Method of producing a resistant starch

The present invention relates to methods of producing modified starch by means of genetically modified plants which contain a foreign nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6-phosphate amidotransferase (GFAT). The present invention furthermore relates to starch obtainable from said plants and to foodstuffs comprising said starch or parts of said plants.

The polysaccharide starch is made up of chemically uniform units, the glucose molecules, but constitutes a complex mixture of different forms of molecules which differ with regard to their degree of polymerization and branching, and thus differ greatly with regard to their physico-chemical properties. One distinguishes between amylose starch, an essentially unbranched polymer of alpha-1 ,4-glycosidically linked glucose units, and amylopectin starch, a branched polymer where the branching points are generated by the occurrence of additional alpha-1 ,6-glycosidic linkages. A further essential difference between amylose and amylopectin is the molecular weight. While amylose, depending on the origin of the starch, has a molecular weight of 5 x 10 ⁵ - 10 ⁶ Da, the molecular weight of amylopectin is between 10 ⁷ and 10 ⁸ Da. The two macromolecules can be distinguished on the bases of their molecular weight and their different physico-chemical properties, and the simplest way of visualizing this is based on their different iodine-binding properties.

Amylose has long been regarded as a linear polymer comprising alpha- 1 ,4-glycosidically linked alpha-D-glucose monomers. More recent studies, however, have revealed the presence of a few alpha-1 ,6-glycosidic branching points (approx. 0.1%) (Hizukuri and Takagi, Carbohydr. Res. 134, (1984), 1-10; Takeda et al., Carbohydr. Res. 132, (1984), 83-92).

The use of resistant starch (RS) gains increasingly in importance in the food industry. Starch is digested mainly in the small intestine, by the alpha-amylase enzyme, which hydrolyzes starch's alpha-1 ,4-glucosidic bonds to give sugars. In contrast, the resistant starch is not digested in the small intestine by alpha-amylases, but travels into the colon, where it behaves similarly to fiber. When RS-containing products are degraded, the organism only gains a small amount of energy. This energy input exclusively relates to the oxidative degradation of absorbed short-chain fatty acids from the colon. These short-chain fatty acids are end products of the carbohydrate metabolism of the intestinal microflora. The uptake of RS-containing foodstuffs means that substrates are provided for the energy metabolism of the intestinal

microflora and of the colon's epithelial cells. The latter rely on the input, via the lumen, of short-chain fatty acids and in particular butyrate to maintain their structure and function. Scientific results have revealed that resistant starch is a factor of preventing diverticulosis and cancer of the colon.

One distinguishes between the following types of resistant starch: RS1 starch which is physically inaccessible to digestion, for example starch which is embedded in a protein or a fiber matrix. When this is broken down physically (for example by mastication) or chemically (for example by degradation of the surrounding matrix), it can be processed in the usual manner by the digestive juices. RS2 indigestible intact (granular) native starch grains, for example uncooked potato or banana starch)

RS3 indigestible retrograded starch which is not granular RS4 indigestible chemically modified starch, for example by crosslinking or esterification (acetylation or the like).

In contrast to RS4, the RS forms 1 to 3 may be made accessible to degradation by alpha-amylase by dissolution in NaOH or dimethyl sulfoxide.

A variety of processes have been described for the production of resistant starch. Most of these processes relate to the production of RS3 starches (EP 564893 A1 ; EP 688872 A1 ; EP 846704 A1 ; US5051271). All of these processes for the production of resistant starch comprise the dispersion and gelatinization of starch in large excesses of water, followed by retrogradation with the use of enzymes or acids. They are based on the opinion that resistant starch is formed when the amylose fraction of starch is retrograded after the gelatinization of starch. It is assumed that the linear amylose molecules, after gelatinization, arrange themselves to form tight double helix configurations bonded by hydrogen bonds, so that the alpha-1 ,4-glucosidic bonds are no longer accessible to alpha-amylases. The above-described processes for the preparation of RS3 starches are labor-intensive, time-consuming and may lead to low yields. Moreover, the high water content, which is required for the production, may make expensive drying steps necessary.

Granular starches of the RS2 type with a high content of resistant starch are found especially in native, uncooked wild-type potato starches which, depending on the method of determination, have an RS content of between 74-85% by weight (Faisant et al., Sciences des Aliments 15, (1995), 83-89; Evans and Thompson, Cereal

Chemistry 81 (1), (2004), 31-37).

A disadvantage of starches of the RS3 and RS4 type is that, once prepared, both have to be added to foodstuffs. This means that these starches can only be accumulated in ready-made products (such as, for example, yoghurts). In food prepared by the end consumer himself from fresh produce (for example boiled potatoes), it is not possible to increase the RS content by adding it. If granular starches are added to foodstuffs, this frequently entails negative effects on the texture of the foodstuff, and, upon consumption, the size of the starch granules inter alia leads to an undesirable, frequently sandy, mouth feel.

Furthermore, a distinction is made between readily digestible starch (RDS) and slowly digestible starch (SDS). RDS refers to the portion of the starch which is digestible within 20 minutes when digested with pancreatin, while SDS means the portion of starch which is digestible within the interval of between 20 minutes and 120 minutes when digested with pancreatin. RS (resistant starch), in contrast, signifies the portion of starch which cannot be digested by pancreatin after 120 minutes (Englyst et al., (1992, European Journal of Clinical Nutrition 46 Suppl. 2, pages 33- 50).

Foodstuffs with a high RDS content, in particular, lead, when consumed, to a rapid rise in the consumer's blood glucose level, whereupon insulin is secreted rapidly. The ongoing consumption of foodstuffs with a high glycemic load, and the secretion of insulin which this entails, is suspected of being a risk factor in the etiology of diseases.

US 5,714,600 describes maize plants which have been generated by means of plant breeding methods and which synthesize a starch with an increased amylose content and an increased RS content. WO 2004 0199942 describes mutants of barley plants which contain a mutation in the gene coding for a starch synthase Il (SSII). These plants synthesize a starch with an increased amylose content, an increased beta- glucan content and an increased RS content.

WO 00 11192 describes the endosperm-specific overexpression of a nucleic acid molecule from maize coding for a protein with the enzymatic activity of a plant GFAT in transgenic maize plants with the aim of synthesizing, in plants, a modified

(cationic) starch which contains 2-amino-anhydroglucose molecules. The described

metabolic pathway, which, in accordance with the description of WO OO 11192, is to lead to the incorporation of 2-amino-anhydroglucose into the starch, comprises, inter alia, incorporation, into the starch, of UDP-glucosamine by means of starch synthases and/or glycogen synthases. It was not possible to detect a cationic starch in flour from endosperm of the respective transgenic maize plants which expressed a nucleic acid molecule coding for a protein with the enzymatic activity of a plant GFAT in translational fusion with a plastidic signal peptide or which expressed a protein with the enzymatic activity of a GFAT without signal peptide.

The object of the present invention was therefore to provide methods of producing modified starch and methods of producing said starches, and foodstuffs or feeding stuffs comprising said modified starch.

A first aspect of the present invention relates to a method of producing a modified starch in genetically modified plants, comprising the following steps: a) introduction, into a plant cell, of a foreign nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6-phosphate amidotransferase (GFAT) b) regeneration of a plant from plant cells obtained as described in step a) c) if appropriate, generation of further plants with the aid of the plants as described in step b) d) extraction of starch from plants obtainable as described in step b) or c).

Surprisingly, it has been found that genetically modified plants, into which a foreign nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6- phosphate amidotransferase (GFAT) has been introduced, synthesize a starch which contains a higher proportion of resistant starch in comparison with starch isolated from corresponding wild-type plants into which no foreign nucleic acid molecule, coding for a protein with the activity of a glutamine:fructose 6-phosphate amidotransferase, has been introduced. In comparison with wild-type plants, such plants have the advantage that they synthesize a starch which has an elevated resistant starch (RS) content even after heating (gelatinization of the starch). Said plants, or parts of these plants, which are heated in order to prepare foodstuffs are therefore distinguished by the fact that they continue to have an elevated RS content, despite the heat treatment. Firstly, they are therefore lower in calories. Secondly, such plants have a reduced readily digestible starch (RDS) content, which is

particularly advantageous since the rapid liberation of substantial amounts of glucose, and its absorption via the small intestine's epithelium, leads to a drastic increase in the blood sugar level and thus to an increased glycemic index. As a consequence, insulin is secreted (insulin response). A continuous consumption of foodstuffs with a high glycemic load, and the secretion of insulin which this entails, is suspected of being a risk factor in the etiology of diseases such as high blood pressure, adipositas, heart disease and type Il diabetes.

In the context of the present invention, the term "foreign nucleic acid molecule" is understood as meaning a molecule which either does not occur naturally in corresponding wild-type plant cells or which does not occur naturally in wild-type plant cells in this specific spatial arrangement, or which is located at a location in the genome of the wild-type plant cell at which it does not occur naturally.

The foreign nucleic acid molecule is preferably a recombinant molecule which comprises various elements (nucleic acid molecules) whose combination, or specific spatial arrangement, does not occur naturally in plant cells.

In the context of the present invention, the term "recombinant nucleic acid molecule" is understood as being a nucleic acid molecule which contains different nucleic acid molecules which do not naturally occur in the combination in which they are present in a recombinant nucleic acid molecule. Thus, recombinant nucleic acid molecules may, for example, contain not only foreign nucleic acid molecules which code for a protein, but also additional nucleic acid sequences which do not occur naturally in combination with said nucleic acid molecules which code for a protein. Said additional nucleic acid sequences which are present on a recombinant nucleic acid molecule in combination with a protein-encoding nucleic acid molecule may be any sequences. They may be, for example, genomic and/or plant nucleic acid sequences. The abovementioned additional nucleic acid sequences preferably take the form of regulatory sequences (promoters, termination signals, enhancers, introns), especially preferably regulatory sequences which are active in plant tissue, particularly preferably tissue-specific regulatory sequences which are active in plant tissue. Methods of generating recombinant nucleic acid molecules are known to the skilled worker and comprise genetic engineering methods such as, for example, the linking of nucleic acid molecules by ligation, genetic recombination or the de-novo synthesis of nucleic acid molecules (see, for example, Sambrok et al., Molecular Cloning, A Laboratory Manual, 3rd edition (2001) Cold Spring Harbour Laboratory Press, Cold

Spring Harbour, NY. ISBN: 0879695773; Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN: 0471250929).

A preferred subject matter of the present invention relates to methods according to the invention of producing modified starch, wherein the foreign nucleic acid molecules coding for a protein with the activity of a GFAT are linked with regulatory elements which initiate transcription in plant cells (promoters). They may take the form of homologous or heterologous promoters. The promoters may take the form of constitutive, tissue-specific, development-specific promoters or promoters which are regulated by external factors (for example after application of chemicals, by the action of abiotic factors such as heat and/or chill, drought, infection with disease or the like).

