DK1951030T3 - IMPROVED METHODS AND MEANS FOR THE PRODUCTION OF hyaluronan - Google Patents

Description

DESCRIPTION

[0001] The present invention relates to plant cells and plants which synthesize an increased amount of hyaluronan, and to methods for preparing such plants, and also to methods for preparing hyaluronan with the aid of these plant cells or plants. Here, plant cells or genetically modified plants according to the invention have hyaluronan synthase activity and additionally an increased glutamine:fructose 6-phosphate amidotransferase (GFAT) activity and an increased UDP glucose dehydrogenase (UDP-GIc-DH) activity, compared to wild-type plant cells or Wild-type plants. The present invention furthermore relates to the use of plants having increased hyaluronan synthesis for preparing hyaluronan and food or feedstuff containing hyaluronan.

[0002] Hyaluronan is a naturally occurring unbranched, linear mucopolysaccharide (glucosaminoglucan) which is constructed of alternating molecules of glucuronic acid and N-acetyl-glucosamine. The basic building block of hyaluronan consists of the disaccharide glucuronic acid-beta-1,3-N-acetyl-glucosamine. In hyaluronan, these repeating units are attached to one another via beta-1,4 linkages.

In pharmacy, use is frequently made of the term hyaluronic acid. Since hyaluronan is in most cases present as a polyanion and not as the free acid, hereinbelow, the term hyaluronan is preferably used, but each term is to be understood as embracing both molecular forms.

[0003] Hyaluronan has unusual physical chemical properties, such as, for example, properties of polyelectrolytes, viscoelastic properties, a high capacity to bind water, properties of gel formation, which, in addition to further properties of hyaluronan, are described in a review article by Lapcik et al. (1998, Chemical Reviews 98(8), 2663-2684).

[0004] Hyaluronan is a component of extracellular connective tissue and bodily fluids of vertebrates. In humans, hyaluronic acid is synthesized by the cell membrane of all body cells, especially mesenchymal cells, and ubiquitously present in the body with a particularly high concentration in the connective tissues, the extracellular matrix, the umbilical cord, the joint fluid, the cartilaginous tissue, the skin and the vitreous body of the eye (Bernhard Gebauer, 1998, Inaugural-Dissertation, Virchow-Klinikum Medizinische Fakultåt Charité der Humboldt Universitåt zu Berlin; Fraser et al., 1997, Journal of Internal Medicine 242, 27-33).

[0005] Recently, hyaluronan was also found in animal non-vertebrate organisms (molluscs) (Volpi and Maccari, 2003, Biochimie 85, 619-625).

[0006] Furthermore, some pathogenic gram-positive bacteria (Streptococcus group A and C) and gram-negative bacteria (Pasteurella) synthesize hyaluronan as exopolysaccharides which protect these bacteria against attack by the immune system of their host, since hyaluronan is a non-immunogenic substance.

[0007] Viruses which infect single-cell green algae of the genus Chlorella, some of which are present as endosymbionts in Paramecium species, bestow upon the single-cell green algae the ability to synthesize hyaluronan after infection by the virus (Graves et al., 1999, Virology 257, 15-23). However, the ability to synthesize hyaluronan is not a feature which characterizes the algae in question. The ability of the algae to synthesize hyaluronan is mediated by an infection with a virus whose genome has a sequence coding for hyaluronan synthase (DeAngelis, 1997, Science 278, 1800-1803). Furthermore, the virus genome contains sequences coding for an UDP-glucose dehydrogenase (UDP-GIc-DH) and a glutamine: fructose 6-phosphate amidotransferase (GFAT). UDP-GIc-DH catalyzes the synthesis of UDP-glucuronic acid used as substrate by hyaluronan synthase. GFAT converts fructose 6-phosphate and glutamine into glucosamine 6-phosphate which is an important metabolite in the metabolic pathway for hyaluronan synthesis in, for example, bacteria. Both algeal genes encode active proteins which, like the hyaluronan synthase of the virus, are transcribed simultaneously in the early phase of the viral infection (DeAngelis et al., 1997, Science 278, 1800-1803, Graves et al., 1999, Virology 257, 15-23). The activity of a protein having glutamine:fructose 6-phosphate amidotransferase (GFAT) activity could be detected neither in extracts from cells not infected by a virus nor in virus-infected cells (Landstein et al., 1998, Virology 250, 388-396). Accordingly, the role of the expression of UDP-GIc-DH and GFAT in virus-infected Chlorella cells for the hyaluronan synthesis, and whether they are required for hyaluronan synthesis, is not known.

[0008] Naturally occurring plants themselves do not have any nucleic acids in their genome which code for proteins catalyzing the synthesis of hyaluronan and, although a large number of plant carbohydrates have been described and characterized, it has hitherto not been possible to detect hyaluronan or molecules related to hyaluronan in non-infected, naturally occurring plants (Graves et al., 1999, Virology 257, 15-23).

[0009] The catalysis of the hyaluronan synthesis is effected by a single membrane-integrated or membrane-associated enzyme, hyaluronan synthase. The hyaluronan synthases which have hitherto been studied can be classified into two groups: hyaluronan synthases of Class I and hyaluronan- synthases of Class II (DeAngelis, 1999, CMLS, Cellular and Molecular Life Sciences 56, 670-682).

[0010] The hyaluronan synthases of vertebrates are further distinguished by the identified isoenzymes. The different isoenzymes are referred to in the order of their identification using Arabic numbers (for example, hsHASI, hsHAS2, hsHAS3).

[0011] The mechanism of the transfer of synthesized hyaluronan molecules across the cytoplasma membrane into the medium surrounding the cell has not yet been fully elucidated. Earlier hypotheses assumed that transport across the cell membrane was effected by hyaluronan synthase itself. However, more recent results indicate that the transport of hyaluronan molecules across the cytoplasma membrane takes place by energy-dependent transport via transport proteins responsible for this action. Thus, Streptococcus strains were generated by mutation in which the synthesis of an active transport protein was inhibited. These strains synthesized less hyaluronan than corresponding wild-type bacteria strains (Ouskova et al., 2004, Glycobiology 14(10), 931-938). In human fibroblasts, it was possible to demonstrate, with the aid of agents specifically inhibiting known transport proteins, that it is possible to reduce both the amount of hyaluronan produced and the activity of hyaluronan synthases (Prehm and Schumacher, 2004, Biochemical Pharmacology 68, 1401-1410). In which amount, if at all, transport proteins capable of transporting hyaluronan are present in plants is not known.

[0012] The unusual properties of hyaluronan offer a wealth of possibilities for application in various fields, such as, for example, pharmacy, the cosmetics industry, in the production of food and feed, in technical applications (for example as lubricants), etc. The most important applications where hyaluronan is currently being used are in the medical and cosmetics field (see, for example, Lapcik et al., 1998, Chemical Reviews 98(8), 2663-2684, Goa and Benfield, 1994, Drugs 47(3), 536-566).

[0013] In the medical field, hyaluronan-containing products are currently used for the intraarticular treatment of arthrosis and in ophthalmics used for eye surgery. Hyaluronan is also used for treating joint disorders in racehorses. In addition, hyaluronic acid is a component of some rhinologics which, for example in the form of eye drops and nasalia, serve to moisten dry mucous membranes. Hyaluronan-containing solutions for injection are used as analgesics and antirheumatics. Patches comprising hyaluronan or derivatized hyaluronan are employed in wound healing. As dermatics, hyaluronan-containing gel implants are used for correcting skin deformations in plastic surgery.

[0014] For pharmacological applications, preference is given to using hyaluronan having a high molecular weight.

[0015] In cosmetic medicine, hyaluronan preparations are among the most suitable skin filler materials. By injecting hyaluronan, for a limited period of time, it is possible to smooth wrinkles or to increase the volume of lips.

[0016] In cosmetic products, in particular in skin creams and lotions, hyaluronan is frequently used as moisturizer by virtue of its high waterbinding capacity.

[0017] Furthermore, hyaluronan-containing preparations are sold as so-called nutraceuticals (food supplements) which can also be used in animals (for example dogs, horses) for the prophylaxis and alleviation of arthrosis.

[0018] Hyaluronan used for commercial purposes is currently isolated from animal tissues (rooster combs) or prepared fermentatively using bacterial cultures.

[0019] US 4,141,973 describes a process for isolating hyaluronan from rooster combs or alternatively from umbilical cords. In addition to hyaluronan, animal tissues (for example rooster combs, umbilical cords) also contain further mucopolysaccharides related to hyaluronan, such as chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate and heparin. Furthermore, animal organisms contain proteins (hyaladherins) which bind specifically to hyaluronan and which are required for the most different functions in the organism, such as, for example, the degradation of hyaluronan in the liver, the function of hyaluronan as lead structure for cell migration, the regulation of endocytosis, the anchoring of hyaluronan on the cell surface or the formation of hyaluronan networks (Turley, 1991, Adv Drug Delivery Rev 7, 257 ff.; Laurent and Fraser, 1992, FASEB J. 6, 183 ff.; Stamenkovic and Aruffo, 1993, Methods Enzymol. 245, 195 ff; Knudson and Knudson, 1993, FASEB 7, 1233 ff.).

[0020] The Streptococcus strains used for the bacterial production of hyaluronan are exclusively pathogenic bacteria. During cultivation, too, these bacteria produce (pyrogenic) exotoxins and hemolysins (streptolysin, in particular alpha- and beta-hemolysin) (Kilian, M.: Streptococcus and Enterococcus. In: Medical Microbiology. Greenwood, D.; Slack, RCA; Peutherer, J.F. (Eds.). Chapter 16. Churchill Livingstone, Edinburgh, UK: pp. 174-188, 2002, ISBN 0443070776) which are released into the culture medium. This renders purification and isolation of the hyaluronan prepared with the aid of Streptococcus strains more difficult. In particular for pharmaceutical applications, the presence of exotoxins and hemolysins in the preparations is a problem.

[0021] US 4,801,539 describes the preparation of hyaluronan by fermentation of a mutagenized bacteria strain (Streptococcus zooedemicus). The mutagenized bacteria strain used no longer synthesizes beta-hemolysin. The yield achieved was 3.6 g of hyaluronan per liter of culture.

[0022] EP 0694616 describes a method for cultivating Streptococcus zooedemicus or Streptococcus equi, where, under the culture conditions employed, no streptolysin, but increased amounts of hyaluronan are synthesized. The yield achieved was 3.5 g of hyaluronan per liter of culture.

[0023] During cultivation, Streptococcus strains release the enzyme hyaluronidase into the culture medium, as a consequence of which, in this production system, too, the molecular weight is reduced during purification. The use of hyaluronidase-negative Streptococcus strains or of methods for the production of hyaluronan where the production of hyaluronidase during cultivation is inhibited are described in US 4,782,046. The yield achieved was up to 2.5 g of hyaluronan per liter of culture, and the maximum mean molecular weight achieved was 3.8 x 106 Da, at a molecular weight distribution of from 2.4 x 106 to 4.0 x 106.

[0024] US 20030175902 and WO 03 054163 describe the preparation of hyaluronan with the aid of heterologous expression of a hyaluronan synthase from Streptococcus equisimilis in Bacillus subtilis. To achieve the production of sufficient amounts of hyaluronan, in addition to heterologous expression of a hyaluronan synthase, simultaneous expression of a UDP-glucose dehydrogenase in the Bacillus cells is also required. US 20030175902 and WO 03 054163 do not state the absolute amount of hyaluronan obtained in the production with the aid of Bacillus subtilis. The maximum mean molecular weight achieved was about 4.2 x 106. However, this mean molecular weight was only achieved for the recombinant Bacillus strain where a gene coding for the hyaluronan synthase gene from Streptococcus equisimilis and the gene coding for the UDP-glucose dehydrogenase from Bacillus subtilis were integrated into the Bacillus subtilis genome under the control of the amyQ promoter, where at the same time the Bacillus si/Mfe-endogenous cxpY gene (which codes for a cytochrome P450 oxidase) was inactivated.

[0025] WO 05 012529 describes the preparation of transgenic tobacco plants which were transformed using nucleic acid molecules encoding for hyaluronan synthases from Chlorella-infecting viruses. In WO 05 012529, use was made, on the one hand, of nucleic acid sequences encoding for hyaluronan synthase of the Chlorella virus strain CVHI1 and, on the other hand, of the Chlorella virus strain CVKA1 for transforming tobacco plants. The synthesis of hyaluronan could only be demonstrated for a plant transformed with a nucleic acid sequence encoding for a hyaluronan synthase isolated from the Chlorella virus strain CVKA1. For tobacco plants transformed with a nucleic acid sequence encoding for a hyaluronan synthase isolated from the Chlorella virus strain CVHI1, it was not possible to detect hyaluronan synthesis in the corresponding transgenic plants. The amount of hyaluronan synthesized by the only hyaluronan-producing transgenic tobacco plant in WO 05 012529 is stated as being about 4.2 pg of hyaluronan per ml of measured volume which, taking into account the description for carrying out the experiment in question, corresponds approximately to an amount of at most 12 pg of hyaluronan produced per gram of fresh weight of plant material.

[0026] Hyaluronan synthase catalyzes the synthesis of hyaluronan from the starting materials UDP-N-acetyl-glucosamine and UDP-glucuronic acid. Both starting materials mentioned are present in plant cells.

[0027] In plant cells, UDP-glucuronic acid serves as metabolite for one of a plurality of possible paths for synthesizing ascorbic acid (Lorence et al., 2004, Plant Physiol 134, 1200-1205) and as a central metabolite for the synthesis of the cell wall components pectin and hemicellulose which are synthesized in the endoplasmatic reticulum of the plant cell (Reiter, 1998, Plant Physiol Biochem 36(1), 167-176). The most important and most frequently occurring monomer of pectin is D-galacturonic acid (2004, H. W. Heldt in "Plant Biochemistry", 3rd Edition, Academic Press, ISBN 0120883910) which is synthesized using UDP-glucuronic acid. Furthermore, it is also possible, inter alia, to synthesize UDP-xylose, UDP-arabinose, UDP-galacturonic acid and UDP-apiose, metabolites for the synthesis of hemicellulose and pectin, using UDP-glucuronic acid (Seitz et al., 2000, Plant Journal, 21(6), 537-546). In plant cells, UDP-glucuronic acid can be synthesized either via the hexose phosphate metabolism comprising, inter alia, the conversion of UDP-glucose into UDP-glucuronic acid by UDP-GIc-DH or by the oxidative myo-inositol metabolism comprising the conversion of glucuronate 1-phosphate into UDP-glucuronic acid by glucuronate 1-phosphate uridilyl transferase. Both metabolic paths for synthesizing glucuronic acid appear to exist independently of one another and alternatively in different tissues/development stages of Arabidopsis plants (Seitz et al., 2000, Plant Journal 21(6), 537-546). The respective contribution of the two metabolic paths mentioned (hexose phosphate or oxidative myo-inositol metabolism) towards the synthesis of UDP-glucuronic acid has not yet been elucidated (Kårkonen, 2005, Plant Biosystems 139(1), 46-49).

[0028] The enzyme UDP-GIc-DH catalyzes the conversion of UDP-glucose into UDP-glucuronic acid. Samac et al. (2004, Applied Biochemistry and Biotechnology 113-116, Humana Press, Editor Ashok Mulehandani, 1167-1182 ) describe the tissue-specific overexpression of a UDP-GIc-DH from soybean in phloem cells of Alfalfa with the aim to increase the pectin content in the stems of these plants. The activity of UDP-GIc-DH, compared to the corresponding wild-type plants, was increased by more than 200%; however, the amount of pectin produced by the corresponding plants was lower than the amount of pectin produced by the corresponding wild-type plants. The amount of xylose and rhamnose monomers in the cell wall fraction of the transgenic plants in question was increased, whereas the amount of mannose monomers in the cell wall fraction was reduced.

[0029] The constitutive overexpression of a UDP-GIc-DH in Arabidosis plants resulted in aberrant growth of the plants in question compared to the corresponding wild-type plants and a dwarf phenotype. The cell wall fraction of the corresponding plants had an increased amount of mannose and galactose and a reduced amount of xylose, arabinose and uronic acids compared to the corresponding wild-type plants (Roman, 2004, "Studies on The Role of UDP-GIc-DH in Polysaccharide Biosynthesis", PhD thesis, Acta Universitatis Upsaliensis, ISBN 91-554-6088-7, ISSN 0282-7476). Thus, these results contradict at least in part the results of Samac et al. (2004, Applied Biochemistry and Biotechnology 113-116, Humana Press, Editor Ashok Mulehandani, 1167-1182 ) who detected a reduced amount of mannose and an increased amount of xylose in the cell wall fraction of corresponding transgenic plants.

[0030] For the synthesis of UDP-N-acetylglucosamine in plant cells, WO 98 35047 describes a metabolic path where glucosamine is converted by a number of successive enzymatically catalyzed reaction steps with formation of the metabolites N-acetyl-glucosamine, N-acetyl-glucosamine 6-phosphate, N-acetyl-glucosamine 1-phosphate into UDP-N-acetylglucosamine. An alternative metabolic path comprises the reaction of fructose 6-phosphate and glutamine giving glucosamine 6-phosphate which is subsequently converted by a number of successive enzymatically catalyzed reaction steps with formation of the metabolites glucosamine 1 -phosphate and N-acetyl-glucosamine 1 -phosphate into UDP-N-acetylglucosamine. The conversion of fructose 6-phosphate and glutamine into glucosamine 6-phosphate is catalyzed by a protein having glutamine:fructose 6-phosphate amidotransferase (GFAT) activity (Mayer et al., 1968, Plant Physiol. 43, 1097-1107).

[0031] WO 00 11192 describes the endosperm-specific overexpression of a nucleic acid molecule of corn encoding for a protein having the enzymatic activity of a GFAT in transgenic corn plants with the aim to synthesize a cationic starch in plants which has 2-amino-anhydroglucose molecules. The metabolic path described which, according to the description ofWO 00 11192, should result in 2-amino-anhydroglucose being incorporated into the starch, comprises inter alia the incorporation of UDP-glucosamine by starch synthases and/or glycogen synthases into the starch. It is stated that increased amounts of UDP-glucosamine could be detected in flour from endosperm of the transgenic corn plants in question overexpressing a nucleic acid molecule encoding for a protein having the (enzymatic) activity of a GFAT translationally fused with a plastid signal peptide. When the protein having the (enzymatic) activity of a GFAT was expressed without signal peptide, it was possible to detect an increased amount of glucosamine 1-phosphate in the corresponding flours from corn endosperm tissue. It was not possible to detect cationic starch in the transgenic plants.

[0032] The production of hyaluronan by fermentation of bacteria strains is associated with high costs, since the bacteria have to be fermented in sealed sterile containers under expensive controlled culture conditions (see, for example, US 4,897,349). Furthermore, the amount of hyaluronan which can be produced by fermentation of bacteria strains is limited by the production facilities present in each case. Here, it also has to be taken into account that fermenters, as a consequence of physical laws, cannot be built for excessively large culture volumes. Particular mention may be made here of homogeneous mixing of the substances fed in from the outside (for example essential nutrient sources for bacteria, reagents for regulating the pH, oxygen) with the culture medium required for efficient production, which, in large fermenters, can be ensured only with great technical expenditure, if at all.

[0033] The purification of hyaluronan from animal organisms is complicated owing to the presence, in animal tissues, of other mucopolysaccharides and proteins which specifically bind to hyaluronan. In patients, the use of hyaluronan-containing medicinal preparations contaminated by animal proteins can result in unwanted immunological reactions of the body (US 4,141,973), in particular if the patient is allergic to animal proteins (for example chicken egg white). Furthermore, the amounts (yields) of hyaluronan which can be obtained from animal tissues in satisfactory quality and purity are low (rooster comb: 0.079% w/w, EP 0144019, US 4,782,046), which necessitates the processing of large amounts of animal tissues. A further problem in the isolation of hyaluronan from animal tissues consists in that the molecular weight of hyaluronan during purification is reduced since animal tissues also contain a hyaluronan-degrading enzyme (hyaluronidase).

[0034] In addition to the hyaluronidases and exotoxins mentioned, Streptococcus strains also produce endotoxins which, when present in pharmacological products, pose risks for the health of the patient. In a scientific study, it was shown that even hyaluronan-containing medicinal products on the market contain detectable amounts of bacterial endotoxins (Dick et al., 2003, Eur J Opthalmol. 13(2), 176-184). A further disadvantage of the hyaluronan produced with the aid of Streptococcus strains is the fact that the isolated hyaluronan has a lower molecular weight than hyaluronan isolated from rooster combs (Lapcik et al. 1998, Chemical Reviews 98(8), 2663-2684). US 20030134393 describes the use of a Streptococcus strain for producing hyaluronan which synthesizes a particularly pronounced hyaluronan capsule (supercapsulated). The hyaluronan isolated after fermentation had a molecular weight of 9.1 x 106 Da. However, the yield was only 350 mg per liter.

[0035] Some of the disadvantages of producing hyaluronan by bacterial fermentation or by isolation from animal tissues can be avoided by producing hyaluronan using transgenic plants; however, the currently achieved amounts of hyaluronan which can be produced using transgenic plants would require a relatively large area under cultivation to produce relatively large amounts of hyaluronan. Furthermore, the isolation or purification of hyaluronan from plants having a lower hyaluronan content is considerably more complicated and costly than the isolation or purification from plants having a higher hyaluronan content.

[0036] Although hyaluronan has unusual properties, it is, owing to its scarcity and the high price, rarely, if at all, used for industrial applications.

[0037] Accordingly, it is an object of the present invention to provide means and methods which permit the provision of hyaluronan in sufficient amounts and quality and which make it possible to provide hyaluronan even for industrial applications and applications in the field of food and feed.

[0038] This object is achieved by the embodiments outlined in the claims.

[0039] Thus, the present invention relates to a genetically modified plant cell which has a nucleic acid molecule coding for a hyaluronan synthase linked to regulatory sequences ensuring transcription in plant cells stably integrated into its genome, wherein said plant cell additionally has a plurality of foreign nucleic acid molecules stably integrated into its genome, where a first foreign nucleic acid molecule codes for a protein having the activity of a glutamine:fructose 6-phosphate amidotransferase-2 (GFAT-2) originating from animals or a glutamine:fructose 6-phosphate amidotransferase (GFAT) originating from bacteria and a second foreign nucleic acid molecule codes for a protein having the activity of a UDP-glucose dehydrogenase (UDP-GIc-DH) and wherein said genetically modified plant cell has an increased activity of a protein having the activity of a glutamine:fructose 6-phosphate amidotransferase (GFAT) and an increased activity of a protein having the activity of a UDP-glucose dehydrogenase (UDP-GIc-DH) compared to corresponding not genetically modified wild-type plant cells.

[0040] The present invention also relates to plants comprising genetically modified plant cells according to the invention.

[0041] Here, the genetic modification of genetically modified plant cells according to the invention or genetically modified plants according to the invention can be any genetic modification resulting in a stable integration of a nucleic acid molecule encoding for a hyaluronan synthase into a plant cell or a plant [0042] In the context of the present invention, the term "wild-type plant cell" is to be understood as meaning plant cells which served as starting material for the preparation of the genetically modified plant cells according to the invention, i.e. their genetic information, apart from the genetic modifications introduced and resulting in a stable integration of a nucleic acid molecule encoding for a hyaluronan synthase and increasing the activity of a protein having the activity of a GFAT and increasing the activity of a protein having the activity of a UDP-GIc-DH, corresponds to that of a genetically modified plant cell according to the invention.

[0043] In the context of the present invention, the term "wild-type plant" is to be understood as meaning plants which served as starting material for the preparation of the genetically modified plants according to the invention, i.e. their genetic information, apart from the genetic modifications introduced and resulting in a stable integration of a nucleic acid molecule encoding for a hyaluronan synthase and increasing the activity of a protein having the activity of a GFAT and increasing the activity of a protein having the activity of a UDP-GIc-DFI, corresponds to that of a genetically modified plant according to the invention.

[0044] In the context of the present invention, the term "corresponding" means that, when a plurality of objects are compared, the objects in question which are compared to one another have been kept under the same conditions. In the context of the present invention, the term "corresponding" in the context of wild-type plant cells or wild-type plants means that the plant cells or plants compared to one another were cultivated under the same cultivation conditions and that they have the same (culture) age.

[0045] In the context of the present invention, the term "hyaluronan synthase" (EC 2.4.1.212) is to be understood as meaning a protein which synthesizes hyaluronan from the substrates UDP-glucuronic acid (UDP-GIcA) and N-acetyl-glucosamine (UDP-GIcNAc). The hyaluronan synthesis is catalyzed according to the reaction schemes below:

[0046] Nucleic acid molecules and corresponding protein sequences coding for hyaluronan synthases have been described, inter alia, for the following organisms: rabbit (Oryctolagus cuniculus) ocFlas2 (EMBL AB055978.1, US 20030235893), ocFlas3 (EMBL AB055979.1, US 20030235893); baboon (Papio anubis) paHasl (EMBL AY463695.1); frog (Xenopus laevis) xlHasl (EMBL M22249.1, US 20030235893), xlHas2 (DG42) (EMBL AF168465.1), xlFlas3 (EMBL AY302252.1); human ( Homo sapiens) hsHASI (EMBL D84424.1, US 20030235893), hsFIAS2 (EMBL U54804.1, US 20030235893), hsFIAS3 (EMBL AF232772.1, US 20030235893); mouse {Mus musculus), mmHasI (EMBL D82964.1, US 20030235893), mmFIAS2 (EMBL U52524.2, US 20030235893), mmFlas3 (EMBL U86408.2, US 20030235893); cattle (Bos taurus) btHas2 (EMBL AJ004951.1, US 20030235893); chicken {Gallus gallus) ggHas2 (EMBL AF106940.1, US 20030235893); rat (Rattus norvegicus) rnFlas 1 (EMBL AB097568.1, Itano et al„ 2004, J. Biol. Chem. 279(18) 18679-18678), rnHas2 (EMBL AF008201.1); rnFlas 3 (NCBI NMJ72319.1, Itano et al„ 2004, J. Biol. Chem. 279(18) 18679-18678), horse (Equus caballus) ecFIAS2 (EMBL AY056582.1, Gl:23428486), pig {Sus scrofa) sscFIAS2 (NCBI NM_214053.1, Gl:47522921), sscHas 3 (EMBLAB159675), zebra fish (Danio rerio) brHasI (EMBL AY437407), brFlas2 (EMBL AF190742.1) brHas3 (EMBL AF190743.1); Pasteurella multocida pmFlas (EMBL AF036004.2); Streptococcus pyogenes spFlas (EMBL, L20853.1, L21187.1, US 6,455,304, US 20030235893) ; Streptococcus equis seFlas (EMBL AF347022.1, AY173078.1), Streptococcus uberis suFlasA (EMBL AJ242946.2, US 20030235893), Streptococcus equisimilis seqFlas (EMBL AF023876.1, US 20030235893); Sulfolobus solfataricus ssFIAS (US 20030235893), Sulfolobus tokodaii stFlas (AP000988.1), Paramecium bursaria Chlorella Virus 1, cvFIAS (EMBL U42580.3, PB42580, US 20030235893).

[0047] In the context of the present invention, the term "UDP-glucose dehydrogenase (UDP-GIc-DFI)" (E.C. 1.1.1.22) is to be understood as meaning a protein which synthesizes, from UDP-glucose (UDP-GIc) and NAD+, UDP-glucuronic acid (UDP-GIcA) and NADH. This catalysis proceeds according to the reaction scheme below:

[0048] In the context of the present invention, the term "glutamine:fructose 6-phosphate amidotransferase (GFAT)" (E.C. 2.6.1.16), in the expert literature also referred to as glucosamine synthase, is to be understood as meaning a protein which synthesizes, from the starting materials glutamine and fructose 6-phosphate (Fruc-6-P), glucosamine 6-phosphate (GlcN-6-P). This catalysis proceeds according to the following reaction scheme: /

[0049] In particular in animal organisms, it was possible to demonstrate two different isoforms of proteins having the (enzymatic) activity of a GFAT (referred to as GFAT-1 and GFAT-2, respectively, in the literature). Flu et al. (2004), J. Biol. Chem. 279(29), 29988-29993 describe differences of the respective proteins from the mouse: in addition to differences in the tissue-specific expression of the proteins in question having the (enzymatic) activity of a glutamine:fructose 6-phosphate amidotransferase 1 (GFAT-1) and a glutamine:fructose 6-phosphate amidotransferase 2 (GFAT-2), it was possible to show that both isoforms are regulated by phosphorylation by a cAMP-dependent protein kinase. The activity of a protein having the (enzymatic) activity of a GFAT-1 is inhibited by phosphorylation of a conserved serine residue (serine 205 in the GFAT-1 from the mouse, GenBank Acc No.: AF334736.1) of the amido acid sequence in question, whereas the activity of a protein having the activity of a GFAT-2 is increased by phosphorylation of a conserved serine residue (serine 202 in the GFAT-2 from the mouse, GenBank Acc No.: NM_013529) of the amino acid sequence in question. Both proteins having the activity of a GFAT-1 and proteins having the activity of a GFAT-2 are inhibited in a concentration-dependent manner by UDP-N-acetylglucosamine; however, for a protein having the activity of a GFAT-2, the inhibition by UDP-N-acetylglucosamine is lower (maximum reduction of activity by UDP-N-acetylglucosamine about 15%) compared to a protein having the activity of a GFAT-1 (maximum reduction of activity by UDP-N-acetylglucosamine about 51% or 80%). There are indications that the inhibition of a protein having the activity of a GFAT-1 in animal organisms is based on the fact that at elevated UDP-N-acetylglucosamine concentrations there is an O-glucose-N-acetylglucosamine glycosylation of the proteins in question. Whether a regulation of the activity of proteins by O-glycosylation also takes place in plant cells is not yet fully understood (Huber and Hardin, 2004, Current Opinion in Plant Biotechnology 7, 318-322).

