGFDGWRLDFAKGYSAAVAKVYVDSTAPTFVVAEIWSSLHYD

GNGEPSSNQDADRQELVNWAQAVGGPAAAFDFTTKGVLQAAVQGELWRMKDGNGKAPG

MIGWLPEKAVTFVDNHDTGSTQNSWPFPSDKVMQGYAYILTHPGTPCIFYDHVFDWNLKQE

ISALSAVRSRNGIHPGSELNILAADGDLYVAKIDDKVIVKIGSRYDVGNLIPSDFHAVAHGNN

YCVWEKHGLRVPAGRHH

Corn alpha-amylase nucleic acid sequence (SEQ ID NO: 52)

ATGGCGAAGCACTTGGCTGCCATGTGCTGGTGCAGCCTCCTAGTGCTTGTACTGCTCTGC

TTGGGCTCCCAGCTGGCCCAATCCCAGGTCCTCTTCCAGGGGTTCAACTGGGAGTCGTGG

AAGAAGCAAGGTGGGTGGTACAACTACCTCCTGGGGCGGGTGGACGACATCGCCGCGAC

GGGGGCCACGCACGTCTGGCTCCCGCAGCCGTCGCACTCGGTGGCGCCGCAGGGGTACA

TGCCCGGCCGGCTCTACGACCTGGACGCGTCCAAGTACGGCACCCACGCGGAGCTCAAG

TCGCTCACCGCGGCGTTCCACGCCAAGGGCGTCCAGTGCGTCGCCGACGTCGTGATCAAC

CACCGCTGCGCCGACTACAAGGACGGCCGCGGCATCTACTGCGTCTTCGAGGGCGGCAC

GCCCGACAGCCGCCTCGACTGGGGCCCCGACATGATCTGCAGCGACGACACGCAGTACT

CCAACGGGCGCGGGCACCGCGACACGGGGGCCGACTTCGCCGCCGCGCCCGACATCGAC

CACCTCAACCCGCGCGTGCAGCAGGAGCTCTCGGACTGGCTCAACTGGCTCAAGTCCGA

CCTCGGCTTCGACGGCTGGCGCCTCGACTTCGCCAAGGGCTACTCCGCCGCCGTCGCCAA

GGTGTACGTCGACAGCACCGCCCCCACCTTCGTCGTCGCCGAGATATGGAGCTCCCTCCA

CTACGACGGCAACGGCGAGCCGTCCAGCAACCAGGACGCCGACAGGCAGGAGCTGGTC

AACTGGGCGCAGGCGGTGGGCGGCCCCGCCGCGGCGTTCGACTTCACCACCAAGGGCGT

GCTGCAGGCGGCCGTCCAGGGCGAGCTGTGGCGCATGAAGGACGGCAACGGCAAGGCG

CCCGGGATGATCGGCTGGCTGCCGGAGAAGGCCGTCACGTTCGTCGACAACCACGACAC

CGGCTCCACGCAGAACTCGTGGCCATTCCCCTCCGACAAGGTCATGCAGGGCTACGCCTA

TATCCTCACGCACCCAGGAACTCCATGCATCTTCTACGACCACGTTTTCGACTGGAACCT

GAAGCAGGAGATCAGCGCGCTGTCTGCGGTGAGGTCAAGAAACGGGATCCACCCGGGG

AGCGAGCTGAACATCCTCGCCGCCGACGGGGATCTCTACGTCGCCAAGATTGACGACAA

GGTCATCGTGAAGATCGGGTCACGGTACGACGTCGGGAACCTGATCCCCTCAGACTTCCA

CGCCGTTGCCCCTGGCAACAACTACTGCGTTTGGGAGAAGCACGGTCTGAGAGTTCCAGC

GGGGCGGCACCACTAG

Amino acid sequence of crude starch binding site (SEQ ID NO: 53)

ATGGTTTTATTTGSGGVTSTSKTTTTASKTSTTTSSTSCTTPTAV

Maize optimized nucleic acid sequence for crude starch binding site (SEQ ID NO: 54)

GCCACCGGCGGCACCACCACCACCGCCACCACCACCGGCTCCGGCGGCGTGACCTCCAC

CTCCAAGACCACCACCACCGCCTCCAAGACCTCCACCACCACCTCCTCCACCTCCTGCAC

CACCCCGACCGCCGTGTC

Pyrococcus furiosus EGLA amino acid sequence (without signal sequence) (SEQ ID NO: 55)

IYFVEKYHTSEDKSTSNTSSTPPQTTLSTTKVLKIRYPDDGEWPGAPIDKDGDGNPEFYIEINL

WNILNATGFAEMTYNLTSGVLHYVQQLDNIVLRDRSNWVHGYPEIFYGNKPWNANYATDG

PIPLPSKVSNLTDFYLTISYKLEPKNGLPINFAIESWLTREAWRTTGINSDEQEVMIWIYYDGL

QPAGSKVKEIVVPIIVNGTPVNATFEVWKANIGWEYVAFRIKTPIKEGTVTIPYGAFISVAANIS

SLPNYTELYLEDVEIGTEFGTPSTTSAHLEWWITNITLTPLDRPLIS

Pyrococcus furiosus EGLA maize optimized nucleic acid sequence (without signal sequence) (SEQ ID NO: 56)