In general, any promoter which is active in plant cells is suitable for expressing a foreign nucleic acid molecule. Examples of suitable promoters are the promoter of the 35S RNA of the cauliflower mosaic virus or the ubiquitin promoter from maize or the Cestrum YLCV promoter (yellow leaf curling virus; WO 01 73087; Stavolone et al., 2003, Plant MoI. Biol. 53, 703-713) for constitutive expression, the patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) for tuber-specific expression in potatoes, or a fruit-specific promoter for tomato such as, for example, the polygalacturonase promoter from tomato (Montgomery et al., 1993, Plant Cell 5, 1049-1062) or the E8 promoter from tomato (Metha et al., 2002, Nature Biotechnol. 20(6), 613-618) or the ACC oxidase promoter from peach (Moon and Callahan, 2004, J. Experimental Botany 55 (402), 1519-1528) or a promoter which ensures expression only in photosynthetically active tissues, for example the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451), or, for endosperm-specific expression, the HMWG promoter from wheat, the USP promoter, the phaseolin promoter, promoters of zein genes from maize (Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., Plant MoI. Biol. 15 (1990), 81-93), a glutelin promoter (Leisy et al., Plant MoI. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993), 357- 366; Yoshihara et al., FEBS Lett. 383 (1996), 213-218), a globulin promoter (Nakase et al., 1996, Gene 170(2), 223-226), a prolamin promoter (Qu and Takaiwa, 2004, Plant Biotechnology Journal 2(2), 113-125), or a Shrunken-1 promoter (Werr et al., EMBO J. 4 (1985), 1373-1380). However, it is also possible to use promoters which are only activated at a point in time which is determined by external factors (see, for example, WO 9307279, whose content is incorporated into the present description by

reference). Promoters which may be of particular interest in this context are promoters of heat-shock proteins, which permit simple induction. It is furthermore possible to use seed-specific promoters, such as, for example, the USP promoter from Vicia faba, which ensures the seed-specific expression in Vicia faba and in other plants (Fiedler et al., Plant MoI. Biol. 22 (1993), 669-679; Baumlein et al., MoI. Gen. Genet. 225 (1991), 459-467).

The use of promoters which are found in the genome of viruses infecting algae may also be suitable for expressing nucleic acid sequences in plants (Mitra et al., 1994, Biochem. Biophys Res Commun 204(1), 187-194; Mitra and Higgins, 1994, Plant MoI Biol 26(1), 85-93, Van Etten et al., 2002, Arch Virol 147, 1479-1516).

In the context of the present invention, the term "tissue-specific" is understood as meaning the predominant limitation of a feature (for example the initiation of transcription) to a particular tissue.

In the context of the present invention, the terms "tuber cell, fruit cell or endosperm cell" are understood as meaning all those cells which are present in a tuber, a fruit or in an endosperm.

In the context of the present invention, the term "homologous promoter" is understood as meaning a promoter which naturally occurs in plant cells which were used for carrying out methods according to the invention (homologous with regard to the plant cell or plant) or a promoter which regulates the expression of a gene in the organism from which the respective foreign nucleic acid molecule which codes for a protein has been isolated (homologous with regard to the nucleic acid molecule to be expressed).

In the context of the present invention, the term "heterologous promoter" is understood as meaning a promoter which does not occur naturally in plant cells which were used for carrying out methods according to the invention (heterologous with regard to the plant cell or plant) or a promoter which does not occur naturally for regulating the expression of said foreign nucleic acid molecule in the organism from which the respective protein-encoding foreign nucleic acid molecule has been isolated (heterologous with regard to the nucleic acid molecule to be expressed).

A termination sequence (polyadenylation signal), which serves to add a poly-A tail to the transcript, may furthermore be present. The poly-A tail is supposed to exert a

function in stabilizing the transcript. Such elements have been described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and are interchangeable.

lntron sequences may also be present between the promoter and the coding region of the foreign nucleic acid molecule. Such intron sequences may lead to stability of the expression and to an increased expression in plants (CaINs et al., 1987, Genes Devel. 1 , 1183-1200; Luehrsen, and Walbot, 1991 , MoI. Gen. Genet. 225, 81-93; Rethmeier et al., 1997; Plant Journal 12(4), 895-899; Rose and Beliakoff, 2000, Plant Physiol. 122 (2), 535-542; Vasil et al., 1989, Plant Physiol. 91 , 1575-1579; XU et al., 2003, Science in China Series C Vol.46 No.6, 561-569). Examples of suitable intron sequences are the first intron of the sh1 gene from maize, the first intron of the poly- ubiquitin gene 1 from maize, the first intron of the EPSPS gene from rice, or one of the first two introns of the PAT1 gene from Arabidopsis.

In the context of the present invention, the term "glutamine:fructose 6-phosphate amidotransferase (GFAT)" (E. C. 2.6.1.16), also referred to as glucosamine synthase in the specialist literature, is understood as meaning a protein which synthesizes glucosamine 6-phosphate (GlcN-6-P) from the starting materials glutamine and fructose 6-phosphate (Fruc-6-P). This catalysis follows the reaction scheme

glutamine + Fruc-6-P → GlcN-6-P + glutamate

In the context of the present invention, the term "glutamine:fructose 6-phosphate amidotransferase (GFAT)" is used as an umbrella term which comprises all known isoforms.

A review article by Milewski (2002, Biochimica et Biophysica Acta 1597, 173-193) describes structural features of proteins with the activity of a GFAT. All known proteins with the activity of a GFAT have, in their amino acid sequence, regions with conserved amino acid sequences. Proteins with the activity of a GFAT have, in their amino acid sequence, an N-terminal glutamine binding domain and a C-terminal fructose 6-phosphate binding domain, which are separated from each other by a sequence of 40 to 90 nonconserved amino acids. When the two domains are present in separate amino acid molecules, both are active. Analyses of the crystal structure of a fragment comprising the N-terminal glutamine binding domain of the protein with the activity of a GFAT from Escherichia coli revealed that the active center of this domain is localized at the N-terminus and that the amino acid Cys1 is involved in the hydrolysis of glutamine. The amino acids Arg73 and Asp123 interact with carboxyl

and amino groups of the glutamine. This interaction is supported by the amino acids Thr76 and His77. The amino acids Gly99 and Trp74 are believed to form hydrogen bonds with the amide group of the glutamine. The amino acids Asn98 and Gly99 stabilize the tetrahedric pocket of the active center. The amino acids 25 to 29 and 73- 80 form flexible loops which, after binding of the substrate glutamine, contribute to the reaction catalyzed by a protein with the activity of a GFAT as the result of a conformation change of the protein. The analysis of the crystal structure of the C- terminal fructose 6-phosphate binding domain of the protein with the activity of a GFAT from Escherichia coli revealed that this domain is made up of two topological^ identical domains (amino acids 241 to 424 and 425 to 592), followed by a domain which is present at the C-terminal end as an irregular loop (amino acids 593 to 608), but only has one active center. The amino acids Ser303, Ser347, Gln348, Ser349 and Thr352 are involved in the binding to the substrate, while the amino acids Glu488, His504 and Lys603 are involved directly in the catalysis of the reaction of the protein with the activity of a GFAT.

Two different isoforms of proteins with the activity of the GFAT (referred in the literature as GFAT-1 and GFAT-2, respectively) have been detected, in particular in animal organisms. Hu et al. (2004), J. Biol. Chem. 279(29), 29988-29993) describe differences of the respective isoforms of proteins with the activity of a GFAT. Besides differences in the tissue-specific expression of the respective isoforms with the activity of a GFAT-1 and a GFAT-2, it has been possible to demonstrate that both isoforms are regulated by phosphorylation by means of a cAMP-dependent protein kinase. The activity of a protein with the enzymatic activity of a GFAT-1 is inhibited by the phosphorylation of a conserved serine residue (Serine 205 in murine GFAT-1 , Gene Bank Ace No.: AF334736.1) of the respective amino acid sequence, whereas the activity of a protein with the activity of a GFAT-2 is increased by the phosphorylation of a conserved serine residue (Serine 202 in murine GFAT-2, Gene Bank Ace No.: NM_013529) of the respective amino acid sequence. Both proteins with the activity of a GFAT-1 and proteins with the activity of a GFAT-2 are inhibited in a concentration-dependent manner by UDP-GIcNAc (UDP-N-acetyl glucosamine), however, the inhibition by UDP-GIcNAc is less for a protein with the activity of a GFAT-2 (maximum reduction of the activity by UDP-GIcNAc by approximately 15%), in comparison with a protein with the activity of a GFAT-1 (maximum reduction of the activity by UDP-GIcNAc by approx. 51% or 80%). There are indications that the inhibition of a protein with the activity of a GFAT-1 in animal organisms can be attributed to the fact that increased UDP-GIcNAc concentrations result in an O-glucose-N-acetylglucosamine glycosylation of the respective proteins. Whether a

regulation of the activity of proteins by means of O-glycosylation also takes place in plant cells is currently not fully elucidated (Huber and Hardin, 2004, Current Opinion in Plant Biotechnology 7, 318-322).

Proteins with the activity of a bacterial GFAT are distinguished by the fact that they are not inhibited by UDP-GIcNAc (Kornfeld, 1967, J. Biol. Chem. 242(13), 3135- 3141).

Proteins with the activity of a GFAT-1 , proteins with the activity of a GFAT-2 and also proteins with the activity of a bacterial GFAT are inhibited by the product generated during their reaction, glucosamine 6-phosphate (Broschat et al., 2002, J. Biol. Chem. 277(17), 14764-14770; Deng et al., 2005, Metabolic Engineering 7, 201-214).

In the context of the present invention, the term "protein with the activity of a glutamine:fructose 6-phosphate amidotransferase isoform I (GFAT-1)" is understood as meaning a protein with the activity of a GFAT and whose activity is inhibited by phosphorylation by means of a cAMP-dependent protein kinase.

In the context of the present invention, the term "protein with the activity of a glutamine:fructose 6-phosphate amidotransferase isoform Il (GFAT-2)" is understood as meaning a protein with the activity of a GFAT and which is activated by phosphorylation by means of a cAMP-dependent protein kinase.

In the context of the present invention, the term "protein with the activity of a bacterial glutamine:fructose 6-phosphate amidotransferase (bacterial GFAT)" is understood as meaning a protein which has the activity of a GFAT and whose activity is not inhibited by UDP-GIcNAc. Alternatively, "proteins with the activity of a bacterial GFAT" may also be referred to as "proteins with the activity of a noneucaryotic GFAT".

In accordance with the invention, the foreign nucleic acid molecule coding for a protein with the enzymatic activity of a GFAT may originate from any organism; said nucleic acid molecule preferably originates from bacteria, fungi, animals, plants or viruses, especially preferably from mammals, plants or bacteria, and in particular from the mouse or Escherichia coli.

As regards viruses, the foreign nucleic acid molecule coding for a protein with the enzymatic activity of a GFAT originates by preference from a virus which infects algae, preferably from a virus which infects algae of the genus Chlorella, especially preferably from a Paramecium bursaria Chlorella virus and particularly preferably from a Paramecium bursaria Chlorella virus of an H 1 strain.

Instead of a naturally occurring nucleic acid molecule coding for a protein with the enzymatic activity of a GFAT it is also possible to introduce, into plant cells, a nucleic acid molecule which has been generated by means of mutagenesis, where said mutagenized foreign nucleic acid molecule is distinguished by the fact that it codes for a protein with the enzymatic activity of a GFAT with a reduced inhibition by metabolites (for example of the glucosamine metabolism). The generation of such mutagenized nucleic acid molecules has been described by way of example for a protein with the enzymatic activity of a GFAT from Escherichia coli by Deng et al. (2005, Metabolic Engineering 7, 201-214; WO 04 003175). Mutants for a protein with the activity of a murine GFAT are described, for example, by Hu et al. (2004, J. Biol. Chem. 279 (29), 29988-29993).