[0050] In the context of the present invention, the term "glutamine:fructose 6-phosphate amidotransferase-1 (GFAT-1)" is to be understood as meaning a protein which has the activity of a GFAT and whose activity is inhibited by phosphorylation by a cAMP-dependent protein kinase.

[0051] In the context of the present invention, the term "glutamine:fructose 6-phosphate amidotransferase-2 (GFAT-2)" is to be understood as meaning a protein which has the activity of a GFAT and which is activated by phosphorylation by a cAMP-dependent protein kinase.

[0052] In the context of the present invention, the term "glutamine:fructose 6-phosphate amidotransferase (GFAT)" is used as a comprehensive term which includes all proteins having the activity of a GFAT. Accordingly, it also comprises proteins referred to in the literature as "glutamine:fructose 6-phosphate amidotransferase-1 (GFAT-1)" or as "glutamine:fructose 6-phosphate amidotransferase-2 (GFAT-2)", but is not limited to these.

[0053] In the context of the present invention, the term "increased activity of a protein having the (enzymatic) activity of a GFAT" means an increased expression of endogenous genes coding for proteins having the activity of a GFAT and/or an increased amount of transcripts coding for proteins having the activity of a GFAT and/or an increased amount of protein having the activity of a GFAT in the cells and/or an increased enzymatic activity of proteins having the activity of a GFAT in the cells.

[0054] In the context of the present invention, the term "increased activity of a protein having the (enzymatic) activity of a UDP-GIc-DH" means an increased expression of endogenous genes coding for proteins having the activity of a UDP-GIc-DH and/or an increased amount of transcripts coding for proteins having the activity of a UDP-GIc-DH and/or an increased amount of protein having the activity of a UDP-GIc-DH in the cells and/or an increased enzymatic activity of proteins having the activity of a UDP-GIc-DH in the cells.

[0055] The genetically modified plant cells according to the invention or the genetically modified plants according to the invention meet in each case at least one of the conditions mentioned above meaning an increased enzymatic activity of a protein for proteins having the (enzymatic) activity of a GFAT and for proteins having the (enzymatic) activity of a UDP-GIc-DH.

[0056] An increased expression can be determined, for example, by measuring the amount of transcripts coding for a protein having the activity of a GFAT or coding for a protein having the activity of a UDP-GIc-DH, for example by Northern blot analysis or RT-PCR. Here, an increase preferably means an increase in the amount of transcripts compared to corresponding not genetically modified wild-type plant cells or not genetically modified wild-type plants by at least 50%, in particular by at least 70%, preferably by at least 85% and particularly preferably by at least 100%. An increase of the amount of transcripts coding for a protein having the activity of a GFAT or coding for a protein having the activity of a UDP-GIc-DH also means that plants or plant cells having no detectable amounts of transcripts coding for a protein having the activity of a GFAT and/or coding for a protein having the activity of a UDP-GIc-DH have, after genetic modification according to the invention, detectable amounts of transcripts coding for a protein having the activity of a GFAT and/or coding for a protein having the activity of a UDP-GIc-DH.

[0057] The increase in the amount of protein having the activity of a GFAT or of proteins having the activity of a UDP-GIc-DH resulting in an increased activity of these proteins in the plant cells in question can be determined, for example, by immunological methods, such as Western blot analysis, ELISA (Enzyme Linked Immuno Sorbent Assay) or RIA (Radio Immune Assay). Methods for preparing antibodies reacting specifically with a particular protein, i.e. binding specifically to said protein, are known to the person skilled in the art (see, for example, Lottspeich and Zorbas (Eds.), 1998, Bioanalytik [Bioanalysis], Spektrum akad. Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4). Some companies (for example Eurogentec, Belgium) offer the preparation of such antibodies as an order service. Here, an increase in the amount of protein preferably means an increase in the amount of protein having the activity of a GFAT and/or of proteins having the activity of a UDP-GIc-DH compared to corresponding not genetically modified wild-type plant cells or not genetically modified wild-type plants by at least 50%, in particular by at least 70%, preferably by at least 85% and particularly preferably by at least 100%. An increase in the amount of protein having the activity of a GFAT and/or of protein having the activity of a UDP-GIc-DH also means that plants or plant cells having no detectable amount of a protein having the activity of a GFAT and/or having no detectable activity of a protein having the activity of a UDP-GIc-DH have, after genetic modification according to the invention, a detectable amount of a protein having the activity of a GFAT and/or a detectable amount of a protein having the activity of a UDP-GIc-DH protein.

[0058] The increased activity of a protein having the activity of a GFAT in plant extracts can be determined by methods known to the person skilled in the art as described, for example, in Samac et al. (2004, Applied Biochemistry and Biotechnology 113-116, Humana Press, Editor Ashok Mulehandani, 1167-1182, ISSN 0273-2289). A preferred method for determining the amount of the activity of a protein having the activity of a GFAT is given in General Methods, item 6.

[0059] The increased activity of a protein having the activity of a UDP-GIc-DH in plant extracts can be described using methods known to the person skilled in the art, as described, for example, in WO 00 11192. A preferred method for determining the amount of the activity of a protein having the activity of a UDP-GIc-DH is given in General Methods, item 7.

[0060] An increased amount of (enzymatic) activity of proteins having the activity of a GFAT or of proteins having the activity of a UDP-GIc-DH preferably means an increase of the activity of such proteins by at least 50%, preferably by at least 70%, especially preferably by at least 85% and particularly preferably by at least 100% compared to corresponding not genetically modified wild-type plant cells or not genetically modified wild-type plants. An increase in the amount of (enzymatic) activity of proteins having the activity of a GFAT and/or of a protein having the activity of a UDP-GIc-DH also means that plants or plant cells having no detectable amount of a protein having the activity of a GFAT and/or having no detectable activity of a protein having the activity of a UDP-GIc-DH have, after genetic modification according to the invention, a detectable amount of a protein having the activity of a GFAT and/or a detectable amount of a protein having the activity of a UDP-GIc-DH.

[0061] In the context of the present invention, the term "genome" is to be understood as meaning the entire genetic material present in a plant cell. It is known to the person skilled in the art that, in addition to the nucleus, other compartments (for example plastids, mitochondria) also contain genetic material.

[0062] In the context of the present invention, the term "stably integrated nucleic acid molecule" is to be understood as meaning the integration of a nucleic acid molecule into the genome of the plant. A stably integrated nucleic acid molecule is characterized in that, during the replication of the corresponding integration site, it is multiplied together with the nucleic acid sequences of the host which border on the integration site, so that the integration site in the replicated DNA strand is surrounded by the same nucleic acid sequences as on the read strand which serves as a matrix for the replication.

[0063] A large number of techniques for stably integrating nucleic acid molecules into a plant host cell is available. 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).

[0064] The use of agrobacterium-mediated transformation of plant cells has been subject to in-depth studies and has been described exhaustively in EP 120516; Hoekema, IN: The Binary Plant Vector System Offsetdrukkerij Kanters B.V. Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4, 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.

[0065] The transformation of monocotyledonous plants using vectors based on Agrobacterium transformation has been described, too (Chan et al., Plant Mol. 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 Mol. 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 corn 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).

[0066] The successful transformation of other cereal species has also been described, for example for barley (Wan and Lemaux, see above; Ritala et al., see above; 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.

[0067] Compared to the prior art, genetically modified plant cells according to the invention or genetically modified plants according to the invention offer the advantage that they produce higher amounts of hyaluronan than plants having only the activity of a hyaluronan synthase. This allows hyaluronan to be produced at little expense since the isolation of hyaluronan from plants having a higher hyaluronan content is less complicated and more cost efficient. Furthermore, compared to the plants described in the prior art, smaller cultivation areas are required to produce hyaluronan using the genetically modified plants according to the invention. This leads to the possibility to provide hyaluronan in sufficient amounts even for industrial applications where it is currently not used owing to its scarcity and the high price. Virus-infected plant organisms of the genus Chlorella are unsuitable for producing relatively large amounts of hyaluronan. In the production of hyaluronan, virus-infected algae have the disadvantage that the genes required for hyaluronan synthase are not stably integrated into their genome (Van Etten and Meints, 1999, Annu. Rev. Microbiol. 53, 447-494 ), so that, for producing hyaluronan, the virus infection has to be repeated. Accordingly, it is not possible to isolate individual Chlorella cells which synthesize continuously the desired quality and quantity of hyaluronan. Furthermore, in virus-infected Chlorella algae, hyaluronan is only produced for a limited period of time, and as a result of the lysis caused by the virus, the algae are killed only about 8 hours after the infection (Van Etten et al., 2002, Arch Virol 147, 1479-1516). In contrast, the present invention offers the advantage that the genetically modified plant cells according to the invention and the genetically modified plants according to the invention can be propagated in an unlimited manner vegetatively or sexually and that they produce hyaluronan continuously.

[0068] The transgenic plants described in WO 05 012529, which have a nucleic acid molecule coding for a hyaluronan synthase, synthesize a relatively small amount of hyaluronan. In contrast, the present invention offers the advantage that genetically modified plant cells according to the invention and genetically modified plants according to the invention synthesize considerably higher amounts of hyaluronan.

[0069] Accordingly, the present invention also provides genetically modified plant cells according to the invention or genetically modified plants according to the invention which synthesize hyaluronan. Genetically modified plant cells according to the invention or genetically modified plants according to the invention preferably synthesize at least 100, with preference at least 600, particularly preferably at least 1000 and especially preferably at least 1500 pg of hyaluronan per g of fresh weight (FW) of plant material.

[0070] Prefrably plant cells according to the invention or plants according to the invention synthesize at most 25000 pg hyaluronan per gram fresh weight, with preference at most 20000 pg hyaluronan per gram fresh weight, particularly preferable at most 15000 pg hyaluronan per gram fresh weight, especially preferable at most 10000 pg hyaluronan per gram fresh weight an mostly preferable at most 6500 pg hyaluronan per gram fresh weight.

[0071] For determining the hyaluronan content with respect to the fresh weight in genetically modified plant cells according to the invention or genetically modified plants according to the invention, preference is given to using the method for work-up of the plant material described under General Methods item 2 and the method for determining the amount of hyaluronan described under General Methods item 4.

[0072] The present invention also provides genetically modified plant cells according to the invention or genetically modified plants according to the invention which synthesize at least 1000, preferably at least 2000, particularly preferably at least 4000, especially preferably at least 5000 pg of hyaluronan per g of dry weight (DW) of plant material. For determining the hyaluronan content with respect to the dry weight in the genetically modified plant cells according to the invention or the genetically modified plants according to the invention, preference is given to using the method for work-up of the plant material described in Example 13 k) and the method for determining the amount of hyaluronan described under General Methods item 4.

[0073] It has been observed that, over the development time, hyaluronan accumulates in plant tissue; accordingly, the amount of hyaluronan with respect to the fresh weight or with respect to the dry weight in the genetically modified plant cells according to the invention or in the genetically modified plants according to the invention is to be determined with particular preference during harvesting or a few (one or two) days before harvesting of the plant cells in question or the plants in question. Here, use is made in particular of plant material (for example tubers, seeds, leaves) with respect to the amount of hyaluronan which is to be used for further processing.

[0074] Genetically modified plant cells according to the invention or genetically modified plants according to the invention which synthesize hyaluronan can be identified by isolating the hyaluronan that is synthesized by them and proving its structure.

[0075] Since plant tissue has the advantage that it does not contain hyaluronidases, a simple and rapid isolation method can be used for confirming the presence of hyaluronan in genetically modified plant cells according to the invention or genetically modified plants according to the invention. To this end, water is added to the plant tissue to be examined and the plant tissue is then comminuted mechanically (with the aid of, for example, a bead mill, a beater mill, a Warring blender, a juice extractor, etc.). If required, more water may then be added to the suspension, and cell debris and water-insoluble components are then removed by centrifugation or sieving. The presence of hyaluronan in the supernatant obtained after centrifugation can then be demonstrated using, for example, a protein which binds specifically to hyaluronan. A method for detecting hyaluronan with the aid of a protein that binds specifically to hyaluronan is described, for example, in US 5,019,498. Test kits for carrying out the method described in US 5,019,498 are commercially available (for example, the hyaluronic acid (HA) test kit from Corgenix, Inc., Colorado, USA, Prod. No. 029-001; see also General Methods item 4). In parallel, it is possible to initially digest an aliquot of the centrifugation supernatant obtained with a hyaluronidase and then to confirm the presence of hyaluronan with the aid of the protein that specifically binds to hyaluronan, as described above. By the action of the hyaluronidase in the parallel batch, the hyaluronan present therein is degraded, so that after complete digestion it is no longer possible to detect significant amounts of hyaluronan.

[0076] The presence of hyaluronan in the centrifugation supernatant can furthermore also be confirmed using other analysis methods, such as, for example, IR, NMR or mass spectroscopy.

[0077] As already discussed above, it is not clear which metabolic path (hexose phosphate or oxidative myo-inositol metabolic path) is mainly used in plant cells for synthesizing UDP-glucuronic acid, and whether both metabolic paths make different quantitative contributions to the synthesis of UDP-glucuronic acid, depending on the tissue and/or development stage of the plant. Furthermore, the overexpression of a UDP-Glc-DH in transgenic plants does not lead to consistent results, and it was not possible to achieve the target to increase the pectin content of the cell wall adopting such an approach. Additionally, the regulation of the activity of proteins having the activity of a UDP-GIc-DH is inhibited by UDP-xylose. This was demonstrated both for relevant proteins originating from prokaryotes (Campbell et al., 1997, J. Biol. Chem. 272(6), 3416-3422; Schiller et al., 1973, Biochim. Biophys Acta 293(1), 1-10), from animal organisms (Balduini et al., 1970, Biochem. J. 120(4), 719-724) and from plants (Hinterberg, 2002, Plant Physiol. Biochem. 40, 1011-1017). Moreover, the reaction products glucuronic acid and NADH originating from the reaction catalyzed by a protein having the activity of a UDP-GIc-DH are inhibitors which regulate the activity of a protein having the activity of a GFAT (Campbell et al., 1997, J. Biol. Chem. 272(6), 3416-3422, Ordman and Kirkwood, 1977, Biochim Biophys Acta 482(1) 25-32; Turner and Botha, 2002, Archives of Biochem. Biophys.407, 209-216).

[0078] The overexpression, in corn, of a protein having the (enzymatic) activity of a GFAT fused translationally with a plastid signal peptide resulted in an increased UDP-glucosamine content, and the cytosolic overexpression, in corn, of a protein having the (enzymatic) activity of a GFAT resulted in an increased glucosamine 1-phosphate content in ground endosperm tissue. Flowever, UDP-glucosamine and glucosamine 1-phosphate are not starting materials for the synthesis of hyaluronan by hyaluronan synthase. Furthermore, it is known that glucosamine has a cytotoxic effect on plant cells (Roberts et al., 1971, Plant Physiol. 48, 36-42) and that, if relatively high concentrations are present in plant cells, it is converted into glucosamine 6-phosphate. Glucosamine 6-phosphate is likewise toxic for plant cells (WO 98 35047, US 6,444,878). Furthermore, it is known that proteins having the activity of a GFAT can be regulated in an inhibitory manner by metabolites which are formed in the further metabolic path for the synthesis of UDP-N-acetyl-glucosamine. Proteins having the activity of a GFAT, isolated from eukaryotes (both with animal and plant organisms) are inhibited, for example, by UDP-N-acetyl-glucosamine, which is one of the two substrates for hyaluronan synthase (Kornfeld, 1967, J. Biol. Chem. 242(13), 3135-3141; Graack et al., 2001, Biochem. J. 360, 401-412; Mayer et al., 1968, Plant Physiol. 43, 1097-1107). Bacterial proteins having the activity of a GFAT are inhibited by glucosamine 6-phosphate, a direct reaction product of the GFAT-catalyzed reaction ( Deng et al., 2005, Metabolic Engineering 7, 201-214). There are no indications in the literature what may limit the amount of hyaluronan synthesized in plant cells.

[0079] Accordingly, it has surprisingly been found that genetically modified plant cells or genetically modified plants having a nucleic acid molecule coding for a hyaluronan synthase and having additionally increased GFAT activity and increased UDP-GIc-DFI activity compared to genetically modified plant cells or genetically modified plants having (only) hyaluronan synthase activity produce significantly higher amounts of hyaluronan.

[0080] In a preferred embodiment, the present invention relates to genetically modified plant cells according to the invention or genetically modified plants according to the invention, wherein they produce an increased amount of hyaluronan compared to genetically modified plant cells or compared to genetically modified plants which (only) have the activity of a hyaluronan synthase or compared to genetically modified plant cells or compared to genetically modified plants having the activity of a hyaluronan synthase and no increased activity of a protein having the activity of a GFAT and no increased activity of a protein having the activity of a UDP-GIc-DH. Preferably, the amount of hyaluronan produced with respect to the fresh weight of the plant material in the genetically modified plant cells according to the invention or in the genetically modified plants according to the invention is at least 1.5 times, preferably at least 5 times, particularly preferably at least 7.5 times and especially preferably at least 10 times higher, compared to corresponding genetically modified plant cells or compared to corresponding genetically modified plants which (only) have the activity of a hyaluronan synthase. To determine the increase of the hyaluronan content with respect to the fresh weight of the plant material in the genetically modified plant cells according to the invention or in the genetically modified plants according to the invention, it is preferred to compare genetically modified plant cells according to the invention or genetically modified plants according to the invention with corresponding plant cells or plants (only) having the activity hyaluronan synthase, where equivalent material (for example leaf, tuber) of plant cells or plants should be compared, the plant cells or plants from which this material is taken should have been cultivated under the same conditions and where the hyaluronan content of plant material having a comparable age (development stage) should be compared. One must not, for example, compare young leaves of a plant with old leaves of another plant or plants.

[0081] In the context of the present invention, the term "plant cell or plant (only) having the activity of a hyaluronan synthase" is to be understood as meaning a genetically modified plant cell or a genetically modified plant where the genetic modification consists in that it comprises a nucleic acid molecule coding for a hyaluronan synthase, compared to corresponding not genetically modified wild-type plant cells or not genetically modified wild-type plants.

[0082] In particular, "plant cells or plants (only) having the activity of a hyaluronan synthase" are characterized in that they synthesize hyaluronan and that they have no additional genetic modifications other than the introduction of a nucleic acid molecule coding for a hyaluronan synthase into not genetically modified wild-type plant cells or not genetically modified wild-type plants. Preferably, such plants do not have an increased activity of a protein having the activity of a GFAT and no increased activity of a protein having the activity of a UDP-GIc-DH.

[0083] The amount of hyaluronan produced by plant cells or plants can be determined with the aid of the methods which have already been described above, for example using a commercial test kit (for example the hyaluronic acid (HA) test kit from Corgenix, Inc., Colorado, USA, Prod. No. 029-001). A method which is preferred in the context of the present invention for determining the hyaluronan content in plant cells or plants is described under General Methods, item 4.

[0084] In a further embodiment of the present invention, the genetically modified plant cells according to the invention or the genetically modified plants according to the invention are plant cells of a green terrestrial plant or green terrestrial plants, respectively, which synthesize hyaluronan.

[0085] In the context of the present invention, the term "green terrestrial plant (Embryophyta)" is to be understood as defined in Strasburger, "Lehrbuch der Botanik" [Textbook of Botany], 34th ed., Spektrum Akad. Verf, 1999, (ISBN 3-8274-0779-6).

[0086] A preferred embodiment of the present invention relates to genetically modified plant cells according to the invention of multicellular plants or genetically modified plants according to the invention which are multicellular organisms. Accordingly, this embodiment relates to plant cells or plants which do not originate from single-cell plants (protists) or which are not protists.

[0087] The genetically modified plant cells according to the invention or the genetically modified plants according to the invention may, in principle, be plant cells and plants, respectively, of any plant species, i.e. both monocotyledonous and dicotyledonous plants. They are preferably crop plants, i.e. plants cultivated by man for the purpose of feeding man and animal or for producing biomass and/or for preparing substances for technical, industrial purposes (for example corn, rice, wheat, alfalfa, rye, oats, barley, manioc, potato, tomato, switchgrass (Panicum virgatum), sago, mung beans, peas, sorghum, carrots, aubergine, radish, oilseed rape, soybeans, peanuts, cucumbers, pumpkins, melons, leek, garlic, cabbage, spinach, sweet potato, asparagus, courgettes, lettuce, artichokes, sweetcorn, parsnip, scorzonera, Jerusalem artichoke, banana, sugarbeet, sugarcane, beetroot, broccoli, cabbage, onion, yellow beet, dandelion, strawberry, apple, apricot, plum, peach, grapevines, cauliflower, celery, bell peppers, swede, rhubarb). Particularly preferred are tomato or potato plants.

[0088] In a preferred embodiment, the present invention relates to genetically modified plant cells according to the invention or genetically modified plants according to the invention where the nucleic acid molecule coding for hyaluronan synthase is characterized in that it codes for a viral hyaluronan synthase. The nucleic acid molecule coding for the hyaluronan synthase preferably codes for a hyaluronan synthase of a virus which infects algae.

[0089] With respect to an algae-infecting virus, the nucleic acid molecule which codes for a hyaluronan synthase preferably codes for a hyaluronan synthase of a C/7/ore//a-infecting virus, particularly preferably a hyaluronan synthase of a Paramecium bursaria Chlorella virus 1 and especially preferably a hyaluronan synthase of a Paramecium bursaria Chlorella virus of an H1 strain.

[0090] In a further preferred embodiment, the present invention relates to genetically modified plant cells according to the invention or genetically modified plants according to the invention where the nucleic acid molecule which codes for the hyaluronan synthase is characterized in that the codons of the nucleic acid molecule coding for a hyaluronan synthase are modified compared to the codons of the nucleic acid molecule coding for the hyaluronan synthase of the organism that the hyaluronan synthase originates from. With particular preference, the codons of the hyaluronan synthase have been modified such that they are adapted to the frequency of the use of the codons of the plant cell or the plant into whose genome they are integrated or to be integrated.

[0091] Owing to the degeneration 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. Suitable 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 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).

[0092] The nucleic acid molecule coding for the hyaluronan synthase is preferably characterized in that it codes for a hyaluronan synthase whose amino acid sequence is at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the amino acid sequence shown under SEQ ID NO 2. Most preferably, the nucleic acid molecule coding for the hyaluronan synthase is characterized in that it codes for a hyaluronan synthase having the amino acid sequence shown under SEQ ID No 2.

[0093] The nucleic acid molecule coding for a hyaluronan synthase may be at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the nucleic acid sequence shown under SEQ ID NO 1 or SEQ ID NO 3. The nucleic acid molecule coding for the hyaluronan synthase may be characterized in that it has the nucleic acid sequence shown under SEQ ID No 3.

[0094] On 8. 25. 2004, the plasmid IC 341-222, comprising a synthetic nucleic acid molecule coding for a Paramecium bursaria Chlorella virus hyaluronan synthase was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Brunswick, Germany, under the number DSM16664, in accordance with the Budapest treaty. The amino acid sequence shown in SEQ ID NO 2 can be derived from the coding region of the nucleic acid sequence integrated into the plasmid IC 341-222 and codes for a Paramecium bursaria Chlorella virus hyaluronan synthase.

[0095] Accordingly, the present description also relates to genetically modified plant cells or genetically modified plants where the nucleic acid molecule which codes for the hyaluronan synthase is characterized in that it codes for a protein whose amino acid sequence can be derived from the coding region of the nucleic acid sequence inserted into plasmid DSM16664 or that it codes for a protein whose amino acid sequence is at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the amino acid sequence which can be derived from the coding region of the nucleic acid sequence inserted into plasmid DSM16664.

[0096] The present description also relates to genetically modified plant cells or genetically modified plants where the nucleic acid molecule coding for hyaluronan synthase is characterized in that it is the hyaluronan-synthase-encoding nucleic acid sequence integrated into plasmid DSM16664 or that it is at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the nucleic acid sequence integrated into plasmid DSM16664.

[0097] The present invention furthermore describes genetically modified plant cells according to the invention or genetically modified plants according to the invention which are characterized in that they have a foreign nucleic acid molecule stably integrated into their genome or a plurality of foreign nucleic acid molecules stably integrated into their genome, said foreign nucleic acid molecule or said foreign nucleic acid molecules increasing the activity of a protein having the activity of a GFAT and increasing the activity of a protein having the activity of a UDP-GIc-DH compared to corresponding not genetically modified wild-type plant cells or corresponding not genetically modified wild-type plants.

[0098] It may be a single foreign nucleic acid molecule which, by integration into the genome of genetically modified plant cells according to the invention or genetically modified plants according to the invention, increases the activity of a protein having the activity of a GFAT and simultaneously increases the activity of a protein having the activity of a UDP-GIc-DFI compared to corresponding wild-type plant cells or to corresponding wild-type plants. Flowever, it may also be a plurality of foreign nucleic acid molecules, one foreign nucleic acid molecule of which increases the activity of a protein having the activity of a UDP-GIc-DFI and another foreign nucleic acid molecule increasing the activity of a protein having the activity of a UDP-GIc-DFI compared to the corresponding wild-type plant cells or to the corresponding wild-type plants. If a plurality of foreign nucleic acid molecules are integrated into the genome of a genetically modified plant cell according to the invention or a genetically modified plant according to the invention, both foreign nucleic acid molecules together may be at one site in the genome of the plant cell or of the plant, or they may be localized in different sites in the genome of the plant cell or the plant (for example on different chromosomes or different chromosome sections). Accordingly, the foreign nucleic acid molecules may either be inherited as a joint locus or as coupled loci according to Mendel's rules, or they may be inherited as separate loci independently of one another according to Mendel's rules.

[0099] In the context of the present invention, the term "foreign nucleic acid molecule" is to be understood as meaning a molecule which either does not naturally occur in the corresponding wild-type plant cells or which does not naturally occur in the concrete spatial arrangement in wild-type plant cells or which is localized at a site in the genome of the wild-type plant cell where it does not naturally occur. Preferably, the foreign nucleic acid molecule is a recombinant molecule comprising various elements whose combination or specific spatial arrangement does not naturally occur in plant cells.

[0100] In the context of the present invention, the term "recombinant nucleic acid molecule" is to be understood as meaning a nucleic acid molecule which has various nucleic acid molecules which are not naturally present in a combination like that present in a recombinant nucleic acid molecule. Thus, recombinant nucleic acid molecules may, in addition to nucleic acid molecules coding for a hyaluronan synthase and/or a protein having the activity of a GFAT and/or a protein having the activity of a UDP-GIc-DFI, additionally have nucleic acid sequences which are not naturally present in combination with the nucleic acid molecules mentioned. The additional nucleic acid sequences mentioned which are present on a recombinant nucleic acid molecule in combination with a nucleic acid molecule encoding for a hyaluronan synthase or a protein having the activity of a GFAT and/or a protein having the activity of a UDP-GIc-DFI may be any sequences. For example, they may be genomic plant nucleic acid sequences. The additional nucleic acid sequences mentioned are preferably regulatory sequences (promoters, termination signals, enhancers), particularly preferably regulatory sequences which are active in plant tissue, especially preferably tissue-specific regulatory sequences which are active in plant tissue. Methods for generating recombinant nucleic acid molecules are known to the person skilled in the art and comprise genetic engineering methods, such as, for example, 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 Flarbour Laboratory Press, Cold Spring Flarbour, NY ISBN: 0879695773, Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN: 0471250929).