ATCTACTTCGTGGAGAAGTACCACACCTCCGAGGACAAGTCCACCTCCAACACCTCCTCC

ACCCCGCCGCAGACCACCCTCTCCACCACCAAGGTGCTCAAGATCCGCTACCCGGACGA

CGGCGAGTGGCCCGGCGCCCCGATCGACAAGGACGGCGACGGCAACCCGGAGTTCTACA

TCGAGATCAACCTCTGGAACATCCTCAACGCCACCGGCTTCGCCGAGATGACCTACAACC

TCACTAGTGGCGTGCTCCACTACGTGCAGCAGCTCGACAACATCGTGCTCCGCGACCGCT

CCAACTGGGTGCACGGCTACCCGGAAATCTTCTACGGCAACAAGCCGTGGAACGCCAAC

TACGCCACCGACGGCCCGATCCCGCTCCCGTCCAAGGTGTCCAACCTCACCGACTTCTAC

CTCACCATCTCCTACAAGCTCGAGCCGAAGAACGGTCTCCCGATCAACTTCGCCATCGAG

TCCTGGCTCACCCGCGAGGCCTGGCGCACCACCGGCATCAACTCCGACGAGCAGGAGGT

GATGATCTGGATCTACTACGACGGCCTCCAGCCCGCGGGCTCCAAGGTGAAGGAGATCG

TGGTGCCGATCATCGTGAACGGCACCCCGGTGAACGCCACCTTCGAGGTGTGGAAGGCC

AACATCGGCTGGGAGTACGTGGCCTTCCGCATCAAGACCCCGATCAAGGAGGGCACCGT

GACCATCCCGTACGGCGCCTTCATCTCCGTGGCCGCCAACATCTCCTCCCTCCCGAACTA

CACCGAGAAGTACCTCGAGGACGTGGAGATCGGCACCGAGTTCGGCACCCCGTCCACCA

CCTCCGCCCACCTCGAGTGGTGGATCACCAACATCACCCTCACCCCGCTCGACCGCCCGC

TCATCTCCTAG

Thermus flavus (Thermus flavus) xylose isomerase amino acid sequence (SEQ ID NO: 57)

MYEPKPEHRFTFGLWTVDNVDRDPFGDTVRERLDPVYVVHKLAELGAYGVNLHDEDLIPRG

TPPQERDQIVRRFKKALDETVLKVPMVTANLFSEPAFRDGASTTRDPWVWAYALRKSLETM

DLGAELGAEIYMFWMVRERSEVESTDKTRKVWDWVRETLNFMTAYTEDQGYGYRFSVEPK

PNEPRGDIYFTTVGSMLALIHTLDRPER

FGLNPEFAHETMAGLNFDHAVAQAVDAGKLFHIDLNDQRMSRFDQDLRFGSENLKAGFFLV

DLLESSGYQGPRHFEAHALRTEDEEGVWTFVRVCMRTYLIIKVRAETFREDPEVKELLAAYY

QEDPATLALLDPYSREKAEALKRAELPLETKRRRGYALERLDQLAVEYLLGVRG

pNOV4800 nucleotide sequence (Amy32B signal sequence and EGLA) (SEQ ID NO: 58)

ATGGGGAAGAACGGCAACCTGTGCTGCTTCTCTCTGCTGCTGCTTCTTCTCGCCGGGTTG

GCGTCCGGCCATCAAATCTACTTCGTGGAGAAGTACCACACCTCCGAGGACAAGTCCAC

CTCCAACACCTCCTCCACCCCGCCGCAGACCACCCTCTCCACCACCAAGGTGCTCAAGAT

CCGCTACCCGGACGACGGTGAGTGGCCCGGCGCCCCGATCGACAAGGACGGCGACGGCA

ACCCGGAGTTCTACATCGAGATCAACCTCTGGAACATCCTCAACGCCACCGGCTTCGCCG

AGATGACCTACAACCTCACTAGTGGCGTGCTCCACTACGTGCAGCAGCTCGACAACATCG

TGCTCCGCGACCGCTCCAACTGGGTGCACGGCTACCCGGAAATCTTCTACGGCAACAAGC

CGTGGAACGCCAACTACGCCACCGACGGCCCGATCCCGCTCCCGTCCAAGGTGTCCAAC

CTCACCGACTTCTACCTCACCATCTCCTACAAGCTCGAGCCGAAGAACGGTCTCCCGATC

AACTTCGCCATCGAGTCCTGGCTCACCCGCGAGGCCTGGCGCACCACCGGCATCAACTCC

GACGAGCAGGAGGTGATGATCTGGATCTACTACGACGGCCTCCAGCCCGCGGGCTCCAA

GGTGAAGGAGATCGTGGTGCCGATCATCGTGAACGGCACCCCGGTGAACGCCACCTTCG

AGGTGTGGAAGGCCAACATCGGCTGGGAGTACGTGGCCTTCCGCATCAAGACCCCGATC

AAGGAGGGCACCGTGACCATCCCGTACGGCGCCTTCATCTCCGTGGCCGCCAACATCTCC

TCCCTCCCGAACTACACCGAGAAGTACCTCGAGGACGTGGAGATCGGCACCGAGTTCGG

CACCCCGTCCACCACCTCCGCCCACCTCGAGTGGTGGATCACCAACATCACCCTCACCCC

GCTCGACCGCCCGCTCATCTCCTAG

Aspergillus niger (Aspergillus niger) maize optimized nucleic acid (SEQ ID NO: 59)