Nucleic acid molecules which code for a protein with the activity of a GFAT are known to the skilled worker and described in the literature. Thus, nucleic acid molecules which code for a protein with the activity of a GFAT have been described from viruses, for example for the Chlorella virus k2 (EMBL ace No AB107976.1), from bacteria, for example for Escherichia coli (Dutka-Malen, 1988, Biochemie 70 (2), 287- 290; EMBL ace No: L10328.1), from fungi for, for example, Saccharomyces cerevisiae (EMBL ace No AF334737.1, Watzele et al., 1989, J. Biol. Chem. 264, 8753-8758), Aspergillus niger (EMBL ace No AY594332.1), Candida albicans (EMBL ace No X94753.1), from insects for, for example, Aedes aegypti (Kato et al., 2002, Insect. Biol. 11 (3), 207,216; EMBL ace No AF399922.1), Drosophila melanogaster (GFAT-1 : EMBL ace No Y18627.1 , GFAT-2: NCBI ace No NMJ43360.2), from algae for Volvariella volvacea (EMBL ace No AY661466.1), from vertebrates for, for example, Homo sapiens (GFAT-1 : EMBL ace No AF334737.1 ; GFAT-2: NCBI ace No BC000012.2, Oki et al., 1999, Genomics 57 (2),227-34), Mus musculυs (GFAT-1 : EMBL ace No AF334736.1 ; GFAT-2: EMBL ace No AB016780.1), or from plants for, for example, Arabidopsis thaliana (EMBLI ace No AP001297.1 ; cds NCBI ace No BAB03027.1).

In a preferred embodiment, a protein with the activity of a GFAT-2 or of a bacterial GFAT is introduced, into a plant cell, in methods according to the invention for the production of modified starch.

In a preferred embodiment, the present invention relates to methods according to the invention for the production of modified starch, wherein the foreign nucleic acid molecule coding for a protein with the activity of a GFAT is selected from the group

comprising a) nucleic acid molecules which code for a protein with the amino acid sequence shown in SEQ ID NO 2 or a protein with the amino acid sequence shown in

SEQ ID NO 5; b) nucleic acid molecules which code for a protein whose sequence has at least

60%, by preference at least 70%, preferably at least 80%, especially preferably at least 90%, particularly preferably at least 95% and specifically preferably at least 97% identity with the amino acid sequence shown in SEQ ID NO 2 and

SEQ ID NO 5; c) nucleic acid molecules which comprise the nucleotide sequence shown in SEQ

ID NO 1 or in SEQ ID NO 3 or in SEQ ID NO 4, or a complementary sequence; d) nucleic acid molecules which have at least 70%, by preference at least 80%, preferably at least 90%, especially preferably at least 95%, particularly preferably at least 97% and specifically preferably at least 98% identity with the nucleic acid sequences described in a) or c); e) nucleic acid molecules which hybridize under stringent conditions with at least one strand of the nucleic acid sequences described under a) or c); f) nucleic acid molecules whose nucleotide sequence deviates from the sequence of the nucleic acid molecules mentioned under a) or c) as the result of the degeneracy of the genetic code; and g) nucleic acid molecules which are fragments, allelic variants and/or derivatives of the nucleic acid molecules mentioned under a), b), c), d), e) or T).

In the context of the present invention, the term "hybridization" means hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, in Sambrock et al., Molecular Cloning, A Laboratory Manual,

2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Especially preferably, "hybridization" means a hybridization under the following conditions: hybridization buffer:

2xSSC; 10xDenhardt solution (Ficoll 400+PEG+BSA; ratio 1 :1 :1); 0.1% SDS; 5 mM

EDTA; 50 mM Na2HPO4; 250 μg/ml herring sperm DNA; 50 μg/ml tRNA; or

25 M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS hybridization temperature: T=65 to 68°C wash buffer: O.ixSSC; 0.1% SDS wash temperature: T=55 to 65°C hybridization time: overnight (approx. 12 to 16 hours)

washing time: 15 minutes

Nucleic acid molecules which hybridize with the abovementioned molecules may be isolated for example from genomic libraries or from cDNA libraries. The identification and isolation of such nucleic acid molecules may be effected using the abovementioned nucleic acid molecules or parts of these molecules, or the reverse complements of these molecules, for example by means of hybridization by standard methods (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), or by amplification by means of PCR. Hybridization probes for isolating a nucleic acid sequence coding for a protein with the activity of a GFAT which may be used are, for example, nucleic acid molecules which have precisely or essentially the nucleotide sequence shown in SEQ ID NO 1 or the nucleotide sequence shown in SEQ ID NO 3 or the nucleotide sequence shown in SEQ ID NO 4, or parts of these sequences. The fragments used as hybridization probe may also take the form of synthetic fragments or oligonucleotides which have been generated with the aid of the current synthetic techniques and whose sequence essentially agrees with that of a nucleic acid molecule described in the context of the present invention. When genes which hybridize with the nucleic acid sequences described in the context of the present invention have been identified and isolated, the sequence should be determined and the properties of the proteins encoded by this sequence should be analyzed in order to find out whether they are proteins with the activity of a GFAT. Methods for determining whether a protein has the activity of a protein with the activity of a GFAT are known to the skilled worker and described, inter alia, in the literature cited (for example Mayer et al., 1968, Plant Physiol. 43, 1097-1107; Deng et al., 2005, Metabolic Engineering 7, 201-214), GFAT-1 or GFAT-2 (for example Hu et al., 2004, J. Biol. Chem. 279 (29), 29988-29993)).

The molecules hybridizing with the nucleic acid molecules described in the context of the present invention comprise in particular fragments, derivatives and allelic variants of the nucleic acid molecules mentioned. In the context of the present invention, the term "derivative" means that the sequences of these molecules differ in one or more positions from the sequences of the nucleic acid molecules described above and are highly identical to these sequences. The differences from the nucleic acid molecules described above may, for example, be due to deletion, addition, substitution, insertion or recombination.

In the context of the present invention, the term "identity" means a sequence identity

over the entire length of the coding region of a nucleic acid molecule or the entire length of an amino acid sequence coding for a protein of, by preference, at least 60%, in particular an identity of at least 70%, preferably of at least 80%, particularly preferably of at least 90% and especially preferably of at least 95% and specifically preferably of at least 98%. In the context of the present invention, the term "identity" is to be understood as meaning the number of amino acids/nucleotides (identity) in agreement with other proteins/nucleic acids, expressed in percent. Preferably, the identity with respect to a protein having the activity of a GFAT is determined by comparisons with the amino acid sequence given under SEQ ID NO 2 or SEQ ID NO 5 and the identity with respect to a nucleic acid molecule coding for a protein having the activity of a GFAT is determined by comparisons of the nucleic acid sequence given under SEQ ID NO 1 or SEQ ID NO 6 or SEQ ID NO 3 or SEQ ID NO 4 with other proteins/nucleic acids with the aid of computer programs. If sequences to be compared with one another are of different lengths, the identity is to be determined by determining the identity in percent of the number of amino acids which the shorter sequence shares with the longer sequence. Preferably, the identity is determined using the known and publicly available computer program ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalW is made publicly available by Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1 , D 69117 Heidelberg, Germany. ClustalW can also be downloaded from various Internet pages, inter alia from IGBMC (Institut de Genetique et de Biologie Moleculaire et Cellulaire, B. P.163, 67404 lllkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and from EBI (ftp://ftp.ebi.ac.uk/pub/software/) and all mirrored Internet pages of the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1 SD, UK).

Preferably, use is made of the ClustalW computer program of version 1.8 to determine the identity between proteins described in the context of the present invention and other proteins. Here, the parameters have to be set as follows: KTUPLE=I , TOPDIAG=5, WIND0W=5, PAIRGAP=3, GAPOPEN=IO, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET,

ENDGAPS(OFF), NOPGAP, NOHGAP. Preferably, use is made of the ClustalW computer program of version 1.8 to determine the identity for example between the nucleotide sequence of the nucleic acid molecules described in the context of the present invention and the nucleotide sequence of other nucleic acid molecules. Here, the parameters have to be set as

follows:

KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=IO,

GAPEXT=5, MAXDIV=40, TRANSITIONS: unweighted.

Identity furthermore means that there is a functional and/or structural equivalence between the nucleic acid molecules in question or the proteins encoded by them. The nucleic acid molecules which are homologous to the molecules described above and represent derivatives of these molecules are generally variations of these molecules which represent modifications having the same biological function. They may be either naturally occurring variations, for example sequences from other species, or mutations, where these mutations may have occurred in a natural manner or were introduced by targeted mutagenesis. Furthermore, the variations may be synthetically produced sequences. The allelic variants may be either naturally occurring variants or synthetically produced variants or variants generated by recombinant DNA techniques. A special form of derivatives are, for example, nucleic acid molecules which differ from the nucleic acid molecules described in the context of the present invention as a result of the degeneracy of the genetic code.

In a further preferred embodiment, the present invention relates to methods according to the invention for the production of modified starch wherein the foreign nucleic acid molecule coding for a protein with the enzymatic activity of a GFAT comprises modifying the codons in said nucleic acid molecule in comparison with the codons of the nucleic acid molecule which code said protein with the enzymatic activity of a GFAT of the starting organism. The codons of the foreign nucleic acid molecule coding for a protein with the enzymatic activity of a GFAT are especially preferably modified in such a way that they are adapted to the frequency of the codon usage of the plant cell or of the plant into whose genome they are integrated or being integrated.

As a result of the degeneracy of the genetic code, amino acids can be encoded by one or more codons. In different organisms, the codons coding for an amino acid are used at different frequencies. Adapting the codons of a coding nucleic acid sequence to the frequency of their use in the plant cell or in the plant into whose genome the sequence to be expressed is to be integrated may contribute to an increased amount of translated protein and/or to the stability of the mRNA in question in the particular plant cells or plants. The frequency of use of codons in the plant cells or plants in question can be determined by the person skilled in the art by examining as many

coding nucleic acid sequences of the organism in question as possible for the frequency with which certain codons are used for coding a certain amino acid. The frequency of the use of codons of certain organisms is known to the person skilled in the art and can be determined in a simple and rapid manner using computer programs. Such computer programs are publicly accessible and provided for free inter alia on the Internet (for example http://gcua.schoedl.de/; http://www.kazusa.or.jp/codon/; http://www.entelechon.com/eng/cutanalysis.html). Adapting the codons of a coding nucleic acid sequence to the frequency of their use in the plant cell or in the plant into whose genome the sequence to be expressed is to be integrated can be carried out by in vitro mutagenesis or, preferably, by de novo synthesis of the gene sequence. Methods for the de novo synthesis of nucleic acid sequences are known to the person skilled in the art. A de novo synthesis can be carried out, for example, by initially synthesizing individual nucleic acid oligonucleotides, hybridizing these with oligonucleotides complementary thereto, so that they form a DNA double strand, and then ligating the individual double-stranded oligonucleotides with one another such that the desired nucleic acid sequence is obtained. The de novo synthesis of nucleic acid sequences including the adaptation of the frequency with which the codons are used to a certain target organism can also be sourced out to companies offering this service (for example Entelechon GmbH, Regensburg, Germany).

A multiplicity of techniques is available for introducing nucleic acid molecules into a plant host cell. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as means of transformation, protoplast fusion, injection, electroporation of DNA, introduction of DNA by the biolistic approach and also further options (review in "Transgenic Plants", Leandro ed., Humana Press 2004, ISBN 1-59259-827-7).

