BR122014007966B1 - Expression cassette comprising an alpha-amylase and a method of obtaining a plant comprising the same - Google Patents

Description

“EXPRESSION CASSETTE UNDERSTANDING AN ALPHA amylase AND METHOD FOR OBTAINING A SAME PLAN” [0001] Application divided from patent application PI0212170-0, filed August 27, 2002.

Related Applications This application claims priority over Application Serial Number 60 / 315,281, filed August 27, 2001, which is incorporated herein by reference.

Field of the Invention The present invention relates generally to the field of plant molecular biology and, more specifically, to the creation of plants expressing a processing enzyme that provides a desired trait to the plant or its products.

Background of the Invention Enzymes are used to process a variety of agricultural products such as wood, fruits and vegetables, starches, juices, wood, fruits and vegetables, starches, juices, and the like. Typically, processing enzymes are produced and recovered on an industrial scale from diverse sources such as microbial fermentation (Bacillus α-amylase), or plant isolation (coffee β-galactosidase or papain from plant parts). Enzyme preparations are used in different processing applications by mixing the enzyme and substrate under appropriate conditions of humidity, temperature, time, and mechanical mixing such that the enzymatic reaction is achieved in a commercially viable manner. The methods involve separate steps of enzyme production, manufacture of an enzyme preparation, mixing of the enzyme and substrate, and subjecting the mixture to appropriate conditions to facilitate the enzymatic reaction. A method that reduces or eliminates the time, energy, mixing, capital, and / or enzyme production costs, or results in improved or unpublished products would be useful and beneficial. An example of where such improvements are needed is in the area of corn milling.

[0005] Nowadays, corn is ground to obtain cornstarch and other corn milling co-products such as corn gluten food, corn gluten flour, and corn oil. Starch obtained from the process is often further processed to other products, such as derivatized starches and sugars, or fermented to prepare a variety of products including alcohols or lactic acid. Corn starch processing often involves the use of enzymes, in particular enzymes that hydrolyze and convert starch to fermentable sugars or fructose (α- and glucosyl amylase, α-glucosidase, glucose isomerase, and the like). The process used commercially today is costly because it requires the construction of very large mills to process maize at the scales required for reasonable economy. Additionally, the process requires the separate manufacture of starch modifying or hydrolyzing enzymes, and the machinery for mixing the enzyme and substrate to produce the hydrolyzed starch products.

The process of recovering starch from corn kernels is well known and involves a wet milling process. Wet milling includes the steps of macerating the corn grain, grinding the corn grain and separating the grain components. Grains are macerated in a maceration tank tank with a countercurrent water flow at about 120 ° F (49 ° C) and the grains remain in the maceration tank for 24 to 48 hours. This macerating water typically contains sulfur dioxide in a concentration of about 0.2% by weight. Sulfur dioxide is employed in the process to help reduce microbial growth and also to reduce disulfide bonds in endosperm proteins to facilitate more efficient starch-protein separation. Typically, about 0.59 gallon (2.23 l) of maize bushel water is used. Maceration water is considered tailings and often contains unwanted levels of residual sulfur dioxide.

The macerated grains are then dewatered and subjected to 'friction' type mill sets. The first set of 'friction' mills breaks the beans releasing the germ from the rest of the grain. An advantageous commercial 'friction' type mill for the wet grinding business is marketed under the Bauer trademark. Centrifugation is used to separate the germ from the rest of the grain. A typical commercial centrifuge separator is the Merco centrifugal separator. Friction mills and centrifugal separators are expensive and large items that use energy to operate.

In the next process step process step, the remaining grain components, including starch, husk, fiber, and gluten are subjected to another set of friction mills and passed through a set of scrubbing screens to separate the fiber components of starch and gluten (endosperm protein). Starch and gluten pass through the screens while other fibers do not. Centrifugation or a third milling, followed by centrifugation, is used to separate starch from endosperm protein. Centrifugation produces a starch syrup that is dewatered, then washed with fresh water and dried to about 12% humidity. Substantially pure starch is typically further processed using enzymes.

Starch separation from other grain components is performed because removal of the seed coat, embryo and endosperm proteins allows the starch to be efficiently contacted with processing enzymes, and the resulting hydrolysis products are relatively free. contaminants from the other grain components. Separation also ensures that other grain components are recovered efficiently and can subsequently be sold as co-products to increase the mill's financial return.

Once the starch is recovered from the wet milling process, it typically undergoes gelatinition, liquefaction and dextrinization processing steps for maltodextrin production, and subsequent saccharification, isomerization and refining steps for glucose production, maltose and fructose.

Gelatinition is employed in starch hydrolysis because currently available enzymes cannot rapidly hydrolyze crystalline starch. To make starch available to hydrolytic enzymes, starch is typically prepared in a syrup with water (20-40% dry solids) and heated to the appropriate gelling temperature. In the case of maize starch, this temperature is between 105-110 ° C. Gelatinized starch is typically very viscous and is therefore diluted in the next step called liquefaction. Liquefaction breaks some of the bonds between starch glucose molecules and is carried out enzymatically or through the use of acid. Heat stable endo α-amylase enzymes are used in this step, and in the subsequent step of dextrinization. The extent of hydrolysis is controlled in the dextrinization step to provide hydrolysis products with the desired percentage of dextrose.

Further hydrolysis of the dextrin products of the liquefaction step is carried out with a quantity of different exoamylases and demystifiers, depending on the desired products. And finally, if fructose is desired, then the immobilized glucose isomerase enzyme is typically employed to convert glucose to fructose. Dry milling processes for the preparation of fermentable sugars (and then ethanol, for example) from corn starch facilitate efficient contact of exogenous enzymes with starch. These processes are less costly than wet milling, but significant economic advantages are still desirable because often the co-products derived from these processes are not as valuable as those derived from wet milling. For example, in dry milling the grain, the grain is ground to a powder to facilitate efficient starch contact by degrading enzymes. After enzyme hydrolysis of the cornmeal, the residual solids have some nutritional value because they contain protein and some other components. Eckhoff recently described the potential for improvement and the relevant issues related to dry milling in a paper entitled "Fermentation and costs of fuel ethanol from corn with quick-germ process" (Appl. Biochem. Biotechnol., 94: 41 (2001)). ). The "quick germ" method allows the separation of the oil-rich germ from starch with the use of reduced maceration time.

An example where regulation and / or level of endogenous processing enzymes in a plant may result in a desirable product is sweet corn. Typical sweet corn varieties are distinguished from field maize varieties in that sweet corn is not capable of normal levels of starch biosynthesis. Genetic mutations in genes encoding enzymes involved in starch biosynthesis are typically employed in sweet corn varieties to limit starch biosynthesis. These mutations are in the genes that encode starch synthases and ADP-glucose pyrophosphorylases (such as sugary and super-sweet mutations). Fructose, glucose and sucrose, which are the simplest sugars needed to produce the palatable sweetening that consumers of fresh edible corn want, accumulate in the developing endosperm of such mutants. However, if the starch buildup level is too high, such as when the corn is allowed to mature for too long (late harvest) or the corn is stored for too long before it is consumed, the product loses sweetness and assumes a taste and an amylaceous sensation in the mouth. The harvest window for sweet corn is therefore quite narrow and shelf life is limited.

Another significant disadvantage for the farmer planting sweet corn varieties is that the utility of these varieties is limited solely to edible food. If a farmer wished to anticipate by harvesting his green corn for use as an edible food during seed development, the crop would have a substantial loss. Grain yield and green corn quality are low for two fundamental reasons. The first reason is that mutations in the starch biosynthesis pathway disable starch biosynthetic 'machinery' and grains do not form completely, compromising yield and quality. Secondly, due to the high levels of sugars present in the grain and the inability to sequester these sugars as starch, the overall seed penetration capacity is reduced, which exacerbates the reduced nutrient storage in the grain. Seed endosperms of the sweet corn variety wither and collapse, lack appropriate desiccation, and are susceptible to disease. The poor quality of sweet corn grain has additional agronomic implications; because low seed viability, low germination, susceptibility to seedling disease, and low vigor of early seedling result from a combination of factors caused by inadequate starch accumulation. Thus, the low quality issues of green corn impact on the consumer, farmer / grower, distributor, and seed producer.

Thus, for dry milling, there is a need for a method that improves process efficiency and / or increases the value of by-products. For wet milling, there is a need for a processing method that does not require the equipment necessary for prolonged maceration, roughing, grinding, and / or separation of grain components. For example, there is a need to modify or eliminate the wet grinding maceration step because this could reduce the amount of wastewater that requires disposal, thus saving energy and time, and increasing mill capacity (grain would remain less time in maceration tanks). There is also a need to eliminate or refine the process of separating starch-containing endosperm from the embryo.

Summary of the Invention The present invention relates to self-processing plants and plant parts and methods of using them. The self-processing plant and plant parts of the present invention are capable of expressing and activating (mesophilic, thermophilic, and / or hyperthermophilic) enzyme (s). Upon activation of the enzyme (s) (mesophilic, thermophilic, or hyperthermophilic) the plant or plant part is able to self-process the substrate upon which it acts to obtain the desired result.

The present invention relates to an isolated polynucleotide a) comprising SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 or its complement, or a polynucleotide that hybridizes to the complement of any of SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41,43, 46, 48, 50 , 52, or 59 under low stringency conditions and encodes a polypeptide having α-amylase, pullulanase, α-glucosidase, glucose isomerase, or glucoamylase activity, or b) encoding a polypeptide comprising SEQ ID NO: 10, 13, 14, 15 , 16, 18, 20, 24, 26, 27, 28, 29, 30, 33, 34, 35, 36, 38, 40, 42, 44, 45, 47, 49, or 51 or an enzymatically active fragment thereof. Preferably, the isolated polynucleotide encodes a fusion polypeptide comprising a first polypeptide and a second peptide, the first polypeptide having α-amylase, pullulanase, α-glucosidase, glucose isomerase, or glucoamylase activity. Most preferably, the second peptide comprises a signal sequence peptide, which can target the first polypeptide to a vacuole, endoplasmic reticulum, chloroplast, starch granule, seed or cell wall of a plant. For example, the signal sequence may be a waxy N-terminal signal sequence, a y-zein N-terminal signal sequence, a starch binding domain, or a C-terminal starch binding domain. Polynucleotides that hybridize to the complement of any of SEQ ID NO: 2, 9, or 52 under low stringency conditions and encode a polypeptide having α-amylase activity; complemented by SEQ ID NO: 4 or 25 under low stringency hybridization conditions and encodes a polypeptide showing pullulanase activity; complemented by SEQ ID NO: 6 and encodes a polypeptide having α-glucosidase activity; complementing any of SEQ ID NO: 19, 21, 37, 39, 41, or 43 under low stringency conditions and encodes a polypeptide exhibiting glucose isomerase activity; complemented by any of SEQ ID NO: 46, 48, 50, or 59 under low stringency conditions and encoding a polypeptide exhibiting glucoamylase activity are further encompassed.

In addition, an expression cassette comprising a polynucleotide a) having SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, or 59 or its complement, or a polynucleotide that hybridizes to the complement of any of SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50 , 52, or 59 or under low stringency conditions and encodes a polypeptide having α-amylase, pullulanase, α-glucosidase, glucose isomerase activity, or glucoamylase activity or b) encoding a polypeptide comprising SEQ ID NO: 10, 13, 14, 15, 16, 18, 20, 24, 26, 27, 28, 29, 30, 33, 34, 35, 36, 38, 40, 42, 44, 45, 47, 49, or 51 or an enzymatically fragment thereof active. Preferably, the expression cassette further comprises a promoter operably linked to the polynucleotide, such as an inducible promoter, tissue specific promoter, or preferably an endosperm specific promoter. Preferably, the endosperm specific promoter is a maize γ-zein promoter or a maize ADP-gpp promoter. In a preferred embodiment, the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12. In addition, in another preferred embodiment the polynucleotide is oriented in sense orientation relative to the promoter. The expression cassette of the present invention may further encode a signal sequence which is operably linked to the polynucleotide encoded polypeptide. The signal sequence preferably targets the polypeptide operably linked to a vacuole, endoplasmic reticulum, chloroplast, starch granule, seed or cell wall of a plant. Preferably signal sequences include a waxy N-terminal signal sequence, a γ-zein N-terminal signal sequence, or a starch binding domain.

The present invention further relates to a vector or cell comprising the expression cassettes of the present invention. The cell may be selected from the group consisting of an Agrobacterium, a monocotyledon cell, a dicotyledon cell, a Liliopsida cell, a Panicoideae cell, a corn cell, and a cereal cell. Preferably, the cell is a corn cell.

Furthermore, the present invention encompasses a plant stably transformed with the vectors of the present invention. A stably transformed plant with a vector comprising an α-amylase is provided having an amino acid sequence of any of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33, or 35, or encoded by a polynucleotide comprising any of SEQ ID NO: 2 or 9. Preferably, α-amylase is hypertermophilic.

In another embodiment, there is provided a plant stably transformed with a vector comprising a pullulanase having an amino acid sequence of either SEQ ID NO: 24 or 34, or encoded by a polynucleotide comprising any of SEQ ID NO: 4 or 25. Also provided is a stably transformed plant with a vector comprising an α-glucosidase having an amino acid sequence of either SEQ ID NO: 26 or 27, or encoded by a polynucleotide comprising SEQ ID NO: 6 Preferably, α-glucosidase is hyperthermophilic. Further described herein is a stably transformed plant with a vector comprising a glucose isomerase having an amino acid sequence of any of SEQ ID NO: 18, 20, 28, 29, 30, 38, 40, 42, or 44. , or encoded by a polynucleotide comprising any of SEQ ID NO: 19, 21,37, 39, 41, or 43. Preferably, the glucose isomerase is hypertermophilic. In another embodiment, a plant stably transformed with a vector comprising a glucose amylase having an amino acid sequence of any of SEQ ID NO: 45, 47, or 49, or encoded by a polynucleotide comprising any of SEQ ID NO, is described. : 46, 48, 50, or 59. Preferably, glucose amylase is hyperthermophilic.

Further provided are plant products such as seeds, fruits or grain from the stably transformed plants of the present invention.

In another embodiment, the invention relates to a transformed plant whose genome is augmented with a recombinant polynucleotide encoding at least one processing enzyme operably linked to a promoter sequence, wherein the sequence of said polynucleotide is optimized for expression. in the plant. The plant can be a monocot, such as corn, or a dicot. Preferably the plant is a cereal plant or a commercially developed plant. The processing enzyme is selected from the group consisting of overnight α-amylase, glucoamylase, glucose isomerase, glucanase, β-amylase, α-glucosidase, isoamylase, pullulanase, neulululase, iso-pululanase, amylopululanase, cellulase. , exo-1,4-p-cellobiohydrolase, exo-1,3-pD-glucanase, β-glucosidase, endoglucanase, L-arabinase, α-arabinosidase, galactanase, galactosidase, mananase, manosidase, xylanase, xylosidase, protease, glucanase , xylanase, esterase, phytase, and lipase. Preferably, the processing enzyme is a starch processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose isomerase, (3-amylase, α-glucosidase, isoamylase, pullulanase, neulululanase, isulululase, More preferably, the enzyme is selected from α-amylase, glucoamylase, glucose isomerase, glucose isomerase, α-glucosidase, and pullulanase. The processing enzyme is additionally and preferably hyperthermophilic. the enzyme may be a non-starch degrading enzyme selected from the group consisting of protease, glucanase, xylanase, esterase, phytase, and lipase. Enzymes of the said type may be additionally hyperthermophilic. In a preferred embodiment, the enzyme accumulates in the vacuole. , endoplasmic reticulum, chloroplast, starch granule, seed, or cell wall of a plant In addition, in another embodiment, the ooid plant genome is further enhanced with a second recombinant polynucleotide comprising a nonhypertermophilic enzyme.

In another aspect of the invention there is provided a transformed plant whose genome is enlarged with a recombinant polynucleotide encoding at least one processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose isomerase, α-glucosidase, and pullulanase, operably linked to a promoter sequence, the sequence of said polynucleotide being optimized for expression in the plant. Preferably, the processing enzyme is hyperthermophilic and maize.

Another embodiment relates to a transformed corn plant whose genome is enlarged with a recombinant polynucleotide encoding at least one processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose isomerase, α-glucosidase, and pululanase, operably linked to a promoter sequence, and the sequence of said polynucleotide is optimized for expression in the corn plant. Preferably, the processing enzyme is hyperthermophilic.

A transformed plant whose genome is enlarged with a recombinant polynucleotide having SEQ ID NO: 2, 9, or 52, operably linked to a promoter and signal sequence is provided. Additionally, a transformed plant whose genome is enlarged with a recombinant polynucleotide having SEQ ID NO: 4 or 25, operably linked to a promoter and signal sequence, is described. In another embodiment, a transformed plant whose genome is augmented with a recombinant polynucleotide having SEQ ID NO: 6, operably linked to a promoter and signal sequence. In addition, a transformed plant whose genome is enlarged with a recombinant polynucleotide having SEQ ID NO: 19, 21, 37, 39, 41, or 43 is described. The transformed plant whose genome is enlarged with a recombinant polynucleotide having SEQ ID NO: 46, 48, 50, or 59.

Products from transformed plants are further considered here. The product, for example, includes seed, fruit, or grain. Alternatively, the product may be the processing enzyme, starch or sugar.

A plant obtained from the stably transformed plants of the present invention are further described. In this regard, a plant can be a hybrid plant or a congenital plant.

A starch composition is a further embodiment of the invention comprising at least one processing enzyme that is a protease, glucanase, or esterase. Preferably, the enzyme is hyperthermophilic.

Grain is another embodiment of the invention comprising at least one processing enzyme, which is an α-amylase, pullulanase, α-glucosidase, glucoamylase, or glucose isomerase. Preferably, the enzyme is hyperthermophilic.

In another embodiment, there is provided a method of preparing starch granules comprising; treating grain comprising at least one non-starch processing enzyme under conditions which activate at least one enzyme, giving a mixture comprising starch granules and non-starch degradation products, wherein the grain is obtained from a transformed plant whose genome is augmented with an expression cassette encoding at least one enzyme; and separating starch granules from the mixture. Here, the enzyme is preferably a protease, glucanase, xylanase, phytase, or esterase. In addition, the enzyme is preferably hyperthermophilic. The grain may be broken again and / or may be treated in conditions of low or medium humidity. Alternatively, the grain may be treated with sulfur dioxide. The present invention may preferably further comprise separating non-starch products from the mixture. Starch products and non-starch products obtained by this method are described in more detail.

In another further embodiment, there is provided a method of producing hyper sweet corn comprising treating processed corn or a portion thereof, whose genome is enlarged with, and expressed in the endosperm, an expression cassette encoding at least one enzyme. starch degrading or starch isomerizing enzyme; under conditions that activate at least one enzyme in order to convert polysaccharides contained in corn to sugar, yielding hyper-sweet corn. The expression cassette preferably further comprises a promoter operably linked to the polynucleotide encoding the enzyme. The promoter may be a constitutive promoter, seed specific promoter, or endosperm specific promoter, for example. Preferably, the enzyme is a hyperthermophilic. More preferably, the enzyme is CC-amylase. The expression cassette used herein may further comprise a polynucleotide encoding a signal sequence operably linked to at least one enzyme. The signal sequence may direct the hyperthermophilic enzyme to the apoplast or endoplasmic reticulum, for example. Preferably, the enzyme comprises any of SEQ ID NO: 13, 14, 15, 16, 33, or 35.

In a more preferred embodiment, a method of producing hyper sweet corn comprising treating processed corn or a part thereof, whose genome is enlarged with, and endosperm expressions, an expression cassette encoding an alpha amylase, is described. , under conditions that activate at least one enzyme in order to convert polysaccharides in maize to sugar, yielding hyper sweet corn. Preferably, the enzyme is hyperthermophilic and the hyperthermophilic α-amylase comprises the amino acid sequence of any one of SEQ ID NO: 10, 13, 14, 15, 16, 33, or 35, or an enzymatically active fragment thereof exhibiting activity. -amylase.

One method of preparing a hydrolyzed starch product solution comprises treating a plant part comprising starch granules and at least one processing enzyme under conditions that activate at least one enzyme, thereby processing the starch granules to form. an aqueous solution comprising hydrolyzed starch product, wherein the plant part is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one starch processing enzyme; and collecting the aqueous solution comprising the hydrolyzed starch product. The hydrolyzed starch product may comprise a dextrin, maltooligosaccharide, glucose and / or mixtures thereof. Preferably, the enzyme is α-amylase, α-glucosidase, glucoamylase, pullulanase, amylopululanase, glucose isomerase, or any combination thereof. In addition, preferably, the enzyme is hyperthermophilic. In another aspect, the plant part genome may be further enlarged with an expression cassette encoding a nonhypertermophilic starch processing enzyme. The non-hyperthermophilic starch processing enzyme may be selected from the group consisting of amylase, glucoamylase, α-glucosidase, pullulanase, glucose isomerase, or a combination thereof. In another additional aspect, the processing enzyme is preferably expressed in the endosperm. Preferably, a plant part is grain, and is corn, wheat, barley, rye, oats, sugar cane or rice. Preferably, the at least one processing enzyme is operably linked to a promoter and signal sequence that targets the enzyme to the starch granule or endoplasmic reticulum, or to the cell wall. The method may further comprise isolating the hydrolyzed starch product and / or fermenting the hydrolyzed starch product.

In another aspect of the invention, a method of preparing hydrolysed starch product comprising treating a plant part comprising starch granules and at least one starch processing enzyme under conditions which activate at least one enzyme is described. that is by processing the starch granules to form an aqueous solution comprising a hydrolysed starch product, whereby the plant part is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one α-amylase; and collecting the aqueous solution comprising hydrolyzed starch product. Preferably, α-amylase is hyperthermophilic, and more preferably hyperthermophilic α-amylase comprises the amino acid sequence of any one of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33, or 35, or an active fragment thereof having α-amylase activity. Preferably, the expression cassette comprises a polynucleotide selected from any one of SEQ ID NO: 2, 9, 46, or 52, a complement thereof, or a polynucleotide that hybridizes to any one of SEQ ID NO: 2, 9, 46, or 52 under low stringency conditions and encodes a polypeptide having α-amylase activity. In addition, the invention further provides the transformed plant genome further comprising a polynucleotide encoding a non-thermophilic starch processing enzyme. Alternatively, a plant part may be treated with a non-hypertermophilic starch processing enzyme.

The present invention further relates to a transformed plant part comprising at least one starch processing enzyme present in the plant cells, wherein the plant part is obtained from a transformed plant whose genome is enlarged with a expression cassette encoding at least one starch processing enzyme. Preferably, the enzyme is a starch processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose isomerase, β-amylase, α-glucosidase, isoamylase, pullulanase, neulululanase, isulululase, and amylopululase. In addition, the enzyme is preferably hyperthermophilic. The plant can be any plant but is preferably corn.

Another embodiment of the invention is a transformed plant part comprising at least one non-starch processing enzyme present in the cell wall or plant cells, wherein the plant part is obtained from a transformed plant whose genome is increased with an expression cassette encoding at least one non-starch processing enzyme or at least one non-starch polysaccharide processing enzyme. Preferably, the enzyme is hyperthermophilic. In addition, the non-starch processing enzyme is preferably selected from the group consisting of protease, glucanase, xylanase, esterase, phytase, and lipase. The plant part can be any plant, but preferably it is an ear, seed, fruit, grain, berry, bark, or pomace.

The present invention also relates to transformed plant parts. For example, a transformed plant part comprising an alpha-amylase having an amino acid sequence of any of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33, or 35, or encoded by a A polynucleotide comprising any one of SEQ ID NO: 2, 9, 46, or 52, a transformed plant part comprising an α-glucosidase having an amino acid sequence of any one of SEQ ID NO: 5, 26 or 27, or encoded by a polynucleotide comprising SEQ ID NO: 6, a transformed plant part comprising a glucose isomerase having the amino acid sequence of any one of SEQ ID NO: 28, 29, 30, 38, 40, 42, or 44, or encoded by a A polynucleotide comprising any of SEQ ID NO: 19, 21, 37, 39, 41, or 43, a transformed plant part comprising a glucoamylase having the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 47, or SEQ ID NO: 49, or encoded by a polynucleotide comprising comprising any of SEQ ID NO: 46, 48, 50, or 59, and a transformed plant part comprising a pullulanase encoded by a polynucleotide comprising any of SEQ ID NO: 4 or 25.

Another embodiment is a method of converting starch to the transformed plant part comprising activating the starch processing enzyme contained therein. Starch, dextrin, maltooligosaccharide or sugar produced according to this method is described in more detail.

The present invention further describes a method of using a transformed plant part comprising at least one non-starch processing enzyme in the cell wall or plant part cell comprising treating a transformed plant part comprising at least one non-starch polysaccharide processing enzyme capable of activating at least one enzyme, thereby digesting non-starch polysaccharide to form an aqueous solution comprising oligosaccharide and / or sugars, wherein the plant part is obtained from a transformed plant, wherein its genome is augmented with an expression cassette encoding at least one non-starch polysaccharide processing enzyme; and collecting the aqueous solution comprising the oligosaccharides and / or sugars. Preferably, the non-starch polysaccharide processing enzyme is hyperthermophilic.

A method of using transformed seeds comprising at least one processing enzyme, comprising treating transformed seeds comprising at least one protease or lipase capable of activating at least one enzyme, giving an aqueous mixture comprising amino acids and fatty acids, being that the seed is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one enzyme; and collect the aqueous mixture. Amino acids, fatty acids or both are preferably isolated. Preferably the at least one protease or lipase is hyperthermophilic.

A method of preparing ethanol comprising treating a plant part comprising at least one polysaccharide processing enzyme capable of activating at least one enzyme, thereby digesting polysaccharide to form fermentable oligosaccharide or sugar, the plant part being is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one polysaccharide processing enzyme; and incubating fermentable sugar under conditions that promote the conversion of fermentable sugar or oligosaccharide to ethanol. Preferably, a plant part is a grain, fruit, seed, stalk, wood, vegetable or root. Preferably, a plant part is obtained from a plant selected from the group consisting of oats, barley, wheat, berries, grapes, rye, corn, rice, potato, sugar beet, sugar cane, pineapple, grass and tree. In another preferred embodiment, the polysaccharide processing enzyme is α-amylase, glucoamylase, α-glucosidase, glucose isomerase, pullulanase, or a combination thereof.

A method of preparing ethanol comprising treating a plant part comprising at least one enzyme selected from the group consisting of α-amylase, glucoamylase, α-glucosidase, glucose isomerase, or pullulanase, or a combination thereof, is provided. heat for a period of time and capable of activating at least one enzyme, thereby digesting the polysaccharide to form fermentable sugar, the plant part being obtained from a transformed plant whose genome is enlarged with an expression cassette encoding the hair. least one enzyme; and incubating fermentable sugar under conditions that promote the conversion of fermentable sugar to ethanol. Preferably, the at least one enzyme is hyperthermophilic or mesophilic.

In another embodiment, there is provided a method of preparing ethanol comprising treating a plant part comprising at least one non-starch processing enzyme capable of activating at least one enzyme, thereby digesting non-starch polysaccharide to oligosaccharide and fermentable sugar, the plant part being obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one enzyme; and incubating fermentable sugar under conditions that promote the conversion of fermentable sugar to ethanol. Preferably, the non-starch processing enzyme is a glucanase, xylanase or cellulase.

Further provided is a method for preparing ethanol comprising treating a plant part comprising at least one enzyme selected from the group consisting of α-amylase, glucoamylase, α-glucosidase, glucose isomerase, or pullulanase, or a combination thereof, capable of activating at least one enzyme, thereby digesting polysaccharide to form fermentable sugar, the plant part being obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one enzyme; and incubating fermentable sugar under conditions that promote the conversion of fermentable sugar to ethanol. Preferably, the enzyme is hyperthermophilic.

In addition, a method for producing a sugary farinaceous food product without the addition of extra sweetener comprising treating a plant part comprising at least one starch processing enzyme under conditions which activate at least one enzyme in this manner is described. processing starch granules in the plant part to sugars to form a sweetened product, the plant part being obtained from a transformed plant whose genome is augmented with an expression cassette encoding at least one enzyme; and process the sweetened product to a farinaceous food product. The farinaceous food product may be formed from the sweetened product and water. In addition, the farinaceous food product may contain malt, flavorings, vitamins, minerals, coloring agents or any combination thereof. Preferably the at least one enzyme is hyperthermophilic. The enzyme may be selected from α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. The plant may additionally be selected from the group consisting of soybeans, rye, oats, barley, wheat, maize, rice and sugar cane. Preferably, the farinaceous food product is a cereal food, a breakfast food, a ready-to-eat food, or a cooked product. Processing may include cooking, boiling, heating, steaming, electric discharge or any combination thereof.

The present invention further relates to a method of sweetening a starch-containing product without added sweetener comprising treating starch comprising at least one starch processing enzyme capable of activating at least one enzyme, thereby digesting the starch. to form a sugar to form a sweetened starch, the starch being obtained from a transformed plant whose genome is augmented with an expression cassette encoding at least one enzyme; and adding sweetened starch to a product to produce a product containing sweetened starch. Preferably, the transformed plant is selected from the group consisting of corn, soybean, rye, oats, barley, wheat, rice and sugar cane. Preferably the at least one enzyme is hyperthermophilic. More preferably, the at least one enzyme is α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof.

A farinaceous food product and sweetened starch-containing product is provided herein.

The invention also relates to a method for sweetening a polysaccharide-containing vegetable or fruit comprising treating a fruit or vegetable comprising at least one polysaccharide processing enzyme under conditions that activate at least one enzyme, thereby processing the polysaccharide in fruit or vegetable to form sugar into a sweetened fruit or vegetable, the fruit or vegetable being obtained from a transformed plant whose genome is augmented with an expression cassette encoding at least one polysaccharide processing enzyme. The fruit or vegetable is selected from the group consisting of potato, tomato, banana, pumpkin, peas, and beans. Preferably the at least one enzyme is hyperthermophilic.

The present invention further relates to a method of preparing an aqueous solution comprising sugar comprising treating starch granules obtained from the plant under conditions which activate at least one enzyme, thereby giving an aqueous solution comprising sugar.

Another embodiment relates to a method of preparing grain starch products which does not involve wet or dry milling the grain prior to the recovery of starch products, comprising treating a plant part comprising starch granules. and at least one starch processing enzyme under conditions which activate the at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising dextrins or sugars, the plant part being obtained from a transformed plant, whose genome is augmented with an expression cassette encoding at least one starch processing enzyme; and collecting the aqueous solution comprising the starch derived product. Preferably, the at least one starch processing enzyme is hyperthermophilic.

A method of isolating α-amylase, glucoamylase, glucose isomerase, α-glucosidase, and pullulanase comprising cultivating the transformed plant and isolating α-amylase, glucose isomerase, α-glucosidase, and pullulanase from the protein is provided. same. Preferably, the enzyme is hyperthermophilic.

A method of preparing maltodextrin comprising mixing transgenic grain with water, heating said mixture, separating solids from the generated dextrin syrup, and collecting maltodextrin. Preferably, the transgenic grain comprises at least one starch processing enzyme. Preferably, the starch processing enzyme is α-amylase, glucoamylase, α-glucosidase, and glucose isomerase. In addition, there is provided maltodextrin produced by the method and also composition produced by this method.

A method of preparing dextrins, or sugars from grain which does not involve mechanical disruption of the grain prior to the recovery of starch-derived [gap] comprising treating a plant part comprising starch granules and at least one enzyme is provided. processing starch under conditions that activate at least one enzyme, thereby processing the starch granules to form an aqueous solution comprising dextrins or sugars, the plant part being obtained from a transformed plant whose genome is increased with a expression cassette encoding at least one processing enzyme; and collecting the aqueous solution comprising sugar and / or dextrins.

The present invention further relates to a method of producing fermentable sugar comprising treating a plant part comprising starch granules and at least one starch processing enzyme under conditions which activate at least one enzyme, thereby processing the starch granules. starch granules to form an aqueous solution comprising dextrins or sugars, wherein the plant part is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one processing enzyme; and collecting the aqueous solution comprising the fermentable sugar.

Further, there is provided herein a stably transformed maize plant with a vector comprising a hypertermophilic α-amylase. For example, preferably a stably transformed maize plant with a vector comprising an alpha-amylase encoding polynucleotide sequence having an identity greater than 60% with SEQ ID NO: 1 or SEQ ID NO: 51 is comprised. .

[0057] Brief Description of the Figures [0058] Figures 1A and 1B illustrate the α-amylase activity expressed on maize grains and on the endosperm of T1 segregating grains from pNOV6201 plants and six pNOV6200 lines.

[0059] Figure 2 illustrates α-amylase activity in segregating T1-segregating pNOV6201 line grains.

[0060] Figure 3 illustrates the amount of ethanol produced upon fermentation of thermostable α-amylase 797GL3 transgenic maize porridge that has undergone liquefaction times of up to 60 minutes at 85 ° C and 95 ° C. This figure illustrates that the ethanol yield at 72 hours of fermentation remained almost unchanged from 15 minutes to 60 minutes of liquefaction. In addition, it shows that porridge produced by liquefaction at 95 ° C produced more ethanol at any time than porridge produced by liquefaction at 85 ° C.

Figure 4 illustrates the amount of residual starch (%) remaining after fermentation of thermostable alpha-amylase-containing transgenic maize mashes which were subjected to a liquefaction time of up to 60 minutes at 85 ° C and 95 ° C. This Figure illustrates that the ethanol yield at 72 hours of fermentation was almost unchanged from 15 [...] minutes to 60 minutes of liquefaction. In addition, it shows that porridge produced by liquefaction at 95 ° C produced more ethanol at any time than porridge produced by liquefaction at 85 ° C.

[0062] Figure 5 illustrates ethanol yields for porridge from a transgenic maize, control maize, and various mixtures thereof prepared at 85 ° C and 95 ° C. This figure illustrates that transgenic maize comprising α-amylase results in a significant improvement in the preparation of starch available for fermentation because there was a reduction in starch left after fermentation.

[0063] Figure 6 illustrates the amount of residual starch measured in dry product after pellet fermentation of a transgenic maize, control maize, and various mixtures thereof prepared at 85 ° C and 95 ° C.

Figure 7 illustrates ethanol yields as a function of fermentation time of a sample comprising 3% transgenic maize over a period of 20-80 hours at various pH ranges of 5.2-6.4. The figure illustrates that fermentation conducted at a lower pH proceeds faster than at a pH of 6.0 or higher.

Figure 8 illustrates the ethanol yields during the fermentation of a porridge comprising various weight percent transgenic maize from 0-12 weight percent over various pH ranges of 5.2-6.4. This Figure illustrates that ethanol yield was independent of the amount of transgenic grain included in the sample.

[0066] Figure 9 shows T2 seed analysis of different events transformed with pNOV 7005. High expression of pullulanase activity compared to non-transgenic control can be detected in several ways.

Figure 10A and 10B show the results of HPLC analysis of starch-expressed pululanase-generated hydrolytic products in transgenic cornmeal. Incubation of pullulanase flour expressing maize in reaction buffer at 75 ° C for 30 minutes results in the production of medium length chain oligosaccharides (DP: 10-30) and short amylose chains ( DP ~ 100-200) of cornstarch. Figures 10A and 10B also show the effect of added calcium ions on pullulanase activity.

Figures 11A and 11B illustrate data generated by HPLC analysis of starch hydrolysis product from two reaction mixtures. The first reaction indicated as 'Amylase' contains a [1: 1 (w / w)] mixture of α-amylase maize flour samples expressing transgenic maize and non-transgenic maize A188; and the second reaction mixture 'Amylase + Pululanase' contains a [1: 1 (wt / wt)] mixture of a-amylase maize flour samples expressing transgenic maize and pululanase expressing transgenic maize.

[0069] Figure 12 illustrates the amount of sugar product in pg in 25 pl of reaction mixture for two reaction mixtures. The first reaction indicated as 'Amylase' contains a [1: 1 (w / w)] mixture of transgenic maize flour samples expressing alpha-amylase and non-transgenic A188 maize; and the second reaction mixture 'Amylase + Pululanase' contains a [1: 1 (w / w)] mixture of samples of transgenic maize expressing a-amylase and transgenic maize expressing pululanase.

Figure 13A and 13B show the starch hydrolysis product of two sets of reaction mixtures after 30 minutes of incubation at 85 ° C and 95 ° C. For each set there are two reaction mixtures; the first reaction indicated as 'Amylase X Pullulanase' contains transgenic maize flour (generated by cross-pollination) expressing both α-amylase and pullulanase, and the second reaction indicated as 'Amylase', mixture of α maize flour samples -amylase expressing transgenic maize and non-transgenic A188 maize in a ratio such as to obtain the same amount of α-amylase activity as observed at the cross (Amylase X Pullulanase).

Figure 14 illustrates glucose starch degradation using non-transgenic (control) maize seed, transgenic maize seed comprising 797GL3 α-amylase, and a combination of 797GL3 transgenic maize seed with Mal A a- glucosidase.

Figure 15 illustrates the conversion of crude starch at room temperature or 30 ° C. In this figure, reaction mixtures 1 and 2 are a combination of water and starch at room temperature and 30 ° C, respectively. Reaction mixtures 3 and 4 are a combination of barley α-amylase and starch at room temperature and 30 ° C, respectively. Reaction mixtures 5 and 6 are combinations of Thermoanaerobacterium glucoamylase and starch at room temperature and 30 ° C, respectively. Reaction mixtures 7 and 8 are combinations of barley α-amylase (sigma) and Thermoanaerobacterium glucoamylase and starch at room temperature and 30 ° C, respectively. Reaction mixtures 9 and 10 are combinations of control barley α-amylase (sigma) and starch at room temperature and 30 ° C, respectively. The degree of polymerization (DP) of Thermoanaerobacterium glucoamylase products is indicated.

Figure 16 illustrates fructose production from transgenic amylase cornmeal using a combination of α amylase, α glucosidase, and glucose isomerase as described in Example 19. Corn amylase flour was mixed with enzyme solutions, more water or buffer. All reactions contained 60 mg of amylase flour and a total of 600 µl of liquid and were incubated for 2 hours at 90 ° C.

Figure 17 illustrates the peak areas of the 100% amylase flour reaction products of a self-processing grain as a function of incubation time from 0-1200 minutes at 90 ° C.

Figure 18 illustrates the peak areas of the reaction products with 10% transgenic amylase flour from one of a self-processing grain and 90% control corn meal as a function of incubation time from 0- 1200 minutes at 90 ° C.

Figure 19 provides the results of HPLC analysis of transgenic amylase flour incubated at 70 °, 80 °, 90 °, or 100 ° C for up to 90 minutes to assess the effect of temperature on starch hydrolysis.

Figure 20 illustrates ELSD peak area for samples containing 60 mg of transgenic amylase flour mixed with enzyme plus water or buffer solutions under various reaction conditions. A set of reactions was buffered with 50 mM MOPS, pH 7.0 at room temperature, plus 10 mM MgSO4 and 1 mM CoCh; In a second set of reactions the metal-containing buffer solution was replaced with water. All reactions were incubated for 2 hours at 90 ° C.

Detailed Description of the Invention According to the present invention, a "self-processing" plant or plant part herein incorporates an isolated polynucleotide encoding a processing enzyme capable of processing, e.g. modification of starches, polysaccharides, lipids, proteins, and analogues in plants, wherein the processing enzyme may be mesophilic, thermophilic or hyperthermophilic, and may be activated by milling, adding water, heating, or, otherwise, providing favorable conditions for enzyme function. Isolated polynucleotide encoding the processing enzyme is integrated into a plant or plant part for expression therein. Upon expression and activation of the processing enzyme, a plant or plant part of the present invention processes the substrate upon which the processing enzyme acts. Therefore, a plant or plant parts of the present invention are capable of self-processing the enzyme substrate upon activation of the processing enzyme contained therein in the absence of, or with reduced external sources normally required to process these substrates. Thus, transformed plants, transformed plant cells, and transformed plant parts have "built-in" processing capabilities for processing desired substrates via the enzymes incorporated therein in accordance with this invention. Preferably, the processing enzyme encoding polynucleotide is "genetically stable", i.e., the polynucleotide is kept stable in the transformed plant or plant parts of the present invention and is stably inherited by progeny through successive generations.