ATGTCCTTCCGCTCCCTCCTCGCCCTCTCCGGCCTCGTGTGCACCGGCCTCGCCAACGTGA

TCTCCAAGCGCGCCACCCTCGACTCCTGGCTCTCCAACGAGGCCACCGTGGCCCGCACCG

CCATCCTCAACAACATCGGCGCCGACGGCGCCTGGGTGTCCGGCGCCGACTCCGGCATC

GTGGTGGCCTCCCCGTCCACCGACAACCCGGACTACTTCTACACCTGGACCCGCGACTCC

GGCCTCGTGCTCAAGACCCTCGTGGACCTCTTCCGCAACGGCGACACCTCCCTCCTCTCC

ACCATCGAGAACTACATCTCCGCCCAGGCCATCGTGCAGGGCATCTCCAACCCGTCCGGC

GACCTCTCCTCCGGCGCCGGCCTCGGCGAGCCGAAGTTCAACGTGGACGAGACCGCCTA

CACCGGCTCCTGGGGCCGCCCGCAGCGCGACGGCCCGGCCCTCCGCGCCACCGCCATGA

TCGGCTTCGGCCAGTGGCTCCTCGACAACGGCTACACCTCCACCGCCACCGACATCGTGT

GGCCGCTCGTGCGCAACGACCTCTCCTACGTGGCCCAGTACTGGAACCAGACCGGCTAC

GACCTCTGGGAGGAGGTGAACGGCTCCTCCTTCTTCACCATCGCCGTGCAGCACCGCGCC

CTCGTGGAGGGCTCCGCCTTCGCCACCGCCGTGGGCTCCTCCTGCTCCTGGTGCGACTCC

CAGGCCCCGGAGATCCTCTGCTACCTCCAGTCCTTCTGGACCGGCTCCTTCATCCTCGCCA

ACTTCGACTCCTCCCGCTCCGGCAAGGACGCCAACACCCTCCTCGGCTCCATCCACACCT

TCGACCCGGAGGCCGCCTGCGACGACTCCACCTTCCAGCCGTGCTCCCCGCGCGCCCTCG

CCAACCACAAGGAGGTGGTGGACTCCTTCCGCTCCATCTACACCCTCAACGACGGCCTCT

CCGACTCCGAGGCCGTGGCCGTGGGCCGCTACCCGGAGGACACCTACTACAACGGCAAC

CCGTGGTTCCTCTGCACCCTCGCCGCCGCCGAGCAGCTCTACGACGCCCTCTACCAGTGG

GACAAGCAGGGCTCCCTCGAGGTGACCGACGTGTCCCTCGACTTCTTCAAGGCCCTCTAC

TCCGACGCCGCCACCGGCACCTACTCCTCCTCCTCCTCCACCTACTCCTCCATCGTGGACG

CCGTGAAGACCTTCGCCGACGGCTTCGTGTCCATCGTGGAGACCCACGCCGCCTCCAACG

GCTCCATGTCCGAGCAGTACGACAAGTCCGACGGCGAGCAGCTCTCCGCCCGCGACCTC

ACCTGGTCCTACGCCGCCCTCCTCACCGCCAACAACCGCCGCAACTCCGTGGTGCCGGCC

TCCTGGGGCGAGACCTCCGCCTCCTCCGTGCCGGGCACCTGCGCCGCCACCTCCGCCATC

GGCACCTACTCCTCCGTGACCGTGACCTCCTGGCCGTCCATCGTGGCCACCGGCGGCACC

ACCACCACCGCCACCCCGACCGGCTCCGGCTCCGTGACCTCCACCTCCAAGACCACCGCC

ACCGCCTCCAAGACCTCCACCTCCACCTCCTCCACCTCCTGCACCACCCCGACCGCCGTG

GCCGTGACCTTCGACCTCACCGCCACCACCACCTACGGCGAGAACATCTACCTCGTGGGC

TCCATCTCCCAGCTCGGCGACTGGGAGACCTCCGACGGCATCGCCCTCTCCGCCGACAAG

TACACCTCCTCCGACCCGCTCTGGTACGTGACCGTGACCCTCCCGGCCGGCGAGTCCTTC

GAGTACAAGTTCATCCGCATCGAGTCCGACGACTCCGTGGAGTGGGAGTCCGACCCGAA

CCGCGAGTACACCGTGCCGCAGGCCTGCGGCACCTCCACCGCCACCGTGACCGACACCT

GGCGC

Claims (234)

1. An isolated polynucleotide a) comprising the nucleotide sequence of SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 or a complement thereof, or a sequence that hybridizes under low stringency hybridization conditions to SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 and encodes a polypeptide having α -amylase, pullulanase, α -glucosidase, glucose isomerase, or glucoamylase activity, or b) a polynucleotide encoding a polypeptide comprising the amino acid 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 an enzymatically active fragment thereof.

2. The isolated polynucleotide of claim 1, wherein the polynucleotide encodes a fusion polypeptide comprising a first polypeptide and a second peptide, wherein the first polypeptide has alpha-amylase, pullulanase, alpha-glucosidase, glucose isomerase, or glucoamylase activity.

3. The isolated polynucleotide of claim 2, wherein said second peptide comprises a signal sequence peptide.

4. The isolated polynucleotide of claim 3, wherein said signal sequence peptide targets said first polypeptide to a vacuole, endoplasmic reticulum, chloroplast, starch granule, seed, or cell wall of a plant.

5. The isolated polynucleotide of claim 3, wherein said signal sequence is the N-terminal signal sequence of waxy, the N-terminal signal sequence of γ -zein, the starch binding domain or the C-terminal starch binding domain.

6. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes under low stringency hybridization conditions to the mature polypeptide of SEQ ID NO: 2, 9, or 52, and encodes a polypeptide having alpha-amylase activity.

7. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes under low stringency hybridization conditions to the mature polypeptide of SEQ ID NO: 4 or 25, and encodes a polypeptide having pullulanase activity.

8. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes to SEQ id no: 6, and encodes a polypeptide having alpha-glucosidase activity.

9. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes under low stringency hybridization conditions to the mature polypeptide of SEQ ID NO: 19, 21, 37, 39, 41 or 43, and encodes a polypeptide having glucose isomerase activity.

10. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes under low stringency hybridization conditions to the mature polypeptide of SEQ ID NO: 46, 48, 50 or 59, and encodes a polypeptide having glucoamylase activity.

11. Comprises the amino acid sequence shown in SEQ ID NO: 2 or 9 or the complement thereof.

12. Comprises the amino acid sequence shown in SEQ ID NO: 4 or 25 or the complement thereof.

13. Comprises the amino acid sequence shown in SEQ ID NO: 6 or the complement thereof.

14. Comprises the amino acid sequence shown in SEQ ID NO: 19, 21, 37, 39, 41 or 43 or the complement thereof.

15. Comprises the amino acid sequence shown in SEQ ID NO: 46, 48, 50, or 59 or the complement thereof.

16. An expression cassette comprising a polynucleotide a) having the sequence of SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 or a complement thereof, or a sequence that hybridizes under low stringency hybridization conditions to SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 and encodes a polypeptide having α -amylase, pullulanase, α -glucosidase, glucose isomerase, or glucoamylase activity, or b) a polynucleotide encoding a polypeptide comprising the amino acid 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 an enzymatically active fragment thereof.

17. The expression cassette of claim 16, which is operably linked to a promoter.

18. The expression cassette of claim 17, wherein the promoter is an inducible promoter.

19. The expression cassette of claim 17, wherein the promoter is a tissue-specific promoter.

20. The expression cassette of claim 19, wherein the promoter is an endosperm-specific promoter.

21. The expression cassette of claim 20, wherein the endosperm-specific promoter is a maize γ -zein promoter or a maize ADP-gpp promoter.

22. The expression cassette of claim 21, wherein the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12.

23. the expression cassette of claim 16, wherein the polynucleotide is oriented in a sense orientation relative to the promoter.

