BRPI0616871A2 - improved methods and means for producing hyaluronan - Google Patents

Abstract

IMPROVED METHODS AND MEANS TO PRODUCE HIALURONAN. The present invention relates to plant cells and plants that synthesize an increase in the amount of hyaluronan, and to methods for preparing these plants, and also to methods for preparing hyaluronan with the aid of those plant or plant cells. Here, plant cells or plants genetically modified according to the invention show hyaluronan synthase activity and additionally an increase in glutamine activity: fructose 6-phosphate amidotransferase (GFAT) and an increase in UDP glucose dehydrogenase activity (UDP-Glc-DH ) compared to wild-type plant cells or wild-type plants. The present invention furthermore relates to the use of plants which show an increase in hyaluronan synthesis for the preparation of hyaluronan and to foodstuffs or animal feed containing hyaluronan.

Description

Descriptive Report of the Invention Patent for "PERFECT METHODS AND MEANS OF PRODUCING HYALURONAN".

The present invention relates to plant cells and plants which synthesize a high amount of hyaluronan, and methods of preparing such plants, and also methods of preparing hyaluronan as an aid to such plant cells or plants. Here, genetically modified plant or plant cells according to the invention exhibit hyaluronan synthase activity and additionally an increase in deglutamine activity: fructose 6-phosphate amidotransferase (GFAT) and an increase in UDP glucose dehydrogenase activity ( UDP-GIc-DH) compared to wild type vegetable cells or wild type plant cells. The present invention furthermore relates to the use of plants which exhibit increased synthesis of hyaluronan for preparation of hyaluronan and food or feed for hyaluronan containing feeds.

Hyaluronan is a naturally occurring branched-branched mucopolysaccharide (glycosaminoglycan) that is constructed of altered molecules of glucuronic acid and N-acetyl glycosamine. The hyaluronan basic building block consists of disaccharide glucuronic acid beta-1,3-N-acetyl glycosamine. In hyaluronan, these repeating units are linked together via beta-1,4 bonds. In pharmacy, use is often made of the term hyaluronic acid. Since hyaluronan is in most cases present as a polyanion and not as free acid, therefore, the term hyaluronan is preferably used, but each term should be understood to encompass both molecular forms.

Hyaluronan exhibits remarkable physicochemical properties, such as, for example, polyelectrolyte properties, viscoelastic properties, a high water-binding ability, gel-forming properties, which, in addition to the additional properties of hyaluronan, are described. in a review article by Lapcik et al. (1998, Chemical Reviews98 (8), 2663-2684).

Hyaluronan is a component of extracellular connective tissue efflected from vertebrates. In humans, hyaluronic acid is synthesized by the cellular 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 cartilage tissue, skin and vitreous body of the eye (Bernhard Gebauer, 1998, Inaugural Dissertation, Virchow-KlinikumMedizinische Fakultät Charité der Humboidt Universitàt zu Berlirr, Fraser et al., 1997, Journal of Internal Medicine 242, 27-33).

Recently, hyaluronan has also been found in non-vertebrate animal organisms (mollusks) (Volpi and Maccari, 2003, Biochimie85, 619-625). In addition, some pathogenic gram-positive bacteria (Streptococcus group A and C) and gram-negative bacteria (PasteurelIa) synthesize hyaluronan as exopolysaccharides that protect these bacteria from attack by their host immune system, since hyaluro-nan is a non-immunogenic substance.

Viruses that infect single cell green algae of the genus Chlo-rella, some of which are present as endosymbionts in Parameeium species, give single cell green algae the ability to synthesize hyaluronan following virus infection (Graves et al., 1999, Virology257 , 15-23). However, the ability to synthesize hyaluronan is not an aspect that characterizes the algae in question. The capacity of hialuronan synthesizing algae is mediated by infection with a virus whose genome has a coding sequence for hialuronan synthase (DeAngelis, 1997, Science 278, 1800-1803). In addition, the viral genome contains coding sequences for a 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 a substrate by hyaluro nan synthase. GFAT converts fructose 6-phosphate and glutamine to glycosamine 6-phosphate which is an important metabolite in the metabolic pathway of dehialuronan synthesis in, for example, bacteria. Both algal genes encode active proteins that, like virus hyaluronan synthase, are transcribed simultaneously in the premature phase of viral infection. <DeAngelis et al., 1997, Science 278, 1S00-1S03, Graves et al., 1999, Virology 257, 15- 23). The activity of a protein that exhibits glutamine-fructose 6-phosphate amidotransferase (GFAT) activity could be detected neither in cell extracts uninfected by a virus nor in cells infected with viruses (Landstein et al., 1998, Virology 250, 388-396 ). Therefore, the role of UDP-GIc-DH and GFAT expression in virus-infected Chlorella cells for hyaluronan synthesis, and whether they are required for hyaluronan synthesis, is not known.

Naturally occurring plants themselves do not have any nucleic acids in their genome that encodes proteins that catalyze the synthesis of hyaluronan - and - although a large number of plant carbohydrates have been described and characterized, until now it has not been possible. detect hyaluronan or hyaluronan-related molecules in naturally occurring uninfected vegetables (Graves et al., 1999, Virology257,15-23).

Catalysis of hyaluronan synthesis is effected by a single membrane-integrated or membrane-associated enzyme, hyaluronan feel. Hyaluronan synthases that have been studied so far can be classified into two groups: Class I hyaluronan synthases and Class II hyaluronan synthases (DeAngelis, 1999, CMLS, Cellular and Molecular Life Sciences 56, 670-682) .

Vertebrate hyaluronan synthases are further distinguished by the identified isoenzymes. The different isoenzymes are referred to in the order of their identification using Arabic numerals (eg hsHASI, hsHAS2, hsHAS3).

The mechanism of transfer of synthesized hyaluronan molecules across the cytoplasm membrane to the surrounding cell environment has not yet been fully understood. It has previously been hypothesized that transport through the cellular membrane was effected by hyaluronan synthase itself. However, more recent results indicate that the transport of hyaluronan molecules across the cytoplasm occurs by energy-dependent transport via protein-transport responsible for this transport. 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 bacterial strains (Ouskova et al., 2004, Glycobiology 14 (10), 931-938). In human fibroblasts, it has been shown, 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 Pharmaco-logy 68,1401-1410). In this amount, if not at all, it is not known that transport proteins capable of carrying hyaluronan are present in vegetables.

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

In the medical field, hyaluronan-containing products are currently used for intra-articular treatment of arthrosis and ophthalmic agents used for eye surgery. Hialuronan is also used to treat joint disorders in racehorses. Furthermore, hyaluronic acid is a component of some rhinological agents which, for example, in the form of eye drops and nasalia, serves to moisten dry mucous membranes. I understand hyialu-ronan injection solutions are used as analgesics and antirheumatics. Plasters comprising hyaluronan or hyaluronan derivatives are employed in wound healing. Likewise, hyaluronan gel implants are used to correct skin deformities in plastic surgery.

For pharmacological applications, preference is given to the use of hyaluronan having a high molecular weight.

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

In cosmetic products, in particular in skin creams and lotions, hyaluronan is often used as a moisturizer because of its high binding capacity in water.

In addition, hyaluronan-containing preparations are sold as nutraceuticals (food supplements) which can also be used on animals (eg dogs, horses) for prophylaxis and relief of arthrosis.

Hialuronan used for commercial purposes is currently isolated from animal kept (rooster ridges) or fermentatively prepared using bacterial cultures.

US 4,141,973 describes a process of isolating hyaluro-nan from rooster ridges or alternatively umbilical cords. In addition to hyaluronan, animal tissues (eg rooster ridges, umbilical cords) also additionally contain hyaluronan related mucopolysaccharides, such as chondroitine sulfate, dermatan sulfate, dequeratan sulfate, heparan sulfate and heparin. In addition, animaiscal organisms contain proteins (hyaladerins) that specifically bind to hyaluronan and which are required for the most diverse functions in the body, such as, for example, the degradation of hyaluronan in the liver, the function of hyaluronan as the main cell migration structure, endocytosis regulation, hyaluronan cell surface uptake or hyaluronan nets formation (Turley, 1991, Adv. Drug Delivery Rev. 7, 257 ff .; Laurent eFraser, 1992, FASEB J. 6, 183 ff .; Stamenkovic and Aruffo, 1993, MethodsEnzymoL 245,195 (Knudson and Knudson, 1993, FASEB 7, 1233 ff.).

Streptococcus strains used for hyaluronan bacterial production are exclusively pathogenic bacteria. During cultivation, too, these bacteria produce exotoxins (pyrogenic) and hemolysins (streptolysin, in particular alpha and beta-hemolysin) (Kilian, M .: Streptoeoeus and Enterococcus. In: Medical Microbiology. Greenwood, D .; Slack1RCA; Peutherer , JF (Eds.) Chapter 16. Churchill Livingstone, Edinburgh, UK: pp. 174-188, 2002, ISBN 0443070776) which are released into the culture medium. This makes purification and isolation of 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.

US 4,801,539 describes the preparation of hyalurorian by defermenting a strain of mutagenized bacteria (Streptococcus zoo-edemicus). The mutagenized bacterial strain used no longer synthesizes beta-hemolysin. The yield obtained was 3.6 g hyaluronan per liter deculture.

EP 0694616 describes a method of cultivating Streptococcus zo-oedemicus or Streptococcus equi, wherein, under the in-grown culture conditions, no streptolysin, but high amounts of synthesized hyaluronansion. The yield obtained was 3.5 g hyaluronan per liter deculture.

Streptococcus strains release hyaluronidase enzyme into the culture medium during cultivation as a consequence of the fact that in this production system, too, the molecular weight is reduced during purification. The use of hyaluronidase-negative Streptococcus strains or methods for hyaluronan production in which hyaluronidase production during cultivation is inhibited is described in US 4,782,046. The yield obtained was 2.5 g hyaluronan per liter of culture, and the average maximum molecular weight obtained was 3.8 χ 106 Da, under a molecular weight distribution of 2.4 χ 106 to 4.0 χ 106 .

US 20030175902 and WO 03 054163 describe the preparation of hyaluronan with the aid of heterologous expression of a Streptococcus equisimilis hyaluronan synthase in Bacillus subtilis. To obtain sufficient amounts of hyaluronan, in addition to the heterologous expression of a hyaluronan synthase, simultaneous expression of a UDP-glucose dehydrogenase in Bacillus cells is also required. US 20030175902 and WO03 054163 do not express the absolute amount of hyaluronan obtained in production with the aid of Bacillus subtilis. The mean maximum molecular weight obtained was approximately 4.2 χ 106. However, this average molecular weight was only obtained for the recombinant Bacillus strain in which a gene co-complication for the Streptococcus equi-similis hyaluronan synthase gene and gene coding for Bacillussubtilis UDP-glucose dehydrogenase were integrated into the Bacillus subtilis genome under the control of the amyQ dopromotor, where at the same time the endogenous cxpY gene Bacillussubtilis (which encodes a cytochrome P450 oxidase) was inactivated.

WO 05 012529 describes a preparation of transgenic tobacco plants that have been transformed using nucleic acid molecules encoding Chlorella infecting virus hyaluronan synthases. In WO 05012529, on the other hand, nucleic acid sequences have been used which co-difficult hyaluronan synthase of the Chlorella virus strain CVHI1 and, on the other hand, the Chlorella virus strain CVKA1 for transformation of seedlings. -spleen. Hyaluronan synthesis could only be demonstrated for a plant transformed with a nucleic acid sequence encoding a hyaluronan synthase isolated from the CVKA1 strain of Chlorella virus. For detabaco plants transformed with a nucleic acid sequence encoding a hyaluronan synthase isolated from the CVHI1 strain of the Chlorella virus, it was not possible to detect hyaluronan synthesis in the corresponding transgenic plants. The amount of hyaluronan synthesized by only the transgenic tobacco plant producing hialuronan in WO 05 012529 is established to be approximately 4.2 pg of hyaluronan per ml measured volume, taking into account the description of the experiment. in question, it corresponds to approximately 12 pg of hyaluro-nan produced per gram of new weight of plant material.

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

In plant cells, UDP-glucuronic acid functions as a me-tabolite for one of a plurality of possible pathways for ascorbic acid synthesis {Lorence et al., 2004, Plant Physioi. 134, 1200-1205) and as a central metabolite for the synthesis of cell wall components pectin and hemicellulose that are synthesized in the plant cell endoplasmic reticulum (Reiter1 1998, Plant Physiol. Biochem. 36 (1), 167-176). The most important and most frequently occurring pectin monomer is D-galacturonic acid (2004, HW Heldt in 'Plant Biochemistiy', 3rd Edition, Academic Press, ISBN 0120883910) which is synthesized using UDP-glycuronic acid. others synthesize UDP-xylose, UDP-arabinose, UDP-galacturonic acid and UDP-apiose, metabolites for hemicellulose and pectin synthesis using UDP-glucuronic acid (Seitz et al. 2000, Plant Journal, 21 (6), 537- In plant cells, UDP-glucuronic acid can be synthesized via hexose phosphate metabolism which comprises, inter alia, the conversion of UDP-glucose to UDP-glycuronic acid by UDP-GIc-DH or the myo-inositol oxidative metabolism which comprises conversion. of glycuronate 1-phosphate to UDP-glucuronic acid by glycoronate 1-uridylyl phosphate transferase. Both metabolic pathways for synthesizing glucuronic acid appear to exist independently of each other and alternatively. at different tissue / developmental stages of Arabidopsis plants (Seitz et al., 2000, Plant Journal 21 (6), 537-546). The respective contribution of the two mentioned metabolic pathways (meta-bolus hexose phosphate or oxidative myo-inositol) towards the synthesis of UDP-glucuronic acid has not yet been elucidated (Kàrkönen, 2005, Plant Bi-osystems 139 (1), 46-49).

The enzyme UDP-GIc-DH catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. Samac et al (2004, Applied Biochemistry and Bio-technology 113-116, Human Press, EditorAshok Mulehandani, 1167-1182) describe the tissue-specific overexpression of a UDP-GIc-DH dislocates in Alfalfa phloem cells with the aim of enhancing pectin content in strains of these plants. UDP-GIc-DH activity compared to corresponding wild type plants was increased by more than 200%; however, the amount of pectin produced by the corresponding plants was less than the amount of pectin produced by the corresponding wild type plants. The amount of xylose and ram-nose monomers in the cell wall fraction of the transgenic plants in question was increased, as the amount of mannose monomers in the cell wall fraction was reduced.

Constitutive overexpression of a UDP-GIc-DH in Arabidosis plants resulted in aberrant growth of the plants in question compared with the corresponding wild-type plants and a non-phenotype. The cell wall fraction of the corresponding plants showed an increase in the amount of mannose and galactose and a reduced amount of xylose, arabinose, and uronic acids compared with the corresponding wild type plants (Roman, 2004, "Studies on the Role ofUDP-GIe- DH in Polysaceharide Biosynthesis ", PhD thesis, Aeta UniversitatisUpsaliensis, ISBN 91-554-6088-7, ISSN 0282-7476). Thus, these results at least partially contradict the results of Samac and others (2004, Applied Biochemistry and Bioteehnology 113-116, HumanaPress, Editor Ashok Mulehandani, 1167-1182) who detected a reduced amount of mannose and an amount xylose in the cell wall fraction of corresponding transgenic plants.

For the synthesis of UDP-N-acetylglycosamine in plant cells, WO 98 35047 describes a metabolic pathway in which glycosamine is converted by several successive enzymatically catalyzed reaction steps with formation of the metabolites N-acetyl glycosamine, N-acetyl glycosamine 6. phosphate, N-acetyl glycosamine 1-phosphate to UDP-N-acetyl glycosamine. An alternative metabolic pathway comprises the reaction of fructose 6-phosphate eglutamine providing glycosamine 6-phosphate which is subsequently converted by several successive enzymatically catalyzed reaction steps with formation of the metabolites glycosamine 1-phosphate and N-acetyl glycosamine1-phosphate to UDP-N -acetylglycosamine. The conversion of fructose 6-phosphate eglutamine to glycosamine 6-phosphate is catalyzed by a protein that exhibits glutaminarfructose 6-phosphate amidotransferase (GFAT) activity (Mayer et al., 1968, Plant Physiol. 43, 1097-1107).

WO 00 11192 describes the endosperm-specific overexpression of a maize nucleic acid molecule encoding a protein that exhibits the enzymatic activity of a GFAT in milhotransgenic plants for the purpose of synthesizing a cationic starch in plants presenting molecules 2. -amino-anhydroglucose. The metabolic pathway described which, according to the description of WO 00 11192, should result in 2-amino anhydroglucose which is incorporated into starch comprises, among others, the incorporation of UDP-glycosamine by starch synthase and / or glycogen. in starch. It is established that high amounts of UDP-glucosamine can be detected in endosperm meal of the transgenic corn plant in question by overexpressing a nucleic acid molecule encoding a protein that exhibits the (enzymatic) activity of a GFAT transactionally fused to a plastid signal peptide. Hate When the protein exhibiting the (enzymatic) activity of umaGFAT was expressed without signal peptide, a high amount of glycosamine 1-phosphate could be detected in the corresponding endosperm maize tissue flours. It was not possible to detect cationic starch in transgenic plants.

The production of hyaluronan by fermentation of bacterial strains is associated with high costs since bacteria must be fermented in sealed sterile containers under controlled expensive culture conditions (see, for example, US 4,897,349). In addition, the amount of hyaluronan that can be produced by fermentation of bacterial strains is limited by the production facilities present in each case. Here it must also be taken into account that fermenters, as a consequence of physical laws, cannot be formed by excessively large crop volumes. Particular mention may be made in this report of the homogeneous mixing of externally fed substances (eg essential nutrient sources of bacteria, pH-regulating reagents, oxygen) with the culture medium required for efficient production, which in large fermenters can only be guaranteed. with great technical expense, if at all.

Purification of hyaluronan from animal organisms is complicated due to the presence in animal tissues of other mucopolysaccharides and proteins that specifically bind to hyaluronan. In patients, the use of medicinal preparations containing hyaluronan contaminated with animal proteins may result in undesirable body immune reactions (US 4,141,973), in particular if the patient is allergic to animal proteins (eg, clear from chicken's egg). In addition, the amounts (yields) of hyaluronan obtainable from animal tissues of satisfactory quality and purity are low (rooster crest: 0.079% w / w, EP 0144019, US 4,782,046), which require processing of large quantities. of animal tissues. An additional problem in the isolation of hyaluronan from animal tissues is that the hyaluronan pesomolecular during purification is reduced since animal tissues also contain a degrading enzyme of hyaluronan (hyaluroni-dase).

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

Some of the disadvantages of producing hyaluronan through bacterial defermentation or isolation of animal tissues can be prevented by producing hyaluronan using transgenic plants, however the currently obtained amounts of hyalurane that can be produced using transgenic plants would require a relatively large area under cultivation to produce quantities. relatively large amounts of hia-luronan. In addition, isolation or purification of hyaluronan from plants with a lower hyaluronan content is considerably more complicated and expensive than isolation or purification of plants which have a higher hyaluronan content.

Although hyaluronan has unusual properties, that is, due to its scarcity and high price, rarely, if at all, used for industrial applications.

Accordingly, it is an object of the present invention to provide means and methods which enable the provision of hyaluronan in sufficient quantities and quality and which make it possible to provide hyaluro-nan for both industrial and food applications.

This objective is achieved by the embodiments described in the general lines in the claims.

Thus, the present invention relates to genetically modified plant cells or genetically modified plants which present a nucleic acid molecule encoding a hyaluronan which is syntactically integrated into their genome, wherein such plant cells additionally exhibit an increase in the activity of a Tna-protein that exhibits the (enzymatic) activity of a glutamine.fructose 6-phosphate amidotransferase (GFAT) and an increase in the activity of a protein that exhibits (enzymatic) activity of a UDP-glucose dehydrogenase (UDP- GIc-DH), compared with the non-genetically modified wild-type plant cells or corresponding non-genetically modified wild-type plants.