The use of Agrobacterium-meώaled transformation of plant cells has been subject to in-depth studies and has been described exhaustively in EP 120516 and Hoekema, IN: The Binary Plant Vector System Offsetdrukkerij Kanters B.V. Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4, (1985), 1-46 and in An et al. EMBO J. 4, (1985), 277-287. For the transformation of potatoes see, for example, Rocha-Sosa et al., EMBO J. 8, (1989), 29-33, for the transformation of tomato plants see, for example, US 5,565,347.

The transformation of monocotyledonous plants using vectors based on Agrobacterium transformation has been described, too (Chan et al., Plant MoI. Biol.

22, (1993), 491-506; Hiei et al., Plant J. 6, (1994) 271-282; Deng et al, Science in China 33, (1990), 28-34; Wilmink et al., Plant Cell Reports 11 , (1992), 76-80; May et al., Bio/Technology 13, (1995), 486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al, Transgenic Res. 2, (1993), 252-265). An alternative system for transforming monocotyledonous plants is the transformation using the biolistic approach (Wan and Lemaux, Plant Physiol. 104, (1994), 37-48; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant MoI. Biol. 24, (1994), 317- 325; Spencer et al., Theor. Appl. Genet. 79, (1990), 625-631), the protoplast transformation, the electroporation of partially permeabilized cells, the introduction of DNA using glass fibers. In particular the transformation of maize has been described several times in the literature (cf., for example, WO95/06128, EP0513849, EP0465875, EP0292435; Fromm et al., Biotechnology 8, (1990), 833-844; Gordon- Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appl. Genet. 80, (1990), 721-726). The transformation of other grasses, such as, for example, switchgrass (Panicum virgatum) has also been described (Richards et al., 2001 , Plant Cell Reporters 20, 48-54). The successful transformation of other cereal species has likewise already been described, for example for barley (Wan and Lemaux, loc. cit.; Ritala et al., loc. cit.; Krens et al., Nature 296, (1982), 72-74) and for wheat (Nehra et al., Plant J. 5, (1994), 285-297; Becker et al., 1994, Plant Journal 5, 299-307). All of the above methods are suitable in the context of the present invention.

Plants into which a foreign nucleic acid molecule coding for a protein with the activity of a GFAT has been introduced can be distinguished in comparison with corresponding wild-type plants which have not been genetically modified, inter alia, by the fact that they comprise a foreign nucleic acid molecule which does not naturally occur in wild-type plants, or by the fact that such a molecule is integrated at a location in the plant genome at which it does not occur in wild-type plants, i.e. in a different genomic environment. Furthermore, such plants can be distinguished from non-genetically-modified wild-type plants by the fact that they comprise at least one copy of the foreign nucleic acid molecule stably integrated into their genome, if appropriate in addition to copies of such a molecule which naturally occur in the wild- type plants. If the foreign nucleic acid molecule which has been introduced into the genetically modified plants takes the form of additional copies of molecules which already naturally occur in the wild-type plants, then the genetically modified plants can be distinguished from wild-type plants in particular by the fact that this additional copy is located at locations in the genome at which it does not occur in wild-type

plants. A foreign nucleic acid molecule which has been introduced into a plant cell can be detected by genetic and/or molecular biology methods. A nucleic acid molecule which has been introduced into a plant cell can be detected, inter alia, with the aid of the Southern blot analysis, the RFLP analysis (Restriction Fragment Length Polymorphism) (Nam et al., 1989, The Plant Cell 1 , 699-705; Leister and Dean, 1993, The Plant Journal 4 (4), 745-750), by PCR-based methods, such as, for example, the analysis of amplified fragment length polymorphisms (AFLP) (Castiglioni et al., 1998, Genetics 149, 2039-2056; Meksem et al., 2001 , Molecular Genetics and Genomics 265, 207-214; Meyer et al., 1998, Molecular and General Genetics 259, 150-160) or by the use of amplified fragments which have been cleaved with restriction endonucleases (Cleaved Amplified Polymorphic Sequences, CAPS) (Konieczny and Ausubel, 1993, The Plant Journal 4, 403-410; Jarvis et al., 1994, Plant Molecular Biology 24, 685-687; Bachem et al., 1996, The Plant Journal 9 (5), 745-753).

The regeneration of the plants in accordance with step b) of the methods according to the invention for the production of modified starch can be effected by methods known to the skilled worker (for example described in "Plant Cell Culture Protocols", 1999, edt. by R. D. Hall, Humana Press, ISBN 0-89603-549-2).

The generation of further plants of the methods according to the invention for the production of modified starch can be effected for example by vegetative propagation (for example via cuttings, tubers or via callus culture and regeneration of intact plants) or by generative propagation. Generative propagation in this context preferably takes place in a controlled manner, that is to say selected plants with specific properties are hybridized with one another and propagated. The selection is preferably carried out in such a manner that the further plants contain the foreign nucleic acid molecules which have been introduced in the preceding steps.

The plant cells used in the methods according to the invention of producing modified starch, into which plants cells a foreign nucleic acid molecule is introduced, may be plant cells of plants of any plant species, i.e. both monocotyledonous and dicotyledonous plants. They are preferably useful plants, i.e. plants which are grown by man for the purposes of human and animal nutrition (for example maize, rice, wheat, rye, oats, barley, cassava, potato, tomato, switch grass (Panicum virgatum), sago, mung bean, pea, sorghum, carrot. They are especially preferably rice, maize, wheat or potato plants.

Processes for extracting starch from plants or from starch-storing parts of plants are known to the skilled worker. In addition, processes are described for extracting the starch from various starch-storing plants, for example in Starch: Chemistry and Technology (Hrsg.: Whistler, BeMiller and Paschall (1994), 2nd edition, Academic Press Inc. London Ltd; ISBN 0-12-746270-8; see, for example, chapter XII, page 412-468: maize and sorghum starches: production; by Watson; chapter XIII, page 469-479: tapioca, arrowroot and sago starches: production; by Corbishley and Miller; chapter XIV, page 479-490: potato starch: production and uses; by Mitch; chapter XV, page 491 to 506: wheat starch: production, modification and uses; by Knight and Oson; and chapter XVI, page 507 to 528: rice starch: production and uses; by Rohmer and Klem; maize starch: Eckhoff et al., Cereal Chem. 73 (1996), 54-57, the extraction of maize starch on an industrial scale is generally achieved by what is termed "wet milling". Devices which are customarily used in methods for extracting starch from plant material are separators, decanters, hydrocyclones, spray dryers and fluidized-bed dryers.

In a preferred embodiment, the method according to the invention of producing a modified starch is a method of producing a resistant starch (RS).

In a further preferred embodiment, methods according to the invention of producing a modified starch additionally comprise a method step in which the starch is first isolated from the plant and then heated or gelatinized.

A preferred embodiment of methods according to the invention of producing a modified starch relates to methods in which the modified starch, after having been heated or gelatinized, has a resistant starch (RS) content of at least 40%, preferably at least 45%, especially preferably at least 50%, particularly preferably at least 55% and specifically preferably at least 60%.

A further preferred embodiment of methods according to the invention of producing a modified starch relates to methods in which the modified starch, after having been heated or gelatinized, has a resistant starch (RS) content of 40% to 80%, preferably 40% to 70%, especially preferably 40% to 60%, and particularly preferably 50% to 60%.

Modified starch produced in a method according to the invention of producing a

modified starch are preferably furthermore distinguished by the fact that they synthesize a starch with a reduced content of readily digestible starch (RDS).

Accordingly, a further preferred embodiment of methods according to the invention of producing a modified starch relates to methods in which the modified starch, after heating or gelatinizing, has a readily digestible starch (RDS) content of not more than 20%, preferably not more than 17%, especially preferably not more than 16% and particularly preferably not more than 15%.

A further preferred embodiment of methods according to the invention of producing a modified starch relates to methods in which the modified starch, after heating or gelatinizing, has a readily digestible starch (RDS) content of 10% to 18%, preferably 12% to 18%, especially preferably 13% to 17% and particularly preferably 15% to 17%.

Modified starch produced in a method according to the invention of producing a modified starch is preferably also distinguished by the fact that it has a reduced slowly digestible starch (SDS) content.

Accordingly, a further preferred embodiment of methods according to the invention of producing a modified starch relates to methods in which the modified starch, after heating or gelatinizing, has a slowly digestible starch (SDS) content of not more than 45%, preferably not more than 40%, especially preferably not more than 35%, particularly preferably not more than 30% and specifically preferably not more than 25%.

A further preferred embodiment of methods according to the invention of producing a modified starch relates to methods in which the modified starch, after heating or gelatinizing, has a slowly digestible starch (SDS content) of 20% to 45%, preferably 20% to 40%, especially preferably 25% to 45% and particularly preferably 25% to 42%.

In the context of the present invention, the term "resistant starch (RS)" is to be understood as meaning the percentage of starch which, after heating or gelatinization and subsequent digestion with pancreatin, has not been digested after 120 minutes.

The skilled worker knows how to prepare a gelatinized starch. To this end, starch is

heated with stirring until the granular structure of the starch disappears. Gelatinization can be effected for example with the aid of apparatuses designed for starch analyses, such as a Rapid Visco Analyzer (Newport Scientific) or a Brabender apparatus (Brabender).

A preferred method of gelatinizing starch is described under General Methods, item 7.

The skilled worker also knows how to determine the content of resistant starch. The content of resistant starch can be determined for example with the AOAC Method 2002.02 or the AACC Method 32-40.

In the context of the present invention, the RS content of starch is preferably determined using the method of Englyst et al. (Europ. J. of Clinical Nutrition 46 (Suppl. 2), (1992), pp. 33-50, see, in particular, the following sections of Englyst et al., page 35-36: "Reagents, Apparatus, Spectrophotometer"; page 36-37, paragraph "Measurement of free glucose (FG)"; page 38, paragraph "Measurement of RDS and SDS"). The resistant starch content (RS) of the starch is understood as meaning the percentage of the starch sample weighed (dry weight) which is not liberated as glucose after 120 minutes by the method described. Accordingly, it can be calculated using the following formula:

RS [%] =

100% - 100% x (glucose liberated after 120 minutes in mg)/(starch dry weight in mg)

In the context of the present invention, the term "readily digestible starch (RDS) content" is understood as meaning the starch content which is liberated as glucose after 20 minutes in the abovementioned method of Englyst et al. for determining the RS content. Here, the data given as percent by weight relates to the starch sample dry weight employed for the determination. Accordingly, the following applies in the context of the present invention:

RDS [% ] =

100% x (glucose liberated after 20 minutes [mg])/(starch dry weight [mg])

In the context of the present invention, the term "slowly digestible starch (SDS) content" is understood as meaning the starch content which is liberated as glucose in

the time interval of between 20 minutes and 120 minutes in the abovementioned method of Englyst et al. for determining the RS content. Here, the data given as percent by weight relates to the starch sample dry weight. Accordingly, the following applies in the context of the present invention:

SDS [% ] =

(glucose liberated after 120 minutes [%])-(glucose liberated after 20 minutes [%])

A modified starch produced by a method according to the invention of producing a modified starch is furthermore preferably distinguished by the fact that it has a reduced granule size in comparison with starch which has been isolated from corresponding wild-type plants.

A further preferred embodiment of methods according to the invention of producing a modified starch therefore relates to methods of producing a modified starch with a reduced granule size in comparison with starch which has been isolated from corresponding wild-type plants. The mean granule size of the starch preferably amounts to not more than 60%, especially preferably not more than 55%, particularly preferably not more than 50% in comparison with the granule size of starch which has been isolated from corresponding wild-type plants.