24. The expression cassette of claim 16, wherein the a) polynucleotide further encodes a signal sequence operably linked to the polypeptide encoded by the polynucleotide.

25. The expression cassette of claim 24, wherein the signal sequence directs the operably linked polypeptide to a vacuole, endoplasmic reticulum, chloroplast, starch granule, seed, or cell wall of a plant.

26. The expression cassette of claim 25, wherein the signal sequence is the N-terminal signal sequence of waxy or the N-terminal signal sequence of γ -zein.

27. The expression cassette of claim 25, wherein the signal sequence is a starch binding domain.

28. The expression cassette of claim 16, wherein the b) polynucleotide is operably linked to a tissue-specific promoter.

29. The expression cassette of claim 28, wherein the tissue-specific promoter is a maize (Zea mays) gamma-zein promoter or a maize ADP-gpp promoter.

30. An expression cassette comprising a polynucleotide, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2 or 9 or a complement thereof.

31. An expression cassette comprising a polynucleotide, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6 or the complement thereof.

32. An expression cassette comprising a polynucleotide, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 19, 21, 37, 39, 41, or 43, or a complement thereof.

33. An expression cassette comprising a polynucleotide, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 46, 48, 50 or 59 or a complement thereof.

34. An expression cassette comprising a polynucleotide, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4 or 25 or the complement thereof.

35. An expression cassette comprising a polynucleotide, wherein said polynucleotide encodes a polypeptide having the sequence of SEQ id no: 10, 13, 14, 15, 16, 24, 26, 27, 28, 29, 30, 33, 34, 35, 36, 38, 40, 42, 44, 45, 47, 49 or 51, or an enzymatically active fragment thereof.

36. An expression cassette comprising a polynucleotide, wherein said polynucleotide encodes a polypeptide having the sequence of SEQ id no: 10, 13, 14, 15, 16, 33, 35, or 51, or an active fragment thereof having alpha-amylase activity.

37. An expression cassette comprising a polynucleotide, wherein said polynucleotide encodes a polypeptide having the sequence of SEQ id no: 3, 24, or 34 or an active fragment thereof having pullulanase activity.

38. An expression cassette comprising a polynucleotide, wherein said polynucleotide encodes a polypeptide having the sequence of SEQ id no: 5, 26, or 27 or an active fragment thereof having alpha-glucosidase activity.

39. An expression cassette comprising a polynucleotide, wherein said polynucleotide encodes a polypeptide having the sequence of SEQ id no: 18, 20, 28, 29, 30, 38, 40, 42 or 44 or an active fragment thereof having glucose isomerase activity.

40. An expression cassette comprising a polynucleotide, wherein said polynucleotide encodes a polypeptide having the sequence of SEQ id no: 45, 47 or 49 or an active fragment thereof having glucoamylase activity.

41. A vector comprising the expression cassette of claim 16.

42. A vector comprising the expression cassette of any one of claims 30-40.

43. A cell comprising the expression cassette of claim 16.

44. A cell comprising the expression cassette of any one of claims 30-40.

45. The cell of claim 44, wherein said cell is selected from the group consisting of: agrobacterium, a monocot plant cell, a dicot plant cell, a Liliopsida cell, a Panicoideae cell, a maize cell, or a cereal cell.

46. The cell of claim 45, wherein the cell is a maize cell.

47. The cell of claim 45, wherein said cell is selected from the group consisting of: agrobacterium, a monocot plant cell, a dicot plant cell, a Liliopsida cell, a Panicoideae cell, a maize cell, or a cereal cell.

48. The cell of claim 47, wherein the cell is a maize cell.

49. A plant stably transformed with the vector of claim 41.

50. A plant stably transformed with the vector of claim 42.

51. A plant stably transformed with a vector comprising an alpha-amylase having the amino acid sequence of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33 or 35, or a polypeptide consisting of an amino acid sequence comprising SEQ ID NO: 2 or 9.

52. The plant of claim 51, wherein said alpha-amylase is hyperthermostable.

53. A plant stably transformed with a vector comprising a pullulanase having the amino acid sequence of SEQ ID NO: 24 or 34, or a polypeptide consisting of a sequence comprising any one of SEQ ID NOs: 4 or 25.

54. A plant stably transformed with a vector comprising an alpha-glucosidase having the amino acid sequence of SEQ ID NO: 26 or 27, or a polypeptide consisting of a sequence comprising any one of SEQ ID NOs: 6.

55. The plant of claim 54 wherein said α -glucosidase is hyperthermophilic.

56. A plant stably transformed with a vector comprising a glucose isomerase enzyme having the amino acid sequence of SEQ ID NO: 18, 20, 28, 29, 30, 38, 40, 42 or 44, or a polypeptide consisting of an amino acid sequence comprising SEQ ID NO: 19, 21, 37, 39, 41 or 43.

57. The plant of claim 56, wherein said α -glucosidase is hyperthermophilic.

58. A plant stably transformed with a vector comprising a glucoamylase having the amino acid sequence of SEQ ID NO: 45, 47 or 49, or a polypeptide consisting of an amino acid sequence comprising SEQ id no: 46, 48, 50 or 59.

59. The plant of claim 58, wherein said glucoamylase is hyperthermophilic.

60. A seed, fruit, or grain from the plant of claim 49.

61. A seed, fruit, or grain from the plant of claim 50.

62. A seed, fruit, or grain from the plant of claim 51.

63. A seed, fruit, or grain from the plant of claim 53.

64. A seed, fruit, or grain from the plant of claim 54.

65. A seed, fruit, or grain from the plant of claim 56.

66. A seed, fruit, or grain from the plant of claim 58.

67. A transformed plant having increased in its genome a recombinant polynucleotide encoding at least one processing enzyme operably linked to a promoter sequence, the sequence of said polynucleotide having been optimized for expression in a plant.