Here, the genetic modification of genetically modified plant cells according to the invention or genetically modified plants according to the invention may be any genetic modification resulting in stable integration of a nucleic acid molecule that encodes a hyaluronan synthase into a cell. increasing the activity of a protein that exhibits the activity (enzymatic) of a GFAT and increasing the activity of a protein that exhibits the activity (enzymatic) of a UDP-GIc-DH in genetically modified plant cells. genetically modified or genetically modified plants compared to non-genetically modified wild-type plant cells or corresponding non-genetically modified wild-type plants.

In the context of the present invention, the term "wild-type plant cell" is to be understood to mean plant cells which have served as a starting material for the preparation of genetically modified plant cells according to the invention, i.e. their genetic formation, except for genetic modifications. introduced and resulting in a stable integration of a nucleic acid molecule encoding a hyaluronan synthase and increasing the activity of a protein having GFAT activity and increasing the activity of a protein which represents the activity of a UDP-GIc-DH, which corresponds to that of a genetically modified plant cell according to the invention.

In the context of the present invention, the term "wild type plant" shall be understood to mean plants which have served as starting materials for the preparation of genetically modified plants according to the invention, i.e. their genetic information, except for the genetic modifications introduced and resulting in the stable integration of a nucleic acid molecule encoding a hyaluronan synthase and increasing the activity of a protein that exhibits the activity of aGFAT and increasing the activity of a protein that exhibits a UDP-GIc-DH which corresponds to that of a genetically modified plant according to the invention.

In the context of the present invention, the term "corresponding" means that when a plurality of objectives are compared, the objectives in question that are compared with each other have been maintained under the same conditions of being. In the context of the present invention, the term "corresponding" in the context of wild-type plant cells or wild-type plants is understood to mean that plant cells or plants compared with each other have been grown under the same cultivation conditions and that they are the same age (culture). In the context of the present invention, the term "hialüronan synthase" (EC 2.4.1.212) is to be understood to mean a protein which synthesizes hialüronan from UDP-glucuronic acid substrates (UDP) -Glyc) and N-acetyl glycosamine (UDP-GIcNAc). The synthesis of hialüronan is catalyzed according to the reaction schemes below:

nUDP-GIcA + nUDP-GIcNAc beta-1,4- [GlcA-beta-1,3-GlcNAc] n + 2 nUDPMNucleic acid molecules and corresponding protein sequences encoding hialüronan synthases are described, among others, in relation to following organisms: rabbit (Oryctolagus cuniculus) ocHas2 (EMBL AB055978.1, US 20030235893), ocHas3 (EMBL AB055979.1, US20030235893); baboon (Papio anubis) paHasl (EMBL AY463695.1); frog (.Xenopus laevis) x1Has1 (EMBL M22249.1, US 20030235893), x1Has2 (DG42) (EMBL AF168465.1), x1Has3 (EMBL AY302252.1); human (Homo sapiens) hsHASI (EMBL D84424.1, US 20030235893), hsHAS2 (EMBL U54804.1, US 20030235893), hsHAS3 (EMBL AF232772.1, US 20030235893); dongo (Mus musculus), mmHasl (EMBL D82964.1, US 20030235893), m-mHAS2 (EMBL U52524.2, US 20030235893), mmHas3 (EMBL U86408.2, US 20030235893); cattle (Bos taurus) btHas2 (EMBL AJ004951.1, US20030235893); chicken (Gallus gallus) ggHas2 (EMBL AFI 06940: 1, US20030235893); rat (Rattus norvegieus) rnHas 1 (EMBL AB097568.1, Itano et al., 2004, J. Biol. Chem. 279 (18) 18679-18678), rnHas2 (EMBL AF008201.1); rnHas 3 (NCBI NM_172319.1, Itano et al., 2004, J. Biol. Chem. 279 (18) 18679-18678), horse (Equus caballus) ecHAS2 (EMBL AY056582.1, G1: 23428486), pig (Sus scrofa) sscHAS2 (NCBI NM_214053.1, Gl : 47522921), sscHas 3 (EMBLAB159675), zebrafish (Danio rerio) brHasl (EMBLAY437407), brHas2 (EMBL AF190742.1) brHas3 (EMBL AF190743.1); Pasteurella multocida pmHas (EMBL AF036004.2); Streptococcus pyogenes s-pHas (EMBL, L20853.1, L21187.1, US 6,455,304, US 20030235893); Strep-tococcus equis seHas (EMBL AF347022.1, AY173078.1), Streptocoeeus u-beris suHasA (EMBL AJ242946.2, US 20030235893), Streptocoeeus equisi-milis seHas (EMBL AF023876.1, US 20030235893); Sulfolobus solfatarieusssHAS (US 20030235893), Sulfolobus tokodaii stHas (AP000988.1), Para-mecium bursaria Chlorella Virus 1, cvHAS (EMBL U42580.3, PB42580, US20030235893).

In the context of the present invention, the term "UDP-glucose dehydrogenase (UDP-GIc-DH)" (EC 1.1.1.22) shall be understood to mean 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:

UDP-GIc + 2 NAD + -> UDP-GIcA + 2 NADH

In the context of the present invention, the term "glutamine: fructose 6-phosphate amidotransferase (GFAT)" (EC 2.6.1.16), in the technical literature also referred to as glycosamine synthase, is to be understood as meaning a protein which synthesizes from of the starting materials glutamine and fructose 6-phosphate (Fruc-6-Ρ), glycosamine 6-phosphate (GlcN-6-Ρ). This catalysis proceeds according to the following reaction scheme:

Glutamine + Fruc-6-Ρ - »· GlcN-6-Ρ + Glutamate

In particular in animal organisms, it is possible to demonstrate two different protein isoforms that exhibit the activity (enzymatic) of a GFAT (referred to as GFAT-1 and GFAT-2, respectively, in the literature). Hu et al. (2004), J. BioL Chem. 279 (29), 29988-29993 describe differences in the respective proteins of the mouse: besides the differences in the expression of the specific tissue proteins in question that presents the (enzymatic) activity of a glutamine: fructose 6-phosphate starch -transferase 1 (GFAT-1) and a glutamine: fructose 6-phosphate amidotransferase2 (GFAT-2), it was shown that both isoforms are regulated by phosphorylation by a cAMP-dependent protein kinase. The activity of a protein that exhibits the (enzymatic) activity of umaGFAT-1 is inhibited by phosphorylation of a conserved serine residue (mouse serine 205 in GFAT-1, GenBank Ac. No. AF334736.1) of the amino acid sequence in question, since the activity of a protein that exhibits the activity of a GFAT-2 is increased by dephosphorylation of a conserved serine residue (serine 202 in GFAT-2 docamundongo, GenBankHo. de Ac .: NM_013529) of the amino acid sequence in question. Both GFAT-1 activity proteins and GFAT-2 activity proteins are inhibited in a concentration-dependent manner by UDP-N-acetylglycosamine, however with respect to a protein that exhibits a GFAT-1 activity. 2, UDP-N-acetylglycosamine inhibition is lower (maximal reduction of activity by UDP-N-acetylglycosamine approximately 15%) compared to a protein that exhibits GFAT-1 activity (maximal reduction of activity by UDP-N- acetylglycosamine approximately 51% or 80%). There are indications that inhibition of a protein that exhibits GFAT-1 activity in animal organisms is based on the fact that high concentrations of UDP-N-acetylglycosamine has an O-glucose-N-acetylglycosamine glycosylation of proteins in question. Whether regulation of protein activity by O-glycosylation also occurs in plant cells is not yet fully understood (Huber and Hardin, 2004, Current Opinion in Plant Biotechnology 7, 318-322).

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 that exhibits the activity of a GFAT and whose activity is inhibited by phosphorylation by a protein kinase. cAMP dependent.

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 that exhibits the activity of a GFAT and is activated by phosphorylation by a protein dependent kinase. of cAMP.

In the context of the present invention, the term "glutamine: fructose6-phosphate amidotransferase (GFAT)" is used as a comprehensive term that includes all proteins exhibiting the activity of a GFAT. Accordingly, it also includes proteins referred to in the literature "glutamine: fructose 6" phosphate amidotransferase-1 (GFAT-1) "or as" glutamine: fructose 6-phosphate amidotransferase-2 (GFAT-2) ", but not limited to these.

In the context of the present invention, the term "increased activity of a protein that exhibits (enzymatic) activity of a GFAT1" means an increase in the expression of endogenous genes encoding proteins that exhibit activity of a GFAT and / or a an increase in the amount of transcripts encoding GFAT activity proteins and / or an increase in the amount of GFAT activity activity in cells and / or an increase in enzymatic activity of GFAT activity proteins. a GFAT in the cells.

In the context of the present invention, the term "increase in the activity of a protein that exhibits (enzymatic) activity of a UDP-Glc-DH" means an increase in the expression of endogenous genes encoding proteins that exhibit the activity of a UDP-Glc-DH. UDP-GIc-DH and / or an increase in the amount of protein-encoding transcripts that exhibit the activity of a UDP-GIc-DH and / or an increase in the amount of protein that exhibits the activity of a UDP-GIc-DH in cells and / or an increase in protein enzymatic activity that exhibits the activity of a UDP-GIc-DH in cells.

Genetically modified plant cells according to the invention or genetically modified plants according to the invention in each case satisfy at least one of the above conditions meaning an increase in the enzymatic activity of a protein relative to proteins that exhibit (enzymatic) activity. of a GFATe in relation to proteins that exhibit the activity (enzymatic) of aUDP-GIc-DH.

An increase in expression can be determined, for example, by measuring the amount of transcripts encoding a protein that exhibits GFAT activity or encoding a protein that exhibits UDP-GIc-DH activity, for example by analysis. Northern blot orTA-PCR. Here, an increase preferably means an increase in transcripts compared to non-genetically modified wild-type plant cells or corresponding non-genetically modified wild-type plants by at least 50%, in particular by at least 70%, preferably by at least 85% and particularly preferably at least 100%. An increase in the amount of transcripts encoding a GFAT activity-encoding protein or a protein encoding a UDP-GIc-DH activity also means that plants or plant cells that have no detectable amount of transcripts which encode a protein which exhibits the activity of a GFAT and / or which encode a protein that exhibits the activity of a UDP-GIc-DH exhibit, upon genetic modification according to the invention, detectable amounts of transcripts which encode a protein which exhibit activity of a GFAT and / or encoding a protein that exhibits the activity of a UDP-GIc-DH.

The increase in the amount of protein that exhibits GFAT activity or the proteins that exhibit the activity of a UDP-GIc-DH resulting in an increase in the activity of these proteins in the plant cells in question may be determined, for example, by immunological methods. , such as Western blot analysis, ELISA (Enzyme-Linked Immunoenzyme Assay) or RIA (Radioimmunoassay). Methods of preparing antibodies that react specifically with a particular protein, i.e., specifically binding to that protein, are known from the art (see, for example, Lottspeich and Zorbas (Eds.), 1998, Bioa-nalytik [ Bioanalysis], Spektrum Akad Verlag, Heidelberg, Berlin, ISBN 3-8274-0041 -4). Some companies (eg Eurogentec, Belgium) offer the preparation of these antibodies as a work order. Here, an increase in the amount of protein preferably means an increase in the amount of protein that exhibits the activity of a GFAT and / or deproteins that exhibit the activity of a UDP-GIc-DH compared to non-genetically modified wild-type plant cells or non-wild-type plants. corresponding genetically modified 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 that shows activity of a GFATe / or protein that shows activity of a UDP-GIc-DH also means that plants or plant cells that have no detectable amount of a protein that show activity of a GFATe / or which exhibits no detectable activity of a protein that exhibits the activity of a UDP-GIc-DH exhibit, upon genetic modification according to the invention, a detectable amount of a protein that exhibits the activity of a GFAT and / or a detectable amount of a protein that exhibits the activity of a UDP-GIc-DH protein.

The increase in activity of a protein that exhibits GFAT activity in plant extracts can be determined by methods known to those skilled in the art as described, for example, inSamac et al. (2004, Applied Biochemistry and Biotechnology 113-116, Humana Press , Editor Ashok Mulehandani, 1167-1182, ISSN 0273-2289).

A preferred method of determining the amount of activity of a protein that exhibits the activity of a GFAT is given in General Methods, item 6.

The increase in activity of a protein that exhibits the activity of a UDP-GIc-DH in plant extracts can be described using methods known to those skilled in the art, as described, for example, in WO 00 11192. A preferred method of determining the amount The age of activity of a protein that exhibits the activity of a UDP-GIc-DH is given in General Methods, item 7.

An increase in the amount of (enzymatic) activity of proteins that exhibit the activity of a GFAT or of proteins that exhibit the activity of a UDP-GIc-DH preferably means an increase in the activity of these proteins by at least at least 50%, preferably at least 70%, especially preferably at least 85% and particularly preferably at least 100% compared to non-genetically modified wild type plant cells or non-genetically wild type plants corresponding changes. An increase in the amount of (enzymatic) activity of proteins that show the activity of a GFAT and / or a protein that shows the activity of a UDP-GIc-DH also means that plants or plant cells that have no detectable amount of a protein that exhibits activity of a GFAT and / or that exhibits no detectable activity of a protein that exhibits the activity of a UDP-GIc-DH exhibit, upon genetic modification according to the invention, a detectable amount of a protein that exhibits the activity of a GFAT and / or a detectable amount of a protein that exhibits the activity of a UDP-GIc-DH.

In the context of the present invention, the term "genome" shall be understood to mean the complete genetic material present in a plant cell. It is well known to one of skill in the art that, in addition to the nucleus, other compartments (eg, plastids, mitochondria) also contain genetic material.

In the context of the present invention, the term "stably integrated nucleic acid molecule" is to be understood to mean the integration of a nucleic acid molecule into the plant genome. A stably integrated nucleic acid molecule is characterized in that, during replication of the corresponding integration site, it is multiplied together with the host nucleic acid sequences that border the integration site, so that the integration site in the replicated DNA strand is surrounding the same sequences of nucleic acid as in the interpreted strand that acts as a matrix for replication.

A large number of nucleic acid molecule techniques stably integrated into a host plant cell are available. These techniques include 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 additionally options (review in "Transgenic Plants", Leandro ed., Humana Press 2004, ISBN 1-59259-827-7).

The use of agro-bacterium-mediated plant cell transformation has been the subject of in-depth studies and has been thoroughly described in EP 120516; Hoekema, IN: The Binary Plant Vector System Offset-drukkeríj Kanters B.V. Alblasserdam (1985), Chapter V; Fraleye Others, Crit.Rev. Plant I know. 4,1-46 and in An and others EMBO J. 4, (1985), 277-287. With regard to potato processing see, for example, Rocha-Sosa et al., EMBO J. 8, (1989), 29-33, for transformation of tomatevide plants, for example, US 5,565,347.

Transformation of monocotyledonous plants using Agrobacterium transformation-based vectors has also been described (Chan et al., PIant Moi Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6, (1994) 271-282; Deng et al., Science in China 33, (1990), 28-34; Wilmink et al., Plant Cell Reports 11, (1992), 76-80; May et al., Bio / Technology 13, (1995), 486 Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al., Transgenic Res. 2 (1993), 252-265). An alternative monocotyledon plant transformation system is transformation using the biolistic approach (Wan and Lemaux, Plant Physiol. 104, (1994), 37-48; Vasil et al., Bio / Technology 11 (1993), 1553). Ritala et al., PlantMoi Biol. 24 (1994), 317-325; Spencer et al., Theor. Appi Genet. 79 (1990), 625-631), protoplast transformation, cell electroporation. partially permeabilized, the introduction of DNA using fiberglass. In particular, corn transformation has been described several times in the literature (as, for example, WO95 / 06128, EP0513849, EP0465875, EP0292435; Fromm et al., Biotechnology 8, (1990), 833-844; Gordon-Kamm and others, PIantCeII 2, (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appi Genet. 80 (1990), 721-726). Transformation of other grasses such as, for example, switchgrass (Panicum virgatum) has also been described (Riehards et al., 2001, Plant Cell Reporters 20, 48-54).

Successful transformation of other cereal species is also described, for example, from barley (Wan and Lemaux, see above; Rita-la and others, see above; Krens and others, Nature 296, (1982), 72-74) and of wheat (Nehra et al., Plant J. 5, (1994), 285-297; Becker et al., 1994, PlantJournal 5, 299-307). All the above methods are suitable in the context of the present invention. 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 larger amounts of hyaluronan than plants. which present only the activity of a hyaluronan synthase. This allows hyaluro-nan to be produced at low cost since the isolation of hyaluro-nan from plants with a higher hyaluronan content is less complicated and more cost efficient. Furthermore, compared to plants described in the prior art, smaller cultivation areas are required to produce hyaluronan using genetically modified plants according to the invention. This leads to the possibility of providing hyaluronan in sufficient quantities even for industrial applications where it is currently unused due to its scarcity and high price. Vegetable organisms infected with the Chlorella genus are inadequate to produce relatively large amounts of hyaluronan. In the production of hyaluro-nan, virus-infected algae have the disadvantage that the genes required by hyaluronan synthase are not stably integrated into their genome (Van Etten and Meints, 1999, Annu. Rev. Microbiol. 53, 447-494). , to produce hyaluronan, the virus infection has to be repeated. Consequently, it is not possible to isolate individual Chlorella cells that continuously synthesize the desired quality and quantity of hyalu-ronan. Also, 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, algae are exterminated only about 8 hours after infection (Van Etten et al., 2002). , Arch. Viroi 147, 1479-1516). 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 can be propagated in a vegetatively or sexually non-limited manner and that they produce hyaluronan continuously.

The transgenic plants described in WO 05 012529, which contain a nucleic acid molecule encoding a hyaluronan synthase, synthesize a relatively small amount of hyalurane. 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 considerably synthesize larger amounts of hyaluronan.

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, preferably at least 600, particularly preferably at least 1000 and especially preferably at least 1500 pg of hyaluronan per g. of fresh pasta (MF) plant material.

Preferably, plant cells according to the invention or plants according to the invention synthesize a maximum of 25,000 pg dehyaluronan per gram of fresh pasta, preferably a maximum of 20,000 pg of hyaluronan per gram of fresh pasta, particularly preferably 15,000 pg of hyaluronan per gram. gram of fresh pasta, especially preferable, a maximum of 10,000 pg of hyaluronan per gram of fresh pasta and mainly preferable, a maximum of 6,500 pg of hyaluronan per gram too fresh.

To determine the hyaluronan content with respect to fresh mass in genetically modified plant cells according to the invention or genetically modified plants according to the invention, preference is given to using the plant material method described below. General Methods item 2 and the method for determining the amount of hyaluronand described under General Methods item 4.

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 1,000, preferably at least 2,000, particularly preferably at least 4,000, especially preferably at least 5,000 pg of hyaluro. -nan per g dry mass (MS) of plant material. To determine the content of hyaluronan with respect to dry weight in genetically modified plant cells according to the invention or genetically modified plants according to the invention, preference is given to using the material processing method described in Example 13 (k) and The method of determining the amount of hyaluronan described under General Methods item 4.

During the developmental time, hyaluro-nan accumulates in plant tissue; therefore, the amount of hyaluro-nan with respect to fresh weight or to dry weight in genetically modified plant cells according to the invention or genetically modified plants according to the invention should be determined with particular preference during harvesting. or a few (one or two) days prior to harvesting the plant cells in question or the plants in question. Here, particular use is made of plant material (eg, tubers, seeds, leaves) with respect to the amount of hyaluronan which should be used for further processing.

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

Since plant tissue has the advantage that it does not contain hyaluronidases, a simple and rapid isolation method can be used to confirm the presence of hyaluronan in genetically modified plant cells according to the invention or genetically modified plants according to the invention. For this purpose, water is added to the plant tissue for examination and the plant tissue is then mechanically broken (with the help of, for example, a bead mill, a whisk mill, a Warring mixer, a juice extractor, etc.) . If required, more water may then be added to the suspension, and cellular 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 that specifically binds hia-luronan. A protein-assisted hyaluronan detector method specifically binds to hyaluronan is described, for example, in US5,019,498. Test kits for carrying out the method described in US 5,019,498 are commercially available (for example, the Corgenix, Inc., Colorado, USA Hyaluronic Acid (HA) Test Kit, Prod. No. 029-001; see also All General item 4). In parallel, it is initially possible to digest an aliquot of the spin supernatant obtained with a hyaluronidase and then confirm the presence of hyaluronan with the aid of the protein that specifically binds to hyaluronan, as described above. By the action of hyaluronidase on the parallel batch, the hyaluronan present in this particular is degraded, so that after complete digestion it is no longer possible to detect significant amounts of hyaluronan.