As regards a starch isolated from a potato plant or from tubers of a potato plant, the mean granule size of the starch, produced by means of a method according to the invention of producing a modified starch, amounts by preference to 15 μm to 30 μm, preferably 17 μm to 28 μm, especially preferably 19 μm to 27 μm and particularly preferably 20 μm to 26 μm.

Methods for determining the granule size of starch are known to the skilled worker. In the context of the present invention, the granule size is preferably determined by means of the method described under General Methods, item 6 e).

In comparison with traditional starches, starches with a reduced granule size have the advantage that they alter the texture of foodstuffs into which they are mixed to a lesser degree. If granular starches are added to foodstuffs, this frequently entails negative effects on the texture of the foodstuff, and, upon consumption, the size of the starch granules frequently leads, to an undesirable, frequently sandy, mouth feel. This negative effect is less pronounced when smaller starch granules are present.

Starches produced in a method according to the invention of producing a modified starch are therefore better suited to the admixture to foodstuffs.

Furthermore, it has surprisingly been found that starches produced in a method according to the invention of producing a modified starch may have altered viscosity properties in comparison with starch which has been isolated from corresponding wild-type plants.

The skilled worker frequently uses different methods of determining the viscosity properties of starches, among which are maximum viscosity and final viscosity. A rapid and effective method of analyzing the gelatinization properties is the RVA analysis.

A protocol of carrying out the RVA analysis is described under General Methods, item 6 c).

Accordingly, the present invention preferably also relates to methods according to the invention of producing a modified starch which has modified viscosity properties in comparison with starch which has been isolated from corresponding wild-type plants.

Starches produced in a method according to the invention of producing a modified starch preferably have, in the RVA analysis, a reduced maximum viscosity (RVA

Max) and/or an increased final viscosity in comparison with starch isolated from corresponding wild-type plants.

The maximum viscosity in the RVA analysis of starch produced in a method according to the invention of producing a modified starch is preferably at least 25%, especially preferably at least 30% and particularly preferably at least 50% lower in comparison with starch which has been isolated from corresponding wild-type plants.

The final viscosity of starch produced in a method according to the invention of producing a modified starch is, in the RVA analysis, preferably at least 20%, especially preferably at least 30% and particularly preferably at least 39% higher in comparison with starch which has been isolated from corresponding wild-type plants.

In the context of the present invention, the term "wild-type plant" means plants whose cells have been used as starting material for introducing a foreign nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6-phosphate amidotransferase (GFAT) in a method according to the invention, i.e. whose genetic information, with the exception of the presence of said foreign nucleic acid molecule, corresponds to that of a plant generated by means of a method according to the

invention.

In the context of the present invention, the term "corresponding" means that, when comparing a plurality of objects, the respective objects which are compared with one another are kept under identical conditions. In the context of the present invention, the term "corresponding" means in connection with wild-type plants that the plants which are compared with one another are grown under identical culture conditions and have the same (culture) age.

Starches, once gelatinized and then cooled, frequently form stable gels. In order to prepare starch gels, the crystalline structure of native starch must first be destroyed by heating in aqueous suspension with constant stirring. This can be carried out by methods known to the skilled worker, for example with the aid of a Rapid Visco Analyzer (Newport Scientific Pty Ltd., Investmet Support Group, Warriewod NSW 2102, Australia). To determine the gel strength, the gelatinized starch suspensions are stored for a certain period of time, and the gels formed as the result of storage are subsequently examined for their strength. The gel strength can be determined by methods known to the skilled worker, for example using what is known as a "Texture Analyzers". A method preferably to be employed in the context of the present invention of determining gel strength is described under General Methods, item 6 d). The property of forming stable starch gels is exploited in many ways in the preparation of foodstuffs.

Starches produced by a method according to the invention are preferably also distinguished by the fact that, once gelatinized, they form gels with an increased strength.

Accordingly, the present invention also preferably relates to methods according to the invention of producing a modified starch which, once gelatinized, forms gels with an increased strength (gel strength) in comparison with starch which has been isolated from corresponding wild-type plants. The gel strength of starch produced in a method according to the invention of producing a modified starch is preferably at least 100%, especially preferably at least 200%, particularly preferably at least 250%, specifically preferably at least 300% higher in comparison with starch which has been isolated from corresponding wild-type plants.

Starches produced in a method according to the invention of producing a modified

starch which have an increased final viscosity in the RVA analysis and which form gels with an increased strength are especially suitable for all those applications in which the thickening power, the gelling properties or the binding properties of added substances is of importance. In order to attain for example a certain thickening power or the formation of a gel in foodstuffs, smaller quantities of starch produced in a method according to the invention of producing a modified starch need to be employed in comparison with starch isolated from wild-type plants. The calorific content of the foodstuff in question can thereby be reduced further, in addition to the reduced percentage which is the result of the increased RS content, which has the above-described advantages for the consumer. The starch according to the invention is therefore particularly suitable for the preparation of foodstuffs such as, for example, baked goods, instant food, blancmange, soups, confectionery, chocolate, ice cream, batters for fish or meat, frozen puddings or extruded snacks.

The transition of starch from the crystalline form into the amorphous form can be shown by means of DSC analysis. The endothermal curves determined by means of

DSC are characterized in greater detail by various parameters (To, Tp, T _e and dH).

The onset temperature To characterizes the beginning of the thermal conversion.

The value for T _P (T _P = DSC peak temperature) indicates the temperature at which the maximum thermal reaction of the crystalline material takes place, while T _e is the temperature at which the conversion process is completed (final temperature).

The DSC conversion enthalpy (deltaH) is determined by calculating the peak area. It represents the total energy required for the transformation.

Starches produced by a method according to the invention of producing a modified starch preferably also show altered thermal stability in comparison with starches isolated from corresponding wild-type plants.

The thermal stability of starches can be determined with the aid of what is known as DSC ("Differential Scanning Calorimetry"). The DSC method is known to the skilled worker. Results of DSC measurements are exploited inter alia for characterizing the thermal stability of RS. The DSC method preferably to be used in the context of the present invention is described under General Methods, item 6 f).

In a further preferred embodiment, the present invention relates to methods according to the invention of producing a modified starch, wherein the thermal stability of the starch has an increased DSC onset temperature (To) and/or an increased DSC peak temperature (T _P ) in comparison with starch isolated from wild-

type plants.

Preferably, starches produced by a method according to the invention for producing a modified starch have a DSC onset temperature (T ₀ ) which is increased by at least 1°C, particularly preferably by at least 2°C and especially by at least 2.5°C in comparison with starch isolated from corresponding wild-type plants.

Preferably, starches produced by a method according to the invention for producing a starch have a DSC peak temperature (T _p ) which is increased by at least TC, particularly preferably by at least 2°C and especially by at least 3.5°C in comparison with starch isolated from corresponding wild-type plants.

After heating or gelatinization and subsequent cooling of starch, more or less crystalline structures form again, at least in part, depending on the nature of the starch (retrogradation). The content of crystalline structures in retrograded starch can also be determined with the aid of DSC analysis by determining the DSC conversion energy (deltaH) required for the transition from the crystalline form into the amorphous form. A method of determining deltaH which is preferred in the context of the present invention is described under General Methods, item 6 f).

In a further preferred embodiment, the present invention relates to methods according to the invention of producing a modified starch in which the thermal stability of retrograded starch produced by a method according to the invention of producing a modified starch has an increased DSC conversion enthalpy (deltaH) in comparison with starch isolated from wild-type plants. Preferably, starches produced by a method according to the invention of producing a starch have, after retrogradation, a DSC conversion enthalpy (deltaH) which is increased by at least 1 J/g, especially preferably by at least 1.5 J/g, particularly preferably by at least 2.0 J/g and specifically preferably by at least 2.5 J/g in comparison with starch which has been isolated from corresponding wild-type plants and then retrograded.

A significant change in the amylose content or the content of phosphate bound at the C-6 position of the starch's glucose molecules in starch produced by a method according to the invention of producing a modified starch in comparison with starch isolated from corresponding wild-type plants has not been detected.

The present invention also relates to starch obtainable by a method according to the invention of producing a modified starch.

The present invention furthermore relates to the use of a nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6-phosphate amidotransferase (GFAT) for producing a modified starch.

Foodstuffs (food) which contain(s) a starch according to the invention or a starch produced by a method according to the invention of producing a modified starch are distinguished by the fact that they have a reduced calorific content. Furthermore, the high RS content of starches produced in a method according to the invention of producing a modified starch leads to a prebiotic effect in the colon, since that is where the growth of beneficial bacteria is promoted. It is additionally known that RS is a preferred substrate for butyrate-producing bacteria. Butyrate is the main energy source of colonocytes. A high butyrate content in the colon inhibits the development of bowel cancer.

Accordingly, the present invention furthermore relates to foodstuffs comprising starch according to the invention or starch produced in a method according to the invention of producing a modified starch. The foodstuff according to the invention preferably takes the form of baked goods (for example bread, bread rolls, baked goods, cake, waffles), cereal products (for example flour, pasta, muesli bars), sweets (for example chocolate, confectionery products, ice cream, wine gum, boiled sweets, puddings), meat products and processed meats (for example meat broth, meat-based soups), sausage products (for example sausage salad, grits, jelly), dairy products (for example yoghurt, whey, milkshakes), vegetable products (for example vegetable broth, vegetable-based soups, salads), potato products (for example potato salad, crisps, French fries, mashed potato).

In the context of the present invention, the term "foodstuffs (food)" are understood as meaning all those substances and products which, if appropriate, having beforehand been prepared, are ingested by mouth by humans for nutritional and/or enjoyment purposes. These include foodstuffs, including luxury foodstuffs, food additives and food supplements.

Foodstuffs also include for example drinks, chewing gum and all substances including water which are added deliberately to the foodstuff when the latter is prepared, worked or processed.

The following are not foodstuffs for the purposes of the present invention: feedstuffs (within the meaning of (EC) Regulation No. 178/2002, 28 January 2002), plants

before harvesting, pharmaceuticals (within the meaning of the Directives 65/65/EEC, 09.02.1965 including the amendments in 93/39/EEC, 24.08.1993 and 92/73/EEC, 13.10.1992) tobacco (within the meanings of Directives 89/622/EEC, 08.12.1989 including the amendments in 92/41 /EEC, 11.06.1992) and narcotics and psychotropic substances (within the meaning of the United Nations' harmonization agreement on addictive drugs, 1961 , and the United Nations' agreement on psychotropic substances, 1971).

The present invention furthermore relates to a method of producing a foodstuff wherein starch according to the invention or starch produced in a method according to the invention of preparing a modified starch is added to a foodstuff.

The present invention furthermore relates to the use of starch according to the invention or starch produced in a method according to the invention of producing a modified starch for the preparation of a foodstuff, preferably a foodstuff with reduced calorific content. Preferred is the use of starch according to the invention or starch produced in a method according to the invention of producing a modified starch for the preparation of a foodstuff which is suitable for the nutrition of diabetics.