68. The plant of claim 67, wherein said plant is a monocot.

69. The plant of claim 68, wherein said monocot is maize.

70. The plant of claim 67, wherein said plant is a dicot.

71. The plant of claim 67, wherein said plant is a cereal plant or a commercially grown plant.

72. The plant of claim 67, wherein said processing enzyme is selected from the group consisting of: alpha-amylase, glucoamylase, glucose isomerase, glucanase, beta-amylase, alpha-glucosidase, isoamylase, pullulanase, neopullulanase, isoamylase, amylopullulanase (amylopullulanase), cellulase, exo-1, 4-beta-cellobiohydrolase, exo-1, 3-beta-D-glucanase, beta-glucosidase, endoglucanase, L-arabinase, alpha-arabinosidase, galactanase, galactosidase, mannanase, mannosidase, xylanase, xylosidase, protease, glucanase, xylanase, esterase, phytase and lipase.

73. The plant of claim 72, wherein said processing enzyme is a starch processing enzyme selected from the group consisting of: alpha-amylase, glucoamylase, glucose isomerase, beta-amylase, alpha-glucosidase, isoamylase, pullulanase, neopullulanase, isoamylase and amylopullulanase.

74. The plant of claim 73, wherein said enzyme is selected from the group consisting of: alpha-amylase, glucoamylase, glucose isomerase, alpha-glucosidase, and pullulanase.

75. The plant of claim 74, wherein said enzyme is hyperthermophilic.

76. The plant of claim 72, wherein said enzyme is a non-starch degrading enzyme selected from the group consisting of: proteases, glucanases, xylanases, esterases, phytases and lipases.

77. The plant of claim 76, wherein said enzyme is hyperthermophilic.

78. The plant of claim 67, wherein said enzyme is accumulated in the vacuole, endoplasmic reticulum, chloroplast, starch granule, seed or cell wall of the plant.

79. The plant of claim 78, wherein said enzyme accumulates in the endoplasmic reticulum.

80. The plant of claim 78, wherein said enzyme accumulates in starch granules.

81. The plant of claim 67, the genome of which is further augmented with a second recombinant polynucleotide comprising a non-hyperthermophilic enzyme.

82. A transformed plant having increased in its genome a recombinant polynucleotide encoding at least one processing enzyme selected from the group consisting of: an alpha-amylase, a glucoamylase, a glucose isomerase, an alpha-glucosidase, and a pullulanase, said polynucleotide operably linked to a promoter sequence, the sequence of said polynucleotide having been optimized for expression in said plant.

83. The transformed plant of claim 82, wherein said processing enzyme is hyperthermophilic.

84. The transformed plant of claim 82, wherein said plant is maize.

85. A transformed maize plant having increased in its genome a recombinant polynucleotide encoding at least one processing enzyme selected from the group consisting of: an alpha-amylase, a glucoamylase, a glucose isomerase, an alpha-glucosidase, and a pullulanase, said polynucleotide operably linked to a promoter sequence, the sequence of said polynucleotide having been optimized for expression in the maize plant.

86. The transformed maize plant of claim 85, wherein said processing enzyme is hyperthermostable.

87. A transformed plant, the genome of which is increased with a promoter having the sequence of SEQ ID NO: 2, 9 or 52, which polynucleotide is operably linked to a promoter and a signal sequence.

88. A transformed plant, the genome of which is increased with a promoter having the sequence of SEQ ID NO: 4 or 25, operably linked to a promoter and a signal sequence.

89. A transformed plant, the genome of which is increased with a promoter having the sequence of SEQ ID NO: 6, operably linked to a promoter and a signal sequence.

90. A transformed plant, the genome of which is increased with a promoter having the sequence of SEQ ID NO: 19, 21, 37, 39, 41 or 43.

91. A transformed plant, the genome of which is increased with a promoter having the sequence of SEQ ID NO: 46, 48, 50 or 59.

92. The product of the transformed plant of claim 82.

93. The product of the transformed plant of claim 85.

94. The product of the transformed plant of any one of claims 87-91.

95. The product of claim 92, wherein the product is a seed, fruit, or grain.

96. The product of claim 92, wherein the product is a processing enzyme, a starch, or a sugar.

97. A plant obtained from the plant of claim 82.

98. A plant obtained from the plant of claim 85.

99. A plant obtained from the plant of any one of claims 87-91.

100. The plant of claim 97, which is a hybrid plant.

101. The plant of claim 98, which is a hybrid plant.

102. The plant of claim 99, which is a hybrid plant.

103. The plant of claim 97, which is an inbred plant.

104. The plant of claim 98, which is an inbred plant.

105. The plant of claim 99, which is an inbred plant.

106. A starch composition comprising at least one processing enzyme which is a protease, a glucanase or an esterase.

107. The starch composition of claim 106, wherein the enzyme is hyperthermophilic.

108. A cereal comprising at least one processing enzyme which is an alpha-amylase, pullulanase, alpha-glucosidase, glucoamylase or glucose isomerase.

109. The grain of claim 108, wherein the enzyme is hyperthermophilic.

110. A method of making starch granules comprising:

a) treating a grain comprising at least one non-starch processing enzyme under conditions effective to activate the at least one enzyme to produce a mixture comprising starch granules and non-starch degradation products, wherein the grain is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one enzyme; and

b) the starch granules are separated from the mixture.

111. The method of claim 110, wherein the enzyme is a protease, a glucanase, a xylanase, a phytase, or an esterase.

112. The method of claim 111, wherein the enzyme is hyperthermophilic.

113. The method of claim 110, wherein the grain is a cracked grain.

114. The method of claim 110 wherein the grain is treated under conditions of low moisture content.

115. The method of claim 110, wherein the grain is treated under conditions of high moisture content.

116. The method of claim 110, wherein the grain is treated with sulfur dioxide.

117. The method of claim 110, further comprising separating the non-starch product from the mixture.

118. Starch obtained according to the method of claim 110.

119. Starch obtained by the method of claim 112.

120. A non-starch product obtained according to the method of claim 110.

121. A non-starch product obtained according to the method of claim 112.

122. A method for producing super-sweet corn comprising treating transformed corn or a portion thereof, which has increased in its genome an expression cassette encoding at least one starch degrading or starch isomerizing enzyme, and expressing the expression cassette in the endosperm, under conditions which activate the at least one enzyme, such that polysaccharides in the corn are converted to sugars, producing super-sweet corn.

123. The method of claim 122, wherein the expression cassette further comprises a promoter operably linked to the polynucleotide encoding the enzyme.