[0101] Genetically modified plant cells and genetically modified plants having a foreign nucleic acid molecule stably integrated into their genome or a plurality of foreign nucleic acid molecules stably integrated into their genome which code for hyaluronan synthase and which increase the activity of a protein having the activity of a GFAT and increase the activity of a protein having the activity of a UDP-GIc-DFI compared to corresponding not genetically modified wild-type plant cells or not genetically modified wild-type plants can be distinguished from said wild-type plant cells and said wild-type plants, respectively, inter alia by the fact that they comprise a foreign nucleic acid molecule which does not naturally occur in wild-type plant cells and wild-type plants, respectively, or that such a molecule is integrated at a site in the genome of the genetically modified plant cell according to the invention or in the genome of the genetically modified plant according to the invention where it does not occur in wild-type plant cells and wild-type plants, respectively, i.e. in a different genomic environment. Furthermore, such genetically modified plant cells according to the invention and genetically modified plants according to the invention can be distinguished from not genetically modified wild-type plant cells and not genetically modified wild-type plants, respectively, in 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 naturally present in the wild-type plant cells or wild-type plants. If the foreign nucleic acid molecule(s) introduced into the genetically modified plant cells according to the invention or the genetically modified plants according to the invention are additional copies of molecules already naturally present in the wild-type plant cells or the wild-type plants, the genetically modified plant cells according to the invention and the genetically modified plants according to the invention can be distinguished from wild-type plant cells and wild-type plants, respectively, in particular by the fact that this additional copy/these additional copies is/are localized at sites in the genome where it/they is/are not present in wild-type plant cells and wild-type plants, respectively.

[0102] The stable integration of a nucleic acid molecule into the genome of a plant cell or a plant can be demonstrated by genetic methods and/or methods of molecular biology. A stable integration of a nucleic acid molecule into the genome of a plant cell or the genome of a plant is characterized in that in the progeny which has inherited said nucleic acid molecule, the stably integrated nucleic acid molecule is present in the same genomic environment as in the parent generation. The presence of a stable integration of a nucleic acid sequence in the genome of a plant cell or in the genome of a plant can be demonstrated using methods known to the person skilled in the art, inter alia with the aid of Southern blot analysis of 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), with methods based on PCR, such as, for example, the analysis of differences in length in the amplified fragment (Amplified Fragment Length Polymorphism, 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 using amplified fragments cleaved with the aid of 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).

[0103] The present invention describes genetically modified plant cells or genetically modified plants which are characterized in that at least one foreign nucleic acid molecule codes for a protein having the (enzymatic) activity of a GFAT or that at least one foreign nucleic acid molecule codes for a protein having the (enzymatic) activity of a UDP-GIc-DH.

[0104] The present invention describes to genetically modified plant cells according to the invention or genetically modified plants according to the invention which are characterized in that a first foreign nucleic acid molecule codes for a protein having the (enzymatic) activity of a GFAT and a second foreign nucleic acid molecule codes for a protein having the (enzymatic) activity of a UDP-GIc-DH.

[0105] The foreign nucleic acid molecule coding for a protein having the (enzymatic) activity of a GFAT may originate from bacteria, or, animals, particularly preferably from mammals or bacteria and especially preferably from the mouse or Escherichia coli.

[0106] With respect to a foreign nucleic acid molecule coding for a protein having the (enzymatic) activity of a GFAT originating from animal organisms, use is preferably to be made of a nucleic acid molecule coding for a protein having the (enzymatic) activity of a GFAT-2; with particular preference, the protein having the (enzymatic) activity of a GFAT-2 originates from the mouse.

[0107] Instead of a naturally occurring nucleic acid molecule coding for a protein having the (enzymatic) activity of a GFAT, it is also possible for a nucleic acid molecule generated by mutagenesis to be introduced into the genetically modified plant cells according to the invention or the genetically modified plants according to the invention, where said mutagenized foreign nucleic acid molecule is characterized in that it codes for a protein having the (enzymatic) activity of a GFAT with reduced inhibition by metabolites (for example of the glucosamine metabolism). The preparation of such mutagenized nucleic acid molecules is described in an exemplary manner for a protein having the (enzymatic) activity of a GFAT from Escherichia coli in Deng et al. (2005, Metabolic Engineering 7, 201-214; WO 04 003175). Mutants for a protein having the activity of a GFAT from the mouse are described, for example, in Hu et al. (2004, J. Biol. Chem. 279 (29), 29988-29993).

[0108] According to the invention, the foreign nucleic acid molecule coding for a protein having the (enzymatic) activity of a UDP-GIc-DH may originate from any organism; preferably, said nucleic acid molecule originates from bacteria, fungi, animals, plants or viruses, particularly preferably from bacteria, plants or viruses, especially preferably from viruses.

[0109] With respect to viruses, the foreign nucleic acid molecule coding for a protein having the (enzymatic) activity of a UDP-GIc-DH preferably originates from a virus which infects algae, with preference from a virus which infects algae of the genus Chlorella, particularly preferably from a Paramecium bursaria Chlorella virus and especially preferably from a Paramecium bursaria Chlorella virus of an H1 strain.

[0110] Instead of a naturally occurring nucleic acid molecule coding for a protein having the (enzymatic) activity of a UDP-GIc-DH, it is also possible for a nucleic acid molecule generated by mutagenesis to be introduced into the genetically modified plant cells according to the invention or the genetically modified plants according to the invention, where said mutagenized foreign nucleic acid molecule is characterized in that it codes for a protein having the (enzymatic) activity of a UDP-GIc-DH with reduced inhibition by metabolites (for example of the glucuronic acid metabolism).

[0111] Nucleic acid molecules coding for a protein having the activity of a GFAT are known to the person skilled in the art and described in the literature. Thus, nucleic acid molecules coding for a protein having the activity of a GFAT are described from viruses, for example for the Chlorella virus k2 (EMBL acc No AB107976.1), from bacteria, for example for Escherichia coli (Dutka-Malen, 1988, Biochemie 70 (2), 287-290; EMBL acc No: L10328.1), from fungi, for example for Saccharomyces cerevisiae (EMBL acc No AF334737.1, Watzele et al., 1989, J. Biol.

Chem. 264, 8753-8758), Aspergillus niger (EMBL acc No AY594332.1), Candida albicans (EMBL acc No X94753.1), from insects, for example for Aedes aegyti (Kato et al., 2002, Insect. Biol. 11 (3),207,216: EMBL acc No AF399922.1), Drosophila melanogaster (GFAT-1: EMBL acc No Y18627.1, GFAT-2: NCBI acc No NM_143360.2), from algae for Volvariella voivacea (EMBL acc No AY661466.1), from vertebrates for example for Homo sapiens (GFAT-1: EMBL acc No AF334737.1; GFAT-2: NCBI acc No BC000012.2, Oki et al., 1999, Genomics 57 (2), 227-34), Mus musculus (GFAT-1: EMBL acc No AF334736.1; GFAT-2: EMBL acc No AB016780.1), or from plants for example for Arabidopsis thaliana (EMBL acc No AP001297.1; cds NCBI acc No BAB03027.1).

[0112] The present description relates to genetically modified plant cells according to the invention and genetically modified plants where the foreign nucleic acid molecule coding for a protein having the activity of a GFAT is selected from the group consisting of 1. a) nucleic acid molecules coding for a protein having the amino acid sequence given under SEQ ID NO 10 or a protein having the amino acid sequence given under SEQ ID NO 12; 2. b) nucleic acid molecules coding for a protein whose sequence is at least 60%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the amino acid sequence given under SEQ ID NO 10 or under SEQ ID NO 12; 3. c) nucleic acid molecules comprising the nucleotide sequence shown under the nucleotide sequence shown under SEQ ID NO 9 or a sequence complementary thereto, the nucleotide sequence shown under SEQ ID NO 11 or a sequence complementary thereto or the nucleotide sequence shown under SEQ ID NO 13 or a sequence complementary thereto; 4. d) nucleic acid molecules which are at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the nucleic acid sequences described under a) or c); 5. e) nucleic acid molecules which hybridize under stringent conditions with at least one strand of the nucleic acid sequences described under a) or c); 6. f) nucleic acid molecules whose nucleotide sequence differs from the sequence of the nucleic acid molecules mentioned under a) or c) owing to the degeneration of the genetic code; and 7. 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 f).

[0113] Nucleic acid molecules coding for a protein having the activity of a UDP-GIc-DFI are described in the literature and known to the person skilled in the art. Thus, nucleic acid molecules coding for a protein having the activity of a UDP-GIc-DFI are described from viruses for example for the Chlorella virus 1 (NCBI acc No NC_000852.3), from bacteria for example tor Escherichia coli (EMBL acc No: AF176356.1), from fungi for example for Aspergillus niger (EMBL acc No AY594332.1), Cryptococcus neoformans (EMBL acc AF405548.1), from insects for example tor Drosophila melanogaster (EMBL acc No AF001310.1), from vertebrates for example tor Homo sapiens (EMBL acc No AF061016.1), Mus musculus (EMBL acc No AF061017.1), Bos taurus (EMBL acc No AF095792.1), Xenopus laevis (EMBL acc No AY762616.1) or from plants for example for poplar (EMBL acc No AF053973.1), Colocasia esculenta (EMBL acc No AY222335.1), Dunaliella salina (EMBL acc No AY795899.1), Glycine max (EMBL acc No U53418.1).

[0114] The present description relates to genetically modified plant cells and genetically modified plants where the foreign nucleic acid molecule coding for a protein having the activity of a UDP-GIc-DFI is selected from the group consisting of 1. a) nucleic acid molecules coding for a protein having the amino acid sequence given under SEQ ID NO 5; 2. b) nucleic acid molecules coding for a protein whose sequence is at least 60%" preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the amino acid sequence given under SEQ ID NO 5; 3. c) nucleic acid molecules comprising the nucleotide sequence shown under SEQ ID NO 4 or a sequence complementary thereto or the nucleotide sequence shown under SEQ ID NO 6 or a sequence complementary thereto; 4. d) nucleic acid molecules which are at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the nucleic acid sequences described under a) or c); 5. e) nucleic acid molecules which hybridize under stringent conditions with at least one strand of the nucleic acid sequences described under a) or c); 6. f) nucleic acid molecules whose nucleotide sequence differs from the sequence of the nucleic acid molecules mentioned under a) or c) owing to the degeneration of the genetic code; and 7. 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 f).

[0115] In the context of the present invention, the term "hybridization" means a hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Flarbor Laboratory Press, Cold Spring Flarbor, NY). With particular preference, "hybridization" means a hybridization under the following conditions:

Flybridization buffer: 2xSSC; 10xDenhardt solution (Fikoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na2FIPQ4; 250 pg/ml of herring sperm DNA; 50 pg/ml of tRNA; or 25 M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS Hybridization temperature:

T=65 to 68°C

[0116] Nucleic acid molecules which hybridize with nucleic acid molecules coding for a protein having the activity of a UDP-GIc-DH may originate from any organism; accordingly, they may originate from bacteria, fungi, animals, plants or viruses.

[0117] Nucleic acid molecules hybridizing with nucleic acid molecules coding for a protein having the activity of a UDP-GIc-DH preferably originate from a virus which infects algae, with preference a virus which infects algae of the genus Chlorella, particularly preferably a Paramecium bursaria Chlorella virus and especially preferably a Paramecium bursaria Chlorella virus of an H1 strain.

[0118] Nucleic acid molecules hybridizing with nucleic acid molecules coding for a protein having the activity of a GFAT preferably originate from mammals, or bacteria and especially preferably from the mouse or Escherichia coli.

[0119] Nucleic acid molecules hybridizing with nucleic acid molecules coding for a protein having the activity of a GFAT-2 originate from an animal organism, preferably from the mouse.

[0120] Nucleic acid molecules which hybridize with the molecules mentioned may be isolated, for example, from genomic or from cDNA libraries. Such nucleic acid molecules can be identified and isolated using the nucleic acid molecules mentioned or parts of these molecules or the reverse complements of these molecules, for example by hybridization according to 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 using PCR.

[0121] As hybridization sample for isolating a nucleic acid sequence coding for a protein having the activity of a UDP-GIc-DH, it is possible to use, for example, nucleic acid molecules which have exactly or essentially the nucleotide sequence given under SEQ ID NO 4 or SEQ ID NO 6, or parts of these sequences.

[0122] As hybridization sample for isolating a nucleic acid sequence coding for a protein having the activity of a GFAT, it is possible to use, for example, nucleic acid molecules having exactly or essentially the nucleotide sequence given under SEQ ID NO 9 or under SEQ ID NO 11 or under SEQ ID NO 13, or parts of these sequences.

[0123] The fragments used as hybridization samples may also be synthetic fragments or oligonucleotides prepared using the customary synthesis techniques, whose sequence is essentially identical to the nucleic acid molecule described in the context of the present invention. Once genes which hybridize with the nucleic acid sequences described in the context of the present invention are identified and isolated, the sequence should be determined and the properties of the proteins coded for by this sequence should be analyzed to determine whether they are proteins having the activity of a GFAT, a GFAT-1 or a GFAT-2 or the activity of a UDP-GIc-DH. Methods of how to determine whether a protein has the activity of a protein having the activity of a GFAT (for example Mayer et al., 1968, Plant Physiol. 43, 1097-1107; Deng et al., 2005, Metabolic Engineering 7, 201-214), a GFAT-1 or a GFAT-2 (for example Hu et al., 2004, J. Biol. Chem. 279 (29), 29988-29993) or a UDP-GIc-DH (for example De Luca et al., 1976, Connective Tissue Research 4, 247-254; Bar-Peled et al., 2004, Biochem. J. 381, 131-136; Turner and Botha, 2002, Archives Biochem. Biophys. 407, 209-216) are known to the person skilled in the art and described, inter alia, in the literature described.

[0124] 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 to the nucleic acid molecules described above may, for example, be due to deletion, addition, substitution, insertion or recombination.

[0125] 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 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%. In the context of the present invention, the term "identity" is to be understood as meaning the number of identical amino acids/nucleotides (identity) with other proteins/nucleic acids, expressed in percent. Preferably, the identity with respect to a protein having the activity of a UDP-GIc-DH is determined by comparison with the amino acid sequence given under SEQ ID NO 5, the identity with respect to a nucleic acid molecule coding for a protein having the activity of a UDP-GIc-DH is determined by comparison with the nucleic acid sequence given under SEQ ID NO 4 or SEQ ID NO 6, the identity with respect to a protein having the activity of a GFAT is determined by comparison with the amino acid sequence given under SEQ ID NO 10 or SEQ ID NO 12 and the identity with respect to a nucleic acid molecule coding for a protein having the activity of a GFAT is determined by comparison with the nucleic acid sequence given under SEQ ID NO 9 or SEQ ID NO 11 or SEQ ID NO 13 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 Génétique et de Biologie Moléculaire 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 1SD, UK).

[0126] 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=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.

[0127] 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, DNAMATRIXIUB, GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS: unweighted.

[0128] 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 owing to the degeneration of the genetic code.

[0129] The various derivatives of the nucleic acid molecules coding for a protein having the activity of a GFAT or a UDP-GIc-DH have certain common characteristics.

[0130] These may, for example, be biological or enzymatic activity, substrate specificity, molecular weight, immunological reactivity, conformation, etc., and also physical properties, such as, for example, the run properties in gel electrophoresis, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum, etc. Preferred properties of proteins having the activity of a GFAT or a UDP-GIc-DH are known to the person skilled in the art, have already been mentioned above and are to apply here in an analogous manner.

[0131] In a further preferred embodiment, the present invention relates to genetically modified plant cells according to the invention or genetically modified plants according to the invention where nucleic acid molecules coding for a protein having the (enzymatic) activity of a GFAT and/or coding for a protein having the (enzymatic) activity of a UDP-GIc-DH are characterized in that the codons of said nucleic acid molecules are different from the codons of the nucleic acid molecules which code for said protein having the (enzymatic) activity of a GFAT or code for a said protein having the (enzymatic) activity of a UDP-GIc-DH of the parent organism. Particularly preferably, the codons of the nucleic acid molecules coding for a protein having the (enzymatic) activity of a GFAT or coding for a protein having the (enzymatic) activity of a UDP-GIc-DH are changed thus that they are adapted to the frequency of use of the codons of the plant cell or the plant into whose genome they are integrated or to be integrated.

[0132] The present invention furthermore provides genetically modified plant cells according to the invention or genetically modified plants according to the invention wherein the foreign nucleic acid molecules stably integrated into the genome of the plant cell or the plant coding for a hyaluronan synthase and/or coding for a protein having the (enzymatic) activity of a GFAT and/or coding for a protein having the (enzymatic) activity of a UDP-GIc-DH are linked to regulatory elements initiating the transcription in plant cells (promoters). These may be homologous or heterologous promoters. The promoters can be constitutive, tissue-specific, development-specific or regulated by external factors (for example after application of chemical substances, by action of abiotic factors, such as heat and/or cold, drought, disease, etc.). Here, nucleic acid molecules coding for a hyaluronan synthase or a protein having the (enzymatic) activity of a GFAT or a protein having the (enzymatic) activity of a UDP-GIc-DH, which nucleic acid molecules are integrated into the genome of a genetically modified plant cell according to the invention or a genetically modified plant according to the invention, may in each case be linked to the same promoter, or the individual sequences may be linked to different promoters. Here, two or three different promoters in any combination may in each case be linked to a relevant foreign nucleic acid molecule coding for a hyaluronan synthase or a protein having the (enzymatic) activity of a GFAT or a protein having the (enzymatic) activity of a UDP-GIc-DH in a genetically modified plant cell according to the invention or a genetically modified plant according to the invention.

[0133] The present description relates to genetically modified plant cells according to the invention or genetically modified plants according to the invention where at least one foreign nucleic acid molecule, particularly preferably at least two foreign nucleic acid molecules, especially preferably three foreign nucleic acid molecules selected from the group consisting of nucleic acid molecules coding for a hyaluronan synthase or a protein having the (enzymatic) activity of a GFAT or a protein having the (enzymatic) activity of a UDP-GIc-DH is (are) linked to a tissue-specific promoter. Preferred tissue-specific promoters are promoters which initiate transcription specifically in plant tuber, fruit or seed cells or leaves.

[0134] To express nucleic acid molecules coding for a hyaluronan synthase or a protein having the (enzymatic) activity of a GFAT or a protein having the (enzymatic) activity of a UDP-GIc-DFI, these are preferably linked to regulatory DNA sequences ensuring the transcription in plant cells. These include in particular promoters. In general, any promoter active in plant cells is suitable for the expression.

[0135] Here, the promoter may be chosen such that expression is constitutively or only in a certain tissue, at a certain point of the development of the plant or at a point of time determined by external factors. Both in respect of the plant and in respect of the nucleic acid molecule to be expressed, the promoter may be homologous or heterologous.

[0136] Suitable promoters are, for example, the promoter of 35S RNAof the cauliflower mosaic virus or the ubiquitin promoter from corn or the Oestrum YLCV (Yellow Leaf Curling Virus; WO 01 73087; Stavolone et al., 2003, Plant Mol. Biol. 53, 703-713) for a constitutive expression, the patatingen promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) for a 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. USA84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451) or for an endosperm-specific expression the HMWG promoter from wheat, the USP promoter, the phaseolin promoter, promoters of zein genes from corn (Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93), the glutelin promoter (Leisy et al., Plant Mol. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993), 357-366; Yoshihara et al., FEBS Lett. 383 (1996), 213-218) or the shrunken-1 promoter (Werr et al., EMBO J. 4 (1985), 1373-1380), a globulin promoter (Nakase et al., 1996, Gene 170(2), 223-226) or a prolamin promoter (Qu und Takaiwa, 2004, Plant Biotechnology Journal 2(2), 113-125). However, it is also possible to use promoters which are only active at a point in time determined by external factors (see, for example, WO 9307279). Of particular interest here may be promoters of heat-shock proteins which permit a simple induction. It is furthermore possible to use seed-specific promoters, such as, for example, the USP promoter from Vida faba which ensures a seed-specific expression in Vida faba and other plants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Baumlein et al., Mol. Gen. Genet. 225 (1991), 459-467).

[0137] The use of promoters present in the genome of algae-infecting viruses are also 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 Mol Biol 26(1), 85-93, Van Etten et al., 2002, Arch Virol 147, 1479-1516).

[0138] In the context of the present invention, the term "tissue specific" is to be understood as meaning the substantial limitation of a manifestation (for example initiation of transcription) to a certain tissue.

[0139] In the context of the present invention, the terms "tuber, fruit or seed cell" are to be understood as meaning all cells present in a tuber, a fruit or in a seed.

[0140] In the context of the present invention, the term "homologous promoter" is to be understood as meaning a promoter which is naturally present in plant cells or plants used for the preparation of genetically modified plant cells according to the invention or genetically modified plants according to the invention (homologous with respect to the plant cell or the plant) or as meaning a promoter which regulates the regulation of the expression of a gene in the organism from which the sequence was isolated (homologous with respect to the nucleic acid molecule to be expressed).

[0141] In the context of the present invention, the term "heterologous promoter" is to be understood as meaning a promoter which is not naturally present in plant cells or plants used for the preparation of genetically modified plant cells according to the invention or genetically modified plants according to the invention (heterologous with respect to the plant cell or plant) or as meaning a promoter which is, in the organism from which a nucleic acid sequence to be expressed was isolated, not naturally present for regulating the expression of said nucleic acid sequence (heterologous with respect to the nucleic acid molecule to be expressed).

[0142] Also present may be a termination sequence (polyadenylation signal) which serves to add a poly-A tail to the transcript. The poly-A tail is thought to act in stabilizing the transcripts. Such elements are described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged as desired.

[0143] It is also possible for intron sequences to be present between the promoter and the coding region. Such intron sequences may lead to stability of expression and an increased expression in plants (Callis et al., 1987, Genes Devel. 1, 1183-1200; Luehrsen, and Walbot, 1991, Mol. 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). Suitable intron sequences are, for example, the first intron of the sh1 gene from corn, the first intron of the poly-ubiquitin gene 1 from corn, the first intron of the EPSPS gene from rice or one of the first two introns of the PAT 1 gene from Arabidopsis.

[0144] The present invention also relates to plants comprising genetically modified plant cells according to the invention. Such plants may be produced by regeneration from genetically modified plant cells according to the invention.

[0145] The present invention also relates to processible or consumable parts of genetically modified plants according to the invention comprising genetically modified plant cells according to the invention.

[0146] In the context of the present invention, the term "processible parts" is to be understood as meaning plant parts which are used for preparing foodstuff or feedstuff, which are used as a raw material source for industrial processes, as a raw material source for the preparation of pharmaceutical products or as a raw material source for the preparation of cosmetic products.

[0147] In the context of the present invention, the term "consumable parts" is to be understood as meaning plant parts which serve as food for man or are used as animal feed.

[0148] The present invention also relates to a propagation material of genetically modified plants according to the invention comprising a genetically modified plant cell according to the invention.

[0149] Here, the term "propagation material" comprises those components of the plant which are suitable for generating progeny via the vegetative or generative route. Suitable for vegetative propagation are, for example, cuttings, callus cultures, rhizomes or tubers. Other propagation material includes, for example, fruits, seeds, seedlings, protoplasts, cell cultures, etc. The propagation material preferably takes the form of tubers, fruits or seeds.

[0150] In a further embodiment, the present invention relates to harvestable plant parts of genetically modified plants according to the invention, such as fruits, storage and other roots, flowers, buds, shoots, leaves or stalks, preferably seeds, fruits or tubers, these harvestable parts comprising genetically modified plant cells according to the invention.

[0151] Preferably, the present invention relates to propagation material according to the invention or harvestable parts of plants according to the invention comprising hyaluronan. Particularly preferred is propagation material according to the invention or harvestable parts of plants according to the invention which synthesize hyaluronan.

[0152] In the context of the present invention, the term "potato plant" or "potato" is to be understood as meaning plant species of the genus Solarium, particularly tuber-producing species of the genus Solarium and in particular Solarium tuberosum.

[0153] In the context of the present invention, the term "tomato plant" or "tomato" is to be understood as meaning plant species of the genus Lycopersicon, in particular Lycopersicon esculentum.

[0154] Afurther advantage of the present invention is that harvestable parts, propagation material, processible parts or consumable parts of genetically modified plants according to the invention comprise more hyaluronan than hyaluronan-synthesizing transgenic plants described in the literature. Accordingly, genetically modified plants according to the invention are not only particularly suitable for use as raw material from which hyaluronan may be isolated but can also be used directly as foodstuff/feedstuff or for preparing foodstuff/feedstuff having a prophylactic or therapeutic character (for example for osteoarthritis prophylaxis, US 6,607,745). Since genetically modified plants according to the invention have a higher hyaluronan content than the plants described in the literature, the preparation of such foodstuff/feedstuff requires lower amounts of harvestable parts, propagation material, processible parts or consumable parts of genetically modified plants according to the invention. If consumable parts of genetically modified plants according to the invention are consumed, for example, directly as a so-called "nutraceutical", it is possible to achieve a positive effect even by ingesting relatively small amounts of substance. This may be of particular significance inter alia in the production of animal feed, since animal feed having too high a content of plant components is unsuitable as feedstuff for various animal species.

[0155] By virtue of the high capacity of hyaluronan to bind water, harvestable parts, propagation material, processible parts or consumable parts of genetically modified plants according to the invention furthermore have the advantage that less thickeners are required when solidified foodstuff/feedstuff is produced. Thus, for example, the production of jelly requires less sugar, which is associated with an additional positive effect on health. In the production of foodstuff/feedstuff requiring the dehydration of the crude plant material, the advantage of using harvestable parts, propagation material, processible parts or consumable parts of genetically modified plants according to the invention consists in the fact that less water has to be removed from the plant material in question, resulting in lower production costs and, owing to more gentle preparation methods (for example lower and/or shorter input of heat), an elevated nutritional value of the foodstuff/feedstuff in question. Thus, for example, in the production of tomato ketchup less energy has to be introduced in order to achieve the desired consistency.

[0156] The present invention furthermore provides a process for preparing a plant which synthesizes hyaluronan, which comprises 1. a) genetically modifying a plant cell, where the genetic modification comprises steps i to iii below 1. i) introduction of a foreign nucleic acid molecule coding for a hyaluronan synthase linked to regulatory sequences ensuring transcription in plant cells into the plant cell 2. ii) introduction of a foreign nucleic acid molecule into the plant cell, the foreign nucleic acid molecule comprising a coding sequence for a protein having the activity of a glutamine:fructose 6-phosphate amidotransferase-2 (GFAT-2) originating from animals or a glutamine:fructose 6-phosphate amidotransferase (GFAT) originating from bacteria 3. iii) introduction of a foreign nucleic acid molecule into the plant cell, the foreign nucleic acid molecule comprising a coding sequence for a protein having the activity of a glutamine:fructose 6-phosphate UDP-glucose dehydrogenase where steps i to iii can be carried out in any order, individually, or any combinations of steps i to iii can be carried out simultaneously 2. b) regenerating a plant from plant cells from step a); 3. c) generating, if appropriate, further plants using the plants according to step b), where, if appropriate, plant cells are isolated from plants according to steps b) or c) and the process steps a) to c) are repeated until a plant is generated which has a foreign nucleic acid molecule coding for a hyaluronan synthase and has an increased activity of a protein having the (enzymatic) activity of a GFAT compared to corresponding not genetically modified wild-type plant cells and an increased activity of a protein having the (enzymatic) activity of a UDP-GIc-DFI compared to corresponding not genetically modified wild-type plant cells.

[0157] The present description preferably relates to processes for preparing a plant which synthesizes hyaluronan which comprises 1. a) genetically modifying a plant cell, where the genetic modification comprises steps to iii below in any order, or any combinations of steps i to iii may be carried out individually or simultaneously, 1. i) introduction of a foreign nucleic acid molecule coding for a hyaluronan synthase linked to regulatory sequences ensuring transcription in plant cells into the plant cell 2. ii) introduction of a foreign nucleic acid molecule into the plant cell, the foreign nucleic acid molecule comprising a coding sequence for a protein having the activity of a glutamine:fructose 6-phosphate amidotransferase-2 (GFAT-2) originating from animals or a glutamine:fructose 6-phosphate amidotransferase (GFAT) originating from bacteria 3. iii) introduction of a foreign nucleic acid molecule into the plant cell, the foreign nucleic acid molecule comprising a coding sequence for a protein having the activity of a glutamine:fructose 6-phosphate UDP-glucose dehydrogenase 2. b) regenerating a plant from plant cells comprising the genetic modification according to steps 1. i) a) i 2. ii) a) ii 3. iii) a) iii 4. iv) a) i and a) ii, 5. v) a) i and a) iii, 6. vi) a) ii and a) iii, or 7. vii) a) i and a) ii and a) iii 3. c) introducing into plant cells of plants according to step 1. i) b) i a genetic modification according to step a) ii, 2. ii) b) i a genetic modification according to step a) iii, 3. iii) b) i a genetic modification according to step a) ii and simultaneously a genetic modification according to step a) iii, 4. iv) b) ii a genetic modification according to step a) i, 5. v) b) ii a genetic modification according to step a) iii, 6. vi) b) ii a genetic modification according to step a) i and simultaneously a genetic modification according to step a) iii, 7. vii) b) iii a genetic modification according to step a) i, 8. viii) b) iii a genetic modification according to step a) ii, 9. ix) b) iii a genetic modification according to step a) i and simultaneously a genetic modification according to step a) ii, 10. x) b) iv a genetic modification according to step a) iii, 11. xi) b) v a genetic modification according to step a) ii, or 12. xii) b) vi a genetic modification according to step a) i and regenerating a plant 4. d) introducing into planet cells of plants according to step 1. i) c) i a genetic modification according to step a) iii, 2. ii) c) ii a genetic modification according to step a) ii, 3. iii) c) iv a genetic modification according to step a) iii, 4. iv) c) v a genetic modification according to step a) ii, 5. v) c) vii a genetic modification according to step a) ii, 6. vi) c) vii a genetic modification according to step a) i, or 7. vii) c) ixa genetic modification according to step a) ii and regenerating a plant 5. e) generating, if appropriate, further plants with the aid of the plants according to any of steps b) vii, c) iii, c) vi, c) x, c) xi, c) xii or according to any of steps d) i to d) vii.