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

As discussed above, it is unclear which metabolic pathway (phosphate hexose or myo-inositol oxidative metabolic pathway) is primarily used in plant cells to synthesize UDP-glucuronic acid, and both metabolic pathways become different quantitative contributions to UDP-glucuronic acid synthesis, depending on the tissue and / or stage of plant development. Furthermore, overexpression of a UDP-GIc-DH in transgenic plants does not lead to consistent results, and it was not possible to obtain the target to increase the cell wall pectin content that adopts such an approach. In addition, regulation of protein activity exhibiting the activity of a UDP-GIc-DH is inhibited by UDP-xylose. This has been demonstrated for both 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), animal organisms (Balduini et al., 1970, Biochem. J. 120 (4), 719-724) and plants (Hinterberg, 2002, Plant Physiol. Biochem. 40,1011-1017). In addition, reaction products with glucuronic acid and NA-DH that originate from a protein-catalyzed reaction that exhibits the activity of a UDP-GIc-DH are inhibitors that regulate the activity of a protein that exhibits the activity of a GFAT (Campbell et al., 1997, J. Bioi Chem. 272 (6), 3416-3422, Ordman and Kirkwood, 1977, Biochim.Biophys Acta 482 (1) 25-32; Turner and Botha 2002 Archives of Biochem. ophys 407, 209-216).

Overexpression in maize of a protein that exhibits the (enzymatic) activity of a GFAT transactionally fused to a plastid signal peptide resulted in an increase in the UDPP-glucosamine content, and cytosolic overexpression of a protein in maize shows activity ( Enzyme) of a GFAT resulted in an increase in glycosamine 1-phosphate content in ground endosperm tissue. However, UDP-glycosamine and glycosamine 1-phosphate are not starting materials for the synthesis of hyaluronan by hyaluronan synthase. In addition, it is known that glycosamine 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 plant cells. glycosamine 6-phosphate. Glycosamine 6-phosphate is also toxic to plant cells (WO 98 35047, US 6,444,878). Furthermore, it is known that proteins that exhibit GFAT activity can be inhibitively regulated by metabolites that are formed in the additional metabolic pathway for the synthesis of UDP-N-acetyl glycosamine. of a GFAT, isolated from eukaryotes (both with animal and plant organisms) are inhibited, for example, by UDP-N-acetyl glycosamine, which is one of two substrates for hyaluronansintase (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 that exhibit umaGFAT activity are inhibited by glycosamine 6-phosphate, a GFAT-catalyzed direct reaction product (Deng et al., 2005, Metabolic Engineering 7,201-214). There are no indications in the literature that could limit the amount of hyaluronan. synthesized in plant cells.

Consequently, it has been surprisingly found that genetically modified plant cells or genetically modified plants which have a nucleic acid molecule encoding a hyalu-ronan synthase and which additionally have increased GFAT activity and increased UDP-GIc activity. -DH compared to genetically modified cells or genetically modified plants that exhibit (only) hyaluronan synthase activity produce significantly higher amounts of hyaluronan.

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 increase in the amount of hyaluronan compared to genetically modified plant cells or compared to genetically modified plants. that (only) exhibit the activity of a hyaluronansynthase or compared to genetically modified plant cells or compared to genetically modified plants that exhibit the activity of a hyaluronan synthase and no increase in the activity of a protein that exhibits the activity of a GFAT and no increase in activity of a protein that exhibits the activity of a UDP-GIc-DH.

Preferably, the amount of hyaluronan produced with respect to the fresh mass of plant material in genetically modified plant cells according to the invention or genetically modified plants according to the invention is at least 1.5 times, preferably at least 5 times. particularly and preferably at least 7.5 times and especially preferably at least 10 times larger compared with corresponding genetically modified plant cells or compared with corresponding genetically modified plants which (only) exhibit the activity of a hyaluronan synthase. In order to determine the increase in hyaluronan content with respect to the fresh mass of plant material in genetically modified plant cells according to the invention or in 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) exhibiting the activity of hyaluronan synthase, where equivalent material (e.g. leaf, tuber) of plant cells or plants should be compared, Plant cells or plants from which this material is taken should be grown under the same conditions and where the hyaluro-nan content of plant material of comparable age (development stage) should be compared. One skilled in the art should not, for example, compare new leaves of one plant with old leaves of another plant or vegetables.

In the context of the present invention, the term "plant cell or plant (only) which exhibits the activity of a hyaluronan synthase" shall be understood to mean a genetically modified plant cell or a genetically modified plant in which the genetic modification consists in the fact that it comprises a nucleic acid molecule that encodes a hyaluronan synthase compared to corresponding non-genetically modified jungle-like plant cells or non-genetically modified wild type plants.

In particular, "plant or plant cells (only) that exhibit the activity of a hyaluronan synthase" are characterized by the fact that they synthesize hyaluronan and that they have no additional genetic modification other than the introduction of a nucleic acid molecule that encodes a hyaluronan synthase in non-genetically modified wild-type plant cells or non-genetically modified wild-type plants. Preferably, these plants do not show an increase in the activity of a protein that exhibits the activity of umaGFAT and no increase in the activity of a protein that exhibits the activity of a UDP-GIc-DH.

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

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

In the context of the present invention, the term "earth-green (Embryophyte) plant" is to be understood as defined in Strasburger, ULehr-buch der Botanik ', [34]. edM Spektrum Akad. Verl., 1999, (ISBN 3-8274-0779-6).

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. Consequently, this modality refers to plant cells or plants that do not originate from single plant cells (protists) or that are not protists.

Genetically modified plant cells according to the invention or genetically modified plants according to the invention may in principle be plant cells and plants, respectively, of any plant species, ie both monocotyledonous and dicotyledonous plants. . They are preferably crop plants, that is, plants grown by man for the purpose of feeding man and animal, or to produce biomass and / or to prepare substances for technical, industrial purposes (eg maize, rice, wheat, alfalfa, rye, oats, barley, cassava, potato, tomato, switchgrass (Panicum virgatum), sago, mung beans, peas, sorghum, carrot, aubergine, radish, rapeseed oil, soy, peanuts, cucumbers, pumpkins, melons, leek, garlic, cabbage, spinach, sweet potato, asparagus, zucchini lettuce, artichoke, green corn, flan, scorching, jerusalem artichoke, bana, sugar beet, sugar cane , beet root, broccoli, lamb, onion, yellow beet, dandelion, strawberry, apple, apricot, plum, peach, vines, cauliflower, celery, bell pepper, turnip, rhubarb).

Particularly preferred are tomato or potato plants.

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 the hyaluronan synthase nucleic acid molecule is characterized by the phage of which it encodes a plant. hyaluronan viral synthase. The hyaluronan synthase encoding nucleic acid molecule preferably encodes a hyaluronan synthase of an algal infecting virus.

With respect to an algal infecting virus, the nucleic acid molecule encoding a hyaluronan synthase preferably encodes a hyaluronan synthase of a Chlorella infecting virus, particularly preferably a hyaluronan synthase of a 1 Chlorella of Paramecium bursaria eespecial and preferably a hyaluronan synthase. synthase of a Chlorella deParamecium bursaria virus from an H1 strain.

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 wherein the hyaluronan-encoding nucleic acid molecule is characterized by: The codons of the nucleic acid molecule that encodes a hyaluronan synthase are modified compared to the codons of the nucleic acid molecule encoding the hyaluronan synthase from which the hyaluronan synthase originates. With particular preference, the codons of hyaluronan synthase must be modified such that they are adapted to the frequency of use of the plant cell codons or plant in whose genome they are integrated or must be integrated.

Due to the degeneration of the genetic code, amino acids may be encoded by one or more codons. In different organisms, codons encoding an amino acid are used at different frequencies. Codon adaptation of a nucleic acid coding sequence to the frequency of its use in the plant cell or plant in whose genome the sequence to be expressed is. Being integrated may contribute to an increase in the amount of translated protein and / or the stability of the mRNA in question in particular plant cells or plants. The frequency of use of the plant or plant cell codons in question can be determined by that in the art by examining as many nucleic acid coding sequences of the organism in question as possible for the frequency with which certain codons are used to encode a certain amino acid. The frequency of codon usage of certain organisms is known to those of skill in the art and can be determined simply and quickly using computer programs. Suitable computer programs are publicly accessible and freely provided, inter alia, on the Internet (eg http: //gcua.schoedl.deA http://www.kazusa.ou.jp/codonf,http://www.entelechon .com / eng / cutanalysis.html).

Adaptation of the codons of a nucleic acid coding sequence to the frequency of its use in the plant cell or plant in which the sequence to be expressed is integrated can be performed by in vitro mutagenesis or, preferably, by de novo synthesis. of the genetic sequence. Methods for de novo synthesis of nucleic acid sequences are known to those skilled in the art. A novel synthesis can be performed, for example, initially by synthesizing individual nucleic acid oligonucleotides, hybridizing them to complementary oligonucleotides, such that they form a double-stranded DNA strand, and then ligating the single-stranded single-stranded oligonucleotides. desired nucleic acid sequence is obtained. The new synthesis of nucleic acid sequences, including the adaptation of the frequency with which codons are used for a given target organism, may also originate externally from companies offering such a service (eg Entelechon GmbH, Regensburg, Germany). ).

The nucleic acid molecule encoding hyaluronan synthase is preferably characterized by the fact that it encodes a hyaluronan synthase whose amino acid sequence is at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 90%. 95%, and most preferably at least 98% identical to the amino acid sequence shown under SEQ ID NO 2. In a particularly preferred embodiment, the nucleic acid molecule encoding the hyaluronan synthase is characterized by the fact that it encodes a hyaluronansynthase. which shows the amino acid sequence shown under SEQ ID NO. 2.

In a further embodiment, the nucleic acid molecule that encodes a hyaluronan synthase is at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95%, and most preferably at least 98% identical to the one below. nucleic acid frequency shown under SEQ ID NO.1 or SEQ ID NO. 3. In a particularly preferred embodiment, the nucleic acid molecule that encodes hyaluronan synthase is characterized by the fact that it has the nucleic acid sequence shown under SEQ ID NO. 3

On 25. 8. 2004, plasmid IC 341-222, which comprises a coding of a synthetic nucleic acid molecule of a hyaluronan feel for Chlorella de Paramecium bursaria virus, was deposited with DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg1b, 38124 Brunswick , Germany, under number DSM16664, according to the Budapest treaty. The amino acid sequence shown in SEQ IDNO. 2, it can be derived from the coding region of the integrated nucleic acid sequence for plasmid IC 341-222 and encodes a Chlorella virus hyaluronansintase from Paramecium bursaría.

Accordingly, the present invention also relates to genetically modified plant cells according to. The invention or genetically modified plants according to the invention wherein the nucleic acid molecule encoding the hyaluronan synthase is characterized by the fact that it encodes a protein whose amino acid sequence may be derived from the coding region of the nucleic acid sequence inserted into the plasmid. DSM16664 or which encodes a protein whose amino acid sequence is at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95%, and more preferably at least 98% identical to the amino acid sequence that can be derived from the nucleic acid sequence coding region inserted in plasmid DSM16664.

The present invention also relates to genetically modified plant cells according to the invention or genetically modified plants according to the invention wherein the nucleic acid molecule encoding hyaluronan synthase is characterized by the fact that it is the sequence of nucleic acid encoding integrated hyaluronan synthase in DSM16664 or at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95% and more preferably at least 98% identical to nucleic acid sequence integrated into plasmid DSM16664.

The present invention further relates to genetically modified plant cells according to the invention or genetically modified plants according to the invention characterized by the fact that they have a stably integrated nucleic acid molecule in their genome or a plurality of foreign nucleic acid molecules stably integrated into their genome, such foreign nucleic acid molecule or foreign nucleic acid molecules which increase the activity of a protein that exhibits the activity of a GFAT and that increases the activity of a protein that exhibits activity of a UDP-GIc-DH compared to corresponding non-genetically modified wild-type plant cells or corresponding non-genetically modified wild type plants.

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, enhances the activity of a protein that exhibits GFAT activity and simultaneously increases the activity of a protein that exhibits the activity of a UDP-GIc-DH compared to corresponding wild type plant cells or corresponding wild type plants. However, it may also be a plurality of foreign nucleic acid molecules, a foreign nucleic acid molecule from which increases the activity of a protein having the activity of a UDP-GIc-DH and another foreign nucleic acid molecule that increases the activity of a protein that exhibits the activity of a UDP-GIc-DH compared to corresponding wild type plant cells or corresponding wild type plants. If a plurality of foreign nucleic acid molecules are integrated into the genome 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 in one site in the cell genome. plant or plant, or they may be located at different sites in the plant or plant cell genome (for example, on different chromosomes or different chromosomal sections).

Consequently, foreign nucleic acid molecules may be inherited as a binding site or as coupled sites (loci) according to Mendel rules, or they may be inherited as separate sites independently of each other according to Mendel

In the context of the present invention, the term "foreign acid molecule" is to be understood to mean that a molecule does not naturally occur in the corresponding wild type plant cells or that does not naturally occur in the concrete spatial arrangement in plant type cells. wild or that is located at a site in the wild-type plant cell genome where it does not naturally occur. Preferably, the foreign nucleic acid molecule is a recombinant molecule comprising several elements whose specific combination or arrangement does not naturally occur in plant cells.

In the context of the present invention, the term "recombinant nucleic acid molecule" is to be understood to mean a nucleic acid molecule having several non-naturally occurring nucleic acid molecules present in a combination similar to that present in a recombinant nucleic acid molecule. . Thus, recombinant nucleic acid molecules may, in addition to nucleic acid molecules encoding a hyaluronan synthase and / or a protein that exhibits the activity of a GFAT and / or a protein exhibiting the activity. of a UDP-GIc-DH additionally have nucleic acid sequences that are not naturally present in combination with the mentioned nucleic acid molecules. The aforementioned additional nucleic acid sequences that are present in a recombinant nucleic acid molecule in combination with a nucleic acid molecule encoding a hyaluronan synthase or a protein that exhibits the activity of a GFAT and / or a protein that exhibits the activity of a UDP -GIc-DH could be any sequences. For example, they may be sequences of plant genomic nucleic acids. The additional nucleic acid sequences mentioned are preferably regulatory sequences (promoters, termination signals, enhancers), particularly and preferably regulatory sequences that are active in plant tissue, especially and preferably tissue-specific regulatory sequences that are active in plant tissue. Methods of generating recombinant nucleic acid molecules are known from that skilled artisan and comprise methods of genetic engineering, such as, for example, ligation of nucleic acid molecules by ligation, genetic recombination or synthesis of of nucleic acid molecules (see, for example, Sambrok et al., Molecular Cloning, A Laboratory Manual, 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773, Ausubel et al., Short Protocols in Molecular Bio-logy, John Wiley &Sons; 5th Edition (2002), ISBN: 0471250929).

Genetically modified plant cells and genetically modified plants that have a strangely integrated stranded nucleic acid molecule in their genome or a plurality of stably integrated stranded nucleic acid molecules in their genome which encodes hyaluronan synthase and which increases activity of a protein that exhibits the activity of a GFAT and enhances the activity of a protein that exhibits the activity of a UDP-GIc-DH compared with corresponding non-genetically modified wild-type plant cells or non-genetically modified wild-type plants can be distinguished from these wild-type plant cells and these wild-type plants, respectively, among others, by the fact that they comprise a foreign nucleic acid molecule that does not naturally occur in wild-type plant cells and wild-type plants, respectively, or that such a molecule is integrated into one s genetically modified plant cell genome according to the invention or genetically modified plant genome according to the invention where this does not occur in wild-type plant cells and wild-type plants, respectively, ie in a genomic environment. - How different. Furthermore, such genetically modified plant cells according to the invention and genetically modified plants according to the invention can be distinguished from non-genetically modified wild-type plant cells and non-genetically modified wild-type plants, respectively, in that they comprise at least one copy of the foreign nucleic acid molecule stably integrated into its genome, if appropriate, in addition to copies of such a molecule naturally present in 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 naturally occurring molecules In wild type plant cells or wild type plants, genetically modified plant cells according to the invention and 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 / additional copies are / are located at sites in the genome where they are not present in wild-type plant cells and plants of the wild. The stable integration of a nucleic acid molecule into the genome of a plant cell or plant can be demonstrated by genetic and / or genetic methods. molecular biology methods. A stable integration of a nucleic acid molecule into the genome of a plant cell or the genome of a plant characterized by the fact that in the progeny that has inherited this nucleic acid molecule, the nucleic acid molecule is stably integrated is present in the same genomic environment as nageration of origin. The presence of stable integration of a nucleic acid sequence into the genome of a plant cell or the genome of a plant can be demonstrated using methods known in the art, among others, with the aid of Southern blot analysis of the analogue. Restriction Fragment Length Polymorphism (RFLP) -lysis (Nam et al., 1989, The Plant Cell 1, 699-705; Leister and Dean, 1993, The Plant Journal 4 (4), 745-750), with PCR-based methods , such as, for example, analysis of length differences in the AmplifiedFragment Length Polymorphism, AFLFf (Castiglioni et al., 1998, Genetics149, 2039-2056; Meksem et al., 2001, Molecular Genetics and Genomics265, 207-214; Meyer et al., 1998, Molecular and General Genetics 259,150-160) or using Cleaved Amplified Polymorphic Sequenees (CAPS) cleaved amplified fragments (Konieczny and Ausubel1 1993, The Plant Journal 4, 403-410 ; Jarvis eoutr 1994, Plant Molecular Biology 24, 685-687; Bachem et al., 1996, The Plant Journal 9 (5), 745-753).

In principle, the foreign nucleic acid molecule may be any nucleic acid molecule that increases, in the plant or plant cell, the activity of a protein that exhibits the activity of a GFATe / or the activity of a protein that exhibits the activity of a UDP-Glc-DH.

In the context of the present invention, genetically modified plant cells according to the invention and genetically engineered plants according to the invention may also be prepared using random insertion genesis (review: Thorneycroft et al., 2001, Journal of Experimental Botany 52 (361 ), 1593-1601). In the context of the present invention, insertion mutagenesis is to be understood to mean in particular the insertion of transposons or transfer DNA (T-DNA) into or around a gene encoding a protein that has GFAT activity and / or encoding a protein that exhibits the activity of a UDP-GIc-DH, thereby increasing the activity of a protein that exhibits the activity of a GFAT and / or a protein exhibits the activity of UDP-GIc-DH in the cell in question.

Transposons may be transposons that occur naturally in the cell (endogenous transposons) or those that are not naturally present in the cell but have been introduced into the cell by genetic engineering, such as, for example, cell transformation (heterologous transposons). . Modification of gene expression by half transposons is known to one of skill in the art. A doubled review of endogenous and heterologous transposons as tools in plant biotechnology is given in Ramachandran and Sundaresan (2001, Plant Physio-Iogy and Biochemistry 39, 234-252).

T-DNA insertion mutagenesis is based on the fact that certain sections (T-DNA) of Agrobacterium Ti plasmids can be integrated into the plant cell genome. The integration site on the plant chromosome is not fixed, integration can be anywhere. If T-DNA is integrated into or around a section of the chromosomock represents a genetic function, it may result in increased gene expression and thus a change in the activity of a protein encoded by the gene in question.

Sequences inserted into the genome (in particular transposons or T-DNA) are characterized by the fact that they comprise sequences that result in activation of regulatory sequences of a gene that encodes a protein that exhibits the activity of a GFAT and / or which co- differs a protein that exhibits the activity of a UDP-GIc-DH ("activation labeling"). Preferably, sequences inserted into the genome (in particular transposons or T-DNA) are characterized by the fact that they are integrated in the vicinity of endogenous nucleic acid molecules in the plant or plant genome encoding a protein that has the activity of a GFAT and / or a protein that exhibits the activity of a UDP-GIc-DH.