In accordance with the present invention, methods employing such plants and plant parts may eliminate the need to grind or otherwise physically disrupt the integrity of plant parts prior to the recovery of starch-derived products. For example, the invention provides improved methods of processing maize and other grain to recover starch products. The invention also provides a method for recovering starch granules containing starch degrading enzyme levels in or on the granules that are suitable for hydrolysis of specific starch bonds without the need to add hydrolyzing enzymes. of exogenously produced starch. The invention also provides improved self-processing plant products or plant parts obtained with the methods of the invention.

Additionally, the transformed "self-processing" plant part, e.g. e.g. grain, and transformed plant avoid major problems with existing technology, i.e. processing enzymes are typically produced by fermentation of microbes, which requires isolating the enzymes from culture supernatants, which costs money; The isolated enzyme must be formulated for the particular application, and processes and machinery must be developed to add, mix and react the enzyme with its substrate. The transformed plant of the invention or a part thereof also constitutes a source of the processing enzyme per se, as well as substrates and products of that enzyme such as starch and non-starch sugars, amino acids, fatty acids and polysaccharides. The plant of the invention may also be employed to prepare progeny plants such as hybrids and congenitals.

Processing Enzymes and Polynucleotides Encoding Them A polynucleotide encoding a processing enzyme (mesophilic, thermophilic, or hypertermophilic) is introduced into a plant or plant part. The processing enzyme is selected based on the desired substrate on which it should act, as found in transgenic plants or plants and / or the desired end product. For example, the processing 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 isomerizing enzymes, including, for example, α-amylase, endo or exo-1,4, or 1,6-aD, glucoamylase, glucose isomerase, β-amylases, α glucosidases, and other exoamylases; and starch debranching enzymes such as isoamylase, pullulanase, neulululanase, isulululase, amylopululanase and the like, glycosyl transferases such as cyclodextrin glycosyl transferase and analogs, cellulases such as exo-1,4-p-cellobiohydrolase, βχο-1, 3-β-D-glucanase, hemicellulase, β-glucosidase and the like; endoglucanases, such as endo-1,3'-glucanase and endo-1,4'-glucanase and the like; L-arabinases, such as endo-1,5-α-L-arabinase, α-arabinosidases and the like; galactanases, such as endo-1,4'-D-galactanase, endo-1,3-β-D-galactanase, β-galactosidase, α-galactosidase and the like; mannanases such as endo-1,4'-D-mannanase, β-mannosidase, α-mannosidase and the like; xylanases, such as endo-1,4'-xylanase, β-D-xylosidase, 1,3'-D-xylanase, and the like; and pectinases; and non-starch processing enzymes including protease, glucanase, xylanase, thioredoxin / thioredoxin reductase, esterase, phytase, and lipase.

In one embodiment, the processing enzyme is a starch degrading enzyme selected from the group consisting of α-amylase, pullulanase, α-glucosidase, glucoamylase, amylopululanase, glucose isomerase, or combinations thereof. According to this embodiment, the starch degrading enzyme is capable of allowing the self-processing plant, or plant part, to degrade starch upon activation of the enzyme contained in the plant or plant part, as will be described more fully herein. The starch degrading enzyme (s) is selected based on the desired end products. For example, a glucose isomerase may be selected to convert glucose (hexose) to fructose. Alternatively, the enzyme may be selected based on the desired starch-derived end product, with various chain lengths based on e.g. eg, a function of the extent of processing or various desired branching patterns. For example, an α-amylase, glucoamylase, or amylopululanase with shorter incubation periods can be used to produce dextrin products, and longer incubation periods to produce sugars or shorter chain length products. A pullulanase may be used to specifically hydrolyze branch points in the starch, giving a high amylose starch, or a neopululanase may be used to produce starch with α1,4 bond lengths with interspersed 1.6 bonds. Glucosidases could be used to produce limiting dextrins, or a combination of different enzymes to prepare other starch derivatives.

In another embodiment, the processing enzyme is a non-starch processing enzyme selected from protease, glucanase, xylanase, and esterase. These non-starch degrading enzymes allow the self-processing plant or plant part of the present invention to incorporate into an object area of the plant, and upon activation disrupt the plant while leaving the starch granule intact therein. . For example, in a preferred embodiment, non-starch degrading enzymes target the plant cell endosperm matrix and, upon activation, disrupt the endosperm matrix while leaving the starch granule intact and more easily recoverable from the resulting material. .

Combinations of processing enzymes are also contemplated by the present invention. For example, starch processing enzymes and non-starch processing enzymes may be used in combination. Combinations of processing enzymes can be obtained by employing multiple gene constructs encoding each of the enzymes. Alternatively, the individual transgenic plants stably transformed with the enzymes may be crossed by known methods to obtain a plant containing both enzymes. Another method includes the use of exogenous enzyme (s) with the transgenic plant.

Processing enzymes may be isolated or derived from any source and the corresponding polynucleotides may be determined by one of ordinary skill in the art. For example, the processing enzyme, preferably α-amylase, is derived from Pyrococcus (e.g. Pyrococcus furiosus), Thermus, Thermococcus (eg Thermococcus hydrothermalis), Sulfolobus (eg Sulfolobus solfataricus). ) Thermotoga (eg Thermotoga maritima and Thermotoga neapolitana), Thermoanaerobacterium (eg

Thermoanaerobacter tengcongensis), Aspergillus (eg Aspergillus shirousaini and Aspergillus niger), Rhizopus (eg Rhizopus oryzae), Thermoproteales, Desulfurococcus (eg Desulfurococcus amylolyochanum, Methanothanum, Methanothanum, Methanothanum, Methanotropicum) Thermosynechococcus elongatus, Thermoplasma acidophiluim, Thermoplasma volcanium, Aeropyrum pernix and plants such as corn, barley, and rice.

The processing enzymes of the present invention are capable of being activated after being introduced and expressed in the genome of a plant. Conditions for activating the enzyme are determined for each individual enzyme and may include various conditions such as temperature, pH, hydration, presence of metals, activation compounds, inactivation compounds, etc. For example, temperature dependent enzymes may include mesophilic, thermophilic, and hyperthermophilic enzymes. Mesophilic enzymes typically exhibit maximum activity at temperatures between 20 ° -65 ° C and are inactivated at temperatures above 70 ° C. Mesophilic enzymes exhibit significant activity at 30 to 37 ° C, activity at 30 ° C is preferably at least 10% of the maximum activity, more preferably at least 20% of the maximum activity.

Thermophilic enzymes exhibit maximum activity at temperatures between 50 and 80 ° C and are inactivated at temperatures above 80 ° C. The thermophilic enzyme will preferably exhibit less than 20% of maximum activity at 30 ° C, more preferably less than 10% of maximum activity.

A "hyperthermophilic enzyme" exhibits activity even at higher temperatures. Hyperthermophilic enzymes exhibit maximum activity at temperatures above 80 ° C and retain activity at temperatures of at least 80 ° C, more preferably retain activity at temperatures of at least 90 ° C and most preferably retain activity at temperatures of at least 80 ° C. 95 ° C. Hyperthermophilic enzymes also exhibit reduced activity at low temperatures. The hyperthermophilic enzyme may exhibit activity at 30 ° C, which is less than 10% of the maximum activity and preferably less than 5% of the maximum activity.

The polynucleotide encoding the processing enzyme is preferably modified to include codons that are optimized for expression in a selected organism such as a plant (see, e.g., Wada et al., Nucl. Acids Res 18: 2367 (1990), Murray et al., Nucl. Acids Res., 17: 477 (1989), US Patents 5,096,825, 5,625,136, 5,670,356 and 5,874,304). Codon optimized sequences are synthetic sequences, ie they do not occur in nature, and preferably encode the identical polypeptide (or an enzymatically active fragment of a full length polypeptide that exhibits substantially the same activity as the full length polypeptide) encoded by the non-codon optimized parental polynucleotide encoding a processing enzyme. It is preferable that the polypeptide be biologically distinct or improved, e.g. via recursive mutagenesis of DNA encoding a particular processing enzyme of the parent source polypeptide in such a way as to enhance its performance in the process application. Preferred polynucleotides are optimized for expression in a target host plant and encode a processing enzyme. Methods of preparing these enzymes include mutagenesis, e.g. eg recursive mutagenesis and selection. Methods of mutagenesis and nucleoside sequence alterations are well known in the art. see, for example, Kunkel, Proc. Natl. Acad. Know. USA, 82: 488, (1985); Kunkel et al., Methods in Enzymol., 154: 367 (1987); U.S. Patent 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 of optimizing the expression of a nucleic acid segment in a target organism or plant are well known in the art. Briefly, a codon utilization table is obtained indicating the optimal codons used by the target organism, the optimal codons are selected to replace those in the target polynucleotide, and the optimized sequence is synthesized chemically. Preferred codons for maize are described in U.S. Patent 5,625,136.

Complementary nucleic acids of the polynucleotides of the present invention are also contemplated. An example of low stringency conditions for complementary nucleic acid hybridization having more than 100 complementary radicals on a filter in a Southern or Northern blot is 50% formamide, e.g. hybridization to 50% formamide, 1 M NaCl, 1% SDS at 37 ° C, and a wash in 0.1 X SSC at 60 ° C to 65 ° C. Exemplary conditions of low stringency include hybridization with a 30 to 35% formamide buffer solution, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C, and a 1X to 2X SSC wash (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50 to 55 ° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37 ° C, and a wash in 0.5X to IX SSC at 55 to 60 ° C.

In addition, polynucleotides encoding an "enzymatically active" fragment of the processing enzymes are further contemplated. As used herein, "enzymatically active" means a polypeptide fragment of the processing enzyme that exhibits substantially the same biological activity as the processing enzyme to modify the substrate on which the processing enzyme normally acts under appropriate conditions.

In a preferred embodiment, the polynucleotide of the present invention is a maize optimized polynucleotide encoding α-amylase as provided in SEQ ID NOS: 2, 9, 46, and 52. In another preferred embodiment, the polynucleotide is a polynucleotide optimized for maize encoding pullulanase as provided in SEQ ID NOS: 4 and 25. In another preferred further embodiment, polynucleotide is a polynucleotide optimized for α-glucosidase encoding as provided in SEQ ID NO: 6. Another preferred polynucleotide is polynucleotide. optimized for corn encoding glucose isomerase having SEQ ID NO: 19, 21, 37, 39, 41, or 43. In another embodiment, the polynucleotide optimized for glucoamylase encoding corn as set forth in SEQ ID NO: 46, 48, or 50. In addition, a maize-optimized polynucleotide for glucanase / mannanase fusion polypeptide is provided in SEQ ID NO: 57. additionally provides such polynucleotide complements, which hybridize under moderate or preferably low stringency conditions, and which encodes a polypeptide having α-amylase, pullulanase, α-glucosidase activity, glucose isomerase, glucoamylase, glucanase, or mannanase as appropriate.

Polynucleotide may be used interchangeably with the terms "nucleic acid" or "polynucleic acid" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof, whether in single or double stranded form, consisting of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or a pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known natural nucleotide analogs, which exhibit binding properties similar to those of the reference nucleic acid and are metabolised in a similar manner to naturally occurring nucleosides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more (or all) selected codons is replaced by mixed base and / or deoxyinosine radicals.

"Variants" or substantially similar sequences are also encompassed herein. For nucleotide sequences, variants include those sequences that, due to the degeneration of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR), hybridization techniques, and ligation assembly techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, using site-directed mutagenesis encoding the native protein, as well as those encoding a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, preferably 70%, more preferably 80%, even more preferably 90%, most preferably 99%, identity, and percent percent. single unit, with the native nucleotide sequence based on these classes. For example, 71%, 72%, 73% and the like, up to at least 90% class. Variants may also include a full length gene corresponding to an identified gene fragment.

Regulatory Sequences: Selectable Promoter / Signal Sequences / Markers [0097] Polynucleotide sequences encoding the processing enzyme of the present invention may be operably linked to polynucleotide sequences encoding localization signals or signal sequences (at the N-terminus or C of a polypeptide), e.g. to target the hyperthermophilic enzyme to a particular compartment within a plant. Examples of such targets include, but are not limited to, the vacuole, endoplasmic reticulum, chloroplast, amyloplast, starch granule, or cell wall, or a particular tissue, e.g. eg seed. Expression of a polynucleotide encoding a processing enzyme presenting a signal sequence in a plant, in particular, in conjunction with the use of an inducible or tissue specific promoter, may provide elevated levels of plant-localized processing enzyme. Numerous signal sequences are known to influence the expression or objectification of a polynucleotide into a particular compartment or out of a particular compartment. Advantageous signal sequences and objectification promoters are known in the art and include, but are not limited to, those provided herein.

For example, where expression in specific organs and tissues is desired, tissue specific promoter may be used. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory elements of scol. Where continuous expression is desired in all cells of a plant, constitutive promoters are used. Additional regulatory sequences upstream and / or downstream of the nucleus promoter sequence may be included in transformation vector expression constructs to provide varying levels of heterologous nucleotide sequence expression in a transgenic plant.

Several plant promoters have been described with various expression characteristics. Examples of some constitutive promoters that have been described include rice actin 1 (Wang et al., Mol. Cell. Biol., 12: 3399 (1992); US Patent 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 the ubiquitin promoters.

Vectors for use in tissue-specific objectification of genes in transgenic plants will typically include the tissue-specific promoter and may also include other tissue-specific control elements, such as enhancer sequences. Promoters that target specific or enhanced expression in certain plant tissues will be well known to those skilled in the art in light of the present disclosure. These include, for example, the green tissue specific rbcS promoter; the ocs promoters, nos and but which present greater activity in injured roots or leaf tissue; a truncated 35S promoter (from -90 to +8) that drives enhanced expression in roots, an α-tubulin gene that drives enhanced expression of cts in roots, an α-tubulin gene that drives expression in roots and promoters derived from zein storage protein genes that drive endosperm expression.

Tissue-specific expression can be functionally performed by introducing a constitutively expressed gene (all tissues) in combination with an antisense [antisense] gene that is expressed only in those tissues in which the gene product is not. is desired. For example, a gene encoding a lipase may be introduced in such a way as to be expressed in all tissues using the Cauliflower Mosaic Virus 35S promoter. Expression of an antisense lipase gene transcription in a corn grain, using, for example, a zein promoter, could prevent the accumulation of lipase protein in the seed. Hence, the protein encoded by the introduced gene could be present in all tissues except the grain.

In addition, various tissue-specific regulated promoters and / or genes have been reported in plants. Some tissue-specific genes reported include genes that encode seed storage proteins (such as napin, cruciferin, beta-conglycinin, and phaseolin) zein or oily body proteins (such as oleosin), or genes involved in acid biosynthesis. fatty acids (including acyl-bearing protein, stearoyl ACP desaturase, and fatty acid desaturases (fad 2-1)), and other genes expressed during embryo development (such as Bce4, see, for example, EP 255378 and Kridl et al. ., Seed Science Research, 1: 209 (1991)). Examples of tissue-specific promoter that have been described include lectin (Vodkin, Prog. Clin. Biol. Res., 138; 87 (1983); Lindstrom et al., Der. Genet., 11: 160 (1990)), corn alcohol dehydrogenase 1 (Vogei et al., 1989; Dennis et al., Nucleic Acids Res., 12: 3983 (1984)), corn light crop complex (Simpson, 1986; Bansal et al., Proc. Natl. Acad. Sci. USA, 89: 3654 (1992)), corn heat shock protein (Odell et al., 1985; Rochester et al., 1986), small pear subunit RuBP carboxylase (Poulsen et al. ., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), Petunia chalcone isomerase (vanTunen et al., EMBO J., 7; 1257 (1988)), bean glycine 1 rich protein (Keller et al., Genes Dev., 3: 1639 (1989)), truncated CaMV 35s (Odell et al., Nature, 313: 810 (1985)), potato patatin (Wenzler et al., Plant Mol. Biol., 13: 347 (1989)), root cell (Y amamoto et al., Nucleic Acids Res., 18: 7449 (1990)), corn zein (Reina et al., Nucleic Acids Res., 18: 6425 (1990); Kriz et al., Mol. Gen. Genet., 207: 90 (1987); Wandelt et al., Nucleic Acids Res., 17: 2354 (1989); Langridge et al., Ceil, 34: 1015 (1983); Reina et al., Nucleic Acids Res., 18: 7449 (1990)), globulin-1 (Belanger et al., Genetics, 129: 863 (1991)), α-tubulin, cab (Sullivan et al., Mol. Gen. Genet., 215: 431 (1989)), PEPCase (Hudspeth & Grula, 1989), R gene complex associated promoters (Chandler et al., Plant Cell, 1: 1175 (1989)), and chalcone promoters synthase (Franken et al., EMBO J., 10: 2605 (1991)). The pear vicicin promoter is particularly useful for seed specific expression (Czako et al., Mol. Gen. Genet., 235: 33 (1992). (See also US Patent 5,625,136, incorporated herein by reference.) Other promoters Useful for expression in mature leaves are those that are activated at the onset of senescence, such as the Arabidopsis SAG promoter (Gan et al., Science, 270: 1986 (1995).

A class of fruit-specific promoters expressed on or during anthesis by fruit development, at least until early ripening, is discussed in US 4,943,674, the disclosure of which is incorporated herein by reference, cDNA clones. which are preferably expressed in cotton fiber have been isolated (John et al., Proc. Natl. Acad. Sci. USA, 89: 5769 (1992). Tomato cDNA clones showing differential expression during fruit development were isolated and characterized (Mansson et al., Gen. Genet., 200: 356 (1985), Slater et al., Plant Mol. Biol., 5: 137 (1985).) The promoter for the polygalacturonase gene is active in ripening of the fruit The polygalacturonase gene is described in US Patent 4,535,060, US Patent 4,769,061, US Patent 4,801,590, and US Patent 5,107,065, the disclosures of which are incorporated herein by reference.

Other examples of tissue-specific promoter include those that drive expression in leaf cells after leaf damage (e.g., by chewing insects), in tubers (e.g., patatin gene promoter), and in fiber cells (an example of a developmentally regulated fibrous cell protein is E6 (John et al., Proc. Natl. Acad. Sci. USA, 89: 5769 (1992). The E6 gene is most active in fiber , although low levels of leaf, egg and flower transcriptions are found.

The tissue-specificity of some "tissue-specific" promoters may not be absolute and can be tested by one of ordinary skill in the art using the diphtheria toxin sequence. Tissue-specific expression with "leaky" expression can also be achieved by a combination of different tissue-specific promoters (Beals et al., Plant Cell, 9: 1527 (1997)) . Other tissue-specific promoters may be isolated by one of ordinary skill in the art (see U.S. 5,589,379).

In one embodiment, the product direction of a polysaccharide hydrolysis gene, such as α-amylase, can be targeted to a particular organelle, such as apoplast, rather than to the cytoplasm. This is exemplified by the use of the maize γ-zein N-terminal signal sequence (SEQ ID NO: 17), which confers objectification of apoplast-specific proteins. Targeting the protein or enzyme to a specific compartment will allow the protein to be localized in a way that will not come into contact with the substrate. In this way the enzymatic action of the enzyme will not occur until the enzyme contacts its substrate. The enzyme may be contacted with its substrate by the milling process (physical disruption of cell integrity), or by heating plant cells or tissues to disrupt the physical integrity of plant cells or organs containing the enzyme. . For example, a mesophilic starch hydrolyzing enzyme may be targeted to the apoplast or endoplasmic reticulum and such as not to come in contact with starch granules in the amyloplast. Grinding the grain will break the grain integrity and then the starch hydrolyzing enzyme will contact the starch granules. In this way, the potential negative effects of co-localization of an enzyme and its substrate can be avoided.

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

In another embodiment of the invention, the polynucleotide encodes a hyperthermophilic processing enzyme that is operably linked to a chloroplast (amyloplast) transit peptide (CTP) and a starch binding domain, e.g. e.g. of the waxy gene. An exemplary polynucleotide in this embodiment encodes SEQ ID NO: 10 (α-amylase bound to the waxy starch binding domain). Other exemplary polynucleotides encode a hyperthermophilic processing enzyme linked to a signal sequence that targets the enzyme to the endoplasmic reticulum and secretion to the apoplast (exemplified by a polynucleotide encoding SEQ ID NO: 13, 27, or 30, which comprises the sequence N γ-zein terminally operatively linked to α-amylase, α-glucosidase, glucose isomerase, respectively), the hypertermophilic processing enzyme linked to a signal sequence that conserves the enzyme in the endoplasmic reticulum (exemplified by a polynucleotide encoding SEQ ID NO: 14, 26, 28, 29, 33, 34, 35, or 36, which comprises the N-terminal sequence of maize γ-zein operably linked to the hyperthermophilic enzyme, which is operably linked to SEKDEL, wherein the enzyme is α-amylase, malA α-glucosidase, T, marine glucose isomerase, T. neapolitana glucose isomerase), a hypertermophilic processing enzyme linked to a N-terminal sequence targeting the enzyme for amyloplast (exemplified by a polynucleotide encoding SEQ ID NO: 15, comprising the targeting sequence of waxy N-terminal amyloplast operatively linked to α-amylase), a hypertermophilic fusion polypeptide that targets the starch granule enzyme (exemplified by a polynucleotide encoding SEQ ID NO: 16, comprising the waxy N-terminal amyloplast objectification sequence operably linked to a α-amylase / waxy fusion polypeptide comprising the starch binding domain waxy), a hyperthermophilic processing enzyme linked to an ER retention signal (exemplified by a polynucleotide encoding SEQ ID NO: 38 and 39). In addition, the hyperthermophilic processing enzyme may be linked to a pure starch binding site having the amino acid sequence (SEQ ID NO: 53), and the polynucleotide encoding the processing enzyme is linked to the optimized nucleic acid sequence. for maize (SEQ ID NO: 54) encoding this binding site.

Several inducible promoters have been reported. Many are described in a review by Gatz, Current Opinion in Biotechnology, 7: 168 (1996) and Gatz, C., Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89 (1997). Examples include tetracycline repressor system, Lac repressor system, copper inducible systems, salicylate inducible systems (such as the PR1a system), glucorticoid inducible system (Aoyama T. et al., NH Plant Journal, 11: 605 (1997)) and ecdysone inducible systems. Other inducible promoters include ABA and turgor inducible promoters, the auxin-binding protein gene promoter (Schwob et al., Plant J., 4: 423 (1993)), the UDP glucose flavonoid glycosyl transferase gene promoter (Ralston et al., Genetics, 119: 185 (1988)), the MPI proteinase inhibitor promoter (Cordero et al., Plant J., 6: 141 (1994)), and the glyceraldehyde-3 gene promoter. phosphate dehydrogenase (Kohler et al., Plant Mol. Biol., 29; 1293 (1995); Quigley et al., J. Mol. Evol., 29: 412 (1989); Martinez et al., J. Mol. Biol., 208: 551 (1989)). Also included are benzene sulfonamide inducible (U.S. 5,364,780) and alcohol inducible (WO 97/06269 and WO 97/06268) systems and glutathione S-transferase promoters.

Other studies have focused on inductively regulated genes in response to stress or environmental stimuli such as increased salinity, drought, pathogen and injury. (Graham et al., J. Biol. Chem., 260: 6555 (1985); Graham et al., J. Biol. Chem., 260: 6561 (1985), Smith et al., Plant, 168: 94 ( 1986)). Accumulation of metallocarboxipeptidase inhibitor protein has been reported in leaves of injured potato plants (Graham et al., Biochem. Biophys. Res. Comm., 101: 1164 (1981)). Other plant genes induced by methyl jasmonate, elicitors, heat shock, anaerobic stress, or herbicidal pesticides have been reported.

Regulated expression of a chimerically-transacting viral replication protein can be further regulated by other genetic strategies, such as Cre-mediated gene activation (Odell et al. Mol.

Gen. Genet., 113: 369 (1990)). Thus, a DNA fragment containing 3 'regulatory sequence linked by lox sites between the promoter and the replication protein coding sequence that blocks the expression of a promoter chimeric replication gene can be removed by Cre-mediated excision. result in the expression of the trans acting replication gene. In this case, the chimeric Cre gene, trans-acting replication gene, or both, may be under the control of inducible or tissue-specific promoters and development. An alternative genetic strategy is the use of the tRNA suppressor gene. For example, regulated expression of a tRNA suppressor gene may conditionally control the expression of a trans-acting replication protein coding sequence containing an appropriate terminating codon (Ulmasov et al. Plant Mol. Biol., 35: 417 (1997)). )). Again, the chimeric tRNA suppressor gene, or the eric trans chemotaxis-acting replication gene, or both may be under the control of inducible or tissue-specific promoters and development.

Preferably, in the case of a multicellular organism, the promoter may also be specific to a particular tissue, organ or stage of development. Examples of such promoters include, but are not limited to, the Zea maize γ-zein promoter and Zea maize ADP-gpp, and the Zea maize globulin promoter.

Expression of a gene in a transgenic plant may be desirable only at a certain time during plant development. Developmental timing is often correlated with tissue-specific gene expression. For example, expression of zein storage proteins is started in the endosperm about 15 days after pollination.

Additionally, vectors can be constructed and employed in the intracellular objectification of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment. This will usually be achieved by conjugating a DNA sequence encoding a signal or transit peptide sequence to the coding sequence of a particular gene. The transit or signal peptide will carry the protein to a particular intracellular or extracellular destination, respectively, and then be removed after translation. Transit or signal peptides act by facilitating protein transport across intracellular membranes, e.g. vacuole, gallbladder, plastid and mitochondrial membranes, because signal peptides direct proteins through the extracellular membrane.

The signal sequence, such as the N-terminal signal sequence of maize γ-zein for endoplasmic reticulum objectification and apoplast secretion, can be operably linked to a polynucleotide encoding a hyperthermophilic processing enzyme according to the present invention. invention (Torrent et al., 1997). For example, SEQ II) NOS: 13, 27, and 30 provide a polynucleotide encoding a hyperthermophilic enzyme operably linked to the N-terminal sequence of the γ-zein protein in maize. Another signal sequence is the SEKDEL amino acid sequence for retention of polypeptides in the endoplasmic reticulum (Munro and Pelham, 1987). For example, a polynucleotide encoding SEQ ID NOS: 14, 26, 28, 29, 33, 34, 35, or 36, which comprises the N-terminal sequence of maize γ-zein operably linked to a processing enzyme that is bound operationally SEKDEL. A polypeptide may also be objectified to the amyloplast by fusion with the waxy amyloplast objectification peptide (Klosgen et al., 1986) or with a starch granule. For example, the polynucleotide encoding a hyperthermophilic processing enzyme may be operably linked to a chloroplast (amyloplast) transit peptide (CTP) and a starch binding domain, e.g. e.g. of the waxy gene. SEQ ID NO: 10 exemplifies α-amylase bound to the waxy starch binding domain. SEQ ID NO: 15 exemplifies the amyloplast objectification sequence of the waxy N-terminal sequence operatively linked to α-amylase. In addition, the polynucleotide encoding the processing enzyme may be fused to target starch granules using the waxy starch binding domain. For example, SEQ ID NO: 16 exemplifies a fusion polypeptide comprising the waxy N-terminal amyloplast objectification sequence operably linked to an α-amylase / waxy fusion polypeptide comprising the waxy starch binding domain.

Polynucleotides of the present invention, in addition to the processing signals, may additionally include other regulatory sequences as known in the art. "Regulatory Sequences" and "Appropriate Regulatory Sequences" each refer to nucleotide sequences located upstream (5 'non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence. , which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences that can be a combination of synthetic and natural sequences.

Selectable markers may also be used in the present invention to allow selection of transformed plants and plant tissue, as is well known in the art. It may be desired to employ a selectable or traceable marker gene as, or in addition to, the expressible gene of interest. "Marker genes" are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells lacking the marker. Said genes may encode a traceable selectable marker, depending on whether the marker confers a selectable trait by chemical means, ie, by the use of a selective agent (eg, a herbicide, antibiotic, or the like). , or if it is simply a trait that can be identified by observation or testing, ie through screening (eg, the locus R trait). Of course, many examples of advantageous marker genes are known in the art and may be employed in the practice of the invention.

The term 'selectable or traceable marker genes' also includes genes encoding a 'secretable marker' whose secretion can be detected as a means of identifying the selected transformed cells.

Examples include markers encoding a secretable antigen that can be identified by antibody interaction, or even secretable enzymes that can be detected by virtue of their catalytic activity. Secretable proteins fall into a number of classes, including detectable small diffuse proteins, e.g. eg by ELISA; detectable small active enzymes in extracellular solution (eg, α-amylase, β-lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or entrapped in the cell wall (e.g., proteins that include a leader sequence, such as that found in the PR-S extensin or tobacco expression unit).

With regard to selectable secretable markers, the use of a gene encoding a protein that becomes sequestered in the cell wall, and that protein comprising a single epitope, is considered particularly advantageous. A secreted antigen marker could ideally employ an epitope sequence that could provide low background in plant tissue, a promoter leader sequence that could confer efficient expression and objectification across the plasma membrane, and could produce cell wall-binding protein. which is still accessible to antibodies. A normally secreted wall protein modified to include a single epitope could satisfy all of these requirements.

An example of a protein capable of modification in this manner is extensin, or hydroxyproline rich glycoprotein (HPRG: hydroxyproline rich glycoprotein). For example, a maize HPRG molecule (Steifel et al., The Plant Cell, 2: 785 (1990)) is well characterized in terms of molecular biology, expression and protein structure. However, any of a variety of glycine-rich extensins and / or wall proteins (Keller et al., EMBO Journal, 8: 1309 (1989)) could be modified by the addition of an antigenic site to create a traceable marker. . The. Selectable Markers Possible selectable markers for use in connection with the present invention include, but are not limited to, a neo or nptll gene (Potrykus et al., Mol.

Gen. Genet., 199: 183 (1985)) encoding kanamycin resistance and which may be selected using kanamycin, G418, and the like; a bar gene that confers resistance to the herbicide fosfinotricin; a gene encoding an altered EPSTP synthase protein (Hinchee et al., Biotech., 6: 915 (1988)) thereby conferring glyphosate resistance; a nitrilase gene such as Klebsiella ozaenae bxn that confers bromoxynil resistance (Stalker et al., Science, 242: 419 (1988)); a mutant acetolactate synthase (ALS) gene that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (European Patent Application 154,204, 1985); a methotrexate resistant DHFR gene (Thillet et al., J. Biol. Chem., 263: 12500 (1988)); a dalapon deshalogenase gene that confers resistance to the dalapon herbicide; a phosphomanose isomerase (PMI) gene; a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan; the hph gene that confers resistance to the hygromycin antibiotic; or the mannose 6-phosphate isomerase gene (also referred to herein as the phosphomanose isomerase gene), which provides the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629). One skilled in the art is able to select a selectable marker gene suitable for use in the present invention. Where a mutant EPSP synthase gene is employed, additional benefit can still be obtained by incorporating a suitable chloroplast transit peptide, CTP [European Patent Application 0,218,571,1987).

An illustrative embodiment of a selectable marker gene capable of being used in systems for selecting transformants are genes encoding the enzyme phosphinothricin acetyltransferase, such as the Streptomyces hygroscopicus bar gene or the Streptomyces viridochromogenes pat gene. The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., Mol. Gen. Genet., 205: 42 (1986); Twell et al., Plant Physiol., 91: 1270 (1989)) causing rapid accumulation of ammonia and death of cell. The success in using this selective system in conjunction with monocotyledons was particularly surprising due to the greater difficulties that have been reported in cereal processing (Potrykus, Trends Biotech., 7: 269 (1989).

Where it is desired to employ a bialaphos resistance gene in the practice of the invention, a particularly useful gene for this purpose is the bar or pat gene which is obtainable from Streptomyces species (eg, ATCC No. 21,705). Cloning of the bar gene has been described (Murakami et al., Mol. Gen. Genet., 205: 42 (1986); Thompson et al., EMBO Journal, 6: 2519 (1987)) as was the use of the gene. bar in the context of non-monocotyledonous plants (De Block et al., EMBO Journal, 6: 2513 (1987); De Block et al., Plant Physiol., 91: 694 (1989)), b. Selectable Markers Selectable markers that may be employed include, but are not limited to, a β-glucuronidase or uidA gene (GUS) that encodes an enzyme for which various chromogenic substrates are known; a locus R gene that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., in Chromosome Structure and Function, pp. 263-282 (1988)); a P-lactamase gene (Sutcliffe, PNAS USA, 75: 3737 (1978)), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., PNAS USA, 80: 1101 (1983)) that encodes a dioxigenase catechol that can convert chromogenic catechols; an α-amylase gene (Ikuta et al., Biotech., 8: 241 (1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol., 129: 2703 (1983)) encoding an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which, in turn, condenses to form the easily detectable compound melanin; a 3-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., Science, 234: 856 (1986)), which allows detection of bioluminescence; or an aequorin gene (Prasher et al., Biochem. Biophys. Res. Comm., 126: 1259 (1985)), which may be employed in the detection of calcium sensitive bioluminescence, or a green fluorescent protein gene (Niedz et al. al., Plant Cell Reports, 14: 403 (1995)).

Genes of the maize R gene complex are considered to be particularly useful as selectable markers. The R gene complex in maize encodes a protein that acts by regulating the production of anthocyanin pigments in most plant and seed tissues. A gene from the R gene complex is suitable for maize transformation, because expression of this gene in transformed cells does not harm cells. Thus, an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be visually classified as a red sector. If the maize line carries dominant alleles for genes encoding enzyme intermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1, and Bz2) but carries a recessive allele in the R locus, transformation of any cell in that line with R will result. in formation of red pigment. Exemplary lines include Wisconsin 22 which contains the rg-Stadler allele and TR112, a derivative of K55 which is r-g, b, P1. Alternatively, any maize genotype can be used if the C1 and R alleles are introduced together. An additional selectable marker considered for use in the present invention is firefly luciferase, encoded by the lux gene. The presence of the lux gene in transformed cells can be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low light video cameras, photon counting cameras or multiwell luminometry. It is also possible to consider that this system can be developed for bioluminescence-based population screening, such as tissue culture plates, or even for whole plant screening.

Polynucleotides used to transform the plant may include, but are not limited to, DNA from plant genes and non-plant genes, such as those from bacteria, yeast, animals, or viruses. The introduced DNA may include modified genes, gene portions, or chimeric genes, including genes from the same or different genotype of maize. The term "chimeric gene" or "chimeric DNA" is defined as a DNA gene or segment or sequence comprising at least two segments or two DNA sequences from species that do not combine DNA under natural conditions, or whose DNA segments or sequences are positioned or linked in a way that does not normally occur in the native genome of the unprocessed plant.

Expression cassettes further comprising the polynucleotide encoding a hypertermophilic processing enzyme, and preferably a codon optimized polynucleotide. It is preferred that the polynucleotide in the expression cassette (the first polynucleotide) is operably linked to regulatory sequences, such as a promoter, enhancer, intron, termination sequence, or any combination thereof, and optionally to a second polynucleotide. which encodes a (N-or C-terminal) signal sequence that directs the enzyme encoded by the first polynucleotide to a particular cellular or subcellular site. Thus, a promoter and one or more signal sequences may provide high levels of enzyme expression at particular sites in a plant, plant tissue or plant cell. Promoters may be constitutive promoters, inducible (conditional) promoters or tissue-specific promoters, e.g. endosperm-specific promoter, such as the corn γ-zein promoter (exemplified by SEQ ID NO: 12) or the maize ADP-gpp promoter (exemplified by SEQ ID NO: 11, which includes an intron sequence and an untranslated 5 '). The invention also provides an isolated polynucleotide comprising a promoter comprising SEQ ID NO: 11 or 12, a polynucleotide that hybridizes to its complement under low stringency conditions, or a fragment thereof that exhibits promoter activity, e.g. at least 10%, and preferably at least 50%, of the activity activity of a promoter of SEQ ID NO: 11 or 12. Also provided are vectors comprising the expression cassette or polynucleotide of the invention and transformed cells comprising the polynucleotide, expression cassette or vector of the invention. A vector of the invention may comprise a polynucleotide sequence encoding more than one hyperthermophilic processing enzyme of the invention, said sequence may be in sense or antisense orientation, and a transformed cell may comprise one or more vectors of the invention. Preferred vectors are those useful for introducing nucleic acids into plant cells.

Transformation The expression cassette, or a vector construct containing the expression cassette can be inserted into a cell. The expression cassette or vector construct can be performed episomatically or integrated into the cell genome. The transformed cell can then be grown into a transgenic plant. Thus, the invention provides the transgenic plant products. Such products may include, but are not limited to, the seeds, fruits, progeny, and progeny products of the transgenic plant.

Several techniques are available and known to those skilled in the art for introducing constructs into a cellular host. Transformation of bacteria and many eukaryotic cells can be accomplished through the use of polyethylene glycol, calcium chloride, viral infection, phage infection, electroporation, and other methods known in the art. Techniques for transforming plant tissue or cells include DNA transformation employing A. tumefaciens or A. rhizogenes as the transforming agent, electroporation, DNA injection, microprojectile bombardment, particle acceleration, etc. (see, for example, EP 295959 and EP 138341).

In one embodiment, binary type vectors of Agrobacterium spp. Ti-derived vectors are used to transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants such as soybeans, cottonseed rape, tobacco, and rice (Pacciotti et al. Bio / Technology, 3: 241 (1985): Byme et Plant Cell Tissue and Organ Culture, 8: 3 (1987); Sukhapinda et al., Plant Mol. Biol., 8: 209 (1987); Lorz et al., Mol. Gen. Genet., 199: 178 (1985) Potrykus Mol. Gen. Genet., 199: 183 (1985); Park et al., J. Plant Biol., 38: 365 (1985): Hiei et al., Plant J., 6: 271 (1994)) . The use of T-DNA to transform plant cells has been the subject of extensive study and is widely described (EP 120516; Hoekema, in: The Binary Plant Vector System. Offset-drukkerij Kanters BV; Alblasserdam (1985), Chapter V; Knauf, et al., Genetic Analysis of Host Range Expression by Agrobacterium in: Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. and  ±, Springer-Verlag, New York, 1983, p. 245; and An. et al. , EMBOJ. 4: 277 (1985)).

Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see EP 295959), electroporation techniques (Fromm et al. Nature (London), 319: 791 (1986)). ), or high-speed biological bombardment of metal particles coated with nucleic acid constructs (Kline et al. Nature (London) 327: 70 (1987), and US Patent 4,945,050).

Once transformed, cells can be regenerated by those skilled in the art. Recently described methods are of particular relevance for turning foreign genes into commercially important crops such as rapeseed (De Block et al., Plant Physiol. 91: 694-701 (1989)), sunflower (Everett et al., Bio / Technology, 5: 1201 (1987)), soybean (McCabe et al., Bio / Technology, 6: 923 (1988); Hinchee et al., Bio / Technology, 6: 915 (1988); Chee et al. , Plant Physiol, 91: 1212 (1989); Christou et al., Proc. Natl. Acad. Sci USA, 86: 7500 (1989) EP 301749), Rice (Hiei et al., Plant J, 6: 271 (1994)) , and corn (Gordon Kamm et al., Plant Cell, 2: 603 (1990); Fromm et al., Biotechnology, 8: 833 (1990)).

Expression vectors containing genomic or synthetic fragments may be introduced into protoplasts or into intact tissues or isolated cells. Preferably, expression vectors are introduced into intact tissue. General methods of cultivating plant tissues are provided, for example, by Maki et al. "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology & Biotechnology, Glich et al. (Eds.), Pp. 67-88 CRC Press (1993); and by Phillips et al. "Cell-Tissue Culture and In-Vitro Manipulation" in Com & Com Improvement, 3rd edition 10, Sprague et al. (Eds.) Pp. 345-387, American Society of Agronomy Inc. (1988).