[0158] The regeneration of the plants according to step b) and, if appropriate, step c) and d) of the processes according to the invention can be carried out using methods known to the person skilled in the art (described, for example, in "Plant Cell Culture Protocols", 1999, edited by R.D. Hall, Humana Press, ISBN 0-89603-549-2).

[0159] The generation of further plants (depending on the process according to step c) or step e)) of the processes can be carried out, for example, by vegetative propagation (for example via cuttings, tubers or via callus culture and regeneration of intact plants) or via generative propagation. In this context, generative propagation generally takes place under controlled conditions, i.e. selected plants with specific characteristics are hybridized with one another and multiplied. The selection preferably takes place in such a manner that the further plants (depending on the process generated according to step c) or step e)) comprise the modifications introduced in the preceding steps.

[0160] In processes according to the invention for preparing plants which synthesize hyaluronan, the genetic modifications for generating the genetically modified plant cells according to the invention can be carried out simultaneously or in successive steps and in any combination. Both wild-type plants and wild-type plant cells may be used as a starting point into which a foreign nucleic acid molecule coding for a hyaluronan synthase has not yet been introduced and into which a genetic modification increasing the activity of a protein having the (enzymatic) activity of a GFAT compared to corresponding not genetically modified wild-type plant cells has not yet been introduced and into which a genetic modification increasing the activity of a protein having the (enzymatic) activity of a UDP-GIc-DH compared to corresponding not genetically modified wild-type plant cells has not yet been introduced, or plant cells or plants which have already been genetically modified and into which a nucleic acid molecule coding for a hyaluronan synthase has already been introduced and/or into which a genetic modification for increasing the activity of a protein having the (enzymatic) activity of a GFAT compared to corresponding not genetically modified wild-type plant cells has already been introduced and/or into which a genetic modification for increasing the activity of a protein having the (enzymatic) activity of a GFAT compared to corresponding not genetically modified wild-type plant cells has already been introduced. Here, it is immaterial whether the same method as for the genetic modification resulting in an increased activity of a protein having the (enzymatic) activity of a UDP-GIc-DH is used for successive genetic modifications resulting in an increased activity of a protein having the (enzymatic) activity of a GFAT, as long as both genetic modifications together result in an increased activity of a protein having the (enzymatic) activity of a GFAT and a protein having the (enzymatic) activity of a UDP-GIc-DH in the same plant cell. It is also immaterial which method is used for introducing a foreign nucleic acid molecule coding for a hyaluronan synthase into the plant cell.

[0161] The processes for preparing a plant which synthesizes hyaluronan, consists in the introduction of at least one foreign nucleic acid molecule or of a plurality of foreign nucleic acid molecules into the genome of the plant cell, where the foreign nucleic acid molecule(s) comprises/comprise a coding sequence for a hyaluronan synthase and a coding sequence for a protein having the (enzymatic) activity of a GFAT and a coding sequence for a protein having the (enzymatic) activity of a UDP-GIc-DH.

[0162] As already described above for the foreign nucleic acid molecules introduced for genetic modification into the plant cell or plant, what is introduced in step a) of the processes according to the invention for preparing a plant which synthesizes hyaluronan may be an individual nucleic acid molecule or a plurality of nucleic acid molecules. Thus, the foreign nucleic acid molecules coding for a hyaluronan synthase and/or coding for a protein having the (enzymatic) activity of a GFAT and/or coding for a protein having the (enzymatic) activity of a UDP-GIc-DH may be present together on a single nucleic acid molecule, or two of the foreign nucleic acid molecules mentioned may be present together on a single nucleic acid molecule and the third foreign nucleic acid molecule may be present on another nucleic acid molecule, in any possible combination, or all three of the foreign nucleic acid molecules mentioned may in each case be present on individual separate nucleic acid molecules.

[0163] Furthermore, to introduce a foreign nucleic acid molecule in the practice of processes according to the invention for preparing a plant which synthesizes hyaluronan, it is possible to use, instead of a wild-type plant cell or wild-type plant, mutant cells or mutants which are distinguished in that they already have an increased activity of a protein having the (enzymatic) activity of a GFAT and/or an increased activity of a protein having the (enzymatic) activity of a UDP-GIc-DH. If the mutant cell or the mutant already has an increased activity of a protein having the (enzymatic) activity of a GFAT or an increased activity of a protein having the (enzymatic) activity of a UDP-GIc-DH compared to the corresponding wild-type plant cells or wild-type plants, it is sufficient for carrying out a process according to the invention for preparing a plant which synthesizes hyaluronan that a foreign nucleic acid molecule coding for a hyaluronan synthase and a genetic modification resulting in an increase in the activity of a protein having the (enzymatic) activity of a UDP-GIc-DH or an increase in the activity of a protein having the (enzymatic) activity of a GFAT, compared to corresponding not genetically modified wild-type plant cells, is introduced into said mutant cell or mutant. If the mutant cell or the mutant already has an increased activity of a protein having the (enzymatic) activity of a GFAT and an increased activity of a protein having the (enzymatic) activity of a UDP-GIc-DH compared to corresponding wild-type plant cells or a corresponding wild-type plant, a foreign nucleic acid molecule coding for a hyaluronan synthase may be introduced into said mutant cell or mutant for carrying out a process according to the invention for preparing a plant which synthesizes hyaluronan.

[0164] All said further above concerning the use of mutants for the preparation of genetically modified plant cells according to the invention or genetically modified plants according to the invention applies here in an analogous manner.

[0165] The present description relates to processes according to the invention for preparing a plant which synthesizes hyaluronan, wherein the nucleic acid molecule coding for a hyaluronan synthase in step a) is selected from the group consisting of: 1. a) nucleic acid molecules characterized in that they code for a viral hyaluronan synthase, 2. b) nucleic acid molecules characterized in that they code for a hyaluronan synthase of a Chlorella-infecting virus, 3. c) nucleic acid molecules characterized in that they code for a hyaluronan synthase of a Paramecium bursaria Chlorella virus 1, 4. d) nucleic acid molecules characterized in that they code for a hyaluronan synthase of a Paramecium bursaria Chlorella virus 1 of strain H1, 5. e) nucleic acid molecules characterized in that the codons of the nucleic acid molecule coding for a hyaluronan synthase are modified compared to the codons of the nucleic acid molecule which code for the hyaluronan synthase in the parent organism of the hyaluronan synthase, 6. f) nucleic acid molecules characterized in that the codons of the hyaluronan synthase have been modified thus that they are adapted to the frequency of the use of the codons of the plant cell or of the plant into whose genome they are to be integrated or are integrated, 7. g) nucleic acid molecules characterized in that they code for a hyaluronan synthase having the amino acid sequence shown under SEQ ID NO 2 or that they code for a hyaluronan synthase whose amino acid sequence is at least 70%, preferably at least 80%, particularly preferably at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the amino acid sequence shown under SEQ ID NO 2, 8. h) nucleic acid molecules characterized in that they code for a protein whose amino acid sequence can be derived from the coding region of the nucleic acid sequence inserted into plasmid DSM16664 or that they code for a protein whose amino acid sequence is at least 70%, preferably at least 80%, particularly preferably at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the amino acid sequence which can be derived from the coding region of the nucleic acid sequence inserted into plasmid DSM16664, 9. i) nucleic acid molecules comprising a nucleic acid sequence shown under SEQ ID NO 1 or SEQ ID NO 3 or being at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the nucleic acid sequence shown under SEQ ID NO 1 or SEQ ID NO 3, 10. j) nucleic acid molecules comprising the nucleic acid sequence inserted into plasmid DSM16664 or being at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the nucleic acid sequence inserted into plasmid DSM16664, 11. k) nucleic acid molecules coding for a hyaluronan synthase, where the nucleic acid sequences coding for the hyaluronan synthase are linked to regulatory elements (promoter) which initiate the transcription in plant cells or 12. I) nucleic acid molecules according to k) where the promoters are tissue-specific promoters, particularly preferably promoters which initiate the initiation of transcription specifically in plant tuber, fruit or seed cells.

[0166] Preferably the nucleic acid molecule coding for a protein having the activity of a GFAT is selected from the group consisting of: 1. a) nucleic acid molecules characterized in that they code for a protein having the activity of a GFAT originating from bacteria or animals, preferably from Escherichia coli or the mouse, 2. b) nucleic acid molecules characterized in that the codons of the nucleic acid molecule coding for a protein having the activity of a GFAT are modified compared to the codons of the nucleic acid molecule coding for the corresponding protein having the activity of a GFAT of the parent organism, 3. c) nucleic acid molecules characterized in that the codons of the protein having the activity of a GFAT are modified thus that they are adapted to the frequency of the use of the codons of the plant cell or of the plant into whose genome they are to be integrated or are integrated, 4. d) nucleic acid molecules coding for a protein having the amino acid sequence shown under SEQ ID NO 10 or for a protein having the amino acid sequence shown under SEQ ID NO 12; 5. e) nucleic acid molecules coding for a protein whose sequence is at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the amino acid sequence shown under SEQ ID NO 10 or under SEQ ID NO 12; 6. f) nucleic acid molecules comprising the nucleic acid sequence shown under SEQ ID NO 9 or a sequence complementary thereto or the nucleic acid sequence shown under SEQ ID NO 11 or a sequence complementary thereto or the nucleic acid sequence shown under SEQ ID NO 13 or a sequence complementary thereto; 7. g) nucleic acid molecules which are at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the nucleic acid sequences described under h); 8. h) nucleic acid molecules which hybridize under stringent conditions with at least one strand of the nucleic acid sequences described under f) or h); 9. i) nucleic acid molecules whose nucleotide sequence differs from the sequence of the nucleic acid molecules mentioned under f) or h) owing to the degeneration of the genetic code; and 10. j) nucleic acid molecules which are fragments, allelic variants and/or derivatives of the nucleic acid molecules mentioned under a), b), c), d), e), f) or h), 11. k) nucleic acid molecules coding for a protein having the activity of a GFAT, where the nucleic acid sequences coding for a protein having the activity of a GFAT are linked to regulatory elements (promoter) which initiate the transcription in plant cells or 12. I) nucleic acid molecules according to m), where the promoters are tissue-specific promoters, particularly preferably promoters which initiate the initiation of transcription specifically in plant tuber, leaf, fruit or seed cells.

[0167] The present description relates to processes according to the invention for preparing a plant which synthesizes hyaluronan, where the foreign nucleic acid molecule coding for a protein having the activity of a UDP-GIc-DFI is selected from the group consisting of: 1. a) nucleic acid molecules characterized in that they code for a protein having the activity of a UDP-GIc-DH originating from viruses, bacteria, animals or plants, 2. b) nucleic acid molecules characterized in that they code for a protein having the activity of a UDP-GIc-DH of a Chlorella-infecting virus, 3. c) nucleic acid molecules characterized in that they code for a protein having the activity of a UDP-GIc-DH of a Paramecium bursaria

Chlorella virus, 4. d) nucleic acid molecules characterized in that the codons of the nucleic acid molecule coding for a protein having the activity of a UDP-GIc-DH are modified compared to the codons of the nucleic acid molecule coding for the corresponding protein having the activity of a UDP-GIc-DH of the parent organism, 5. e) nucleic acid molecules characterized in that the codons of the protein having the activity of a UDP-GIc-DH are modified thus that they are adapted to the frequency of the use of the codons of the plant cell or of the plant into whose genome they are to be integrated or are integrated, 6. f) nucleic acid molecules coding for a protein having the amino acid sequence shown under SEQ ID NO 5; 7. g) nucleic acid molecules coding for a protein whose sequence is at least 70%, preferably at least 80%, with preference at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the amino acid sequence shown under SEQ ID NO 5; 8. h) nucleic acid molecules comprising the nucleotide sequence shown under SEQ ID NO 4 or a sequence complementary thereto or the nucleotide sequence shown under SEQ ID NO 6 or a sequence complementary thereto; 9. i) nucleic acid molecules which are at least 70%, preferably at least 80%, with preference at least 90%, especially preferably 95% and most preferably at least 98% identical to the nucleic acid sequences described under h); 10. j) nucleic acid molecules which hybridize under stringent conditions with at least one strand of the nucleic acid molecules described under f) or h); 11. k) nucleic acid molecules whose nucleotide sequence differs from the sequence of the nucleic acid molecules mentioned under f) or h) owing to the degeneration of the genetic code; and 12. I) nucleic acid molecules which are fragments, allelic variants and/or derivatives of the nucleic acid molecules mentioned under a), b), c), d), e), f) or h), 13. m) nucleic acid molecules coding for a protein having the activity of a UDP-GIc-DH where the nucleic acid sequences coding for a protein having the activity of a UDP-GIc-DH are linked to regulatory elements (promoter) which initiate the transcription in plant cells or 14. n) nucleic acid molecules according to m), where the promoters are tissue-specific promoters, particularly preferably promoters which initiate the initiation of transcription specifically in plant tuber, leaf, fruit or seed cells.

[0168] In a preferred embodiment of the present invention, the processes for preparing a plant which synthesizes hyaluronan relate to processes for preparing a plant synthesizing at least 100, preferably at least 600, particularly preferably at least 1000, especially preferably at least 1500, pg of hyaluronan per g of fresh weight (FW) of plant material.

[0169] In a further preferred embodiment, processes according to the invention for preparing a plant which synthesizes hyaluronan are used for preparing genetically modified plants according to the invention.

[0170] The present invention also provides plants obtainable by a process according to the invention for preparing a plant which synthesizes hyaluronan.

[0171] The present invention furthermore relates to a process for preparing hyaluronan which comprises the step of extracting hyaluronan from genetically modified plant cells according to the invention, from genetically modified plants according to the invention, from propagation material according to the invention, from harvestable plant parts according to the invention or from plants or parts of these plants obtainable by a process according to the invention for preparing plants which synthesize hyaluronan.

[0172] Preferably, such a process also comprises the step of harvesting the cultivated genetically modified plant cells according to the invention, the genetically modified plants according to the invention, the propagation material according to the invention, the harvestable plant parts according to the invention, the processible plant parts according to the invention prior to extracting the hyaluronan, and particularly preferably furthermore the step of cultivating genetically modified plant cells according to the invention or genetically modified plants according to the invention prior to harvesting.

[0173] In contrast to bacterial or animal tissues, plant tissues have no hyaluronidases and do not contain any hyaladherins. Accordingly, as already described above, extraction of hyaluronan from plant tissues is possible using relatively simple methods. If required, the aqueous extracts, described above, of plant cells or tissues containing hyaluronan can be purified further using methods known to the person skilled in the art, such as, for example, repeated precipitation with ethanol. A preferred method for purifying hyaluronan is described under General Methods item 3.

[0174] The processes already described for extracting hyaluronan from genetically modified plant cells according to the invention or genetically modified plants according to the invention are also suitable for isolating hyaluronan from propagation material according to the invention, from harvestable plant parts according to the invention or from plants or parts of these plants obtainable by a process according to the invention for preparing plants which synthesize hyaluronan.

[0175] The present invention also provides the use of genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, harvestable plant parts according to the invention, processible plant parts according to the invention or plants obtainable by a process according to the invention for preparing hyaluronan.

[0176] The present invention furthermore describes compositions comprising genetically modified plant cells according to the invention. Here, it is immaterial whether the plant cells are intact or no longer intact because they have been destroyed, for example, by processing. The compositions are preferably foodstuff or feedstuff, pharmaceutical or cosmetic products.

[0177] The present invention further describes compositions comprising components of genetically modified plant cells according to the invention, of genetically modified plants according to the invention, of propagation material according to the invention, of harvestable plant parts according to the invention or of plants obtainable by a process according to the invention and comprising recombinant nucleic acid molecules, where the recombinant nucleic acid molecules are characterized in that they comprise nucleic acid molecules coding for a hyaluronan synthase and proteins having the (enzymatic) activity of a GFAT and proteins having the (enzymatic) activity of a UDP-GIc-DH.

[0178] A stable integration of foreign nucleic acid molecules into the genome of a plant cell or plant results in the foreign nucleic acid molecules being flanked after integration into the genome of the plant cell or plant by genomic plant nucleic acid sequences. Compositions described by herein are characterized in that the recombinant nucleic acid molecules present in the composition are flanked by genomic plant nucleic acid sequences.

[0179] Here, the genomic plant nucleic acid sequences may be any sequences naturally present in the genome of the plant cell or plant used for preparing the composition.

[0180] The recombinant nucleic acid molecules present in the compositions described herein may be individual or various recombinant nucleic acid molecules in which nucleic acid molecules coding for a hyaluronan synthase and proteins having the (enzymatic) activity of a GFAT and proteins having the (enzymatic) activity of a UDP-GIc-DH are present on a nucleic acid molecule, or those where the nucleic acid molecules mentioned may be present on separate recombinant nucleic acid molecules. Nucleic acid molecules coding for a hyaluronan synthase or coding for a protein having the (enzymatic) activity of a GFAT or coding for a protein having the (enzymatic) activity of a UDP-GIc-DH may be present together on a single recombinant nucleic acid molecule, or two of the nucleic acid molecules mentioned may be present together on a single recombinant nucleic acid molecule and the third nucleic acid molecule may be present on another recombinant nucleic acid molecule in any possible combination, or all three nucleic acid molecules mentioned may in each case be present on individual separate recombinant nucleic acid molecules. Depending on how the nucleic acid molecules coding for a hyaluronan synthase or coding for a protein having the (enzymatic) activity of a GFAT or coding for a protein having the (enzymatic) activity of a UDP-GIc-DH are present in a composition , they may be flanked by identical or different genomic plant nucleic acid sequences.

[0181] That compositions described herein which comprise recombinant nucleic acid molecules may be demonstrated using methods known to the person skilled in the art, such as, for example, methods based on hybridization or, preferably, using methods based on PCR (polymerase chain reaction).

[0182] Preferably, compositions described herein comprise at least 0.005%, with preference at least 0.01%, particularly preferably at least 0.05% and especially preferably at least 0.1 % of hyaluronan.

[0183] Preferably, compositions described herein comprise at most 5%, with preference at most 2%, particularly preferably at most 1% and especially preferably at least 0,5% of hyaluronan.

[0184] As already mentioned above, it is possible to use genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, harvestable plant parts according to the invention, processible plant parts according to the invention, consumable plant parts according to the invention or plants obtainable by a process according to the invention to prepare foodstuff or feedstuff. However, use as raw materials for industrial applications is also possible, without hyaluronan having to be isolated. Thus, for example, genetically modified plants according to the invention or parts of genetically modified plants according to the invention can be applied to areas under agricultural cultivation to achieve increased water binding of the soil. Furthermore, genetically modified plants according to the invention or genetically modified plant cells according to the invention can be used for preparing drying agents (for example for use when shipping moisture-sensitive items) or as absorbers of liquids (for example in nappies or for absorbing spilt aqueous liquids). For such applications, it is possible to use entire genetically modified plants according to the invention, parts of genetically modified plants according to the invention or comminuted (for example ground) genetically modified plants according to the invention or plant parts according to the invention, as required. Suitable for applications in which ground plants or plant parts are used are in particular plant parts containing hyaluronan, but only a low proportion of water. These are preferably grains of cereal plants (corn, rice, wheat, rye, oats, barley, sago or sorghum). Since genetically modified plant cells according to the invention and genetically modified plants according to the invention have a higher hyaluronan content than transgenic plants described in the literature, compared to these less material has to be used for industrial applications when use is made of genetically modified plant cells according to the invention or genetically modified plants according to the invention.

[0185] The present invention also provides processes for preparing a composition according to the invention, where genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, harvestable plant parts according to the invention, processible plant parts according to the invention, consumable plant parts according to the invention or plants obtainable by a process according to the invention for preparing a plant which synthesizes hyaluronan are used. The processes for preparing a composition according to the invention are preferably processes for preparing foodstuff or feedstuff, processes for preparing a pharmaceutical product or processes for preparing a cosmetic product.

[0186] Processes for preparing foodstuff or feedstuff are known to the person skilled in the art. Processes for using genetically modified plants according to the invention or plant parts according to the invention in industrial areas are also known to the person skilled in the art and include inter alia comminuting or grinding of genetically modified plants according to the invention or plant parts according to the invention; however, they are not exclusively limited thereto. Some of the advantages resulting from using subject-matters according to the invention for preparing foodstuff/feedstuff or for use in industrial areas have already been described above.

[0187] A process according to the invention for preparing a composition is particularly preferably a process for preparing a composition which comprises hyaluronan.

[0188] Compositions obtainable by a process for preparing a composition according to the invention are likewise provided by the present description [0189] The present invention also relates to the use of genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, harvestable plant parts according to the invention, processible plant parts according to the invention, consumable plant parts according to the invention or plants obtainable by a process according to the invention for preparing a plant which synthesizes hyaluronan for preparing a composition according to the invention. Preference is given to the use of genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, harvestable plant parts according to the invention, processible plant parts according to the invention, consumable plant parts according to the invention or of plants obtainable by a process according to the invention for preparing a plant which synthesizes hyaluronan for preparing foodstuff or feedstuff, for preparing a pharmaceutic or for preparing a cosmetic product.

Description of the sequences [0190] SEQ ID NO 1:

Nucleic acid sequence coding for a hyaluronan synthase of Paramecium bursaria Chlorella virus 1. SEQ ID NO 2:

Amino acid sequence of a hyaluronan synthase of the Paramecium bursaria Chlorella virus 1. The amino acid sequence shown can be derived from SEQ ID NO 1. SEQ ID NO 3:

Synthetic nucleic acid sequence coding for a hyaluronan synthase of Paramecium bursaria Chlorella virus 1. The synthesis of the codons of the sequence shown was carried out such that it is adapted to the use of codons in plant cells. The nucleic acid sequence shown codes for a protein having the amino acid sequence shown under SEQ ID NO 2. SEQ ID NO 4:

Nucleic acid sequence coding for a protein having the activity of a UDP-GIc-DH of Paramecium bursaria Chlorella virus 1. SEQ ID NO 5:

Amino acid sequence of a protein having the activity of a UDP-GIc-DH of Paramecium bursaria Chlorella virus 1. The amino acid sequence shown can be derived from SEQ ID NO 4. SEQ ID NO 6:

Synthetic nucleic acid sequence coding for a protein having the activity of a UDP-GIc-DH of Paramecium bursaria Chlorella virus 1. The synthesis of the codons of the sequence shown was carried out such that it was adapted to the use of codons in plant cells. The nucleic acid sequence shown codes for a protein having the amino acid sequence shown under SEQ ID NO 5. SEQ ID NO 7:

Nucleic acid sequence coding for a protein having the activity of a GFAT-1 from the mouse. SEQ ID NO 8:

Amino acid sequence of a protein having the activity of a GFAT-1 from the mouse. The amino acid sequence shown can be derived from SEQ ID NO 7. SEQ ID NO 9:

Nucleic acid sequence coding for a protein having the activity of a GFAT-2 from the mouse. SEQ ID NO 10:

Amino acid sequence of a protein having the activity of a GFAT-2 from the mouse. The amino acid sequence shown can be derived from SEQ ID NO 9. SEQ ID NO 11:

Nucleic acid sequence coding for a protein having the activity of a GFAT from Escherichia coli. SEQ ID NO 12:

Amino acid sequence of a protein having the activity of a GFAT from Escherichia coli. The amino acid sequence shown can be derived from SEQ ID NO 11. SEQ ID NO 13:

Synthetic nucleic acid sequence coding for a protein having the activity of a GFAT from Escherichia coli. The synthesis of the codons of the sequence shown was carried out such that it was adapted to the use of codons in plant cells. The nucleic acid sequence shown codes for a protein having the amino acid sequence shown under SEQ ID NO 12. SEQ ID NO 14:

Synthetic Oligonucleotide used as primer in Example 1 SEQ ID NO 15:

Synthetic Oligonucleotide used as primer in Example 1 Description of the figures [0191]

Fig. 1:

Shows a calibration curve and the corresponding equation of the regression line used for calculating the hyaluronan content in plant tissue. The calibration curve was established with the aid of the commercial test kit (hyaluronic acid (HA) test kit from Corgenix, Inc., Colorado, USA, Prod. No. 029-001) and the standard solutions supplied therewith.

General methods [0192] Methods which can be used in connection with the present invention are described below. 1. Transformation of potato plants [0193] Potato plants were transformed with the aid of Agrobacterium, as described in Rocha-Sosa et al. (EMBO J. 8, (1989), 23-29). 2. Isolation of hyaluronan from plant tissue [0194] To detect the presence of hyaluronan and to determine the hyaluronan content in plant tissue, plant material was worked up as follows: 200 pi of water (demineralized, conductivity = 18 ΜΩ) were added to about 0.3 g of tuber material, and the mixture was comminuted in a laboratory oscillating ball mill (MM200, from Retsch, Germany) (30 sec at 30 Flz). Afurther 800 μΙ of water (demineralized, conductivity = 18 ΜΩ) was then added, and the mixture was mixed well (using, for example, a Vortex mixer). Cell debris and insoluble components were separated from the supernatant by centrifuging at 16 000 xg for 5 minutes. 3. Purification of hyaluronan [0195] About 100 grams of tubers were peeled, cut into pieces of a size of about 1 cm3 and, after addition of 100 ml of water (demineralized, conductivity = 18 ΜΩ), comminuted in a Warring blender at maximum speed for about 30 seconds. The cell debris was then removed using a tea sieve. The cell debris that had been removed was resuspended in 300 ml of water (demineralized, conductivity = 18 ΜΩ) and again removed using a tea sieve. The two suspensions obtained (100 ml + 300 ml) were combined and centrifuged at 13 000 xg for 15 minutes. NaCI was added to the centrifugation supernatant obtained until a final concentration of 1% had been reached. After the NaCI had gone into solution, precipitation was carried out by addition of twice the volume of ethanol followed by thorough mixing and incubation at -20°C overnight. The mixture was then centrifuged at 13 000 xg for 15 minutes. The sedimented precipitate obtained after this centrifugation was dissolved in 100 ml of buffer (50 mM TrisHCI, pH 8, 1 mM CaCl2) and proteinase Kwas then added to a final concentration of 100 pg/ml and the solution was incubated at 42°C for 2 hours. This was followed by 10 minutes of incubation at 95°C. Once more, NaCI was added to this solution until a final concentration of 1% had been reached. After the NaCI had gone into solution, another precipitation was carried out by addition of twice the volume of ethanol, thorough mixing and incubation at -20°C for about 96 hours. This was followed by 15 minutes of centrifugation at 13 000 xg. The sedimented precipitate obtained after this centrifugation was dissolved in 30 ml of water (demineralized, conductivity = 18 ΜΩ), and once more, NaCI was added to a final concentration of 1%: By adding twice the volume of ethanol, thorough mixing and incubation at -20°C overnight, another precipitation was carried out. The precipitate obtained after subsequent centrifugation at 13 000 xg for 15 minutes was dissolved in 20 ml of water (demineralized, conductivity = 18 ΜΩ).