Genetically modified plant cells according to the invention and genetically modified plants according to the invention can be generated, for example, using the activation labeling method (see, for example, Walden et al., Plant J. (1991), 281 -288; Walden et al., Plant Moi Biol. 26 (1994), 1521-1528). This method is based on activating endogenous promoters by enhancer sequences, such as, for example, the cauliflower mosquito virus 35S RNA promoter enhancer or the octopin synthase enhancer.

In the context of the present invention, the term "T-DNA activation labeling" is to be understood to mean a T-DNA fragment comprising enhancer sequences and, by integrating into the stem cell of a plant cell, increases the activity of a protein that exhibits the activity of a GFAT and / or a protein that exhibits the activity of a UDP-GIc-DH.

In the context of the present invention, the term "transposon activation labeling" shall be understood to mean a transposon comprising enhancer sequences and, by integrating into the genome of a plant cell, increases the activity of a protein that exhibits GFAT activity and / or a protein that exhibits the activity of a UDP-GIc-DH.

A preferred embodiment of the present invention relates to genetically modified plant cells according to the invention or genetically modified plants according to the invention which are characterized by the fact that at least one foreign nucleic acid molecule encodes a protein having (enzymatic) activity of a GFAT or that at least one foreign nucleic acid molecule encodes a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH. One embodiment of the present invention is particularly preferred to genetically modified plant cells according to The invention or genetically modified plants according to the invention are characterized in that a first foreign nucleic acid molecule encodes a protein that exhibits the (enzymatic) activity of a GFAT and a second foreign nucleic acid molecule encodes a protein that exhibits the (enzymatic) activity of a U DP-GIc-DH.

According to the invention, the foreign nucleic acid molecule encoding a protein that exhibits the (enzymatic) activity of a GFAT may originate from any organism; preferably, such a nucleic acid molecule originates from bacteria, fungi, animals, plants or viruses, particularly and preferably from mammals or special bacteria and preferably from mouse or Escherichia coli.

With respect to a foreign nucleic acid molecule encoding a protein that exhibits the (enzymatic) activity of a GFAT that originates from animal organisms, use should preferably be made of a nucleic acid molecule encoding a protein that has the (enzymatic) activity of a GFAT. a GFAT-2; particularly preferably, the protein that exhibits the (enzymatic) activity of a GFAT-2 originates from the mouse.

With respect to viruses, the foreign nucleic acid molecule that encodes a protein that exhibits the (enzymatic) activity of a GFAT preferably originates from an algal infecting virus, preferably a particular Chlorella algae infecting virus, preferably a Chlorella Paramecium bursaria virus and especially, preferably, a Chlorella Paramecium bursaria virus of a strain.

Instead of a naturally occurring nucleic acid molecule encoding a protein that exhibits the (enzymatic) activity of a GFAT, it is also possible for a mutagenically generated nucleic acid molecule to be introduced into genetically modified plant cells accordingly. according to the invention or the genetically modified plants according to the invention, wherein such a mutagenized foreign nucleic acid molecule is characterized by the fact that it encodes a protein that exhibits the (enzymatic) activity of a re-inhibiting GFAT. - induced by metabolites (e.g., from glycosamine metabolism). The preparation of such mutagenized nucleic acid molecules is described in an exemplary manner by a protein that exhibits the (enzymatic) activity of an Escherichia coli in Deng et al. (2005, MetabolicEngineering 7, 201-214; WO 04 003175). Mutants of a protein that exhibit the activity of a mouse GFAT are described, for example, in Hu et al. (2004, J. Biol Chem. 279 (29), 29988-29993).

According to the invention, the foreign nucleic acid molecule encoding a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH may originate from any organism; preferably, such a nucleic acid molecule originates from bacteria, fungi, animals, plants or viruses, particularly and preferably from bacteria, vegetables or viruses, especially and preferably from viruses.

With respect to viruses, the foreign nucleic acid molecule that encodes a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH preferably originates from an algal infecting virus, comprising a virus infecting the genus Chlorella, particularly preferably from a Chlorella Paramecium bursaria virus and especially preferably from a Chlorella Paramecium bursaria virus from an HepaHI.

Instead of a naturally occurring nucleic acid molecule encoding a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH, it is also possible for a mutagenically generated nucleic acid molecule to be introduced into cells. genetically modified plants according to the invention or genetically modified plants according to the invention, wherein such a mutagenized foreign nucleic acid molecule is characterized by the fact that it encodes a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH with reduced inhibition by metabolites (eg, from glucuronic acid metabolism).

Nucleic acid molecules encoding a protein that exhibit the activity of a GFAT are known from that skilled artisan and described in the literature. Thus, nucleic acid molecules encoding a protein that exhibits the activity of a GFAT are described from viruses, eg from the Chlorella k2 virus (EMBL No. ac.AB107976.1), from bacteria, eg from Escherichia coli ( Dutka-Malen, 1988, Biochemie 70 (2), 287-290; EMBL Ac. No .: L10328.1), Fungi, for example, from Saecharomyces eerevisiae (EMBL, Ac. AF.3434737.1, Watzele et al. , 1989, "J. Bioi Chem. 264, 8753-8758), Aspergillus niger (EMBL ac.AY594332.1), Candida albieans (EMBL ac. No. X94753.1), insect, e.g. Aedes aegyti (Kato et al., 2002, Inseet. Bioi 11 <3), 207, 216; EMBL Ac. No. AF399922.1), Drosophila melanogaster (GFAT-1: EMBL.Ac. Y18627.1, GFAT- 2: NCBI Ac. No. NM_143360.2), from algae of Volvariella volvaeea (EMBL Ac. No. AY661466.1), for example, Homo sapiens vertebrates (GFAT-1: EMBL Ac. No. AF334737. 1; GFAT-2: NCBI Ac. No. BC000012.2, Oki et al., 1999, Genomies 57 (2), 227-34), Mus museulus ( GFAT-1: EMBL Ac. AF334736.1; GFAT-2: EMBL Ac. AB016780.1), or from vegetables, for example from Arabidopsis thaliana (EMBL Ac. No. AP001297.1; cds. NCBI Ac. No. BAB03027.1).

In a preferred embodiment, the present invention relates to genetically modified plant cells according to the invention and genetically modified plants according to the invention wherein the foreign nucleic acid molecule encoding a GFAT-activating protein is selected. of the group consisting of:

a) Nucleic acid molecules encoding a protein which has the amino acid sequence given under SEQ ID NO. 8 or a protein having the amino acid sequence given under SEQ ID NO. 10or a protein having the amino acid sequence given under SEQ ID NO. 12;

b) nucleic acid molecules encoding a protein whose sequence is at least 60%, preferably at least 80%, preferably at least 90%, especially preferably at least 95% and more preferably at least 98% identical to the sequence. of amino acids given under SEQ ID NO. 8, under SEQ ID NO. 10 or under SEQ ID NO. 12;

c) nucleic acid molecules comprising the denucleotide sequence shown under SEQ ID NO. 7 or a sequence will complement this, 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 to this or the denucleotide sequence shown under SEQ ID NO. 13 or a sequence complementary to it;

d) nucleic acid molecules which are at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95% and more preferably at least 98% identical to the nucleic acid sequences described below. ) or c);

e) nucleic acid molecules that hybridize under stringent conditions to at least one strand of the nucleic acid sequences described under a) or c);

f) nucleic acid molecules whose nucleotide sequence differs from the sequence of nucleic acid molecules mentioned under a) or

c) due to the degeneration of the genetic code; and

g) nucleic acid molecules which are fragments, variantselelic and / or derived from the nucleic acid molecules mentioned under (a), (b), (c), (d), (e) or (f).

Nucleic acid molecules encoding a protein that exhibits the activity of a UDP-GIc-DH are described in the literature and known to those skilled in the art. Thus, nucleic acid molecules encoding a protein that exhibits the activity of a UDP-GIc-DH are described from viruses, for example Chlorella 1 virus (NCBI ac. NC_000852.3), from bacteria. eg Escherichia coli (EMBLNo Ac .: AF176356.1), fungus e.g. Aspergillus niger (EM-BL Ac. No. AY594332.1), Cryptococcus neoformans (EMBL Ac. AF405548.1), from insects eg from Drosophila melanogaster (EMBL Ac. No. AF001310.1), from vertebrates, for example from Homo sapiens (EMBL Ac. No. AF061016.1), Mus musculus ( EMBL Ac. No. AF061017.1), Bostaurus (EMBL Ac. No. AF095792.1), Xenopus Iaevis (EMBL Ac.AY762616.1) or from plants such as poplar (EMBL ac.AF053973.1), Colocasia esculenta (EMBL Ac. No. AY222335.1), Dunaliel-la Saline (EMBL Ac. No. AY795899.1), Glycine max (EMBL Ac.U53418.1) .

In a further preferred embodiment, the present invention relates to genetically modified plant cells according to the invention and genetically modified plants according to the invention, wherein the foreign protein-encoding nucleic acid molecule exhibits the activity of a UDP-GIc -DH is selected from the group consisting of:

(a) nucleic acid molecules encoding a protein which has the amino acid sequence given under SEQ ID NO 5;

b) nucleic acid molecules encoding a protein whose sequence is at least 60%, preferably at least 80%, preferably at least 90%, especially preferably at least 95% and more preferably at least 98% identical to amino acid sequence given under SEQ ID NO. 5;

c) nucleic acid molecules comprising the nucleotide sequence shown under SEQ ID NO. 4 or a sequence complementary to this or the nucleotide sequence shown under SEQ ID NO. 6 or a sequence complementary to this;

d) nucleic acid molecules which are at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95% and more preferably at least-98% identical to the described nucleic acid sequences. under a) or c);

e) nucleic acid molecules that hybridize under stringent conditions to at least one strand of the nucleic acid sequences described under a) or c);

f) nucleic acid molecules whose nucleotide sequence differs from the sequence of nucleic acid molecules mentioned under a) or c) due to degeneration of the genetic code; and

g) nucleic acid molecules which are fragments, variantselelic and / or derived from the nucleic acid molecules mentioned under (a), (b), (c), (d), (e) or (f).

In the context of the present invention, the term "hybridization" means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, ϊηSambrooke others; Molecular Cloning, A Laboratory Manual, 2-. ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). With particular preference, "hybridization" means hybridization under the following conditions:

Hybridization Buffer:

2 χ SSC; 10 χ Denhardt's solution (Fikoll 400 + PEG + BSA; 1: 1: 1 ratio); 0.1% SDS; 5 mM EDTA; 50 mM Na 2 HPO 4; 250 pg / ml herring sperm DNA; 50 pg / ml tRNA; or 25 M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% of

SDS

Hybridization Temperature: T = 65 to 68 ° C Wash Buffer: 0.1 χ SSC; 0.1% SDST Washing temperature: T = 65 to 68 ° C.

Nucleic acid molecules that hybridize to nucleic acid molecules that encode a protein that exhibits the activity of a UDP-GIc-DH or that exhibits GFAT activity may originate from any organism; consequently, they may originate from bacteria, fungi, animals, vegetables or viruses.

Nucleic acid molecules that hybridize to nucleic acid molecules encoding a protein that exhibits the activity of a UDP-GIc-DH preferably originate from an al-gas-infecting virus, preferably a Chlorella, particulate, and algae-infecting virus. preferably a Chlorella virus of Paramecium bursaria and especially and preferably a Chlorella virus of Paramecium bursaria de a strain H1.

Nucleic acid molecules that hybridize to nucleic acid molecules encoding a protein that exhibits the activity of a particular GFAT and preferably originates from mammals, plants or bacteria, and especially preferably from mouse or Escherichiacoli.

Nucleic acid molecules that hybridize to nucleic acid molecules encoding a protein that exhibits the activity of a GFAT-1 or a GFAT-2 preferably originates from a particular eukaryotic organism and preferably they originate from an animal organism, especially and preferably from the mouse.

Nucleic acid molecules that hybridize to the mentioned molecules may be isolated, for example, from genomic or cDNA libraries. Such nucleic acid molecules may be identified by using the aforementioned nucleic acid molecules or parts thereof or the inverse complements of such molecules, for example, by hybridization according to standard methods (see, for example, Sambrooke et al., 1989, Molecular Cloning, A Laboratory Manual, 2- Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or by amplification using PCR.

As a hybridization sample for isolation of a nucleic acid sequence that encodes a protein that exhibits the activity of a UDP-GIc-DH, it is possible to use, for example, nucleic acid molecules that present exactly or essentially the sequence of a UDP-GIc-DH. nucleotides given under SEQ ID NO. 4 or SEQ ID NO. 6, or parts of these sequences.

As a hybridization sample for isolation of a nucleic acid sequence encoding a protein that exhibits the activity of a GFAT, it is possible to use, for example, nucleic acid molecules that accurately or essentially present the nucleotide sequence given under SEQ ID NO. 7 or under SEQ ID NO. 9 or under SEQ ID NO. 11 or under SEQ IDNO. 13, or parts of these sequences.

Fragments used as hybridization samples may also be synthetic fragments or oligonucleotides prepared using standard synthesis techniques, the sequence of which is essentially identical to the nucleic acid molecule described in the context of the present invention. Since genes that hybridize to the nucleic acid sequences described in the context of the present invention are identified and isolated, the sequence must be determined and the properties of the proteins encoded with respect to this sequence must be analyzed to determine whether they are proteins. which show the activity of a GFAT, a GFAT-1 or a GFAT-2 or the activity of a UDP-GIc-DH. Methods of determining whether a protein has the activity of a protein that exhibits GFAT activity (eg, Mayer et al., 1968, Plant Physio 43, 1097-1107; Deng et al., 2005, Metabolic Engineering 7, 201-214), a GFAT-1 or a GFAT-2 (e.g. Hu et al., 2004, J. Biol Chem. 279. (29), 29988-29993) or a UDP-GIc-DH (e.g. 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. 216) are known to those skilled in the art and described, among others, in the described literature.

Molecules which hybridize to 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 at one or more positions from the sequences of the nucleic acid molecules described above and are highly identical to those sequences. Differences to the nucleic acid molecules described above may, for example, be due to deletion, addition, substitution, insertion or recombination.

In the context of the present invention, the term "identity" means an identity sequence over the total length of the coding region of a nucleic acid molecule or the total length of an amino acid sequence encoding a protein of at least 60% by weight. particularly an identity of at least 70%, preferably at least 80%, particularly preferably at least 90% and especially preferably at least 95%. In the context of the present invention, the term "identity" shall be understood to mean the number of identical amino acids / nucleotides (identity) with other proteins / other nucleic acids, expressed as a percentage. Preferably, the identity with respect to a protein displaying UDP-GIc-DH activity is determined by comparison with the amino acid sequence given under SEQ ID NO. 5, identity with respect to a nucleic acid molecule encoding a protein displaying UDP-GIc-DH activity is determined by comparison with the nucleic acid sequence under SEQ ID NO. 4 or SEQ ID NO. 6, identity with respect to a protein that exhibits GFAT activity is determined by comparison with the amino acid sequence given under SEQ ID NO. 8 or SEQ IDNO. 10 or SEQ ID NO. 12 and identity with respect to a nucleic acid molecule encoding a protein that exhibits umaGFAT activity is determined by comparison with the nucleic acid sequence coded under SEQ ID NO. 7 or SEQ ID NO. 9 or SEQ ID NO. 11 or SEQ ID NO.13 with other proteins / other nucleic acids with the aid of computer programs. If sequences are compared to each other at different lengths, identity must be determined by determining identity in percent of the amino acid number that the shorter sequences share with the longer sequence.

Preferably, identity is determined using the known and publicly available computer program CIustaIW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). CIustaIW is made publicly available by Julie Thompson (Thompson @ EMBL-HeideIberg DE) and Toby Gibson (Gibson © EMBL-HeideIberg DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany. CIustaIW may also be downloaded from various websites, inter alia, from IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulire, BP163, 67404 Illkirch Cedex, France; ftp: //ftp-igbmc.u-strasbg .fr / pubή and EBI {ftp://ftp.ebi.ac.uk/pub/software/) and all European Bioinformatics Institute, Wellcome TrustGenome Campus, Hinxton, Cambridge CB10 1SD , UK).

Preferably, the Clus-talW version 1.8 computer program is used to determine the identity between the described proteins in the context of the present invention and other proteins. Here, the parameters have to be adjusted as follows: KTUPLE = I, TOPDIAG = 5, WINDOW = 5, PAIR-GAP = 3, GAPOPEN = 10, GAPEXTEND = 0.05, GAPDIST = 8, MAXDIV = 40, MATRIX = GONNET, ENDGAPS ( OFF), NOPGAP, NOHGAP.

Preferably, the ClusterW version 1.8 computer program is used 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 sequence of nucleotides of other nucleic acid molecules. Here the parameters must be adjusted as follows: KTUPLE = 2, T0PDIAGS = 4, PAIRGAP = 5, DNAMATRIX: IUB, GAPOPEN = 1 (), GAPEXT = 5, MAXDIV = 40, TRANSITIONS: Unweighted.

Identity, moreover, is understood to be that there is a functional and / or structural equivalence between the nucleic acid molecules in question or the proteins that encode them. Nucleic acid molecules that are homologous to the molecules described above and which represent derivatives of these molecules are generally variations of these molecules that represent modifications having the same biological function. They may be naturally occurring servariations, for example sequences from other species, or mutations, where these mutations may have occurred naturally or were introduced by directed mutagenesis. In addition, variations may be synthetically produced sequences. Allelic variants may be naturally occurring variants synthetically produced listeners or variants generated by recombinant DNA techniques. A special form of derivatives is, for example, nucleic acid molecules that differ from the nucleic acid molecules described in the context of the present invention due to the degeneration of the genetic code.

The various derivatives of nucleic acid molecules that encode a protein that exhibits the activity of a GFAT or a UDP-GIc-DH have certain common characteristics.

These may for example be biological or enzymatic activity, substrate specificity, molecular weight, immunological reactivity, conformation, etc., as well as physical properties such as, for example, gel electrophoresis processing, chromatographic behavior , sedimentation coefficients, solubility, spectroscopic properties, stability, optimal pH, optimal temperature, etc. Preferred protein properties which exhibit the activity of a GFAT or a-UDP-Glc-DH are known to those skilled in the art, have already been mentioned above and are to be applied here analogously.

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 wherein nucleic acid molecules encoding a protein that has the (enzymatic) activity of a GFAT and / or encoding a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH is characterized by the fact that the codons of these nucleic acid molecules are different from the codons of the nucleic acid molecules encoding this protein that exhibits the (enzymatic) activity of a GFAT or encodes a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH from the source organism. Particularly and preferably, the nucleic acid molecule codons that encode a protein that exhibits the (enzymatic) activity of a GFAT or that encodes a protein that exhibits (enzymatic) activity of a UDP-GIc-DH they are altered as soon as they are adapted to the frequency of use of plant cell or plant codons in whose genome they are or should be integrated.

The present invention further 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 hyaluronan-encoding plant or plant cell synthase and / or encoding a protein that exhibits the (enzymatic) activity of a GFAT and / or that co-complicates a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH are linked to regulatory elements initiating transcription in vegetable cells (promoters). These may be homologous or heterologous promoters. Promoters may be constitutive, tissue-specific, development-specific, or regulated by external factors (for example after chemical application, abiotic factors such as heat and / or cold, drought, disease, etc.). Here, nucleic acid molecules that encode a hyaluronan synthase or a protein that exhibits the (enzymatic) activity of a GFAT or a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH, nucleic acid molecules that are integrated as such. in 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 to individual sequences, may be linked to different promoters. . Here, two or three different promoters in any combination may in each case be linked to an extraneous stranded nucleic acid molecule encoding a hyaluronan synthase or a protein that exhibits (enzymatic) activity of a GFAT or a protein that exhibits activity. (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.

A preferred embodiment of the present invention relates to genetically modified plant cells according to the invention or genetically modified plants according to the invention wherein at least one foreign nucleic acid molecule, particularly preferably at least two acidic molecules. foreign nucleic acids, especially preferably three foreign nucleic acid molecules selected from the group consisting of nucleic acid molecules encoding a hyaluronan synthase or a protein that exhibits the (enzymatic) activity of a GFAT or a protein that exhibits the (enzymatic) activity of a UDP- GIc-DH is (are) bound to a tissue specific promoter. Preferred tissue-specific promoters are promoters that initiate transcription specifically in tuber cells or leaves, fruit or vegetable seeds.