Genetically modified plants into which a foreign nucleic acid molecule coding for a protein with the activity of a GFAT has been introduced or parts of these plants are distinguished by the fact that they synthesize a starch which has the already- described properties. Such plants are preferably furthermore distinguished by an increased content in N-acetylated glucosamine derivatives. N-acetylglucosamine has a stimulating effect on the growth of bifidobacteria (Liepke et al., 2002, Eur. J. Biochem. 269, 712-718). Furthermore, it has been demonstrated that N- acetylglucosamine acts as a substrate for fish gut lactobacilli (for example Lactobacillus casei subspecies paracasei) (Adolfo Bucio Galindo, 2004, Proefschrift, Wageningen Universiteit, ISBN 90-5808-943-6). Accordingly, N-acetylglucosamine has a positive effect on probiotic bacteria. Plants with increased contents of N-acetylglucosamine should have a positive effect on the growth of probiotic bacteria. Furthermore, N-acetylated glucosamine derivatives are sold for example as food additives which are supposed to have a prophylactic effect on the development of joint diseases (for example arthroses). Genetically modified plants into which a foreign nucleic acid molecule coding for a protein with the activity of a GFAT has been introduced or parts of these plants are therefore firstly distinguished by the fact that they synthesize a starch with an

increased RS content which, upon consumption, has a positive effect on the gut flora. The N-acetylated glucosamine derivatives, which are additionally preferably synthesized in said plants, may firstly have a further positive effect on the gut flora of the consumer, but, as the result of being continuously ingested, may also bring about a prophylactic effect for preventing joint diseases.

Said plants or their parts are therefore firstly particularly suitable for the preparation of foodstuffs with a reduced calorific content. Secondly, as has already been mentioned, they may have a positive effect on the consumer's stomach and gut flora and/or may prevent the development of joint diseases. N-Acetylglucosamine is furthermore distinguished by the fact that it has a fresh, sweet taste, whereas for example glucosamine tastes bitter. Genetically modified plants into which a foreign nucleic acid molecule coding for a protein with the activity of a GFAT has been introduced, or parts of these plants, are therefore additionally distinguished by the fact that they comprise N-acetylglucosamine derivatives which mediate sweetening power. In contrast to traditional substances which mediate sweetening power (for example sucrose), the additional advantage of said plants or parts of these plants is that they have no negative effects on the consumer, such as, for example, the development of tooth decay or the generation of an increased glycemic index.

Accordingly, the present invention furthermore relates to foodstuffs comprising genetically modified plant cells or parts of genetically modified plants which contain a foreign nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6-phosphate amidotransferase (GFAT), where said plant cells or plants synthesize a modified starch and/or have an increased content of N-acetylated glucosamine derivatives in comparison with corresponding wild-type plant cells or wild-type plants.

Foodstuffs according to the invention preferably comprise a resistant starch which has the properties of starch produced by a method according to the invention of preparing a modified starch.

In the context of the present invention, the term "N-acetylated glucosamine derivatives" is understood as meaning all derivatives of glucosamine (2-amino- 2-deoxyglucose), which also includes epimers such as, for example, galactosamine (2-amino-2-deoxygalactose) or mannosamine (2-amino-2-deoxymannose) which are measured by the method described in General Methods, item 3. The N-acetylated glucosamine derivatives preferably take the form of N-acetylglucosamine phosphate

(N-acetylglucosamine 1 -phosphate and/or N-acetylglucosamine 6-phosphate), N-acetylglucosamine and/or UDP-N-acetylglucosamine.

N-Acetylated glucosamine derivatives can be detected by methods known to the skilled worker (Morgan and Elson (1934, Biochem J. 28(3), 988-995). In the context of the present invention, the method of determining the N-acetylated glucosamine derivative content described under General Methods, item 3, is preferably used.

Foodstuffs according to the invention preferably also contain increased amounts of glucosamine phosphate (glucosamine 1 -phosphate and/or glucosamine 6-phosphate). These substances can be detected by the skilled worker using known methods, for example with the aid of mass spectroscopy.

In the context of the present invention, the term "parts of plants" is understood as meaning for example parts of plants capable of being processed which are used in the preparation of foodstuffs, which are employed as a feedstock for industrial processes (for example for the isolation of starch), as a feedstock for the preparation of pharmaceutical products or as a feedstock for the preparation of cosmetic products (for example for the isolation of N-acetylated glucosamine derivatives). In the context of the present invention, the term "parts of plants" is understood furthermore as meaning for example "consumable plant parts" which act as foodstuffs for humans.

Preferred "parts of plants" are fruits, storage roots, roots, flowers, buds, shoots, leaves or stems, especially preferably seeds, fruits, grains or tubers.

Foodstuffs according to the invention preferably comprise, in addition to modified starch according to the invention, by preference at least 0.05%, preferably at least 0.1%, especially preferably at least 0.5%, particularly preferably at least 1.0%, of N-acetylated glucosamine derivatives per gram of dry weight. Compositions according to the invention comprise, by preference, additionally to modified starch according to the invention no more than 10%, preferably no more than 5%, especially preferably no more than 3%, particularly preferably no more than 2%, of N-acetylated glucosamine derivatives per gram of dry weight.

Foodstuffs according to the invention comprising genetically modified plant cells or parts of genetically modified plants which contain a foreign nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6-phosphate

amidotransferase (GFAT) have the advantage over the prior art that they still have an increased RS and SDS content even after heating, which may lead to gelatinization of the starch. Accordingly, genetically modified plant cells or parts of genetically modified plants which contain a foreign nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6-phosphate amidotransferase (GFAT) are therefore especially suitable for the preparation of foodstuffs which require heating for their preparation.

Accordingly, a further subject matter relates to foodstuffs according to the invention which are heated during their preparation. This includes for example canned foods, but also boiled vegetables (for example sweetcorn, soups) and potato products (for example boiled potatoes, potato salad, crisps, French fries).

The subject of the present invention furthermore relates to methods of preparing a foodstuff according to the invention wherein genetically modified plant cells or genetically modified plants or parts of genetically modified plants which comprise a foreign nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6-phosphate amidotransferase are used for the preparation of the foodstuff.

Methods according to the invention for preparing a foodstuff preferably serve for the preparation of a foodstuff according to the invention or for the preparation of a foodstuff which has the properties of a foodstuff according to the invention.

The present invention furthermore relates to the use of genetically modified plant cells, genetically modified plants, parts of genetically modified plants which comprise a foreign nucleic acid molecule coding for a protein with the activity of a glutamine:fructose 6-phosphate amidotransferase for the preparation of a foodstuff. The use for the preparation of a foodstuff preferably takes the form of the preparation of a foodstuff with a reduced calorific content and/or an increased RS content and/or which serves for the prevention of joint diseases and/or which brings about a positive (prebiotic) effect on the consumer's gut flora.

Sequence description

SEQ ID NO 1 : Nucleic acid sequence coding for a protein with the activity of a bacterial GFAT from Escherichia coli.

SEQ ID NO 2: Amino acid sequence of a protein with the activity of a GFAT from Escherichia coli. The amino acid sequence shown can be derived from SEQ ID NO 1.

SEQ ID NO 3: Synthetic nucleic acid sequence coding for a protein with the activity of a GFAT from Escherichia coli. The synthesis of the codons of the sequence shown was performed in such a way that it is adapted to the codon usage in plant cells. The nucleic acid sequence shown codes for a protein with the amino acid sequence shown under SEQ ID No 2.

SEQ ID NO 4: Nucleic acid sequence coding for a protein with the activity of a murine GFAT-2. SEQ ID NO 5: Amino acid sequence of a protein with the activity of a murine

GFAT-2. The amino acid sequence shown can be derived from SEQ ID NO 4.

Description of the figures

FIG 1 : shows the content of starch which is stainable by means of Lugol's solution in boiled slices of potato tubers following digestion with pancreatin (see example 12).

The content of all publications cited, including the respective "accession numbers" of nucleic acid molecules and amino acid sequences mentioned for sequence databases, is incorporated into the description of the application by reference. In what follows, methods will be described which may be used in the context of the present invention. These methods represent specific use forms, but do not limit the invention to these methods. The skilled worker knows that the invention can be practiced in the same way by modification of the methods described and/or by replacing individual methods or parts of methods by alternative methods or alternative parts of methods.

General methods

1. Transformation of potato plants

Potato plants were transformed with the aid of Agrobacteriυm as described by Rocha-Sosa et al. (EMBO J. 8, (1989), 23-29).

2. Extraction of starch from potato plants

Potato tubers are processed in a commercially available juicer (Multipress automatic MP80, Braun). The starch-containing juice is collected in a vessel into which some tap water comprising sodium disulfite has been introduced. Thereafter, the vessel is filled completely with tap water. After the starch has settled for approximately 2 hours, the supernatant is decanted off, and the starch is resuspended in tap water and passed through a sieve of mesh size 125 μm. After approximately 2 hours (starch has again settled at the bottom of the bucket), the aqueous supernatant is again decanted off. This washing process is repeated three more times so that the starch is resuspended in fresh tap water five times in total. Thereafter, the starches are dried at 37°C to a water content of 12% to 17% and homogenized in a mortar. The starches are now available for analyses. Typical quantitative ratios for the processing method described: starch-containing juice of 4 to 5 kg potato tubers is transferred into a 10-liter vessel into which approximately 200 ml of tap water comprising 3 to 4 g of sodium disulfite has been placed. Before the starch settles, the volume is made up in each case to approximately 10 liters, and the starch is resuspended. Depending on the amount of potato tubers employed, the specified quantities can be adapted to suit the conditions.

3. Determination of the content of N-acetylated glucosamines

N-Acetylated glucosamine derivatives which have a reducing terminus were determined via a method similar to that of Elson and Morgan (1933, J Biochem. 27,1824) and the colorimetric determination method improved by Reissig et al. (1955, Biol. Chem. 217, 959-966). The colorimetric determination method is based on the reaction of chromogen III (Muckenschnabel et al., 1998, Cancer Letters 131 , 13-20) with p-dimethylaminobenzaldehyde (DMAB, Ehrlich's reagent) to give a red product whose concentration can be determined photometrically.

a) Processing of the plant material

First, harvested plant material was comminuted. Depending on the amount of the plant material used, the comminution was performed in a laboratory mixer mill (MM200, Retsch, Germany) for 30 seconds at 30 Hz or by means of a Waring

blender for approximately 30 seconds at maximum speed. As a rule, approximately 0.5 g of the comminuted plant material (for example leaf, tuber or rice grain) were mixed with 1 ml of a solution consisting of 7% strength perchloric acid, 5 mM EGTA and incubated for 20 minutes on ice. This was followed by centrifugation (5 minutes at 16000xg, 4°C). The supernatant obtained after centrifugation was removed and neutralized with a solution consisting of 5M KOH, 1 M TEA (adjusted to pH 7.0) and then the mixture was recentrifuged (5 min at 16000xg, 4°C). When the centrifugation had ended, the supernatant was removed, its volume was determined and the amount of N-acetylated glucosamine derivatives with a reducing terminus was determined using the method described under b).

b) Determination of the content of N-acetylated glucosamine derivatives with reducing termini

100 μl of plant extract obtained by the method described under a) are treated with 20 μl of a solution consisting of 0.8 M K ₂ B ₄ O ₇ , pH 9.6 and, after mixing thoroughly, the mixture was heated for 5 minutes at 95 ⁰ C. After the mixture had cooled to room temperature, it is treated with 0.7 ml of Ehrlich's reagent (solution, diluted with glacial acetic acid 1 :10, consisting of 10 g of DMAB in 12.5 ml of cone. HCI, 87.5 ml of glacial acetic acid), mixed again and incubated at 37°C for a further 30 minutes. Thereafter, the mixture is centrifuged for 1 minute at 16000xg, and the optical density (OD) of the supernatant obtained after centrifugation is then determined in the photometer at 585 nm.

c) Calculating the concentration of N-acetylated glucosamine derivatives

First, a calibration curve was established using defined amounts of

N-acetylglucosamine 6-phosphate. To this end, the ODs of solutions comprising 0 mM, 0.1 mM, 0.5 mM, 1 mMA, 5 mM and 10 mM N-acetylglucosamine 6-phosphate were determined by the method described under b).