124. The method of claim 123, wherein the promoter is a constitutive promoter.

125. The method of claim 123, wherein the promoter is a seed-specific promoter.

126. The method of claim 123, wherein the promoter is an endosperm-specific promoter.

127. The method of claim 123, wherein the enzyme is hyperthermophilic.

128. The method of claim 127, wherein the enzyme is an alpha-amylase.

129. The method of claim 122, wherein the expression cassette further comprises a polynucleotide encoding a signal sequence operably linked to the at least one enzyme.

130. The method of claim 129, wherein the signal sequence directs the hyperthermophilic enzyme to an apoplast.

131. The method of claim 129, wherein the signal sequence directs the hyperthermophilic enzyme to the endoplasmic reticulum.

132. The method of claim 122, wherein said enzyme comprises SEQ ID NO: 13, 14, 15, 16, 33 or 35.

133. A method of producing super-sweet corn comprising treating transformed corn or a portion thereof having increased in its genome an expression cassette encoding an alpha-amylase and expressing the expression cassette in the endosperm under conditions which activate the at least one enzyme such that polysaccharides in the corn are converted to sugars, producing super-sweet corn.

134. The method of claim 133, wherein the enzyme is hyperthermophilic.

135. The method of claim 134, wherein said hyperthermophilic alpha-amylase comprises the amino acid sequence of SEQ ID NO: 10, 13, 14, 15, 16, 33 or 35, or an enzymatically active fragment thereof having α -amylase activity.

136. The method of claim 134, wherein the expression cassette comprises a nucleic acid sequence selected from the group consisting of SEQ id nos: 2, 9 or 52 or the complement thereof, or a polynucleotide that hybridizes under low stringency hybridization conditions to the nucleic acid sequence of SEQ ID NO: 2, 9 or 52, and encodes a polypeptide having alpha-amylase activity.

137. A process for preparing a hydrolyzed starch product solution comprising

a) Treating a plant part comprising starch granules and at least one processing enzyme under conditions that activate the at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising a hydrolyzed starch product, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one starch processing enzyme; and

b) the aqueous solution containing the hydrolyzed starch product was collected.

138. The method of claim 137, wherein said hydrolyzed starch product comprises dextrins, maltooligosaccharides, sugars, and/or mixtures thereof.

139. The method of claim 137, wherein the enzyme is an alpha-amylase, an alpha-glucosidase, a glucoamylase, a pullulanase, a starch pullulanase, a glucose isomerase, a beta-amylase, an isoamylase, a neo-pullulanase, an iso-pullulanase, or a combination thereof.

140. The method of claim 137, wherein said at least one processing enzyme is hyperthermophilic.

141. The method of claim 139, wherein the at least one processing enzyme is hyperthermophilic.

142. The method of claim 137, wherein the genome of said plant part is further augmented by an expression cassette encoding a non-hyperthermophilic starch processing enzyme.

143. The method of claim 142, wherein said non-hyperthermophilic starch processing enzyme is selected from the group consisting of: an amylase, a glucoamylase, an alpha-glucosidase, a pullulanase, a glucose isomerase, or a combination thereof.

144. The method of claim 137, wherein the at least one processing enzyme is expressed in the endosperm.

145. The method of claim 137, wherein the plant part is grain.

146. The method of claim 137, wherein said plant part is from maize, wheat, barley, rye, oats, sugarcane or rice.

147. The method according to claim 137, wherein the at least one processing enzyme is operably linked to a promoter and a signal sequence that directs the enzyme to the starch granule or endoplasmic reticulum or cell wall.

148. The method of claim 137, further comprising isolating the hydrolyzed starch product.

149. The method of claim 137, further comprising fermenting the hydrolyzed starch product.

150. A process for preparing a hydrolyzed starch product comprising

a) Treating a plant part comprising starch granules and at least one starch processing enzyme under conditions to activate the at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising a hydrolyzed starch product, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding at least one alpha-amylase; and

b) the aqueous solution containing the hydrolyzed starch product was collected.

151. The method of claim 150, wherein the alpha-amylase is hyperthermophilic.

152. The method of claim 151, wherein the hyperthermophilic alpha-amylase comprises the amino acid sequence of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33 or 35 or an active fragment thereof having alpha-amylase activity.

153. The method of claim 151, wherein the expression cassette comprises an amino acid sequence selected from the group consisting of SEQ id nos: 2, 9, 46 or 52 or a complement thereof, or a polynucleotide that hybridizes under low stringency hybridization conditions to SEQ ID NO: 2, 9, 46 or 52 and encodes a polypeptide having alpha-amylase activity.

154. The method of claim 150, wherein the genome of the transformed plant further comprises a polynucleotide encoding a non-thermostable starch processing enzyme.

155. The method of claim 150, further comprising treating the plant part with a non-hyperthermophilic starch processing enzyme.

156. A transformed plant part comprising at least one starch processing enzyme present within a plant cell, wherein said plant part is obtained from a transformed plant in which an expression cassette encoding said at least one starch processing enzyme has been increased in the genome.

157. The plant part of claim 156, wherein the enzyme is a starch processing enzyme selected from the group consisting of: alpha-amylase, glucoamylase, glucose isomerase, beta-amylase, alpha-glucosidase, isoamylase, pullulanase, neopullulanase, isoamylase and amylopullulanase.

158. The plant part of claim 156, wherein the enzyme is hyperthermophilic.

159. The plant part of claim 156, wherein the plant is maize.

160. A transformed plant part comprising at least one non-starch processing enzyme present in a plant cell wall or cell, wherein said plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding said at least one non-starch processing enzyme or at least one non-starch polysaccharide processing enzyme.

161. The plant part of claim 160, wherein the enzyme is hyperthermophilic.

162. The plant part of claim 160, wherein said non-starch processing enzyme is selected from the group consisting of: proteases, glucanases, xylanases, esterases, phytases and lipases.

163. The plant part of claim 156 or 160 which is an ear, seed, fruit, grain, straw, chaff or slag crumb.

164. A transformed plant part comprising an alpha-amylase having the amino acid sequence of seq id NO: 1, 10, 13, 14, 15, 16, 33 or 35, or a polypeptide consisting of an amino acid sequence comprising SEQ ID NO: 2, 9, 46 or 52.