[0196] Further purification was carried out by centrifugal filtration. To this end, in each case 5 ml of the dissolved precipitate were applied to a membrane filter (CentriconAmicon, pore width 10 000 NMWL, Prod. No. UCF8 010 96), and the sample was centrifuged at 2200 xg until only about 3 ml of the solution above the filter remained. Two more times, in each case 3 ml of water (demineralized, conductivity = 18 ΜΩ) were then added to the solution above the membrane and in each case re-centrifuged under identical conditions until, at the end, only about 3 ml of the solution above the filter remained. The solutions still present above the membrane after centrifugal filtration were taken off, and the membrane was rinsed repeatedly (three to five times) with about 1.5 ml of water (demineralized, conductivity = 18 ΜΩ). All solutions which were still present above the membrane and the solutions obtained from rinsing were combined, NaCI was added to a final concentration of 1%, after the NaCI had gone into solution, twice the volume of ethanol was added, the sample was mixed and a precipitate was obtained by storage at -20°C overnight. The precipitate obtained after subsequent centrifugation at 13 000 xg for 15 minutes was dissolved in 4 ml of water (demineralized, conductivity = 18 ΜΩ) and then freeze-dried (24 hours under a pressure of 0.37 mbar, freeze drying apparatus Christ Alpha 1-4 from Christ, Osterode, Germany). 4. Detection of hyaluronan and determination of the hyaluronan content [0197] Hyaluronan was detected using a commercial test (hyaluronic acid (HA) test kit from Corgenix, Inc., Colorado, USA, Prod. No. 029-001) according to the instructions of the manufacturer which are herewith incorporated into the description by way of reference. The test principle is based on the availability of a protein which binds specifically to hyaluronan (HABP) and is carried out similarly to an ELISA, where a color reaction indicates the hyaluronan content in the sample examined. Accordingly, for the quantitative determination of hyaluronan, the samples to be measured should be employed in a concentration such that they are within the stated limits (for example: dilution of the sample in question or use of less water for extracting hyaluronan from plant tissue, depending on whether a limit was exceeded or not reached).

[0198] In parallel batches, aliquots of the samples to be determined were initially subjected to hyaluronidase digestion and then measured using the commercial test (hyaluronic acid (HA) test kit from Corgenix, Inc., Colorado, USA, Prod. No. 029-001). Hyaluronidase digestion was carried out using 400 pi of potato tuber extract in hyaluronidase buffer (0.1 M potassium phosphate buffer, pH 5.3; 150 mM NaCI) by adding 5 pg (-3 units) of hyaluronidase (hyaluronidase type III from Sigma, Prod. No. H 2251) and incubating at 37°C for 30 min.

[0199] In each case in a dilution of 1:10, all samples were then used for determining the hyaluronan content. 5. Calculation of standard deviations [0200] The stated standard deviations were calculated using the formula below: square root

where x is the value of individual measured values and n is the sum of all measured values used for determining the standard deviation in question.

6. Determination of the activity of a GFAT

[0201] The activity of a protein having the activity of a GFAT is determined as described in Rachel et al. (1996, J. Bacteriol. 178 (8), 2320-2327).

[0202] To distinguish whether a protein has the activity of a GFAT-1 or GFAT-2, the method described in Hu et al. (2004, J. Biol. Chem. 279 (29), 29988-29993) is used.

7. Determination of the activity of a UDP-GIc-DH

[0203] The activity of a protein having the activity of a UDP-GIc-DH is determined as described in Spicerl et al. (1998, J. Bacteriol. 273 (39), 25117-25124). 8. Transformation of tomato plants [0204] Tomato plants were transformed with the aid of Agrobacterium using the method described in US 5,565,347.

Examples 1. Preparation of the plant expression vector IR 47-71 [0205] The plasmid pBinAR is a derivative of the binary vector plasmid pBin19 (Bevan, 1984, Nucl Acids Res 12: 8711-8721) which was constructed as follows:

Afragment of a length of 529 bp which comprised the nucleotides 6909-7437 of the 35S promoter of the cauliflower mosaic virus was isolated as EcoR IIKpn I fragment from the plasmid pDI-151 (Pietrzak et al, 1986 Nucleic Acids Res. 14, 5858) and ligated between the EcoR I and Κρη I restriction sites of the polylinker of pUC18. In this manner, the plasmid pUC18-35S was formed. Using the restriction endonucleases Hind III and Pvu II, a fragment of a length of 192 bp which included the polyadenylation signal (3' terminus) of the Octopin Synthase gene (gene 3) of the T-DNAof the Ti plasmid pTiACH5 ( Gielen et al, 1984, EMBO Journal 3, 835-846) (nucleotides 11 749-11 939) was isolated from the plasmid pAGV40 (Herrera-Estrella et al, 1983 Nature, 303, 209-213). Following addition of Sph I linkers to the Pvu II restriction site, the fragment was ligated between the Sph I and Hind III restriction sites of pUC18-35S. This gave the plasmid pA7. Here, the entire polylinker comprising the 35S promoter and Ocs terminator was removed using EcoR I and Hind III and ligated into the appropriately cleaved vector pBin19. This gave the plant expression vector pBinAR (Hofgen and Willmitzer, 1990, Plant Science 66, 221-230).

[0206] The promoter of the patatin gene B33 from Solanum tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) was, as Dra I fragment (nucleotides -1512 - +14), ligated into the Ssf l-cleaved vector pUC19 whose ends had been blunted using T4-DNA polymerase. This gave the plasmid pUC19-B33. From this plasmid, the B33 promoter was removed using EcoR I and Sma I and ligated into the appropriately restricted vector pBinAR. This gave the plant expression vector pBinB33.

[0207] To facilitate further cloning steps, the MCS (Multiple Cloning Site) was extended. To this end, two complementary oligonucleotides were synthesized, heated at 95°C for 5 minutes, slowly cooled to room temperature to allow good fixation (annealing) and cloned into the Sal I and Κρη I restriction sites of pBinB33. The oligonucleotides used for this purpose had the following sequence:

[0208] The plasmid obtained was named IR 47-71. 2. Preparation of the plant expression vector pBinARHyg [0209] The fragment comprising the 35S promoter, the Ocs terminator and the entire Multiple Cloning Site was removed from pA7 using the restriction endonucleases EcoR I and Hind III and cloned into the vector pBIBHyg (Becker, 1990, Nucleic Acids Res. 18, 203) which had been cut using the same restriction endonucleases. The plasmid obtained was named pBinARHyg. 3. Preparation of the cloning vector IC 317-204 [0210] Nucleic acid fragments comprising the OCS terminator were isolated from the plasmid IR 47-71 using the restriction endonucleases Xho I and Hind III and cloned into the vector pBlueScript KS (from Stratagene, Prod. No. 212207) which had been cut with the same restriction endonucleases. The plasmid obtained was named IC 306-204 [0211] Nucleic acid fragments comprising the B33 promoter were isolated from the plasmid IR 47-71 using the restriction endonucleases Bam HI and Eco Rl and cloned into the vector pBlueScript KS (from Stratagene, Prod. No. 212207) which had been cut with the same restriction endonucleases. The plasmid obtained was named IC 314-204.

[0212] The OCS terminator was isolated from IC 306-204 using the restriction endonuclease Bam HI and cloned into the plasmid IC 314-204 which had been cut with the same restriction endonuclease. The plasmid obtained was named IC 317-204. 4. Synthesis of nucleic acid molecules a) Synthesis of nucleic acid molecules coding for a hyaluronan synthase of Paramecium bursaria Chlorella virus 1 [0213] The nucleic acid sequence coding for a hyaluronan synthase (HAS) of Paramecium bursaria Chlorella virus 1 was synthesized by Medigenomix GmbH (Munich, Germany) and cloned into the vector pCR2.1 from Invitrogen (Prod. No. K2000-01). The plasmid obtained was named IC 323-215. The synthetic nucleic acid sequence coding for the HAS protein from Paramecium bursaria Chlorella virus 1, is shown under SEQ ID NO 3. The corresponding nucleic acid sequence originally isolated from the Paramecium bursaria Chlorella virus 1 is shown under SEQ ID NO 1. b) Synthesis of nucleic acid molecules coding for a protein having the activity of a UDP-GIc-DH of Paramecium bursaria Chlorella virus 1 [0214] The nucleic acid sequence coding for a protein having the activity of a UDP-GIc-DH from Paramecium bursaria Chlorella virus 1, was synthesized by Entelechon GmbH and cloned into the vector pCR4Topo from Invitrogen (Prod. No. K4510-20). The plasmid obtained was named IC 339-222. The synthetic nucleic acid sequence coding for the UDP-GIc-DH protein from Paramecium bursaria Chlorella virus 1, is shown under SEQ ID NO 6. The corresponding nucleic acid sequence originally isolated from Paramecium bursaria Chlorella virus 1 is shown under SEQ ID NO 4. c) Synthesis of nucleic acid molecules coding for a protein having the activity of a GFAT from Escherichia coli [0215] The nucleic acid sequence coding for a protein having the activity of a GFAT 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 IC 373-256. The synthetic nucleic acid sequence coding for a protein having the activity of a GFAT from Escherichia coli, is shown under SEQ ID NO 13. The corresponding nucleic acid sequence originally isolated from Escherichia coli is shown under SEQ ID NO 11. 5. Origin of further nucleic acid molecules a) Nucleic acid molecules coding for a protein having the activity of a GFAT-1 from the mouse [0216] The nucleic acid sequence coding for a protein having the activity of a GFAT-1 was purchased from BioCat GmbH, Heidelberg (Art.No. MMM1013-65346, cDNA clone MGC:58262, IMAGE:6742987). This is a clone produced by I.M.A.G.E. Konsortium (http://image.llnl.gov) and distributed by BioCat GmbH. Here, the cDNA coding for a protein having the activity of a GFAT-1 was cloned into the vector pCMV Sport 6 from Invitrogen. The plasmid obtained was named IC 365-256. The nucleic acid sequence, inserted into IC 365-256, coding for a protein having the activity of a GFAT-1 from Mus musculus has, compared to the nucleic acid sequence shown under SEQ ID NO 7, a base exchange from T to C in position 1090 and a base exchange from G to Ain position 2027. These base exchanges do not result in amido acid exchanges of the amino acid sequences coded for by the two different nucleic acid molecules.

[0217] The coding nucleic acid sequence for the protein having the activity of a GFAT-1 from the mouse is shown in SEQ ID NO 8.

[0218] To facilitate subsequent cloning steps, the sequence coding for a protein having the activity of a GFAT-1 was isolated using the restriction endonucleasesXho I and Eco RV from IC 365-256 and cloned into the plasmid pME9 (pBlueSkript vector from Stratagene, Prod. No. 212207) having a modified multiple cloning site which additionally has a Pac I restriction site at both ends, which plasmid had been cut with the same restriction endonucleases. The plasmid obtained was named IC 367-256. b) Nucleic acid molecules coding for a protein having the activity of a GFAT-2 from the mouse [0219] Nucleic acid molecules coding for a protein having the activity of a GFAT-2 from the mouse were purchased from Invitrogen (Clone ID 4167189, cDNA clone MGC:18324, IMAGE:4167189). This is a clone which is produced by I.M.A.G.E. Konsortium (http://image.llnl.gov) and distributed by Invitrogen. Here, the cDNA coding for a protein having the activity of a GFAT-2 is cloned into the vector pCMV Sport 6 from Invitrogen. The plasmid was named IC 369-256. The nucleic acid sequence coding for the protein having the activity of a GFAT-2 from Mus musculus is shown under SEQ ID NO 9. 6. Preparation of the plant expression vector IC 341-222 which comprises a coding nucleic acid sequence for a hyaluronan synthase of Paramecium bursaria Chlorella virus 1 [0220] Using restriction digestion with ESamH I and Xho I, nucleic acid molecules comprising the coding sequence of hyaluronan synthase were isolated from the plasmid IC 323-215 and cloned into the ESamH I and Xho I restriction sites of the plasmid IR 47-71. The plant expression vector obtained was named IC 341-222. 7. Preparation of the plant expression vectors 1C 370-256 and 1C 376-256 comprising coding nucleic acid sequences for a protein having the activity of a GFAT-1 from the mouse and for a protein having the activity of a UDP-GIc-DH from Paramecium bursaria Chlorella virus 1 [0221] Using restriction digestion with ESamH I and Κρη I, nucleic acid molecules comprising the coding sequence for a protein having the activity of a UDP-GIc-DH of Paramecium bursaria Chlorella virus 1 were isolated from the plasmid IC 339-222 and cloned into the plasmid pA7 which had been cut with the same restriction endonucleases. The plasmid obtained was named IC 342-222.

[0222] By restriction digestion with Xba I and Κρη I, nucleic acid molecules comprising the coding sequence for a protein having the activity of a UDP-GIc-DH of Paramecium bursaria Chlorella virus 1 were isolated from the plasmid IC 342-222 and cloned into the expression vector pBinAR Hyg which had been restricted with Xba I and Κρη I. The plasmid obtained was named IC 349-222.

[0223] In the next step, a nucleic acid fragment comprising the B33 promoter and the OCS terminator, which fragment had been isolated from IC 317-204 by restriction digestion using Eco IR, was cloned into the Eco IR restriction site of IC 349-222. Here, it was made sure that the promoters (35S and B33) were oriented head-to-head. The vector obtained was named IC 354-222.

[0224] In a further cloning step, a nucleic acid fragment comprising the coding sequence of the protein having the activity of a GFAT-1 from the mouse was isolated by restriction digestion with Xho I and Eco RV from IC 367-256 and cloned into the plasmid IC 354-222, which had been restricted with Xho I and Ed 136 II. The plant expression vector obtained was named IC 370-256.

[0225] Following a sequence analysis of the plasmid IC 370-256, it was found that the coding nucleic acid sequence of the protein having the activity of a GFAT-1 from the mouse had modifications in two positions compared to the nucleic acid sequence inserted into plasmid IC 365-256. Compared to the nucleic acid sequence shown under SEQ ID NO 7, the nucleic acid sequence coding for the protein having the activity of a GFAT-1 from the mouse.contained in the plasmid IC 370-256 has a base exchange from G to Ain position 1160, a base exchange from T to C in position 1190, a base exchange from T to C in position 1245 and from G to Ain position 2027. This modified nucleic acid sequence codes for a protein which, with respect to the amino acid sequence shown under SEQ ID NO 8, has a modification of the amino acids in position 304 from R to Q and in position 366 from C to R.

[0226] To obtain a plant expression vector which comprises the correct nucleic acid sequence coding for a protein having the activity of a GFAT-1 from the mouse, the coding sequence of the protein having the activity of a GFAT-1 from the mouse was again isolated from IC 365-256 by restriction digestion with Xho I and Eco RV and cloned into the plasmid IC 354-222, restricted with Xho I and Ec/136 II. The plant expression vector obtained was named IC 376-256.

[0227] The nucleic acid sequence coding for the protein having the activity of a GFAT-1 from the mouse which is contained in the plasmid IC 376-256 is identical to the coding sequence for a protein having the activity of a GFAT-1 from the mouse inserted into plasmid 365-256. The amino acid sequence coded for by this nucleic acid molecule is shown under SEQ ID NO 8. 8. Preparation of the plant expression vector IC 372-256 comprising coding nucleic acid sequences for a protein having the activity of a GFAT-2 from the mouse and for a protein having the activity of a UDP-GIc-DH from Paramecium bursaria Chlorella virus 1 [0228] A nucleic acid fragment comprising the coding sequence of the protein having the activity of a GFAT-2 from the mouse was isolated from IC 369-256 by restriction digestion with Xho I and Eco RV and cloned into the plasmid IC 354-222, restricted with Xho I and Ed 136 II. The plant expression vector obtained was named IC 372-256. 9. Preparation of the plant expression vector IC 375-271 comprising coding nucleic acid sequences for a protein having the activity of a GFAT from Escherichia coli and for a protein having the activity of a UDP-GIc-DH of Paramecium bursaria Chlorella virus 1 [0229] A nucleic acid fragment comprising the coding sequence of the protein having the activity of a GFAT from Escherichia coli was isolated from IC 373-256 by restriction digestion with Xho I and Eco RV and cloned into the plasmid IC 354-222, restricted by Xho I and Ed 136 II. The plant expression vector obtained was named IC 375-271. 10. Transformation of potato plants with plant expression vectors comprising nucleic acid molecules coding for a hyaluronan synthase [0230] Potato plants were transformed using the plant expression vector IC 341-222, which comprises a coding nucleic acid sequence for a hyaluronan synthase from Paramecium bursaria Chlorella virus 1 under the control of the promoter of the patatin gene B33 from Solarium tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) using the method given under General Methods item 1. The transgenic potato plants obtained, which were transformed with the plasmid IC 341-222, were named 365 ES. 11. Analysis of the transgenic plants transformed with plant expression vectors comprising nucleic acid molecules coding for a hyaluronan synthase a) Construction of a calibration curve [0231] A calibration curve was constructed using the standard solutions supplied with the commercial test kit (hyaluronic acid (HA) test kit from Corgenix, Inc., Colorado, USA, Prod. No. 029-001), according to the methods described by the manufacturer. To determine the extinction at 1600 ng/ml of hyaluronan, double the amount, based on the amount of supplied standard indicated by the manufacturer, comprising 800 ng/ml of hyaluronan was used. In each case, three independent measurement series were carried out, and the corresponding mean was determined. This gave the following calibration curve:

Table 1: Values for constructing a calibration curve for the quantitative determination of the hyaluronan content in plant tissue. With the aid of software (Microsoft Office Excel 2002, SP2), the measured values obtained were entered into a diagram and the equation of the function of the trend line was determined (see Fig 1). E45onm refers to the extinction at a wavelength of 450 nm, s.d. is the standard deviation of the calculated mean of the individual values.

b) Analysis of potato tubers of lines 365 ES

[0232] In a greenhouse, individual plants of the line 365 ES ware cultivated in soil in 6 cm pots. In each case about 0.3 g of material of potato tubers of the individual plants was processed according to the method described under General Methods item 2.

[0233] Using the method described under General Methods item 4, the amount of hyaluronan present in the respective plant extracts was determined, with the aid of the calibration curve shown in Example 10a) and Fig. 1. Here, the supernatant obtained after centrifugation was used in a dilution of 1:10 for determining the hyaluronan content. The following results were obtained for selected plants:

Table 2: Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced by independent selected transgenic plants of the line 365 ES. Column 1 refers to the plant from which tuber material was harvested (here, "wild-type" refers to untransformed plants which, however, have the genotype used as starting material for the transformation). Column 2 indicates the amount of tuber material of the plant in question used for determining the hyaluronan content. Column 3 contains the measured extinction of a 1:10 dilution of the respective plant extract. Column 4 was calculated with the aid of the regression line equation (see Fig. 1) taking into account the dilution factor, as follows: ((value column 3 - 0.149)/0.00185) x 10. Column 5 indicates the amount of hyaluronan based on the fresh weight used and was calculated as follows: (value column 4/value column 2)/1000. "n.d." means not detectable.

12. Transformation of hyaluronan-synthesizing plants with plant expression vectors comprising coding nucleic acid sequences for a protein having the [0234] activity of a GFAT and for a protein having the activity of a UDP-GIc-DH Potato plants of the lines 365 ES 13 and 365 ES 74 were in each case transformed with the plant expression vectors IC 370-256, IC 376-256, IC 372-256 and IC 375-271 using the method given under General Methods item 1.

[0235] The transgenic potato plants obtained of line 365 ES 13 which had been transformed with the plasmid IC 370-256 were named 393 ES.

[0236] The transgenic potato plants obtained of line 365 ES 74 which had been transformed with the plasmid IC 370-256 were named 394 ES.

[0237] The transgenic potato plants obtained of line 365 ES 13 which had been transformed with the plasmid IC 372-256 were named 395 ES.

[0238] The transgenic potato plants obtained of line 365 ES 74 which had been transformed with the plasmid IC 372-256 were named 396 ES.

[0239] The transgenic potato plants obtained of line 365 ES 13 which had been transformed with the plasmid IC 375-271 were named 403 ES.

[0240] The transgenic potato plants obtained of line 365 ES 74 which had been transformed with the plasmid IC 375-271 were named 404 ES.

[0241] The transgenic potato plants obtained of line 365 ES 13 which had been transformed with the plasmid IC 376-256 were named 408 ES.

[0242] The transgenic potato plants obtained of line 365 ES 74 which had been transformed with the plasmid IC 376-256 were named 409 ES.

13. Analysis of transgenic hyaluronan-synthesizing potato plants additionally transformed with plant expression vectors comprising coding nucleic acid sequences for a protein having the activity of a GFAT and for a protein having the activity of a UDP-GIc-DH

[0243] In a greenhouse, individual plants of the lines 393 ES, 394 ES, 395 ES, 396 ES, 403 ES, 404 ES and 409 ES were cultivated in soil in 6 cm pots. In each case about 0.3 g of material of potato tubers or leaves of the individual plants was processed according to the method described under General Methods item 2. Using the method described under General Methods item 4, the amount of hyaluronan contained in the respective plant extracts was determined, with the aid of a calibration curve generated according to Example 10a), which calibration curve was generated new for each individual measurement series. Here, for determining the hyaluronan content, the supernatant obtained after centrifugation was in each case diluted with water (demineralized, conductivity = 18 ΜΩ) such that the measured extinction values of the individual samples were in the linear range of the calibration curve. The results for plants originating from original transformations with various plasmids are shown below.

a) Analysis of tubers of the line 393 ES

[0244] For each tuber of individual transgenic plants of the lines named 393 ES, two independent samples were taken and the hyaluronan content was in each case determined separately. The mean and the standard deviation of the values obtained for the individual measurements of each tuber were then calculated using the formula given under General Methods item 5. The stated means and standard deviations for plants named wild-type and plants named 365 ES 13 were calculated by calculating the amount of hyaluronan in tubers of in each case ten different plants which are vegetative progeny of the wild-type (Solarium tuberosum cv. Désirée) and of the line 365 ES 13, respectively. The following results were obtained for selected plants:

Table 3: Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in tubers of independently selected transgenic plants of the line 393 ES. Column 1 contains the name of the plant from which tuber material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 13 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 370-256 for generating the lines 393 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined.

b) Analysis of tubers of the line 394 ES

[0245] For each tuber of individual transgenic plants of the lines named 394 ES, two independent samples were taken and the hyaluronan content was in each case determined separately. The mean and the standard deviation of the values obtained for the individual measurements of each tuber were then calculated using the formula given under General Methods item 5. The stated means and standard deviations for plants named wild-type and plants named 365 ES were calculated by calculating the amount of hyaluronan in tubers of in each case 18 different wild-type plants and 26 different plants of the line 365 ES 74 which are vegetative progeny of the wild-type (Solarium tuberosum cv. Désirée) and of the line 365 ES 74, respectively. The following results were obtained for selected plants:

Table 4 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in tubers of independently selected transgenic plants of the line 394 ES. Column 1 contains the name of the plant from which tuber material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 74 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 370-256 for generating the lines 394 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined.

c) Analysis of tubers of the line 395 ES

[0246] For each tuber of individual transgenic plants of the lines named 395 ES, if possible, two independent samples were taken and the hyaluronan content was in each case determined separately. The mean and the standard deviation of the values obtained for the individual measurements of each tuber were then calculated using the formula given under General Methods item 5. The stated means and standard deviations for plants named wild-type and plants named 365 ES were calculated by calculating the amount of hyaluronan in tubers of in each case 10 different wild-type plants and 18 different plants from the line 365 ES 13 which are vegetative progeny of the wild-type (Solanum tuberosum cv. Désirée) and of the line 365 ES 13, respectively. The following results were obtained for selected plants:

Table 5 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in tubers of independently selected transgenic plants of the line 395 ES. Column 1 contains the name of the plant from which tuber material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 13 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 372-256 for generating the lines 395 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined. In the case of plants where no standard deviation is stated, the hyaluronan content of only one tuber sample was determined.

d) Analysis of leaves of the line 395 ES

[0247] The hyaluronan content of individual leaves of plants of the lines having the name 395 ES were determined. The stated means and standard deviations for plants having the name wild-type and plants having the name 365 ES were calculated by calculating in each case the amount of hyaluronan in leaves of 4 different wild-type plants and 9 different plants of the line 365 ES 13, which are vegetative progeny of the wild-type (Solanum tuberosum cv. Désirée) and line 365 ES 13, respectively. The following results were obtained for selected plants:

Table 6 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in leaves of independently selected transgenic plants of the line 395 ES. Column 1 contains the name of the plant from which tuber material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 13 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 372-256 for generating the lines 395 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined. In the case of plants where no standard deviation is stated, the hyaluronan content of only one leaf sample was determined.

e) Analysis of tubers of the line 396 ES

[0248] For each tuber of individual transgenic plants of the lines named 396, two independent samples were taken and the hyaluronan content was in each case determined separately. The mean and the standard deviation of the values obtained for the individual measurements of each tuber were then calculated using the formula given under General Methods item 5. The stated means and standard deviations for plants named wild-type and plants named 365 ES were calculated by calculating the amount of hyaluronan in tubers of in each case 12 different wild-type plants and 14 different plants of the line 365 ES 74 which are vegetative progeny of the wild-type (Solarium tuberosum cv. Désirée) and of the line 365 ES 74, respectively. The following results were obtained for selected plants:

Table 7 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in tubers of independently selected transgenic plants of the line 396 ES. Column 1 contains the name of the plant from which tuber material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 74 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 372-256 for generating the lines 396 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined.

f) Analysis of leaves of the line 396 ES

[0249] The hyaluronan content of individual leaves of plants of the lines having the name 396 ES was determined. The stated means and standard deviations for plants having the name wild-type and plants having the name 365 ES were calculated by calculating in each case the amount of hyaluronan in leaves of 4 different wild-type plants and 6 different plants of the line 365 ES 13, which are vegetative progeny of, the wild-type (Solarium tuberosum cv. Désirée) and line 365 ES 13, respectively. The following results were obtained for selected plants:

Table 8 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in leaves of independently selected transgenic plants of the line 396 ES. Column 1 contains the name of the plant from which leaf material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 74 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 372-256 for generating the lines 396 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined. In the case of plants where no standard deviation is stated, the hyaluronan content of only one leaf sample was determined.

g) Analysis of tubers of the line 403 ES

[0250] For each tuber of individual transgenic plants of the lines named 403 ES, two independent samples were taken if possible and the hyaluronan content was in each case determined separately. The mean and the standard deviation of the values obtained for the individual measurements of each tuber were then calculated using the formula given under General Methods item 5. The stated means and standard deviations for plants named wild-type and plants named 365 ES were calculated by calculating the amount of hyaluronan in tubers of in each case 10 different wild-type plants and 10 different plants of the line 365 ES 13 which are vegetative progeny of the wild-type (Solarium tuberosum cv. Désirée) and of the line 365 ES 13, respectively. The following results were obtained for selected plants:

Table 9 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in tubers of independently selected transgenic plants of the line 403 ES. Column 1 contains the name of the plant from which tuber material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 13 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 375-271 for generating the lines 403 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined, "n.d." means that it was not possible to detect hyaluronan in the tubers. In the case of plants where no standard deviation is stated the hyaluronan content of only one tuber sample was determined.

h) Analysis of leaves of the line 403 ES

[0251] The hyaluronan content of individual leaves of plants of the lines having the name 403 ES were determined. The stated means and standard deviations for plants having the name wild-type and plants having the name 365 ES were calculated by calculating in each case the amount of hyaluronan in leaves of 5 different wild-type plants and 5 different plants of the line 365 ES 13, which are vegetative progeny of the wild-type (Solarium tuberosum cv. Désirée) and line 365 ES 13, respectively. The following results were obtained for selected plants:

Table 10 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in leaves of independently selected transgenic plants of the line 403 ES. Column 1 contains the name of the plant from which leaf material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 13 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 375-271 for generating the lines 403 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined. In the case of plants where no standard deviation is stated, the hyaluronan content of only one leaf sample was determined.

i) Analysis of tubers of the line 404 ES

[0252] For each tuber of individual transgenic plants of the lines named 404 ES, two independent samples were taken if possible and the hyaluronan content was in each case determined separately. The mean and the standard deviation of the values obtained for the individual measurements of each tuber were then calculated using the formula given under General Methods item 5. The stated means and standard deviations for plants named wild-type and plants named 365 ES were calculated by calculating the amount of hyaluronan in tubers of in each case 10 different wild-type plants and 12 different plants of the line 365 ES 74 which are vegetative progeny of the wild-type (Solarium tuberosum cv. Désirée) and of the line 365 ES 74, respectively. The following results were obtained for selected plants:

Table 11 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in tubers of independently selected transgenic plants of the line 404 ES. Column 1 contains the name of the plant from which tuber material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 74 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 375-271 for generating the lines 404 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined, "n.d." means that it was not possible to detect hyaluronan in the tubers. In the case of plants where no standard deviation is stated the hyaluronan content of only one tuber sample was determined.

j) Analysis of leaves of the line 404 ES

[0253] The hyaluronan content of individual leaves of plants of the lines having the name 404 ES were determined. The stated means and standard deviations for plants having the name wild-type and plants having the name 365 ES were calculated by calculating in each case the amount of hyaluronan in leaves of 7 different wild-type plants and 9 different plants of the line 365 ES 74, which are vegetative progeny of the wild-type (Solarium tuberosum cv. Désirée) and line 365 ES 74, respectively. The following results were obtained for selected plants:

Table 12 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in leaves of independently selected transgenic plants of the line 404 ES. Column 1 contains the name of the plant from which leaf material was harvested (here, "wild-type" refers to plants which have not been transformed; 365 ES 74 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 375-271 for generating the lines 404 ES). Column 2 shows the mean of the amount of hyaluronan determined for the leaves in question. Column 3 shows the standard deviation of the means determined. In the case of plants where no standard deviation is stated, the hyaluronan content of only one leaf sample was determined.

k) Analysis of tubers of the line 409 ES

[0254] For each tuber of individual transgenic plants of the lines named 409 ES, samples were taken and the hyaluronan content was in each case determined. The stated means and standard deviations for plants named wild-type and plants named 365 ES were calculated by calculating the amount of hyaluronan in tubers of in each case 4 different wild-type plants and 6 different plants of the line 365 ES 74 which are vegetative progeny of the wild-type (Solarium tuberosum cv. Désirée) and of the line 365 ES 74, respectively. The mean and the standard deviation for the values obtained for the individual measurements of each tuber were calculated using the formula given under General Methods item 5. The following results were obtained for selected plants:

Table 13 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in tubers of independently selected transgenic plants of the line 409 ES. Column 1 contains the name of the plant from which tuber material was harvested (here, "wild-type" refers to plants which have not been transformed, 365 ES 74 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 375-271 for generating the lines 409 ES). Column 2 shows the mean of the amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the means determined, "n.d." means that it was not possible to detect hyaluronan in the tubers. In the case of plants where no standard deviation is stated, the hyaluronan content of only one tuber sample was determined.

l) Analysis of leaves of the line 409 ES

[0255] The hyaluronan content of individual leaves of plants of the lines having the name 409 ES was determined. The stated means and standard deviations for plants having the name wild-type and plants having the name 365 ES were calculated by calculating in each case the amount of hyaluronan in leaves of 4 different wild-type plants and 6 different plants of the line 365 ES 74, which are vegetative progeny of the wild-type (Solarium tuberosum cv. Désirée) and line 365 ES 74, respectively. The following results were obtained for selected plants:

Table 14 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in leaves of independently selected transgenic plants of the line 409 ES. Column 1 contains the name of the plant from which leaf material was harvested ("wild-type" refers to plants which have not been transformed; 365 ES 74 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 376-256 for generating the lines 409 ES). Column 2 shows the amount of hyaluronan determined for the leaves in question. Column 3 shows the standard deviation of the means determined, "n.d." means that no hyaluronan could be detected in the tubers. In the case of plants where no standard deviation is stated, the hyaluronan content of only one leaf sample was determined.

m) Determination of the hyaluronan content with respect to fresh weight and with respect to dry weight [0256] Individual leaves of plants of lines 395 ES and 396 ES were, before harvesting of the tubers of the plants in question, removed from the plants and divided in the middle. One half of each individual leaf was in each case frozen in liquid nitrogen, the corresponding other half was freeze-dried overnight.