In one embodiment, expression vectors may be introduced into maize or other plant tissues using a method of direct gene transformation, such as micro-projectile mediated delivery, DNA injection, electroporation, and the like. Expression vectors are introduced into plant tissues using micro-projectile medium delivery using the biolistic device. See, for example, Tomes et al. "Direct DNA transfer into intact plant cells via microprojectile bombardment" in Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer Verlag, Berlin (1995). However, the present invention contemplates the transformation of plants with a hyperthermophilic processing enzyme according to known transformation methods. See also, Weissinger et al., Annual Rev. Genet., 22: 421 (1988); Sanford et al., Particulate Science and Technology, 5:27 (1987) (onion); Christou et al., Plant Physiol., 87: 671 (1988) (soy); McCabe et al., Bio / Technology, 6: 923 (1988) (soy); Datta et al., Bio / Technology, 8: 736 (1990) (rice); Klein et al., Proc. Natl. Acad. Know. 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., BioTechnology, 8: 833 (1990) (maize); and Gordon-Kamm et al., Plant Cell, 2,603 (1990) (maize); Svab et al., Proc. Natl. Acad. Know. USA, 87: 8526 (1990) (tobacco chloroplast); Koziel et al., Biotechnology, 11: 194 (1993) (maize); Shimamoto et al., Nature, 338: 274 (1989) (rice); Christou et al., Biotechnology, 9: 957 (1991) (rice); European patent application EP 0 332 581 (gram of the species Dacatylis and other Pooideae); Vasil et al., Biotechnology, 11: 1553 (1993) (wheat); Weeks et al., Plant Physiol., 102: 1077 (1993) (wheat). Methods in Molecular Biology, 82, Arabidopsis Protocols Ed. Martinez-Zapater and Salinas 1998 Humana Press (Arabidopsis).

Plant transformation can be undertaken with a single DNA molecule or with multiple DNA molecules (i.e., co-transformation), and both of these techniques are suitable for use with the expression cassettes and constructs of the present invention. Numerous transformation vectors are available for plant transformation, and the expression cassettes of this invention may be used in conjunction with any such vector. Vector selection will depend on the preferred transformation technique and the target species for transformation.

Finally, the most desirable DNA segments for introduction into a monocotyledon genome may be homologous genes or gene families encoding a desired trait (e.g., protein, lipid or polysaccharide hydrolysis) and which are introduced under control of unprecedented promoters or enhancers, etc., or perhaps even tissue-specific or homologous control elements or promoters (e.g., root-specific, collar / sheath, petals, stem, spike stem, grain or leaf). Indeed, it is contemplated that a particular use of the present invention will be the objectification of a gene in a constitutive or inducible manner.

Examples of Suitable Transformation Vectors Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention may be used in conjunction with any vectors. of the said type known in the art. Selection of the vector will depend on the preferred transformation technique and the target species for transformation. The. Suitable Vectors for Agrobacterium Transformation Many vectors are available for transformation using Agrobacterium tumefaciens. Typically, these carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Below we describe the construction of two typical vectors suitable for Agrobacterium transformation.

[00141] pCI3200 and pCIB2001 [00142] The binary vectors pclB200 and pCIB2001 are used for constructing recombinant vectors for use with Agrobacterium and are constructed as follows. pTJS75kan is created by pTJS75 Narl digestion (Schmidhauser & Helinski, J. Bacteriol., 164: 446 (1985)) allowing excision of the tetracycline resistance gene, followed by insertion of a pUC4K Accl fragment carrying an NPTII (Messing & Vierra, Gene, 19: 259 (1982): Bevan et al., Nature, 304: 184 (1983): McBride et al., Plant Molecular Biology, 14: 266 (1990)). XhoI ligands are ligated to the PCIB7 EcoRV fragment containing the left and right edges of T-DNA, a plant selectable nos / npt11 chimeric gene, and the pUC polyligand (Rothstein et al., Gene, 53: 153 (1987)) , and the XhoI digested fragment are cloned into SalI digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following single polyligand restriction sites as follows: EcoRI, Sstl, Kpnl, Bglll, Xbal, and SalI. pCIB2001 is a pCIB200 derivative created by inserting additional restriction sites into the poly-ligand. Single restriction sites on the pCIB2001 poly-ligand are EcoRI, SstI, KpnI, Bglll, XbaI, SalI, Mlul, Bell, Avrll, Apal, Hpal, and Stul. pCIB2001, besides containing these unique restriction sites also features plant and bacterial kanamycin selection, T-DNA left and right edges for Agrobacterium-mediated transformation, RK2-derived trfA function for mobilization between E. coli and other hosts , and the functions of OriT and OriV also of RK2. The pCIB2001 poly-ligand is suitable for cloning plant expression cassettes containing their own regulatory signals.

PCIB 10 and Hygromycin selection of derivatives thereof: The binary vector pCIB 10 contains a gene that encodes kanamycin resistance for selection in plant and T-DNA right and left edge sequences and incorporates plasmid sequences. with wide host range, pRK252, allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene, 53: 153 (1987)). Several pCIB10 derivatives that incorporate the hygromycin B phosphotransferase gene described by Gritz et al. (Gene, 25: 179 (1983)). These derivatives allow selection of transgenic plant cells in hygromycin (pCIB743) only, or hygromycin and kanamycin (pCIB715, pCIB717).

[00145] b. Suitable Vectors for Non-Agrobacterium Transformation Transformation without the use of Agrobacterium tumefaciens avoids the requirement for T-DNA sequences in the selected transformation vector and, consequently, vectors that do not have these sequences can be used in addition to vectors such as those described. above which contain T-DNA sequences. Non-Agrobacterium transformation techniques include transformation via particle bombardment, protoplast uptake (eg, PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Non-limiting examples of the construction of typical vectors suitable for transformation without the use of Agrobacterium are described further.

[00147] pCIB3064 [00148] pCIB3064 is a pUC-derived vector suitable for direct gene transformation techniques in combination with sufficient herbicide (or phosphinothricin) selection. Plasmid pCIB246 comprises the CaMV 35S promoter fusion operable with the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in PCT published application WO 93/07278. The 35S promoter of this vector contains two 5 'ATG sequences from the initial site. These sites are mutated using standard PCR techniques in order to remove ATGs and generate the Sspl and Pvull restriction sites. The new restriction sites are 96 and 37 bp from the Sall single site and 101 and 42 bp from the effective initiation site. The resulting derivative of pCIB246 is called pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with SalI and Saci, the blunt ends blown back to generate plasmid pCIB3060. Plasmid pJIT82 can be obtained from the John Innes Center, Norwich, and the Smal fragment of a [sic] 400 bp containing the Streptomyces viridochroinogenes bar gene is excised and inserted into the HP1 site of pCIB3060 (Thompson et al., EMBO J, 6 : 2519 (1987)). This generated pCIB3064, which comprises the bar gene under the control of the CaMV 35S promoter and herbicide selection terminator, an ampicillin resistance gene (for selection in E. coli) and a poly-ligand with the unique Sphl, Pstl sites. , HindIII, and BamHI. This vector is suitable for cloning plant expression cassettes containing their own regulatory signals.

PSOG19 and pSOG35: Plasmid pSOG35 is a transformation vector that uses the E. coli gene dihydrofolate reductase (DHFR) as a selectable marker that confers methotrexate resistance. PCR is used to amplify the 35S promoter (-800 bp), maize Adh1 gene intron 6 (-550 bp) and 18 bp of the pSOGIO untranslated GUS leader sequence. A 250 bp fragment encoding the E. coli gene, type II dihydrofolate reductase, is also amplified by PCR and these two PCR fragments are assembled with the SacB-Pstl fragment from pB1221 (Clontech) comprising the spine. backbone of the pUC19 vector and the nopaline synthase terminator. Assembly of these fragments generates pSOG19 containing the 35S promoter in fusion with intron 6 sequence, GUS leader, DHFR gene and nopaline synthase terminator. Replacing the GUS leader in pSOG19 with the Maize Chlorotic Mottle Virus (MCMV) leader sequence generates the pSOG35 vector. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlll, Aphl, Pstl and EcoRI sites available for cloning foreign substances.

C. Suitable vector for chloroplast transformation For expression of a nucleotide sequence of the present invention in plant plastids, the plastid transformation vector pPH143 (WO 97/32011, example 36) is used. The nucleotide sequence is inserted into pPH143 thus replacing the PROTOX coding sequence. This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance. Alternatively, the nucleotide sequence is inserted into pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.

Plant Host Subject to Transformation Methods Any plant tissue capable of subsequent clonal propagation, either by organogenesis or embryogenesis, can be transformed with a construct of the present invention. The term organogenesis means a process whereby shoots and roots are developed sequentially from meristematic centers, while the term embryogenesis means a process whereby shoots and roots develop together in a concerted (not sequential) manner, either gametes or somatic cells. The particular tissue selected will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include differentiated and undifferentiated plants or tissues, including, but not limited to, leaf discs, roots, stems, buds, leaves, pollen, seeds, embryos, cotyledons, hypocotyledones, megagametophytes, callus tissue, existing meristematic tissue ( apical meristems, axillary shoots, and root meristems), and induced meristem tissue (eg, cotyledon meristem and hypocotyledon meristem), tumor tissue, and various forms of cells and culture such as cells singles, protoplast, embryos, and callus tissue. Plant tissue may be in plants or in organ, tissue or cell culture.

Plants of the present invention may come in a variety of forms. Plants may be transformed and untransformed cell chimeras; plants may be clonal transformants (e.g., all cells transformed to contain the expression cassette); plants may comprise both transformed and unprocessed tissue grafts (e.g., a stock of transformed roots grafted into an unprocessed region of citrus species). Transformed plants can be propagated by various means, such as by classical clonal propagation or crossbreeding techniques. For example, first-generation (or T1) transformed plants can be selfed by giving homozygous second-generation (or T2) transformed plants, and even more propagated T2 plants by classical breeding techniques. A dominant selectable marker (such as npt II) may be associated with the expression cassette to assist in creation.

[00156] The present invention may be used for transformation of any plant species, including monocotyledons or dicotyledons, including, but not limited to, maize (Zea mays), Brassica sp. (eg, B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (eg pearl millet (Pennisetum glaucum), prose millet (Panicum miliaceum), fox-tailed millet (Setaria italica), finger millet (Eleusine coracana)) , sunflower (Helianthus annuus), saffron (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum) ), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea ( Camellia sinensis), Banana (Musa spp.), Avocado (Persea americana), Fig (Ficus casica), Guava (Psidium guajava), Mango (Mangifera indica), Oli va (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.) oats, barley, greens, ornamentals, woody plants such as conifers and deciduous trees, pumpkin, pumpkin, hemp, cucumber, apple, pear, cidonia, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, soybean, sorghum, sugar cane, rapeseed, clover, carrot, and Arabidopsis thaliana.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans; peas (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, and members of the genus Cucumis, such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and melon (C. melo). Ornamentals include azalea (Rhododendron spp.), Hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), Tulips (Tulipa spp.), Daffodil (Narcissus spp.), Petunias (Petunia hybrida), carnation ( Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Conifers which may be employed in the practice of the present invention include, for example, pine such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine ( Pinus radiata), Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver spruce (Abies amabilis) and balsam spruce (Abies balsamea); and cedars, such as Western red cedar (Thuja plicata) and Alaskan yellow cedar (Chamaecyparis nootkatensis). Legume plants include beans and peas. Beans include guar, carob, Trigonella foenum-graecum, soybeans, garden beans, Vigna unguiculata, Vigna radiata, Phaseolus limensis, fava beans, lentils, Cicer arietinum, etc. Vegetables include, but are not limited to, Arachis, p. eg peanut, Vicia, p. Coronilla varia, hairy pea, adzuki bean, Vigna radiata, and Cicer arietinum, Lupinus, p. eg, lupine, trifolium, Phaseolus, p. eg common beans and lima beans, Pisum, p. eg field bean, Melilotus, p. wheat, Medicago, e.g. eg alfalfa, Lotus, p. e.g. trifolium, lentils, e.g. eg lentil, and fake indigo. Preferred grass and forage for use in the methods of the invention include alfalfa, orchard grass, large straw, perennial rye, spreading curved grass, and Agrostis gigantea.

Preferably plants of the present invention include cultivated plants, for example corn, alfalfa, sunflower, brassica, soybean, cotton, saffron, peanut, sorghum, wheat, millet, tobacco, barley, rice, tomato, potato, pumpkin , melons, vegetable crops, etc. Other preferred plants include Liliopsida and Panicoideae.

Once a desired DNA sequence has been transformed into a particular plant species, it can be propagated in that species or moved to other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.

Below are descriptions of representative techniques for transforming both monocotyledonous and monocotyledonous plants, as well as a representative technique of plastid transformation. The. Dicotyledon Transformation Dicotyledon transformation techniques are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium techniques involve the capture of exogenous genetic material directly through protoplasts or cells. This can be accomplished with electroporation or PEG mediated uptake, particle bombardment delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J, 3: 2717 (1984), Potrykus et al., Mol. Gen.

Genet., 199: 169 (1985), Reich et al., Biotechnology, 4: 1001 (1986), and Klein et al., Nature, 327: 70 (1987). In each case, transformed cells are regenerated to whole plants using conventional techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique for dicotyledon transformation because of its high transformation efficiency and its broad utility with many different species. Transformation with Agrobacterium typically involves the transfer of the binary vector carrying the foreign DNA of interest (eg pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend on the complement of genes carried by the appropriate Agrobacterium strain either on the plasmid. Ti co-resident or chromosomally (eg strain CIB542 to pCIB200 and pCIB2001 (Uknes et al., Plant Cell, 5: 159 (1993)). Transfer of the recombinant binary vector to Agrobacterium is performed by a procedure E. coli carrying the recombinant binary vector, an auxiliary E. coli strain carrying a plasmid such as pRK2013 and capable of mobilizing the recombinant binary vector for the target strain of Agrobacterium. Recombinant binary can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res., 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of Agrobacterium with plant explants and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium bearing the antibiotic or herbicide resistance marker present between the T-DNA edges of the binary plasmid. Vectors can be introduced into plant cells in known ways. Preferred cells for transformation include Agrobacterium, monocotyledon cells and dicotyledon cells, including Liliopsida cells and Panicoideae cells. Preferred monocotyledon cells are cereal cells, e.g. e.g. maize, barley, and wheat, and starch accumulating dicotyledonous cells, e.g. e.g. potato.

Another approach to transforming a plant cell with a gene involves propelling inert or biologically active particles into plant cells and tissues. This technique is disclosed in U.S. Patents 4,945,050, 5,036,006, and 5,100,792. Generally, this procedure involves propelling inert or biologically active particles into cells under conditions effective to penetrate the outer surface of the cell and to provide incorporation therein. When using inert particles, the vector may be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell may be enveloped by the vector such that the vector is carried to the cell by the particle mat. Biologically active particles (eg, dried yeast cells, dried Bacterium or a bacteriophage, each containing DNA to be introduced) can also be propelled into plant cell tissue.

B. Transformation of monocotyledons Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer to protoplasts using electroporation or polyethylene glycol (PEG) techniques, and particle bombardment in callus tissue. Transformations can also be undertaken with a single DNA species or with multiple DNA species (i.e., co-transformation) and both of these techniques are suitable for use with this invention. Co-transformation can have the advantage of avoiding full vector construction and generating transgenic plants with unlinked loci for the gene of interest and selectable marker, allowing removal of the selectable marker in subsequent generations, if deemed desirable. However, a disadvantage of using co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al., Biotechnology, 4: 1093 1986)).

EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite congenital maize line, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (Plant Cell, 2: 603 (1990)) and Fromm et al. (Biotechnology, 8: 833 (1990)) published techniques for transformation of A 188-derived corn line using particle bombardment. In addition, WO 93/07278 and Koziel et al. (Biotechnology, 11: 194 (1993)) describe techniques for transforming elite congenital maize lines by particle bombardment. This technique uses immature 1.5-2.5 mm long maize embryos excised from a maize spike 14-15 days after pollination and a PDS-1000He Biolistics bombing device. Rice transformation can also be undertaken by direct gene transfer techniques using protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japanese and Indica types (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 are also routinely transformable using particle bombardment (Christou et al., Biotechnology, 9: 957 (1991)). In addition, WO 93/21335 describes techniques for the transformation of rice via electroporation. EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. In addition, wheat transformation has been described by Vasil et al. (Biotechnology, 10: 667 (1992)) using particle bombardment on type C long-term regenerable callus cells, and also by Vasil et al. (Biotechnology, 11: 1553 (1993)) and Weeks et al. (Plant Physiol., 102: 1077 (1993)) using particle bombardment of immature embryos and callus derived from immature embryo. However, a preferred technique for wheat transformation involves wheat transformation by particle bombardment of immature embryos and includes either a high sucrose step or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) is plated on MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum, 15: 473 (1962)) and 3 mg / l of 2 , 4-D for induction of somatic embryos, which is allowed to proceed in the dark. On the day selected for the bombardment, embryos are removed from the induction medium and placed in the osmotic (i.e. sucrose or maltose induction medium added at the desired concentration, typically 15%). Embryos are allowed to plasmaize for 2-3 hours and then bombarded. Twenty embryos per target plate is typical, though not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer sized gold particles using standard procedures. Each plate of embryos is targeted with the DuPont Biolistics® helium device using a blowing pressure of about 1000 psi (6900 kPa) employing a standard 80 mesh screen. After bombardment, the embryos are placed back in the dark for recovery for about 24 hours (still on osmotic). After 24 hours, the embryos are removed from the osmotic and placed back on induction media where they remain for about one month before regeneration. Approximately one month later, developing embryogenic callus embryo explants are transferred to regeneration medium (MS + 1 mg / liter NAA, 5 mg / liter GA), additionally containing the appropriate selection agent (10 mg / l sufficient for pCIB3064 and 2 mg / l methotrexate for pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as "GA7s" which contain half concentration MS, 2% sucrose, and the same concentration of selection agent.

Transformation of monocotyledons using Agrobacterium has also been described. See, WO 94/00977 and U.S. Patent 5,591,616, both incorporated herein by reference.

C. Plastid transformation [00170] Nicotiana tabacum c.v. 'Xanthi nc' are germinated on a per-plate basis in a 1 "circular arrangement on T agar medium and bombarded 12-14 days after being seeded with 1 pm tungsten particles (M10, Biorad, Hercules, CA) coated with Plasmid DNA pPH143 and pPH145 essentially as described (Svab and Maliga, PNAS. 90: 913 (1993).) Bombarded seedlings are incubated on T medium for two days, after which leaves are excised and placed abaxial side up under bright light (350-500 pmol photons / m2 / s) on RMOP medium plates (Svab, Hajdukiewicz and Maliga, PNAS, 87: 8526 (1990)) containing 500 pg / ml spectinomycin dihydrochloride (Sigma, St. Louis (MO) Resistant shoots appearing under the bleached leaves three to eight weeks after bombardment are subcloned onto the same selective medium, are allowed to form callus, and secondary shoots are isolated and subcloned.Full segregation of transformed plastid genome copies (homoplasmic) in Independent subclones are evaluated by conventional Southern staining techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1989)). BamHI / EcoRI digested total cellular DNA (Mettler, IJ Plant Mol Biol Reporter, 5: 346 (1987)) is separated over 1% Tris-borate (TBE) agarose gels, transferred to nylon membranes (Amersham) and probed with 32 P-labeled random primed DNA sequences corresponding to a 0.7 kb BamHI / HindIII DNA fragment of pC8 containing a portion of the rps7 / 12 plastid targeting sequence. Homoplasmic shoots are aseptically rooted in MS / IBA medium containing spectinomycin (McBride et al., PNAS, 91: 7301 (1994)) and transferred to the greenhouse.

Production and Characterization of Stably Transformed Plants Transformed plant cells are then placed in a selective medium suitable for selection of transgenic cells, which are then grown at callus. Sprouts are developed from corns to sprouts generated from rooting development. The various constructs will normally be conjugated with a marker for selection in plant cells. Advantageously, the label may be biocide resistance (particularly an antibiotic such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like). The particular marker used will allow the selection of transformed cells compared to cells that do not have the DNA was introduced. Components of DNA constructs, including transcription / transcription cassettes of this invention, may be prepared from sequences, which are native (endogenous) or foreign (exogenous) to the host. By "foreign" is meant that the sequence is not found in the wild type host into which the construct is introduced. Heterologous constructs will contain at least one region, which is not native to the gene from which the transcription-initiation region was derived.

To confirm the presence of transgenes in transgenic plants and cells, a Southern blot analysis can be performed using methods known to those skilled in the art. Integration of a polynucleic acid segment into the genome can be detected and quantified by Southern blotting, because it can be easily distinguished from constructs containing the segments by the use of appropriate restriction enzymes. Transgenic expression products can be detected in any of a variety of ways, depending on the nature of the product, and include Western blotting and enzyme assay. One particularly useful way to quantify protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants are obtained, they can be developed to produce plant tissues or parts having the desired phenotype. Plant tissue or plant parts may be harvested and / or seed collected. The seed can serve as a source for the development of additional plants with tissues or parts having the desired characteristics.

Thus, the invention provides a transformed plant or plant part, such as an ear, seed, fruit, grain, berry, bark, or bagasse comprising at least one polynucleotide, expression cassette or vector of the invention, methods of preparing a such a plant, and methods of using such a plant or a part thereof. The transformed plant or plant part expresses a processing enzyme, optionally located in a particular cell or subcellular compartment of a given tissue or in the developing grain. For example, the invention provides a transformed plant part comprising at least one starch processing enzyme present in plant cells, the plant part being obtained from a transformed plant whose genome is enlarged with an expression cassette encoding the at least one starch processing enzyme. The processing enzyme does not act on the target substrate unless activated by methods such as heating, milling, or other methods that allow the enzyme to contact the substrate under conditions where the enzyme is active.

Preferred Methods of the Present Invention The self-processing plants and plant parts of the present invention may be used in a variety of methods by employing the processing enzymes (mesophilic, thermophilic, or hyperthermophilic) expressed and activated therein. . In accordance with the present invention, a transgenic plant part of a transgenic plant whose genome is increased with at least one processing enzyme is obtained, placed under conditions in which the processing enzyme is expressed and activated. Upon activation, the processing enzyme is activated and works by acting on the substrate on which it normally acts to obtain the desired result. For example, starch processing enzymes act on starch to degrade, hydrolyze, isomerize, or otherwise modify to achieve the desired result upon activation. Non-starch processing enzymes can be used to disrupt the plant cell membrane to facilitate the extraction of starch, lipids, amino acids, or other plant products. In addition, non-hyperthermophilic and hyperthermophilic enzymes may be used in combination in the self-processing plant or plant parts of the present invention. For example, a non-starch-degrading mesophilic enzyme may be activated to break the plant cell membrane for starch extraction, and subsequently a starch-degrading hyperthermophilic enzyme may then be activated in the self-processing plant to degrade the starch. starch.

Enzymes expressed in the grain may be activated by substituting the plant or plant part containing them under conditions such that their activity is promoted. For example, one or more of the following techniques may be used: The plant part may be contacted with water, which provides a substrate for a hydrolytic enzyme and thus activating the enzyme. The plant part can be contacted with water, which will allow the enzyme to migrate from the compartment in which it was deposited during the development of the plant part and thus to associate with its substrate. Enzyme movement is possible because compartmentalization is violated during maturation, grain drying and rehydration. The intact or broken grain can be contacted with water, which will allow the enzyme to migrate from the compartment in which it was deposited during plant part development and thus to associate with its substrate. Enzymes can also be activated by adding an activating compound. For example, a calcium-dependent enzyme may be activated by the addition of calcium. Other activating compounds may be determined by those skilled in the art. Enzymes can be activated by removing an inactivator. For example, there are known peptide inhibitors of amylase enzymes, [wherein] amylase could be co-expressed with an amylase inhibitor and then activated by addition of a protease. Enzymes can be activated by changing the pH to a [pH] where the enzyme is most active. Enzymes can also be activated by increasing the temperature. An enzyme usually increases its activity to the maximum temperature for that enzyme. The mesophilic enzyme will increase its activity from one level of activity at room temperature to the temperature at which it loses activity, which is typically less than or equal to 70 ° C. Similarly, thermophilic and hyperthermophilic enzymes can be activated by increasing the temperature. Thermophilic enzymes can be activated by heating them to temperatures up to the maximum temperature of activity or stability. For a thermophilic enzyme, maximum stability and activity temperatures will generally be between 70 and 85 ° C. Hyperthermophilic enzymes will exhibit even greater relative activation than mesophilic or thermophilic enzymes due to the greater change in temperature potential from 25 ° C to 85 ° C to 95 ° C or even 100 ° C. The increased temperature may be obtained by any method, for example by heating, such as cooking, boiling, heating, steaming, electric discharge or any combination thereof. In addition, in plants expressing mesophilic or thermophilic enzyme (s), activation of the enzyme can be accomplished by grinding, thus allowing the enzyme to contact the substrate.

[00178] The optimal conditions, p. e.g., temperature, hydration, pH, etc. may be determined by one of ordinary skill in the art and may depend on the individual enzyme being employed and the desired application of the enzyme.

The present invention further provides for the use of exogenous enzymes which may contribute to a particular process. For example, the use of a self-processing plant or plant part of the present invention may occur in combination with an exogenously provided enzyme to facilitate the reaction. As an example, transgenic corn α-amylase may be used in combination with other starch processing enzymes, such as pullulanase, α-glucosidase, glucose isomerase, mannanases, hemicellulases, etc., to hydrolyze starch to produce ethanol. Indeed, it has been found that combinations of transgenic maize α-amylase with enzymes of this type unexpectedly provided higher degrees of starch conversion relative to the use of transgenic maize α-amylase alone.

Example of methods found to be advantageous herein are provided. The. Plant Starch Extraction The invention provides a method for facilitating plant starch extraction. In particular, at least one polynucleotide encoding a processing enzyme that disrupts the physically restricting matrix of the endosperm (cell walls, non-starch polysaccharide, and protein matrix) is introduced into a plant so that the enzyme is preferably present. in close physical proximity to starch granules in the plant. Preferably, in this embodiment of the invention, transformed plants express one or more protease, glucanase, xylanase, thioredoxin / thioredoxin reductase, esterase and the like, but not enzymes which exhibit any starch degrading activity, in order to maintain the integrity of the starch granules. The expression of these enzymes in a plant part, such as grain, thus promotes the process characteristics of the grain. The processing enzyme may be mesophilic, thermophilic, or hyperthermophilic. In one example, grain from a transformed plant of the invention is heat dried, probably inactivating non-hyperthermophilic processing enzymes and improving seed integrity. Grain (or broken grain) is macerated at low temperatures or at high temperatures (where time is of the essence) under low or high humidity conditions or contents (see Primary Cereal Processing, Gordon and Willm, eds., Pp. 319- 337 (1994), the disclosure of which is incorporated herein), with or without sulfur dioxide. When high temperatures are reached, optionally under certain humidity conditions, the integrity of the endosperm matrix is disrupted by enzyme activation, e.g. proteases, xylanases, phytase or glucanases that degrade the non-starch proteins and polysaccharides present in the endosperm, leaving the starch granule intact and more easily recoverable from the resulting material. In addition, non-starch polysaccharide proteins in the effluent are at least partially degraded and highly concentrated, and thus can be used for animal feed, food, or as components of medium for fermentation of microorganisms. The effluent is considered a maize bleach of improved composition.

Thus, the invention provides a method of preparing starch granules. The method comprises treating grain, e.g. broken grain, comprising at least one non-starch processing enzyme under conditions that activate at least one enzyme, giving a mixture comprising starch granules and non-starch degradation products, P. e.g., digested endosperm matrix products. The non-starch processing enzyme may be mesophilic, thermophilic, or hyperthermophilic. After activation of the enzyme, the starch granules are separated from the mixture. The grain is obtained from a transformed plant whose genome comprises (is enlarged with) an expression cassette encoding at least one processing enzyme. For example, the processing enzyme may be a protease, glucanase, xylanase, phytase, tiroredoxin / thioredoxin reductase, or esterase. Preferably, the processing enzyme is hyperthermophilic. The grain can be treated in conditions of high or low humidity, in the presence or absence of sulfur dioxide. Depending on the level of activity and expression of the processing enzyme in the transgenic plant grain, the transgenic grain may be mixed with market grain before or during processing. Also provided are products obtained by this method, such as starch, non-starch products and improved macerating water comprising at least one additional component.

B. Starch Processing Methods Transformed plants or plant parts of the present invention may comprise starch degrading enzymes as described herein, and which degrade starch granules to dextrins, other modified starches, or hexoses (e.g. amylase, pullulanase, α-glucosidase, glucoamylase, amylopululanase) or convert glucose to fructose (e.g. glucose isomerase). Preferably, the starch degrading enzyme is selected from α-amylase, α-glucosidase, glucoamylase, pullulanase, neopululanase, amylopululanase, glucose isomerase, and combinations thereof used to transform the grain. In addition, preferably, the enzyme is operably linked to a promoter and signal sequence that targets the enzyme to the starch granule, an amyloplast, the apoplast, or the endoplasmic reticulum. Most preferably, the enzyme is expressed in the endosperm, and particularly in the corn endosperm, and located in one or more cell compartments, or within the starch granule itself. The preferred part of the plant is the grain. Preferred plant parts are those of corn, wheat, barley, rye, oats, sugar cane, or rice.

According to a starch degradation method of the present invention, the transformed grain accumulates the starch degrading enzyme in starch granules, is leached at conventional temperatures of 50 ° C-60 ° C, and wet milled as know yourself in art. Preferably, the starch degrading enzyme is hyperthermophilic. Due to the subcellular objectification of the enzyme to the starch granule, or due to the association of the enzyme with the starch granule, by contacting the enzyme and starch granule during the wet milling process at conventional temperatures at The processing enzyme is co-purified with the starch granules to obtain the enzyme / starch mixture granules. Subsequent to the recovery of the enzyme / starch granule mixture, the enzyme is then activated providing favorable conditions for enzyme activity. For example, processing may be performed under various conditions of humidity and / or temperature to facilitate partial hydrolysis (to prepare derivatized starches or dextrins) or complete starch to hexoses. Syrups containing high dextrose or fructose equivalents are obtained in this manner. This method effectively reduces time, energy, and enzyme costs, and increases the efficiency with which starch is converted to the corresponding hexose, and the efficiency of producing products such as high sugar maceration water and syrups. equivalents with higher dextrose content.

In another embodiment, a plant, or a product of the plant, such as a fruit or grain, or prepared flour of the enzyme-expressing grain, is treated to activate the enzyme and convert polysaccharides expressed and contained within the plant to sugars. . Preferably, the enzyme is fused to a signal sequence that targets the enzyme to a starch granule, an amyloplast, the apoplast or the endoplasmic reticulum, as disclosed herein. The sugar produced can then be isolated or recovered from the plant or plant product. In another embodiment, the processing enzyme capable of converting polysaccharides to sugars is placed under the control of an inducible promoter according to methods known in the art and disclosed herein. The processing enzyme may be mesophilic, thermophilic or hyperthermophilic. The plant is grown to a desired stage, and the promoter is induced causing enzyme expression and conversion of polysaccharides within the plant or plant product to sugars. Preferably, the enzyme is operably linked to the signal sequence that targets the enzyme to a starch granule, an amyloplast, an apoplast or to the endoplasmic reticulum. In another embodiment, a transformed plant expressing a processing enzyme capable of converting starch to sugar is produced. The enzyme is fused with a signal sequence that targets the enzyme to a starch granule within the plant. Starch is then isolated from the transformed plant containing the enzyme expressed by the transformed plant. The enzyme contained in the isolated starch can then be activated to convert the starch to sugar. The enzyme may be mesophilic, thermophilic, or hyperthermophilic. Examples of hyperthermophilic enzymes capable of converting starch to sugar are provided herein. The methods may be used with any plant which produces a polysaccharide and which may express an enzyme capable of converting a polysaccharide to sugars or hydrolyzed starch product, such as dextrin, maltooligosaccharide, glucose and / or mixtures thereof.

The invention provides a method of producing altered dextrins and starches from a plant, or a product from a plant, which has been transformed with a processing enzyme that hydrolyzes certain covalent bonds of a polysaccharide to form a polysaccharide derivative. In one embodiment, a plant, or plant product, such as a fruit or grain, or prepared flour of the enzyme-expressing grain, is placed under conditions sufficient to activate the enzyme and convert polysaccharides contained within the plant to low molecular weight polysaccharides. . Preferably, the enzyme is fused to a signal sequence that targets the enzyme to a starch granule, an amyloplast, the apoplast or the endoplasmic reticulum as disclosed herein. The dextrin or starch derivative produced can then be isolated or recovered from the plant or plant product. In another embodiment, the processing enzyme capable of converting polysaccharides to dextrins or altered starches is placed under the control of an inducible promoter according to methods known in the art and disclosed herein. The plant is grown to a desired degree and the promoter is induced causing enzyme expression and conversion of polysaccharides within the plant or plant product to altered dextrins or starches. Preferably, the enzyme is Î ± -amylase, pullulanase, iso or neulululanase and is operably linked to a signal sequence that targets the enzyme in which the starch granule, an amyloplast, the apoplast or the endoplasmic reticulum. In one embodiment, the enzyme is targeted to the apoplast or endoreticulum. In another additional embodiment, a transformed plant expressing an enzyme capable of converting starch to dextrins or altered starches is produced. The enzyme is fused with a signal sequence that targets the enzyme to a starch granule within the plant. Starch is then isolated from the transformed plant containing the enzyme expressed by the transformed plant. The enzyme contained in the isolated starch may then be activated under conditions sufficient for activation to convert the starch to dextrins or altered starches. Examples of hyperthermophilic enzymes are provided herein, for example, capable of converting starch to hydrolyzed starch products. The methods can be used with any plant that produces a polysaccharide and can express an enzyme capable of converting a polysaccharide to sugar.

In another embodiment, transformed plant grains of the invention that accumulate starch degrading enzymes that degrade starch granule bonds to dextrins, modified starches or hexose (e.g., α-amylase, pullulanase, α-glucosidase, glucoamylase , amylopululanase) are macerated under conditions that favor the activity of starch degrading enzyme over various time periods. The resulting mixture may contain high levels of the starch product. The use of such a grain: 1) eliminates the need to grind the grain, or otherwise process the grain to first obtain starch granules, 2) makes the starch more accessible to enzymes due to its being placed enzymes directly into the grain endosperm tissue, and 3) eliminates the need for microbially produced starch hydrolyzing enzymes. Thus, the entire wet milling process is eliminated prior to hexose recovery by simply heating the grain, preferably corn grain, in the presence of water to allow the enzymes to act on the starch.

[00189] This process may also be employed for the production of ethanol, high fructose syrups, hexose (glucose) containing fermentation medium, or any other use of starch that does not require refining of grain components.

The invention further provides a method of preparing dextrin, maltooligosaccharides, and / or sugar involving treating a plant part comprising starch granules and at least one starch processing enzyme under conditions such as to activate at least one enzyme thereof. forms by digesting starch granules to form an aqueous solution comprising sugars. The plant part is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one processing enzyme. The aqueous solution comprising dextrins, maltooligosaccharides, and / or sugar is then collected. In one embodiment, the processing enzyme is α-amylase, α-glucosidase, pullulanase, glucoamylase, amylopululanase, glucose isomerase, or any combination thereof. Preferably, the enzyme is hyperthermophilic. In another embodiment, the method further comprises isolating dextrins, maltooligosaccharides, and / or sugar.

C. Improved maize varieties The invention also provides for the production of improved maize varieties (and varieties from other crops) that exhibit normal levels of starch accumulation, and which accumulate sufficient levels of amylolytic enzyme (s) in their endosperm, or starch accumulating organ, such that by activating the enzyme contained therein, such as by boiling or heating the plant or a part thereof, in the case of a hyperthermophilic enzyme, the enzyme (s) is activated and facilitates the rapid conversion of starch to simple sugars. These simple sugars (primarily glucose) will provide sugar to the treated corn. The resulting maize plant is an improved variety for dual use, as a grain producing hybrid and as sweet corn. Thus, the invention provides a method of producing hyper-sweet corn, comprising treating transformed corn or a portion thereof, whose genome is enlarged with, and expressed in the endosperm, an expression cassette comprising a promoter operably linked to a first polynucleotide encoding by least one amylolytic enzyme, conditions that activate at least one enzyme in order to convert polysaccharides in maize to sugar, giving hyper sweet maize. The promoter may be a constitutive promoter, a seed specific promoter, or an endosperm specific promoter that is linked to a polynucleotide sequence encoding a processing enzyme, such as α-amylase, e.g. one comprising SEQ ID NO: 13, 14, or 16. Preferably, the enzyme is hyperthermophilic. In one embodiment, the expression cassette further comprises a second polynucleotide encoding a signal sequence operably linked to the enzyme encoded by the first polynucleotide. Exemplary signal sequences in this embodiment of the invention direct the enzyme to the apoplast, the endoplasmic reticulum, a starch granule, or to an amyloplast. The corn plant is developed in such a way that the ears with grains are formed and then the promoter is induced causing the enzyme to be expressed, converting polysaccharide contained in the plant to sugar.

D. Self-Fermenting Plants In another embodiment of the invention, plants such as maize, rice, wheat, or sugar cane are manipulated to accumulate large amounts of processing enzymes on their own walls, e.g. xylanases, cellulases, hemicellulases, glucanases, pectinases and the like (non-starch polysaccharide degrading enzymes). After harvesting the grain component (or sugar in the case of sugar cane), the berry, rind, or bagasse is used as a source of the enzyme, which was intended for expression and accumulation in the cell walls, and as a source of biomass. Berry (or other residual tissue) is used as a feed material in a process for recovering fermentable sugars. The process of obtaining the fermentable sugars consists of activating the non-starch polysaccharide degrading enzyme. For example, activation may comprise heating plant tissue in the presence of water for suitable time periods for hydrolysis of non-starch polysaccharide in the resulting sugars. Thus, this self-processing berry produces the enzymes required for conversion of polysaccharides to monosaccharides, essentially at no additional cost because they are a component of the feed material. In addition, temperature-dependent enzymes do not exert any detrimental effects on plant growth and development, and cell wall objectification, even objectification to polysaccharide microfibrils, due to protein-fused cellulose / xylose binding domains , improving substrate accessibility to the enzyme.

Thus, the invention also provides a method of using a transformed plant part comprising at least one non-starch polysaccharide processing enzyme in the cell wall of the plant part cells. The method comprises treating a transformed plant part comprising at least one non-starch polysaccharide processing enzyme under conditions that activate at least one enzyme, thereby digesting starch granules to form an aqueous solution comprising sugars, wherein the part of plant is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one non-starch polysaccharide processing enzyme; and collect the aqueous solution comprising the sugars. The invention also includes a transformed plant or plant part comprising at least one non-starch polysaccharide processing enzyme present in the cell or cell wall of the plant cells or plant part. The plant part is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one non-starch processing enzyme, e.g. e.g., a xylanase, a cellulase, a glucanase, a pectinase, or any combination thereof.

E.g. High Protein and Sugar Aqueous Phase In another further embodiment, proteases and lipases are engineered to accumulate in seeds, e.g. eg soybean seeds. After activation of the protease or lipase, such as by heating, these seed enzymes hydrolyze the lipid and storage proteins present in soybean seeds during processing. In this way soluble products can be obtained comprising amino acids, which can be used as feed, food or fermentation medium, and fatty acids. Polysaccharides are typically found in the insoluble fraction of processed grain. However, by combining polysaccharide degrading enzyme expression and accumulation in seeds, proteins and polysaccharides can be hydrolyzed and are found in the aqueous phase. For example, corn and storage protein zeins and non-starch soy polysaccharides may be solubilized in this manner.

Components of the aqueous and hydrophobic phases can be easily separated by extraction with organic solvent or supercritical carbon dioxide. Thus provided is a method of producing an aqueous grain extract that contains higher levels of protein, amino acids, sugars or saccharides.

F. Self-Processing Fermentation The invention provides a method of producing ethanol, a fermented beverage, or other fermentation-derived product (s). The method involves obtaining a plant, or the product or part of a plant, or plant derivative, as grain flour, in which the processing enzyme that converts polysaccharides to sugar is expressed. The plant or product thereof is treated in such a way that sugar is produced by converting the polysaccharide as described above. Sugars and other plant components are then fermented to form ethanol or a fermented beverage, or other fermentation products, according to methods known in the art. See, for example, U.S. Patent 4,929,452. Briefly, sugar produced by polysaccharide conversion is incubated with yeast under conditions that promote sugar to ethanol conversion. Suitable yeast includes highly alcohol-tolerant and highly sugar-tolerant yeast strains such as, for example, S. cerevisiae ATCC No. 20867. This strain was deposited with the American Type Culture Collection, Rockville, MD, on 17 October. September 1987 and received ATCC designation No. 20867. The fermented product or fermented beverage may then be distilled to isolate ethanol or a distilled beverage, or the fermentation product may be otherwise recovered. The plant used in this method can be any plant that contains a polysaccharide and is capable of expressing an enzyme of the invention. Many such plants are disclosed here. Preferably, the plant is one that is commercially developed. More preferably, the plant is one commonly used to produce ethanol or fermented beverages or fermented products such as wheat, barley, corn, rye, potato, grapes or rice. Most preferably, the plant is corn.