The calibration curve was established in Microsoft Excel by drawing a second-order polynomic trend/regression line through the data points obtained for the individual concentrations, in accordance with the formula y = ax ² + bx +c and y = x ² + px +q, respectively.

To calculate the values, the equation obtained was resolved toward x, so that x = -p/2 - square root (p ² / 4 - q) where p = b / a, q = (c-y) / a and y is the measured OD of the unknown sample.

Taking into consideration the fresh weight employed, the volume employed and

taking into consideration a dilution factor which may have been used, the contents were calculated as μmol (in the measuring solution) or as μmol per g fresh weight.

4. Determination of the activity of a GFAT

The determination of the activity of a protein with the activity of a GFAT is carried out as described by Rachel et al. (1996, J. Bacteriol. 178 (8), 2320-2327).

To distinguish whether a protein has the activity of a GFAT-1 or a GFAT-2, the method described by Hu et al. (2004, J. Biol. Chem. 279 (29), 29988-29993) is employed.

5. Detection of N-acetylated glucosamine derivatives by means of mass spectroscopy

To detect N-acetylated glucosamine derivatives by means of mass spectroscopy, plant tissue was processed as described under General Methods, item 3 a). In order to obtain an extract which is as free from salt as possible, the respective samples were first frozen at -20 ⁰ C and defrosted during a centrifugation (16000xg at room temperature) before being analyzed by mass spectroscopy. For the measurement, the supernatant was diluted 1 :20 with a 1 :1 (volume/volume) methanohwater mixture. MS spectra were recorded with three different detector sensitivities in order to increase the detection sensitivity for weak signals (peaks). In this case, however, the response of the detector is no longer linear, which is noticeable when the signal intensities (peak areas) of different metabolites are compared, and which should be taken into consideration. In order to ensure the comparability of the measurements, care was taken that the individual samples gave the same signal intensities (in cps, counts per second) with the same detector settings. The areas of the signals obtained (peak areas) which were assigned to the different metabolites are stated in % in relation to the peak area of hexoses (m/z = 179). By comparing the signal intensities (peak areas) in different samples, a statement can be made about the concentration ratios of the corresponding N-acetylated glucosamine derivatives in relation to the concentration of hexoses in the relevant sample. MS-MS measurements of the individual samples and of individual corresponding reference substances (glucosamine, N-acetylglucosamine, glucosamine 6-phosphate, glucosamine 1 -phosphate, N-acetylglucosamine 6-phosphate, N-acetylglucosamine 1 -phosphate, UDP-N-acetylglucosamine) were carried out in parallel. This allows an estimate to be made as to whether the signal (peak) which is

used for determining the area is a signal which has been generated exclusively by a specific metabolite, or by specific isomeric metabolites with the same mass, or whether the signal in question can only be assigned partially to the corresponding metabolite, or the corresponding specific isomeric metabolites with the same mass. MS and MS-MS spectra were recorded in the negative mode in a Q-STAR Pulsar i hybrid mass spectrometer from Applied Biosystems, which is equipped with a nano- electrospray source. Here, it was mostly deprotonated single-charged ions which were detected.

The measurements were carried out under the following conditions: Mass range 50-700 Da

Sensitivity of the detector: 2000, 2050 or 2100

With each of the three detector settings, care was taken that the signal intensities (in cps, counts per second) obtained for the samples were as similar as possible.

6. Starch analyses

a) Determination of the amylose content and of the amylose/amylopectin ratio

Starch was isolated from potato plants by standard methods, and the amylose content and the amylose:amylopectin ratio were determined by the method described by Hovenkamp-Hermelink et al. (Potato Research 31 , (1988), 241-246).

b) Determination of the phosphate content in the C6 position of the starch's glucose molecules

In starch, the positions C2, C3 and C6 of the glucose units may be phosphorylated. To determine the C6-P content of the starch, 50 mg of starch are hydrolyzed for 4 hours at 95°C in 500 μl of 0.7 M HCI. The mixtures are then centrifuged for 10 minutes at 15500 g and the supernatants are removed. 7 μl of the supematants are mixed with 193 μl of imidazole buffer (100 mM imidazole, pH 7.4; 5 mM MgCI ₂ , 1 mM EDTA and 0.4 mM NAD). The measurement was carried out in a photometer at 340 nm. After the base absorption had been established, the enzyme reaction was started by addition of 2 units of glucose 6-phosphate dehydrogenase (from Leuconostoc mesenteroides, Boehringer Mannheim). The change in absorption is directly proportional to the concentration of the G6-P content of the starch.

c) Determination of the viscosity properties by means of a Rapid Visco Analyzer (RVA)

2 g of starch (DW) are taken up in 25 ml H ₂ O (fully demineralized water, conductivity at least 15 mega Ohm) and used for the analysis in a Rapid Visco Analyzer (Newport Scientific Pty Ltd., Investmet Support Group, Warriewod NSW 2102, Australia). The apparatus is operated as specified by the manufacturer. Here, the viscosity values are given in RVUs as specified in the manufacturer's operating instructions, which are herewith incorporated into the description by reference. To determine the viscosity of the aqueous starch solution, the starch suspension is first heated for one minute at 50 ⁰ C (step 1), then heated from 50 ⁰ C to 95°C at a rate of 12°C per minute (step 2). Thereafter, the temperature is held for 2.5 min at 95 ⁰ C (step 3). The solution is then cooled from 95°C to 50°C at a rate of 12°C per minute (step 4). The viscosity is determined over the entire duration.

After the program has ended, the stirrer is removed and the cup is covered. The gelatinized starch is now available for analyzing the gel strength (see below) after 24 h. There are characteristic parameters in the profile of the RVA analysis which are shown for comparison of different measurements and substances. In the context of the present invention, the following terms are to be understood as follows: Maximum viscosity (RVA Max) The maximum viscosity is understood as meaning the highest viscosity value, measured in RVUs, which is achieved in step 2 or 3 of the temperature profile. Minimum viscosity (RVA Min)

The minimum viscosity is understood as meaning the lowest viscosity value, measured in RVUs, which is observed in the temperature profile after the maximum viscosity. This is normally done in step 3 of the temperature profile. Final viscosity (RVA Fin)

The final viscosity is understood as meaning the viscosity value, measured in RVUs, which is observed at the end of the measurement.

Setback (RVA Set)

What is known as setback is calculated by subtracting the value of the final viscosity from that of the minimum which is observed in the curve after the maximum viscosity is reached.

Gelatinization temperature (pasting temperature)

The gelatinization temperature is understood as meaning the temperature in the RVA profile at which the viscosity climbs rapidly for the first time within a short period. Peak Time (RVA T)

The peak time is understood as meaning the point in time in the temperature profile at which the viscosity has reached its maximum.

d) Determination of the gel strength (texture analyzer)

2 g of starch (DW) are gelatinized in 25 ml of an aqueous suspension in the RVA apparatus (temperature program: see above) and then stored for 24 h at room temperature in a sealed container. The samples are fixed underneath the probe (cylindrical stamp with a plane surface) of a TA-XT2 Texture Analyzer from Stable Micro Systems (Surrey, UK) ₁ and the gel strength is determined by setting the following parameters on the apparatus: test speed 0.5 mm/s penetration depth 7 mm contact area 113 mm ² pressure 2 g

e) Particle size determination

Starch was extracted from potato tubers by standard methods (see above).

Then, the particle size determination was carried out using a laser diffractometer of the "Mastersizer 2000" type from Malvern Instruments Ltd. using the software version

5.22, which was included.

Laser diffractometry is based on the diffraction of the light, where, depending on the shape and size of the particle at which the light is diffracted, different diffraction patterns are generated. Large particles generate a small diffraction angle, while small particles generate a large diffraction angle. On the basis of the diffraction angles obtained, the particle size is determined by means of the abovementioned computer program (software version 5.22).

The following settings were used for determining the particle size of potato starches: dispersion medium: water (refractive index 1.33) obscuration: 12% dispersing time 1 min measurement range: 0.02 - 2000 μm light sources: red light - helium neon laser blue light semiconductor light source measuring cycles: 5 measuring time: 6 sec. background: 6 sec. interval between the measurements: 5sec. stirrer speed: 3500rpm

evaluation method: Fraunhofer approximation

f) Analysis of starch by means of differential scanning calorimetry (= DSC)

Amounts of approximately 10 mg (dry weight) maize or wheat flour or maize or wheat starch were treated with an excess, preferably a 3-fold excess, of demineralized water (preferably 20 μl) in stainless steel pans (Perkin Elmer, "Large Volume Stainless Steel Pans" [03190218], volume 60 μl) and sealed hermetically with the aid of a press. The sample was heated from 20 ⁰ C to 120 ⁰ C in a Diamond DSC apparatus (Perkin Elmer) at a heating rate of 10°C/min. An empty, sealed stainless steel pan was used as reference. The system was calibrated with defined amounts of indium and octadecane.

The data analysis was carried out using the software program from Pyris (Perkin Elmer, version 7.0). Raw data capable of being evaluated were processed by analyzing the individual peaks of the 1st-order phase transitions on T-onset ( ⁰ C), T-peak ( ⁰ C), T-end ( ⁰ C) and dH (J/g) (the standard being the straight baseline). Here, DSC T-onset is characterized as the point of intersection between the continuation of the baseline and the tangent applied at the ascending flank of the peak across the inflection point. It characterizes the beginning of the phase conversion. The maximum temperature DSC T-peak refers to the maximum temperature at which the DSC curve has reached a maximum (i.e. the temperature at which the first derivation of the curve is zero).

In the case of the function used in Pyris (calc-peak Area), a start temperature and a final temperature are input manually in order to determine the baseline.

7. Determination of the resistant starch content (digestibility) Before the resistant starch content was determined, in each case approximately 10 mg of starch were suspended in 10 μl of demineralized water and incubated for 10 minutes at 90°C, with shaking. The resistant starch content was then determined directly.

The resistant starch content is determined by the method described in Englyst et al. ((Europ. J. of Clinical Nutrition 46 (Suppl. 2), (1992), pp. 33-50)) (see, in particular, the following sections of Englyst et al., page 35-36: "Reagents, Apparatus, Spectrophotometer"; page 36-37, section "Measurement of free glucose (FG)"; page 38, section "Measurement of RDS and SDS").

As an alternative, the method of Englyst et al. may be carried out as described by Zhang et al. (Biomacromolecules 7, (2006), 3252-3258, in particular page 3253: Methods. Enzymatic Starch Hydrolysis).

The method of Englyst et al. may be carried out in the following manner on the laboratory scale:

To prepare the enzyme solution, 1.2 g of pancreatin (Merck) are extracted for 10 minutes at 37°C into 8 ml of water. After centrifugation (10 min, 3000 U/min; RT), 5.4 ml of the supernatant are mixed with 84 U amyloglucosidase (Sigma-Aldrich, Taufkirchen) and made up with water to a final volume of 7 ml. In parallel, 10 mg (dry weight) of starch or starch which has already been gelatinized, per sample, are treated with 0.75 ml of sodium acetate buffer (0.1 M sodium acetate pH 5.2; 4 mM CaCI ₂ ) in a 2 ml reaction vessel and incubated for 5 minutes at 37°C in order to warm the mixture.