[0257] About 0.3 g of leaf material of the frozen or about 0.02 g of the freeze-dried leaf samples were comminuted with a laboratory oscillating ball mill (MM200, from Retsch, Germany) (30 sec. at 30 HZ). 300 pi of water (demineralized, conductivity = 18 ΜΩ) were then added to each individual comminuted sample, which were then mixed well using a vortex mixer, and cell debris and insoluble components were then separated from the supernatant by centrifugation (5 minutes at 16 OOOxg). The supernatant was removed, and each sample was made up to 500 pi with water (demineralized, conductivity = 18 ΜΩ). Aliquots of the samples prepared in this manner were used for determining the hyaluronan content using the method described under General Methods item 4. The means and the standard deviations were calculated using the formula given under General Methods item 5. For selected plants, the following results were obtained:

Table 15 Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in leaves of independently selected transgenic plants of lines 395 ES and 396 ES. Column 1 names the plant from which leaf material was harvested. 365 ES 13 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 372-256 for generating the lines 395 ES. 365 ES 74 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 372-256 for generating lines 396 ES. Column 2 states the amount of hyaluronan determined for different leaves of the plants in question. Column 3 states the mean of the amounts of hyaluronan measured in different leaves of a plant. Column 4 states the standard deviation for the means determined.

Table 16 Amount of hyaluronan (in pg of hyaluronan per g of dry weight) produced in leaves of independently selected transgenic plants of lines 395 ES and 396 ES. Column 1 names the plant from which leaf material was harvested. 365.ES 13 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 372-256 for generating the lines 395 ES. 365 ES 74 refers to plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 372-256 for generating lines 396 ES. Column 2 states the amount of hyaluronan determined for different leaves of the plants in question. Column 3 states the mean of the amounts of hyaluronan measured in different leaves of a plant. Column 4 states the standard deviation for the means determined

14. Transformation of tomato plants with plant expression vectors comprising nucleic acid molecules coding for a hyaluronan synthase [0258] Tomato plants were initially transformed using the plant expression vector IC 341-222 comprising a coding nucleic acid sequence for an HAS protein from Paramecium bursaria Chlorella virus 1 under the control of the promoter of the patatin gene B33 from Solarium tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) using the method given under General Methods item 8. The transgenic tomato plants obtained, which had been transformed with the plasmid IC 341-222, were named 367 ES.

[0259] Tomato plants of lines 367 ES 25 and 367 ES 42 were then transformed with the plant expression vector IC 341-222 using the method given under General Methods item 8. The transgenic tomato plants obtained of the line 367 ES 25, which had been transformed with the plasmid IC 341-222, were named 399 ES. The transgenic tomato plants obtained of the line 367 ES 42, which had been transformed with the plasmid IC 341-222, were named 400 ES.

[0260] Tomato plants of lines 367 ES 25 were then transformed with the plant expression vector IC 375-271 using the method given under General Methods item 8. The transgenic tomato plants obtained of the line 367 ES 25, which had been transformed with the plasmid IC 375-271, were named 405 ES.

15. Analysis of transgenic hyaluronan-synthesizing tomato plants additionally transformed with plant expression vectors comprising the coding nucleic acid sequences for a protein having the activity of a GFAT and for a protein having the activity of a UDP-GIc-DH

a) Leaves of tomato plants of lines 399 ES and 400 ES

[0261] From different selected tomato plants of lines 399 ES and 400 ES, which had been cultivated in soil in a greenhouse, in each case 1 leaf was harvested and frozen in liquid nitrogen. Further work-up and the determination of the hyaluronan content were carried out as described under Example 11b) for leaves of potato plants. The following results were obtained:

Table 17: Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in leaves of independently selected transgenic plants of lines 399 ES and 400 ES. Column 1 refers to the plant from which the leaf material was harvested. 367 ES 25 and 367 ES 42 refer to different plants which express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 372-256 for generating the lines 399 ES and 400 ES, respectively. Column 2 states the value of the amount of hyaluronan determined in the leaves of the plants in question. Wild-type refers to plants which were not transformed.

b) Fruits of tomato plants of lines 399 ES and 400 ES

[0262] Of different selected tomato plants of lines 399 ES and 400 ES, which had been cultivated in soil in a greenhouse, in each case ripe fruits were harvested, comminuted, centrifuged, and the supernatant was, after centrifugation, filtered. Further work-up of the filtrate and the determination of the hyaluronan content were carried out as described under Example 11 b) for leaves of potato plants. The following results were obtained:

Table 18: Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in ripe fruits of independently selected transgenic plants of lines 399 ES and 400 ES. Column 1 refers to the plant from which the leaf material was harvested. 367 ES 25-1 and 267 ES-2 refer to different clonal progeny of the plant 367 ES 25, and 367 ES 42-1 and 267 ES 42-2 refer to different clonal progeny of the plant 367 ES 42. Column 2 states the mean of the amount of hyaluronan determined in fruits of the plants in question. To this end, the hyaluronan content in in each case 3 (lines 399 ES-1, 400 ES 3), 5 (lines 367 ES-25-1,325 ES-2, 367 ES 42-1, 367 ES 42-2) or 6 (line 399 ES-11) different fruits of the lines in question were determined. Column 3 states the standard deviation of the means determined.

c) Fruits of tomato plants of lines 405 ES

[0263] Of different selected tomato plants of lines 405 ES, which had been cultivated in soil in a greenhouse, in each case ripe fruits were harvested, comminuted, centrifuged, and the supernatant was, after centrifugation, filtered. Further work-up of the filtrate and the determination of the hyaluronan content were carried out as described under Example 11 b) for leaves of potato plants. The following results were obtained:

Tabelle 19: Amount of hyaluronan (in pg of hyaluronan per g of fresh weight) produced in ripe fruits of independently selected transgenic plants of lines 405 ES. Column 1 refers to the plant from which the leaf material was harvested. 367 ES 25-8 and 367 ES 25-9 refer to different clonal progeny of the plant 367 ES 25. Exstensions by Latin numbers refer to different fruits of the respective plant, ("wt" refers to plants which were not transformed) Column 2 states the mean of the amount of hyaluronan determined in different fruits of the plants in question. Column 3 states the standard deviation of the means determined.

16) Concluding remarks [0264] When the hyaluronan content of different leaves of a plant was determined, it was found that older leaves of the same plant generally contained more hyaluronan than younger leaves of the same plant. Accordingly, the content of hyaluronan in leaves seems to increase with increasing age of the leaf, so that it may be assumed that hyaluronan accumulates over time. This phenomenon may explain different amounts of hyaluronan found in independent measurements for progeny of the same line.

SEQUENCE LISTING

[0265]

<110> Bayer CropScience GmbH <120> Improved methods and means for producing hyaluronan

<130> BCS 05-5010 PCT <150> EP05090277.4 <151 > 2005-10-05 <150> EP06090053.7 <151 >2006-04-07 <150> US60/725,530 <151 > 2005-10-11 <160> 15 <170> Patentln version 3.3

<210> 1 <211>1707 <212> DNA <213> Paramecium bursaria Chlorella virus 1 <220> <221 > CDS <222> (1)..(1707) <300> <308> PB42580 <309> 1995-12-24 <313> (50903)..(52609) <400> 1 atg ggt aaa aat ata ate ata atg gtt teg tgg tac ace ate ata act 48

Met Gly Lys Asn lie He lie Met val ser Trp Tyr Thr He He Thr 15 10 15 tea aat eta ate geg gtt gga gga gee tet eta ate ttg get ccg gca 96

Ser Asn Leu Ile Ala val Gly Gly Ala ser Leu lie Leu Ala Pro Ala 20 25 30 att act ggg tat gtt eta cat tgg aat att get etc teg aca ate tgg 144

Ile Thr Gly Tyr val Leu His Trp Asn lie Ala Leu Ser Thr lie Trp 35 40 45 gga gta tea get tat ggt att ttc gtt ttt ggg ttt ttc ett gca caa 192

Gly val Ser Ala Tyr Gly lie Phe Val Phe Gly Phe Phe Leu Ala Gin 50 55 60 gtt tta ttt tea gaa ctg aac agg aaa egt ett ege aag tgg att tet 240

Val Leu Phe Ser Glu Leu Asn Arg Lys Arg Leu Arg Lys Trp lie Ser 65 70 75 80 etc aga ect aag ggt tgg aat gat gtt cgt ttg get gtg ate att get 288

Leu Arg Pro Lys Gly Trp Asn Asp Val Arg Leu Ala val tie lie Ala 85 90 95 gga tat ege gag gat cct tat atg ttc cag aag tgc etc gag tet gta 336

Gly Tyr Arg Glu Asp Pro Tyr Met Phe Gin Lys Cys Leu Glu Ser val 100 105 110 cgt gac tet gat tat ggc aac gtt gee cgt ctg att tgt gtg att gac 384

Arg Asp ser Asp Tyr Gly Asn val Ala Arg Leu He Cys Val He Asp 115 120 125 ggt gat gag gac gat gat atg agg atg get gee gtt tac aag geg ate 432

Gly Asp Glu Asp Asp Asp Met Arg Met Ala Ala val Tyr Lys Ala lie 130 135 140 tac aat gat aat ate aag aag ccc gag ttt gtt ctg tgt gag tea gac 480

Tyr Asn Asp Asn lie Lys Lys Pro Glu Phe val Leu Cys Glu Ser Asp 145 150 155 160 gac aag gaa ggt gaa ege ate gac tet gat ttc tet ege gac att tgt 528

Asp Lys Glu Gly Glu Arg lie Asp Ser Asp Phe Ser Arg Asp lie Cys 165 170 175 gtc etc cag cct cat cgt gga aaa egg gag tgt ett tat act ggg ttt 576 val Leu Gin Pro His Arg Gly Lys Arg Glu cys Leu Tyr Thr Gly Phe 180 185 190 caa ett gca aag atg gac ccc agt gtc aat get gtc gtt ctg att gac 624

Gin Leu Ala Lys Met Asp Pro Ser val Asn Ala val val Leu lie Asp 195 200 205 age gat acc gtt etc gag aag gat get att ctg gaa gtt gta tac cca 672

Ser Asp Thr Val Leu Glu Lys Asp Ala lie Leu Glu Val Val Tyr Pro 210 215 220 ett gca tgc gat ccc gag ate caa gee gtt gca ggt gag tgt aag att 720

Leu Ala Cys Asp Pro Glu lie Gin Ala val Ala Gly Glu cys Lys lie 225 230 235 240 tgg aac aca gac act ett ttg agt ett etc gtc get tgg egg tac tat 768

Trp Asn Thr Asp Thr Leu Leu ser Leu Leu val Ala Trp Arg Tyr Tyr 245 250 255 tet geg ttt tgt gtg gag agg agt gee cag tet ttt ttc agg act gtt 816

Ser Ala Phe Cys val Glu Arg Ser Ala Gin Ser Phe Phe Arg Thr val 260 265 270 cag tgc gtt ggg ggg cca ctg ggt gee tac aag att gat ate att aag 864

Gin Cys val Gly Gly Pro Leu Gly Ala Tyr Lys lie Asp lie lie Lys 275 280 285 gag att aag gac ccc tgg att tee cag ege ttt ett ggt cag aag tgt 912

Glu lie Lys Asp pro Trp lie ser Gin Arg Phe Leu Gly Gin Lys cys 290 295 300 act tac ggt gac gac ege egg eta acc aac gag ate ttg atg cgt ggt 960

Thr Tyr Gly Asp Asp Arg Arg Leu Thr Asn Glu lie Leu Met Arg Gly 305 310 315 320 aaa aag gtt gtg ttc act cca ttt get gtt ggt tgg tet gac agt ccg 1008

Lys Lys val val Phe Thr pro Phe Ala val Gly Trp Ser Asp Ser Pro 325 330 335 acc aat gtg ttt egg tac ate gtt cag cag acc ege tgg agt aag teg 1056

Thr Asn val Phe Arg Tyr lie val Gin Gin Thr Arg Trp Ser Lys Ser 340 345 350 tgg tgc ege gaa att tgg tac acc etc ttc gee geg tgg aag cac ggt 1104

Trp Cys Arg Glu lie Trp Tyr Thr Leu Phe Ala Ala Trp Lys His Gly 355 360 365 ttg tet gga att tgg ctg gee ttt gaa tgt ttg tat caa att aca tac 1152

Leu Ser Gly lie Trp Leu Ala Phe Glu Cys Leu Tyr Gin lie Thr Tyr 370 375 380 ttc ttc etc gtg att tac etc ttt tet ege eta gee gtt gag gee gac 1200

Phe Phe Leu val lie Tyr Leu Phe ser Arg Leu Ala val Glu Ala Asp 385 390 395 400 cct cgc gcc cag aca gcc acg gtg att gtg age acc aeg gtt gca ttg 1248

Pro Arg Ala Gin Thr Ala Thr vat ile vat Ser Thr Thr Val Ala Leu 405 410 415 att aag tgt gag tat ttt tea ttc ega gcc aag gat att egg geg ttt 1296

Ile Lys Cys Gly Tyr Phe Ser Phe Arg Ala Lys Asp Ile Arg Ala Phe 420 425 430 tac ttt gtg ett tat aca ttt gtt tac ttt ttc tgt atg att ccg gcc 1344

Tyr Phe vaT Leu Tyr Thr phe Val Tyr Phe Phe Cys Met lie Pro Ala 435 440 445 agg att act gca atg atg acg ett tgg gac att gac tgg ggt act cgc 1392

Arg lie Thr Ala Met Met Thr Leu Trp Asp lie Gly Trp Gly Thr Arg 450 455 460 ggt gga aac gag aag cct tcc gtt ggc acc egg gtc get ctg tgg gca 1440

Gly Gly Asn Glu Lys Pro Ser Val Gly Thr Arg val Ala Leu Trp Ala 465 470 475 480 aag caa tat etc att gca tat atg tgg tgg gcc geg gtt gtt ggc get 1488

Lys Gin Tyr Leu lie Ala Tyr Met Trp Trp Ala Ala val val Gly Ala 485 490 495 gga gtt tac age ate gtc cat aac tgg atg ttc gat tgg aat tet ett 1536

Gly val Tyr Ser lie val His Asn Trp Met Phe Asp Trp Asn Ser Leu 500 505 510 tet tat cgt ttt get ttg gtt ggt att tgt tet tac att gtt ttt att 1584

Ser Tyr Arg Phe Ala Leu val Gly lie Cys Ser Tyr lie val Phe lie 515 520 525 gtt att gtg ctg gtg gtt tat ttc acc ggc aaa att acg act tgg aat 1632

Val lie Vat Leu VaT val Tyr Phe Thr Gty Lys lie Thr Thr Trp Asn 530 535 540 ttc acg aag ett cag aag gag eta ate gag gat cgc gtt ctg tac gat 1680

Phe Thr Lys Leu Gin Lys Glu Leu lie Glu Asp Arg val Leu Tyr Asp 545 550 555 560 gca act acc aat get cag tet gtg tga 1707

Ala Thr Thr Asn Ala Gin ser vat 565

<210> 2 <211 >568 <212> PRT <213> Paramecium bursaria Chlorella virus 1 <400> 2

Met Gly Lys Asn lie lie lie Met Val Ser Trp Tyr Thr lie lie Thr 15 10 15

Ser Asn Leu lie Ala val Gly Gly Ala ser Leu lie Leu Ala Pro Ala 20 25 30 lie Thr Gly Tyr val Leu His Trp Asn lie Ala Leu ser Thr lie Trp 35 40 45

Gly Val ser Ala Tyr Gly lie Phe Val Phe Gly Phe Phe Leu Ala Gin 50 55 60

Val Leu Phe Ser Glu Leu Asn Arg Lys Arg Leu Arg Lys Trp ile ser 65 70 75 80

Leu Arg Pro Lys Gly Trp Asn Asp val Arg Leu Ala val ile ile Ala 85 90 95

Gly Tyr Arg Glu Asp Pro Tyr Met Phe Gin Lys Cys Leu Glu Ser val 100 105 110

Arg Asp Ser Asp Tyr Gly Asn val Ala Arg Leu ile cys val ile Asp 115 120 125

Gly Asp Glu Asp Asp Asp Met Arg Met Ala Ala val Tyr Lys Ala ile 130 135 140

Tyr Asn Asp Asn Ile Lys Lys Pro Glu Phe Val Leu Cys Glu Ser Asp 145 150 155 160

Asp Lys Glu Gly Glu Arg ile Asp ser Asp Phe ser Arg Asp Ile Cys 165 170 175

Val Leu Gin Pro His Arg Gly Lys Arg Glu Cys Leu Tyr Thr Gly Phe 180 185 190

Gir Leu Ala Lys Met Asp Pro ser Val Asn Ala Val Val Leu Ile Asp 195 200 205

Ser Asp Thr val leu Glu Lys Asp Ala Ile Leu Glu val val Tyr Pro 210 215 220

Leu Ala Cys Asp Pro Glu Ile Gin Ala val Ala Gly Glu cys Lys Ile 225 230 235 240

Trp Asn Thr Asp Thr Leu Leu ser Leu Leu val Ala Trp Arg Tyr Tyr 245 250 255

Ser Ala Phe Cys Val Glu Arg ser Ala Gin ser phe Phe Arg Thr val 260 265 270

Gin Cys Val Gly Gly Pro Leu Gly Ala Tyr Lys Ile Asp Ile Ile Lys 275 280 285

Glu Ile Lys Asp Pro Trp Ile ser Gin Arg Phe Leu Gly Gin Lys Cys 290 295 300

Thr Tyr Gly Asp Asp Arg Arg Leu Thr Asn Glu Ile Leu Met Arg Gly 305 310 315 320

Lys Lys Val Val Phe Thr Pro Phe Ala val Gly Trp Ser Asp Ser Pro 325 330 335

Thr Asn val Phe Arg Tyr ile val Gin Gin Thr Arg Trp ser Lys ser 340 345 350

Trp Cys Arg Glu ile Trp Tyr Thr Len Phe Ala Ala Trp Lys His Gly 355 360 365

Leu ser Gly ile Trp Leu Ala Phe Glu cys Leu Tyr Gin ile Thr Tyr 370 375 380

Phe Phe Leu Val Ile Tyr Leu Phe ser Arg Leu Ala val Glu Ala Asp 385 390 395 400

Pro Arg Ala Gin Thr Ala Thr val ile val ser Thr Thr val Ala Leu 405 410 415

Ile Lys Cys Gly Tyr Phe Ser Phe Arg Ala Lys Asp ile Arg Ala Phe 420 425 430

Tyr Phe val Leu Tyr Thr Phe val Tyr Phe Phe Cys Met ile Pro Ala 435 440 445

Arg Ile Thr Ala Met Met Thr Leu Trp Asp ile Gly Trp Gly Thr Arg 450 455 460

Gly Gly Asn Glu Lys Pro Ser val Gly Thr Arg Val Ala Leu Trp Ala 465 470 475 480

Lys Gin Tyr Leu Ile Ala Tyr Met Trp Trp Ala Ala val val Gly Ala 485 490 495

Gly Val Tyr ser ile val His Asn Trp Met Phe Asp Trp Asn Ser Leu 500 505 510

Ser Tyr Arg Phe Ala Leu val Gly Ile Cys Ser Tyr ile Val Phe Ile 515 520 525

Val Ile val Leu val val Tyr Phe Thr Gly Lys Ile Thr Thr Trp Asn 530 535 540

Phe Thr Lys Leu Gin Lys Glu Leu ile Glu Asp Arg val Leu Tyr Asp 545 550 555 560

Ala Thr Thr Asn Ala Gin ser val 565

<210> 3 <211>1707 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence encoding Paramecium bursaria Chlorella virus Hyaluronansynthase protein <400> 3 atgggtaaga acattatcat tatggtgtcc tggtacacaa ttattacaag taatctcatc 60 gcagttggtg gtgcatctct tattctcgct ccagctatca ctggatatgt tcttcactgg 120 aacatcgccc tctcaactat.ttggggagtt tccgcatatg gtatttttgt tttcgggttc 180 tttttggctc aggttctgtt ctcagagctc aatcgtaaga gactcaggaa gtggattagc 240

cttagaccaa aggggtggaa tgacgttcgt ctcgctgtca ttatcgctgg ctaccgtgaa BOO gatccttaca tgtttcaaaa gtgcttggaa tcagttaggg atagtgatta tggcaacgtc 360 gctagactga tctgtgtgat tgatggagat gaggacgacg atatgaggat ggcagctgtt 420 tataaggcta tctataatga taacattaag aagcctgaat ttgttctttg cgagtctgat 480 gacaaggaag gagaacggat tgattcagat ttctcacgtg atatctgcgt tctccaacct 540 catcgtggga agcgtgaatg tctttataca ggtttccaac tcgccaaaat ggacccatca 600 gtgaacgctg tggttcttat cgatagtgat actgtgctgg agaaagatgc tatcttggag 660 gttgtttacc ctcttgcctg tgatcctgaa attcaagctg tggctggaga gtgcaagatc 720 tggaacacag atactcttct ttctctgctt gtcgcatgga gatattactc cgcattctgt 780 gtggagagga gcgctcaatc ctttttccgt accgttcaat gcgttggtgg tcctttggga 840 gcttacaaaa ttgatatcat caaggagatt aaggacccat ggattagtca aaggtttctt 900 ggtcagaagt gcacttatgg cgatgatcgt agattgacta acgaaatcct tatgaggggc 960 aagaaagtcg tttttactcc atttgctgtc ggatggtctg attcacctac aaatgttttc 1020 cgttatattg tgcaacaaac acgttggagt aagagctggt gtagggagat ctggtacact 1080 ttgttcgctg cttggaagca cgggcttagc ggaatttggc ttgcttttga atgcctttac 1140 cagattacat actttttctt ggtgatctat ttgttttcac gtcttgccgt cgaggctgac 1200 cctagagcac agactgcaac tgtgattgtt tctactacag tcgcacttat taagtgtggc 1260 tatttcagtt ttagagcaaa agatattaga gccttctatt ttgttttgta cacatttgtt 1320 tatttctttt gcatgattcc agctcgtatt accgctatga tgaccttgtg ggacatcgga 1380 tggggaacta gaggtggtaa cgaaaagcct tctgtgggaa caagggtggc cctttgggca 1440 aaacaatatc tcatcgccta catgtggtgg gccgctgtcg ttggtgccgg agtgtactca 1500 atcgttcata actggatgtt tgactggaac tctttgagct atcgtttcgc tcttgtgggt 1560 atttgttctt acattgtttt catcgtgatt gtgctcgttg tgtatttcac tggtaaaatc 1620 acaacctgga atttcactaa acttcaaaag gaattgattg aagacagggt tctgtatgat 1680 gctactacca acgcccagtc agtttaa 1707

<210> 4 <211 >1260 <212> DNA <213> Paramecium bursaria chlorella virus 1 <220>

<221 > CDS <222> (62)..(1228) <300> <308> U42580.4 <309> 2004-09-20 <313> (291749.)..(292918) <400> 4 atcaacgtga tttatatttt aaacaaagac cattcacatc tttagtactt aattaattat 60 a atg tea ega ate gca gtc gtt ggt tgt ggt tac gtc gga acc get tgt 109

Met Ser Arg ile Ala val val Gly cys Gly Tyr val Gly Thr Ala cys 15 10 15 gca gta ett ett get caa aaa aac gaa gtc ate gtg ett gat att age 157

Ala Val Leu Leu Ala Gin Lys Asn Glu Val lie Val Leu Asp Ile Ser 20 25 30 gaa gac cgt gtt caa eta ate aag aac aag aag agt cca ate gag gac 205

Glu Asp Arg val Gin Leu lie Lys Asn Lys Lys Ser pro lie Glu Asp 35 40 45 aag gaa ate gaa gag ttt etc gaa aeg aaa gac ctg aac ctg acc geg 253

Lys Glu lie Glu Glu Phe Leu Glu Thr Lys Asp Leu Asn Leu Thr Ala 50 55 60 aeg act gac aag gtt ett gca tac gaa aac gee gaa ttt gtc ate ate 301

Thr Thr Asp Lys Val Leu Ala Tyr Glu Asn Ala Glu Phe val He lie 65 70 75 80 gca acc ccg act gac tat gac gtg gtt act agg tat ttt aac aeg aaa 349

Ala Thr Pro Thr Asp Tyr Asp val val Thr Arg Tyr Phe Asn Thr Lys 85 90 95 tet gtg gaa aac gtc att ggg gac gtg ate aaa aat aca cag acc cat 397

Ser val Glu Asn val lie Gly Asp val lie Lys Asn Thr Gin Thr His 100 105 110 cca act ate gtg att aaa tet acc ate ccc att gga ttt gtt gat aag 445

Pro Thr lie val lie Lys Ser Thr lie Pro lie Gly Phe val Asp Lys 115 120 125 gtt cgt gag caa ttc gac tac caa aat ate att ttc tee cca gaa ttt 493 val Arg Glu Gin Phe Asp Tyr Gin Asn lie He Phe ser Pro Glu Phe 130 135 140 ctg cgt gaa ggt aga gee ttg tat gat aat etc tac cca tee cgt ate 541

Leu Arg Glu Gly Arg Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg lie 145 150 155 160 ate gta gga gat gat tee ccc att geg ett aag ttc gca aac ett etc 589 lie Val Gly Asp Asp Ser Pro lie Ala Leu Lys Phe Ala Asn Leu Leu 165 170 175 gtt gaa ggt tet aaa act ccg ett gee ect gtc ctg aeg atg gga act 637

Val Glu Gly ser Lys Thr Pro Leu Ala Pro val Leu Thr Met Gly Thr 180 185 190 ege gaa gee gag gee gtc aaa eta ttc tet aac aeg tat ett gca atg 685

Arg Glu Ala Glu Ala val Lys Leu Phe Ser Asn Thr Tyr Leu Ala Met 195 200 205 ega gtt gca tac ttc aac gaa eta gat aca ttc gca atg tet cac ggt 733

Arg val Ala Tyr Phe Asn Glu Leu Asp Thr Phe Ala Met Ser His Gly 210 215 220 atg aat geg aaa gaa ate att gat ggt gtg act ctg gag cct ege att 781