165. A transformed plant part comprising an alpha-glucosidase having the amino acid sequence of SEQ ID NO: 5, 26, or 27, or a polypeptide consisting of a sequence comprising any one of SEQ ID NOs: 6.

166. A transformed plant part comprising a glucose isomerase enzyme having the amino acid sequence of SEQ ID NO: 28, 29, 30, 38, 40, 42, or 44, or a polypeptide consisting of any one of the amino acid sequences comprising SEQ ID NO: 19, 21, 37, 39, 41 or 43.

167. A transformed plant part comprising a glucoamylase having the amino acid sequence of SEQ ID NO: 45, or SEQ ID NO: 47 or SEQ ID NO: 49, or by an amino acid sequence comprising SEQ ID NO: 46, 48, 50, or 59.

168. A transformed plant part comprising a pullulanase consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or 25.

169. A method of transforming starch in the transformed plant part of claim 156, comprising activating a starch processing enzyme contained therein.

170. A method for converting starch in the transformed plant part of any one of claims 164-168 to a starch-derived product comprising activating the enzyme contained therein.

171. A starch, dextrin, maltooligosaccharide or sugar produced according to the method of claim 169.

172. A starch, dextrin, maltooligosaccharide or sugar produced according to the method of claim 170.

173. A method of using a transformed plant part containing at least one non-starch processing enzyme in its cell wall or cells, comprising

a) Treating a transformed plant part comprising at least one non-starch polysaccharide processing enzyme, wherein said plant part is obtained from a transformed plant to which an expression cassette encoding said at least one non-starch polysaccharide processing enzyme has been added, under conditions capable of activating said at least one enzyme, thereby digesting non-starch polysaccharides to form an aqueous solution comprising oligosaccharides and/or sugars; and

b) Collecting the aqueous solution containing the oligosaccharides and/or sugars.

174. The method of claim 173, wherein the non-starch polysaccharide processing enzyme is hyperthermophilic.

175. A method of using a transformed seed containing at least one processing enzyme comprising

a) Treating transformed seed comprising at least one protease or lipase under conditions capable of activating said at least one enzyme to produce an aqueous mixture comprising amino acids and fatty acids, wherein said seed is obtained from a transformed plant having increased in its genome an expression cassette encoding said at least one enzyme; and

b) the aqueous mixture was collected.

176. The method of claim 175, wherein the amino acid, fatty acid, or both are isolated.

177. The method of claim 175, wherein the at least one protease or lipase is hyperthermostable.

178. A process for producing ethanol comprising

a) Treating a plant part containing at least one polysaccharide processing enzyme under conditions capable of activating said at least one enzyme, thereby digesting polysaccharides to form oligosaccharides or fermentable sugars, wherein said plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding said at least one polysaccharide processing enzyme; and

b) Incubating the fermentable sugar under conditions that promote conversion of the fermentable sugar or oligosaccharide to ethanol.

179. The method of claim 178, wherein the plant part is a grain, fruit, seed, stem, wood, vegetable, or root.

180. The method of claim 178, wherein said plant part is obtained from a plant selected from the group consisting of: oats, barley, wheat, berries, grapes, rye, corn, rice, potatoes, sugar beets, sugarcane pineapples, grasses and trees.

181. The method of claim 178, wherein the polysaccharide processing enzyme is: an alpha-amylase, a glucoamylase, an alpha-glucosidase, a glucose isomerase, a pullulanase, or a combination thereof.

182. The method of claim 178, wherein said polysaccharide processing enzyme is hyperthermophilic.

183. The method of claim 178, wherein said polysaccharide processing enzyme is mesophilic.

184. The method of claim 181, wherein the polysaccharide processing enzyme is hyperthermophilic.

185. A process for producing ethanol comprising

a) Heat treating a plant part containing at least one enzyme selected from the group consisting of: an alpha-amylase, glucoamylase, alpha-glucosidase, glucose isomerase, or pullulanase, or a combination thereof, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one enzyme; and

b) Incubating the fermentable sugar under conditions promoting conversion of the fermentable sugar to ethanol.

186. The method of claim 185, wherein the at least one enzyme is hyperthermophilic.

187. The method of claim 185, wherein said at least one enzyme is mesophilic.

188. The method of claim 185, wherein the alpha-amylase has the amino acid sequence of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33 or 35, or a polypeptide consisting of a sequence comprising seq id NO: 2 or 9.

189. The method of claim 185, wherein the α -glucosidase has the amino acid sequence of SEQ id no: 5, 26, or 27, or a polypeptide consisting of a sequence comprising any one of SEQ ID NOs: 6.

190. The method of claim 185, wherein the glucose isomerase enzyme has the amino acid sequence of seq id NO: 28, 29, 30, 38, 40, 42, or 44, or a polypeptide consisting of any one of the amino acid sequences comprising SEQ ID NO: 19, 21, 37, 39, 41 or 43.

191. The method of claim 185, wherein the glucoamylase has the amino acid sequence of SEQ id no: 45, or an amino acid sequence consisting of SEQ ID NO: 46, 48 or 50.

192. The method of claim 185, wherein the pullulanase has the amino acid sequence of SEQ id no: 24 or 34, or a polypeptide consisting of an amino acid sequence comprising SEQ ID NO: 4 or 25.

193. A process for producing ethanol comprising

a) Treating a plant part comprising at least one non-starch processing enzyme under conditions capable of activating said at least one enzyme, thereby digesting non-starch polysaccharides into oligosaccharides and fermentable sugars, wherein said plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding said at least one enzyme; and

b) incubating the fermentable sugar under conditions promoting conversion of the fermentable sugar to ethanol.

194. The method of claim 193, wherein the non-starch processing enzyme is a glucanase, xylanase or cellulase.

195. A process for producing ethanol comprising

a) Treating a plant part containing at least one enzyme selected from the group consisting of: an alpha-amylase, glucoamylase, alpha-glucosidase, glucose isomerase, pullulanase, or a combination thereof, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one enzyme; and

b) incubating the fermentable sugar under conditions promoting conversion of the fermentable sugar to ethanol.