Starch digestion is initiated by adding in each case 0.25 ml of enzyme solution per mixture. A mixture into which water is added instead of enzyme solution acts as the control. After 20, 60 and 120 minutes, aliquot portions of 100 μl are withdrawn and added directly into four times the volume of ethanol, whereby the enzymes are inactivated. This dilution is used for measuring the glucose content.

To this end, 2 μl of dilute sample are mixed with 200 μl of measuring buffer (100 mM imidazole/HCI pH 6.9, 5 mM MgCI ₂ , 1 mM ATP, 2 mM NADP), and the absorption of the sample is recorded at 340 nm. The conversion of the glucose is initiated by addition of 2 μl of enzyme mix (10 μl hexokinase, 10 μl glucose 6-phosphate dehydrogenase, 80 μl measuring buffer), and the equimolar conversion of NADP into NADPH is monitored at 340 nm until the point when a plateau is reached. The amounts of glucose determined are related to the initial weight and give the part of the sample which has been liberated as glucose after the interval in question.

Examples

1. Obtaining a nucleic acid sequence coding for a protein with the activity of a murine GFAT-2

The nucleic acid sequence coding for a protein with the activity of a GFAT-2

(glutamine:fructose 6-phosphate amidotransferase or glucosamine 6-phosphate synthase, EC 2.6.1.16), was obtained commercially from Invitrogen (Clone ID 4167189, cDNA clone MGC:18324, IMAGE:4167189). It is a clone which is produced

by I.M.A.G.E. Konsortium (http://image.llnl.gov) and sold by Invitrogen. The cDNA coding for a protein with the activity of a GFAT-2 was cloned into the vector pCMV Sport 6 from Invitrogen. The plasmid was named IC 369-256. The nucleic acid sequence coding for the protein with the activity of a GFAT-2 from Mus muscυlus is shown in SEQ ID NO 4.

2. Synthesis of the nucleic acid sequences coding for a protein with the activity of a bacterial GFAT from Escherichia coli

The nucleic acid sequence coding for a protein with the activity of a bacterial GFAT (glutamine:fructose 6-phosphate amidotransferase or glucosamine 6-phosphate synthase, glms, EC 2.6.1.16 ) from Escherichia coli was synthesized by Entelechon GmbH and cloned into the vector pCR4Topo from Invitrogen (Prod. No. K4510-20). The plasmid obtained was named as IC 373-256. The synthetic nucleic acid sequence coding for the protein with the activity of a bacterial GFAT from Escherichia coli is shown in SEQ ID NO 3. The corresponding nucleic acid sequence which has originally been isolated from Escherichia coli ls shown in SEQ ID NO 1.

3. Preparation of the plasmid pBinB33

The promoter of the patatin gene B33 from Solarium tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) was ligated as a Dra I fragment (Nucleotides -1512 to +14) into the SST /-cleaved vector pUC19 which has been made blunt-ended with the aid of T4-DNA ligase. This gave rise to the plasmid pUC19-B33. The B33 promoter was excised from this plasmid using the restriction endonucleases Eco Rl and Sma I and ligated into the correspondingly cut vector pBinAR (Hδfgen and Willmitzer, 1990, Plant Science 66, 221-230). The resulting vector was named pBinB33.

4. Preparation of the plasmid pBinB33Hyg Starting from pBinB33, a fragment comprising the B33 promoter, part of the polylinker and the ocs terminator was excised by means of the restriction endonucleases EcoRI and Hindlll and cloned into the correspondingly cut vector pBIB-Hyg (Becker, 1990, Nucleic Acids Res. 18, 203). The resulting plasmid was named pBinB33Hyg.

5. Preparation of the plant expression vector IC 398-311 comprising a coding nucleic acid sequence for a protein with the activity of a bacterial GFAT

The coding sequence of the protein with the activity of a bacterial GFAT from E. coli

was isolated from plasmid IC 373-256 by means of restriction digestion with Ec1 136 I and Xho I and ligated into the Sma I and Sal I cleavage sites of the vector pBinB33Hyg. The resulting plant expression vector was named IC 398-311.

6. Potato plants which contain a nucleic acid molecule coding for a protein with the activity of a bacterial GFAT

a) Transformation of potato plants

Potato plants (cultivar Desiree) were transformed with the plant expression vector IC 398-311 comprising a coding nucleic acid sequence for a protein with the activity of a bacterial GFAT from Escherichia coli under the control of the promoter of the patatin gene B33 from Solanum tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) following the method detailed under General Methods, item 1. The resulting transgenic lines which are transformed with the plasmid IC 398-311 were named 432 ES.

b) Analysis of the lines 432 ES

Plants of line 432 ES were grown in the greenhouse in soil in 6-cm pots. Approximately in each case 0.3 g to 0.8 g leaf material harvested from individual plants were processed by the method described under General Methods, item 3, and the content in N-acetylated glucosamine derivatives was determined. The following results were obtained for individual plants with an increased content of N-acetylated glucosamine derivatives:

Table 1 : Amount of N-acetylated glucosamine derivatives (in μmol per gram fresh weight) which has been determined in leaves or tubers of independent transgenic plants of line 432 ES. Column 1 identifies in each case the plant which has independently been obtained in the transformation and of which material has been harvested (wt here identifies corresponding wild-type plants which are not transformed).

7. Determination of the resistant starch content in plants of line 432ES

Plants of line 432 ES were grown in soil in the greenhouse in pots. Mature tubers of these lines were harvested. Thereafter, starch was isolated by the method described under General Methods, item 2, and the RDS, SDS and RS contents of the starch in question were determined by the method described under General Methods, item 7. The data, which are shown in the table hereinbelow, were determined as follows: RDS: amount of the glucose liberated from starch after 20 minutes' digestion (GIc 20 min) in percent by weight [%] based on the amount of the dry weight (DW) of the starch employed for the digestion: RDS [%] = GIc 20 min/DW x 100

SDS: difference between the amount of the glucose liberated from the starch after 120 minutes' digestion (GIc 120 min) and the amount of the glucose liberated from starch after 20 minutes' digestion (GIc 20 min), in each case in percent by weight [%] based on the amount of the dry weight [DW] of the starch employed for the digestion: SDS [%] = GIc 120 min [%] - GIc 20 min [%]

RS: difference between the amount of the starch employed for the digestion based on the dry weight in percent (=100%) and the amount of the glucose liberated from the starch after 120 minutes' digestion in percent by weight [%] based on the amount of the dry weight [DW] of the starch employed for the digestion: RS [%] = 100% - GIc 120 min [%]

To determine the values for RDS (after 20 minutes and after 120 minutes) which are shown in the table hereinbelow, in each case 3 independent samples of the starches in question were measured with in each case 2 replications, and the respective mean was determined. The standard deviation (SD) was determined by the general formula:

SD = square root [(n∑x ² - (∑x) ² ) / n(n-1)]: The following results were obtained:

Table 2: Amount of RDS, SDS and RS in starch isolated from tubers of the plants

432ES22, 432ES9 and 432ES 40 and corresponding untransformed wild-type plants (wt). Novelose ^® 330 denotes a commercially available RS product from National Starch.

8. Determination of the thermal stability of starches isolated from plants of line 432ES by means of DSC analysis

Starches from independent plants of line 432ES were prepared as described in example 7 and subsequently analyzed by the method described in General Methods, item 6 f)- To determine the values which are shown in the table hereinbelow, in each case 2 independent samples of the respective starches were measured, the means were determined, and the standard deviation (SD) was determined using the formula shown in example 6 b). The following results were obtained:

Table 3: Determination of the thermal stability of starches isolated from the plants 432ES09, 432ES33, 432ES40 and corresponding untransformed wild-type plants (wt) by means of DSC analysis.

In a further analysis, starches which, following gelatinization in a first DSC analysis, had been stored (retrograded) for 7 days at room temperature in the sealed stainless steel pans were reanalyzed by means of DSC. The table shows the means of in each case 2 independent samples of the starches in question. The standard deviation (SD) was determined using the formula shown in example 6 b). The following results were obtained:

Table 4: Determination of the thermal stability of retrograded starches isolated from

the plants 432ES09, 432ES33, 432ES40 and corresponding untransformed wild-type plants (wt) by means of DSC analysis.

9. Determination of the viscosity properties of starches isolated from plants of line 432ES by means of RVA analysis

Starches of independent plants of line 432ES were prepared as described in example 7 and subsequently analyzed by the method described in General Methods, item 6 c). The following results were obtained:

Table 5: Determination of the viscosity properties of starches isolated from the plants 432ES09, 432ES33, 432ES40 and corresponding untransformed wild-type plants (wt) by means of RVA analysis. The data are shown in absolute units (RVUs, minutes or ⁰ C).

Table 6: Representation of the data shown in table 5 in relative units in percent based on the data obtained for starch isolated from corresponding untransformed wild-type plants (wt = 100%).

10. Determination of the gel strength of starches isolated from plants of line 432ES by means of a texture analyzer

The gel strength was analyzed by the method described in General Methods, item 6 d). The following results were obtained:

Table 7: Determination of the gel strength of starches isolated from the plants 432ES09, 432ES33, 432ES40 and corresponding untransformed wild-type plants (wt) by means of texture analyzer. The data shown are the absolute values of the force in gram.

Table 8: Representation of the data shown in table 7 in relative units in percent based on the data obtained for starch isolated from corresponding untransformed wild-type plants (wt = 100%).

11. Determination of the granule size of starches isolated from plants of line 432ES

Starches from independent plants of line 432ES were prepared as described in example 7, and the granule size of the starch granules in question was subsequently determined by the method described in General Methods, item 6 e). The following results were obtained:

Table 9: Mean starch granule size of starches isolated from the plants 432ES09, 432ES33, 432ES40 and corresponding untransformed wild-type plants (wt).

12. Digestibility of boiled potatoes

Cylinders were punched from tubers of independent plants of line 432ES using a

cork bore (diameter 4 mm). Thereafter, slices approximately 1-2 mm thick were cut from these cylinders using a surgical blade. In each case 10 slices of a cylinder were transferred into a reaction vessel, approximately 1 ml of water was added, and the slices were boiled in water for 5 minutes. Directly after cooling to room temperature, the boiled samples are incubated for 4 hours at 37°C with 1 ml of pancreatin solution. After the digestion with pancreatin solution had ended, the individual samples were stained with Lugol's solution to visualize the amount of undigested starch. The result of this experiment is shown in fig. 1. The dark areas which become visible after staining with Lugol's solution demonstrate that there are still significant amounts of starch present in these areas. This is RS. Light areas, or areas which do not stain, in contrast, show that starch is no longer present in these areas, i.e. that most of the starch has been digested.

Preparation of the pancreatin solution: 3.75 g of pancreatin (from porcine pancreas, Merck, Product No. 1.07130.1000) were shaken for 10 minutes at 37°C in 25 ml of demineralized water and the mixture was subsequently centrifuged for 10 minutes at 2000xg. 21.06 ml of the supernatant obtained were treated with 3.85 ml of demineralized water, 54.6 μl of amyloglucosidase (6000 U/ml, Sigma, Product No. A-3042) and 100 ml of 0.1 M sodium acetate buffer pH 5.2 + 4 mM CaCI ₂ .