To express nucleic acid molecules encoding a hyaluronan synthase or a protein that exhibits the (enzymatic) activity of a GFAT or a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH, these are preferably linked to regulatory sequences of a DNA ensuring transcription in plant cells. These include in particular promoters. And generally, any active promoter in vegetable cells is heated for expression.

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

Suitable promoters are, for example, the cauliflower mosaic virus 35S RNA promoter or maize ubiquitin promoter or OCTestrum YLCV (Yellow Leaf Curling Virus \ WO 01 73087; Stavolone et al., 2003, Plant Moi Biol. 53 703-713) of a constitutive expression, patatingen B33 opromotor (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) of a tuber-specific expression in potatoes or a tomato-specific promoter such as such as the tomato polygalacturonase promoter (Montgomery et al., 1993, Plant Cell 5, 1049-1062) or tomato E8 opromotor (Metha et al., 2002, Nature Biotechnol. 20 (6), 613-618 ) or the peach ACC oxidase promoter (Moon and Callahan, 2004, J. Experimental Botany 55 (402), 1519-1528) or a promoter that guarantees expression only in photosynthetically active tissues, for example the ST- LS1 (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451) or from an expr endosperm-specific species, the wheat HMWG promoter, the USP promoter, the phaseolin promoter, maize zein gene promoters (Peder-sen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., Plant Mol. Biol.15 (1990), 81-93), the glutelin promoter (Leisy et al., Plant Moi Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993). ), 357-366; Yoshihara et al., FEBS Lett. 383 (1996), 213-218) or the shrunken-1 promoter (Werr et al., EMBO J. 4 (1985), 1373-1380), a promoter globulin (Nakase et al., 1996, Gene 170 (2), 223-226) or a prolamine promoter (Qu und Ta-kaiwa, 2004, Plant Biotechnology Journal2 (2), 113-125). However, it is also possible to use promoters that are only active at a point in time determined by external factors (see, for example, WO 9307279). Of particular interest in this report may be promoters of thermal shock proteins that allow simple induction. It is furthermore possible to use seed-specific promoters, such as, for example, the Vicia fabaUSP promoter which ensures seed-specific expression in Vicia faba and other vegetables (Fiedler et al., Plant Moi Biol. 22 (1993)). , 669-679; Baumlein et al., Mol. Gen. Genet. 225 (1991), 459-467).

The use of promoters present in the genome of algal-infecting viruses is also suitable for expressing nucleic acid sequences in plants (Mitra et al., 1994, Biochem. Biophys. Res. Commun204 (1), 187-194; Mitra and Higgins, 1994, PIantMoIBioI 26 (1), 85-93, Van Etten et al., 2002, Arch. Virol. 147, 1479-1516).

In the context of the present invention, the term "tissue specific" is to be understood to mean the substantial limitation of a manifestation (e.g., transcriptional initiation) to a certain tissue.

In the context of the present invention, the terms "tuber cell, fruit or seed" shall be understood to mean all cells present in a tuber, fruit or seed.

In the context of the present invention, the term "homologous promoter" is understood to mean a promoter that is naturally present in plant or plant cells used for the preparation of genetically modified plant cells according to the invention or genetically modified plants. according to the invention (homologous to plant or plant cell) or as meaning a promoter that adjusts the expression of a gene in the organism from which the sequence has been isolated (homologous to the expressed nucleic acid molecule) .

In the context of the present invention, the term "hetero-go promoter" is understood to mean a promoter that is not naturally present in plant cells or plants used for the preparation of genetically modified plant cells according to the invention or plants. genetically modified according to the invention (heterologous to plant cell or plant) or as meaning a promotor is in the organism from which a nucleic acid sequence that is expressed has been isolated, not naturally present to regulate the expression of that sequence. nucleic acid (heterologous to the expressed nucleic acid molecule).

Also present may be a termination sequence (polyadenylation signal) that functions to add a transcribed poly-A tail. The poly-A tail is thought to act in stabilizing transcripts. These elements are described in the literature (as per Gielen et al., EM-BO J. 8 (1989), 23-29) and may be altered as desired.

It is also possible for intron sequences to be present between the engine and the coding region. Such intron sequences may lead to stability of expression and increased expression in vegetables (Callise et al., 1987, Genes Devi. 1, 1183-1200; Luehrsen and Walbot, 1991, MoiGen. Genet. 225, 81-93; Rethmeier et al. 1997; Plant Journan (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 maize sh1 gene, the first intron of the maize genepoliubiquitin 1, the first intron of the rice EPSPS gene, or that of the first two introns of the Arabidopsis PAT1 gene.

The present invention also relates to plants comprising genetically modified plant cells according to the invention. Such plants may be produced by regenerating genetically modified plant cells according to the invention.

The present invention also relates to processable or consumable parts of genetically modified plants according to the invention comprising genetically modified plant cells according to the invention.

In the context of the present invention, the term "processable parts" shall be understood to mean parts of vegetables that are used to prepare food or feed, which are used as a source of raw material for industrial processes, as a source of raw material for the preparation of pharmaceuticals or as a source of raw material for the preparation of cosmetic products.

In the context of the present invention, the term "consumable parts" is to be understood to mean plant parts that serve as food for man or are used as animal feed.

The present invention also relates to a genetically modified plant propagation material according to the invention comprising a genetically modified plant cell according to the invention.

Here, the term "propagating material" includes those plant components that are suitable for generating progeny via rotavegetative or generative. Suitable for vegetative propagation are, for example, cuts, corns, rhizomes or tubers. Other payment material includes, for example, fruits, seeds, seedlings, protoplasts, cell cultures, etc. The propagating material preferably takes the form of tubers, fruits or seeds.

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, buds, leaves or stems, preferably seeds, fruits or tubers, such harvestable parts comprising genetically modified plant cells according to the invention. Preferably, the present invention relates to crop material according to the invention or harvestable parts according to the invention. which comprise hyaluronan. Particularly preferred is propagating material according to the invention or harvestable vegetable parts according to the invention which synthesize hyaluronan.

In the context of the present invention, the term "potato plant" or "potato" is to be understood to mean plant species of the genus Solanum, particularly species producing tubers of the genus So-lanum, and in particular Solanum tuberosum.

In the context of the present invention, the term "tomato plant" or "tomato" is to be understood to mean plant species of the genus Lycopersicon, in particular Lycopersicon esculentum.

A further advantage of the present invention is that harvestable parts, propagating material, processable parts or consumable parts of genetically modified plants according to the invention comprise more hyaluronan than transgenic plants synthesizing hyaluronan described in the literature. Accordingly, genetically modified vegetables according to the invention are not only particularly suited for use as raw material from which hyaluronan may be isolated but may also be used directly as food / feed or for preparing foodstuffs. / ration for animals that have a prophylactic or therapeutic character (eg for osteoarthritis prophylaxis, US 6,607,745). Provided that genetically modified plants according to the invention have a higher hyaluronan content than the plants described in the literature, the preparation of such food / feed requires lesser quantities of harvestable parts, propagating material, processable parts. or consumable parts of genetically modified plants according to the invention. If consumable parts of genetically modified vegetables according to the invention are consumed, for example, directly as an alleged "nutraceutical", a positive effect can be obtained by ingesting relatively small amounts of sub-substance. This may be of particular significance, among others, in the production of feed, provided that feed which also has a high content of plant components is inadequate as animal feed of various animal species.

Because of the high ability of hyaluronan to bind to water, harvestable parts, propagating material, processable parts or consumable parts of genetically modified vegetables according to the invention further have the advantage that less thickeners are required when food / feed for solidified animals are produced. Thus, for example, jelly production requires less sugar, which is associated with an additional positive effect on health. In the production of food / feed requiring the dehydration of raw vegetable material, the advantage of using harvestable parts, propagating material, processable parts or consumable parts of genetically modified plants according to the invention is that water has to be removed from the plant material in question, resulting in lower production costs and, due to smoother preparation methods (eg lower and / or more limited thermal input, high nutritional value of the foodstuff / Thus, for example, in the production of ketchup, less energy has to be introduced in order to obtain the desired advice.

The present invention further provides a process for preparing a vegetable which synthesizes hyaluronan, which process comprises

a) genetically modifying a plant cell, wherein the genetic modification comprises steps i to iii below

i) introduction of a foreign nucleic acid molecule encoding a hyaluronan synthase into the plant cell

ii) introduction of a genetic modification in the plant cell, genetic amodification resulting in an increased activity of a protein that exhibits the (enzymatic) activity of a GFAT compared to corresponding non-genetically modified wild-type plant cells.

iii) introduction of a genetic modification in the plant cell, genetic amodification resulting in an increased activity of a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH compared to non-genetically modified wild-type plant cells corresponding wherein steps ia iii may be performed in any order individually, or any combinations of steps ia iii may be performed simultaneously

b) regenerate a plant from plant cells of step a);

c) generating, if appropriate, additional plants using plants according to step b), wherein, if appropriate, plant cells are isolated from plants according to steps b) or c) and process steps a) c) are repeated until a plant is generated which has a foreign nucleic acid molecule encoding a hyaluronan synthase and an increase in activity of a protein that exhibits the (enzymatic) activity of a GFAT compared to plant-type cells. corresponding non-genetically modified and increased activity of a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH compared to corresponding non-genetically modified wild-type plant cells.

The present invention preferably relates to processes for preparing a vegetable which synthesizes hyaluronan, which process comprises

(a) genetically modifying a plant cell, wherein the genetic modification comprises steps i to iii below in any order, or any combination of steps i to iii may be performed individually or simultaneously;

i) introduction of a foreign nucleic acid molecule that encodes a hyaluronan synthase into the plant cell

ii) introduction of a genetic modification in the plant cell, genetic amodification resulting in an increased activity of a protein that exhibits the (enzymatic) activity of a GFAT compared to corresponding non-genetically modified wild-type plant cells.

iii) introduction of a genetic modification in the plant cell, genetic amodification resulting in an increased activity of a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH compared to non-genetically modified wild-type plant cells corresponding

b) regenerate a plant from plant cells that comprise genetic modification according to the steps

i) a) i

ii) a) ii

iii) a) iii

iv) a) i and a) ii,

v) a) i and a) iii,

vi) a) ii and a) iii, or

vii) a) i and a) ii and a) iii

c) introduce into plant plant cells according to step

i) b) i a genetic modification according to step a) ii,

ii) b) i a genetic modification according to step a) iii,

(iii) (b) a genetic modification according to step (a) and simultaneously a genetic modification according to step (a) iii;

(iv) (b) (ii) a genetic modification according to step (a) (i);

v) b) ii a genetic modification according to step a) iii,

vi) b) ii a genetic modification according to step a) ie simultaneously a genetic modification according to step a) iii,

vii) b) iii a genetic modification according to step a) i,

viii) b) iii a genetic modification according to step a) ix) b) iii a genetic modification according to step a) ie simultaneously a genetic modification according to step a) ii,

x) b) iv a genetic modification according to step a) iii,

xi) b) ν a genetic modification according to step a) ii, or

xii) b) saw a genetic modification as per step a) ie regenerate a plant

(d) introduce into plant plant cells according to step

i) c) i a genetic modification according to step a) iii,

ii) c) ii a genetic modification according to step a) ii,

(iii) c) iv a genetic modification according to step a) iii,

iv) c) ν a genetic modification according to step a) ii,

(v) c) vii a genetic modification according to step a) ii,

vi) c) vii a genetic modification according to step-a) i, or

vii) c) ix a genetic modification according to step a) i regenerate a plant

(e) generate, as appropriate, additional plants with the aid of vegetables in accordance with any of steps b) vii, c) iii, c) vi, c) x, c) xi, c) xii or any of the following of steps d) iad) vii.

The genetic modifications introduced (according to step a) to the plant cell could, in principle, be any kind of modification resulting in an increase in the activity of a protein that exhibits (enzymatic) activity of a GFAT and an increase in activity. of a protein that exhibits the (enzymatic) activity of a UDP-glucose dehydrogenase.

Regeneration of plants according to step b) and, if appropriate, step c) and d) of the processes according to the invention may be carried out using methods known to one skilled in the art (described, for example, in "Plant Cell Culture"). Protocols, 1999, edited by RD Hall, Humana Press, ISBN 0-89603-549-2).

The generation of additional plants (depending on the process according to step c) or step e)) of the processes according to the invention may be carried out, for example, by vegetative propagation (eg via cuts, tubers or callus culture and regeneration). deplants intact) or via generative propagation. In this context, generative propagation usually occurs under controlled conditions, that is, selected plants with specific characteristics are hybridized to each other and multiplied. Selection preferably takes place such that additional plants (depending on the process generated according to step c) or step e)) comprise the modifications introduced in the preceding steps.

In processes according to the invention for preparing hyaluronan synthesizing plants, genetic modifications to generate the genetically modified vegetable cells according to the invention may be carried out simultaneously or in successive steps and in any combination. Both wild-type plants and wild-type plant cells could be used as a starting point for which a foreign nucleic acid molecule encoding a hyaluronan synthase has not yet been introduced and for which a genetic modification that increases protein activity presenting the (enzymatic) activity of a GFAT compared to corresponding non-genetically modified wild-type plant cells has not yet been introduced and for which a genetic modification that increases the activity of a protein that exhibits the (enzymatic) activity of a UDP-GIc -DH compared to corresponding non-genetically modified wild-type vegetable cells has not yet been introduced, or plant cells or plants that have already been genetically modified and for which a nucleic acid molecule encoding a hyaluronan synthase has already been introduced and / or a genetic modification to increase the activity of a protein that exhibits (enzymatic) activity of a GFAT compared to corresponding non-genetically modified wild-type plant cells that have already been introduced and / or for which a genetic modification to increase the activity of a protein that exhibits (enzymatic) activity of a GFAT compared to corresponding non-genetically modified wild-type plant cells that have already been introduced. Here, it is immaterial that the same method as for the genetic modification that Jesuits increase in the activity of a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH is used for successive genetic modifications resulting in an increase in activity. of a protein that exhibits the (enzymatic) activity of a GFAT, provided that both genetic modifications together result in an increase in activity of a protein that exhibits the (enzymatic) activity of a GFAT and a protein that exhibits the activity ( UDP-GIc-DH in the same plant cell. It is also immaterial that a method is used to introduce a foreign nucleic acid molecule encoding a hyaluronan synthase into the cellvegetal.

In a further embodiment of the invention according to the invention for preparing a plant which synthesizes hyaluronan, the genetic modification consists of introducing at least one foreign nucleic acid molecule into the plant cell genome, wherein the presence or expression of of the foreign nucleic acid molecule (s) results in an increase in the activity of a protein that exhibits the activity (enzymatic) of a GFAT and a protein that exhibits the activity (enzymatic) of a UDP-GIc -DH in the same plant cell.

In a further embodiment of the invention according to the invention for preparing a hyaluronan synthesizing plant, the genetic modification consists of introducing at least one foreign nucleic acid molecule or a plurality of foreign nucleic acid molecules into the cell genome. in which the foreign nucleic acid molecule (s) comprises / comprises a coding sequence for a hyaluronan synthase and a coding sequence for a protein (enzymatic) ) of a GFAT and a protein coding sequence that exhibits the (enzymatic) activity of a UDP-GIc-DH.

As already described above with respect to the genetically modified foreign introduced nu-clicic acid molecule into the plant cell or plant which is introduced in step a) of the processes according to the invention for the preparation of a hyaluronan synthesizing plant may be a individual nucleic acid molecule or a plurality of nucleic acid molecules. Thus, foreign nucleic acid molecules that encode a hyaluronan synthase and / or encode a protein that exhibits the (enzymatic) activity of a GFAT and / or that encodes a protein exhibits the (enzymatic) activity of a UDP-GIc. -DH may be present together in a single nucleic acid molecule, or two of the mentioned foreign nucleic acid molecules may be present together in a single nucleic acid molecule and the third foreign nucleic acid molecule may be present in another nucleic acid molecule. in any possible combination, or all of the above mentioned foreign nucleic acid molecules, may be present if present in separate individual nucleic acid molecules.

In addition, 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 cell or wild-type plant, mutant cells or v mutants that are distinguished by the fact that they already show an increase in the activity of a protein that exhibits the (enzymatic) activity of a GFAT and / or an increase in the activity of a protein that exhibits (enzymatic) activity of a UDP- GIc-DH. If the mutant cell or mutant already shows an increase in activity of a protein that exhibits (enzymatic) activity of a GFAT or an increase in activity of a protein that exhibits (enzymatic) activity of a UDP-GIc-DH With the corresponding wild-type plant cells or jungle-like plants, a process according to the invention is sufficient to prepare a plant which synthesizes hyaluronan such that a foreign nucleic acid molecule encoding a hyaluronan synthase and a modification genetics resulting in an increase in the activity of a protein that exhibits the (enzymatic) activity of a UDP-GIc-DH or an increase in the activity of a protein that exhibits the (enzymatic) activity of a GFAT compared to cells corresponding non-genetically modified wild-type plant is introduced into that mutant or mutant cell. If the mutant cell or mutant already shows an increase in the activity of a protein that exhibits the (enzymatic) activity of a GFAT and an increase in the activity of a protein that exhibits 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 encoding a hyaluro nan synthase may be introduced into that mutant or mutant cell to carry out a process according to the invention for preparing a mutant. plant that synthesizes hyaluronan.

All of the above with reference to the use of mutants for the preparation of genetically modified plant cells according to the invention or genetically modified plants according to the invention apply in a similar manner in this report.

In preferred embodiments, the present invention relates to processes according to the invention for the preparation of a hyaluronan synthesizing plant, wherein the nucleic acid molecule encoding a hyaluronan synthase in step a) is selected from the group consisting of:

(a) nucleic acid molecules characterized by the fact that they encode a viral hyalurane synthase,

b) nucleic acid molecules characterized by the fact that they encode a hyaluronan synthase of an infectious virus Chlorellal

c) nucleic acid molecules characterized by the fact that they encode a hyaluronan synthase of a Chlorella 1 Parameciumbursaria virus, d) nucleic acid molecules characterized by the fact that they encode a Chlorella 1 Parameciumbursaria strain H1 virus,

e) nucleic acid molecules characterized in that the codons of the nucleic acid molecule encoding a hyaluronan synthase are modified compared to the codons of the nucleic acid molecule encoding hialuronan synthase in the source organism of hyaluronan synthase,

f) Nucleic acid molecules characterized by the fact that the hyaluronan synthase codons are modified as soon as they are adapted to the frequency of use of the plant cell or plant codons so that they must be integrated or integrated,

g) Nucleic acid molecules characterized by the fact that they encode a hyaluronan synthase having the amino acid sequence shown under SEQ ID NO. 2 or that they encode 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,

(h) nucleic acid molecules characterized in that they encode for a protein whose amino acid sequence may be derived from the coding region for the nucleic acid sequence inserted in plasmid DSM16664 or that they are encoded with 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 more preferably at least 98% identical to the amino acid sequence which may be derived from the decoding region of the inserted nucleic acid sequence. on plasmidDSM16664,

(i) nucleic acid molecules comprising a nucleic acid sequence shown under SEQ ID NO. 1 or SEQ ID NO. Or at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95% and more preferably at least 98% identical to the nucleic acid sequence shown under SEQ ID NO. . 1 or SEQ ID NO. 3,

j) nucleic acid molecules that comprise the nucleic acid sequence inserted into plasmid DSM16664 or which is at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95% and preferably at least 98%. identical to the nucleic acid sequence inserted into plasmid DSM16664,

k) nucleic acid molecules encoding a hyaluronan synthase, wherein the nucleic acid sequences encoding the hyaluronansynthase are linked to regulatory elements (promoters) that initiate transcription in plant cells or

I) nucleic acid molecules according to k) wherein the promoters are tissue-specific promoters, particularly and preferably promoters that initiate transcription initiation specifically in tuber, fruit or vegetable seed cells.