The method comprises treating a plant part comprising at least one polysaccharide processing enzyme capable of activating at least one enzyme, thereby digesting polysaccharide in the plant part to form fermentable sugar. The polysaccharide processing enzyme may be mesophilic, thermophilic, or hyperthermophilic. Preferably, the enzyme is hyperthermophilic. The plant part is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one polysaccharide processing enzyme. Plant parts for 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, berry, grapes, rye, corn, rice, potato, sugar beet, sugar cane, pineapple, grass and trees. The plant part may be combined with commercial grain or other commercially obtainable substrates; The source of the processing substrate may be a source other than the self-processing plant. Fermentable sugar is then incubated under conditions that promote the conversion of fermentable sugar to ethanol, e.g. eg with yeast and / or other microbes. In a preferred embodiment, the plant part is derived from α-amylase transformed maize, which, as it turned out, reduces the amount of time and cost of fermentation.

The amount of residual starch has been found to be reduced when transgenic maize prepared according to the present invention expressing a thermostable α-amylase, for example, is used in fermentation. This indicates that more starch is solubilized during fermentation. The reduced amount of residual starch results in the distillers grains having higher protein content by weight and higher value. In addition, the fermentation of the transgenic corn of the present invention allows the liquefaction process to be performed at a lower pH, resulting in cost savings of the chemicals used to adjust the pH at a higher temperature, for example above 85 ° C. ° C, preferably greater than 90 ° C, more preferably 95 ° C or higher, resulting in shorter liquefaction times and more complete solubilization of starch, and reduction in liquefaction times, all resulting in efficient fermentation reactions. with higher ethanol yields.

Furthermore, it has been found that contacting conventional plant parts with even a small portion of the transgenic plant prepared in accordance with the present invention can reduce the fermentation time and costs associated with it. As such, the present invention relates to reducing fermentation time for plants comprising subjecting a transgenic plant part of a plant comprising a polysaccharide processing enzyme that converts polysaccharides to sugar, relative to the use of a non-plant part. comprises the polysaccharide processing enzyme.

[00203] g. Crude Starch Processing Enzymes and Polynucleotides Encoding them A polynucleotide encoding a mesophilic processing enzyme (s) is introduced into a plant or plant part. In a preferred embodiment, the polynucleotide of the present invention is a maize optimized polynucleotide as provided in SEQ ID NOS: 48, 50, and 59 encoding a glucoamylase as provided in SEQ ID NOS: 47 and 49. In another preferred embodiment The polynucleotide of the present invention is a maize optimized polynucleotide as provided in SEQ ID NO: 52 encoding an α-amylase as provided in SEQ ID NO: 51. In addition, enzyme fusion products are also considered. processing In a preferred embodiment, the polynucleotide of the present invention is a maize optimized polynucleotide as provided in SEQ ID NO: 46 encoding a glucoamylase and α-amylase fusion as provided in SEQ ID NO: 45. Combinations of processing enzymes also are further contemplated by the present invention. For example, a combination of starch processing enzymes and non-starch processing enzymes is contemplated herein. Such combinations of processing enzymes may be obtained by using multiple gene constructs encoding each of the enzymes. Alternatively, the individual transgenic plants stably transformed with the enzymes may be crossed by known methods to obtain a plant containing both enzymes. Another method includes the use of exogenous enzyme (s) with the transgenic plant.

The source of starch processing enzymes and non-starch processing enzymes may be isolated or derived from any source, and the corresponding polynucleotides thereof may be determined by one of ordinary skill in the art. Preferably, α-amylase is derived from Aspergillus (e.g., Aspergillus shirousami and Aspergillus niger), Rhizopus (e.g. Rhizopus oryzae), and plants such as corn, barley, and rice. Preferably, the glucoamylase is derived from Aspergillus (e.g. Aspergillus shirousami and Aspergillus niger), Rhizopus (e.g. Rhizopus oryzae), and Thermoanaerobacter (eg Thermoanaerobacter thetmosaccharolyticum).

In another embodiment of the invention, the polynucleotide encodes a mesophilic starch processing enzyme that is operably linked to a maize optimized polynucleotide as provided in SEQ ID NO: 54 encoding a crude starch binding domain as provided in SEQ ID NO: 53.

In another embodiment, a tissue-specific promoter includes endosperm-specific promoters, such as the corn γ-zein promoter (exemplified by SEQ ID NO: 12) or the corn ADP-gpp promoter (exemplified by SEQ ID NO: 11, which includes an intron sequence and an untranslated 5 '). Thus, the present invention includes an isolated polynucleotide comprising a promoter comprising SEQ ID NO: 11 or 12, a polynucleotide that hybridizes to its complement under low stringency conditions, or a fragment thereof that exhibits promoter activity, e.g. at least 10%, and preferably at least 50%, of the activity of a promoter having SEQ ID NO: 11 or 12.

In one embodiment, the product of a starch hydrolysis gene, such as α-amylase, glucoamylase, or α-amylase / glucoamylase fusion, may be targeted to a particular site or organelle, such as the endoplasmic reticulum or apoplast, rather than for cytoplasm. This is exemplified by the use of the maize γ-zein N-terminal signal sequence (SEQ ID NO: 17), which confers objectification of apoplast-specific proteins, and the use of the γ-zeine N-terminal signal sequence. zein (SEQ ID NO: 17) which is operably linked to the processing enzyme that is operably linked to the SEKDEL sequence for retention in the endoplasmic reticulum. Targeting the protein or enzyme to a specific compartment will allow the enzyme to be localized in such a way that it does not come into contact with the substrate. In this way, the enzymatic action of the enzyme will not occur until the enzyme contacts its substrate. The enzyme can be contacted with its substrate through the milling process (physical disruption of cell integrity) and hydration. For example, a mesophilic starch hydrolyzing enzyme may be targeted to the apoplast or endoplasmic reticulum and thus will not come into contact with starch granules in the amyloplast. Grinding the grain will disrupt the grain integrity and the starch hydrolyzing enzyme will then contact the starch granules. In this way, it is possible to avoid the potential negative effects of co-localization of an enzyme and its substrate.

[H]. Foods Without Addition of Sweetener A method of producing a sweetened farinaceous food product without the addition of additional sweetener is also provided. Examples of farinaceous products include, but are not limited to, breakfast food, ready-to-eat food, baked food, pasta and cereal products such as breakfast cereals. The method comprises treating a plant part comprising at least one starch processing enzyme under conditions that activate the starch processing enzyme, thereby processing starch granules in the plant part to sugars to form a sweetened product, e.g. for the product produced by processing starch granules from a plant part that does not comprise the hyperthermophilic enzyme. Preferably, the starch processing enzyme is hyperthermophilic and is activated by heating, such as by cooking, boiling, heating, steaming, electric discharge, or any combination thereof. The plant part is obtained from a transformed plant, such as processed soybean, rye, oats, barley, wheat, maize, rice or processed sugarcane, whose genome is enlarged with an expression cassette encoding at least a hyperthermophilic starch processing enzyme, e.g. α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. The sweetened product is then processed to a farinaceous food product. The invention also provides a farinaceous food product, e.g. a cereal food, a breakfast food, a ready-to-eat food, or a cooked food produced by said method. The farinaceous food product may be formed from the sweetened product and water, and may contain malt, flavorings, vitamins, minerals, coloring agents or any combination thereof.

The enzyme may be activated to convert polysaccharides contained in the plant material to sugar prior to inclusion of the plant material in the cereal product or during processing of the cereal product. Thus, polysaccharides contained in the plant material can be converted to sugar upon activation of the material, such as by heating, in the case of a hyperthermophilic enzyme, prior to inclusion in the farinaceous product. Sugar-containing plant material produced by converting the polysaccharides is then added to the product to produce a sweetened product. Alternatively, polysaccharides may be converted to sugars by the enzyme during processing of the farinaceous product. Examples of processes used to prepare cereal products are well known in the art and include heating, baking, boiling and the like, as described in U.S. Patent Nos. 6,183,788; 6,159,530; 6,149,965; 4,988,521 and 5,368,870.

Briefly, pasta can be prepared by mixing various dry ingredients together with water and baking to gelatinize the starchy components and develop a cooked aroma. The baked material can then be mechanically processed to form a baking dough such as cereal dough. Dry ingredients may include various additives such as sugars, starch, salt, vitamins, minerals, colorants, flavorings, salt and the like.

In addition to water, it is possible to add various liquid ingredients such as corn syrup (plus) or malt. Flouraceous material may include cereal grains, chopped, chopped or floured wheat, rice, maize, oats, barley, rye, or other cereal grains and mixtures thereof, from a transformed plant of the invention. Thereafter, the dough may be processed to a desired shape by a process such as extrusion or stamping, and further cooked using means such as a James cooker cooker, an oven or an electric discharge device.

Further provided is a method of sweetening a starch-containing product without adding sweetener. The method comprises treating starch comprising at least one starch processing enzyme capable of activating at least one enzyme, thereby digesting the starch to form a sugar, thereby forming a treated (sweetened) starch, e.g. for the product produced by treating starch which does not comprise the hyperthermophilic enzyme. The starch of the invention is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one processing enzyme. Preferred enzymes include α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. Preferably, the enzyme is hyperthermophilic and is heat activated. Preferred transformed plants include corn, soybeans, rye, oats, barley, wheat, rice and sugar cane. Thereafter, the treated starch is added to a product to produce a product containing sweetened starch, e.g. eg a farinaceous food product. A sweetened starch-containing product produced by the method is also provided.

The invention further provides a method of sweetening a polysaccharide-containing fruit or vegetable comprising: treating a fruit or vegetable comprising at least one polysaccharide processing enzyme under conditions that activate at least one enzyme, thereby processing the polysaccharide in fruit or vegetable to form sugar, giving a fruit or vegetable to form sugar, giving a sweetened fruit or vegetable, e.g. for a fruit or vegetable of a plant which does not comprise the polysaccharide processing enzyme. The fruit or vegetable of the invention is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one polysaccharide processing enzyme.

Preferred fruits and vegetables include potatoes, tomatoes, bananas, squash, peas, and beans. Preferred enzymes include (α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. Preferably, the enzyme is hypertermophilic.

Sweetening a Plant or Plant Product Containing Polysaccharide The method involves obtaining a plant that expresses a polysaccharide processing enzyme that converts a polysaccharide to a sugar as described above. Thus, the enzyme is expressed in the plant and in plant products, such as in a fruit or vegetable. 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 types of inducible constructs and promoters are well known in the art and are described herein. Expression of the enzyme within the plant or product thereof causes the polysaccharide contained within the plant or product thereof to be converted to sugar and will sweeten the plant or product thereof. In another embodiment, the polysaccharide processing enzyme is constitutively expressed. Thus, a plant or product thereof can be activated under conditions sufficient to activate the enzyme to convert polysaccharides to sugar by the action of the enzyme to sweeten the plant or product thereof. As a result, this self-processing of the polysaccharide in the fruit or vegetable to form sugar gives a sweetened fruit or vegetable, e.g. for a fruit or vegetable of a plant that does not comprise the polysaccharide processing enzyme. The fruit or vegetable of the invention is obtained from a transformed plant whose genome is enlarged with an expression cassette encoding at least one polysaccharide processing enzyme. Preferred fruits and vegetables include potatoes, tomatoes, bananas, squash, peas, and beans. Preferred enzymes include α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. Preferably, the polysaccharide processing enzyme is hyperthermophilic.

Isolation of transformed grain starch containing an endosperm matrix-breaking enzyme The invention provides a method of isolating starch from a transformed grain in which an endosperm matrix-breaking enzyme is expressed. The method involves obtaining a plant expressing an enzyme that disrupts the endosperm matrix by modifying, for example, cell walls, non-starch polysaccharides and / or proteins. Examples of such enzymes include, but are not limited to, proteases, glucanases, thioredoxin, thioredoxin reductase, and esterase. Enzymes of the said type do not include any enzyme which exhibits starch degrading activity in order to maintain the integrity of the starch granules. Preferably, the enzyme is fused to a signal sequence that targets the enzyme to the starch granule. In one embodiment the grain is heat dried to activate the enzyme and inactivate the endogenous enzymes contained in the grain. Heat treatment causes activation of the enzyme, which acts by breaking the endosperm matrix which is then easily separated from the starch granules. In another embodiment, the grain is macerated at a low or high temperature, high or low moisture content, with or without sulfur dioxide. The grain is then heat treated to break the endosperm matrix and allow easy separation of the starch granules. In another embodiment, appropriate temperature and humidity conditions are created to allow proteases to enter starch granules and degrade protein contained within the granules. Treatment of the said type could produce high yield starch granules and little contaminating protein.

Syrup Featuring a High Sugar Equivalent and Using Syrup to Produce Ethanol or a Fermented Drink [00221] The method involves obtaining a plant that expresses a polysaccharide processing enzyme that converts a polysaccharide to a sugar as described above. . The plant, or product thereof, is macerated in an aqueous stream under conditions such that the expressed enzyme converts polysaccharide contained within the plant, or product thereof, to dextrin, maltooligosaccharide, and / or sugar. The aqueous stream containing the dextrin, maltooligosaccharide, and / or sugar produced by polysaccharide conversion is then separated to produce a syrup having a high sugar equivalent content.

The method may or may not include an additional wet milling step of the plant or product thereof to obtain starch granules. Examples of enzymes that may be used in the method include, but are not limited to, α-amylase, glucoamylase, pullulanase and α-glucosidase. Preferably, the enzyme is hyperthermophilic. Sugars produced according to the method include, but are not limited to, hexose, glucose and fructose. Examples of plants that may be used with the method include, but are not limited to, corn, wheat or barley. Examples of plant products that may be used include, but are not limited to, fruit, grain and vegetables. In one embodiment, the polysaccharide processing enzyme is placed under the control of an inducible promoter. Thus, before or during the maceration process, the promoter is induced to cause expression of the enzyme, which then provides for the conversion of polysaccharide to sugar. Examples of inducible promoters and constructs containing them are well known in the art and are provided herein. Thus, where polysaccharide processing is hyperthermophilic, maceration is performed at a high temperature to activate the hypertermophilic enzyme and inactivate endogenous enzymes found within the plant or product thereof. In another embodiment, the hyperthermophilic enzyme capable of converting polysaccharide to sugar is constitutively expressed. This enzyme may or may not be objectified to a compartment within the plant through the use of a signal sequence. The plant, or product thereof, is macerated under high temperature conditions to cause the conversion of polysaccharides contained within the plant to sugar.

A method of producing ethanol or a fermented beverage from syrup containing a high sugar equivalent content is also provided. The method involves incubating the syrup with yeast under conditions that allow conversion of sugar contained within the syrup to ethanol or a fermented beverage. Examples of fermented beverages of the said type include, but are not limited to, beer and wine. Fermentation conditions are well known in the art and are described in U.S. Patent 4,929,452 and herein. Preferably, the yeast is a high alcohol tolerant and high sugar tolerant yeast strain such as S. cerevisiae ATCC No. 20867. The fermented product or fermented beverage may be distilled to isolate ethanol or a distilled drink. 1. Accumulation of Hyperthermophilic Enzyme on the Cell Wall of a Plant The invention provides a method of accumulating a hyperthermophilic enzyme on the cell wall of a plant. The method involves expressing, within a plant, a hyperthermophilic enzyme that is fused to a cell wall objectification signal such that the objectified enzyme accumulates on the cell wall. Preferably, the enzyme is capable of converting polysaccharides to monosaccharides. Examples of objectification sequences include, but are not limited to, a cellulose or xylose binding domain. Examples of hyperthermophilic enzymes include those listed in SEQ ID NO: 1, 3, 5, 10, 13, 14, 15 or 16. Plant material containing cell walls may be added as a desired enzyme source in a process for recovering sugars from feed material or as a source of enzymes for the conversion of polysaccharides originating from other sources to monosaccharides. Additionally, cell walls may serve as a source from which enzymes can be purified. Methods of purifying enzymes are well known in the art and include, but are not limited to, gel filtration, ion exchange chromatography, chromatofocus, isolating focus, affinity chromatography, FPLC, HPLC, salt precipitation, dialysis, and the like. Thus, the invention also provides purified enzymes isolated from plant cell walls.

Method of preparing and isolating processing enzymes In accordance with the present invention, recombinantly produced processing enzymes of the present invention may be prepared by transforming plant tissue or plant cells comprising the processing enzyme of the present invention. capable of being activated in the plant, selected for the transformed plant cell or tissue, developing the transformed plant cell or tissue into a transformed plant, and isolating the processing enzyme from the transformed plant or part thereof. Preferably, the recombinantly produced enzyme is an α-amylase, glucoamylase, glucose isomerase, α-glucosidase, and pullulanase. Most preferably, the enzyme is encoded by the polynucleotide selected from any of SEQ ID NOS: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41.43, 46, 48, 50, 52, or 59.

The invention will be further described with the following examples, which are not intended to limit the scope of the invention in any way.

Examples [00229] Example 1 [00230] Corn-optimized gene construction for starch isomerization / processing hyperthermophilic enzymes [00231] Enzymes, α-amylase, pullulanase, α-glucosidase, and glucose isomerase, involved starch degradation or glucose isomerization were selected based on their desired activity profiles. These include, for example, minimal activity at room temperature, activity / stability at high temperature, and activity at low pH. Corresponding genes were then engineered using preferred corn codons as described in U.S. Patent 5,625,136 and synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).

[00232] The 797GL3 α-amylase, featuring the amino acid sequence of SEQ ID NO: 1, was selected based on its hypertermophilic activity. This nucleic acid sequence of the enzyme was derived and optimized for maize as represented in SEQ ID NO: 2. Similarly, 6gp3 pululanase was selected by presenting the amino acid sequence shown in SEQ ID NO: 3. The nucleic acid sequence for 6gp3 pululanase was deduced and optimized for maize as represented in SEQ ID NO: 4.

[00233] The amino acid sequence for Sulfolobus solfataricus malA α-glucosidase was obtained from the literature, J. Bact. 177: 482-485 (1995); J. Bact. 180: 1287-1295 (1998). Based on the published protein amino acid sequence (SEQ ID NO: 5), the maize optimized synthetic gene (SEQ ID NO: 6) encoding malA α-glucosidase was designed.

Several glucose isomerase enzymes were selected. The amino acid sequence (SEQ ID NO: 18) for maritime Thermotoga derived glucose isomerase was predicted based on the published DNA sequence accession No. NC_000853 and a synthetic maize optimized gene (SEQ ID NO: 19) was designed. ). Similarly, the amino acid sequence (SEQ ID NO: 20) for Thermotoga neapolitan-derived glucose isomerase was predicted based on the DNA sequence published in Appl. Send Microbiol. 61 (5): 1867-1875 (1995), accession number L38994. A maize-optimized synthetic gene encoding Thermotoga neapolitan glucose isomerase (SEQ ID NO: 21) was designed.

Example 2 Fusion Expression of 797GL3 α-Amylase and Starch Encapsulating Region in E. coli [00237] A hyperthermophilic 797GL3 α-Amylase encoding construct fused to the starch encapsulating region of granule-bound starch synthase (waxy [maize granule-bound starch synthase]) was introduced and expressed in E. coli. The maize granule-linked starch synthase cDNA (SEQ ID NO: 7) encoding the amino acid sequence (SEQ ID NO: 8) (Klosgen RB, et al. 1986) has been cloned as a source of a bite binding domain. starch, or starch encapsulating region (SER). Full length cDNA was amplified by RT-PCR from maize seed prepared RNA using SV57 (5 'AGCGAATTCATGGCGGCTCTGGCCACGT 3') primers (SEQ ID NO: 22) and SV58 (5 'AGCTAAGCTTCAGGGCGCGGCCACGTTCT 3') (SEQ ID NO: 23) designed from GenBank Accession No. X03935. The complete cDNA was cloned into pBluescript as an EcoRI / HindIII fragment and the plasmid was named pNOV4022.

The C-terminal (encoded by bp 919-1818) portion of the waxy cDNA, including the starch binding domain, was amplified from pNOV4022 and fused in-frame to the 3 'end of the 797GL3 full-length maize optimized gene (SEQ ID NO: 2). The fused gene product, 797GL3 / waxy, presenting the nucleic acid with SEQ ID NO: 9 and encoding the amino acid sequence, SEQ ID NO: 10, was cloned as an Ncol / Xbal fragment in pET28b (NOVAGEN, Madison, Wl) which was cut with Ncol / Nhel. The 797GL3 gene alone was also cloned into the pET28b vector as an NcoI / XbaI fragment.

The pET28 / 797GL3 and pET28 / 797GL3 / waxy vectors were transformed into E. coli BL21 / DE3 (NOVAGEN) cells and developed and induced according to the manufacturer's instruction. PAGE / Stain analysis Coomassie revealed an induced protein in both extracts, corresponding to the predicted fused and unfused amylase sizes, respectively.

Total cell extracts were analyzed for hyperthermophilic amylse activity as follows: 5 mg of starch was suspended in 20 µl of water, then diluted with 25 µ of ethanol. Positive standard amylase control or the sample to be tested for amylase activity was added to the mixture and water was added to a final reaction volume of 500 μΙ. The reaction was performed at 80 ° C for 15-45 minutes. Then the reaction was cooled to room temperature, and 500 μΙ of o-dianisidine and glucose oxidase / peroxidase (Sigma) mixture were added. The mixture was incubated at 37 ° C for 30 minutes. 500 μΙ of 12 N sulfuric acid was added to stop the reaction. Absorbance at 540 nm was measured to quantify the amount of glucose released by the amylase / sample. Analysis of both fused and non-fused amylase extracts provided similar levels of hyperthermophilic amylase activity, while control extracts were negative. This indicated that 797GL3 amylase was still active (at high temperatures) when fused to the C-terminal portion of the waxy protein.

Example 3 Isolation of Promoter Fragments for Endosperm-Specific Expression in Corn.

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

The Zea mays γ-zein gene promoter was amplified as a 673 bp fragment (SEQ ID NO: 12) from plasmid pGZ27.3 (obtained from Dr. Brian Larkins). The γ-zein promoter has been shown to be endosperm specific (Torrent etal. 1997).

Example 4 [00246] Transformation Construction Vectors for the 797GL3 a-amylse Hyperthermophilic Expression cassettes were constructed to express the 797GL3 hyperthermophilic amylase in the corn endosperm with various objectification signals as follows: [ 00248] pNOV6200 (SEQ ID NO: 13) comprises the N-terminal maize γ-zein signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to synthetic amylase 797GL3 as described above in Example 1 for objectification to endoplasmic reticulum and apoplast secretion (Torrent et al. 1997). The fusion was cloned behind the maize ADP-gpp promoter for expression specifically in endosperm.

[00249] pNOV6201 (SEQ ID NO: 14) comprises the N-terminal signal sequence of synthetic 797GL3-fused γ-zein with a C-terminal addition of the SEKDEL sequence for objectification to and retention in the endoplasmic reticulum (ER). ) (Munro and Pelham, 1987). The fusion was cloned behind the maize ADP-gpp promoter for expression specifically in endosperm.

[00250] pNOV7013 comprises the N-terminal signal sequence of synthetic 797GL3-fused γ-zein with a C-terminal addition of the SEKDEL sequence for objectification to and retention in the endoplasmic reticulum (ER). PNOV7013 is the same as pNOV6201 except that the γ-maize zein promoter (SEQ ID NO: 12) was used in place of the maize ADP-spp promoter for the purpose of expressing endosperm fusion.

PNOV4029 (SEQ ID NO: 15) comprises the waxy amyloplast objectifying peptide (Klosgen et al., 1986) fused to synthetic amylase 797GL3 for amyloplast objectification. The fusion was cloned behind the maize ADP-gpp promoter for expression specifically in endosperm.

[00252] pNOV4031 (SEQ ID NO: 16) comprises the waxy amyloplast objectification peptide fused to the synthetic waxy / 797GL3 fusion protein aimed at objectification for starch granules. The fusion was cloned behind the maize aDP-gpp promoter for expression specifically in the endosperm.

Additional constructs were prepared with these cloned fusions behind the maize γ-zein promoter to obtain higher levels of enzyme expression. All expression cassettes were moved to a binary vector for transformation in maize via Agrobacterium infection. The binary vector contained the phosphomanosis isomerase (PMI) gene that allows selection of transgenic mannose cells. Transformed corn plants were either self-pollinated or exogenously crossed and seed was harvested for analysis.

Additional constructs were prepared with the fusion targeting signals described above, either 6gp3 pululanase or 340g12 α-glucosidase in precisely the same manner as described for α-amylase. These fusions were cloned behind the maize ADP-gpp promoter and / or the γ-zeine promoter and transformed into maize as described above. Transformed corn plants were either self-pollinated or exogenously crossed and seed was collected for analysis.

Combinations of the enzymes may be produced, either by crossing plants expressing the individual enzymes or by cloning several expression cassettes into the same binary vector to allow co-transformation.

Example 5 Construction of Plant Transformation Vectors for Thermophilic Pululanase 6GP3 An expression cassette for expressing thermophilic 6GP3 pulanase in the maize endosperm endoplasmic reticulum was constructed as follows: [00259] pNOV7005 (SEQ ID NOS: 24 and 25) comprises the N-terminal signal sequence of maize γ-zein fused to synthetic 6GP3 pululanase with a C-terminal addition of the SEKDEL sequence for targeting and retention in the endoplasmic reticulum ( ER, in English). The SEKDEL amino acid peptide was fused to the C-terminal enzymes using PCR primers designed to amplify the synthetic gene and simultaneously add the 6 amino acids to the C-terminal protein. The fusion was cloned behind the corn γ-zein promoter for expression specifically in endosperm.

[00260] Example 6 [00261] Construction of Plant Transformation Vectors for HyperAmophilic α-glucosidase malA

Expression cassettes were constructed to express the Sulfolobus solfataricus hyperthermophilic α-glucosidase malA in the maize endosperm with various objectification signals as follows: [00263] pNOV4831 (SEQ ID NO: 26) comprises the N-signal sequence γ-zein-terminal protein (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to synthetic α-glucosidase malA with a C-terminal addition of the SEKDEL sequence for targeting and retention in the endoplasmic reticulum (ER) (Munro) and Pelham, 1987).

The fusion was cloned behind the corn γ-zein promoter for expression specifically in the endosperm.

[00265] pNOV4839 (SEQ ID NO: 27) comprises the N-terminal maize γ-zein signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to synthetic α-glucosidase targeting endoplasmic reticulum and secretion in the apoplast (Torrent et al. 1997). The fusion was cloned behind the corn γ-zein promoter for expression specifically in endosperm.

[00266] pNOV4837 comprises the maize γ-zein (MRVLLVALALLALAASATS) N-terminal signal sequence (SEQ ID NO: 17) fused to synthetic α-glucosidase malA with a C-terminal addition of the SEKDEL sequence for objectification to, and retention in the endoplasmic reticulum. The fusion was cloned behind the maize ADP-gpp promoter for expression specifically in endosperm. The amino acid sequence for this clone is identical to that of pNOV4831 (SEQ ID NO: 26).

Example 7 [00268] Construction of Plant Transformation Vectors for the Thermotoga maritima and Thermotoga neapolitan Hyperthermophilic Glucose Isomerases [00269] Expression cassettes have been constructed to express the hyperthermophilic glucose isomerases of Thermotoga maritima and Thermotoga neapolitan neapolitan neapolitan. maize with various objectification signals as follows: [00270] pNOV4832 (SEQ ID NO: 28) comprises the N-terminal maize γ-zein signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to glucose isomerase Thermotoga maritima synthesis with a C-terminal addition of the SEKDEL sequence for objectification to and retention in the endoplasmic reticulum. The fusion was cloned behind the corn γ-zein promoter for expression specifically in endosperm.

[00271] pNOV4833 (SEQ ID NO: 29) comprises the N-terminal maize γ-zein signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to the Neapolitan synthetic glucose isomerase with a C-terminal addition. SEKDEL sequence aiming at objectification to and retention in the endoplasmic reticulum. The fusion was cloned behind the maize g-zein promoter for expression specifically in endosperm.

[00272] pNOV4840 (SEQ ID NO: 30) comprises the N-terminal maize γ-zein signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to Thermotoga neapolitan glucose isomerase aiming at endoplasmic reticulum and secretion in the apoplast (Torrent et al. 1997). The fusion was cloned behind the corn γ-zein promoter for expression specifically in endosperm.

[00273] pNOV4838 comprises the N-terminal maize γ-zein (MRVLLVALALLALAASATS) signal sequence (SEQ ID NO: 17) fused to Neapolitan glucose isomerase with a C-terminal addition of the SEKDEL sequence for objectification to, and retention no, ER. The fusion was cloned behind the maize ADP-gpp promoter for expression specifically in endosperm. The amino acid sequence for this clone is identical to that of pNOV4833 (SEQ ID NO: 29).

Example 8 [00275] Construction of Plant Transformation Vectors for Expression of Hyperthermophilic Glucanase EgIA [00276] pNOV4800 (SEQ ID NO: 58) comprises barley a-amylase signal sequence (MGKNGNLCCFSLLLLLLAGLASGHQ) (SEQ ID NO: 31) fused to the mature EgIA protein sequence for apoplast localization. The fusion was cloned behind the corn γ-zein promoter for expression specifically in endosperm.

Example 9 Construction of Plant Transformation Vectors for Expression of Multiple Hyperthermophilic Enzymes [00279] pNOV4841 comprises a double gene construct of a 797GL3 α-amylase fusion and a 6GP3 pullulanase fusion. Both the 797GL3 fusion (SEQ ID NO: 33) and the 6GP3 fusion (SEQ ID NO: 34) had the maize γ-zein N-terminal signal sequence and SEKDEL sequence for objectification to and retention in the endoplasmic reticulum. . Each fusion was cloned behind a maize γ-zein promoter for expression specifically in the endosperm.

[00280] pNOV4842 comprises a double gene construct of a 797GL3 α-amylase fusion and a α-glucosidase malA fusion. Both 797GL3 fusion polypeptide (SEQ ID NO: 35) and α-glucosidase malA fusion polypeptide (SEQ ID NO: 36) have the N-terminal maize γ-zein signal sequence and SEKDEL sequence for objectification. and retention in the endoplasmic reticulum. Each fusion was cloned behind a separate maize γ-zein promoter for expression specifically in endosperm.

[00281] pNOV4843 comprises a double gene construct of a 797GL3 α-amylase fusion and a α-glucosidase malA fusion. Both the 797GL3 fusion and the αA glucosidase malA fusion have the N-terminal maize γ-zein signal sequence and SEKDEL sequence for objectification to and retention in the endoplasmic reticulum. Fusion 797GL3 was cloned behind maize γ-zein promoter and malA fusion was cloned behind maize ADP-gpp promoter for expression specifically in endosperm. The amino acid sequences of fusion 797GL3 and fusion malA are identical to those of pNOV4842 (SEQ ID Nos: 35 and 36, respectively).

[00282] pNOV4844 comprises a triple gene construct of a 797GL3 α-amylase fusion, a 6GP3 pullulanase fusion, and an α-glucosidase fusion. 797GL3, malA, and 6GP3 all have the N-terminal maize γ-zein signal sequence and SEKDEL sequence for objectification to and retention in the endoplasmic reticulum. The 797GL3 and malA fusions were cloned behind 2 separate maize γ-zein promoters, and the 6GP3 fusion was cloned behind the maize ADP-gpp promoter for expression specifically in the endosperm. Amino acid sequences for 797GL3 and malA fusions are identical to those of pNOV4842 (SEQ ID Nos: 35 and 36, respectively): The amino acid sequence for fusion 6GP3 is identical to that of fusion 6GP3 in pNOV4841 (SEQ ID NO: 34) .

All expression cassettes were moved to binary vector pNOV2117 for transformation in maize via Agrobacterium infection. pNOV2117 contains the phosphomanose isomerase (PMI) gene allowing selection of transgenic mannose cells. pNOV2117 is a binary vector with both pVS1 and ColE1 replication sources. This vector contains the constitutive VirG gene of pAD1289 (Hansen, G., et al., PNAS USA 91: 7603-7607 (1994)) and a Tn7 spectinomycin resistance gene. The ubiquitin promoter, PMI coding region, and pNOV117 nopaline synthase terminator (Negrotto, D., et al., Plant Cell Reports 19: 798-803 ( 2000)). Transformed maize plants will either be self-pollinated or exogenously crossed and seed collected for analysis. Combinations of the different enzymes can be produced by crossing plants expressing the individual enzymes or by transforming a plant with one of the multiple gene cassettes.

Example 10 Construction of Bacterial and Pichia Expression Vectors Expression cassettes were constructed to express glucose isomerases and hypertermophilic α-glucosidase in Pichia or bacteria as follows: [00287] pNOV4829 (SEQ ID NOS: 37 and 38) comprises a maritime Thermotoga synthetic glucose isomerase fusion with ER retention signal in the bacterial expression vector pET29a. The glucose isomerase fusion gene was cloned into pET29a NcoI and Saci sites, resulting in the addition of an N-terminal S [S-tag] marker for protein purification.

PNOV4830 (SEQ ID NOS: 39 and 40) comprises a fusion of synthetic Thermotoga neapolitan glucose isomerase with ER retention signal in the bacterial expression vector pET29a. The glucose isomerase fusion gene was cloned into pET29a NcoI and Saci sites, resulting in the addition of an N-terminal S-tag for protein purification.

PNOV4835 (SEQ ID NO: 41 and 42) comprises the maritime Thermotoga synthetic glucose isomerase gene cloned into the BamHI and EcoRI sites of the pET28C bacterial expression vector. This resulted in the fusion of a His-tag (for protein purification) at the N-terminal end of glucose isomerase.

PNOV4836 (SEQ ID NO: 43 and 44) comprises the synthetic Neapolitan Thermotoga glucose isomerase gene cloned into the BamHI and EcoRI sites of the pET28C bacterial expression vector. This resulted in the fusion of a His-tag (for protein purification) at the N-terminal end of glucose isomerase.

[00291] ExampleH

Transformation of immature maize embryos was performed essentially as described in Negrotto et al., Plant Cell Reports 19: 798-803.

For this example, all media constituents are as described in Negrotto et al., Above. However, various media constituents described in the literature may be substituted. A. Plasmid Transformation and Selectable Marker The genes used for transformation were cloned into a suitable vector for maize transformation. Vectors used in this example contained the phosphomanose isomerase (PMI) gene for selection of transgenic lines (Negrotto et al. (2000) Plant Cell Reports 19: 798-803).

B. Preparation of Agrobacterium tumefaciens [00295] Agrobacterium LBA4404 (pSB1) strain containing the plant transformation plasmid was developed on YEP (yeast extract (5 g / l), peptone (10 g / l), NaCI (5 g / l), 15 g / l agar, pH 6.8) solid medium for 2-4 days at 28 ° C. Approximately 0.8 X 10 9 Agrobacterium were suspended in LS-inf medium supplemented with 100 μΜ As (Negrotto et al., (2000) Plant Cell Rep 19: 798-803). Bacteria were pre-induced in this medium for 30-60 minutes.

C. Inoculation Immature embryos from A188 or other suitable genotype were excised from 8-12 day-old ears in liquid LS-inf + 100 μΜ As Embryos were rinsed once with fresh infection medium. Agrobacterium N-terminal solution was added and embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. Embryos were N-terminally transferred, scutellum upwards, to LSAs medium and grown in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri dish were transferred to LSDc medium supplemented with cefotaxime (250 mg / l) and silver nitrate (1.6 mg / l) and cultured in the dark at 28 ° C for 10 days.

D. Selection of transformed cells and regeneration of transformed plants Immature embryos producing embryogenic callus were transferred to LSD1M0.5S medium. Cultures were selected on this medium for 6 weeks with a 3 week subculture step. Surviving calli were transferred to mannose supplemented Regí medium. After cultivation under light (16 hours light / 8 hours dark), green tissues were transferred to Reg2 medium without growth regulators and incubated for 1-2 weeks. Little plants are transferred to Magenta GA-7 boxes (Magenta Corp., Chicago III.) Containing Reg3 medium and grown in light. After 2-3 weeks, plants were tested for the presence of PMI genes and other genes of interest by PCR. Positive PCR assay plants were transferred to the greenhouse.

Example 12 T1 Seed Analysis of Corn Plants Expressing Targeted A-Amylase for Endoplasmic Apoplast or Reticulum T1 seeds from self-pollinated maize plants transformed with pNOV6200 or pNOV6201 were obtained. described in Example 4. Starch accumulation in these grains appeared to be normal based on visual inspection and normal starch staining with an iodine solution prior to any exposure to high temperature. Immature grains were dissected and purified endosperms were placed individually in microfuge tubes and immersed in 200 μΙ of 50 mM NaPCU buffer. The tubes were placed in a 85 ° C water bath for 20 minutes, then cooled on ice. Twenty microliters of 1% iodine solution was added to each tube and mixed. Approximately 25% of the segregating grains normally stained for starch. The remaining 75% failed to stain, indicating that the starch had been degraded to low molecular weight sugars that did not stain with iodine. The T1 grains of pNOV6200 and pNOV6201 were found to self-hydrolyze in cornstarch. There was no detectable reduction in starch after incubation at 37 ° C.

Amylase expression was further analyzed by isolation of the endosperm hyperthermophilic protein fraction, followed by Coomassie / PAGE staining. A segregating protein band of the appropriate molecular weight (50 kD) was observed. These samples are subjected to an α-amylase assay using commercially obtainable dyed amylose (AMYLAZYME from Megazyme, Ireland). High levels of hyperthermophilic amylase activity correlated with the presence of the 50 kD protein.

It has also been found that starch in grains of most transgenic maize expressing hypertermophilic α-amylase targeted for amyloplast is sufficiently active at room temperature to hydrolyze most starch if the enzyme is left in direct contact with a starch granule. Of the eighty lines presenting the targeted α-amylase, four lines accumulating starches in the grains were identified. Three of these lines were analyzed for thermostable α-amylase activity using a colorimetric amylase assay (Megazyme). Amylase enzyme assay indicated that these three lines had low levels of thermostable amylase activity. When purified starch from these 3 lines was treated under appropriate conditions of humidity and heat, the starch hydrolyzed indicating the presence of adequate levels of α-amylase to facilitate self-hydrolysis of the starch prepared from these lines.

Independent multiple row T1 seeds were obtained from both transformants pNOV6200 and pNOV6201. Individual grains from each row were dissected and purified endosperms were homogenized individually in 300 µl of 50 mM NaP04 buffer. Fractions of endosperm suspensions were analyzed for α-amylase activity at 85 ° C. Approximately 80% of the lines secrete for hyperthermophilic activity (see Figures 1A, 1B, and 2).

Grains of wild-type plants or plants transformed with pNOV6201 were heated at 100 ° C for 1, 2, 3, or 6 hours and then stained for starch with an iodine solution. Little or no starch was detected in mature grains after 3 or 6 hours, respectively. Thus, starch in mature grains of transgenic corn expressing hypertermophilic amylase, which is targeted for endoplasmic reticulum, was hydrolyzed when incubated at high temperature.

In another experiment, partially purified starch from mature T1 grains of pNOV6201 plants that were macerated at 50 ° C for 16 hours was hydrolyzed after heating at 85 ° C for 5 minutes. This illustrated that the targeted α-amylase for the endoplasmic reticulum binds to starch after grinding the grain, and is able to hydrolyze the starch upon heating. Iodine staining indicated that starch remains intact in mature seeds after maceration for 16 hours at 50 ° C.

In another experiment, segregating mature grains of plants transformed with pNOV6201 were heated at 95 ° C for 16 hours and then dried. In seeds expressing hyperthermophilic α-amylase, hydrolysis of sugar starch resulted in a wrinkled appearance upon drying.

Example 13 T1 Seed Analysis of Corn Plants Expressing Targeted Amyloplast A-Amylase T1 seeds from self-pollinated maize plants transformed with pNOV4029 or pNOV4031 were obtained as described in Example 4. Starch accumulation in grains from these lines was clearly not normal. All lines segregated, with some severity variation, to a phenotype with very little or no starch. Purified immature grain endosperm stained only weakly with iodine before exposure to high temperatures. After 20 minutes at 85 ° C, no staining was observed. When the ears were dried, the grains withered. This particular amylase clearly showed sufficient activity at greenhouse temperatures to hydrolyze starch if left in direct contact with the granule.