Met Asn Ala Lys Glu lie lie Asp Gly val Thr Leu Glu Pro Arg lie 225 230 235 240 ggt cag ggg tac tea aac cct teg ttc ggt tat gga get tat tgc ttt 829

Gly Gin Gly Tyr Ser Asn Pro ser Phe Gly Tyr Gly Ala Tyr Cys Phe 245 250 255 cca aag gat aeg aag caa ctg ctg get aat ttc gag gga gtg cct caa 877

Pro Lys Asp Thr Lys Gin Leu Leu Ala Asn phe Glu Gly Val Pro Gin 260 265 270 gat ate ate gga gca att gta gaa tea aat gag act ege aag gaa gtg 925

Asp lie lie Gly Ala lie Val Glu Ser Asn Glu Thr Arg Lys Glu val 275 280 285 att gtg agt gaa gta gaa aat cgt ttc ccc aeg act gtt ggt gtg tat 973 lie val Ser Glu val Glu Asn Arg Phe Pro Thr Thr val Gly val Tyr 290 295 300 aag etc gee get aaa geg ggt tet gat aat ttt egg agt tet gca att 1021

Lys Leu Ala Ala Lys Ala Gly Ser Asp Asn Phe Arg ser ser Ala lie 305 310 315 320 gta gac ata atg gag ega ett gca aac aag ggt tat cac att aag att 1069 val Asp lie Met Glu Arg Leu Ala Asn Lys Gly Tyr His lie Lys lie 325 330 335 ttc gaa cca act gtg gaa caa ttc gaa aac ttt gaa gtt gat aac aac 1117

Phe Glu Pro Thr val Glu Gin Phe Glu Asn Phe Glu val Asp Asn Asn 340 345 350 ctg aca aca ttt geg act gag age gat gta att ate gca aac aga gtt 1165

Leu Thr Thr Phe Ala Thr Glu Ser Asp val lie lie Ala Asn Arg val 355 360 365 ccc gtt gaa cat ege att etc ttt ggt aaa aaa tta ate aca cgt gat 1213

Pro val Glu His Arg lie Leu Phe Gly Lys Lys Leu He Thr Arg Asp 370 375 380 gta tat ggc gat aac taaaatgttt tcaatatgat gttgttaatg at 1260 val Tyr Gly Asp Asn 385

<210> 5 <211 >389 <212> PRT <213> Paramecium bursaria Chlorella virus 1 <400> 5

Met Ser Arg ile Ala val val Gly Cys Gly Tyr val Gly Thr Ala Cys 15 10 15

Ala Val Leu Leu Ala Gin Lys Asn Glu val ile val Leu Asp ile Ser 20 25 30

Glu Asp Arg val Gin Leu Ile Lys Asn Lys Lys ser pro ile Glu Asp 35 40 45

Lys Glu ile Glu Glu Phe Leu Glu Thr Lys Asp Leu Asn Leu Thr Ala 50 55 60

Thr Thr Asp Lys val Leu Ala Tyr Glu Asn Ala Glu Phe val ile ile 65 70 75 80

Ala Thr Pro Thr Asp Tyr Asp val val Thr Arg Tyr Phe Asn Thr Lys 85 90 95

Ser val Glu Asn Val Ile Gly Asp val ile Lys Asn Thr Gin Thr His 100 105 110

Pro Thr Ile val ile Lys Ser Thr Ile Pro ile Gly Phe val Asp Lys 115 120 125 val Arg Glu Gin Phe Asp Tyr Gin Asn ile Ile Phe Ser Pro Glu Phe 130 135 140

Leu Arg Glu Gly Arg Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg ile 145 150 155 160

Ile val Gly Asp Asp Ser Pro ile Ala Leu Lys Phe Ala Asn Leu Leu 165 170 175 val Glu Gly ser Lys Thr Pro Leu Ala Pro val Leu Thr Met Gly Thr 180 185 190

Arg Glu Ala Glu Ala val Lys Leu Phe ser Asn Thr Tyr Leu Ala Met 195 200 205

Arg val Ala Tyr Phe Asn Glu Leu Asp Thr Phe Ala Met Ser His Gly 210 215 220

Met Asn Ala Lys Glu ile ile Asp Gly Val Thr Leu Glu Pro Arg ile 225 230 235 240

Gly Gin Gly Tyr Ser Asn Pro ser Phe Gly Tyr Gly Ala Tyr Cys Phe 245 250 255

Pro Lys Asp Thr Lys Gin Leu Leu Ala Asn Phe Glu Gly val Pro Gin 260 265 270

Asp Ile Ile Gly Ala Ile val Glu ser Asn Glu Thr Arg Lys Glu val 275 280 285 ile val ser Glu val Glu Asn Arg Phe Pro Thr Thr val Gly val Tyr 290 295 300

Lys Leu Ala Ala Lys Ala Gly ser Asp Asn Phe Arg ser ser Ala Ile 305 310 315 320

Val Asp Ile Met Glu Arg Leu Ala Asn Lys Gly Tyr His Ile Lys Ile 325 330 335

Phe Glu Pro Thr val Glu Gin Phe Glu Asn Phe Glu Val Asp Asn Asn 340 345 350

Leu Thr Thr Phe Ala Thr Glu ser Asp val ile ile Ala Asn Arg val 355 360 365

Pro val Glu His Arg ile Leu Phe Gly Lys Lys Leu ile Thr Arg Asp 370 375 380

Val Tyr Gly Asp Asn 385

<210> 6 <211>1170 <212>DNA <213> Artificial Sequence <220>

<223> Synthetic sequence encoding a Paramecium bursaria chlorella virus protein having the activity of a UDP-GIc-DH <400> 6 atgtctcgca tagctgttgt aggatgtggc tatgtgggaa ctgcatgtgc ggttctactt 60 gctcaaaaga acgaagttat tgtgcttgat attagtgaag accgtgttca acttattaag 120 aacaagaagt ctcctattga ggataaggaa atcgaagagt tcttggaaac aaaggatctt 180 aatcttactg cgactacaga taaggttctt gcctacgaga acgctgagtt tgtgataatc 240 gctacaccaa ccgattacga cgttgtgact cgatatttca ataccaaatc cgtggaaaac 300 gttataggag atgttatcaa gaacactcaa acccacccta ctatcgtcat caagtccaca 360 attcccatcg gtttcgttga taaggtcaga gagcagtttg attatcaaaa cattatcttc 420 tcacctgagt tcttaaggga gggtcgtgct ctctacgata atttgtatcc gtcccgtatt 480 atcgttggcg acgattctcc tatcgctctc aagttcgcaa atctcttagt tgagggtagt 540 aagacccctt tggctcctgt tttgacaatg ggaaccagag aagcagaagc tgtcaagcta 600 ttctctaata cctaccttgc catgagggta gcatacttta acgaacttga tacatttgct 660 atgtcgcatg gtatgaatgc caaggagatt atagatggtg tcactttaga gcccaggatc 720 ggtcaaggat attctaaccc atcattcggc tatggagctt actgctttcc taaggacact 780 aagcagttgc tggcaaactt cgagggagtt cctcaagaca tcataggcgc tattgtggag 840 tcaaacgaaa caaggaaaga ggtgatagtt agtgaggtag agaatcgttt cccaacgaca 900 gtcggtgttt acaaactggc agctaaagct ggtagcgata acttcaggtc aagtgctatt 960 gtcgacarca tggaacgcct ggctaacaaa ggttaccaca ttaagatctt tgagccaact 1020 gtagagcagt tcgaaaattt cgaagttgac aataacttga caacgtttgc tactgagtca 1080 gacgttatta tcgcaaatcg tgtccctgtg gaacatagaa tcctatttgg aaagaagctc 1140 attaccagag atgtttacgg tgataattaa 1170 <210> 7 <211 >2298

<212> DNA <213> Mus musculus <220> <221 > CDS <222> (150)..(2192) <220> <221 > mutation <222> (1060)..(1060) <223> sequnce inserted in plasmid IC 370-256 contains an exchnage from G to A at position 1060 <220> <221 > allele <222> (1190)..(1190) <223> sequnce inserted in plasmid IC 365-256 contains a base exchnage from T to C at position 1190 <220> <221 > mutation <222> (1245)..(1245) <223> sequnce inserted in plasmid IC 370-256 contains an exchnage from T to C at position 1245 <220> <221 > allele <222> (2027)..(2027) <223> sequnce inserted in plasmid IC 365-256 contains a base exchnage from G to A at position 2027 <300> <308> BC050762.1 <309> 2005-03-08 <313> (150)..(2195) <400> 7 gagagcgaag cgagcgctga gtcggactgt cgggtctgag ctgtcgcatc ccagagtcct 60 ctcattgcca ccaccccggc ccgagctcac cctcgcttct gaagctctcc gcgcgcccga 120 cagctcagcc ctcgcccgtg accaacatc atg tgc ggt ata ttt get tat tta 173

Met Cys Gly Ile Phe Ala Tyr Leu 1 5 aat tac cat gtt cct ega aca aga ega gaa ate ttg gag aca eta ate 221

Asn Tyr His val pro Arg Thr Arg Arg Glu lie Leu Glu Thr Leu lie 10 15 20 aaa ggc ett cag aga ctg gaa tac aga gaa tat gat tet get ggt gtg 269

Lys Gly Leu Gin Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala Gly Val 25 30 35 40 gga ett gac gga ggc aat gac aaa gac tgg gaa gee aac gee tgc aaa 317

Gly Leu Asp Gly Gly Asn Asp Lys Asp Trp Glu Ala Asn Ala Cys Lys 45 50 55 ate cag etc att aag aag aaa gga aaa gtt aag gca ctg gat gaa gaa 365

He Gin Leu lie Lys Lys Lys Gly Lys val Lys Ala Leu Asp Glu Glu 60 65 70 gtt cac aaa caa caa gat atg gac ttg gat ata gaa ttt gat gtg cat 413

Val His Lys Gin Gin Asp Met Asp Leu Asp lie Glu Phe Asp Val His 75 80 85 ett gga ata get cat acc cgt tgg geg aca cat gga gaa ccc aat cct 461

Leu Gly lie Ala His Thr Arg Trp Ala Thr His Gly Glu Pro Asn Pro 90 95 100 gtc aat agt cac ccc cag egc tet gat aaa aat aat gaa ttc att gtt 509 val Asn ser His Pro Gin Arg Ser Asp Lys Asn Asn Glu Phe lie val 105 110 115 120 att cat aat gga ate ate acc aac tac aaa gac ttg aaa aag ttt ctg 557 lie His Asn Gly lie lie Thr Asn Tyr Lys Asp Leu Lys Lys Phe Leu 125 130 135 gaa age aaa ggc tat gac ttt gaa tet gaa aca gac aca gaa acc att 605

Glu Ser Lys Gly Tyr Asp Phe Glu Ser Glu Thr Asp Thr Glu Thr lie 140 145 150 gee aag etc gtc aag tac atg tat gac aac tgg gag age cag gac gtc 653

Ala Lys Leu val Lys Tyr Met Tyr Asp Asn Trp Glu ser Gin Asp val 155 160 165 agt ttt acc acc ttg gtg gag aga gtt ate caa caa ttg gaa ggc gee 701 ser Phe Thr Thr Leu vaT Glu Arg val lie Gin Gin Leu Glu Gly Ala 170 175 180 ttt get ett gtg ttt aaa agt gtc cat ttt ccc ggg caa gca gtt ggc 749

Phe Ala Leu val Phe Lys ser val His Phe pro Gly Gin Ala val Gly 185 190 195 200 aca agg ega ggt age cct etc ttg att ggt gtg egg agt gaa cat aag 797

Thr Arg Arg Gly Ser pro Leu Leu lie Gly val Arg ser Glu His Lys 205 210 215 ett tet aca gat cac att ccg att ctg tac aga aca ggc aaa gac aag 845

Leu Ser Thr Asp His lie Pro lie Leu Tyr Arg Thr Gly Lys Asp Lys 220 225 230 aaa gga age tgc ggt ett tee cgt gtg gac age aeg aca tgc ctg ttc 893

Lys Gly ser Cys Gly Leu Ser Arg val Asp ser Thr Thr cys Leu Phe 23S 240 245 cct gtt gag gaa aag gca gtt gaa tat tac ttt get tet gat gca agt 941

Pro val Glu Glu Lys Ala val Glu Tyr Tyr Phe Ala ser Asp Ala ser 250 255 260 gee gtg ata gag cac acc aat cgt gtc ate ttt ctg gaa gat gat gat 989

Ala val lie Glu His Thr Asn Arg val lie Phe Leu Glu Asp Asp Asp 265 270 275 280 gtt gca gca gtg gtg gat ggc cgt etc tet ate cac ega att aaa ega 1037

Val Ala Ala val val Asp Gly Arg Leu Ser lie His Arg lie Lys Arg 285 290 295 act gca gga gac cat cct ggc ega get gtg caa act etc cag atg gag 1085

Thr Ala Gly Asp His Pro Gly Arg Ala val Gin Thr Leu Gin Met Glu 300 305 310 etc cag cag ate atg aag ggc aac ttt agt tea ttt atg cag aag gaa 1133

Leu Gin Gin lie Met Lys Gly Asn Phe ser ser Phe Met Gin Lys Glu 315 320 325 att ttt gag cag cca gaa tet gtt gtg aac aca atg aga gga aga gtc 1181

He Phe Glu Gin pro Glu ser val val Asn Thr Met Arg Gly Arg val 330 335 340 aat ttt gat gac tac act gtg aat ttg gga ggt ttg aaa gat cac att 1229

Asn Phe Asp Asp Tyr Thr val Asn Leu Gly Gly Leu Lys Asp His lie 345 350 355 360 aag gag ate cag egg tgt egg egg ttg att ett att get tgt ggc aca 1277

Lys Glu ile Gin Arg cys Arg Arg Leu il'e Leu ile Ala cys Gly Thr 365 370 375 agt tac cac get ggt gtg gea acc egt cag gtc ctg gag gag ctg acc 1325

Ser Tyr His Ala Gly val Ala Thr Arg Gin val Leu Glu Glu Leu Thr 380 385 390 gag ctg ccc gtg atg gtg gag ett gcc agt gac ttc ttg gat aga aac 1373

Glu Leu Pro val Met Val Glu Leu Ala ser Asp Phe Leu Asp Arg Asn 395 400 405 act cca gtc ttt ega gat gat gtt tgc ttt ttc att agt caa tea ggc 1421

Thr Pro Val Phe Arg Asp Asp val Cys Phe Phe ile ser Gin ser Gly 410 415 420 gag aca get gac acc ctg atg gga ett egt tac tgt aag gag aga gga 1469

Glu Thr Ala Asp Thr Leu Met Gly Leu Arg Tyr Cys Lys Glu Arg Gly 425 430 435 440 gcc tta act gtg ggg atc aca aat aca gtc ggc agt tet ata tea agg 1517

Ala Leu Thr val Gly Ile Thr Asn Thr val Gly Ser Ser ile ser Arg 445 450 4S5 gag aca gat tgc ggg gtt cat att aat get ggt cct gag att ggc gtg 1565

Glu Thr asp Cys Gly val His Ile Asn Ala Gly Pro Glu ile Gly val 460 465 470 gcc agt aca aag gea tac acc age cag ttt gtg tee etc gtg atg ttt 1613

Ala ser Thr Lys Ala Tyr Thr ser Gin Phe val ser Leu val Met Phe 475 480 485 get etc atg atg tgt gat gac agg atc tee atg caa gag aga ege aaa 1661

Ala Leu Met Met Cys Asp Asp Arg ile ser Met Gin Glu Arg Arg Lys 490 495 500 gag atc atg etc gga ctg aag ega ctg ccg gac ttg att aag gaa gtg 1709

Glu ile Met Leu Gly Leu Lys Arg Leu Pro Asp Leu Ile Lys Glu Val 505 510 515 520 ctg age atg gat gat gaa atc cag aag ctg gcg aeg gag ett tac cac 1757

Leu Ser Met Asp Asp Glu ile Gin Lys Leu Ala Thr Glu Leu Tyr His 525 530 535 cag aag teg gtc ctg ata atg gag egg ggc tac cat tat get aca tgc 1805

Gin Lys Ser val Leu ile Met Gly Arg Gly Tyr His Tyr Ala Thr Cys 540 545 550 ett gaa ggg get ctg aaa ate aag gag att act tat atg cat teg gaa 1853

Leu Glu Gly Ala Leu Lys Ile Lys Glu ile Thr Tyr Met His Ser Glu 555 560 565 ggc ate ett get ggt gag etc aag cac ggc cct ctg gcc ttg gtg gac 1901

Gly ile Leu Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu val Asp 570 575 580 aag ttg atg cct gtc ate atg ate ate atg ega gac cac act tat gcc 1949

Lys Leu Met Pro val ile Met ile ile Met Arg Asp His Thr Tyr Ala 585 590 . 595 600 aag tgc cag aac get ett cag cag gtg gtt gea egg cag ggg egt cca 1997

Lys cys Gin Asn Ala Leu Gin Gin Val Val Ala Arg Gin Gly Arg Pro 605 610 615 gtc gtg ate tgt gat aag gag gat act gag acc att aag aat aca aaa 2045 val val Ile Cys Asp Lys Glu Asp Thr Glu Thr ile Lys Asn Thr Lys 620 625 630 agg aca ate aag gtg ccc cac tea gtg gac tgc ttg cag ggc att etc 2093

Arg Thr Ile Lys val Pro His ser val Asp cys Leu Gin Gly ile Leu 635 640 645 agt gtg att ccc ctg cag ctg ctg get ttc cac ctg get gtg ctg aga 2141

Ser val ile Pro Leu Gin Leu Leu Ala Phe His Leu Ala val Leu Arg 650 655 660 ggc tac gat gtt gat ttt cca egg aat ett gcc aaa tet gta aca gta 2189

Gly Tyr Asp val Asp Phe Pro Arg Asn Leu Ala Lys ser val Thr val 665 670 675 680 gag taacagacac ctgaaactta agaeagttaa gcaacacgag ataccttttg 2242

Glu tatttaaatt tttgatttaa actatcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2298 <210> 8 <211 >681

<212> PRT <213> Mus musculus <400> 8

Met Cys Gly Ile Phe Ala Tyr Leu Asn Tyr His val Pro Arg Thr Arg 15 10 15

Arg Glu lie Leu Glu Thr Leu Ile Lys Gly Leu Gin Arg Leu Glu Tyr 20 25 30

Arg Gly Tyr Asp ser Ala Gly val Gly Leu Asp Gly Gly Asn Asp Lys 35 40 45

Asp Trp Glu Ala Asn Ala Cys Lys lie Gin Leu lie Lys Lys Lys Gly 50 55 60

Lys Val Lys Ala Leu Asp Glu Glu val His Lys Gin Gin Asp Met Asp 65 70 75 80

Leu Asp lie Glu Phe Asp val His Leu Gly lie Ala His Thr Arg Trp 85 90 95

Ala Thr His Gly Glu Pro Asn Pro val Asn Ser His Pro Gin Arg ser 100 105 110

Asp Lys Asn Asn Glu Phe lie val lie His Asn Gly lie lie Thr Asn 115 120 125

Tyr Lys Asp Leu Lys Lys Phe Leu Glu ser Lys Gly Tyr Asp Phe Glu 130 135 140

Ser Glu Thr Asp Thr Glu Thr lie Ala Lys Leu val Lys Tyr Met Tyr 145 150 155 160

Asp Asn Trp Glu Ser Gin Asp val Ser Phe Thr Thr Leu Val Glu Arg 165 170 175 val lie Gin Gin Leu Glu Gly Ala Phe Ala Leu val Phe Lys ser val 180 185 190

His Phe Pro Gly Gin Ala val Gly Thr Arg Arg Gly Ser Pro Leu Leu 195 200 205 lie Gly val Arg Ser Glu His Lys Leu ser Thr Asp His lie Pro lie 210 215 220

Leu Tyr Arg Thr Gly Lys Asp Lys Lys Gly Ser Cys Gly Leu Ser Arg 225 230 235 240 val Asp Ser Thr Thr Cys Leu Phe Pro Val Glu Glu Lys Ala val Glu 245 250 255

Tyr Tyr Phe Ala ser Asp Ala Ser Ala val lie Glu His Thr Asn Arg 260 265 270 val He Phe Leu Glu Asp Asp Asp val Ala Ala val val Asp Gly Arg 275 280 285

Leu Ser lie His Arg lie Lys Arg Thr Ala Gly Asp His Pro Gly Arg 290 295 300

Ala val Gin Thr Leu Gin Met Glu Leu Gin Gin lie Met Lys Gly Asn 305 310 315 320

Phe Ser Ser Phe Met Gin Lys Glu lie Phe Glu Gin Pro Glu Ser val 325 330 335

Val Asn Thr Met Arg Gly Arg val Asn Phe Asp Asp Tyr Thr val Asn 340 345 350

Leu Gly Gly Leu Lys Asp His lie Lys Glu lie Gin Arg Cys Arg Arg 355 360 365

Leu lie Leu lie Ala Cys Gly Thr Ser Tyr His Ala Gly Val Ala Thr 370 375 380

Arg Gin val Leu Glu Glu Leu Thr Glu Leu Pro val Met Val Glu Leu 385 390 395 400

Ala Ser Asp Phe Leu Asp Arg Asn Thr Pro val Phe Arg Asp Asp val 405 410 415

Cys Phe Phe lie Ser Gin Ser Gly Glu Thr Ala Asp Thr Leu Met Gly 420 425 430

Leu Arg Tyr cys Lys Glu Arg Gly Ala Leu Thr val Gly lie Thr Asn 435 440 445

Thr val Gly ser ser ile ser Arg Glu Thr Asp cys Gly val His Ile 450 455 460

Asn Ala Gly Pro Glu Ile Gly val Ala ser Thr Lys Ala Tyr Thr Ser 465 470 475 480

Gin Phe Val Ser Leu Val Met Phe Ala Leu Met Met Cys Asp Asp Arg 485 490 495

Ile Ser Met Gin Glu Arg Arg Lys Glu ile Met Leu Gly Leu Lys Arg 500 505 510

Leu Pro Asp Leu ile Lys Glu val Leu ser Met Asp Asp Glu ile Gin 515 520 525

Lys Leu Ala Thr Glu Leu Tyr His Gin Lys ser val Leu ile Met Gly 530 535 540

Arg Gly Tyr His Tyr Ala Thr Cys Leu Glu Gly Ala Leu Lys ile Lys 545 550 555 560

Glu ile Thr Tyr Met His ser Glu Gly ile Leu Ala Gly Glu Leu Lys 565 570 575

His Gly Pro Leu Ala Leu val Asp Lys Leu Met Pro Val Ile Met Ile 580 585 590

Ile Met Arg Asp His Thr Tyr Ala Lys Cys Gin Asn Ala Leu Gin Gin 595 600 605

Val Val Ala Arg Gin Gly Arg Pro val val ile cys Asp Lys Glu Asp 610 615 620

Thr Glu Thr Ile Lys Asn Thr Lys Arg Thr ile Lys val Pro His Ser 625 630 635 640 val Asp cys Leu Gin Gly Ile Leu Ser val ile Pro Leu Gin Leu Leu 645 650 655

Ala Phe His Leu Ala val Leu Arg Gly Tyr Asp val Asp Phe Pro Arg 660 665 670

Asn Leu Ala Lys ser val Thr val Glu 675 680 <210> 9 <211 >2049

<212> DNA <213> Mus musculus <220> <221 > CDS <222> (1)..(2046) <300> <308> BC031928.1 <309> 2003-10-07 <313> (51)..(299) <400> 9 atg tgc gqa ate ttt gee tac atg aat tac aga gtt.ccc aag aca agg 48

Met Cys Gly ile Phe Ala Tyr Met Asn Tyr Arg val Pro Lys Thr Arg 15 10 15 aaa gag att ttc gaa ace ett ate agg gqt ctg cag egg ctg gag tac 96

Lys Glu lie Phe Glu Thr Leu lie Arg Gly Leu Gin Arg Leu Glu Tyr 20 25 30 egg gac tat gac tet geg gag gtt gee att gat ggg aat aac cac gaa 144

Arg Gly Tyr Asp ser Ala Gly val Ala lie Asp Giy Asn Asn His Glu 35 40 45 gtc aaa gaa aga cac ate cat ett gtq aag aaa agg ggg aaa gta aag 192

Val Lys Glu Arg His lie His Leu val Lys Lys Arg Giy Lys val Lys 50 55 60 get ctg gat gaa gaa ett tac aag caa gat age atg gac ttg aag gtq 240

Ala Leu Asp Glu Glu Leu Tyr Lys Gin Asp Ser Met Asp Leu Lys val 65 70 75 80 gag ttt gag aca cac ttc ggc att gee cac aca cgt tgg gee ace cac 288

Glu Phe Glu Thr His Phe Gly lie Ala His Thr Arg Trp Ala Thr His 85 90 95 ggg gtt ccc aat get gtc aac agt cac ccg cag cgt teg gac aaa gac 336

Gly val Pro Asn Ala val Asn Ser His Pro Gin Arg Ser Asp Lys Asp 100 105 110 aat gaa ttt gtt gtc ate cac aac ggg ate ate act aat tac aag gat 384

Asn Glu Phe val val lie His Asn Gly He He Thr Asn Tyr Lys Asp 115 120 125 eta agg aag ttt ctg gaa age aaa ggc tac gag ttt gag tea gaa aca 432

Leu Arg Lys Phe Leu Glu Ser Lys Gly Tyr Glu Phe Glu Ser Glu Thr 130 135 140 gac aeg gag acc ate gee aag ctg att aaa tat gta ttt gac aac aga 480

Asp Thr Glu Thr lie Ala Lys Leu lie Lys Tyr val Phe Asp Asn Arg 145 150 155 160 gag act gag gac ata aeg ttt tee aca ttg gtc gaa aga gtc att cag 528

Glu Thr Glu Asp He Thr Phe Ser Thr Leu val Glu Arg val lie Gin 165 170 175 cag ttg gaa ggc gee ttt gca ctg gtt ttc aag agt att cac tac ccg 576

Gin Leu Glu Gly Ala Phe Ala Leu val Phe Lys ser lie His Tyr Pro 180 185 190 gga gaa get gtc gee aeg agg aga ggc age ccc ttg etc ate ggg gta 624

Gly Glu Ala val Ala Thr Arg Arg Gly Ser Pro Leu Leu lie Gly val 195 200 205 ega age aaa tac aaa etc tee aca gag cag ate ccc gtc tta tat ccg 672

Arg Ser Lys Tyr Lys Leu Ser Thr Glu Gin lie Pro val Leu Tyr Pro 210 215 220 aca tgc aat ate gag aat gtq aag aat ate tgc aag act agg atg aag 720

Thr cys Asn lie Glu Asn val Lys Asn lie cys Lys Thr Arg Met Lys 225 230 235 240 aga ctg gac age tee ace tgc ctg cac get gtg gac gat aaa get gtg 768

Arg Leu Asp ser Ser Thr Cys Leu His Ala Val Gly Asp Lys Ala val 245 250 255 gaa ttc ttc ttt get tet gat gca agt gee ate ata gaa cac ace aac 816

Glu Phe Phe Phe Ala Ser Asp Ala Ser Ala lie lie Glu His Thr Asn 260 265 270 egg gtc ate ttc tta gaa gat gat gat ate get gea gtg get gat ggg 864

Arg Val lie Phe Leu Glu Asp Asp Asp lie Ala Ala val Ala Asp Gly 275 280 285 aaa etc tee att cac ega gtc aag ege tea get act gat gac ccc tee 912

Lys Leu Ser He His Arg val Lys Arg ser Ala Thr Asp Asp Pro ser 290 295 300 ega gee ate cag ace ttg cag atg gaa ctg cag caa ata atg aaa ggt 960

Arg Ala lie Gin Thr Leu Gin Met Glu Leu Gin Gin lie Met Lys Gly 305 310 315 320 aac ttc age gca ttt atg cag aag gag ate ttc gag cag cca gaa tea 1008

Asn Phe ser Ala Phe Met Gin Lys Glu lie Phe Glu Gin Pro Glu Ser 325 330 335 gtt ttt aat acc atg aga gat egg gtg aat ttt gag ace aac aca gtg 1056

Val Phe Asn Thr Met Arg Gly Arg Val Asn Phe Glu Thr Asn Thr Val 340 345 350 etc ctg ggt gac ttg aag gac cat ttg aaa gag ate ega ega tgc ega 1104

Leu Leu Gly Gly Leu Lys Asp His Leu Lys Glu lie Arg Arg cys Arg 355 360 365 agg etc att gtg att ggc tgt gga acc age tac cat gee get gtg get 1152

Arg Leu ile val ile Gly Cys Gly Thr Ser Tyr His Ala Ala val Ala 370 375 380 aca egg caa gtc tta gag gaa ctg acc gag ctg cct gtg atg gtt gaa 1200