In preferred embodiments, the present invention relates to processes according to the invention for preparing a plant synthesizing hyaluronan, wherein the protein-encoding nucleic acid molecule exhibits the activity of a GFAT is selected from the group consisting of:

(a) nucleic acid molecules characterized by the fact that they encode a protein which exhibits the activity of a GFAT which is derived from bacteria, animals or plants, preferably from Escherichiacoliou mouse,

b) nucleic acid molecules characterized by the fact that they encode a protein that exhibits the activity of a Chlorella infecting virus GFAT;

c) nucleic acid molecules characterized by the fact that they encode a protein that exhibits the activity of a Chlorella Paramecium bursaria GFAT, d) nucleic acid molecules characterized by the fact that the codons of the nucleic acid molecule encoding a protein that has the activity of a GFAT are modified compared to nucleic acid molecule codons encoding the corresponding protein that exhibits the activity of a GFAT from the source organism,

e) nucleic acid molecules characterized by the fact that the codons of the protein displaying the activity of a GFAT are modified in such a way that they are adapted to the frequency of use of the codons of the plant cell or plant in whose genome they are. must be integrated or integrated,

f) nucleic acid molecules encoding a protein which has the amino acid sequence shown under SEQ ID NO. 8 or a protein having the amino acid sequence shown under SEQID NO. 10 or a protein having the amino acid sequence shown under SEQ ID NO. 12;

g) Nucleic acid molecules encoding a protein whose sequence is at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95%, and most preferably at least 98% identical to the sequence. of amino acids shown under SEQ ID NO. 8 or under SEQ ID NO. 10 or under SEQ ID NO. 12;

h) nucleic acid molecules comprising the nucleic acid sequence shown under SEQ ID NO. 7 or a sequence complementary to this or the nucleic acid sequence shown under SEQ ID NO. 9or a sequence complementary to this or the nucleic acid sequence shown under SEQ ID NO. 11 or a sequence complementary to this or nucleic acid sequence shown under SEQ ID NO. 13 or a sequence complementary to it;

(i) nucleic acid molecules which are at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95% and more preferably at least 98% identical to the nucleic acid sequences described below. H);

j) nucleic acid molecules that hybridize under stringent conditions to at least one strand of the nucleic acid sequences described under f) or h);

k) nucleic acid molecules whose nucleotide sequence differs from the sequence of nucleic acid molecules mentioned under f) or h) due to the degeneration of the genetic code; and

(I) nucleic acid molecules which are fragments, variantsalelic and / or derived from the nucleic acid molecules mentioned above (b), (c), (d), (e), (f) or (h);

m) Nucleic acid molecules encoding a protein that exhibits GFAT activity, wherein the nucleic acid sequences encoding a GFAT protein are linked to regulatory elements (promoters) that initiate transcription in plant cells. or

n) nucleic acid molecules according to m), wherein the promoters are tissue-specific promoters, particularly and preferably promoters that initiate transcription initiation specifically into plant tuber, leaf, fruit or seed cells.

In preferred embodiments, the present invention relates to processes according to the invention for preparing a plant synthesizing hyaluronan, wherein the foreign nucleic acid molecule encoding a protein displaying UDP-GIc-DH activity is selected from the group consisting of in:

(a) nucleic acid molecules characterized by the fact that they encode a protein that exhibits the activity of a UDP-GIc-DH originating from viruses, bacteria, animals or plants;

b) nucleic acid molecules characterized by the fact that they encode a protein that exhibits the activity of a UDP-GIc-DH of an Chlorella infecting virus;

c) nucleic acid molecules characterized by the fact that they encode a protein that exhibits the activity of a UDP-GIc-DH of a Chlorella virus of Paramecium bursaria,

d) Nucleic acid molecules characterized in that the codons of the nucleic acid molecule encoding a protein that has the activity of a UDP-GIc-DH are modified compared to the codons of the nucleic acid molecule encoding the corresponding protein that presents the activity of a UDP-GIc-DH from the source organism,

e) Nucleic acid molecules characterized by the fact that the codons of the protein displaying the activity of a UDP-GIc-DH are modified in such a way that they are adapted to the frequency of use of the codons of the plant cell or plant in whose genome they should be. integrated or integrated,

f) nucleic acid molecules encoding a protein which has the amino acid sequence shown under SEQ ID NO. 5;

g) nucleic acid molecules encoding a protein whose sequence is at least 70%, preferably at least 80%, preferably at least 90%, especially preferably at least 95%, and most preferably at least 98% identical to the sequence. of amino acids shown under SEQ ID NO. 5;

h) nucleic acid molecules comprising the nucleotide sequence shown under SEQ ID NO. 4 or a sequence complementary to this or the nucleotide sequence shown under SEQ ID NO. 6 or a sequence complementary to this;

i) nucleic acid molecules which are at least 70%, preferably at least 80%, preferably at least 90%, especially preferably 95% and more preferably at least 98% identical to the nucleic acid sequences described under h);

j) nucleic acid molecules that hybridize under stringent conditions to at least one strand of the nucleic acid molecules described under f) or h);

k) nucleic acid molecules whose nucleotide sequence differs from the sequence of nucleic acid molecules mentioned under f) or h) due to the degeneration of the genetic code; and

I) nucleic acid molecules which are fragments, variantselelic and / or derived from the nucleic acid molecules mentioned above), b), c), d), e), f) or h),

m) Nucleic acid molecules encoding a protein that exhibits the activity of a UDP-GIc-DH wherein the nucleic acid sequences encoding a protein that exhibits the activity of aUDP-GIc-DH are linked to regulatory elements (promoters) that initiate transcription in plant cells or

n) nucleic acid molecules according to m), wherein the promoters are tissue specific, particularly and preferably promoters, that initiate transcription initiation specifically in plant cells, leaf, fruit or plant seeds.

In a preferred embodiment of the present invention, the processes for preparing a hyaluronan synthesizing plant refer to processes for preparing a plant which synthesizes at least 100, preferably at least 600, particularly and preferably at least 1. .000, especially and preferably at least 1,500 pg of hyaluronan per g of fresh pasta (MF) of plant material.

In a further preferred embodiment, processes according to the invention for preparing a hyaluronon synthesizing plant are used to prepare genetically modified plants in accordance with the invention.

The present invention also provides plants obtainable by a process according to the invention for preparing a hyaluronan synthesizing plant.

The present invention further relates to a hyaluronan preparation process comprising the step of extracting hyaluro-nan from genetically modified plant cells according to the invention, from genetically modified plants according to the invention, from propagating material. according to the invention, from harvestable plant parts according to the invention or from plants or parts thereof obtainable by a process according to the invention for preparing hyaluronan synthesizing plants. Preferably, this process also comprises harvesting of genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagating material according to the invention, harvestable plant parts according to the invention, processable plant parts of according to the invention prior to extraction of hia-luronan, and particularly preferably preferably the step of cultivating genetically modified plant cells according to the invention or genetically modified plants according to the invention prior to harvesting.

In contrast to bacterial or animal tissues, plant tissues have no hyaluronidases and do not contain any hyaladerins. 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 decayed hyaluronan-containing plant cells can be further purified using methods known to those skilled in the art, such as for example repeated precipitation with ethanol. A preferred method for purifying dehyaluronan is described under General Methods item 3.

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 propagating material according to the invention from plant parts capable of harvesting according to the invention or from plants or parts thereof obtained by a process according to the invention to prepare hyaluronan synthesizing plants.

The present invention also provides for 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, Processable plant parts according to the invention or plants obtainable by a process according to the invention for preparing hyaluronan. The present invention furthermore relates to compositions comprising genetically modified plant cells according to the invention. Here it is immaterial whether plant cells are intact or no longer intact because they have been destroyed, for example by processing. The compositions are preferably animal food or feed, pharmaceuticals or cosmetics.

The present invention preferably provides compositions comprising genetically modified plant cell components according to the invention, genetically modified plants according to the invention, propagating material according to the invention, harvestable plant parts according to the invention or from plants obtainable by a process according to the invention comprising recombinant nucleic acid molecules, wherein the recombinant nucleic acid molecules are characterized in that they comprise nucleic acid molecules encoding a hyaluronansintase and proteins that exhibit (enzymatic) activity of a GFAT and proteins that exhibit (enzymatic) activity of a UDP-GIc-DH.

A stable integration of foreign nucleic acid molecules into the genome of a plant or plant cell results in foreign nucleic acid molecules that are flanked after integration into the genome plant or plant cell via plant nucleic acid genomic sequences.

Accordingly, in a preferred embodiment, compositions according to the invention are characterized in that the recombinant nucleic acid molecules present in the composition according to the invention are flanked by plant nucleic acid genomic sequences.

Here the plant nucleic acid genomic sequences may be any sequences naturally present in the plant cell or plant genome used to prepare the composition.

Recombinant nucleic acid molecules present in the compositions according to the invention may be single or multiple recombinant nucleic acid molecules wherein nu-cyclic acid molecules encoding a hyaluronan synthase and proteins displaying (enzymatic) activity of a GFAT and proteins exhibiting The (enzymatic) activity of a UDP-GIc-DH is present in a nucleic acid molecule, or those in which the mentioned nucleic acid molecules may be present in separate recombinant nucleic acid molecules. Nucleic acid molecules encoding a hyaluronan synthase or encoding a protein that exhibits (enzymatic) activity of a GFAT or encoding a protein that exhibits (enzymatic) activity of a UDP-GIc-DH may be present together in one a single recombinant nucleic acid molecule, or two of the mentioned nucleic acid molecules may be present together in a single recombinant nucleic acid molecule and the third nucleic acid molecule may be present in another recombinant nucleic acid molecule in any possible combination, or all three mentioned nucleic acid molecules may in each case be present in separate separate recombinant nucleic acid molecules. Depending on how nucleic acid molecules encoding a hyaluranosynthase or encoding a protein displaying the (enzymatic) activity of a GFAT or encoding a protein displaying the (enzymatic) activity of a UDP-GIc-DH are present. in a composition according to the invention, they may be flanked by identical or different nucleic acid plant genomic sequences.

Such compositions according to the invention which comprise recombinant nucleic acid molecules may be demonstrated using methods known to one of skill in the art, such as, for example, hybridization based methods or, preferably, PCR (chain reaction) based methods. polymerase).

Preferably compositions according to the invention comprise at least 0.005%, preferably at least 0.01%, particularly preferably at least 0.05% and especially preferably at least 0.1% hyaluronan. Preferably compositions according to the invention. with the invention comprise at most 5%, preferably at most 2%, particularly preferably at most 1% and especially at least 0.5% hyaluronan.

As already mentioned above, genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagating material according to the invention, harvestable plant parts according to the invention, plant parts may be used. processable according to the invention, consumable plant parts according to the invention or plants obtainable by a process according to the invention for preparing foodstuffs or animal feed. 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 may be applied to areas under agricultural cultivation to achieve increased soil binding water. In addition, genetically modified plants according to the invention or genetically modified plant cells according to the invention may be used to prepare drying agents (e.g. for use when moisture sensitive articles) or as water absorbers. liquids (eg in diapers or for absorption of spilled aqueous liquids). For such applications, genetically modified whole plants according to the invention, parts of genetically modified plants according to the invention or fragmented plants (e.g., ground) genetically modified plants according to the invention or parts of the plant according to the invention may be used. the invention as required. Suitable for applications where ground plants or plant parts are used are in particular parts of the plant containing hyaluronan, but only a low proportion of water. These are preferably cereal plant grains (maize, rice, wheat, cenote, 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 genetically modified plant cells according to the invention or genetically modified plants according to the invention are used.

The present invention also provides processes for preparing a composition according to the invention using 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, processable 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. Processes for preparing a composition according to the invention are preferably processes for preparing food or animal feed, processes for preparing a pharmaceutical product or processes for preparing a cosmetic product.

Processes for preparing food or animal feed are well known to those skilled in the art. Processes for using genetically modified plants according to the invention or parts of the plant according to the invention in industrial areas are also known to those skilled in the art and include, but are not limited to, comminution or milling of genetically modified plants according to the invention. or parts of the plant according to the invention; however, they are not exclusively limited to this. Some of the advantages of using subject matter according to the invention for the preparation of food / feed or for use in industrial areas have already been described above.

A process according to the invention for preparing a composition is particularly preferably a process for preparing a composition comprising hyaluronan. Compositions obtainable by a process for preparing a composition according to the invention are also provided by the present invention.

The present invention also relates to the use of genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagating material according to the invention, harvestable plant parts according to the invention. invention, processable plant parts according to the invention, consumable plant parts according to the invention or plants obtainable by a process according to the invention for the preparation of a hyaluronan synthesizing plant for the preparation of a color composition with 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, plant parts harvestable according to the invention, parts of the processable plants according to the invention, consumable plant parts according to the invention or plants obtainable by a process according to the invention for preparing a hyaluronan synthesizing plant for preparing food or para-animal feed, for preparing a pharmaceutical or for the preparation of a cosmetic product.

SEQ ID NO. 1: Sequence of nucleic acids encoding a hyaluronan

Chlorella 1 synthase from Paramecium bursaria.SEQ ID NO. 2; Amino acid sequence of a hyaluronan synthase of the Chlorella 1 Paramecium bursaria virus. The amino acid sequence shown may be derived from SEQ ID NO 1.SEQ ID NO 3: Synthetic nucleic acid sequence encoding a Paramecium bursaria Chlorella 1 virus hyaluronan synthase. The codon synthesis of the sequence shown was performed in detail to be adapted to the use of codons in vegetable cells. The nucleic acid sequence shown encodes a protein that has the amino acid sequence shown under SEQ ID NO 2.

SEQ ID NO 4: Nucleic acid sequence encoding a protein that has the activity of a Chlorella virus UDP-GIc-DH

1 Paramecium bursaria.

SEQ ID NO 5: Amino acid sequence of a protein that has the activity of a Chlorella 1 Parame-cium bursaria virus UDP-GIc-DH. The amino acid sequence shown may be derived from SEQ ID NO 4.

SEQ ID NO 6: Synthetic nucleic acid sequence encoding a protein that has the activity of a ChecoreIIa 1 Paramecium bursaria UDP-GIc-DH. The codon synthesis of the sequence shown was performed in such a way that it was adapted to the use of codons in plant cells. The nucleic acid sequence shown encodes a protein having the amino acid sequence shown under SEQ ID NO 5.

SEQ ID NO. 7: Nucleic acid sequence encoding a protein that has the activity of a mouse GFAT-1. ID ID NO 8: Amino acid sequence of a protein that has

the activity of a mouse GFAT-1. The amino acid sequence shown may be derived from SEQ ID NO 7.

SEQ ID NO 9: Nucleic acid sequence encoding a protein that has the activity of a mouse GFAT-2.

SEQ ID NO 10: 'Amino acid sequence of a protein that has the λactivity of a mouse GFAT-2. The amino acid sequence shown may be derived from SEQ ID NO 9.

SEQ ID NO 11: Nucleic acid sequence encoding a protein that has the activity of an Escherichia coli GFAT.

SEQ ID NO 12: Amino acid sequence of a protein displaying the activity of an Escherichia coli GFAT. The amino acid sequence shown may be derived from SEQ ID NO 11. SEQ ID NO 13: Synthetic nucleic acid sequence encoding a protein that exhibits the activity of an Escherichiacoli GFAT. The codon synthesis of the sequence shown was performed in such a way that it was adapted to the use of codons in plant cells. The nucleic acid sequence shown encodes a protein that has 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

Figure 1: Shows a calibration curve and the corresponding regression line equation used to calculate the vegetable tissue hyaluronan content. The calibration curve was established with the aid of the commercial test kit (Corgenix Hyaluronic Acid (HA) Test Kit, Inc., Colorado, USA, Prod. No. 029-001) and the standard solutions supplied with it.

All literature cited including, but not limited to accession numbers of nucleic acid and amino acid sequences are incorporated in the description by reference.

General Methods

Methods that may be used in connection with the present invention are described below. These methods are specific modalities; however, the present invention is not limited to such methods. It is well known in the art that the invention may be carried out in the same manner by modifying the methods described and / or by substituting individual methods or parts of methods for alternative methods or alternative parts of methods.

1. Potato Plant Transformation

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

To detect the presence of hyaluronan and to determine the hyaluronan content in plant tissue, plant material was processed as follows: 200 μl of water (demineralized, conductivity = 18 M Ω) was 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 seconds under 30 Hz). An additional 800 μΙ of water (demineralised, conductivity = 18 ΜΩ) was then added, and the mixture mixed well (using, for example, a Vortex mixer). Cell debris and insoluble components were separated from the supernatant by centrifugation under 16,000 xg for 5 minutes.

3. Hyaluronan Purification

Approximately 100 grams of tubers were peeled, cut into pieces of a size of approximately 1 cm3 and, after addition of 100 ml of water (demineralized, conductivity = 18 ΜΩ), comminuted in Warring mixer at full speed for approximately 30 seconds Cell debris was then removed using a tea sieve. Cell debris that had been removed was resuspended in 300 ml water (demineralized, conductivity = 18 ΜΩ) and removed again using a tea sieve. The obtained two suspensions (100 ml + 300 ml) were combined and centrifuged at 13000 xg for 15 minutes. NaCl was added to the centrifugation supernatant obtained until a final concentration of 1% was reached. After NaCl had penetrated the solution, precipitation was performed by adding twice the volume of ethanol followed by complete mixing and incubation at -20 ° C overnight. The mixture was then centrifuged at 13,000 xg for 15 minutes. The pelleted precipitate obtained after such centrifugation was dissolved in 100 ml buffer (50 mM JrisHCI, pH 8, 1 mM CaCl 2) and Kproteinase was then added to a final concentration of 100μg / ml and the solution was incubated at 42 ° C for 2 hours. This was followed by 10 minutes of incubation at 95 ° C. Once again, NaCl was added to this solution until a final concentration of 1% was reached. After the NaCl solution had entered the solution, further precipitation was performed by adding twice the volume of ethanol, complete mixing and incubation at -20 ° C for approximately 96 hours. This was followed by 15 minutes of centrifugation at 13,000 xg. The pelleted precipitate obtained after this centrifugation was dissolved in 30 ml of water (demineralized, conductivity = 18 ΜΩ), and once again NaCl was added to a final concentration of 1%. By adding twice the volume of ethanol, complete mixing and incubation at -20 ° C overnight, another precipitation was performed. The precipitate obtained after subsequent centrifugation at 13,000 xg for 15 minutes was dissolved in 20 ml of water (demineralized, conductivity = 18 ΜΩ).

Further purification was performed by centrifugal filtration. For this purpose, in each case 5 ml of the dissolved precipitate was 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 about 3 ml of the membrane. solution above remain in the filter. Twice more, in each case 3 ml of water (demineralized, conductivity = 18 ΜΩ) was then added to the above membrane solution and in freshly centrifuged under identical conditions until only about 3 ml of the above solution remained in the filter at the end. Solutions still present above the membrane after centrifugal filtration were removed, and the membrane was rinsed repeatedly (three to five times) with approximately 1.5 ml of water (demineralized, conductivity = 18 ΜΩ). All solutions that were still present above the membrane and the obtained rinse solutions were combined, NaCl was added to a final concentration of 1%, after NaCl had entered the solution, twice the volume of ethanol was added. In addition, 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 37 Pa (0.37 mbar). ChristAIpha 1-4 freezing of Christ, Osterode, Germany).

4. Hyaluronan detection and determination of hyaluronan content

Hialuronan was detected using a commercial assay (Corgenix, Inc., Colorado, USA, Prod. No. 029-001) Hyaluronic Acid (HA) Test Kit according to the manufacturer's instructions which are hereby incorporated in the description. by way of reference. The test principle is based on the availability of a protein that specifically binds to hyaluronan (HABP) and is performed similarly to an ELISA, in which the color reaction indicates the content of hyaluronan in the sample examined. Consequently, for quantitative determination of hyaluronan, the samples to be measured should be employed at a concentration such that they are within the established limits (eg dilution of the sample in question or use of water to extract hyaluronan from plant tissue, depending on whether a limit was exceeded or not reached).

In parallel batches, aliquots of samples to be determined were initially subjected to hyaluronidase digestion and then measured using the commercial test (Corgenix, Inc., Colorado, USA, Prod. No. 029-001). Hyaluro-nidase digestion was performed using 400 μΙ of potato tuber extract in hyaluronidase buffer (0.1 M potassium phosphate buffer, pH 5.3; 150 mM NaCl) by addition of 5 pg (~ 3 units) hyaluronidase (Sigma hyaluronidase type III, Prod. No. H 2251) and incubation at 37 ° C for 30 minutes.