Example 14 [00313] Fermentation of a-Amylase-expressing Corn Plant Grain 100% transgenic grain 85 ° C. 95 ° C, varying liquefaction time.

[00315] Transgenic corn (pNOV6201) containing a thermostable α-amylase performs well in fermentation without the addition of exogenous α-amylase, requires much less time for liquefaction and results in more complete solubilization of starch. Laboratory scale fermentations were performed using a protocol with the following steps (detailed below): 1) milling, 2) moisture analysis, 3) preparation of a syrup containing ground corn, water, backset and a-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. Additionally, transgenic maize was liquefied with and without exogenous α-amylase and the ethanol production performance was compared with commercially obtainable α-amylase treated control maize.

The transgenic maize used in this example was prepared according to the procedures set forth in Example 4 using a vector comprising the α-amylase gene and the selectable marker PMI, nominally pNOV6201. Transgenic maize was produced by pollination of a commercial hybrid (N303OBT) with pollen from a transgenic line expressing a high level of thermostable a-amylase. The corn was dried at 11% humidity and stored at room temperature. The α-amylase content of transgenic maize flour was 95 units / g, with 1 enzyme unit generating 1 micromol of reducing tips per min. of cornmeal at 85 ° C in MES buffer pH 6.0. The control corn that was used was a yellow indentation corn known to perform well in ethanol production. 1) Grinding: Transgenic corn (1180 g) was ground in a Perten 3100 hammer mill equipped with a 2.0 mm screen thus generating transgenic cornmeal. Control corn was ground in the same mill after careful cleaning to prevent contamination by transgenic corn. 2) Moisture analysis: Samples (20 g) of transgenic corn and control corn were weighed on aluminum weighing plates and heated at 100 ° C for 4 h. The samples were again weighed and the moisture content calculated from the weight loss. The moisture content of transgenic flour was 9.26%; that of control flour was 12.54%. 3) Preparation of syrups: The syrup composition is designed to provide a 36% solids porridge at the beginning of SSF. Control samples were prepared in 100 ml plastic flasks containing 21.50 g of control cornmeal, 23 ml deionized water, 6.0 ml backset (8 wt.% Solids), and 0, 30 ml of a commercially obtainable α-amylase diluted 1/50 with water. The α-amylase dose was selected as representative of industrial use. When analyzed under the conditions described above for analysis of transgenic α-amylase, the control α-amylase dose was 2 U / g corn meal. The pH was adjusted to 6.0 by the addition of ammonium hydroxide. Samples were prepared in the same manner but contained 20 g of cornmeal due to the lower moisture content of the transgenic flour. Transgenic flour syrups were prepared either with α-amylase at the same dose as control samples or without exogenous α-amylase. 4) Liquefaction: Flasks containing transgenic cornmeal syrups were immersed in water baths at 85 ° C or 95 ° C for periods of 5, 15, 30, 45 or 60 min. Control samples were incubated for 60 min. at 85 ° C. During high temperature incubation the syrups were vigorously mixed by hand every 5 min.

After the high temperature step the syrups were cooled on ice. 5) Simultaneous Saccharification and Fermentation: The liquefied porridge was mixed with glucoamylase (0.65 ml of a 1/50 dilution of a commercially obtainable L-400 glucoamylase), protease (0.60 ml of a 1,000 fold of a commercially obtainable protease), 0.2 mg Lactocide & urea (0.85 ml of a 10 fold dilution of 50% urea liquor). A hole was cut in the lid of the 100 ml flask containing the porridge to allow CO2 to vent. The porridge was then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) adjusted water bath. After 24 hours of fermentation the temperature was reduced to 90 F (30 ° C); at 48 hours it was set at 90 F (28 ° C).

Yeast for inoculation was propagated by preparing a mixture containing yeast (0.12 g) with 70 grams maltodextrin, 230 ml water, 100 ml backset, glucoamylase (0.88 ml 10-fold dilution). a commercially obtainable glucoamylase), protease (1.76 ml of a 100-fold dilution of a commercially obtainable enzyme), urea (1.07 grams), penicillin (0.67 mg) and zinc sulfate (0.13 g ). Propagation culture was started the day before its use and was incubated with mixing at 90 ° F (32 ° C).

At 24, 48 & 72 hours samples were taken from each fermentation vessel, filtered through 0.2 µm filters and analyzed by HPLC for ethanol & sugars. At 72 h samples were analyzed for total dissolved solids and residual starch.

HPLC analysis was performed on a binary gradient system equipped with Bio-Rad Aminex HPX-87H refractive index detector, column heater & column heater. The system was equilibrated with 0.005 M H2SO4 in water at 1 ml / min. The column temperature was 50 ° C. The injection sample volume was 5 μΙ; the elution was in the same solvent. The IR response was calibrated by injection of known standards. Ethanol and glucose were both measured with each injection.

Residual starch was measured as follows. Standard samples were dried at 50 ° C in an oven, then ground to a powder in a sample mill. The powders (0.2 g) were weighed in a 15 ml graduated centrifuge tube. The powder was washed 3 times with 10 ml of aqueous ethanol (80% v / v) by vortexing by centrifugation and the supernatant was discarded. DMSO (2.0 ml) was added to the pellet, followed by 3.0 ml of a thermostable α-amylase (300 units) in MOPS buffer. After vigorous mixing, the tubes were incubated in a 85 ° C water bath for 60 min. During incubation, the tubes were mixed four times. The samples were cooled and 4.0 ml of sodium acetate buffer (200 mM, pH 4.5) was added, followed by 0.1 ml of glucoamylase (20 U). Samples were incubated at 50 ° C for 2 hours, mixed, then centrifuged for 5 min. at 3,500 rpm. The supernatant was filtered through a 0.2 µm filter and analyzed for glucose by the HPLC method described above. An injection size of 50 μΙ was used for samples with low residual starch content (<20% solids).

Results Transgenic corn showed good performance in fermentation without addition of α-amylase. The 72 hour ethanol yield was essentially the same with or without exogenous α-amylase, as shown in Table I. These data also show that a higher ethanol yield is achieved when the liquefaction temperature is higher; The present enzyme expressed in transgenic maize has activity at higher temperatures than other commercially used enzymes, such as Bacillus liquefaciens α-amylase.

When the liquefaction time was varied, it was found that the liquefaction time required for efficient ethanol production was much shorter than the time required by the conventional process. Figure 3 shows that the ethanol yield at 72 hours of fermentation was almost unchanged from 15 min. at 60 min. of liquefaction. In addition, liquefaction at 95 ° C gave more ethanol at all times than with liquefaction at 85 ° C. This observation demonstrates the improvement of the process provided with the use of a hyperthermophilic enzyme.

Control corn provided a higher final ethanol yield than transgenic corn, but control was selected because it has very good fermentation performance. In contrast, transgenic maize has a genetic background selected to facilitate transformation. The introduction of an α-amylase trait into elite maize germplasm by well-known rearing techniques should eliminate this difference.

Examination of residual starch levels of beer brewed at 72 hours (Figure 4) shows that transgenic α-amylase results in significant improvement in starch preparation available for fermentation; much less starch left after fermentation.

Using both ethanol and residual starch levels the optimum liquefaction times were 15 min. at 95 ° C and 30 min. at 85 ° C. In the present experiments, these times were the total time that the reaction vessels remained in the water bath and thus include a time period during which the temperature of the samples increased from room temperature to 85 ° C or 95 ° C. Shorter liquefying times can be optimal in large-scale industrial processes that quickly heat the pore using equipment such as jet cookers. Conventional industrial liquefaction processes require storage tanks to allow the porridge to be incubated at high temperature for one or more hours. The present invention eliminates the need for such storage tanks and will increase the productivity of liquefaction equipment.

[00360] An important function of α-amylase in fermentation processes is to reduce the viscosity of the porridge. At all times, samples containing transgenic cornmeal were markedly less viscous than the control sample. Additionally, transgenic samples did not appear to pass through the gel phase observed in all control samples; Gelatinition usually occurs when cooking corn syrups. Thus, since α-amylase is distributed throughout all endosperm fragments, beneficial physical properties are obtained for the porridge during cooking by preventing the formation of large gels which slow the diffusion rate and increase mixing energy costs. and pumping the pope.

The high dose of α-amylase in transgenic maize may also contribute to the favorable properties of transgenic porridge. At 85 ° C, transgenic maize α-amylase activity was often greater than the exogenous α-amylase dose activity used in controls. The latter was selected as representative of commercial utilization rates.

[00362] Example 15 [00363] Effective Function of Transgenic Corn When Mixed with Control Corn [00364] Transgenic cornmeal was mixed with control cornmeal at various levels, from 5% to 100% transgenic cornmeal. These were treated as described in Example 14. The transgenically expressed α-amylase-containing papes were liquefied at 85 ° C for 30 min. or at 95 ° C for 15 min; Control porridge was prepared as described in Example 14 and these were liquefied at 85 ° C for 30 or 60 min. (once each) or at 95 ° C for 15 or 60 min. (once each).

Data for 48 and 72 hour ethanol and residual starch are given in Table 2. 48 hour ethanol levels are reproduced in Figure 5; The residual starch determinations are shown in Figure 6. These data show that transgenically expressed thermostable α-amylase provides good yield in ethanol production even when the transgenic grain constitutes only a small portion (as low as 5%) of the total corn in the porridge. . The data also show that the residual starch content is markedly lower than in the control porridge when the transgenic grain comprises at least 40% of the total corn.

[00366] Table 2 • Control samples. Values are the average of 2 determinations Example 16 Ethanol Production as a Function of Liquefaction pH Using Transgenic Corn at a Rate of 1.5 to 12% of Total Corn Because Transgenic Corn showed a good performance at a level of 5-10% of the total corn in a fermentation, an additional series of fermentations was carried out in which the transgenic maize comprised 1.5 to 12% of the total corn. The pH ranged from 6.4 to 5.2 and the α-amylase enzyme expressed in transgenic maize was optimized for activity at a lower pH than conventionally used in industry.

[00436] The experiments were performed as described in Example 15 with the following exceptions: 1). Transgenic flour was mixed with control flour as a percentage of total dry weight at levels ranging from 1.5% to 12.0%.

[00438] 2). Control corn was N3030BT which is more similar to transgenic corn than the control used in examples 14 and 15.

[00439] 3). No exogenous α-amylase was added to samples containing transgenic flour.

[00440] 4). Samples were adjusted to pH 5.2, 5.6, 6.0 or 6.4 before liquefaction. At least 5 samples ranging from 0% transgenic cornmeal to 12% transgenic cornmeal were prepared at each pH.

5). Liquefaction for all samples was performed at 85 ° C for 60 min.

The change in ethanol content as a function of fermentation time is shown in Figure 7. This Figure shows data obtained from samples containing 3% transgenic corn. At the lowest pH, fermentation proceeds faster than at pH 6.0 and above; Similar behavior was observed in samples with other transgenic grain doses. The pH profile of the transgenic enzyme activity combined with the high expression levels will allow lower pH liquefactions, resulting in faster fermentations and therefore higher flow than is possible with the conventional pH 6.0 process.

The 72 hour ethanol yields are shown in Figure 8. As can be seen, based on the ethanol yield, the results showed little dependence on the amount of transgenic grain included in the sample. Thus, the grain contains abundant amylase to facilitate fermentative ethanol production. It is also shown that lower liquefaction pH results in higher ethanol yield.

The viscosity of the samples after liquefaction was monitored and it was observed that at a pH of 6.0, 6% transgenic grain is sufficient for adequate viscosity reduction. At pH 5.2 and 5.6, the viscosity is equivalent to that of control at 12% transgenic grain, but not at lower percentages of transgenic grain.

Example 17 [00446] Fructose Production From Corn Flour Using Thermophilic Enzymes [00447] Corn expressing hyperthermophilic α-amylase, 797GL3, has been shown to facilitate fructose production when mixed with an α-glucosidase (MalA) and a xylose isomerase (XylA).

Seeds of transgenic pNOV6201 plants expressing 797GL3 were ground to a flour in a Kleco cell, thereby creating amylase flour. Non-transgenic corn kernels were ground in the same manner to generate control flour.

Α-Glucosidase, MalA (from S. solfataricus), was expressed in E. coli. Harvested bacteria were suspended in 50 mM potassium phosphate phosphate buffer pH 7.0 containing 1 mM 4- (2-aminoethyl) benzenesulfonyl chloride, then lysed in a French pressure cell. The lysate was centrifuged at 23,000 x g for 15 min. at 4 ° C. The supernatant solution was removed, heated to 70 ° C for 10 min, cooled on ice for 10 min, then centrifuged at 34,000 x g for 30 min. at 4 ° C. The supernatant solution was removed and MalA concentrated twice on centricon 10 devices. The filtrate from step centricon 10 was retained for use as a negative control for MalA.

Xylose (glucose) isomerase was lost upon expression of the T. neapolitana xylA gene in E. coli. Bacteria were suspended in 100 mM sodium phosphate pH 7.0 and lysed by passage through a French pressure cell. After precipitation of the cell debris, the extract was heated at 80 ° C for 10 min. then centrifuged. The supernatant solution contained the enzymatic activity of XylA. An empty vector control extract was prepared in parallel with the XylA extract.

Cornmeal (60 mg per sample) was mixed with buffer and E. coli extracts. As indicated in Table 3, samples contained amylase (amylase) or control (control) cornmeal, 50 pl of MalA (+) or filtered (-) extract, and 20 pl of XylA (+) extract. or empty vector control (-). All samples also contained 230 μΙ of 50 mM MOPS, 10 mM MgSCXi, and 1 mM COCl2; Buffer pH was 7.0 at room temperature.

Samples were incubated at 85 ° C for 18 hours. At the end of the incubation time, samples were diluted with 0.9 ml of 85 ° C water and centrifuged to remove insoluble material. The supernatant fraction was then filtered through a Centricon3 ultrafiltration device and analyzed by ELSD detection HPLC.

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

HPLC results also indicated the presence of larger maltooligosaccharides in all samples containing α-amylase. These results demonstrate that the three thermophilic enzymes can work together to produce fructose from cornmeal at a high temperature.

Example 18 [00511] Isomerase Amylase Flour In another example, amylase flour was mixed with purified malA and each of two bacterial xylose isomerases: T. maritime XylA, and an enzyme called BD8037 obtained from Diverse. Amylase flour was prepared as described in Example 18.

S. solfataricus malA with a 6His purification marker was expressed in E. coli. Cell lysates were prepared as described in Example 18, then purified to apparent homogeneity using a nickel afinidae resin (Probond, Invitrogen) and following the manufacturer's instructions for native protein purification.

T. maritime xylA with the addition of an S-tag and an ER retention signal was expressed in E. coli and prepared in the same manner as the T. neapolitana xylA described in Example 18.

Xylose isomerase BD8037 was obtained as a lyophilized powder and was resuspended in 0.4 x the original volume of water.

Amilse cornmeal was mixed with enzyme solutions plus water or buffer. All reactions contained 60 mg of amylase flour and a total of 600 pl of liquid. One reaction set was buffered with 50 mM MOPS, pH 7.0 at room temperature, plus 10 mM MgSO4 and 1 mM CoCl2; In a second set of reactions the metal-containing buffer solution was replaced with water. Amounts of isomerase enzyme were varied as indicated in Table 4. All reactions were incubated for 2 hours at 90 ° C. Reaction supernatant fractions were prepared by centrifugation. The pellets were washed with an additional 600 μΙ H20 and centrifuged again. The supernatant fractions of each reaction were combined, filtered through a Centricon 10, and analyzed by ELSD detection HPLC as described in Example 17. The amounts of glucose and fructose observed are shown in Figure 15.

With each of the isomerases, fructose was produced from corn meal in a dose dependent manner when α-amylase and α-glucosidase were present in the reaction. These results demonstrate that grain-expressed 797GL3 amylase can work with MalA and a variety of thermophilic isomerases, with or without added metal ions, to produce fructose from maize flour at an elevated temperature. In the presence of added divalent metal ions, isomerases may provide the predicted fructose: glucose balance at 90 ° C of approximately 55% fructose. This would be an improvement over the current process using mesophilic isomerases, which requires chromatographic separation to increase fructose concentration.

Example 19 Expression of a Pululanase in Corn Transgenic plants that were homozygous for pNOV7013 or pNOV7005 were crossed to generate transgenic maize seed expressing both α-amylase 797GL3 and pululanase 6GP3.

T1 or T2 seeds were obtained from self-pollinated maize plants transformed with pNOV 7005 or pNOV 4093. pNOV4093 is a fusion of the 6GP3 maize-optimized synthetic gene (SEQ ID: 3,4) with the targeting sequence of amyloplast (SEQ ID NO: 7.8) for localization of the amyloplast fusion protein. This fusion protein is under the control of the ADP-gpp promoter (SEQ ID NO: 11) for expression specifically in endosperm. The pNOV7005 construct aims at the expression of pullulanase in the endoplasmic reticulum of the endosperm. The localization of this enzyme in the endoplasmic reticulum allows normal starch accumulation in the grains. Normal starch staining with an iodine solution was also observed prior to any exposure to high temperature.

As described in the case of α-amylase, expression of the target amyloplast pullulanase (pNOV4093) resulted in abnormal starch accumulation in the grains. When the ears of corn were dried, the grains withered. Apparently, this thermophilic pullulanase is sufficiently active at low temperatures and hydrolyzes starch if left in direct contact with the starch granules in the seed endosperm.

Enzyme preparation or enzyme extraction from maize flour: The pullulanase enzyme was extracted from the transgenic seeds by milling them in a Kleco mill, followed by incubation of the flour in the 50 mM NaOAc buffer pH 5.5 for 1 h at room temperature with continuous stirring. The incubated mixture was then centrifuged for 15 min. at 14000 rpm. The supernatant was used as an enzyme source.

Pullulanase analysis: The assay reaction was performed in a 96-well plate. The enzyme extracted from the cornmeal (100 pl) was diluted 10 times with 900 μ 50 of 50 mM NaOAc pH 5.5 buffer containing 40 mM CaCl2. The mixture was vortexed, 1 tablet of Limit-Dextrizyme (Megazyme azurine-retululate-pullulan) was added to each reaction mixture and incubated at 75 ° C for 30 min. (or as indicated). At the end of the incubation, the reaction mixtures were centrifuged at 3500 rpm for 15 min. The supernatants were diluted 5-fold and transferred to a 96-well flat bottom plate for absorbance measurement at 590 nm. Azurine-retululate-pullulan substrate hydrolysis by the pullulanase process produces water-soluble dye fragments and their release rate (measured as the increase in absorbance at 590 nm) is directly related to enzyme activity.

[00558] Figure 9 shows T2 seed analysis of different events transformed with pNOV 7005. High expression of pullulanase activity compared to non-transgenic control can be detected in a number of events.

To a measured amount (-100 pg) of dry transgenic (expressing pullulanase, or amylase or both enzymes) and / or control (non-transgenic) cornmeal was added 1000 µl of 50 mM buffer. NaOAc pH 5.5 containing 40 mM CaCl 2. The reaction mixtures were vortexed and incubated on a shaker for 1 h. The enzymatic reaction was initiated by transferring the incubation mixtures at high temperature (75 ° C, optimum reaction temperature for pullulanase or as indicated in the figures) over a period of time as indicated in the figures. Reactions were stopped by cooling on ice. The reaction mixtures were then centrifuged for 10 min. at 14000 rpm. A fraction (100 μΙ) of the supernatant was diluted three times, filtered through a 0.2 micron filter for HPLC analysis.

Samples were analyzed by HPLC using the following conditions: Column: Alltech Prevail Carbohydrate ES 5 microns 250 X 4.6 mm Detector: Alltech ELSD 2000 Pump: Gilson 322 Injector: Gilson 215 injector / diluter solvents: HPLC class acetonitrile (Fisher Scientific) and water (purified by the Waters Millipore System) [00566] Gradient used for low polymerization oligosaccharides (DP 1 -15).

% Water% acetonitrile% [00568] 0 15 85 [00569] 5 15 85 [00570] 25 50 50 [00571] 35 50 50 [00572] 36 80 20 [00573] 55 80 20 [00574] 56 15 85 [00575] 76 15 85 [00576] Gradient used for high polymerization saccharides (DP 20 -100 and above).

% Water% acetonitrile% water [00578] 0 35 65 [00579] 60 85 15 [00580] 70 85 15 [00581] 85 35 65 [00582] 100 35 65 [00583] System used for data analysis: Gilson Unipoint Software System version 3.2 Figures 10A and 10B show HPLC analysis of hydrolytic products generated by starch-expressed pullulanase in transgenic cornmeal. Incubation of pullulanase flour expressing maize in reaction buffer at 75 ° C for 30 minutes results in the production of medium chain oligosaccharides (DP ~ 10-30) and medium amylose chains (DP ~ 100-200) from soybean starch. corn. This figure also shows the dependence of pullulanase activity on the presence of calcium ions.

Pullulanase-expressing transgenic corn can be used to produce dextrin / modified starch that is branched (cleaved Î ± 1-6 bonds) and thus will have a high level of straight chain amylose / dextrin. Also depending on the type of starch (eg waxy, high amylose etc.) used, the distribution of the pullulanase-generated amylose / dextrin chain length will vary, and so will the property of the modified dextrin / starch.

Hydrolysis of an α 1-6 bond has also been demonstrated using pullulan as the substrate. Pululanase isolated from cornmeal efficiently hydrolyzed pullulan. HPLC analysis (as described) of the product generated at the end of incubation showed maltotriose production as expected due to hydrolysis of α 1-6 bonds in pullulan molecules by the corn enzyme.

Example 20 Pululanase Expression in Corn Pulphanase expression 6gp3 was further analyzed by cornmeal extraction followed by Coomassie and PAGE staining. Cornmeal was prepared by comminuting the seeds for 30 s in the Kleco mill. The enzyme was extracted from about 150 mg of flour with 1 ml of 50 mM NaOAc pH 5.5 buffer. The mixture was vortexed with a stirrer at room temperature for 1h followed by an additional 15 min. incubation at 70 ° C. The mixture was then centrifuged to sediment (14000 rpm for 15 min. At room temperature) and the supernatant was used as SDS-PAGE analysis. A protein band of the appropriate molecular weight (95 kDal) was observed. These samples are subjected to pullulanase analysis using commercially obtainable dye-conjugated limit dextrins (LIMIT-DEXTRIZYME from Megazyme, Ireland). High levels of thermophilic pullulanase activity correlated with the presence of the 95 kD protein.

Western blotting and ELISA analysis of transgenic corn seed also demonstrated expression of the ~ 95 kD protein that reacted with antibody produced against pullulanase (expressed in E. coli).

Example 21 Increased Starch Hydrolysis Rate and Improved Performance of Small-Chain (Fermentable) Oligosaccharides Through Addition of Corn Expressing Pululanase The data shown in Figures 11A and 11B were generated from analysis. HPLC assay as described above of the starch hydrolysis products from two reaction mixtures. The first reaction indicated as 'Amylase' contains a [1: 1 (w / w)] mixture of α-amylase-expressing transgenic maize flour samples prepared according to the method described in Example 4, for example, and non-maize. transgenic A188; and the second reaction mixture 'Amylase + Pululanase' contains a [1: 1 (w / w)] mixture of α-amylase-expressing transgenic maize flour samples and pululanase-expressing transgenic maize prepared according to the method described in Example 19. The results obtained support the benefit of the use of pullulanase in combination with α-amylase during starch hydrolysis processes. Benefits include increased starch hydrolysis rate (Figure 11A) and increased yield of low-DP fermentable oligosaccharides (Figure 11B).

It has been found that α-amylase alone or α-amylase and pullulanase (or any other combination of starch hydrolytic enzymes) expressed in maize can be used to produce maltodextrin (straight or branched chain oligosaccharides) (Figures 11 A, 11B, 12, and 13A). Depending on the reaction conditions, the type of hydrolytic enzymes and their combinations, and the type of starch used, the composition of the produced maltodextrins and therefore their properties will vary.

Figure 12 illustrates the results of an experiment performed in a manner similar to that described for Figure 11. The different temperature and time schemes followed during the incubation of the reactions are shown in the figure. The optimum reaction temperature for pullulanase is 75 ° C and for α-amylase is> 95 ° C. Therefore, the indicated schemes were followed to provide scope for catalysis with pullulanase and / or α-amylase at their respective optimal reaction temperatures. From the result shown it can be clearly deduced that the combination of α-amylase and pullulanase showed better performance in corn starch hydrolysis at the end of a 60 min incubation period.

HPLC analysis as described above (except that ~ 150 mg of cornmeal was used in these reactions) of the starch hydrolysis product of two sets of reaction mixtures after 30 min. of incubation is shown in Figures 13A and 13B.

The first reaction set was incubated at 85 ° C and the second was incubated at 95 ° C. For each set there are two reaction mixtures; the first reaction indicated as 'Amylase X Pululanase' contains transgenic maize flour (generated by cross-pollination) expressing both α-amylase and pullulanase, and the second reaction indicated as 'Amylase' mixture of maize flour samples. a transgenic maize expressing α-amylase and non-transgenic maize Α188 in a ratio such that the same amount of α-amylase activity is obtained as observed at the cross (Amylase X Pululanase). Total yield of low DP oligosaccharides was higher in the case of α-amylase and pullulanase crossover compared to maize expressing α-amylase only, when the cornmeal samples were incubated at 85 ° C. The incubation temperature of 95 ° C (at least partially) inactivates the pullulanase enzyme, so that little difference can be observed between 'Amylase X Pululanase' and 'Amylase'. However, those regarding incubation temperatures show a significant improvement in the amount of glucose produced (Figure 13B) at the end of the incubation period when using α-amylase and pullulanase maize flour compared to maize expressing α- amylase only. Therefore, the use of maize expressing both α-amylase and pullulanase may be especially beneficial for the process where complete starch hydrolysis to glucose is important.

The above examples largely corroborate that pullulanase expressed in maize seeds, when used in combination with α-amylase, enhances the starch hydrolysis process. Pululanase enzyme activity, being specific for α1-6 binding, degrades starch much more efficiently than α-amylase (a α1-4 binding specific enzyme) thereby reducing the amount of branched oligosaccharides (eg limit dextrin , panose; these are usually not fermentable) and increasing the amount of short straight chain oligosaccharides (easily fermentable to ethanol etc.). Secondly, fragmentation of starch molecules by pullulanase-catalyzed deramification increases substrate accessibility to α-amylase, thus resulting in increased efficiency of the α-amylase catalyzed reaction.

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

Example 23 The utility of Thermoanaerobacterium glucoamylase in the hydrolysis of crude starch was determined. As shown in Figure 15, the conversion by hydrolysis of crude starch was tested with water, barley α-amylase (Sigma commercial preparation), Thermoanaerobacterium glucoamylase, and combinations thereof were determined at room temperature and 30 ° C. As shown, the combination of barley α-amylase as Thermoanaerobacterium glucoamylase was able to hydrolyze crude starch to glucose. In addition, the amount of glucose produced by the thermoanaerobacter barley amylase and GA is significantly greater than that produced by either of these enzymes alone.

Example 24 [00604] Genes Optimized for Corn and Sequences for Crude Starch Hydrolysis and Plant Transformation Vectors [00605] Enzymes have been selected based on their ability to hydrolyze raw starch at temperatures ranging from approximately 20 ° - 50 ° C. Corresponding genes or gene fragments were then designed using preferred corn codons for the construction of synthetic genes as indicated in Example 1.

Aspergillus shirousami glucoamylase / α-amylase fusion peptide (no signal sequence) was selected and has the amino acid sequence as set forth in SEQ ID NO: 45 as identified in Biosci. Biotech Biochem., 56: 884-889 (1992); Agric. Biol. Chem. 545: 1905-14 (1990); Biosci Biotechnol. Biochem. 56: 174-79 (1992). Corn-optimized nucleic acid was designed and is represented in SEQ ID NO: 46.

Similarly, Thermoanaerobacterium thermosaccharolyticum glucoamylase having the amino acid of SEQ ID NO: 47 as published in Biosci was selected. Biotech Biochem. 62: 302-308 (1998). Corn-optimized nucleic acid (SEQ ID NO: 48) was designed.

Rhizopus oryzae glucoamylase having the amino acid sequence (no signal sequence) (SEQ ID NO: 50) was selected as described in the literature (Agric. Biol. Chem. (1986) 50, pp. 957-964 ). Corn-optimized nucleic acid was designed and is represented in SEQ ID NO: 51.

In addition, maize α-amylase was selected, and the amino acid sequence (SEQ ID NO: 51) and nucleic acid sequence (SEQ ID NO: 52) were obtained according to the literature, see, P. Plant Physiol. 105: 759-760 (1994).

Expression cassettes are constructed to express the Aspergillus shirousami α-amylase / glucoamylase fusion polypeptide from maize optimized nucleic acid, as depicted in SEQ ID NO: 46, Thermoanaerobacterium thermosaccharolyticum glucoamylase was designed from of corn-optimized nucleic acid as represented in SEQ ID NO: 48, Rhizopus oryzae glucoamylase was selected by presenting the amino acid sequence (without signal sequence) (SEQ ID NO: 49) of corn-optimized nucleic acid [e] was designed from maize-optimized nucleic acid and is represented in SEQ ID NO: 50, and maize α-amylase.

A plasmid comprising the N-terminal maize γ-zein signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) is fused to the synthetic gene encoding the enzyme. Optionally, the SEKDEL sequence is fused to the C-tip of the synthetic gene for objectification to and retention in the endoplasmic reticulum. The fusion is cloned behind the corn γ-zein promoter for expression specifically in endosperm in a plant transformation plasmid. Fusion is provided to the grain tissue via Agrobacterium transfection.

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

Example 26 Construction of Optimized Corn Genes and Plant Transformation Vectors The genes or gene fragments were designed using preferred corn codons for the construction of synthetic genes as set forth in Example 1.

Pyrococcus furiosus EGLA, hypertermophilic endoglucacanase amino acid sequence (no signal sequence) has been selected and has the amino acid sequence as shown in SEQ ID NO: 55, as identified in the Journal of Bacteriology (1999) 181, pp. 284-290). Corn-optimized nucleic acid was designed and is represented in SEQ ID NO: 56.

Thermus flavus xylose isomerase has been selected and has the amino acid sequence as set forth in SEQ ID NO: 57 as described in Applied Biochemistry and Biotechnology 62: 15-27 (1997).

Expression cassettes are constructed to express the corn optimized nucleic acid Pyrococcus furiosus EGLA (endoglucanase) (SEQ ID NO: 56) and Thermus flavus xylose isomerase from an amino acid sequence encoding nucleic acid optimized nucleic acid for maize SEQ ID NO: 57. A plasmid comprising the N-terminal maize γ-zein signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) is fused to the synthetic maize optimized gene encoding the enzyme. Optionally, the SEKDEL sequence is fused to the C-tip of the synthetic gene for objectification to and retention in the endoplasmic reticulum. The fusion is cloned behind the corn γ-zein promoter for expression specifically in endosperm in a plant transformation plasmid. Fusion is provided to corn tissue via Agrobacterium transfection.

Example 27 [00621] Corn Flour Glucose Production Using Corn Express Thermophilic Enzymes [00622] Expression of hypertermophilic α-amylase, 797GL3 and α-glucosidase (MalA) has been shown to result in glucose production when mixed with an aqueous solution and incubated at 90 ° C.

A transgenic maize line (line 168A10B, pNOV4831) expressing MalA enzyme was identified by measuring α-glucosidase activity as indicated by hydrolysis of p-nitrophenyl-a-glucoside.

Corn kernels from transgenic plants expressing 797GL3 were ground to a flour in a Kleco cell, thereby creating amylase flour. Corn grains from transgenic plants expressing MalA were ground to a flour in a Kleco cell thus creating malA flour. Non-transgenic corn kernels were ground in the same manner to generate control flour.

The buffer consisted of 50 mM pH 6.0 MES buffer.

Cornmeal hydrolysis reactions: Samples were prepared as indicated in Table 5 below. Cornmeal (about 60 mg per sample) was mixed with 40 ml of 50 mM MES buffer pH 6.0. Samples were incubated in a 90 ° C water bath for 2.5 and 14 hours. At the end of the indicated incubation periods, samples were removed and analyzed for glucose content.

The samples were analyzed for glucose by a glucose oxidase / radish peroxidase based assay. GOPOD reagent contained: 0.2 mg / ml o-dianisidine, 100 mM Tris pH 7.5, 100 U / ml glucose oxidase & 10 U / ml radish peroxidase. 20 µl of sample or diluted sample was placed in a 96 well plate along with glucose standards (ranging from 0 to 0.22 mg / ml). 100 µl of GOPOD reagent was added to each well with mixing and the plate was incubated at 37 ° C for 30 min. 100 æl sulfuric acid (9M) was added and the absorbance at 540 nm was measured. The glucose concentration of the samples was determined by reference to the standard curve. The glucose concentration of the samples was determined by reference to the standard curve. The amount of glucose observed in each sample is shown in Table 5.

These data demonstrate that when the expression of α-glucosidase and hypertermophilic α-amylase in maize results in a corn product that will generate glucose when hydrated and heated under appropriate conditions.

Example 28 Production of Maltodextrins Grain expressing thermophilic α-amylase was used to prepare maltodextrins. The exemplified process does not require prior starch isolation nor does it require addition of exogenous enzymes.

With grains of transgenic plants expressing 797GL3 they were ground to a flour in a Kleco cell to create "amylase flour". The mixture of 10% transgenic grains / 90% non-transgenic grains was ground in the same manner to create "10% amylase flour".

Amylase flour and 10% amylase flour (approximately 60 mg / sample) were mixed with water at a rate of 5 μΙ water per mg flour. The resulting mixtures were incubated at 90 ° C for up to 20 hours as indicated in Table 6. Reactions were stopped by adding 0.9 ml of 50 mM EDTA at 85 ° C and mixed by pipetting. 0.2 ml syrup samples were removed, centrifuged to remove insoluble material and diluted 3x in water.

Samples were analyzed by HPLC by detection of ELSD for sugars and maltodextrins. The gradient HPLC system was equipped with an Astec Polymer Amino Column, 5 micron particle size, 250 X 4.6 mm and an Alltech ELSD 2000 detector. The system was pre-equilibrated with a water: acetonitrile mixture. at 15:85. The flow rate was 1 ml / min. Initial conditions were maintained for 5 min. after injection, followed by a 20 min gradient. in water: 50:50 acetonitrile followed by 10 minutes of the same solvent. The system was washed with 20 min. of water: 80:20 acetonitrile and then re-equilibrated with the starting solvent.

The resulting peak areas were normalized to flour volume and weight. The ELSD response factor per μg of carbohydrate decreases with increasing DP, so that maltodextrins with higher SD represent a higher percentage of the total than indicated by the peak area.

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

These data demonstrate that it is possible to produce a variety of maltodextrin mixtures by varying the heating time. The level of α-amylase activity can be varied by mixing transgenic maize expressing α-amylase with wild type maize in order to alter the maltodextrin profile.

The hydrolysis reaction products described in this example may be concentrated and purified to provide food and other applications using a variety of well-defined methods, including: centrifugation, filtration, ion exchange, gel permeation, ultrafiltration. nanofiltration, reverse osmosis, carbon particle decolorization, spray drying and other conventional techniques known in the art.

Example 29 [00688] Effect of Time and Temperature on Maltodextrin Production [00689] The composition of grain self-hydrolysis maltodextrin products containing thermophilic α-amylase may be altered by varying time and temperature. of the reaction.

In another experiment, amylase flour was produced as described in Example 28 above and mixed with water at a rate of 300 µl water per 60 mg flour. Samples were incubated at 70 °, 80 °, 90 °, or 100 ° C for up to 90 minutes. Reactions were stopped by the addition of 900 ml of 50 mM EDTA at 90 ° C, centrifuged to remove insoluble material and filtered through 0.45 pm nylon filters. Filtrates were analyzed by HPLC as described in Example 28.

[00691] The result of this analysis is presented in Figure 19. The nomenclature of DP numbers refers to the degree of polymerization. DP2 is maltose; DP3 is maltotriosis, etc. Larger PD maltodextrins eluted at a single peak near the end of the elution are labeled "> DP12". This aggregate includes dextrins that have passed through 0.45 μίτι filters and through the guard column and do not include any very large starch fragments trapped by the filter or guard column.

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

Example 30 Production of Maltodextrin The composition of transgenic maltodextrin products containing thermophilic α-amylase may also be altered by the addition of other enzymes such as α-glucosidase and xylose isomerase as including salts. in the aqueous flour mixture before heat treatment.

Another amylase flour prepared as described above was mixed with purified malA and / or a bacterial xylose isomerase called BD8037. S. sulfotaricus malA with a 6His purification marker was expressed in E. coli. Cell lysate was prepared as described in Example 28, then purified to apparent homogeneity using a nickel affinity resin (Probond, Invitrogen) and following the manufacturer's instructions for native protein purification. Xylose isomerase BD8037 was obtained as a lyophilized powder from Diversa and resuspended in 0.4x the original volume of water.

Amylase cornmeal was mixed with enzyme solutions plus water or buffer. All reactions contained 60 mg of amylase flour and a total of 600 pl of liquid. One reaction set was buffered with 50 mM MOPS, pH 7.0 at room temperature, plus 10 mM MgSO4 and 1 mM CoCl2; In a second set of reactions the metal-containing buffer solution was replaced with water. All reactions were incubated for 2 hours at 90 ° C. Reaction supernatant fractions were prepared by centrifugation. The pellets were washed with an additional 600 μΙ H2O and re-centrifuged. The supernatant fractions of each reaction were combined, filtered through a Centricon 10, and analyzed by ELSD detection HPLC as described above.

The results are shown in Figure 20. They demonstrate that the grain expressed amylase, 797GL3, can work with other thermophilic enzymes, with or without the addition of metal ions, to produce a variety of maltodextrin mixtures from flour. of corn at a high temperature. In particular, inclusion of a glucoamylase or α-glucosidase may result in a product with more glucose and other products with low DP. Inclusion of an enzyme with glucose isomerase activity results in a product that has fructose and would therefore be sweeter than that produced by amylase alone or by α-glucosidase amylase. In addition, the data indicate that the proportion of DP5, DP6 and DP7 maltooligosaccharides may be increased by including divalent cationic salts such as COCl2 and MgSO4.

Other means of altering the composition of maltodextrin produced by a reaction such as that described herein include: varying the pH of the reaction, varying the type of starch in the transgenic or non-transgenic grain, varying the ratio of solids, or by the addition of organic solvents.

Example 31 Preparation of Dextrins, or Grain Sugars Without Mechanical Breakage of Grain Prior to Recovery from Starch Derived Products Sugars and maltodextrins were prepared by contacting the transgenic grain expressing α-amylase, 797GL3 , with water and heating at 90 ° C overnight (> 14 hours). Then the liquid was separated from the grain by filtration. The liquid product was analyzed by HPLC according to the method described in Example 15. Table 6 shows the profile of detected products.

[00703] Table 6 • Quantification of DP3 includes maltotriose and may include maltotriose isomers having an α (1-> 6) bond rather than an α (1 ^ 4) bond. Similarly, the quantification of DP4 to DP7 includes linear maltooligosaccharides of a given chain length and also with isomers having one or more α (1-6) bonds rather than one or more α bonds. [00723] These data demonstrate that sugars and maltodextrins can be prepared by contacting intact grain by expressing α-amylase with water and heating this. The products may then be separated from the intact grain by filtration or centrifugation or by gravitational deposition.

Example 32 Raw Corn Starch Fermentation Expressing Rhizopus oryzae Glucoamylase.

Transgenic corn kernels are harvested from transgenic plants prepared as described in Example 29. The kernels are ground to a flour. Corn kernels express a protein that contains an active fragment of Rhizopus oryzae glucoamylase (Sequence ID NO: 49) targeting the endoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 15. Then a porridge containing 20 g of cornmeal, 23 ml of deionized water, 6.0 ml of backset (8%) is prepared. solids by weight). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the porridge: protease (0.60 ml of a 1,000-fold dilution of a commercially obtainable protease), 0.2 mg of Lactocida & urea (0.85 ml of a 10-fold dilution of urea liquor). at 50%). Cut a hole in the lid of the 100 ml flask containing the porridge to allow CO2 to vent. The porridge is then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) water bath. After 24 hours of fermentation the temperature is decreased to 86 F; at 48 hours it was set at 90 F (28 ° C).