Thr Arg Gin val Leu Glu Glu Leu Thr Glu Leu Pro val Met val Glu 385 390 395 400 ett gee agt gac ttt ctg gac agg aac aca cct gtg ttc agg gat gac 1248

Leu Ala Ser Asp Phe Leu Asp Arg Asn Thr pro val phe Arg Asp Asp 405 410 415 gtt tgc ttt ttc ata age caa tea ggt gag act gca gac aeg etc ctg 1296 val Cys Phe Phe lie ser Gin Ser Gly Glu Thr Ala Asp Thr Leu Leu 420 425 430 geg ctg ega tac tgt aag gat ega ggt geg ctg acc gtg ggc ate acc 1344

Ala Leu Arg Tyr Cys Lys Asp Arg Gly Ala Leu Thr vat Gty lie Thr 435 440 445 aac acc gtg ggt age tee ate tee egg gag act gac tgt ggc gtc cac 1392

Asn Thr vat Gty ser ser lie ser Arg Glu Thr Asp Cys Gty val His 450 455 460 ate aac gca ggg ccc gag att ggg gtg gee age acc aag geg tac acc 1440

He Asn Ala Gty pro Glu lie Gty vat Ala Ser Thr Lys Ala Tyr Thr 465 470 475 480 age cag ttc ate tet ctg gtg atg ttt ggt ttg atg atg tet gaa gat 1488

Ser Gin Phe He Ser Leu vat Met Phe Gly Leu Met Met Ser Glu Asp 485 490 495 ega att tet eta cag aac agg aga caa gag ate ate cgt ggc etc aga 1536

Arg lie ser Leu Gin Asn Arg Arg Gin Glu lie lie Arg Gty Leu Arg 500 505 510 tet tta ccg gag ctg ate aaa gaa gtg ctg tee ctg gat gag aag ate 1584

Ser Leu Pro Glu Leu lie Lys Glu vat Leu Ser Leu Asp Glu Lys lie 515 520 525 cat gac ttg gee ctg gag etc tac aca caa agg tet etc etc gtg atg 1632

His Asp Leu Ala Leu Glu Leu Tyr Thr Gin Arg ser Leu Leu vat Met 530 535 540 gaa egg gaa tat aac tat gee aca tgt ctg gaa ggt gee ttg aaa att 1680

Gty Arg Gty Tyr Asn Tyr Ala Thr Cys Leu Glu Gly Ala Leu Lys lie 545 550 555 560 aag gag ata acc tac atg cat tea gaa gat ate eta gee gga gag ctg 1728

Lys Glu lie Thr Tyr Met His ser Glu Gty lie Leu Ala Gty Glu Leu 565 570 575 aag cac ggg ccc ett get etc gtc gac aag cag atg cca gtc ate atg 1776

Lys His Gty Pro Leu Ala Leu val Asp Lys Gin Met Pro val He Met 580 585 590 gtc ate atg aag gat cct tgc ttt gee aag tgc cag aat gee ctg cag 1824 val He Met Lys Asp Pro Cys Phe Ala Lys Cys Gin Asn Ala Leu Gin 595 600 605 cag gtc act gee ege cag ggt ege cca ate ata ctg tgt tee aag gat 1872

Gin val Thr Ala Arg Gin Gly Arg pro He He Leu cys Ser Lys Asp 610 615 620 gac acc gag age tee aag ttt gca tat aaa acc att gaa ett ccc cac 1920

Asp Thr Glu ser Ser Lys Phe Ala Tyr Lys Thr lie Glu Leu Pro His 625 630 635 640 aca gtg gac tgt etc cag ggt ate ctg age gtg att cca etc cag ett 1968

Thr val Asp cys Leu Gin Gly lie Leu ser val lie Pro Leu Gin Leu 645 650 655 ctg tee ttc cac ctg get gtc etc ega ggt tat gat gtt gac ttc ccc 2016

Leu Ser Phe His Leu Ala val Leu Arg Gly Tyr Asp val Asp Phe Pro 660 665 670 aga aac eta gee aag tet gtc act gtg gaa tga 2049

Arg Asn Leu Ala Lys ser Val Thr val Glu 675 680

<210> 10 <211 >682 <212> PRT <213> Mus musculus <400> 10

Met Cys Gly ile Phe Ala Tyr Met Asn Tyr Arg val Pro Lys Thr Arg 15 10 15

Lys Glu lie Phe Glu Thr Leu lie Arg Gly Leu Gin Arg Leu Glu Tyr 20 25 30

Arg Gly Tyr Asp Ser Ala Gly Val Ala lie Asp Gly Asn Asn His Glu 35 40 45 val Lys Glu Arg His lie His Leu val Lys Lys Arg Gly Lys val Lys 50 55 60

Ala Leu Asp Glu Glu Leu Tyr Lys Gin Asp ser Met Asp Leu Lys val 65 70 75 80

Glu Phe Glu Thr His Phe Gly lie Ala His Thr Arg Trp Ala Thr His 85 90 95

Gly val Pro Asn Ala val Asn ser His Pro Gin Arg Ser Asp Lys Asp 100 105 110

Asn Glu Phe Val Val lie His Asn Gly lie lie Thr Asn Tyr Lys Asp 115 120 125

Leu Arg Lys Phe Leu Glu Ser Lys Gly Tyr Glu Phe Glu Ser Glu Thr 130 135 140

Asp Thr Glu Thr lie Ala Lys Leu lie Lys Tyr Val Phe Asp Asn Arg 145 150 155 160

Glu Thr Glu Asp lie Thr Phe ser Thr Leu val Glu Arg val lie Gin 165 170 175

Gin Leu Glu Gly Ala Phe Ala Leu val Phe Lys Ser lie His Tyr Pro 180 185 190

Gly Glu Ala val Ala Thr Arg Arg Gly ser pro Leu Leu He Gly val 195 200 205

Arg Ser Lys Tyr Lys Leu Ser Thr Glu Gin lie Pro val Leu Tyr Pro 210 215 220

Thr Cys Asn lie Glu Asn val Lys Asn lie Cys Lys Thr Arg Met Lys 225 230 235 240

Arg Leu Asp Ser Ser Thr Cys Leu His Ala val Gly Asp Lys Ala Val 245 250 255

Glu Phe Phe Phe Ala Ser Asp Ala Ser Ala He He Glu His Thr Asn 260 265 270

Arg val lie Phe Leu Glu Asp Asp Asp lie Ala Ala Val Ala Asp Gly 275 280 285

Lys Leu ser He His Arg val Lys Arg ser Ala Thr Asp Asp Pro Ser 290 295 300

Arg Ala lie Gin Thr Leu Gin Met Glu Leu Gin Gin lie Met Lys Gly 305 310 315 - 320

Asn Phe Ser Ala Phe Met Gin Lys Glu lie Phe Glu Gin Pro Glu Ser 325 330 335 val Phe Asn Thr Met Arg Gly Arg Val Asn Phe Glu Thr Asn Thr val 340 345 350

Leu Leu Gly Gly Leu Lys Asp His Leu Lys Glu ile Arg Arg cys Arg 355 360 365

Arg Leu ile val Ile Gly Cys Gly Thr ser Tyr His Ala Ala val Ala 370 375 380

Thr Arg Gin val Leu Glu Glu Leu Thr Glu Leu Pro val Met Val Glu 385 390 395 400

Leu Ala Ser Asp Phe Leu Asp Arg Asn Thr Pro Val Phe Arg Asp Asp 405 410 415

Val cys Phe Phe Ile Ser Gin ser Gly Glu Thr Ala Asp Thr Leu Leu 420 425 430

Ala Leu Arg Tyr Cys Lys Asp Arg Gly Ala Leu Thr Val Gly Ile Thr 435 440 445

Asn Thr Val Gly Ser Ser ile Ser Arg Glu Thr Asp Cys Gly Val His 450 455 460

Ile Asn Ala Gly Pro Glu ile Gly val Ala ser Thr Lys Ala Tyr Thr 465 470 475 480

Ser Gin Phe ile ser Leu val Met Phe Gly Leu Met Met Ser Glu Asp 485 490 495

Arg Ile Ser Leu Gir Asn Arg Arg Gin Glu ile Ile Arg Gly Leu Arg 500 505 510

Ser Leu Pro Glu Leu ile Lys Glu val Leu ser Leu Asp Glu Lys ile 515 520 525

His Asp Leu Ala Leu Glu Leu Tyr Thr Gin Arg ser Leu Leu Val Met 530 535 540

Gly Arg Gly Tyr Asn Tyr Ala Thr Cys Leu Glu Gly Ala Leu Lys ile 545 550 555 560

Lys Glu Ile Thr Tyr Met His Ser Glu Gly Ile Leu Ala Gly Glu Leu 565 570 575

Lys His Gly Pro Leu Ala Leu Val Asp Lys Gin Met Pro val Ile Met 580 585 590

Val Ile Met Lys Asp Pro Cys Phe Ala Lys Cys Gin Asn Ala Leu Gin 595 600 605

Gin val Thr Ala Arg Gin Gly Arg Pro Ile ile Leu Cys Ser Lys Asp 610 615 620

Asp Thr Glu Ser ser Lys Phe Ala Tyr Lys Thr ile Glu Leu Pro His 625 630 635 640

Thr val Asp cys Leu Gin Gly Ile Leu Ser val ile Pro Leu Gin Leu 645 650 655

Leu Ser Phe His Leu Ala val Leu Arg Gly Tyr Asp val Asp Phe Pro 660 665 670

Arg Asn Leu Ala Lys Ser val Thr Val Glu 675 680 <210>11 <211 >1830

<212> DNA <213> Escherichia coli <220> <221 > CDS <222> (1)..(1827) <300> <308> U00096.2 <309> 2005-09-08 <313> (3909862)..(3911691) <400> 11 atg tgt goa att gtt ggc gcg ate geg caa cgt gat gta gca gaa ate 48

Met Cys Gly ile val Gly Ala ile Ala Gin Arg Asp val Ala Glu lie 15 10 15 ett ett gaa ggt tta egt egt ctg gaa tac ege gga tat gac tet gee 96

Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp ser Ala 20 25 30 ggt ctg gee gtt gtt gat gca gaa ggt cat atg acc ege ctg cgt ege 144

Gly Leu Ala val val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 etc ggt aaa gtc cag atg ctg gca cag gca gcg gaa gaa cat cct ctg 192

Leu Gly Lys val Gin Met Leu Ala Gin Ala Ala Glu Glu His Pro Leu 50 55 60 cat ggc ggc act ggt att get cac act ege tgg gcg acc cac ggt gaa 240

His Gly Gly Thr Gly lie Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 cct tea gaa gtg aat gcg cat ccg cat gtt tet gaa cac att gtg gtg 288

Pro ser Glu val Asn Ala His Pro His val Ser Glu His lie val val 85 90 95 gtg cat aac ggc ate ate gaa aac cat gaa ccg ctg cgt gaa gag eta 336

Val His Asn Gly lie lie Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 aaa gcg cgt ggc tat acc ttc gtt tet gaa acc gac acc gaa gtg att 384

Lys Ala Arg Gly Tyr Thr Phe val Ser Glu Thr Asp Thr Glu val lie 115 120 125 gee cat ctg gtg aac tgg gag ctg aaa caa ggc ggg act ctg cgt gag 432

Ala His Leu val Asn Trp Glu Leu Lys Gin Gly Gly Thr Leu Arg Glu 130 135 140 gee gtt ctg cgt get ate ccg cag ctg cgt ggt gcg tac ggt aca gtg 480

Ala val Leu Arg Ala lie Pro Gin Leu Arg Gly Ala Tyr Gly Thr val 145 150 155 160 ate atg gac tee cgt cac ccg gat acc ctg ctg gcg gca cgt tet ggt 528 lie Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg ser Gly 165 170 175 agt ccg ctg gtg att ggc ctg ggg atg ggc gaa aac ttt ate get tet 576

Ser Pro Leu Val lie Gly Leu Gly Met Gly Glu Asn Phe lie Ala Ser 180 185 190 gac cag ctg gcg ctg ttg ccg gtg acc cgt ege ttt ate ttc ett gaa 624

Asp Gin Leu Ala Leu Leu Pro val Thr Arg Arg Phe lie Phe Leu Glu 195 200 205 gag ggc gat att gcg gaa ate act ege cgt teg gta aac ate ttc gat 672

Glu Gly Asp lie Ala Glu lie Thr Arg Arg Ser Val Asn lie Phe Asp 210 215 220 aaa act ggc gcg gaa gta aaa cgt cag gat ate gaa tee aat ctg caa 720

Lys Thr Gly Ala Glu val Lys Arg Gin Asp lie Glu ser Asn Leu Gin 225 230 235 240 tat gac gcg ggc gat aaa ggc att tac cgt cac tac atg cag aaa gag 768

Tyr Asp Ala Gly Asp Lys Gly lie Tyr Arg His Tyr Met Gin Lys Glu 245 250 255 ate tac gaa cag ccg aac gcg ate aaa aac acc ett acc gga ege ate 816 lie Tyr Glu Gin Pro Asn Ala lie Lys Asn Thr Leu Thr Gly Arg lie 260 265 270 age cac ggt cag gtt gat tta age gag ctg gga ccg aac gee gac gaa 864

Ser His Gly Gin Val Asp Leu ser Glu Leu Gly Pro Asn Ala Asp Glu 275 280 285 ctg ctg teg aag gtt gag cat att cag ate etc gee tgt ggt act tet 912

Leu Leu Ser Lys val Glu His lie Gin lie Leu Ala Cys Gly Thr ser 290 295 300 tat aac tee ggt atg gtt tee ege tac tgg ttt gaa teg eta gca gat 960

Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu ser Leu Ala Gly 305 310 315 320 att ccg tgc gac gtc gaa ate gee tet gaa ttc ege tat ege aaa tet 1008 lie Pro Cys Asp val Glu lie Ala ser Glu Phe Arg Tyr Arg Lys ser 325 330 335 gee gtg cgt cgt aac age ctg atg ate acc ttg tea cag tet ggc gaa 1056

Ala vat Arg Arg Asn ser Leu Met lie Thr Leu Ser Gin ser Gly Glu 340 345 350 acc gcg gat acc ctg get gac ctg cgt ctg teg aaa gag ctg ggt tac 1104

Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 ett gat tea ctg gca ate tgt aac gtt ccg ggt tet tet ctg gtg ege 1152

Leu Gly Ser Leu Ala lie Cys Asn val Pro Gly Ser Ser Leu val Arg 370 375 380 gaa tee gat ctg gcg eta atg acc aac gcg ggt aca gaa ate ggc gtg 1200

Glu ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu lie Gly val 385 390 395 400 gca tcc act aaa gca ttc acc act cag tta act gtg ctg ttg atg ctg 1248

Ala Ser Thr Lys Ala Phe Thr Thr Gin Leu Thr val Leu Leu Met Leu 405 410 415 gtg gcg aag ctg tct cgc ctg aaa ggt ctg gat gcc tcc att gaa cat 1296

Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser lie Glu His 420 425 430 gac ate gtg cat got ctg cag gcg ctg ccg age cgt att gag cag atg 1344

Asp lie val His Gly Leu Gin Ala Leu Pro Ser Arg lie Glu Gin Met 435 440 445 ctg tct cag gac aaa cgc att gaa gcg ctg gca gaa gat ttc tct gac 1392

Leu ser Gin Asp Lys Arg lie Glu Ala Leu Ala Glu Asp phe ser Asp 450 455 460 aaa cat cac gcg ctg ttc ctg gac cgt gac gat cag tac cca ate gcg 1440

Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gin Tyr pro lie Ala 465 470 475 480 ctg gaa ggc gca ttg aag ttg aaa gag ate tct tac att cac get gaa 1488

Leu Glu Gly Ala Leu Lys Leu Lys Glu lie Ser Tyr lie His Ala Glu 485 490 495 gcc tac get get ggc gaa ctg aaa cac ggt ccg ctg gcg eta att gat 1536

Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu lie Asp 500 505 510 gcc gat atg ccg gtt att gtt gtt gca ccg aac aac gaa ttg ctg gaa 1584

Ala Asp Met Pro val lie val val Ala Pro Asn Asn Glu Leu Leu Glu 515 520 525 aaa ctg aaa tcc aac att gaa gaa gtt cgc gcg cgt ggc ggt cag ttg 1632

Lys Leu Lys Ser Asn lie Glu Glu val Arg Ala Arg Gly Gly Gin Leu 530 535 540 tat gtc ttc gcc gat cag gat gcg ggt ttt gta agt age gat aac atg 1680

Tyr Val Phe Ala Asp Gin Asp Ala Gly Phe Val Ser Ser Asp Asn Met 545 550 555 560 cac ate ate gag atg ccg cat gtg gaa gag gtg att gca ccg ate ttc 1728

His Ile He Glu Met pro His val Glu Glu val He Ala pro lie Phe 565 570 575 tac acc gtt ccg ctg cag ctg ctg get tac cat gtc gcg ctg ate aaa 1776

Tyr Thr val Pro Leu Gin Leu Leu Ala Tyr His val Ala Leu lie Lys 580 585 590 ggc acc gac gtt gac cag ccg cgt aac ctg gca aaa teg gtt aeg gtt 1824

Gly Thr Asp val Asp Gin Pro Arg Asn Leu Ala Lys ser val Thr val 595 600 605 gag taa 1830

Glu <210> 12 <211 >609

<212> PRT <213> Escherichia coli <400> 12

Met Cys Gly lie Val Gly Ala lie Ala Gin Arg Asp val Ala Glu lie 15 10 15

Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30

Gly Leu Ala val val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45

Leu Gly Lys val Gin Met Leu Ala Gin Ala Ala Glu Glu His Pro Leu 50 55 60

His Gly Gly Thr Gly lie Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80

Pro Ser Glu val Asn Ala His Pro His val ser Glu His lie val val 85 90 95 val His Asn Gly lie lie Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110

Lys Ala Arg Gly Tyr Thr Phe val ser Glu Thr Asp Thr Glu val lie 115 120 125

Ala His Leu val Asn Trp Glu Leu Lys Gin Gly Gly Thr Leu Arg Glu 130 135 140

Ala val Leu Arg Ala He Pro Gin Leu Arg Gly Ala Tyr Gly Thr val 145 150 155 160 lie Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg ser Gly 165 170 175

Ser Pro Leu Val lie Gly Leu Gly Met Gly Glu Asn Phe lie Ala Ser 180 185 190

Asp Gin Leu Ala Leu Leu Pro val Thr Arg Arg Phe lie Phe Leu Glu 195 200 205

Glu Gly Asp lie Ala Glu lie Thr Arg Arg Ser val Asn lie Phe Asp 210 215 220

Lys Thr Gly Ala Glu val Lys Arg Gin Asp lie Glu Ser Asn Leu Gin 225 230 235 240

Tyr Asp Ala Gly Asp Lys Gly lie Tyr Arg His Tyr Met Gin Lys Glu 245 250 255 lie Tyr Glu Gin Pro Asn Ala lie Lys Asn Thr Leu Thr Gly Arg lie 260 265 270

Ser His Gly Gin val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu 275 280 285

Leu Leu Ser Lys val Glu His Ile Gin ile Leu Ala cys Gly Thr Ser 290 295 300

Tyr Asn Ser Gly Met val Ser Arg Tyr Trp phe Glu Ser Leu Ala Gly 305 310 315 320

Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335

Ala val Arg Arg Asn ser Leu Met Ile Thr Leu Ser Gin Ser Gly Glu 340 345 350

Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365

Leu Gly Ser Leu Ala ile Cys Asn val Pro Gly ser ser Leu val Arg 370 375 380

Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu ile Gly val 385 390 395 400

Ala Ser Thr Lys Ala Phe Thr Thr Gin Leu Thr val Leu Leu Met Leu 405 410 415

Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser ile Glu His 420 425 430

Asp ile val His Gly Leu Gin Ala Leu Pro Ser Arg ile Glu Gin Met 435 440 445

Leu Ser Gin Asp Lys Arg ile Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460

Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gin Tyr pro Ile Ala 465 470 475 480

Leu Glu Gly Ala Leu Lys Leu Lys Glu ile ser Tyr ile His Ala Glu 485 490 495

Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp 500 505 510

Ala Asp Met pro val ile val val Ala pro Asn Asn Glu Leu Leu Glu 515 520 525

Lys Leu Lys Ser Asn ile Glu Glu val Arg Ala Arg Gly Gly Gin Leu 530 535 540

Tyr val Phe Ala Asp Gin Asp Ala Gly Phe val Ser ser Asp Asn Met 545 550 555 560

His ile ile Glu Met Pro His val Glu Glu val ile Ala Pro Ile Phe 565 570 575

Tyr Thr val Pro Leu Gin Leu Leu Ala Tyr His val Ala Leu ile Lys 580 585 590

Gly Thr Asp Val Asp Gin Pro Arg Asn Leu Ala Lys Ser val Thr-val 595 600 605

Glu

<210> 13 <211 >1830 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence encoding an Escherichia coli protein having the activity of a GFAT <400> 13 atgtgcggaa ttgttggtgc tatcgcccaa agagacgttg ctgagatttt gttagagggt 60 ctgcgaaggc tagagtatag aggatatgac tccgctggtc tggctgtcgt tgatgctgag 120 ggtcatatga caaggctaag aaggttagga aaggttcaga tgcttgctca ggcagctgag 180 gaacatccat tgcatggagg tactggtatt gcacatacca ggtgggctac tcatggggag 240 ccatcagaag ttaatgctca tccacatgtg agtgagcata tcgttgtagt tcacaatggg 300 ataattgaaa accacgaacc attgagggaa gagttaaagg caagaggata tacttttgtg 360 agtgagactg acactgaggt tattgcacat ttagtgaact gggaactcaa acaggggggc 420 acattgcgtg aggctgtgtt aagagctatt cctcaactta gaggtgcata cggtactgtt 480 attatggatt caagacaccc agatactctc cttgcagcta gatcaggtag tcccttggtc 540 ataggacttg gaatgggtga aaattttatc gctagcgacc aattggcctt attgccagtt 600 acaagacgat ttattttcct tgaagagggc gatattgctg agattactag aaggtctgtg 660 aacatctttg ataagactgg cgctgaggtt aaacgtcagg atatcgagtc taaccttcaa 720 tacgatgctg gtgataaagg aatttacagg cattatatgc aaaaggaaat ttatgaacaa 780 ccaaatgcta tcaaaaacac acttactggc cgtatttctc atggacaggt cgatttaagc 840 gagcttggtc ctaatgcaga cgaactgcta tcaaaagttg agcacataca gatactggca 900 tgcggaacta gttataattc aggaatggtc tctagatact ggttcgaaag cttggcaggt 960 ataccttgtg atgtagagat cgcttctgag tttaggtata gaaagtctgc tgtgcgtaga 1020 aattcattaa tgattacatt atctcaatcc ggagaaacag cagatacact ggctggattg 1080 aggctttcta aggaactcgg atatctgggt tcacttgcta tttgtaatgt accaggttcc 1140 tcattggttc gtgaatcaga tctagcactt atgacaaatg caggaactga aataggtgtg 1200 gcaagtacca aggctttcac aacccaactg accgtacttt taatgttggt agcaaaactc 1260 agtcgattaa aggggctaga tgcatctatc gaacatgata ttgttcacgg gcttcaagct 1320 ctcccttcaa gaattgaaca aatgctttca caagataaga gaatagaggc attggctgaa 1380 gatttttccg acaaacatca cgcattgttt cttggacgtg gcgatcaata tccaattgca 1440 ttggaaggag ctttgaagtt gaaagaaata agttacattc acgcagaagc atatgcagct 1500 ggagaactca agcatggtcc tttggcactc atcgacgctg acatgcccgt gatcgtagtg 1560 gctcctaata acgaactgct cgaaaagctt aaatcaaata tcgaagaggt tcgagctaga 1620 ggaggtcagc tttacgtttt cgctgaacaa gatgctggat tcgtgtcaag cgataatatg 1680 catataattg aaatgcctca cgttgaagaa gtgattgcac ctatatttta tacagtccca 1740 ttgcaacttc tagcttacca tgttgcactt attaaaggaa ctgatgttga tcagcctaga 1800 aacctagcaa aatctgtaac agtcgaataa 1830 <210> 14 <211 >48 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 14 tcgacaggcc tggatcctta attaaactag tctcgaggag ctcggtac 48 <210> 15 <211 >40 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 15 cgagctcctc gagactagtt taattaagga tccaggcctg 40

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description • IJS4141973A Γ00191 [00331 • US4S01539A10821 . EF0694616Å ?0022[

• US4782046A £00231 .EMSSI . US20030175S02AF00241 Γ00241 • WOO3054163A 10324: 10024· • WQ05012529A Γ00251[00251 [00251 ΓΟΟββΊ • WO9835047A [00301 [0078] • VVO0011192A [00311 :08311 :0053: • US4897349A Γ00321 • EP0144019A [0033: . US20030134393A Γ00341 • US20030235893A [0046] [0046} £00461 [0046] [0046] £00461 [0046] [0046] [0046] 10040].1004¾ [0046] [0046] I0046J £00461 [0046] . IJS64553Q4B Γ00461 • EP120516A [0064] . US5565347A 100641 Γ02041 • WQ9506128A [0065] • EP0513849A [00651 • EP0465875A [6065] • EP0292435A 100651 • US5019498A Γ00751 [0075] • USS444878B ]0078[ . WO04QQ3175A Γ01071 • WO0173087A [0136] . WO9307279A Γ01361 • US6607745B ]0154[ • EP0e090053A [0265] • US60725530B [0265]

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Claims (10)

A genetically modified plant cell having a nucleic acid molecule encoding a hyaluronan synthase linked to regulatory sequences ensuring transcription in plant cells stably integrated into its genome, wherein the plant cell further has a number of foreign nucleic acid molecules stably integrated into its genome, wherein first foreign nucleic acid molecule encodes a protein with the activity of a glutamine: fructose-6-phosphatamidotransferase-2 (GFAT-2) derived from animals, or a glutamine: fructose-6-phosphatamidotransferase (GFAT) derived from bacteria; and another foreign nucleic acid molecule encodes a protein with the activity of a UDP-glucose dehydrogenase (UDP-Glc-DH) and wherein the genetically modified plant cell has an increased activity of a protein with the activity of a glutamine: fructose-6-phosphate amidotransferase (GFAT) and an increased activity of a protein with the activity of a UDP-glucose dehydrogenase (UDP-Glc-DH) compared to corresponding non-genetically modified ildtypeplanteceller. The genetically modified plant cell of claim 1, which synthesizes an increased amount of hyaluronan compared to plant cells having the activity of a hyaluronan synthase and no increased activity of a glutamine: fructose-6-phosphatamidotransferase (GFAT) and no increased activity of a UDP-glucose dehydrogenase. . A plant comprising genetically modified plant cells according to any one of claims 1 or 2. Propagating material of a plant according to claim 3, comprising genetically modified plant cells according to any one of claims 1 or 2. A harvestable plant portion of a plant according to claim 3, comprising genetically modified plant cells according to any one of claims 1 or 2. A method of producing a plant that synthesizes hyaluronan comprising: a) gene modification of a plant cell, the gene modification comprising steps i) to iii) below: i) introducing a foreign nucleic acid molecule encoding a hyaluronan synthase which is linked to regulatory sequences ensuring transcription in plant cells, in the plant cell; ii) introducing a foreign nucleic acid molecule into the plant cell, which foreign nucleic acid molecule comprises a coding sequence for a protein having the activity of a glutamine: fructose-6-phosphatamidotransferase-2 (GFAT-2) derived from animals, or a glutamine: fructose-6-phosphatamidotransferase (GFAT) derived from bacteria; iii) introducing a foreign nucleic acid molecule into the plant cell, which foreign nucleic acid molecule comprises a coding sequence for a protein having the activity of a glutamine: fructose-6-phosphate-UDP-glucose dehydrogenase; wherein steps i) to iii) can be performed in any order individually, or any combination of steps i) to iii) can be performed simultaneously; b) regenerating a plant from plant cells of step a). A method of producing hyaluronan comprising the step of extracting hyaluronan from genetically modified plant cells according to any one of claims 1 or 2, from plants of claim 3, from propagating material of claim 4 or from harvestable plant parts of claim 5. Use of a genetically modified plant cell according to any one of claims 1 or 2, plant according to claim 3, propagating material according to claim 4 or harvestable plant parts according to claim 5 for the production of hyaluronan. A method of preparing a composition comprising hyaluronan using genetically modified plant cells according to any one of claims 1 or 2, plants according to claim 3, propagating material according to claim 4 or harvestable plant parts according to claim 5. Use of genetically modified plant cells according to any one of claims 1 or 2, of plants according to claim 3, of propagating material according to claim 4 or of harvestable plant parts according to claim 5 for the preparation of a composition.