In each case at a 1:10 dilution, all samples were then used to determine the hyaluronan content.

5. Calculation of standard deviations

The established standard deviations were calculated using the formula below: square root [nXx2- (£ x) 2 / n (n-1)] where χ is the value of measured individual values and is the sum of all measured values used to determine the standard deviation in question.

6. Determining the Activity of a GFAT

The activity of a protein that exhibits umaGFAT activity is determined as described in Rachel et al. (1996, J. Bacteri-Ol 178 (8), 2320-2327).

To distinguish whether a protein has the activity of umaGFAT-1 or GFAT-2, the method described in Hu et al. (2004, J. Biol. Chem. 279 (29), 29988-29993) .7 is used. Determination of activity of a UDP-GIc-DH

The activity of a protein that exhibits the activity of a UUDP-GIc-DH is determined as described in Spicerl et al. (1998, J. Bacterioi. 273 (39), 25117-25124).

8. Transformation of tomato plants

Tomato plants were transformed with the aid of Agro-bacterium using the method described in US 5,565,347. .

Examples

1. Preparation of the IR 47-71 Plant Expression Vector Plasmid pBinAR is a derivative of the binary vector plasmid pBin19 (Bevan, 1984, NucI Acids Res 12: 8.711-8.721) which was constructed as follows:

a 529 base-length (bp) fragment that comprised nucleotides 6,909-7,437 of the cauliflower mosaic virus 35S promoter was isolated as the EcoR UKpn I fragment of plasmid pDH51 (Pietrzak et al., 1986, Nucleie Aeids Res 14, 5,858) and linked between the Eco R I and Kpn I restriction sites of the pUC18 polylinker. In this way, pUC18-35S oplasmid was formed. Using the restriction endonucleases Hind III and Pvu II, a 192 bp length fragment that included the signaling-degeneration signal (3 'terminus) of the gene for Oetopine Synthase (gene 3) from the Ti pTiACH5 T-DNA plasmid (Gielen et al, 1984, EMBO Journal 3,835-846) (nucleotides 11,749-11,939) was isolated from plasmid pAGV40 (Herrera-Estrella et al., 1983, Nature, 303, 209-213). After addition of Sph I ligands to the Pvu II restriction site, the fragment was ligated between the pUC18-35S Sph I and HindIII restriction sites. This provided the plasmid, pA7. Here, all polylinker comprising the 35S promoter and Ocs terminator was removed using EcoR I and Hind Ille linked to the appropriately cleaved vector pBin19. This provided the pBinAR vegetable expression vector (Höfgen and Willmitzer, 1990, Plant Science 66, 221-230).

The Solanum tuberosum patatin B33 gene promoter (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) was, as a Dra I fragment (nucleotides -1.512 - +14), ligated to the Sst I cleaved vector. pUC19 whose ends had been blunted using T4-DNA polymerase. This provided plasmid pUC19-B33. From this plasmid, the B33 promoter was removed using EeoR I and Sma I and ligated to the appropriately restricted pBinAR vector. This provided the pBinB33 vegetable expression vector.

To facilitate additional cloning steps, the Multiple Cloning Site (MCS) has been 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 pBinB33 Sal I and Kpn I restriction sites. The oligonucleotides used for this purpose were as follows: 5'-TCg ACA ggC CTg gAT CCT TAA TTA AAC TAg TCT CgA ggA gCT Cgg TAC-3'5'-CgA gCT CCT CgA gAC TAg TTT AAT TAA ggA TCC Agg CCT g-3 '

The plasmid obtained was named IR 47-71.

2. Preparation of pBinARHyg Vegetable Expression Vector

The fragment comprising the 35S promoter, the Ocse Multiple Cloning Site terminator was removed from pA7 using the EcoR I and Hind III restriction endonucleases and cloned into the pBIBHyg vector (Becker, 1990, Nucleic Acids Res. 18, 203). had been cut using the same restriction en-donucleases. The plasmid obtained was named pBinARHyg.

3. Preparation of IC 317-204 cloning vector

Nucleic acid fragments comprising the OCS terminator were isolated from plasmid IR 47-71 using the Xho I and Hind III restriction endonucleases and cloned into the pBlueScript KS vector (from Stratagene, Prod. No. 212207) which had been cut with the same restriction endonucleases. The plasmid obtained was named IC 306-204.

Nucleic acid fragments comprising the B33 promoter were isolated from plasmid IR 47-71 using the restriction endonucleases Bam H1 and Eco R1 and cloned into the pBlueScript KS vector (from Stratage-ne, Prod. No. 212,207) which had been cut with the same restriction endonucleases. The plasmid obtained was named IC 314-204.

The OCS terminator was isolated from IC 306-204 using the Bam H1 restriction endonuclease and cloned into plasmid IC 314-204 which had been cut with the same restriction endonuclease. The obtained plasmid was called IC 317-204.

4. Synthesis of nucleic acid molecules

(a) Synthesis of nucleic acid molecules encoding a virus hyaluronan synthase 1 Chlorella de Paramecium bursaria

The nucleic acid sequence encoding a Chlorella Paramecium bursaria virus 1 hyaluronansynthase (HAS) was synthesized by Medigenomix GmbH (Munich, Germany) and cloned into Invi-trogen vector pCR2.1 (Prod. No. K2000-01). The plasmid obtained was named IC 323-215. Synthetic sequence of nucleic acids encoding Paramecium bursaria 1Chlorella virus HAS protein is shown under SEQ ID NO 3. The corresponding nucleic acid sequence originally isolated from Parameeium bursaria 1 C-hlorella virus is shown under SEQ ID NO 1. .

b) Synthesis of nucleic acid molecules encoding a protein that exhibits the activity of a virus UDP-GIc-DH 1 Chlorellade Paramecium bursaria

The sequence of nucleic acids encoding a protein that shows the activity of a Chlorella de Parameei-a bursaria 1 UDP-GIc-DH was synthesized by Enteleehon GmbH and cloned into the p-CR4Type Invitrogen vector (Prod. No. K4510-20 ). The obtained plasmid was called IC 339-222. The synthetic nucleic acid sequence encoding the UDP-GIc-DH protein of Parameeium bursaria 1 Chlorella virus is shown under SEQ ID NO 6. The correspondingly isolated nucleic acid sequence of Parameeium bursaria 1 Chlorella virus is shown under SEQ ID NO. 4.

c) Synthesis of nucleic acid molecules encoding a protein that exhibits Eseheriehia eoli GFAT activity

The nucleic acid sequence encoding a protein that exhibits the activity of an Eseheriehia eoli GFAT has been synthesized by Enteleehon GmbH and cloned into the pCR4Tope Invitrogen vector (Prod.No. K4510-20). The plasmid obtained was named IC 373-256. The nucleic acid sequence encoding the protein displaying the activity of an Escherichia coli GFAT is shown under SEQ ID NO 13. The corresponding nucleic acid sequence originally isolated from Esche-richia coli shown under SEQ ID NO 11.

5. Origin of additional nucleic acid molecules

a) Nucleic acid molecules encoding a protein exhibit the activity of a mouse GFAT-1

The nucleic acid sequence encoding a protein that exhibits 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 IMAGE Konsortium (http://image.llnl.gov) and distributed by BioCat GmbH. Here, the cDNA encoding a protein that exhibits the activity of a GFAT-1 has been cloned into the pCMV Sport 6 vector from Invitrogen. The plasmid obtained was named IC 365-256. The nucleic acid sequence, inserted in IC 365-256, which encodes a protein that shows the activity of a Mus musculus GFAT-1 presents, in comparison with the nucleic acid sequence shown under SEQ ID NO 7, a base exchange. from T to C at position 1,090 and a base change from Gem A to position 2,027 These base exchanges do not result in amino acid exchanges of the amino acid sequences encoded by the two different nucleic acid molecules.

The sequence of nucleic acids encoding the protein that shows the activity of a mouse GFAT-1 is shown in SEQID NO 8.

To facilitate subsequent cloning steps, the sequence encoding a protein displaying GFAT-1 activity was isolated using the Xho I and Eco RV restriction endonucleases of IC 365-256 cloned into plasmid pME9 (Stratagene pBlueSkript vector, Prod. No. 212207) which has a modified multiple cloning site which additionally has a Pac I restriction site at the ends, which plasmid had been cut with the same restriction endonucleases. The obtained plasmid was named IC 367-256.

b) Nucleic acid molecules encoding a protein exhibit the activity of a mouse GFAT-2

Nucleic acid molecules encoding a protein that exhibit the activity of a mouse GFAT-2 were purchased from Invitro-gen (Clone ID 4167189, cDNA clone MGC: 18324, IMAGE: 4167189). This is a clone that is produced by I.M.A.G.E. Konsortium (http://image.llnl.gov) and distributed by Invitrogen. Here, cDNA encoding a protein that exhibits the activity of a GFAT-2 is cloned into Invi-trogen's pCMV Sport 6 vector. The plasmid was named IC 369-256. The sequence of nu-clicic acids encoding the protein displaying the activity of a GFAT-2 deMus musculus is shown under SEQ ID NO 9.

6. Preparation of Plant Expression Vector IC 341-222 which comprises a nucleic acid coding sequence for a Chlorella de Paramecium bursaria 1 hyaluronan synthase.

Using restriction digestion with BamH I and Xho I, nucleic acid molecules comprising the hyaluronon synthase coding sequence were isolated from plasmid IC 323-215 and cloned into the BamH I and Xho I restriction sites of plasmid IR 47-71. The vegetable expression vector obtained was. called IC 341 -222.

7. Preparation of the plant-expression vectors IC 370-256 and IC 376-256 which comprise nucleic acid coding sequences of a protein that exhibits the activity of a mouse GFAT-1 and a protein that has the activity of a UDP-GIc-DH virus 1 Chlorella of Paramecium bursaria.

Using restriction digestion with BamH I and Kpn I, nucleic acid molecules that comprise the coding sequence of a protein displaying the activity of a Chlorellade Paramecium bursaria virus 1 UDP-GIc-DH were isolated from plasmid IC 339-222 and clone- plasmid pA7 which had been cut with the same restriction endonucleases. The plasmid obtained was named IC 342-222.

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

In the next step, a nucleic acid fragment comprising the B33 promoter and OCS terminator, which fragment had been isolated from IC 317-204 by restriction digestion using Eco IR, was cloned into the IC 349- Eco IR restriction site. 222 Here, he ensured that the engines (35S and B33) were oriented head to head. The obtained vector was called IC 354-222.

In an additional cloning step, a nucleic acid fragment comprising the protein coding sequence that exhibits the activity of a mouse GFAT-1 was isolated by restriction digestion with IC 367- Xho I and Eco RV. 256 and cloned into plasmid IC 354-222, which had been restricted with Xho I and Ec / 136 II. The expression vector obtained in vegetation was called IC 370-256.

Following a sequence analysis of plasmid IC 370-256, it was found that the nucleic acid coding sequence of the protein which has the activity of a mouse GFAT-1 was modified in two positions compared to the sequence of nucleic acids inserted into plasmid IC 365-256. Compared to the nucleic acid sequence shown under SEQ ID NO 7, the nucleic acid sequence that encodes the protein that exhibits the activity of a mouse GFAT-1 contained in plasmid IC 370-256 exhibits a base exchange. Gem A at position 1.160, a base change from T to C at position 1.190, a base change from T to C at position 1.245 and from G to / A at position2027. This modified nucleic acid sequence encodes a protein with respect to the amino acid sequence shown under SEQ ID NO 8, has an amino acid modification at position 304 of R in Q and position 366 of C in R.

To obtain a vegetable expression vector that comprises the correct nucleic acid sequence that encodes a protein that exhibits the activity of a mouse GFAT-1, the protein coding sequence that exhibits the activity of a mouse GFAT-1 was isolated from IC 365-256 by restriction digestion with Xho I and EcoRV and cloned into plasmid IC 354-222, restricted with Xho I and Ec / 136 II. Vegetable expression ovator obtained was named IC 376-256.

The protein-encoding nucleic acid sequence that shows the activity of a mouse GFAT-1 contained in IC 376-256 is identical to the protein-encoding sequence of a mouse GFAT-1 inserted activity. noplasmid 365-256. The amino acid sequence encoded by this nucleic acid molecule is shown under SEQ ID NO 8.

8. Preparation of Plant Expression Vector IC 372-256 comprising Nucleic Acid Coding Sequences for a Protein has the activity of a mouse GFAT-2 and a Protein for the activity of a Chlorella virus UDP-GIc-DH Para-mecium bursaria.

A nucleic acid fragment comprising the protein coding sequence the activity of a mouse GFAT-2 was isolated from IC 369-256 by restriction digestion with Xho I and Eco RV and cloned into plasmid IC 354-222, restricted with Xho I and Ec / 136 II. The vegetable expression vector obtained was called IC 372-256.

9. Preparation of Vegetable Expression Vector IC 375-271 which comprises nucleic acid coding sequences for a protein has the activity of an Escherichia coli GFAT and a protein has the activity of a 1 Chlorella virus UDP-GIc-DH Para-mecium bursaria.

A nucleic acid fragment comprising the coding sequence of the protein displaying the activity of an Es-cherichia coli GFAT was isolated from IC 373-256 by restriction digestion with Xho Ie Eco RV and cloned into restricted plasmid IC 354-222. by Xho I and Ec / 136II. The vegetable expression vector obtained was called IC 375-271.

10. Transformation of potato plants with expression vectors into vegetables comprising nucleic acid molecules encoding a hyaluronan synthase.

Potato plants were transformed using vegetable expression vector IC 341-222, which comprises a nucleic acid coding sequence of a Chlorella deParamecium bursaria 1 hyaluronan synthase under the control of the patatinade B33 gene promoter Solanum tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) using the method provided under General Methods, item 1. The obtained transgenic potato plants, which were transformed with plasmid IC 341- 222, were named 365 ES.

11. Analysis of transgenic plants transformed with expression vectors in plants comprising nucleic acid molecules encoding a hyaluronan synthase.

a) Constructing a calibration curve A calibration curve was constructed using the standard solutions provided with the commercial test kit (Hygenic Acid Test Kit (HA) from Corgenix, Inc., Colorado, USA, Prod. No. 029 -001) according to the methods described by the manufacturer. To determine extinction under 1,600ng / ml hyaluronan, twice the amount, based on the manufacturer's supplied standard amount, comprising 800 ng / ml hia-luronan was used. In each case, three independent measurement series were performed and the corresponding average determined. This provided the following calibration curve:

<table> table see original document page 90 </column> </row> <table> Table 1: Values for constructing a calibration curve for the quantitative determination of hyaluronan content in plant tissue. With the help of software (Microsoft Office Excel 2002, SP2), the measured values obtained were entered into a diagram and the trend line function equation was determined (see Fig 1). E4SOnm refers to extinction at a wavelength of 450 nm, d.p. is the standard deviation of the calculated mean of the individual values. ι

b) Analysis of potato tubers from 365 ES strains In a greenhouse, individual plants of 365 ES strains were grown in soil in 6 cm pots. In each case, about 0.3 g of potato tuber material from 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 present in the respective plant extracts was determined with the aid of the calibration curve shown in Example 10a) and Figure 1. Here, the supernatant obtained after centrifugation was used at a 1:10 dilution to determine the hyaluronan potent. The following results were obtained for selected plants:

Plant name Weight of plant material employed [g] Extinction E450 Amount of hyalurane [ng / ml] Hialuronan based on fresh mass of plant material [pq / ql365 ES 13 0.297 2,746 14,038 47365 ES 74 0,306 4,000 20,816 68Wild type 0.305 0.111 ndnd

Table 2: "Amount of hyaluronan (in pg of hyaluronan per g of fresh mass) produced by selected independent transgenic plants from 365 ES. Column 1 refers to the plant from which tubers material was harvested (here," wild type "refers to unprocessed plants which, however, have the genotype used as the starting material for the transformation). Column 2 indicates the amount of tuber material from the plant in question used to determine the content of hyaluronan. Column 3 contains the extinction. measured as a 1:10 dilution of the respective plant extract Column 4 was calculated using the linear regression equation (see Figure 1) taking into account the dilution factor as follows: ((value 3 - 0.149 ) / 0.00185) χ 10. Column 5 indicates the amount of hyaluro-nan based on the fresh mass used and was calculated as follows: (column value 4 / column 2 value) / 1,000. "Nd" means undetectable .

12. Transformation of plants that synthesize hyaluronan expression vectors in plants comprising nucleic acid coding sequences of a protein that exhibits the activity of aGFAT and a protein that exhibits the activity of a UDP-GIc-DH.

Potato plants of lines 365 ES 13 and 365 ES 74 were each case transformed with the vegetable expression vectors IC370-256, IC 376-256, IC 372-256 and IC 375-271 using the method provided under General Methods, item 1. .

The transgenic potato plants obtained from strain 365 ES13 that were transformed with plasmid IC 370-256 were named 393 ES.

Transgenic potato plants obtained from strain 365 ES74 that were transformed with plasmid IC 370-256 were named 394 ES.

The transgenic potato plants obtained from strain 365 ES13 that were transformed with plasmid IC 372-256 were named 395 ES.

The transgenic potato plants obtained from strain 365 ES74 that were transformed with plasmid IC 372-256 were named 396 ES.

Transgenic potato plants obtained from strain 365 ES13 that were transformed with plasmid IC 375-271 were named 403 ES.

The transgenic potato plants obtained from strain 365 ES74 that were transformed with plasmid IC 375-271 were named 404 ES.

The transgenic potato plants obtained from strain 365 ES13 that were transformed with plasmid IC 376-256 were named 408 ES.

The transgenic potato plants obtained from strain 365 ES74 that were transformed with plasmid IC 376-256 were named 409 ES.

13. Analysis of transgenic potato plants synthesizing hyaluronan further transformed with vector expression vectors comprising nucleic acid coding sequences of a protein that exhibits the activity of a GFAT and a protein that has the activity of a UDP-GIc- DH.