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 33 Example of Fermentation of Raw Corn Starch Expressing Rhizopus oryzae Glucoamylase Transgenic corn kernels are harvested from transgenic plants prepared as described in Example 28. The kernels are ground to a flour. Corn kernels express a protein that contains an active Rhizopus oryzae glucoamylase fragment (Sequence ID NO: 49) targeting the endoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 15. Then the porridge is prepared containing 20 g corn flour, 23 ml deionized water, 6.0 ml backset (8% solids). by weight). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the porridge: protease (0.60 ml of a 1,000-fold dilution of a commercially obtainable protease), 0.2 mg of Lactocida & urea (0.85 ml of a 10-fold dilution of 50% urea). A hole was cut in the lid of the 100 ml flask containing the porridge to allow CO2 to vent. Then the porridge is inoculated with yeast (1.44 ml) and incubated in a 90 F water bath. After 24 hours of fermentation the temperature is decreased to 86 F; at 48 hours it is set at 82 F.

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 34 Example of Fermentation of Raw Starch in Whole Corn Grains Expressing Rhizopus oryzae Glucoamylase With Addition of Exogenous α-Amylase Transgenic corn kernels are harvested from transgenic plants prepared as described in Example 28. Corn kernels express a protein that contains an active fragment of Rhizopus oryzae glucoamylase (Sequence ID NO: 49) targeting the endoplasmic reticulum.

Corn kernels are contacted with 20 g corn meal, 23 ml deionized water, 6.0 ml backset (8 wt.% Solids). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added: Sigma-acquired barley α-amylase (2 mg), protease (0.60 ml of a 1,000-fold dilution of a commercially obtainable protease), 0.2 mg of Lactocida & urea (0 , 85 ml of a 10-fold dilution of 50% urea liquor). A hole is cut in the lid of the 100 ml vial containing the mixture to allow CO 2 ventilation. The mixture is then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) adjusted water bath. After 24 hours of fermentation the temperature is reduced to 90 F (30 ° C); at 48 hours it is set at 90 F (28 ° C).

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 35 [00743] Corn Starch Fermentation Expressing Rhizopus oryzae Glucoamylase and Zea mays Amylase [00744] Transgenic corn kernels are harvested from transgenic plants prepared as described in Example 28. Corn kernels express a protein that contains an active fragment of Rhizopus oryzae glucoamylase (Sequence ID NO: 49) targeting the endoplasmic reticulum. The kernels also express crude starch binding domain corn amylase as described in Example 28.

The corn kernels are ground to a flour as described in Example 14. Then a porridge containing 20 g of cornmeal, 23 ml of deionized water, 6.0 ml of backset (8% of solids by weight). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the porridge: protease (0.60 ml 1000-fold dilution of a commercially obtainable protease), 0.2 mg Lactocide & urea (0.85 ml 10-fold dilution of 50% urea). A hole is cut in the lid of the 100 ml flask containing the porridge to allow CO 2 ventilation. The porridge is then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) water bath. After 24 hours of fermentation the temperature is reduced to 90 F (30 ° C); at 48 hours it is set at 90 F (28 ° C).

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 36 Example of Raw Corn Starch Fermentation Expressing Thermoanaerobacter thermosaccharolyticum Glucoamylase.

[00750] Transgenic corn kernels are harvested from transgenic plants prepared as described in Example 28. Corn kernels express a protein containing an active Thermoanaerobacter thermosaccharolyticum glucoamylase fragment (Sequence ID NO: 47) targeted for the endoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 15. Then a porridge containing 20 g of cornmeal, 23 ml of deionized water, 6.0 ml of backset (8% of solids by weight). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the porridge: protease (0.60 ml 1000-fold dilution of a commercially obtainable protease), 0.2 mg Lactocide & urea (0.85 ml 10-fold dilution of 50% urea). A hole is cut in the lid of the 100 ml flask containing the porridge to allow CO 2 ventilation. The porridge is then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) water bath. After 24 hours of fermentation the temperature is reduced to 90 F (30 ° C); at 48 hours it is set at 90 F (28 ° C).

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 37 Example of Raw Corn Starch Fermentation Expressing Aspergillus niger Glucoamylase Transgenic corn kernels are harvested from transgenic plants prepared as described in Example 28. Corn kernels express a protein containing a Aspergillus niger glucoamylase active fragment (Fiil, NP "Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs" EMBO J. 3 (5), 1097-1102 (1984), accession number P04064). Maize optimized nucleic acid encoding glucoamylase has SEQ ID NO: 59 and is targeted for the endoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 14. Then a porridge containing 20 g of cornmeal, 23 ml of deionized water, 6.0 ml of backset (8% of solids by weight). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the porridge: protease (0.60 ml 1000-fold dilution of a commercially obtainable protease), 0.2 mg Lactocide & urea (0.85 ml 10-fold dilution of 50% urea). A hole is cut in the lid of the 100 ml flask containing the porridge to allow CO 2 ventilation. The porridge is then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) water bath. After 24 hours of fermentation the temperature is reduced to 90 F (30 ° C); at 48 hours it is set at 90 F (28 ° C).

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 39 [00761] Example of Raw Corn Starch Fermentation Expressing Aspergillus niger Glucoamylase and Zea mays Amylase Transgenic maize kernels are harvested from transgenic plants prepared as described in Example 28. Corn kernels express a protein that contains an active Aspergillus niger glucoamylase fragment (Fiil, NP "Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs" EMBO J. 3 (5), 1097-1102 (1984): number P04064) (SEQ ID NO: 59, maize optimized nucleic acid) and is targeted for the endoplasmic reticulum. The kernels also express the crude starch binding domain corn amylase as described in Example 28. The corn kernels are ground to a flour as described in Example 14. Then a porridge containing 20 g of corn flour is prepared. corn, 23 ml deionized water, 6.0 ml backset (8 wt.% solids). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the porridge: protease (0.60 ml 1000-fold dilution of a commercially obtainable protease), 0.2 mg Lactocide & urea (0.85 ml 10-fold dilution of 50% urea). A hole is cut in the lid of the 100 ml flask containing the porridge to allow CO2 to vent. The porridge is then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) water bath. After 24 hours of fermentation the temperature is reduced to 90 F (30 ° C); at 48 hours it is set at 90 F (28 ° C).

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 39 [00766] Example of Raw Corn Starch Fermentation Expressing Thermoanaerobacter thermosaccharolyticum Glucoamylase and Barley Amylase Transgenic corn kernels are harvested from transgenic plants prepared as described in Example 28. Corn kernels express a protein containing an active fragment of Thermoanaerobacter thermosaccharolyticum glucoamylase (Sequence ID NO: 47) targeting the endoplasmic reticulum. The grains also express the low pl-barley amylase amyl amylase gene (Rogers.JC and Milliman.C. "Isolation and sequence analysis of a barley alpha amylase cDNA clone" J. Biol. Chem. 258 (13), 8169-8174 (1983) modified to target protein expression to the endoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 14. Then a porridge containing 20 g of cornmeal, 23 ml of deionized water, 6.0 ml of backset (8% of solids by weight). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the porridge: protease (0.60 ml 1000-fold dilution of a commercially obtainable protease), 0.2 mg Lactocide & urea (0.85 ml 10-fold dilution of 50% urea). A hole is cut in the lid of the 100 ml flask containing the porridge to allow CO2 to vent. The porridge is then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) water bath. After 24 hours of fermentation the temperature is reduced to 90 F (30 ° C); at 48 hours it is set at 90 F (28 ° C).

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 40 Example of Raw Starch Fermentation in Whole Corn Kernels Expressing Thermoanaerobacter thermosaccharolyticum Glucoamylase and Barley Amylase.

Transgenic corn kernels are harvested from transgenic plants prepared as described in Example 28. Corn kernels express a protein containing an active fragment of Thermoanaerobacter thermosaccharolyticum (Sequence ID NO: 47) targeted for the endoplasmic reticulum. The grains also express the low pl-barley amylase amyl gene (Rogers, JC and Milliman C. "Isolation and sequence analysis of a barley alpha-amylase cDNA clone" J. Biol. Chem. 258 (13), 8169- 8174 (1983) modified to target protein expression to the endoplasmic reticulum.

Corn kernels are contacted with 20 g corn meal, 23 ml deionized water, 6.0 ml backset (8% solids by weight). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the mixture: protease (0.60 ml of a 1000-fold dilution of a commercially obtainable protease), 0.2 mg of Lactocida & urea (0.85 ml of a 10-fold dilution of 50% urea). A hole is cut in the lid of the 100 ml flask containing the porridge to allow CO2 to vent. The mixture is then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) adjusted water bath. After 24 hours of fermentation the temperature is reduced to 90 F (30 ° C); at 48 hours it is set at 90 F (28 ° C).

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

Example 41 Example of Raw Corn Starch Fermentation Expressing a Fusion of α-Amylase and Glucoamylase.

Transgenic corn kernels are harvested from transgenic plants prepared as described in Example 28. Corn kernels express a corn optimized polynucleotide as provided in SEQ ID NO: 46 encoding a α-amylase and glucoamylase fusion as provided in SEQ ID NO: 45, which are objectified to the endoplasmic reticulum.

The corn kernels are ground to a flour as described in Example 14. Then a porridge containing 20 g of cornmeal, 23 ml of deionized water, 6.0 ml of backset (8% of solids by weight). The pH is adjusted to 6.0 by the addition of ammonium hydroxide. The following components are added to the porridge: protease (0.60 ml 1000-fold dilution of a commercially obtainable protease), 0.2 mg Lactocide & urea (0.85 ml 10-fold dilution of 50% urea). A hole is cut in the lid of the 100 ml flask containing the porridge to allow CO 2 ventilation. The porridge is then inoculated with yeast (1.44 ml) and incubated in a 90 F (32 ° C) water bath. After 24 hours of fermentation the temperature is reduced to 90 F (30 ° C); at 48 hours it is set at 90 F (28 ° C).

Inoculation yeast is propagated as described in Example 14.

Samples are removed as described in Example 14 and then analyzed by the methods described in Example 14.

All publications, patents and patent applications are incorporated herein by reference. Although, in the foregoing description, this invention has been described with respect to certain preferred embodiments thereof, and many details have been offered for illustration purposes, one skilled in the art will be able to realize that the invention is amenable to further embodiments and that certain The details described herein may be varied considerably without departing from the basic principles of the invention.

Α-Amylase Amino Acid Sequence 797GL3 (SEQ ID NO: 1) MAKYLELEEGGVItvíQFYWDVPSGGIViTVDTtRQKIPEWYDAGISAIWlPPASKGMSGGYSM

GYDPYDYFDLGEYYQKGWETRFGSKQELIMNTAHAYGIKVIADIVINARY <jDLE \ VNPF

VGPYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDICHDKSWDQYWLWASQES

YAAYLRSIGroAWRFDYVKGYGAWVVKDmNWWGGWAVGEYWDTNVDALLNWAYSSG

AKTTFDFPLYYKMDAAEDNKNIPALVEALKNGGlYVSRDPFKAVTFVANHDTDEroT ^ KYPAY

AFILTYEGQFTIPYRDYEEWLNKDKLENLrWIHDNLAGGSTSIVYYDSDEMIFVRNGy GS KPG

LFTYTNLGSSKVGRWVYVPKFAGACMEYTGNLGGWVDKYVYSSGWYYLEAPAYPPANGQ

YGYSVWSYCGVG

797GL3 Maize Optimized Nucleic Acid Sequence (SEQ ID NO: 2) ATGGCCAAGTACCTGGAGCTGGAGGAGGGCGGCGTGATCATGCAGGCGTTCTACTGGGA

CGTCCCGAGCGGAGGCATCTGGTGGGACACCATCCGCCAGAAGATCCCCGAGTGGTACG

ACGCCGGCATCTCCGCXjATCTGGATACCGCCAGCTTCCAAGGGCATGTCCGGGGGCTACT

CGATGGGCTACGACCCGTACGACTACTTCGACCTCGGCGAGTACTACCAGAAGGGCACG

GTGGAGACGCGCTTCGGGTCCAAGCAGGAGCTCATCAACATGATCAACACGGCGCACGC

CTACGGCATCAAGGTCATCGCGGACATCGTGATCAACCACAGGGCCGGCGGCGACCrGG

AGTGGAACCCGTTCGTCGGCGACTACACCTGGACGGACrTCrCCAAGGTCGCCTCCGGCA

AGTACACCGCCAACTACCTCGACrrCCACXAACGAGCTGCACGCGGGCGACTCCGGC

ACGTTCGGCGGCTACCCGGACATCTGCCACGACAAGTCCTGGGACCAGTACTGGCTCTGG

GCCTCGG \ GGAGTCCTACGCGGCCTACCTGCGCTCCATCGGCATCGACGCGTGGCGCTTC

GACTACGTCAAGGGCTACGGGGCCTGGGTGGTCAAGGACTGGCTCAACTGGTGGGGCGG

CTGGGCGGTGGGCGAGTACTGGGACACCAACGTCGACGCGCTGCTCAACTGGGCCTACT

CCTCCGGCGCCAAGGTGTTCGACTTCCCCCTGTACTACAAGATGGACGCGGCCTTCGACA

ACAAGAACATCCCGGCGCTCGTCGAGGCCCTGAAGAACGGCGGCACGGTGGTCTCCCGC

GACCCGTTCAAGGCCGTGACCrrCGTCGCCAACCACGACACGGACATCATCTGGAACAA

GTACCCGGCGTACGCCTTCATCCTCACCTACGAGGGCCAGCCCACGATCTTCTACCGCGA

CTACGAGGAGTGGCTGAACAAGGACA / VGCTCAAGAACCTGATCTGGATTCACGACAACC

TCGCGGGCGGCTCCACTAGTATCGTGTACTACGACTCCGACGAGATGATCITCGTCCGCA

ACGGCTACGQCTCCAAGCCCGGCCTGATCACGTACATCAACCTGGGCTCCTCCAAGGTGG

GCCGCTGGGTGTACGTCCCGAAGTTCGCCGGCGCGTGCATCCACGAGTACACCGGCAAC

CrCGGCGGCTGGGTGGACAAGTACGTGTACTCCTCCGGCTGGGTCTACCTGGAGGCrCCG

GCCTACGACCCCGCCAACGGCCAGTACGGCrACTCCGTGTGGTCCTACTGCGGCGTCGGC

Pullulanase Amino Acid Sequence 6gp3 (SEQ ID NO: 3) MGHWYKHQRAYQFIOEDDFGKVAVVKLPMDLTKVQIIVRLNBWQAKDVAKDRHEIKDGK

AEVWILQGVEEIFYEEPDTSPRIFFAQARSNKV] LEAFLTNPVDTKKKff'KVTVDGKHIPVSRV

EKADPTDIDVTM ^ VRIVLSESLKEEDLRKDVELIIEGYKPARVIMMEILDDYYYDGELGAVYS

PEKTIFR VWSPV SKW VKVLLFKNGEDTEPYQWNME YKGNGVWEAVVEGDLDGVFYI-Y Q3L

ENYGKIRTTVDPYSKAVYANNQESAWlSlLARTNPEGWENDRGPKIEGYEDAIIYEIHI

LENSGVKNKGLYLGLTEENTKGPGGVTTGLSHLVELGVTHVHÍLPFFDFYTGDELDKDFEKY

YIWGYDPYLFMWEGRYSTOPKNPHTRIREVKEMYKALHKHGIGVIMDMVFPHTYGIGELS

AFDQTVPYYFYRtDKTGAYLNESGCGNVIASERPMMRKFiVDTVTYWVKEYfflDGFRFDQM

GLroKKIMLEVERALHKIDPTnLYGEPWGGWGAPIRFGKSDVAGTHVAAFNDEERDATRGSV

FNPSVKGFVMGGYGKETKIKRGVVGSINYDGKL1KSFALDPEETINYAACHDNHTLWDKNY

LAAKADKKKEWTEEELKjNAQKLAGAILLTSQGVPFLHGGQDFCRTTNFNDNSYNAPISINGF

DYERKLQFIDVFNYIIKGLIIÍLRKEHPAFRLKNAEEIKKIILEFLPGGRRIVAFMI ^ KDHAGGDP

WKDWVIYNGM.EKTTmJEGKWNWVNSQlSAGTEVIETVEGTIELDPLSAYVLYRE

Nucleic acid sequence optimized for pullulanase maize 6gp3 (SEQ ID NO: 4) ATGGGCCÀCTGGTACAAGCACCAGCGCGCCTACCAGTTCACCGGCGAGGACGACTTCGG

GAAGGTGGCCGTGGTGAAGCTCCCGATGGACCTCACCAAGGTGGGCATCATCGTGCGCC tcaacgagtggcaggcgaaggacgtggccaaggaccgcttcatcgagatcaaggacggc aaggccgaggtgtggatactccagggcgtggaggagatcttctacüagaagccggacac ctccccgcgcatcttcttcgcccaggcccgctccaacaaggtgatcgaggccttcctcac caacccggtggacaccaagaagaaggagctgitcaaggtgaccgtcgacggcaaggag atcccggtgtcccgcgtggagaaggccgacccgaccgacatcgacgtgaccaactacgt GCGCATCGTGCTCrCCGAGTCCCTCAAGGAGGAGGACCrcCGCAAGGACGTGGAGCTGA

TCATCGAGGGCTACAAGCCGGCCCGCGTGATCATGATGGAGATCCTCGACGACTACTACT

ACGACGGCGAGCTGGGGGCGGTGTACTCCCCGGAGAAGACCATCTTCCGCGTGTGGTCC coggtgtccaagtgggtgaaggtgctcctcitcaagaacggcggacaccgagccgta CCAGGTGGTGAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGAGG

GACCTCGACGGCGTGTTCTACCTCTACCAGCTGGAGAACTACGGCAAGATCCGCACCACC

GTGGACCCGTACTCCAAGGCCGTGTACGCCAACAACCAGGAGTCTGCAGTGGTGAACCT

CGCCCGCACCAACCCGGAGGGCTGGGAGAACGACCGCGGCCCGAAGATCGAGGGCTAC

GAGGACGCCATCATCTACGAGATCCACATCGCCGACATCACCGGCCTGGAGAACTCCGG

CGTGAAGAACAAGGGCCTCTACCTCGGCCCCACACGGGAGAAGACACAGAGCCCCGGGCG gcgtgaccaccggcctctcccacctcgtggagctgggcgtgacccacgtgcacatcctcc CGTTCTTCGACTGGACACAGGACCACTGACACAGGACCACGAGC

GGGGCTACGACCCGTACCTCTTCATGGTGCCGGAGGGCCGCTACTCCACCGACCCGAAG

AACCCGCACACCCCCATATTCGCGAGGTGAAGGAGATGGTGAAGGCCCTCCACAAGCACGG

CAIX ^ GGCGTGATCATGGACATGGTGTTCCCGCACACCTACGGCATCGGCGAGCTGTCCGC

CTTCGACCAGAjGCGTGCX ^ GTACTACTTCTACCGCATCGACAAGACCGGCGCCTACCTCAA

CGAGTCCGGCTGCGGCAACGTGATCGCCrCCGAGCGCCCGATGATGCGCAAGITCATCGT

GGACACCGTGACCTACTGGGTGAAGGAGTACCACATCGACGGCTTCCGCrTCGACCAGA

TGGGCCTCATCGACAAGAAGACCATGCTGGAGGTGGAGCGCGCCCTCCACAAGATCGAC

CCGACCATCATCCTCTACGGCGAGCCGTGGGGCGGCTGGGGGGCCCCGATCCGCITCGG

CAAGTCCGACGTGGCCGGCACCCACGTGGCCGCCrrCAACGACGAGTTCCGCGACGCCA

TCCGCGGCTCCGTGTTCAACCCGTCCGTGAAGGGCTTCGTGATGGGCGGCTACGGCAAGG

AGACCAAGATCAAGCGCGGCGTGGTGGGCTCCATCAACTACGACGGCAAGCTCATCAAG

TCCTTCGCCXTCGACCCGGAGGAGACXIATCAACTACGCCGCCrGCCACGACAACCACACC

CTCTGGGACAAGAACTACCTCGCCGCCAAGGCCGACAAGAAGAAGGAGTGGACCGAGG

AGGAGCTGAAGAACGCCCAGAAGCICGCCGGCGCCATCCTCCrCACTAGTCAGGGCGTG

CCGTTCCTCCACGGCGGCCAGGAGTTCTGCCGCACCACCAACTTCAACGACAACTCCTAC

AACGCCCCGATCTCCATCAACGGCITCGACrACGAGCGCAAGCrOCAGTrCATCGACGTG

TTCAACTACCACAAGGGCCTCATCAAGCTCCGC ^ VAGGAGCACCCGGCCTTCCGCCTCAAG

AACGCCGAGGAGATCAAGAAGCACCTGGAGTTCCTCCCGGGCGGGCGCCGCATCGTGGC

CTTCATGCTCAAGGACCACGCCGGCGGCGACGCGTGGAAGGACATCGTGGTGATCTACA

ACGGCAACCTGGAGAAGACCACCTACAAGCTCCCGGAGGGCAAGTGGAACGTGGTGGTG aactcccagaaggccggcaccgaggtgatcgagaccgtggagggcaccatcgagctgga CCCGCTCTCCGCCTACGTGAGC

Amino acid sequence and α-glucosidase malA from Sulfolobus solfataricus (SEQ ID NO: 5) METIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIVQQGNKVIVEKSLDLKEHnG

LGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISVKDGVATGYFPNSASKVIFDV

GLEEYDKVTVITPEDSVEFYVÍEGPRIEDVLEKYTELTGKPFLPPMWAFGYMISRYSYYPQDK

WELVDMQKEGFRVAGVFLDn-IYMDSYKLFTWHPYRPPEPKKLIDELHKRNVKLmVDHGI

RVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFREDTREWWAGL1SEWLSQGVD

GIWLDMNEPTDFSRAIEÍRDVLSSLPVQFRDDRLVTTFPDNVVHYLRGKRVKHEKVRNAYPL

YEAMATFKGFRTSHRNEMLSRAGYAGIQRYAFIWTGDNlPSWDDLKLQIjQLVLGLSISGW

FVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKATDGIDTEPVELPDYYKEKVK

ELVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRIEDEYMVGKYLLYAPIVSKEE

SRLVTLPRGKWYNYWNGEDNGKSVVKSTHELPIYLREGSUPLEGDELIVYGETSFKRYDNAE

ITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQNT ^ VAKINQKIRGKINLE

Corn-optimized nucleic acid sequence of Sulfolobus solfataricus α-glucosidase malA (SEQ ID NO: 6) ATGGAGACCATCAAGATCTACGAGAACAAGGGCGTGTACAAGGTGGTGATCGGCGAGCC

GTTCCCGCCGATCGAGTTCCCGCTCGAGGAGAAGATCTCCTCCAACAAGTCCCTCTCCGA

GCTGGGCCTCACCATCGTGCAGCAGGGCAACAAGGTGATCGTGGAGAAGTCCCTCGACC

TCAAGGAGCACATCATCGGCCTCGGCGAGAAGGCCTTCGAGCTGGACCGCAAGCGCAAG

CGCTACGTGATGTACAACGTGGACGCCGGCGCCTACAAGAAGTACCAGGACCCGCTCTA

CGTGTCCATCCCGCTCrrCATCTCCGTGAAGGACGGCGTGGCCACCGGCTACTTClTCAA

CTCCGCCTCCAAGGTGATCTTCGACGTGGGCCTCGAGGAGTACGACAAGGTGA1CGTGA

CCATCCXrGGAGGACrCCGTGGAGTlOTACGTGATCGAGGGCCCGCGCATCGAGGACGTG

CTCGAGAAGTACACCGAGCTGACCGGCAAGCCGTTCCTCCCGCCGATGTGGGCCTTCGGC

TACATGATCTCCCGCTACrCCTACTACCCGCAGGACAAGGTGGTGGAGCIGGTGGACATC

ATGCAGAAGGAGGGCTTCCGCGTGGCCGGCGTGTTCCTCGACATCCACTACATGGACTCC

TACAAGCTCITCACCTGGCACCCGTACCGCTTCCCGGAGCCGAAGAAGCTCATCGACGAG

CTGCACAAGCGCAACGTGAAGCTCATCACCATCGTGGACCACGGCATCCGCGTGGACCA

GAACTACTCCX: CGTTCCTCTCCGGCATGGGCAAGlTCrGCGAGATCGAGTCCGGCGAGCT

GTTCGTGGGCAAGATGTGGGCGGGCACCACCGTGTACCCGGACTTCTTCCGCGAGGACA

CCCGCGAGTGGTGGGCTGGCCTCATCrCCXJAGTGGCTCTCCCAGGGCGTGGACGGCATCT

GGCTCGACATGAACGAGCCGACCGACTTCTCCCGCGCCATCGAGATCCGCGACGTGCTCT

CCrCCCTCCCGGTG € AGTTCCGCGACGACCGCCTCGTGACCACCTl'CCCGGACAACGTGG

TGCACTACCTCCGCGGCAAGCGCGTGAAGCACGAGAAGGTGCGCAACGCCTACCCGCTC

TACGAGGCGATGGCCACCTTCAAGGGCTTCCGCACCTCCCACCGCAACGAGATCTTCATC

CTCTCOCGCGCCGGCTACGCCGGCATCCAGCGCTACGCCTTCATCrGGACCGGCGACAAC

ACCCCGTCCTGGGACGACCTCAAGCTCCAGCTCCAGCrCGTGCTCGGCCTCrCCAlGTCC

GGCGTGCCGTTCGTGGGCTGCGACATCGGCGGCTTCCAGGGCCGCAACTTCGCCGAGATC

GACAACTCGATGGACCTCCTCGTGAAGTACTACGCCCTCaCCCTCITCrTCCCGTTCTACC

GC1 '€ CCACAAGGCCACCGACGGCATCGACACCGAGCCGGTGTTCCTCCCGGACTACTAC

AAGGAGAAGGTGAAGGAGATCGTGGAGCTGCGCTACAAGITCCrCCCGTACATCTACTC

CCTCGCCCTCGAGGCCTCCGAGAAGGGCCACCCGGTGATCCGCGCGCrCTTCTACGAGTT ccaggacgacgacgacatgtaccgcatcgaggacgagtacatggtgggcaagtacctcc TCTACGCCCCGATGGCGCCGCCGCCGCCGCCGCCGCCGCCGCCCC

GGTACAACTACTGGAACGGCGAGATCATCAACGGCAAGTCCGTGGTGAAGTCCAC (XAC

GAGCTGCCGATCTACCTCCGCGAGGGCTCCATCATCCCGCTCGAGGGCGACGAGCTGATC

GTGTACGGCGAGACCTOCITCAAGCGCTACGACAACGCCGAGATCACCTCCrccrCCAAC

GAGATCAAGTTCTCCCGCGAGATCTACGTGTGCAAGCrCACCATCACCTCCXSAGAAGCCG

GTGTCCAAGATCATCGTGGACGACTCCAAGGAGATGCAGGTGGAGAAGACCATGCAGAA

CACCTACGTGGCCAAGATCAACCAGAAGATCCGCGGCAAGATCAACCTCGAGTGA

Waxy cDNA sequence (SEQ ID NO: 7) ATGGCGGCTCTGGCCACGTCGCAGCTCGTCGCAACGCGCGCCGGCCTGGGCGTCCCGGA

CGCGTCCACGTTCCGCCGCGGCGCCGCGCAGGGCCTGAGGGGGGCCCGGGCGTCGGOGG

CGGCGGACACX3CTCAGCATGCGGACCAGCGCGCGCGCGGCGCCCAGGCACCAGCACCAG

CAGGCGCGCCGCGGGGCCAGGTTCGCXjTCGCTCGTCXjTGTGCGCCAGCGCCGGCATGAA

CGTCGTCTTCGTCGGCGCCGAGATGGCGCCGTGGAGCAAGACCGGAGGCCTCGGCGACG

TCCTCGGCGGCCTGCCGCCGGCCATGGCCGCGAACGGGCACCGTGTCATGGTCGTCrCTC

CCCGCTACGACXAGTACAAGGACGCCTGGGACACCAGCGTCGTGTCCGAGATCAAGATG

GGAGACGGGTACGAGACGGTCAGGTTCTTCCACTGCTACAAGCGCGGAGTGGACCGCGT

GTTCGTTGACCACCCACTGTTGCTGGAGAGGGTTTGGGGAAAGACCGAGGAGAAGATCT

ACGGGCCTGTCGCTGGAACGGACTACAGGGACAACCAGCTGCGGTTCAGCCTGCTATGC

CAGGCAGCACTTGAAGCTCCAAGGATCCTGAGCCTCAACAACAACCCATACITCTCCGG

ACCATACGGGGAGGACGTCGTGTTCGTCTGCAACGACTGGCACACCGGCCCTCTCTCGTG

CTACCTCAAGAGCAACTACCAGTCCCACGGCATCTACAGGGACGCAAAGACCGCTTTCT

GCATCCACAACATCroCTACCAGGGCGGGTTCGCCTTCTCCGACTACCCGGAGCTGAACC

TCCCCGAGAGATTCAAGTCGTCCITCGA'TTTCATCGACGGCTACGAGAAGCCCGTGGAAG

GCCGGAAGATCAACTGGATGAAGGCCGGGATCCTCGAGGCCGACAGGGTCCTCACCGTC

AGCCCCTÀCTACGCCGAGGAGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTCGACAA

CATCATGCGCCTCACCGGCATCACCGGCATCGTCAACGGCATGGACGTCAGCGAGTGGG

ACCCCAGCAGGGACAAGTACATCGCCGTGAAGTACGACGTGTCGACGGCCGTGGAGGCC

AAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACA

TCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGCCCCGACGTCATGGCG

GCGGCGATCCCGCAGCTCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCAC

GGGCAAGAAGAAGTTCGAGCGCATGCTCATGAGCGCCGAGGAGAAGTTCCCAGGCAAG gtgcgcgccgtggtcaagttcaacgcgggctggcgcaccacatcatggccggcgccga CGTGCTCGCCGTCGAGGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCG

ATACGGAACGCCCTGCGCCTGCGCGTCCACCGGTGGACTCGTCGACACCATCATCGAAG

GCAAGACCGGGTTCCACATGGGCCGCCTCAGCGTCGACTGCAACXjTCGTCGAGCCGGCG

GACGTCA, AGAAGGTGGCCACCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGC

GTACGAGGAGATGGTGAGGAACTGCATGATCCAGGATCTCrCCTGGAAGGGCCCTGCCA

AGAACrGGGAGAACGTGCrGCTCAGCCTCGGGGTCGCCGGCGGCGAGCCAGGGGTTGAA

GGCGAGGAGATCGCGCCGCTCGCCAAGGAGAACGTGGCCGCGCCC

Waxy amino acid sequence (SEQ ID NO: 8) MAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRtlQHQQ

ARRGARFPSLWCASAGMNWFVGAEMAPWSKTOGLGDVLGGLPPAMAANGHRVMWSP

RYDQYKDAWDTSVYSEIKMGDGYETVRFFHCYKRGVDRVFVDHPLFLERVWGKTEEiaYG

PVAGTDYRDNQLRFSLLCQAALEAPRÍLSLNNNPYFSGPYGEDWFVCNDWIITGPLSCYLKS

NY QSI-IGIYRD.AKTAFCIHNISY QGRFAFSDYPELNLPERFKSSFDFIDGYEKPWGRKliNWMK

AGILEADRVLTVSPYYAAELISGIARGCELDNIMRLTGITGR ^ GMDVSEWDPSRDKYIAVKY

DVSTAVEAKALNKEALQAEVGLPVDRNIPLVAHGRLEEQKGPDVMAAAIPQLMEMVEDVQ

rVLLGTGKKKFERMLMSAEEKFPGKVRAVVKENAALAHHIMAGADVLAVTSRFEPCGLIQL

QC ^ IRRYGTPCACASTGGLVDTIJEGKTGFHMGRLSVDCNYVEPADVKKVATTLQRAIKVVGT

PAYEEMVRNCMIQDLSWKGPAKNWENVLLSLGVAGGBPGVEGEEIAPLAKENVAAP 797GL3 / waxy nucleic acid sequence (SEQ ID NO: 9) ATGGCCAAGTACCTGGAGCrGGAGGAGGGCGGCQTGATCATGCAGGCGTTCTACT

CGTCCCGAGCXJGAGGCATCTGGTGGGAGACCATCCGCCAGAAGATCCCCGAGTGGTACG

ACGCCGGCATCTCCGCGATCTGGATACCGCCAGCTTCCAAGGGCATGTCCGGGGGCTACT

CGATGGGCTACXJACCCGTACGACrACTTCGACCTCGGCXiAGTACTACCAGAAGGGCACG

GTGGAGACGCGCTTCGGGTCCAAGCAGGAGCTCATCAACATGATCAACACGGCGCACGC

CTACGGCATCAAGGTCATCGCGGACATCGTGATCAACCACAGGGCCGGCGGCGACCTGG

AGTGGAACCCGTTCGTCGGCGACTACACCTGGACGGACTrCTCCAAGGTCGCCTCCGGCA

AGTACACCGCCAACTACCTCGACTTCCACCCCAACGAGCTGCACGCGGGCGACTCCGGC

ACGTTCGGCGGCTACXXGGACATCiXSCCACGACAAGlXXTGGGACCAGTACTGGCTCTGG

GCCTCGCAGGAGTCCTACGGGGCCTACCTGCGCTCCATCGGCATCGACGCGTGGCGCTTC

GACTACGTCAAGGGCTACX3GGGCCIGGGTGGTCAAGGACrGGCTGAACTGGTGGGGCGG CTGGGCGGTGGGCGAGTACTGGGACACCAACGTCGACGCGCTGCTCAACTGGGCCACACT.

(XrrcCGGCGCGAAGGTGTTCGACITCCCSXTGTACTACAAGATGGACGCGGCCTTCGACA

ACAAGAACATCOCGGCGCTCGTCGAGGCCCTGAAGAACGGCGGCACGGTGGTCnCCCGC

GACCCGTTCAAGGCCGTGACCTTCGTCGCCAACCACGACACGGACATCATCIXJGAACAA

GTACCCGGCGTACGCCrrCATCCTCACCTACGAGGGCCAGCCCACGATCTTCTACCGCGA

CTACGAGGAGTGGCTGAACAAGGACAAGCTCAAGAACCTGATCTGGATrCACGACAACC

TCGCGGGCGGCTCCACTAGTATCGTGTACTACGACTCCGACGAGATGATCTTCGTCCGCA

ACGGCTACGGCTCCAAGCCCGGCCTGATCACGTACATCAACCTGGGCTCCTCCAAGGTGG

GCCGCTGGGTGTACGTCCCGAAGTTCGCCGGCGCGTGCATCCACGAGTACACCGGCAAC

CTCGGCGGCTGGGTGGACAAGTACGTGTACTCCTCCGGCTGGGTCIACCTGGAGGCCCCG

GCCTACGACCCCGCCAACGGCCAGTACGGCTACTCCGTGTGGTCCTACTGCGGCGTCGGC

ACATCGATTGCTGGCATCCTCGAGGCCGACAGGGTCCTCACCGTCAGCCCCTACTACGCC

GAGGAGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTCGACAACATCATGCGCCTCAC cggcatcaccggcatcgtcaacggcatggacgtcagcgagtgggaccccagcagggaca AGTACATCGCCGTGAAGTACGACGTGTCGACGGCCGTGGAGGCCAAGGCGCTGAACAAG GAGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACATCCCGCTGGTGGCGTr CATCGGCAGGCTGGAAGAGCAGAAGGGCCCCGACGTCATGGCGGCCGCCATCCCGCAGC

TCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAAGAAGATC

GAGCGCATGCTCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCGTGGTCAA

GTTCAACGCGGCGCTGGCGCACCACATCATGGCCGGCGCCGACGUGCTCGCCGTCACCA

GCCGCTTCGAGCCCTGCGGCCTCATCCAGCTGCAGGGGATGCGATACGGAACGCCCTGC

GCCTGCGCGTCCACCGGTGGACTCGTCGACACCATCATCGAAGGCAAGACCGGTTCCA

CATGGGCCGCCTCAGCGTCGACTGCAACGTCGTGGAGCCGGCGGACGTCAAGAAGGTGG

CCACCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGAGGAGATGGTG

AGGAACTGCATGATGCAGGATCTCTCCTGGAAGGGCCCTGCCAAGAACTGGGAGAACGT

GGTGCTCAGCCTCGGGGTCGCCGGCGGCGAGCCAGGGGTTGAAGGCGAGGAGATCGCGC

CGCTCGCCAAGGAGAACGTGGCCGCGCCC

797GL3 / waxy Amino Acid Sequence (SEQ ID) NO: 10) MAKYLELEEGGVIMQAFYWDVPSGG1WWDTIRQKIPEWYDAGISAIWIPPASKGMSGGYSM

GYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAUAYGIKVJADIVI ^ HRAGGDLEWNPF

VGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDÍCHDKSWDQYWLWASQES

YAAYLRSIGIDAWREDYVKGYGAWW35D ^ 7LNWWGGWAYGEYWDTNVDALLNWAYSSG

AKVFDFPLYYOtDAAFDNKMPALVEALKNGGTVVSRDPFKAVTFVANHDTOIPiVNKYPAY

AJíILTYEGQPTlFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSrVYYDSDEMIFvTlNGYGSKPG

LITYINLGSSKVGRWVYVPKFAGACIHEYTGNIGGWVDKYVYSSGWVYLEAPAYDPANGQ

YGYSVWSYCGVGTSIAGILEADRVLTVSPYYAEELISGIARGCELDMMRLTGITGIVNGMDV

SEWDPSRDKYIAVKYDVSTAVEAKALNKEALQAEVGI-PVDRNIPLVAFIGRLEEQKGPDVM AAAlPQLMETsFVEDVQrVLLGTGKíCKFERMLMSAEEKFPGKVRAVVKFNAALAIll-GAGVGGVVGVVGVVGVGVVGVGVVGT

KVAm, QRAIKVVGTPAYEBMVRNCMIQDLSWKGPAKNWENVLLSLGVAGGEPGVEGEEIA

PLAKENVAAP

Zea mays ADP-gpp Promoter Nucleic Acid Sequence (SEQ ID NO: 11) GGAGAGCTATGAGACGTATGTCCrCAAAGOCACnTGCATrGTGTGAAACCAATATCGAT

CTTTGTTACTTCAIGATGCATGAACATTTGTGGAAACTACTAGCTTACAAGCATTAGTGA

CAGCTCAGAAAAAAGTTATCTATGAAAGGTTTCATGTGTACCGTGGGAAATGAGAAATG

TrGCCAACTCAAACACCTTCAATATGTTGTTTGCAGGCAAACTCTTCTGGAAGAAAGGTG

TCTAAAACrATGAACGGGTTACAGAAAGGrATAAACCACGGCTGTGCATnTGGAAGTA

TCATCTATAGATGTCTGTTGAGGGGAAAGCCGTACGCCAACGTIATITACTCAGAAACAG

CTTCAACACACAGTTGTCTGCrTTATGATGGCATCrCCACCCAGGCACCCACCATCACCT

ATCrCTCGTGCCTGTTTATTTTCITGCCCTrTCTGATCATAAAAAACATTAAGAGTlTGC

AAACATGCATAGGCATATC AATATGCTCATTTATT ATTTGCT AGC AGATC ATCTTCCTAC

TCTITACTTTATTTATTGTTTGAGAAAATATGTCGTGCACCTAGGGAGCTCGTATACAGTAC

CAATGCATCTTCACTAAATGTGAAllTCAGAAAGGAAGTAGGAACCTATGAGAGTATTTT

TCAAAATTAATTAGCGGCrrCTAlTATGTlTATAGCAAAGGCCAAGGGCAAAATTGGAAC

ACrAATGATGGTrGGTTGCATGAGTCTGTCGATTACrTGCAAGAAATGTQÂACCTITGTT

TCTGTGCGTGGGCATAIVACACAAACAGCTTCTAGCCTCTTTTACGGTACTTGCACTTGCAA

GAAATGTGAACTCCTTITCATTTCTGTATGTGGACATAATGCCAAAGCATCCAGGCTITTT

CATGGTTGTTGAIGTCm'ACACAGTTCATCTCCACCAGTATGCCCTCCTCATACTCTATA

TAAACACATCAACAGCATCGCAATTAGCCACAAGATCACTTCGGGAGGCAAGTGCGATT tgttagtttaatttgtaattgggaagtattagtggaaagaggatgagatgctatcatcta TGTACTCTGCAAATGCATCTGACGTTATATGGATACTGCATATGCATA