196. The method of claim 195, wherein the at least one enzyme is hyperthermophilic.

197. A method of producing a sweetened farinaceous food product without the addition of an additional sweetener comprising

a) Treating a plant part comprising at least one starch processing enzyme, wherein the plant part is obtained from a transformed plant in which an expression cassette encoding the at least one enzyme has been added to its genome, under conditions that activate the at least one enzyme, thereby processing starch granules in the plant part to sugars, thereby forming a sweetened product; and

b) processing the sweetened product into a farinaceous food.

198. The method of claim 197 wherein the flour food product is made from a sweetened product and water.

199. The method of claim 197, wherein the farinaceous food product comprises malt, flavoring agents, vitamins, minerals, coloring agents, or any combination thereof.

200. The method of claim 197, wherein the at least one enzyme is hyperthermophilic.

201. The method of claim 197, wherein the enzyme is: an alpha-amylase, an alpha-glucosidase, a glucoamylase, a pullulanase, a glucose isomerase, or any combination thereof.

202. The method of claim 197, wherein the plant is selected from the group consisting of: soybean, rye, oat, barley, wheat, corn, rice and sugarcane.

203. The method of claim 197 wherein the farinaceous food product is a cereal.

204. The method of claim 197 wherein the farinaceous food product is a breakfast food product.

205. The method of claim 197 wherein the farinaceous food product is a ready-to-eat food product.

206. The method of claim 197 wherein the farinaceous food product is a baked good.

207. The method of claim 197, wherein the processing is baking, boiling, heating, steaming, discharging, or any combination thereof.

208. A method of sweetening a starch-containing product without the addition of a sweetener comprising

a) Treating a starch comprising at least one starch processing enzyme under conditions capable of activating the at least one enzyme, thereby digesting the starch to form sugars to form a sweetened starch, wherein the starch is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one enzyme; and

b) adding the sweetened starch to a product to make a sweetened starch-containing product.

209. The method of claim 208, wherein said transformed plant is selected from the group consisting of: corn, soybean, rye, oat, barley, wheat, rice and sugarcane.

210. The method of claim 208, wherein said at least one enzyme is hyperthermophilic.

211. The method of claim 208, wherein the at least one enzyme is an alpha-amylase, an alpha-glucosidase, a glucoamylase, a pullulanase, a glucose isomerase, or any combination thereof.

212. A farinaceous food product obtained by the method of claim 197.

213. A sweetened starch-containing product obtained by the method of claim 208.

214. A method of sweetening a polysaccharide-containing fruit or vegetable, comprising treating a fruit or vegetable containing at least one polysaccharide processing enzyme under conditions that activate the at least one enzyme, thereby processing polysaccharides in the fruit or vegetable to form sugars, resulting in a sweetened fruit or vegetable, wherein the fruit or vegetable is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one polysaccharide processing enzyme.

215. The method of claim 214, wherein said fruit or vegetable is selected from the group consisting of potatoes, tomatoes, bananas, squash, peas and beans.

216. The method of claim 214, wherein said at least one enzyme is hyperthermophilic.

217. The method of claim 214, wherein the enzyme is an alpha-amylase, an alpha-glucosidase, a glucoamylase, a pullulanase, a glucose isomerase, or any combination thereof.

218. A method of preparing an aqueous solution comprising sugars, comprising treating starch granules obtained from the plant part of claim 156 under conditions that activate the at least one enzyme, thereby producing an aqueous solution comprising sugars.

219. A process for preparing a starch-derived product from grain without involving wet milling or dry milling of the grain prior to recovery of the starch-derived product, comprising

a) Treating a plant part comprising starch granules and at least one starch processing enzyme under conditions that activate the at least one enzyme, whereby starch granules are processed to form an aqueous solution comprising a dextrin or sugar, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one starch processing enzyme; and

b) collecting the aqueous solution containing the starch-derived product.

220. The method of claim 219, wherein the at least one starch processing enzyme is hyperthermophilic.

221. A method of isolating alpha-amylase, glucoamylase, glucose isomerase, alpha-glucosidase and pullulanase comprising culturing the transformed plant of claim 82 and isolating alpha-amylase, glucoamylase, glucose isomerase, alpha-glucosidase and pullulanase.

222. The method of claim 221, wherein the α -amylase, glucoamylase, glucose isomerase, α -glucosidase and pullulanase are hyperthermostable.

223. A process for preparing maltodextrin comprising

a) Mixing the transgenic grain with water;

b) heating the mixture;

c) separating solids from the dextrin syrup produced in (b), and

d) and collecting the maltodextrin.

224. The method of claim 223, wherein the transgenic grain comprises at least one starch processing enzyme.

225. The method of claim 224, wherein the starch processing enzyme is an alpha-amylase, glucoamylase, alpha-glucosidase, and glucose isomerase.

226. The method of claim 225, wherein the at least one starch processing enzyme is hyperthermophilic.

227. Maltodextrin produced by the method of any one of claims 223-226.

228. A maltodextrin composition produced by the method of any one of claims 223-226.

229. A process for preparing dextrins or sugars from cereals without involving mechanical disruption of the cereals prior to recovery of a starch-derived product, comprising

a) Treating a plant part comprising starch granules and at least one starch processing enzyme under conditions to activate the at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising dextrin or sugar, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one processing enzyme; and

b) Collecting the aqueous solution containing the sugar and/or dextrin.

230. The method of claim 229, wherein the starch processing enzyme is an alpha-amylase, glucoamylase, alpha-glucosidase, and glucose isomerase.

231. A process for the preparation of fermentable sugars comprising

a) Treating a plant part comprising starch granules and at least one starch processing enzyme under conditions that activate the at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising a dextrin or sugar, wherein the plant part is obtained from a transformed plant having increased in its genome an expression cassette encoding the at least one processing enzyme; and

b) collecting the aqueous solution containing fermentable sugars.

232. The method of claim 249, wherein the starch processing enzyme is an alpha-amylase, glucoamylase, alpha-glucosidase, and glucose isomerase.

233. A corn plant stably transformed with a vector comprising an hyperthermophilic alpha-amylase.

234. A corn plant stably transformed with a vector comprising a polynucleotide sequence encoding an alpha-amylase that hybridizes to SEQ ID NO: 1 or SEQ ID NO: consistency of 51 is higher than 60%.