In a greenhouse, individual plants of the 393 ES1 394ES, 395 ES, 396 ES, 403 ES, 404 ES and 409 ES strains were grown in soil with 6 cm pots. In each case, about 0.3 g of debatata tuber material or individual plant leaves was processed according to the method described under General Methods, item 2. Using the method described under General Methods, item 4, the amount of The hyaluronan contained in the respective plant extracts was determined with the aid of a calibration curve generated according to Example 10a), a calibration curve generated for each series of individual measurements. Here, to determine 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 within the linear range of the calibration curve. Results for plants originating from original multi-plasmid transformations are shown below.

a) Analysis of tubers of strain 393 ES7

For each tuber of individual transgenic plants of the lines named 393 ES, two independent samples were taken and the hyaluronan content in each case determined separately. The mean and standard deviation of the values obtained for the individual measurements of each tuber were then calculated using the formula given under General Method, item 5. The means and standard deviations established for wild-type plants 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 progenies of the wild type (Solanum tuberosum cv. Désirée) and of the strain 365 ES 13, respectively. The following results were obtained for selected plants:

<table> table see original document page 94 </column> </row> <table>

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

b) Analysis of tubers of strain 394 ES

For each tuber of individual transgenic plants from the 394 ES strains, two independent samples were taken and the hyaluronan content in each case determined separately. The mean and standard deviation of the values obtained for the individual measurements of each tuber were then calculated using the formula given under General Method, item 5. The means and standard deviations established for wild-type plants and plants named 365 ES were calculated using calculating the amount of hyaluronan} in tubers of in each case 18 different wild-type plants and 26 different plants of the 365 ES 74 strain which are wild-type (Sola-num tuberosum cv. Désirée) and 365 ES 74 strain respectively. The following results were obtained for selected plants:

<table> table see original document page 95 </column> </row> <table>

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

c) Analysis of tubers of strain 395 ES

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

<table> table see original document page 96 </column> </row> <table> Table 5: Amount of hyaluronan (in μρ of hyaluronan per g of fresh mass) produced in independently selected transgenic plant tubers of the 395 ES line. Column 1 contains the name of the plant from which tuber material was harvested (here "wild type" refers to plants that have not been transformed; 365 ES 13 refers to plants that express a hyaluronan synthase and have been used as starting material for transformation with the vegetable expression vector IC 372-256 (to 395 strains ES). Column 2 shows the average amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the determined means. In the case of plants in which no standard deviation is established, the hyaluronan content of only one tuber sample was determined.

d) Analysis of leaves of strain 395 ES

The hyaluronan content of individual plant leaves of the lines bearing the name 395 ES was determined. The averages and standard deviations established for plants bearing the name wild type and plants bearing the name 365 ES were calculated by calculating, in each case, the amount of hyaluronan in leaves of 4 different wild type and 9 different 365 ES 13 strain. , which are wild type vegetative progenies (Solanum tuberosum cv. Désirée) and strain 365 ES 13, respectively. The following results were obtained for selected plants:

<table> table see original document page 97 </column> </row> <table> <table> table see original document page 98 </column> </row> <table>

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

e) Analysis of tubers of strain 396 ES

For each tuber of individual transgenic plants of the 396 strains, two independent samples were taken and the hyaluronan content in each case determined separately. The mean and standard deviation of the values obtained for the individual measurements of each body were then calculated using the formula given under General Methods, item 5. The averages and standard deviations established for wild type denominated plants and plants named 365 ES were calculated using Calculation of the amount of hyaluronan in tubers of 12 different wild type plants and 14 different 365ES 74 lineage plants which are wild type (Sòlanumtuberosum cv. Désirée) and 365 ES 74 lineages, respectively. The following results were obtained for selected plants:

<table> table see original document page 99 </column> </row> <table>

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

f) Analysis of leaves of strain 396 ES

The hyaluronan content of individual leaves of the plant lines bearing the name 396 ES was determined. The averages and standard deviations established for plants bearing the name wild type and plants bearing the name 365 ES were calculated by calculating, in each case, the amount of hyaluronan in leaves of 4 different wild type and 6 plants of 365 ES 13. , which are wild type vegetative progenies (Solanum tuberosum cv. Désirée) and strain 365 ES 13, respectively. The following results were obtained for selected plants:

<table> table see original document page 100 </column> </row> <table> <table> table see original document page 101 </column> </row> <table>

Table 8: Amount of hyaluronan (in pg of hyaluronan per g of fresh mass) produced in independently selected transgenic plant leaves 396 ES. Column 1 contains the name of the plant from which leaf material was collected (here, "wild type" refers to plants that have not been transformed; 365 ES 74 refers to plants expressing a hyaluronan synthase and were used as starting material for transformation). with the vegetable expression vector IC 372-256 to generate strains 396 ES). Column 2 shows the average amount of hyaluro-nan determined for the tubers in question. Column 3 shows the standard deviation of the determined means. In the case of plants in which no standard deviation is established, the hyaluronan content of only one leaf sample was determined.

g) Analysis of tubers of strain 403 ES

For each tuber of individual transgenic plants of the lines named 403 ES, two independent samples were taken, if possible, and the hyaluronan content in each case determined separately. The mean and standard deviation of the values obtained for individual measurements of each tuber were then calculated using the formula given under General Methods, item 5. The means and standard deviations established for wild-type plants and 365-ES plants were calculated by calculating the amount of hyaluronan. in each case 10 different wild-type plants and 10 different 365 ES 13 seedlings which are vegetative progenies of the jungle-type (Solanum tuberosum cv. Désirée) and of the 365 ES 13 strain, respectively. . The following results were obtained for selected plants: <table> table see original document page 102 </column> </row> <table>

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

h) Leaf analysis of 403 ES strain

The hyaluronan content of individual leaves of the plant lines bearing the name 403 ES was determined. The averages and standard deviations established for plants bearing the name wild type and plants bearing the name 365 ES were calculated by calculating, in each case, the amount of hyaluronan in leaves of 5 different wild type and 5 365 ES 13 strain. , which are wild type vegetative progenies (Solanum tuberosum cv. Désirée) and strain 365 ES 13, respectively. The following results were obtained for selected plants:

<table> table see original document page 103 </column> </row> <table>

Table 10: Amount of hyaluronan (in pg of hyaluronan per g of fresh mass) produced in leaves of independently selected transgenic plants of 403 ES. Column 1 contains the name of the plant from which leaf material was collected (here "wild type" refers to plants that have not been transformed; 365 ES 13 refers to plants expressing a hyaluronan synthase and were used as starting material for transformation). with the vegetable expression vector IC 375-271 to generate the 403 ES strains). Column 2 shows the average amount of hyaluro-nan determined for the tubers in question. Column 3 shows the standard deviation of the determined means. In the case of plants in which no standard deviation is established, the hyaluronan content of only one leaf sample was determined.

(i) Analysis of tubers of strain 404 ES

For each tuber of individual transgenic plants of the so-called 404 ES1 lines two independent samples were taken, if possible, and the hyaluronan content in each case determined separately. The mean and standard deviation of the values obtained for individual measurements of each tuber were then calculated using the formula given under General Methods, item 5. The means and standard deviations established for wild-type plants and 365-ES plants were calculated by calculating the amount of hyaluronan. in each case 10 different wild-type plants and 12 different 365 ES 74 outlining plants which are vegetative progenies of the jungle-type (Solanum tuberosum cv. Désirée) and of the 365 ES 74 lineage, respectively. . The following results were obtained for selected plants:

<table> table see original document page 104 </column> </row> <table>

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

j) Leaf analysis of 404 ES strain

The hyaluronan content of individual leaves of the plant lines bearing the name 404 ES was determined. The averages and standard deviations established for plants bearing the name wild type and plants bearing the name 365 ES were calculated by calculating, in each case, the amount of hyaluronan in leaves of 7 different wild type plants and 9 365 ES 74 different lineage plants. , which are wild type vegetative progenies (Solanum tuberosum cv. Désirée) and strain 365 ES 74, respectively. The following results were obtained for selected plants:

<table> table see original document page 105 </column> </row> <table> <table> table see original document page 106 </column> </row> <table>

Table 12: Amount of hyaluronan (in pg of hyaluronan per g of fresh mass) produced in independently selected transgenic leaves of the 404 ES strain. Column 1 contains the name of the plant from which leaf material was collected (here, "wild type" refers to plants that have not been transformed; 365 ES 74 refers to plants expressing a hyaluronan synthase and were used as starting material for transformation). with the vegetable expression vector IC 375-271 to generate the 404 ES strains). Column 2 shows the average amount of hyaluro-nan determined for the leaves in question. Column 3 shows the standard deviation of the determined means. In the case of plants where no standard deviation is established, the hyaluronan content of only one leaf sample has been determined.

k) Analysis of tubers of strain 409 ES

For each tuber of individual transgenic plants of the lines named 409 ES, samples were taken and the hyaluro-nan content in each case determined. The means and standard deviations established for wild-type plants and 365-ES plants were calculated by calculating the amount of hyaluronan in tubers of in each case 4 different wild-type plants and 6 365ES 74-different plants which are wild-type vegetative progenies. (Solanumtuberosum cv. Désirée) and 365 ES 74, respectively. The mean and standard deviation of the values obtained for the individual measurements of each body were calculated using the formula given under General Methods, item

5. The following results were obtained for selected plants:

<table> table see original document page 107 </column> </row> <table> Table 13: Amount of hyaluronan (in μς of hialüronan per g of fresh mass) produced in independently selected transgenic plant tubers of the 409 ES line. Column 1 contains the name of the plant from which tuber material was harvested (here "wild type" refers to plants that have not been transformed; 365 ES 74 refers to plants that express a hyaluronan synthase and have been used as starting material for transformation with the vegetable expression vector IC 375-271 (409 ES). Column 2 shows the average amount of hyaluronan determined for the tubers in question. Column 3 shows the standard deviation of the determined means, "n.d." This means that it was not possible to detect hyaluronan in the tubers. In the case of plants where no standard deviation is established, the hyaluronan content of only one tuber sample was determined.

I) Leaf analysis of 409 ES strain

The hyaluronan content of individual leaves of the plant lines bearing the name 409 ES was determined. The averages and standard deviations established for pianias bearing the name wild type and plants bearing the name 365 ES were calculated by calculating, in each case, the amount of hyaluronan in leaves of 4 different wild type and 6 plants of 365 ES 74. , which are wild type vegetative progenies (Solanum tuberosum cv. Désirée) and strain 365 ES 74, respectively. The following results were obtained for selected plants:

<table> table see original document page 108 </column> </row> <table> <table> table see original document page 109 </column> </row> <table>

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

(m) Determination of hyaluronan content in relation to fresh mass and mass $ eca

Individual leaves of plants from 395 ES and 396 ES strains were removed before harvesting the tubers of the plants in question, removed from the plants and split in half. Half of each individual leaf was frozen frozen in liquid nitrogen, the other half was frozen by overnight freezing.

About 0.3 g of leaf material from the frozen leaf samples or about 0.02 of the freeze-dried leaf samples was flushed with a laboratory oscillating ball mill (MM200, deRetsch, Germany) (30 s to 30 HZ). 300 μl of water (demineralized, conductivity = 18 ΜΩ) was then added to each individual fragment sample, which was therefore mixed thoroughly using a vortex mixer, and cell fragments and insoluble components were then separated from the supernatant by centrifugation (5 minutes at 16,000 χg). The supernatant was removed and each sample completed to 500 μl with water (demineralized, conductivity = 18 ΜΩ). Aliquots of samples prepared in this manner were used to determine hyaluro-nan content using the method described under General Methods, item 4. Mean and standard deviation were calculated using the formula given under General Methods, item 5. For selected plants , the following results were obtained:

<table> table see original document page 110 </column> </row> <table> Table 15: Amount of hyaluronan (in pg of hyaluronan per g of fresh mass) produced in independently selected transgenic plant leaves from 395 ES and 396 ES strains . Column 1 names the plant from which leaf material was harvested. 365 ES 13 refers to plants expressing a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 372-256 to generate the 395 ES strains. 365 ES 74 refers to plants expressing a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 372-256 to generate 396 ES lines. Column 2 sets out the amount of hyaluronan determined for different leaves of the plants concerned. Column 3 gives an average of the amounts of hyaluronan measured on different leaves of a plant. Column 4 establishes the standard deviation for the determined means.

<table> table see original document page 111 </column> </row> <table> <table> table see original document page 112 </column> </row> <table>

Table 16: Amount of hyaluronan (in μς of hyaluronan per g of massa -eca) produced in independently selected transgenic plant leaves from 395 ES and 396 ES strains. Column 1 names the plant from which leaf material was harvested. 365 ES 13 refers to plants expressing a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 372-256 to generate the 395 ES strains. 365 ES 74 refers to plants expressing a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 372-256 to generate 396 ES line-genes. Column 2 sets out the amount of hyaluronan determined for different leaves of the plants concerned. Column 3 gives an average of the amounts of hyaluronan measured on different leaves of a plant. Column 4 establishes the standard deviation for the determined means.

14. Transformation of tomato plants with expression vector into vegetables comprising nucleic acid molecules encoding a hyaluronan synthase.

Tomato plants were initially transformed using the IC 341-222 vegetable expression ovator which comprises a nucleic acid coding sequence for a Paramecium bursaria Chlo-rella virus HAS 1 protein under the control of the B33 pathogen gene promoter. Solanum tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8,23-29) using the method provided under General Methods, item 8. The obtained transgenic tomato plants, which were transformed with plasmid IC 341-222, were named 367 ES.

Tomato plants of 367 ES 25 and 367 ES 42 lines were then transformed with the vegetable expression vector IC 341-222 using the method provided under General Methods, item 8. Tomato plants obtained from 367 ES 25, which were transformed as plasmid IC 341-222, were named 399 ES. The tomatetransgenic plants obtained from strain 367 ES 42, which were transformed into plasmid IC 341-222, were named 400 ES.

Tomato plants from 367 ES, 25 strains were then transformed with the vegetable expression vector IC 375-271 using the method provided under General Methods, item 8. Transgenic tomato plants obtained from 367 ES 25 strain, which were transformed with plasmid IC 375-271, were named 405 ES.

15. Analysis of transgenic tomato plants synthesizing hyaluronan further transformed with expression vectors in plants comprising nucleic acid coding sequences of a protein that exhibits the activity of a GFAT and a protein that has the activity of a UDP-GIc- DH.

a) Leaves of pianias u and lOniciifc! UB umiIayerib o »» co e ^ fUU to

From different tomato plants selected from 399 ES and 400 ES genotypes, which were grown in soil in a greenhouse, each case 1 leaf was harvested and frozen in liquid nitrogen. Further processing and determination of hyaluronan content were performed as described under Example 11b) for potato plant leaves. The following results were obtained:

<table> table see original document page 113 </column> </row> <table>

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

(b) Fruits of tomato plants of 399 ES and 400 ESA strains from different tomato plants selected from 399 ES and 400 ES strain lines, which were grown in soil in a greenhouse, but in case ripe fruits were harvested, fragmented, centrifuged and the supernatant, after centrifugation, filtered. Further filtration processing and determination of hyaluronan content were performed as described under Example 11 b) for potato plant leaves. The following results were obtained:

<table> table see original document page 114 </column> </row> <table>

Table 18: Amount of hyaluronan (in pg of hyaluronan per g of fresh mass) produced in independently selected mature fruits of transgenic plants 399 ES and 400 ES. Column 1 refers to the plant from which 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 267ES 42-2 refer to different clonal progeny of the 367 ES 42 plant. -in 2 establishes the average quantities of hyaluronan determined from fruit of the plants concerned. For this purpose, the hyaluronan content in each case 3 (strains 399 ES-1, 400 ES 3), 5 (strains 367 ES-25-1, 325ES-2, 367 ES 42-1, 367 ES 42-2) or 6 (lineage 39'9jES-11), different fruits of the lineages in question, was determined. Column 3 establishes the standard deviation of the determined means.

c) Fruits of tomato plants from 405 ES strains

From different tomato plants selected from 405 ES row-lines, which were grown in soil in a greenhouse, each mature castrate was harvested, fragmented, centrifuged and the supernatant, after centrifugation, filtered. Further processing of the filtrate and determination of hyaluronan content was performed as described under Example 11 b) for potato plant leaves. The following results were obtained:

<table> table see original document page 115 </column> </row> <table>

Table 19: Amount of hyaluronan (in pg of hyaluronan per g of fresh mass) produced in mature fruits of independently selected transgenic plants from 405 ES strains. Column 1 refers to the plant from which leaf material was harvested. 367 ES 25-8 and 367 ES 25-9 refer to different clonal progeny of the plant 367 ES 25. Extensions in Latin numbers refer to different fruits of the respective plant, ("weight" refers to plants that were not transformed). Column 2 sets out the average quantities of hyaluronan determined on different fruits of the plants concerned. Column 3 establishes the standard deviation of the determined means.

16) Concluding note

When the hyaluronan content of different leaves of a plant was determined, older leaves of the same plant were generally found to contain more hyaluronan than younger leaves of the same plant. Consequently, the hyaluronan content in leaves seems to increase with increasing leaf age, so it may be assumed that hyalu-ronan accumulates over time. This phenomenon may explain the different amounts of hyaluronan found in independent measurements for progeny of the same lineage. INTERNATIONAL FORMAT

Bayer CropScience GmbHBrüningstr. 50

65929 Frankfurt

FEASIBILITY STATEMENT Issued under Rule 10.2 by the INTERNATIONAL DEPOSIT AUTHORITY

identified below

<table> table see original document page 117 </column> </row> <table>

1 Indicates the original filing date or, when a new deposit or transfer is made, the date

relevant date (new deposit date or transfer date

2 In cases related to Rule 10.2 (a) (ii) and (iii), refer to the most recent feasibility test.

3 X mark the applicable field

4 Fill in if required and negative test results.

DSMZ-BP / 9 Format (Single Page) 0196 INTERNATIONAL FORMAT

<table> table see original document page 118 </column> </row> <table>

Claims (13)

1. Genetically modified plant cell that has a nucleic acid molecule encoding a hyaluronan synthase stably integrated into its genome, where this plant cell additionally exhibits increased activity of a protein that exhibits glutamine activity: fructose 6 -phosphate amidotransferase (GFAT) and an increase in activity of a protein that exhibits the activity of a UDP-glucose dehydrogenase (UDP-GIc-DH) compared to corresponding non-genetically modified wild-type plant cells. Genetically modified plant cell according to claim 1, which has a strangely integrated nucleic acid molecule in its genome or a plurality of stably integrated stranded nucleic acid molecules in its genome, wherein such a foreign nucleic acid molecule or these molecules of foreign nucleic acids increase the activity of a protein that exhibits glutamine activity: fructose 6-phosphate amidotransferase (GFAT) increases the activity of a protein that presents activity of a UDP-glucose dehydrogenase (UDP-GIc-DH) compared to cells corresponding non-genetically modified wild-type vegetables. Genetically modified plant cell according to claim 2, wherein a first foreign nucleic acid molecule encodes a protein that exhibits glutamine activity: fructose 6-phosphate amidotransferase (GFAT) and a second nucleic acid molecule encode a protein. which presents the activity of a UDP-glucose dehydrogenase (UDP-Gluc-DH). Genetically modified plant cell according to any one of claims 1, 2 or 3 which synthesizes an increase in the amount of hyaluronan compared to plant cells that exhibit activity of a hyaluronan synthase and no increase in glutamine activity: fructose 6-phosphate amidotransferase ( GFAT) and no increase in the activity of a UDP-glucose dehydrogenase. Plant comprising genetically modified plant cells as defined in any one of claims 1 to 4. Plant propagating material as defined in claim 5 which comprises genetically modified plant cells as defined in any one of claims 1 to 4. Part of a plant harvestable plant as defined in claim 5 which comprises genetically modified plant cells as defined in any one of claims 1 to 4. A process for preparing a plant which synthesizes hyalu-ronan, which process comprises) genetically modifying a plant cell, wherein the genetic modification comprises steps ia iii below (i) introducing a foreign nucleic acid molecule encoding a hyaluronan synthase. introduction of a genetic modification in the plant cell, the genetic modification resulting in an increase in the activity of a glutamine-like protein: fructose 6-phosphate ami-dotransferase (GFAT) compared with plant-type cells non-genetically modified, wild-type (iiii) introduction of a genetic modification in the plant cell, the genetic modification resulting in an increase in the activity of a protein that exhibits glutamine activity: fructose 6-phosphateUDP-glucose dehydrogenase compared to jungle-type plant cells corresponding genetically modified non-genetically modified steps ia ii may be performed in any order individually, or any combinations of steps ia iii may be performed simultaneously b) regenerate a plant of plant cells from step a) c) generate, if appropriate, additional plants using the plants according to according to step b), wherein, if appropriate, plant cells are isolated from plants according to steps b) or c) and process steps a) to c) are repeated until a plant is generated which has a A foreign nucleic acid molecule encoding a hyaluronan feels and exhibits an increase in the activity of a glutamine activating protein: fructose 6-phosphate amidotransferase (GFAT) with non-genetically modified, wild-type plant cells and an increase in the activity of a protein presenting glutamine activity: fructose 6-phosphate UDP-glucose dehydrogenase compared to non-wild type plant cells the corresponding genetically modified. A process for the preparation of hyaluronan which comprises the step of extracting hyaluronan from genetically modified plant cells as defined in any one of claims 1 to 4 from plants as defined in claim 5 from material of propagation as defined in claim 6 from harvestable plant parts as defined in claim 7 or from plants obtainable by a process according to claim 8. Use of a genetically modified plant cell as defined in any one of claims 1 to 4, a plant as defined in claim 5, propagating material as defined in claim 6, harvestable plant parts, as defined in claim 7, or from plants obtainable by a process as defined in claim 8 for preparing hyaluronan. A composition comprising genetically modified plant cells as defined in any one of claims 1 to 4. A process for preparing a composition comprising hyaluronan, in which genetically modified plant cells as defined in any one of claims 1 to 4, plants as defined in claim 5, propagating material as defined in claim 6, parts of the plant subject to Harvesting plants according to claim 7 or plants obtainable by a process according to claim 8 are used. Use of genetically modified plant cells as defined in any one of claims 1 to 4 of plants as defined in claim 5 of propagating material as defined in claim 6 of harvestable plant parts; according to claim 7 or plants obtainable by a process as defined in claim 8 for preparing a composition as defined in claim 11.