TCTTGCCGACAATATATTGCAAGGTATATGCXTAGTTCCATCAAAAGTrCTGTTriTrCAT

TCTAAAAGCATTTTAGTGGCACACAATrTTTGTCCATGAGGGAAAGGAAATCTGTlTTGG

TrACTTTGCTTGAGGTGCATTCTTCATATGTCCAGTTTTÀTGGAAGTAATAAACTTCAGTT

TGGTCATAAGATGTCATATTAAAGGGCAAACATATATTCAATGTTCAATTCATCGTAAAT

GTTCCCTITTTGTAAAAGATTGCATACTCATTTATITGAGTTGCAGGTGTATCTAGTAGTT

GGAGGAG

Zea mays γ-zein promoter nucleic acid sequence (SEQ ID NO: 12) GATCATCCAGGTGCAACCGTATAAGTCCTAAAGTGGTGAGGAACACGAAACAACCATGC

À'ITGGCATGTAAAGCTCCAAGAATTTGTTGTATCCrTAACAACTCACÀGAACATCAACCA

AAATTGCACGTCAAGGGTATTGGGTAAGAAACAATCAAACAAATCCTCTCTGTGTGCAA

AGAAACACGGTGAGTCATGCCGAGATCATACTCATCTGATATACATGCTTÂCAGCTCACA

AGACATTACAAACAACTCATATTGCATrACAAAGATCXjTTrCATGAAAATAAAATAGG

CCGGACAGGACAAAAATCCITGACGTGTAAAGTAAATITACAACAAAAAAAAGCCATA

TGTCAAGCTAAATCrÀATTCGlTTTACGTAGATCAACAACCTGTAGAAGGC / VACAAAACT

GAGCCACGCAGAAGTACAGAATGATTCCAGATGAACCATCGACXjTGCTACGTAAAGAGA

GTGACGAGTCATATACATTTGGCAAGAAACCATGAAGCTGCCTACAGCCGTCTCGGTGG

CATAAGAACACAAGAAATTGTGTTAATTAATCAAAGCTATAAATAACGCTCGCATGCCT

GTGCACITCrCCATCACCACXACTGGGTCrrCAGACCATTAGCTTTATCTACrCCAGAGCG

CAGAAGAACCCGATCGACA

Amylase Fusion Amino Acid Sequence pNOV6200 (SEQ ID NO: 13) MRVLLVALALLALAASATSAKYLELBEGGVMQAFYWDVPSGGIWWDTIRQKIPEWDAGI

SAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELMMINTAHAYGIKVI

ADIV1NHRAGGDLBWNPFVGDYTWTDFSKVASGKYTANYLDPHPNELHAGDSGTFGGYPDI

CHDKSWDQYWLWASQESYAAYLRSIGIDWRFDYVKGYGAWVYKDWLN \ VWGGWAVGE

YWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTVVSRGPFKAV tfvanhdtdhwnkypayafiltyegqptifyrdybewlnkdklknliwihdnlaggstsivy YDSDEMIFA ^GYLVYYYY

SGWVYLEAPAYDPANGQYGYSVWSYCGVG

Amylase Fusion Amino Acid Sequence pNOV6201 (SEQ ID NO: 14) mrvllvalallalaasatsakyleleeggvimqafywdvpsggiwwdtirqkipewydagi SAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVELRGINETKYKHAKY

ADIVINIIRAGGDLEWNPFVGDYTWTOFSKVASGKYTANYLDFHPNELHAGDSGTFGGYPDI

CHDKSWDQYWLWASQESYAAYLRSIGIDAWREDYVKGYGAWVVKDWLNWWGGWAVGE

YWDTNVDALLNWAYSSGAKVFDEPLYYKMDAAFDNKNIPALVEALKNGGTVVSRDPFKAV

TFVANHDTDnWNKYPAYAHLTYEGQPTlFYRDYEEWI.NKDKLKKLIWIHDNLAGGSTSIVY

YDSDEMIFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYS

SGWV YLEAPAYDPANGQYGYS VWS Y CGV GSEKDEL

Sequence pNOV4029 amylase fusion amino acid sequence (SEQ ID NO: 15) MLAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRHQHQ qarrgarfpslwcasagamakyleleeggvimqafywdvpsggiwwdtirqkipewyga GISAIWIPPASKGMSGGYSMGYDPYDYEDLGEYYQKGTVETi ^ FGSKQELINMINTAHAYGIK

VIADIVINHRAGGDLEWNPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYP

DICHDKSWDQYWLWASQESYAAYLRSIGTOAWRFDYVKGYGAWWKDWLNWWGGWAV geywdtnvdallnwayssgakvfdfplyykmdaafdnknipalvealknggtwsrdpfk avtfvam-idtdiiwnkypayapiltyegqptifyrdyeewlnkdklknliwihdnlaggstsi vyydsdemifvrngygskpglityinlgsskvgrwvyvpkfagacihbytgnlggwvdkyv yssgwvyleapaydpangqygysvwsycgvgtsi amylase fusion amino acid sequence of pNOV4031 (SEQ ID NO: 16) MLAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAADTLSMRTSARAAPRHQHQ QARRGARFPSLVVCASAGAMAKYLELEEGGVIMQAFyWDVPSGGIWWDTIRQKIPEWYDA GIS AIWIPPASKGMSGGY SMGYDPYD YFDLGE Y YQKGTVETOFGSKQELINMÍNT AHAY GIK VIADIV1NHRAGGDLEWMPFVGDYTWTDFSKVASGKYTANYLDFHPNELHAGDSGTFGGYP DICHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWWKDWLNWWGGWAV geywdtnvdallnwayssgakvfdfplyykmdaafdnknipalvealknggtvvsrdpfk AVIFVAWIl> roilWNKYPAYAITLTYEGQPTEFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSI

VYYDSDEMIFVRNGYGSKPGL1TYINI.GSSKVGRWVYVPKFAGACIHEYTGNLGGWDKYV

YSSGWVYLEAPAYDPANGQYGYSVWSYCGVGTSIAGILEADRVLTVSPYYAEELISGIARGC

ELDNIMRLTGITGIVNGMDVSEWDPSRDKYIAVKYDVSTAVEAKALNKEALQAEVGLPVDR

NIPLVAFIGRLEEQKGPDVMAAAIPQLMEMVEDVQIVLLGTGKKKEERMLMSAEEKPPGKVR

AVVKFNAALAHHIMAGADVLAVTSRFBPCGLIQLQGMRYGTPCACASTGGLVDTIIEGKTGF

HMGRLSVDCNWEPADVKKVATTLQRAIKVVGTPAYEEMVRNCMIQDLSWKGPAKNWEN

VLLSLGVAGGEPGVEGEEI

APLAKENVAAP

Maize γ-zein N-terminal Signal Sequence (SEQ ID NO: 17) (MRVLLVALALLALAASATS) Thermotoga maritima Glucose Isomerase Amino Acid Sequence (SEQ ID NO: 18) MAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFNTFVFWRT

AERPWNRFSDPMDKAFARVDALFEFCEKLmYFCmDRDlAPEGKTLRETNIGJ.DKVVTiRM

ERMKDSNVKLLWGTANLFSHPRYlvn-IGAATTCSADVFAYAAAQVKKALEITKELGGEGYVP

WGGREGYETLLNTDLGLELENLARELRMAVEYAKKIGFTGQELIÊPICPKEPTKHQYDFDVAT

AYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGTOTDQ

FPTNIYDTTLAMYEVIKAGGFTKGGLNFDAKVKRASYKVEDLF1GHIAGMDTFALGFKIAYK

LAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKEDIELPSGKQEYLESLLNSYIV

KTIAELR

Nucleic acid sequence optimized for Thermotoga maritima glucose isomerase maize (SEQ ID NO: 19)

ACCACCTCAAGTTCTCCGTGGCCrTCrGGCACACCTTCGTGAACGAGGGCXXJCGACCCGT

TCGGCGACCCGACCGCCGAGCGCCCGTGGAACCGCrrCTCCGACCCGATGGACAAGGCC

TTCGCCCGCGTGGACGCCXTCTTCGAGTTCTGCGAGAAGCTCAACATCGAGTACTTCTGC rrCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCr CGACAAGGTGGTGAGAGGAGCAGCAGCAGCAGCAGCAGTAGG

GGGGCACCGCCAACCTCrrcrcCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCT

CCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAG GAGCrGGGCGGCGAGGGCTACGTGTTCrGGGGCGGCCGCGAGGGCTACGAGACCCTCCr CAACACCCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCG

GTACGCCAAGAAGATCGGCrrCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGC

CGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCCTACGCCITCCTCAAGAACCAC

GGCCTCGAC

GAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAG

CACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGG

CGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACATCTACGACACCACGCT

CGCCATGTACGAGGTGATCAAGGCCGGCGGCrTCACCAAGGGCGGCCTCAACITCGACG

CCAAGGTGCGCCGCGCCTCCTACAAGGTGGAGGACCTCTTCAIGGGCCACATCGCCGGC

ATGGACACCTTCGCCCTCGGCrrCAAGATCGCCTACAAGCTCGCCAAGGACGGCGTGTTC

GACAAGTTCATCGAGGAGAAGTACCGCTCCmCAAGGAGGGCATCGGCAAGGAGATCGT

GGAGGGCAAGACCGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGGACATC

GAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCCTCAACTCCTACATCGTGAAG

ACCATCGCCGAGCTGCGCTGA

Thermotoga neapolitan glucose isomerase amino acid sequence (SEQ ID NO: 20) MAEEmaPKVQFEGKESTNPLAFKFYDPBEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPT

ADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNIGLDKVVBRI

KERMKDSNVKLLWGTANLFSKPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYV

FWGGREGYEIXvLNTDLGFELENLARFLRMAVDYAKRIGFTGQFLIEPKPKEPTKHQYDFDVA

TAYAI ^ KSHGLDEYPKFNffiANHATLAGim? QHELRMAREJGKLGSD [) ANQGDLLLGWDTD QFPTNVYDYTLAMYEVIKAGGFTKGGLNFDAKVRRASYICVEDLFLYYGYKYKYKYKYKYKYKKYKYKKYKYKYKYKYKKYKYKYKYKYKKY

IVKTILELR

Optimized Nucleic Acid Sequence for Neapolitan Thermotoga Glucose Isomerase (SEQ ID NO: 21)

ACCACCTCAAGITCTCCGTGGOCTTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGT

TCGGCX3ACCCGA (XGCCGACCGCCX; GTGGAACCGC1‘ACACCGACCCGATGGACAAGGCC

TTCGCCCGCGTGGACGCCCTCrrCGAGTTCTGCGAGAAGCrcAACATCGAGTACTrCTGC

TrCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCT

CGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCT

GGGGCACCGCCAACCrCTTCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCT

CCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAG

GAGCTGGGCGGCGAGGGCrACGTGTrCrGGGGCGGCCGCGAGGGCTACGAGACCCTCCT

CAACACCGACCTCGGCTTCGAGCTGGAGAACCTCGCCCGdTCCTCCGCATGGCCGTGGA

CTACGCCAAGCGCATCGGCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGAGC

CGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCCTACGCCTTCCTCAAGTCCCACG

GCCTCGACGAGTACTTCAAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCAC

ACCrrcCAGCACGAGCTGCGCATGGCCCGCATCCTCGGCAAGCrCGGCTCCATCGACGCC

AACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCAGTTCCCGACCAACGTGTACGA

CACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCA

ACTTCGACGCCAAGGTGCGCCGCGCCTCXTACAAGGTGGAGGACCTCITCATCGGCCACA

TCGCCGGCATGGACACCTTCGCCCTCGGCTTCAAGGTGGCCTACAAGCTCGTGAAGGACG

GCGTGCTCGACAAGTTCATCGAGGAGAAGTACCGCTCCTTCCGCGAGGGCATCGGCCGC

GACATCGTGGAGGGCAAGGTGGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGG

AGACCATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTGGAGTCCCTCATCAACTCCTAC

ATGGTGAAGACCATCCTGGAGCTGCGCTGA SV57 (SEQ ID NO: 22) (S ^ AGCGÂATTCATGGCGGCTCTGOCCACGT 3f) SV58 (SEQ ID NO: 23) (5'AGCTAAGCrTCAGGGCGCGGOCACGTTCT 35) gGP3 fusion amino acid sequence of pNOV7005 (SEQ ID NO: 24) MRVLLVALALLALAASATSAGHWYÍCHQRAYQFTGEDDFGKVAVVKLPMDLTKVGIIVRLN

EWQAICDVAKDRFIEIKDGKAEVWTLQGVEElFYEKPDTSPRIFFAQARSNKVIEAFLmPVDT

KKXELPKVTVDGKEIPVSR ^ KADPTDIDVTNYVRIVLSESLKEEDLRIEDVELIIEGYKPARVI

MMEELDD Y Y YDGELGAV YSPEKTIFRV WSPV SKWVKVLLFKNGEDTEPY QV NMEYKGN

VWEAVVEGDLDGVFYLYQLENYGKIRTTVDPYSKAVYANNQESAVVNLARTNPEGWENDR

GPKIEGYEDAnYEIHIADrrGLENSGVKNKGLYLGLTEENTKAPGG \ TTGLSHLVEX5VTMVH

R.PFFDFYTGDELDKDFEKYYNW GYDPYLFMVPEGRY STDPKNPHTR1RE VKEMVKALHKH GIGVTMDMVPKITYGIGEI-SAFDQTVPYYFYRIDKTGAYLNESGCGNVIASBRPMMRICFÍVDT: VTYWVKEYHIDGFRFDQMGUDKKTMI.EVERALHKlDPTIILYGEPWGGWGAPmFGKSDVA GTHVAAFNDEFRDAJRGSVFNPSVKGPVMGGYGKETKIKRGVVGSINYDGKLIKSFALDPEE · ^ TJMYAAaTONmWDKNYIAAKADKKKE TEBELKNAQKLAGAIlJ TSQGVPFLHGGQDF CRTTNFNDN-S YN APISIN GFD YERKLQFID VFN YHICGLEELRKEHPAFRLKNAEEIKJKHLEFl GGRÍGVAFMLKDÍIAGGDIWKDlVVIYNGNLEKTrYKÍ.PEGKWN \ rWNSQKAGTEVIETVEG

TIELDPLSAYVLYRESBKDEL

Optimized Nucleic Acid Sequence for Fusion 6GP3 pNOV7005 (SEQ ID NO: 25) ATGAGGGTGTTGCTCGTTGCCCrCGCTCTCCTGGCTCTCGCTGCGAGCGCCACCAGCGCT

GGCCACTGGTACAAGCACCAGCGCGCCTACCAGTTCACCGGCGAGGACGACTTCGGGAA

GGTGGCCGTGGTGAAGCTCCCGATGGACCTCACCAAGGTGGGCATCATCGrGa3CCrCA

ACGAGTGGCAGGCGAAGGACGTGGCCAAGGACCGCTTCATCGAGATCAAGGACGGCAA

GGCCGAGGTGTGGATACTCCAGGGCGTGGAGGAGATCTTCTACGAGAAGCCGGACACCT

ACCORGCGCATCITCTTCGCCCAGGCCCGCTCCAACAAGGTGATCGAGGCCTTCCTCACCA

ACCCGGTGGACACCAAGAAGAAGGAGCTGTTCAAGGTGACCGTCGACGGCAAGGAGAT

CCCGGTGYCCCGCGTGGAGAAGGCCGACCCGACCGACATCGACGTGACCAACTACGTGC

GCATCGTGCrCTCCGAGTCCCTCAAGGAGGAGGACCTCCGCAAGGACGTGGAGCTGATC

ATCGAGGGCTACAAGCCGGCCCGCGTGATCATGATGGAGATCCTCGACGACTACTACTA

CGACGGCGAGCTGGGGGCGGTGTACTCCCCGGAGAAGACCATCrrCCGCGTGTGGTCCC

CGGTGTCCAAGTGGGTGAAGGTGCTCCrCTTCAAGAACGG € GAGGACACCGAG (XGTAC

CAGGTGGTGAACATGGAGTACAAGGGCAACGGCGTGTGGGAGGCCGTGGTGGAGGGCG

ACCTCGACGGCGTGTTCTACCTCTACCAGCTGGAGAACTACGGCAAGATCCGCACCACCG

TGGACCCGTACTCCAAGGCCGTGTACGCCAACAACCAGGAGTCTGCAGTGGTGAACCTC

GCCCGCACCAACCCGGAGGGCTGGGAGAACGACCGCGGCCCGAAGATCGAGGGCTACG

AGGACGCCATCATCTACGAGATCCACA'1'CGCCGACATCACCGGCCTGGAGAACTCCGGC

GTGAAGAACAAGGGCCrCTACCTCGGCCTCACCGAGGAGAACACCAAGGCCCCGGGCGG

CGTGACCACCGGCCTCTCCCACCTCGTGGAGCTGGGCGTGACCCACGTGCACATCCTCCC gttcttcgacttctacaccggcgacgagctggacaaggacttcgagaagtactacaactg gggctacgacccgtacctcttcatgütgccggagggccgctactccacggacccgaaga acccgcacacccgaattcgcgaggtgaaggagatggtgaaggccctccacaagcacggc ATCGGCGTCATCATGGACATGGTGTnXCGCACACCTACGGCATCGGCGAGCTGTCCGCC

11'CGACCAGACCGTGC € GTACTACTTCTACCGCATCGACAAGACCGGCGCCTACCTCA \ C

GAGTCCGGCTGCGGCAACGTGATCGCCrCCGAGCGCCCGATGATGCGCAAGTTCATCGT

GGACACCGTGACCTACTGGGTGAAGGAGTACCACATCGACGGCTTCCGdTCGACCAGA

TGGGCCTCATCGACAÂGAAGACGATGCrGGAGGTGGAGCGCGCCCTCCACAAGATCGAC

CCGACCATCATCCTCTÀCGGCGAGCCGTGGGGCGGCTGGGGGGCCCCGATCCGCTTCGG

CAAGTCCGACGTGGCCGGCACCCACGTGGCCGCCrrCAACGACGAGTTCCGCGACGCGA

TCCGCGGCTCCGTGTTCAACCGGTCCGTGAAGGGCTTCGTGATGGGCGGCTÀCGGCAAGG

AGACCAAGATCAAGCGCGGCGTGGTGGG € T <XATCAACTACGACGGCAAGCTCATCAAG

TCCTTCGCCCTCGACXXGGAGGAGACCATCAACrACGCCGCCTGCCACGACAACCACACC

CTCTGGGACAAGAACrACCTCGCCGCC

AAGGCCGACAAGAAGAAGGAGTGGACCGAGGAGGAGCTGAAGAACGCCCAGAAGCTCG

CCGGCGCCATCCTCCTCACTAGTCAGGGCGTGCCGTTCCTCCACGGCGGCCAGGACTTCT

GGCGCACCACCAACTTCAACGACAACTCCTACAACGCCCCGATCTCCATCAACGGCrrCG

ACTACGAGCGCAAGCTCCAGTTCATCGACGTGTTCAACTACCACAAGGGCCTCATCAAGC

TCCGCAAGGAGCACCXGGCCTTCCGCCTCAAGAACGCCGAGGAGATCAAGAAGCACCTG

GAGTTCCrCCCGGGCGGGCGCCGCATCGTGGCCTTCATGCTCAAGGACCACGCCGGCGG

CGACX ^ CGTGGAAGGACÀTCGTGGTGATCTACAACGGCAACCTGGAGAAGACCACCTACA

AGCTCCCGGAGGGCAAGTGGAACGtGGTGGTGAACTCCCAGAAGGCCGGCACCGAGGTG atcgagaccgtggagggcaccatcgagctggacccgctctccgcctacgtgctctaccgc GAGTCCGAGGAGG

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

QQGNKVIVEKSLDLKEI-fflGLGEKAFELDRKRKRYVMYNVDAGAyKICYQDPLYVSIPLFISV

KDGVATGYEFNSASKVIFDVGLEEYDKVIVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPP

MWAFGYMISRYSYYPQDKVVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPK

KLIDELHKRNVKLrnVDHGIRVDQNYSPFLSGMGKPCBlESGELFVGKMWPGTTVYPDFFRE

DTREWWAGLISEWLSQGVDGrvSTLDMNEPTDPSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVV

HYLRGKRVKHEKVRNAYPLYBAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPS

WDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKA

TDGEDTEPVHJPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRI

EDEYMVGKYLLYAPIVSKEESRI-VTLPRGKWYNYWNGEIINGKSWKSTHBLPIYLRBGSIEP

LEGDELIVYGETSFKRYDNAErrSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQ

OTYVAKINQIvIRGKINLESEKDEL

Fusion amino acid sequence malA pNOV4839 (SEQ ID NO: 27) MRVLLVALALLALAASATSMETEKIYENKGVYKVVIGEPFPPIEFPLEQK1SSNKSLSELGLTIV

QQGNKVIVEKSLDLKEHnGLGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISV

KPGVATGYFFNSASKVlFDVGLEEYDKVIVTBPEDSVEFYVIEGPRIEDVLEiCYTELTGKPFLPP

MWAFGYMISRYSYYPQDKWELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPK

KLlDELHKRKVKLtTIVDHGIRVDQNYSPFLSGMGKFCEIESGELFVGKAPiYPGTTVYPDFFRE

DTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDViLSSLPVQFRDDRLVTTFPDNVV

HYLRGKRVKHEKVRKAYPLYEAMATFKGFRTSHRNEIPILSRAGYAGIQRYAFIWTGDNTPS

WDDLKLQLQLVLGLSISGVPFVGCBIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKA

TDGIDIEPVHJPDYYKEKVKErVELRYKFLPYIYSLALEASBKGHPVIRPLFYEFQDDDDMYRI

EDEYM V GKYLLY APIVSKEESRLVTLPRGKWYNYWNGBnNGKS WKSTHELPIYLREGSHP

LEGDELÍVYGEISFKRYDNAEITSSSNEIKFSREIYVSKLTITSEKPVSKHVDDSKEIQVEKTMQ

NTYVAKINQKIRGK1NLE

Sequence of pNOV4832 glucose isomerase fusion amino acid sequence (SEQ ID NO: 28) MRVIXVALALLALAASATSMAEFFPEIPKIQFEGKESTNPLAFRFYDPNEV1DGKI3LKDHLKFS vafwhtfvnegrdpfgdptaerpwkrfsdpmdkapartoalfefceklnieyfcfhdrdiap EGKTLRETNKILDKVVHRIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAA

QVKKALElTKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIGFrGQF

LIEPKPKEPTKHQYDFDVATAYAFLKNHGLOEYFKFNIEANHATLAGHTFQHELRMARILGK

LGSID AN QGDUXGWDITXJFPTMY DTTLAMYEVIKAGGFTKGGLMFDAKVRRAS YK VEDL

FIGIEAGMDTFALGFKXAYKLAKDGVEDKHEEKYRSFKEGIGKEIVEGKTDFBKLEEYIIDKE

DBBLPSGKQEYLESLLNSYIVKTIAELRSEKDEL

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

VAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCBKLNIEYFOFHDRDIA

PEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAA

AQVKKALEITKELGGEGYVFWGGREGYETLLMTDLGFBLEMLARFLRMAVDYAKRIGFTGQ

H, IEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILG

ÍCLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFrKGGLNPmKVRRASYKVE

DLFIGHIAGMDTFALGFKVAYKLVKDGVLDICFIEEKYRSFREGIGRDIVEGKVDFBKI ^ EEYIID

KETIELPS GKQEYL & SLlNSYrV KTELELRSEKDEL

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

VAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDALFEFCBKLNIEYFCFHDRDJA

PEGKTLRETNKILPKWERIICERMKDSNVKLLWGTANLFSHPRYlvniGAAITCSADVFAYAA

AQVKXALElTKELGGEGYVFWGGREGYETLLNTDLGFiLENLARFLRMAVDYAKRIGFTGQ

FLIEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQIIEI-.RMARILG

KLGSIDANQGDLLLGWiyi'DQFPTISÍVYDTTLAMYEVIKAGGFrKGGLNFDAK.VRRASYKVE

DLFIGHÍAGMDTFALGFKVAYKLVKDGVLDKEIEEKYRSFREGIGRDIVEGKVDFEKLEEY1ID

KETIELPSGKQEYLESL1N SY ÍVKT1LELR AMY32b-sequence signal α barley amylase (SEQ ID NO: 31) (MGKNGNLCCFSLLLLLLAGLASGHQ) PR1a Sequence Signal (SEQ ID NO: 32) (MGFVLFSQLPSFLLVSnXLFLVISHSCRA) 797GL3 fusion (SEQ ID NO: 33) MRVLLVALALLALAASATSAKYLELEKGGVIMQAFYWDVPSGGimVDmQKIPEWYDAGI S AIWIPPASKGMSGGYSMGYDP YD YFDLGEY YQKGTVETRFGSKQELMMINTAHAY GltCVI ADIVINHRAGGDLEWNPFVGDY1 "WIDFSKVASGKYTANYLDFHP1S0EI-HAGDSGTPGGYPDI CHDKSWDQYWLWASQESYAAYLRSIGIDAWRFDYVKGYGAWWKDWLNWWGGWAVGE YWDTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTWSRDPFKAV TFV ANHDTDIIWNKYPAY AFILTYEGQPTIFYRDYEEWLNKDKLKNLIWIHDNLAGGSTSIV Y YDSDEMBFVRNGYGSKPGLITYINLGSSKVGRWVYVPKFAGACIHEYTGNLGGWVDKYVYS SGWVYLEAPAYDPANGQYGYSYWSYCGVGSEKDEL

6GP3 Fusion (SEQ ID NO: 34) MRVLLVALALLALAASATSAGHWYKHQRAYQPTGEDDFGKVAWKLPMDLTKVGHVRLN

EWQAKDVAKDRHErKDGKAEV ^ TLQGVEEIPYEKPDlBPRJFFAQARSNKVlEAFLTNPVDT

KÍGG2LEKVTVDGKE1PV SRVEKADPTDEDVTNYVRIVLSESLKEEDLRKJD VELEIEG YKP ARVI

MMEILDDYYYDGELGAVYSPEKTaFRVWSPVSKWVKVIXFKNGEDTEPYQVVNMEYKGNG

YWEAYVEGDEDGVFYLYQLENYGKIRITVDPYSKAVYANNQESAVVNLARTNPEGWENDR

GPKIEGYEDAnYErfflADrTGLENSGVKNKGLYLGLTEENTKAPGGVTTGLSHLVELGVTHVH

ILPFFDFYrGDELDKDFEKYYNWGYDPYLFMVPEGRYSTDPKNPHTRIREVKEMVKALHKH

GIGVIMDMVFPHTYGIGELSAFDQTVPYYFYRIDKTGAYLNESGCXSNVIASERPMMRKFIVDT

VTYWVKEYHIDGFRFDQMGLIDKKTMLEVERALHKIDPTnLYGEPWGGWGAPIRFGKSDVA GTHVAAFNDEFRDAIRGSVFNPSVKGFVMGGYGKETKIKRGWGSINYDGKLIKSFALDP ^ ^ TINYAACHDNHTLWDKNYLAAKADKKKEWTEEELKNAQKI AGATLLTSQGVPFLHGGQDF

CRTTNFNDNSYNAPISINGFDYERKLQFIDVFNYHKGLIKLRKEIIPAFRLKNAEEIKKHLEFLP

GGRRWAIMLKDHAGGDPWKDIVVrYNGNLEKTIYKLPEGKWNVVVNSQKAGTEVIEWEG

TIELDPLSAYVLYRESEKDEL

Fusion 797GL3 (SEQ ID NO: 35) MRVLLVALALLALAASATSAKYLELEEGGVIMQAFYWDWSGGIWWDTIRQKIPEWYDAGI

SAIWIPPASKGMSGGYSMGYDPYDYFDLGEYYQKGTVETRFGSKQELINMINTAHAYGIKVI

ADmNHRAGGDLEWNPWGDYTWTDFSKYASGKYTANYLDFHPNELHAGDSGTFGGYPDI

CHDKSWDQY ^ T-WASQESYAAYLRSIGIDAWRPDYVKGYGAWV VKDWLíWWGGWAVGE

YVsTtTNVDALLNWAYSSGAKVFDFPLYYKMDAAFDNKNIPALVEALKNGGTWSRDPFKAV

TFVANHDTDHWNKYPAYAHLTYEGQPTIFYRDYEEW ^ LNKDKLKNLIWIHDNLAGGSTSIVY

YDSDEMIFVRNGYGSKPGLITYWLGSSKVGRWVYWKFAGACIHEYTGNLGGWDKYVYS

SGWVYLEAPAYDPANGQYGYSVWSYCGVGSEKDEL

FusionA (SEQ ID NO: 36) MRVLLVALALLALAASATSMETIKIYENKGVYKVVIGEPFPPIEFPLEQKJSSNKSLSELGLTW

QQGNKVIVEKSLDLKEHIIGLGEKAFELDRKRKRYVMYKVDAGAYKKYQDPLYVSIPLFISV

KDGVATGYFFNSASKVIFDVGLEEYDKVrVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPP

MWAFGYMISRYSYYPQDKWELVDMQKEGFRVAGVFLDIHYMDSYKI.FTWHPYRFPEPK

KLIDELHKRNVKLrnVDHGIRVDQNYSPFLSGMGKFCBIESGELFVGKMWPGITVYPDEFRE

DTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTHPDN \ fV

HYLRGKRVICHEICVRNAYPLYEAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPS

WDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRJSDFAEIDNSMDLLVKYYALALFFPFYRSHKA

TDGDYTEPVFLPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRI

EDEYMVGKYLLYAPIVSKEESRLVTLPRGKWYNYWNGEIINGKSVVKSTHELPIYLREGSnP

LEGDELrVYGETSFKRYDNAErrSSSNEIKFSREIYVSKLTrrSEKPVSiOIVDDSKEIQVEKTMQ

NT YV AKINQK3RGKMLESEKDBL

Glucose Isomerase Fusion Nucleotide Sequence pNOV4829 (SEQ ID NO: 37) ATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGOQ

TACCCTGGTGCCACGCGGTTCCATGGCCGAGTTCTTCCCGGAGATCCCGAAGATCCAGTT

CGAGGGCAAGGAGTCCACCAACCCGCTCGCCTTCCGCTTCrACGACCCGAACGAGGTGA

TCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCCGTGGCCTTCTGGCACACCTTCG

TGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGAGCGCCCGTGGAACCGCITC

TCCGACCCGATGGACAAGGGCTTCGCCCGCGTGGACGCCCTCTTCGAGTTCTGCGAGAAG

CTCAACATCGAGTACTTCTGCITCCACGACCGCGACATCGCCCCGGAGGGCAAGACCCTC

CGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCGCATCAAGGAGCGCATGAAGG

ACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATGC

ACGGCGXGCCACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAG

AAGGCCCTGGAGATCACCAAGGAGCrGGGCGGCGAGGGCTACGTGrTCTGGGGCGGCCG

CGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCCrGGAGCTGGAGAACCTCGCCC

GCTTCCTCCGCATGGCCGTGGAGTACGCCAAGAAGATCGGCTTCACCGGCCAGTTCCTCA

TCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGACTTCGACGTGGCCACCGCC

TACGCCTTCCTCAAGAACCACGGCCTCGACGAGTACTTCAAGTrCAACATGGAGGCrAAC

CACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGCATGGCCCGCATCCTCGGC

AAGCTCGGCTCCATCGACXSCCAACCAGGGCGACCrCCTCCrCGGCTGGGACACXGACCA

GTTCCCGACCAACATCTACGACACCAXEXCGCCATGTACGAGGTGATCAAGGCCGGCG

GCTTCACCAAGGGCGGCCTCAACITCGACGCCAAGGTGCGCCGCGCCrCCTACAAGGTG

GAGGACCTCTTCATCXKKXACATCGCCGGCATGGACACCITCGCCCTCGGCITCAAGATC

GCCTACAAGCTCGCCAAGGACGGCGTGTTCGACAAGTTCATCGAGGAGAAGTACCGCrC

CTTCAAGGAGGGCATCGGCAAGGAGATCGTGGAGGGCAAGACCGACTTCGAGAAGCTG

GAGGAGTACATCATCGACAAGGAGGAGATCGAGCTGCCGTCCGGCAAGCAGGAGTACCT

GGAGTCCCTCCTCAACrCCTACATCGTGAAGACCATCGCCGAGCTGCGCTCCGAGAAGG

ACGAGCTGTGA

Sequence of pNOV4829 glucose isomerase fusion amino acid sequence (SEQ ID NO: 38) MKBTAAAKHERQHMDSPDLGTLVPRGSMABFPPEIPKIQFBGKESTNPLAFRFYDPNEVIDGK PLKDHLKFSVAFWHTFVNEGRDPPGDPTAERPWNRFSDPMDKAFARVDALFEFCEIvLNIBYI 'CFHDRDIAPEGKTLRETNmDKWERIKERIvlKDSNVKLLWGTANLFSHPRYMHGAATTCS

ADVFAYAAAQVKKALErrKELGGEGYVFWGGREGYETLLNTDLGLELENLARELRMAWY

AKXIGFTGQFLIEPKPKHPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQH elrmarilgklgsidafiqgdlllgwdtdqfptniydttlamyevikaggftkgglnfdakv RRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDF EKLEEYIIDKEDIELPSGKQEY1.ES LLNSYIVKTIAELRSEKDEL

Glucose Isomerase Fusion Nucleotide Sequence pNOV4830 (SEQ ID NO: 39) ATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGG

TACCCTGGTGCCACGCGGTrCCATGGCCGAGTTCTrCCCGGAGATCCCGAAGGTGCAGTT

CX3AGGGCAAGGAGTGCAjCCAACCCGCTCGCCITCAAGTTCTACGACCCGGAGGAGCA

TCGACGGCAAGCCGCrCAAGGACXACCTCAAGTTCTCCGTGGCCrrCrGGCACACCITCG

TGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCCGACCGCCCGTGGAACCGCTAC

ACCGACCCGATGGACAAGGCCrrCGCCCGCGTGGACGCCCTCrrCGAGITCTGCGAGAA GCTCAACATCGAGTACTTCrGCTTCCACGACXrGCGACATCGCCCCGGAGGGCAAGACCCr CCGCGAGACCAACAAGATGGAGCAGGAGCAG

GACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCTTCTCCCACCCGCGCTACATG

CÀCGGCGCCGCGACCACCTGCTCCGCCGACGTGTTCGCCTACGCCGCCGCCCAGGTGAAG

AAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGGCTACGTGTTCTGGGGCGGCCG

CGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCTTCGAGCTGGAGAACCTCGCCC

GCTTCCTCCGCATGGCCGTGGACTACGCCAAGCGCATCGGCTCACCGGCCAGTTCCTCA

TCGAGCCGAAGCCGAAGGAGCCGACCAAGCACCAGTACGACTrCGACGTGGCCACCGCC

TACGCCTrCCrCAAGTCCCACGGCCTCGACGAGTACTTCAAGfTTCAACATCGAGGCCAAC

CACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTGCGGATGGCCCGCATCCrCGGC

AAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTCCTCGGCTGGGACACCGACCA

GTTCCCGACCAACGTGTACGACACCACCCTCGCCATGTACGAGGTGATCAAGGCCGGCG

GCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCGCCGCGCCTCCTACAAGGTG

GAGGACCXCTTCATCGGCCACATCGCCGGCATGGACACCTTCGCCCrCGGCTTCAAGGTG

GCCTACAAGCTCGTGAAGGACGGCGTGCTCGACAAGTTCATCGAGGAGAAGTACCGCTC

CTTCCGCGAGGGCATCGGCCGCGACATCGTGGAGGGCAAGGTGGACTrcGAGAAGCTGG

AGGAGTACATCATCGACAAGGAGACCATCGAGCTGCCGTCCGGCAAGCAGGAGTACCTG

GAGTCCCTCATCAACr <XTACATCGTGAAGACCATCd’GGAGCTGCGCTCCGAGAAGGAC

GAGCTGTGA

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

PLKDHLKFSYAFWHTFVNEGKDPFGDPTADRPWNRYTDPMDKAFARTOALFEFCEKLNIEY

FCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCS

ADVFAYAAAQVKKALErrKELGGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAYDY

AKRIGFrGQFUEPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHIFQHE

LRÍVlARELGKLGSroj-VNQGDLLLGYTDIDQFPTNVyDTTLAMYEVIKAGGFTKGGLNFDAKVR rasykvedlhghiagmdtfalgfkvayklvkdgvldkfibekyrsfregigrdivegiIlEKIEKIElElIYEKIEKIEKIEKIEKIEKIEKIEKIEKYE

Glucose Nucleotide Fusion Nucleotide Sequence of Marine Thermotoga PNOV4835 (SEQ ID NO: 41) ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCA

TATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGATCCCCATGGCCGAGTTCTiCCC

GGAGATCCCGAAGATCCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCrrCCGCT

TCTACGACCCGAACGAGGTGATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCC

GTGGCCrTCTGGCACACCTTCGTGAACGAGGGCCGCGACCCGTTCGGCGACCCGACCGCC

GAGCGCCCGTGGAACCGCTTCTCCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGC cctcttcgagttctgcgagaagctcaacatcgagtactttctgcttccacgaccgcgacat

CATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCT

TCTCCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCGCCT

ACGCCGCCGCCCAGGTG ^ GAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGG

CTACGTGTTCTGGGGCXjGCXXjCGAGGGCTàCGAGACCCTCCTCAACACCGACCTCGGCCT

GGAGCTGGAGAACCTCGCCCGCITCCTCCGCATGGCCGTGGAGTACXjCCAAGAAGATCG

GCTTCACCGGCCAGTTCCTCATCGAGCCGAAGCCGAAGGÀGCCGACCAAGCACCAGTAC

GCXTCGACGTGGCCACCGCCTACGCXTrCCTGAAGAACCACGGCCTCGACGAGTACTTCA

AGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCTG

CGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCTC

CTCGGCTGGGACACCGACCAGTTCCCGACCAACATCTACGACACCACCCTCGCCATGTAC

GAGGTGATCAAGGCCGGCGGCrrCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGCG

CCGCGCCTCCTACAAGGTGGAGGACCTCTTCATCGGCCACATCGCCGGCATGGACACCTT

CGCCCTCGGCTTCAAGATCGCCTACAAGCTCGCCAAGGACGGCGTGnCGACAAGTTCAT

CGAGGAGAAGTACCGCrCCrrCAAGGAGGGCATCGGCAAGGAGATCGTGGAGGGCAAG

ACCGACTTCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGGACATCGAGCTGCCGTC

CGGCAAGCAGGAGTACCTGGAGTC (XTCCTCAACTCCTACATCGTGAAGACCATCGCCG

AGCTGCGCTGA

Sequence glucose isomerase fusion amino acid pNOV4835 Thermotoga maritima (SEQ ID NO: 42) MGSSHHHHHHSSGLVPRGSH3V1ASMTGGQQMGRIPMAEFFPBIPKIQEEGKES1M> LAFRFYD pnbvbdgkplkdhlkfsvafwfiwnegrdpfgdptaerpwnrfsdpmdkafarvdalfef CBICLMEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYM

HGAATTCSADVFAYAAAQVKKÀLErrKELGGEGYVFWGGREGYETIXNTDLGLBLENLARF

LRMA \ rEYAKKIGFTGQFLIBPKPKEPTKHQYDFDVATAYAFLKNHGLDBYFKJ} NIEANHAl'L

AGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAÜGFTKGG

LNFDAKVRRANSYKVEDLFIGHIAGMDTFALGFKIAYKLAICDGVFDKHEEKYRSFKEG1GKEI

VEGKTDFEKLEEYIlDKEDIELPSGKQEYLESLLNSYIVKnAELR

Glucose Fusion Nucleotide Sequence of Marine Thermotoga Isomerase PNOV4836 (SEQ ID NO: 43) ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCA

TATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGATCCCCATGGCCGAGTTCTTCCC

GGAGATCCCGAAGGTGCAGTTCGAGGGCAAGGAGTCCACCAACCCGCTCGCCITCAAGT

TCTACGACCCGGAGGAGATCATCGACGGCAAGCCGCTCAAGGACCACCTCAAGTTCTCC

GTGGCCTTCTGGCACACCtTCGTGAACGAGGGCCGCGACCCGITCGGCXjACCCGACCGCC

GACCGCCCGTGGAACCGCTACACCGACCCGATGGACAAGGCCTTCGCCCGCGTGGACGC

(XnxrrrGGAGTTCTGCGAGAAGCrCAACATCGAGTACTTCTGCTTCCACGACCGCGACAT

ACCGGAGGGCAAGACCCTCCGCGAGACCAACAAGATCCTCGACAAGGTGGTGGAGCG

CATCAAGGAGCGCATGAAGGACTCCAACGTGAAGCTCCTCTGGGGCACCGCCAACCTCT

TCrrcCCACCCGCGCTACATGCACGGCGCCGCCACCACCTGCTCCGCCGACGTGTTCXjCCT

ACGCCGCCGCCCAGGTGAAGAAGGCCCTGGAGATCACCAAGGAGCTGGGCGGCGAGGG

CTACGTGITCTGGGGCGGCCGCGAGGGCTACGAGACCCTCCTCAACACCGACCTCGGCIT

CGAGCTGGAGAACCTCGGCCGCTlOCrCCGCATGGCCGTGGACTACGCCAAGCGCATCG

GCTTCACCGGCCAGTTCCTCATCGAGCCGAÀGCCGAAGGAGCCGACCAAGCACCAGTAC

GACTTCGACGTGGCCACCGCCTACXjCCTTCCTCAAGTCCCACGGCCTCGACGAGTACITC

AAGTTCAACATCGAGGCCAACCACGCCACCCTCGCCGGCCACACCTTCCAGCACGAGCT

GCGCATGGCCCGCATCCTCGGCAAGCTCGGCTCCATCGACGCCAACCAGGGCGACCTCCT

CCrCGGCTGGGACACCGACCAGTTCCCGACCAACGTGTACGACACCACCCTCGCCATGTA

CGAGGTGATCAAGGCCGGCGGCTTCACCAAGGGCGGCCTCAACTTCGACGCCAAGGTGC GCCGCGCCTCCTACAAGGTGGAGGACCTClTCATCGGCCACATCGCCGGCATGGACACCr TCGCCCIGGGCTTCAAGGAGGAGGGGCGGCGGTGCATA

TCGAGGAGAAGTACCGCTCCTTCCGCGAGGGCATCGGCCGCGACATCGTCiGAGGGCAAG

GTGGACriCGAGAAGCTGGAGGAGTACATCATCGACAAGGAGACCATCGAGCTGCCGTC 1 CGGCAAGCAGGAGTACCTGGAGTCCCTCATCAACTCCTACATCGTGAAGACCATCCTGG

AGCTGCGCTGA

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

PEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTADRPWNRYTDPMDKAFARVDAJ ^ FEFC

EKIJ ^ YFCFHDRDIAPEGKTiJ ^ TNSÜLDKVVERIKERMKDSNVKLLWGTANLFSHPRYMH

GAATTCSADVFAYAAAQVKKALEITKELGGEGYVIWGGREGYETLLNTDLGFELENLARFL

RMAVDYAKRIGFTGQFL1EPKPKEPTKHQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLA

GIITFQHELRMARD-GKLGSIDANQGDLLLGWDTDQFPTNVYDTTLAMYEV1KAGGFTKGGL

NFDAKVRRASYKVEDLFIGHIAGMDTFALGFKVAYKLVKDGVLDKFIEEJCYRSRREGIGRDI

VEGKVDFEKLEEYHDKBTIHLPSGKQEYLESLINSYIVKTILELR

Aspergillus shirousami α-amylase / glucoamylase fusion amino acid sequence (no signal sequence) (SEQ ID NO: 45) ATPAD ^ VRSQSIYFLLTDRFARTDGSTTATCNTADQKYCGGTV.i'QG] IDKLDYIQGMGFTAIWI

'rPVTAQLPQTTAYGDAYHGYWQQDIYSLNBNYGTADDLKALSSALHERGMYIMVDWAN

HMGYDGAGSSVDYSVFKPFSSQDYFHPFCFIQNYEDQTQVEDCWLGDNTVSLPDLDTrKDV

VKNBWYDWVGSLVSNYSroGLRIDTVKHVQKDFWPGYNKAAGVYaGEVLDVDPAYTCPY

QNVMDGVLNYPmTLLNAFKSTSGSMDDLYNMINTVKSDCPDSTLLGTFVENHDNPRFASY

TNDIALAKNVAAFIILNDGMIYAGQEQHYAGGNDPANREATWLSGYPTDSELYKLLASANAI

RNYAISKDTGFVTYKNWPIYKDDTTIAmKGTOGSQIVTR ^ NKGASGDSYTLSLSGAGYTA

GQQLTEVIGCTTVTVGSDGNVPVPMAGGLPRVLYFrEKLAGSKICSSSKPATLDSWLSNEAT

VARTAE.NNIGADGAWVSGADSGIVVASPSTDNPDYFYTWTRDSGIVLKTLVDLFRNGDTDL

LSTIEHYISSQAnQGVSNPSGDLSSGGLGEPKFNVDETAYAGSWGRPQRDGPALRATAMIGF

GQWLLDNGYTSAATEIYWHvVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTíAVQHRALVE

GSAFATAVGSSCSWCDSQAPQILCYLQSFWTGSYILANFDSSRSGKDTNTLLGSIHTFDPEAG

CDDSTFQPCSPRAI-ANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDSYYNGNPWFLCTLA

AAEQLYDALYQWDKQGSLEriDVSLDFFKALYSGAATGTYSSSSSTYSSIVSAVKTFADGFVS

IVETHAASNGSLSEQFDKSDGDELSARDLTWSYAALLTANNRRNSWmWGETSASSVPGT

CAATSASGTYSSVTVTSWPSIVATGGTTTTATTTGSGGVTSTSKTTTTASKTSTTTSSTSCTrP

TAVAVTFDLTATnYGENIYLVGSlSGLGDWETSDGIALSADKYTSSNPPWYVTVTLPAGESF

EYKFIRVESDDSVEWESDPNREYTVPQACGESTATVTDTWR

Amino acid sequence optimized for Aspergillus shirousami a-amyase / glucoamylase fusion maize (no signal sequence) (SEQ ID NO: 46) GCCACCCCGGCCGACTGGCGCTCCCAGTCCÀTCTACTTCCTCCTCACCGACCGCTTCGCC

CGCACCGACGGCTCCACCACCGCCACCTGCAACÀCCGCCGACCAGAAGTACrGCGGCGG

CACCTGGCAGGGCATCATCGACAAGCTCGACTACATCCAGGGCATGGGCTTCACCGCCA

TCTGGATCACCCCGGTGACCGCCCAGCrCCCGCAGACCACCGCCTACGGCGACGCCTACC

ACGGCTACTGGCAGCAGGACATCTACTCCCTCAACGAGAACTACGGCACCGCCGACGAC

CTCAAGGCCCTCTCCTCCGCCCTCCACGAGCGCGGCATGTACCTCATGGTGGACGTGGTG

GCCAACCACATGGGCTACGACGGCGCCGGCTCCTCCGTGGACTACTCCGTGTTCAAGCCG

TTCrCCTCCCAGGACTACrrcCACCCGrrcTGClTCATCCAGAACTACGAGGACCAGACC

CAGGTGGAGGACTGCTGGCrCGGCGACAACACCGTGTCCCTCCCGGACCTCGACACCAC

CAAGGACGTGGTGAAGAACGAGTGGTACGACTGGGTGGGCTCCCrCGTGTCCAACTACT

CCATCGACGGCCTCCGCATCGACACCGTGAAGCACGTGCAGAAGGACTTCTGGCCGGGC

TACAACAAGGCCGCCGGCGTGTACTGCATCGCICGAGGTGCTCGACGTGGACCCGGCCTA

CACCTGCCCGTACCAGAACGTGATGGACGGCGTGCTCAACrACCCGATCTACrACCCGCT

CCrCAACGCCTTCAAGTCCACCTCCGGCTCGATGGACGACXjrCTACAACATGATCAACAC

CGTGAAGTCCGACTGCC03GACTCCACCCTCCTCGGCACCXrCGTGGAGAACCACGACAA

CCCGCGCTTCGCCTCCTACACCAACGACATCGCCCTCGCCAAGAACGTGGCCGCCTTCAT

CATCCrCAACGACGGCATCCCGATCATCTACGCCGGCCAGGAGCAGCACrACGCCGGCG

GCAACGACCCGGCCAACCGCGAGGCCACCTGGCTCTCCGGCTACCCGACCGACTCCGAG

CTGTACAAGCTCATCGCCTCCGCCAACGCCATCCGCAACTACGCCATCTCCAAGGACACC

GGCTTCGTGACCTACAAGAACTGGCCGATCTACAAGGACGACACCACCATCGCCATGCG

CAAGGGCÀCCGACGGCTCCCAGATCGTGACCATCCTCTCCAACAAGGGCGCCTCCGGCG

ACTCCTACACCCTCTCCCTCTCCGGCGCCGGCTACACCGCCGGCCAGCAGCrCACCGAGG

TGATCGGCTGCACCACCGTGACCGTGGGCTCCGACGGCAACGTGCCGGTGCCGATGGCC

GGCGGCCTCCTGCGCGTGCTCTACCCGACCGAGAAGCTCGCCGGCrCCAAGATATGCrCC

TCCTCCAAGCCGGCX: ACCCTCGACT (XTGGCTCTCCAACGAGGCCACCGTGGCCCGCACC

GCCATCCTCAACAACATCGGCGCCGACGGCGCCTGGGTGTCCGGCGCCGACTCCGGCAT

CGTGGTGGCCTCCCCGTCCACCGACAACCCGGACTACTTCTACACCTGGACCCGCGACTC

CGGCATCGTGCTCAAGACCCTCGTGGACCTCTTCCGCAACGGCGACACCGACCTCCTCTC

CACCATCGAGCACTACATCTCCTCCCAGGCCATCATCCAGGGCGTGTCCAACCCGTCCGG

CGACCTCTCCTCCGGCGGCCTCGGCGAGCCGAAGTTCAACGTGGACGAGACCGCCTACG

CCGGCTCCTGGGGCGGCCCGCAGCGCGACGGCCCGGCCCTCCGCGCCACCGCCATGATC

GGCTTCGGCCAGTGGCTCCTCGACAACXjGCTACACCTCCGCCGCCACCGAGATCGTGTGG

CCGCrCGTGCGCAACGACCTCTCCrACGTGGCCCAGTACTGGAACCAGACCGGCTACGAC

CTCrGGGAGGAGGTGAACGGCTCCTCCTTCITCACCATCGCCGTGGAGCACCGCGCCCTC

GTGGAGGGCTCCGCCrrCGCCACCGCCGTGGCiCrCCTCCTGCTCCrGGTGCGACTCCCAG

GCCCCGCAGATCCTCTGCTACCTCCAGTCCITCTGGACCGGCTCCTACATCCTCGCCAACT

TCGACTCXJrCCCGCTCCGGCAAGGACACCAACACCCTCCTCGGCTCCATCCACACCTTCG

ACCCGGAGGCCGGCTGCGACGACTCCACCTTCCAGCCGTGCTCCCCGCGCGCCCTCGCCA

ACCACAAGGAGGTGGTGGACTCCiTCCGCrCCATCTACACCCrCAACGACGGCCTCrCCG

ACTCCGAGGCCGTGGCCGTGGGCCGCTACCCGGAGGACTCCTACTACAACGGCAACCCG

TGGTTCCTCTGCACCCTCGCCGCCGCCGAGCAGCTCTACGACGCCCTCTACCAGTGGGAC

AAGCAGGGCTCCCTrGGAGATCACGGACGTGTCCCTCGACTrCTTCAAGGCCCrcrACrcC

GGCGCCGCCACCGGCACCTACTCCTCCTCCTCCTCCACCTACTCCTCCATCGTGTCCGCCG

TGAAGACCTTCGCCGACGGCTTCGTGTCCATCGTGGAGACCCACGCCGCCTCCAACGGCT

CCCTCTCCGAGCAGTTCGACAAGTCCGACGGCGACGAGCTGTCCGCCCXjCGACCTGACCT

GGTCCTACGCCGGCCTCCTCACCGCCAACAACCGCCGCAACTCCGTGGTGCCGCCGTCCT

GGGGCGAGACCTCCGCCTCCTCCGTGCCGGGCACCTGCGCCGCCACCrCCGCCTCCGGCA

CCTACTCCTCCGTGACCGTGACCTCCTGGCCGTCCATCGTGGCCACCGGCGGCACCACCA ccaccgccaccaccaccggctccggcogcgtgacctccacctccaagaccaccaccacc GCCTCCAAGACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC

GTGACCTTCGACCTCACCGCCACCACCACCrACGGCGAGAACATCTACCTCGTGGGCTCC

ATCTCCCAGCTCGGCGACTGGGAGACCTCCGACGGCATCGCCCTCrCCGCCGACAAGTAC

ACClOCTCCAACCCGCCGTGGTACGTGACXGTGACCCrCCCGGCCGGCGAGTCCTTCGAG

TACAAGTrCATCCGCGTGGAGTCCGACGACTXXGTGGAGTGGGAGTCCGACCCGAACCG

CGAGTAC

ACCGTGCCGCAGGCCTGCGGCGAGTCCACCGCCACCGTGACCGACACCTGGCGC

Thermoanaerobacterium thermosaccharolyticum Glucoamiasis Amino Acid Sequence (No Signal Sequence) (SEQ ID NO: 47)

YYl ^ IDTADVKEIKFIVTDGKSFVSDETKDAISKVEKFIOKSLGYKLVNTDKIIGRYRITKEIFT

DVKRNSLIMKAKFEALEGSIHDYKLYLAYDPHIKNQGSYNEGYVIKANNNEMLMAKRDNV

YTALSSNIGWKGYSIGYYKVNDMTDLDENKQMTKHYDSARGNflEGAEIDLTKNSEFEIVLS

FGGSDSEAAKTALETLGEDYNNr.KNNYTOEWTKYCNTLNNFNGKANSLYYNSMM] LKASED

KTNKGAYIASLSIPWGDGQRDDNTGGYHLVWSRDLYHVANAFIAAGDVDSANRSLDYLAK

VVKJ ^ NGMIPQNTWISGKPYWTSIQLDEQADPIILSYRLKRYDLYDSLVKPLADFIIKIGPKTGQ

ERVvEEIGGYSPAIMAAEVAGLTCAAYXAEQNKDYBSAQKYQBKADNWQKLIDNLTYTENG

PLGNGQYYIRIAGLSDPNADFMIMANGGGVYDQKE1VDPSFLELVRLGVKSADDPKILNTLK

WDSTTKVDTPKGPSWYRYNHDGYGEPSKTELYHGAGKGRLWPLLTGERGIVIYEIAAGKDA

TPYVKAMEKFANEGGUSEQVWEDTGLPTDSASPLNWAHAEYVILFASNIEHKVLDMPDrVY

Optimized amino acid sequence for Thermoanaerobacterium thermosaccharolyticum glucoamylase corn (no signal sequence) (SEQ ID NO: 48) GTGCTCTCCGGCTGCrCCAACAACGTGTCCTCCATCAAGATCGACCGCTTCAACAACATC

TCCGCCGTGAACGGCCCGGC3CGAGGAGGACACCTGGGCCTCCGCCCAGAAGCAGGGCGT

GGGCACCGCCAACAACTACGTGTCCCGCGTGTGGTTCACCCTCGCCAACGGCGCCATCTC

CGAGGTGTACTACCCGACCATCGACACCGCCGACGTGAAGGAGATCAAGTTCATCGTGA

CCGACGGCAAGTCCITCGTGTCCGACGAGACCAAGGACGCCATCTCCAAGGTGGAGAAG

TTCACCGACAAGTCCCTCGGCTACAAGCTCGTGAACACCGACAAG.AAGGGCCGCTACCG

CATCACCAAGGAAATCTTCACCGACGTGAAGCGCAACrcCCTCATCATGAAGGCCAAGT

TCGAGGCCCTCGAGGGCTCCATCCACGACTACAAGCTCrACCrCGCCTACGACCCGCACA

TCAAGAACCAGGGCTCCIACAACGAGGGCTACGTGATCAAGGCCAACAACAACGAGATG

CTCATGGCCAAGCGCGACAACGTGTACACCGCCCTCTCCTCCAAGATCGGCTGGAAGGG

CTACTCCATCGGCTACTACAAGGTGAACGACATCATGACCGACCTCGACGAGAACAAGC

AGATGACCAAGGACTACGACTCCGCCCGCGGCAACATCATCGAGGGCGCCGAGATCGAC

CTCACCAAGAACTCCGAGTTCGAGATCGTGCTCTCCrrCGGCGGCTCCGACTCCGAGGCC

GCCAAGACCGCCCTCGAGACCCTCGGCGAGGACTACAACAACCTCAAGAACAACTACAT

CGACGAGTGGACXTAAGTACTGCAACACXXTrCAACAACTTCAACGGCAAGGCCAACTCCC

TCTACTACAACTCCATGATGATGCTCAAGGCCTCCGAGGACAAGACCAACAAGGGCGCC

TACATCGCCrCCCTCTCCATCCCGTGGGGCGACGGCCAGCGCGACGACAACACCGGCGG

CTACCACCTCGTGTGGTCCCGCGACCTCTACCACGTGGCCAACGCCTTCATCGCCGCCGG

CGACGTGGACTCCGCCAACCGCTCCCTCGACTACCTCGCCAAGGTGGTGAAGGACAACG

GCATGATCCCGCAGAACACCTGGATCTCCGGCAAGCCGTACTGGACCTCCATCCAGCTCG

ACGAGCAGGCCGACCCGATCATCCrCTCCTACCGCCTCAAGCGCTACGACCTCTACGACT

CCCTCGTGAAGCCGCTCGCCGAC1TCATCATCAAGATCGGCXXGAAGACCGGCCAGGAG

CGCTGGGAGGAGATCGGCGGCTACTCCCCGGCCACGATGGCCGCCGÀGGTGGCCGGCCT

CACCTGCGCCGCCTACATCGCCGAGCAGAACAAGGACTACGAGTCCGCCCAGAAGTACC

AGGAGAAGGCCGACAACTGGCAGAAGCTCATCGACAACCTCACCTACACCGAGAACGGC

(XGCTCGGCAACGGCCAGTACrACATCCGCATCGCCGGCCTCTCCGACCCGAACGCCGAC

TTCATGATCAACATCGCCAACGGCGGCGGCGTGTACGACCAGAAGGAGATCGTGGACCC

GTCCTTCCTCGAGCTGGTGCGCCTCGGCGTGAAGTCCGCCGACGACCCGA ^ GATCCTCAA

CACCCTCAAGGTGGTGGACTCCACCATCAAGGTGGACACCOCGAAGGGCCCGTCCTGGT

ATCGCTACAACCACGACGGCTACGGCGAGCCCjrcCAAGACCGAGCTGTACCACGGCGCC

GGCAAGGG (XGCXjrCTGGCCGCTCCrCACCGGCGAGCGCGGCATGTACGAGATCGCCGC

CGGCAAGGACGCCACCCCGTACGTGAAGGCGATGGAGAAGTTCGCCAACGAGGGCGGC

ATCATCTCCGAGCAGGTGTGGGAGGACACCGGCCrrCCCGACCGACIGCGCCTCCCCGCTC

AACTGGGCCCACGCCGAGTACGTGATCCTCnGGCCTCCAACATCGAGCACAAGGTGCTC

GACATGCCGGACATCGTGTAC

Rhizopus oryzae Glucoamylase Amino Acid Sequence (No Signal Sequence) (SEQ ID NO: 49) ASJPSSASVQLDSYNYDGSTFSGKIYVKNIAYSKKWVIYADGSDNWNNNGNTIAASYSAPIS

GSNYEYWTKASINGIKBFYIKYEVSGKTYYDNN ^ ANYQVSTSKPTTTTATATTTTAPSTST

TrPPSRSEPATEPTGNSTÍSSWn <XQEGISRFAMLRNINPPGSATGPIAASLSTAGPDY ^ AWTR

DAALTSNVIVYEYNTTLSGNKTILNVLICDYVTFSVKTQSTSWCNCLGEPKI-NPDASGYTGA

WGRPQNDGPAERATTFELFADSYLTQTKDASYVTGTLKPAIFKDLDYVVNVWSNGCFDLWE

EVNGVHFYTLMVMRKGLLLGADFAKRNGDSTRASTYSSTASTÍANKISSFWVSSiNNWIQVSQ

SVTGGVSKKGLDVSTliAANLGSVDDGFFIPGSEIGLATAVAVEDSFASLYPINKNLPSYLGN sigryfedtyngngnsqgnswelavtgyablyyraikewignggvtvssislpffkkfdssat SGKKYTVGTSDFNNLAQGALWHALNNLQLWLGNG

ASUTASYAKAGAPAA

Nucleic acid sequence optimized for Rhizopus oryzae glucoamylase maize (no signal sequence ID NO: 50) GCCTrCATCCCGTCCTCrGCCrcCGTGCAGCrcGACTCCTACAACrACGACGGCTCCACC

TTCrCCGGCAAAATCTACGTGAAGAACATCGCCTACTCCAAGAAGGTGACCGTGATCTAC

GCCGACGGCTCCGACAACTGGAACAACAACGGCAACACCATCGCCGCCTCCrACTCCGC

CCCGATCTCCGGCTCCAACTACGAGTACTGGACCTTCTCCGCCTCCATCAACGGCATCÀA

GGAGTTCTACATCAAGTACGAGGTGTCCGGCÀAGACCTACTACGACAACAACAACTCCG

CCAACTACCAGGTGTCCACCTCCAAGCCGACCACCACCACCGCCACCGCCACCACCACC

ACCGCCCCGTCCACCTCCACCACCACCCCGCCGTCCCGCTCCGAGCCGGCCACCTTCCCG

ACCGGCAACTCCACCATCTCCTCCTGGATCAAGAAGCAGGAGGGCATCTCCCGCTTCGCC

ATGCTCCGCAACATCAACCCGCCGGGCrCCGCCACCGGCITCATCGCCGCCTCCCrcrCC

ACCGCCGGCCCGGACTACTACTACGCCTGGACCCGCGACGCCGCCCTCACCTCCAACGTG

ATCGTGTACXjAGTACAACACCACCCTCTCCGGCAACAAGACCATCCTCAACGTGCTCAAG

GACTACXjrTGACCTTCTCCGTGAAGACCXIAGTCCACCTCCACCGTGTGCAACTGCCTCGGC

GAGCCGAAGITCAACXXGGACGCCTCCGGCTACACCGGCGCCTGGGGCCGCCCGCAGAA

CGACGGCCCGGCCGAGCGCG € CACCACCTTCATCCrCTTCGC € GACTCCTACCrCACX: CA

GACCAAGGACGCCTCCTACGTGACCGGCACCCTCAAGCCGGCCATCTTCAAGGACCTCG

ACTACGTGGTGAACGTGTGGTCCAACGGCTGCTTCGACCTCTGGGAGGAGGTGAACGGC

GTGCACrrCTACACCCrCATGGTGATGCGCAAGGGCCrCCTCCTCGGCGCCGAClTCGCC

AAGCGCAACGGCGACTCCACOCGCGCCTCCACCTACTCCTCCACCGCCTCCACCATCGCC

AACAAAATCTCCTCCITCrGGGTGTCCrCCAACAACTGGATACAGGIXJrCCCAGTCCGTG

ACCGGCGGCGT GT CC AGAÀGGG CCTCG ACGTGT CCACCCTCCTCGCCGCC \ CCTCGGC

TCCGTGGACGACGGCrrCTTCACCCCGGGCTCCGAGAAGATCCTCGCCACCGCCGTGGCC

GTGGAGGACTCCTTCGGCTCCCTCTACCCGAlGAACAAGAACCrCCCGTCCTACCrcGGC

AACTCCATCGGCCGCTACCCGGAGGACACCTACAACGGCAACGGCAACTCCCAGGGGAA

CTCCTGGTTCCrCGCCGTGACCGGCrACGCCGAGCTGTACTACCGCGCCATCAAGGAGTG

GATCGGCAACGGCGGCGTGACCGTGTCCTCCATCTCCCrCCCGTTCTTCAAGAAGTrCGA

CTCCTCXIGCCACCTCCGGCAAGAAGTACACCGTGGGCACCTCCGACnTCAACAACCTCGC

CX ^ AGAACATCGCCCTCGCCGCCGACCGCTrCCTCrcCACCGTGCAGCTCCACGCCCACAA

CAACGGCTCCCTCGCCGAGGAGTIGGACCGCCCACCGGCCTCTCCACCGGCGCCCGCG

ACCTCACCTGGTCCCACGCCrCCCrCATCACCGCCTOCTACGCCAAGGCCGGCGCCCCGG

CCGCC

Corn α-Amylase Amino Acid Sequence (SEQ ID NO: 51) MAKHLAAMCWCSLLVLVLLCLGSQLAQSQVLFQGFNWESWKKQGGWYNYLLGRVDDIAA

TGATIIVWIJQPSHSVAPQGYMPGRLYDLDASKYGTHAEIXSLTAAFHAKGVQCVADVVIN

HRCADYKDGRGIYCVFEGGTPDSRLDWGPDMICSDDTQYSNGRGHRDTGADFAAAPDIDHL

NPRVQQELSDWLNWLKSDLGFDGWRLDFAKGYSAAVAKVYYDSTAFEFVVAEIWSSLHYD

GNGEPSSNQDADRQELVNWAQAVGGPAAAFDFTTKGVLQAAVQGELWRMKDGNGKAPG migwlpekavtfvdnhdtgstqnswpepsdkvmqgyayilthpgtpcifydhvfdwnlkqe ISALIAVKVNGHVDKNGVHVNG

YCVWEKHGLRVPAGRHH

Corn α-Amylase Nucleic Acid Sequence (SEQ ID NO: 52) ATGGCGAAGCACTTGGCTGCCATGTGCrGGTCiCAGCCIXXTAGTGCTrGTACTGCTCTGC

TTGGGCTCCCAGCTGGCCCAATCCCAGGTCCTCTTCCAGGGGTTCAACTGGGAGTCGTGG

AAGAAGCAAGGTGGGTGGTACAACTACCTCCTGGGGCGGGTGGACGACATCGCCGCGAC

GGGGGCCACGCACGTCrGGCTCCCGCAGCCGTCGCACTCGGTGGCGCCGCAGGGGTACA

TGCCCGGCCGGCTCTACGACXrrGGACGCGTCCAAGTACGGCACCCACGCGGAGCTCAAG

TCGCTCACCGCGGCGTTCCACGCCAAGGGCGTCCAGTGCGTCGCCGACGTCGTGATCAAC

CACCGCTGCGCCGACTACAAGGACGGCCGCGGCATCTACTGCGTCTTCGAGGGCGGCAC

GCCCGACAGCCGCCTCGACTGGGGCCCCGACATGATCTGCAGCGACGACACGCAGTACT

CCAACGGGCGCGGGCACCGCGACACGGGGGCCGACTTCGCCGCCGCGCCCGACATCGAC

CACCTCAACCCGCGCGTGCAGCAGGAGCTCTCGGACTGGCTCAACTGGCTCAAGTCCGA

CCTCGGCTTCGACGGCrGGCGCCTCGACTTCGCCAAGGGCTACTCCGCCGCCGTCGCCAA ggtgtacgtcgacagcaccgcccxx: accttcgtcgtcgccgagatatggagctccctcca CTACGACGGCAACGGCGAGCCGTCCAGCAACCAGGACX3CCGACAGGCAGGAGCTGGTC aactgggcgcaggcggtgggcggccccgccgcggcgttcgacttcaccaccaagggcgt gctgcaggcggcGgtccagggcgagctgtggcgcatgaaggacggcaacggcaaggcg cccgggatgatcggctggctgccggagaaggccgtcacgttcgtcgacaaccacgacac CGGCTCCACGCAGAACTCGTGGCCArrCCCCTGCGACAAGGTCATGCAGGGCTACGCCTA

TATCCTCACGCACCCAGGAACrCCATGCATCTTCTACGACCACGTTTTCGACTGGAACCT gaagcaggagatcagcgcgctgtctgcggtgaggtcaagaaacgggatccacccgggg agcgagctgaacatcctcgccgccgacggggatctctacgtcgccaagattgacgacaa ggtcatcgtgaagatcgggtcacggtacgacgtcgggaacctgatcccctcagacttcca CGCCGTTGCCCCTGGCAACAACTACIGCGTrrGGGAGAAGCACGGTCrGAGAGTTCCAGC

GGGGCGGCACCACTAG

Raw Starch Binding Site Amino Acid Sequence (SEQ ID NO: 53) ATGGTTTrATTTGSGGVTSTSKlTrrASKTSTTTSSTSCTTPTAV

Nucleic acid sequence optimized for crude starch binding site maize (SEQ ID NO: 54) I / 5 / I I GCCA0CGGCGGCACCCACCACC <3CCACCACCACX: GGGTCCGGCGGCGTGACCTCCAC

CTO ^ AGACCACCACCACCGCCTCCAAGACXTCCACCACCACCIXXTC ^ ACCIXXTGCAC

CACCCCGACCGCCGTGTC

Pyrococcus furiosus EGLA amino acid sequence (no signal sequence) (SEQ ID NO: 55) lYFVEKYHTSBDKSTSNTSSTPPQTn ^ ITKVLKIRYPDDGEWPGAPIDKDGDGNPHFyiEINL

WNE.NATGFAEMTYNLTSGVLHYVQQLDNIVLRDRSNWVHGYPEIFYGNKPWNANYAIDG

PIPLPSKVSNLTDFYLTISYKLEPKNGLPINFAIESWLTREAWRTTGrNSDEQEVMIWIYYDGL

QPAGSKVKEIYWnVNGlTVNATFEVWICANIGWEYVAFRIKTPlKEGTVTIPYGAFISVAANlS

SlPlTYTBLYLEDVSGTEFGTPSTrSAHI ^ WWmOTLTPLDRjPUS

Nucleic acid sequence optimized for maize Eglà Pyrococcus furiosus (without Signal sequence) (SEQ ID NO: 56) atctacttcgtggagaagtaccacacctcx: gaggacaagtccacctccaacacctcctcc ACOCCGCCGCAGACCACCXTCTCCACrACCAAGGTGCTCAAGATCCGCrACCCGGACGA

CGGCGAGTGGCCCGGCGCCCCGATCGACAAGGACGGCGACGGCAACCCGGAGTTCTACA

TCGAGATCAACCTCTGGAACATCCTCAACXjCX: AC € GG € TTCGCCGAGATGA € CTACAACC

TCACTAGTGGCGTGCTCCACrACGTGCAGCAGCTCGACAACATCGTGCTCCGCGACCGCT ccaactgggtgcacggctacccggaaatcttctacggcaacaagccgtggaacgccaac tacgccaccgacggcccgatcccgctcccgtcoaaggtgtccaacxtcaccgacttctac ctcaccatctcctacaagctcgagccgaagaacggtctcccgatcaacttcggcatcgag tcctggctcacccgcgaggcctggcgcaccaccggcatcaactccgacgagcaggaggt gatgatctggatctactacgacggcctccagcccgcgggctccaaggtgaaggagatcg tggtgccgatcatcgtgaacggcaccccggtgaacgccaccttcgaggtgtggaaggcc aacatcggctgggagtacgtggccttccgcatcaagaccccgatcaaggagggcaccgt gaccatcccgtacggcgccttcatctccgtggccgccaacatctcctccctcccgaacta caccgagaagtacctcgaggacgtggagatcggcaccgagttcggcaccxxgtccacca CCTCCGCCCACCTCGAGTGGTGGATCACCAACATCACCCrCACCCJCGCTCGACCGCCCGC

TCATCTCCTAG

xylose isomerase amino acid sequence of Thermus flavus (SEQ ID 'NO: 57) MYEPKPEH1ÍFTFGL \ WVDNVDRDPFGDTVRERLDPVYWHKLAELGAYGVNLHDEDLIPRG TPPQERDQIVRSFKKALDETVLKVPMVTANLFSEPAFRDGASTTRDPWVWAYALRKSLBTM DLGAELGAEIYMFWMVRERSEVESTDKTRfCVWDWVRETLNFMTAYTEDQGYGYRI'SVEPK PNEPRGDIYFTT V GSML ALIHTLDRFER

FGLNPEEAHETMAGLNFDHAVAQAVDAGKLEHIDLNDQRMSRFDQDLRFGSENLKAGFFLV

DLLESSGYQGPRHFEAHALRTEDEEGVWTFVRVCMRTYLIIKVRAETFREDPEVKELLAAYY

QEDPATLALLDPYSREKAEALICRAELPLETKRRRGYALERLDQLAVEYLLGVRG

PNOV4800 Nucleotide Sequence (AmY32B-EGLA Signal Sequence) (SEQ ID NO: 58) ATGGGGAAGAACGGCAACCTGTGCrGCTTCrCTCrGCTGCrGCTTCTTCrCGCXrGGGTTG

GCGTCCGGCCATCAAATCTACTTCGTGGAGAAGTACCACACCTCCGAGGACAAGTCCAC

CICCAACACCTCCTCCACCCCGCCGCAGACCACCCTCTCCACCACCAAGGTGCTCAAGAT

CCGCTACCCGGACGACGGTGAGTGGCCCGGCGCCCCGATCGACAAGGACGGCGACGGCA

ACCCGGAGTTCTACATCGAGATCAACCTCTGGAACATCCTCAACGCCACCGGCTrCGCCG

AGATGACCTACAACCTCACTAGTGGCGTGCTCCACTACGTGCAGCAGCTCGACAACATCG

TGCTCCGCGACCGCTCCAACTGGGTGCACGGCrACCCGGAAATCITCTACGGCAACAAGC

CGTGGAACGCCAACTACGCCACXXjACGGCCCGATCCCGCTCCCGTCCAAGGTGTCCAAC

CTCACrGACTTCTACCTCACCATCrCCTACAAGCrCGAGCCGAAGAACGGTCTCCCGATC

AACTTCGCCATCGAGTCCTGGCTCACCCGCGAGGCCTGGCGCACCACCGGCÀTCAACTCC

GACGAGCAGGAGGTGATGATCTGGATCTACTACGACGGCCTCCAGCCCGCGGGCTCCAA

GGTGAAGGAGATCGTGGTGCCGATCATCGTGAACGGCACCCCGGTGAACGCCACCTTCG

AGGTGTGGAAGGCCAACATCGGCTGGGAGTACGTGGCCTTCCGCATCAAGACCCCGATC

AAGGAGGGCACCGTGACCATCCCGTACGGCGCCTTCATCTCCGTGGCCGCCAACATCTCC

TCCCTCCCGAACTACACCGAGAAGTACCTCGAGGACGTGGAGATCGGCACCGAGTTCGG

CACCCCGTCCACCACCTGCGCCCACCTCGAGTGGTGGATCACCAACATCACCCTCACCCC

GCTCGAOCGCCCGGTCATCTCCTAG Aspergillus niger Corn Optimized Nucleic Acid (SEQ ID NO: 59) ATGTCXTTCCGCTCCGTCCTCGCCCTCTCCGGCCTCGTGTGCACCGGCCTCGCCAACGTGA

TCrCCAAGCGCGCCACCCTCGACTCCTGGCTCTCCAACGAGGCCACCGTGGCCCGCACCG

CCATCCTCAACAACATCGGCGCCGACGGCGCCTGGGTGTCCGGCGCCGACTCCGGCATC

GTGGTGGCCTCCCCGTCCACCGACAACCCGGACTACTTCTACACCTGGACCCGCGACICC

GGCCTCGTGCTCAAGACCCTCGIGGACCTCTTCCGCAACGGCGACACCTCCCTCCTCTCC

ACCATCGAGAACTACATCTCCGCCCAGGCCATCGTGCAGGGCATCrCCAACCCGTCCGGC

GACCTCTCCTCCGGCGCCGGCCrCGGCGAGCCGAAGTTCAACXjTGGACGAGACCGCCTA

CACCGGCTCCTGGGGCCGCCCGCAGCGCGACGGXCGGCCCrCCGCGCCACCGCCATGA

TCGGCTTCGGCCAGTGGCrCCTCGACAACGGCTACACCTCCACCGCCACCGACATCGTGT

GGOCGCTCGTGCGCAACGACCrCTCCTACGTGGCCCAGTACTGGAACCAGACCGGCTAC

GACCTCTGGGAGGAGGTGAACGGCTCCTCCITCTTCACGATCGCCGTGCAGCACCGCGCC

CTCGTGGAGGGCTCCGCCITCGCCACCGCCG7'GGGCTCCrCCTGCrCCTGGTGCGACTCC

CAGGCCCCGGAGATCCTCTGCTACCTCCAGTCCITCrGGACCGGCTCCTTCATCCTCGCCA AClTCGACTCCTCCCGCTCCGGCAAGGACGCCAACACCCTCCTCGGCrCCATCCACACXJr TCGACCCGGAGGCCGCCTGCGCCCCCCCCCCCCCCCCCCCCCCCCGCC

CCAACCACAAGGAGGTGGTGGACTCCTTCCGCrCCATCTACACCCTCAACGACGGCCTCT

CCGACTCCGAGGCCGTGGCCGTGGGCCGCTACCCGGAGGACACCTACTACAACGGCAAC

CCGTGGTTCCrCTGCACCCTCGCCGCCGOCGAGCAGCTCTACGACGCCCTCTACCAGTGG

GACAAGCAGGGCTCCCTCGAGGTGACCGACGTGTCCCTCGACTTCTTCAAGGCCCTCTAC i TCCGACGCCGCCACCGGCACCTACrCCTCCTCCTCCTCCACCTACTCCrCCATCGTGGACAC

CCGIGAAGACCITCGCCGACGGCTTCGTGTOCATCGTGGAGACCCACGCCGCCTCCAACG

GCTCCATGTCCGAGCAGTACGACAAGTCCGACGGCGAGCAGCTCTCCGCCCGCGACCTC

ACCTGGTCCrACGCCGCCCTCCTCACCGCCAACAACCGCCGCAACTCCGTGGTGCCGGCC

TCCTGGGGCGAGACCTCCGCCTCCTCCGTGCCGGGCACCTGCGCCGCCAGCTCCGCCATC

GGCACCTACTCCTCCGTCACCGTGACCTCCTGGCCGTCCATCGTGGCCACCGGCGGCACC ACCACCACCGCCACCCCGACCXjGCTCCGGCTCCGTGACCTCCACCTCCAAGACCACCGCX: A (XGCCTCCCACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCT

GCCGTGACCTTCGACCTCACCGCCACCACCACCTACGGCGAGAACATCTACCTCGTGGGC

TCCATCrCCCAGCrCGGCGACTGGGAGACCrCCGACGGCATCGCCCTCTCCGCCGACAAG

TACACCTCCTCCGACCCGCTCTGGTACGTGACCGTGACCCTCCCGGCCGGCGAGTCCTTC

GAGTACAAGTTCATCCGCATCGAGTCCGACGACTCCGTGGAGTGGGAGTCCGACCCGAA

CCGCGAGTACACCGTGCCGCAGGCCTGCGGCACCTCCACCGCCACCGTGACCGACACCT

GGCGC

Claims (3)

Expression cassette comprising a polynucleotide having SEQ ID NO: 2 and a heterologous signal sequence of maize y-zein (SEQ ID NO: 17) and SEKDEL (combined with 797GL3 amylase called pNOV6201 f and SEQ ID NO: 14). A method for obtaining a transgenic plant or plant part comprising the expression cassette as defined in claim 1, comprising: (a) transforming a plant cell or tissue with the expression cassette as defined in claim 1; (b) selecting transforming cells comprising the expression cassette as defined in claim 1; and (c) regenerate and grow plant cells or tissues transformed under plant growth conditions in a transgenic plant, Method according to claim 2, characterized in that the plant or plant part is selected from a group consisting of monocotyledons, dicotyledons, Liliopsida and Panicoideae.