ES2536916T3 - Improved procedures and means to produce hyaluronan - Google Patents

Abstract

A genetically modified plant cell having a nucleic acid molecule encoding a hyaluronan synthase linked to regulatory sequences that ensure transcription in plant cells stably integrated into its genome, wherein said plant cell additionally has a plurality of acid molecules foreign nucleic acid stably integrated into its genome, in which a first foreign nucleic acid molecule encodes a protein having the activity of a glutamine:fructose 6-phosphate amidotransferase-2 (GFAT-2) that originates from animals or a glutamine:fructose 6-phosphate amidotransferase (GFAT) originating from bacteria and a second foreign nucleic acid molecule encoding a protein having the activity of a UDP-glucose dehydrogenase (UDP-Glc-DH) and wherein said genetically modified plant cell has an increased activity of a protein having the activity of a glutamine:fructose 6-phosphate amidotransferase (GFAT) and an increased activity of a protein having the activity of a UDP-glucose dehydrogenase (UDP-Glc-DH) compared to the corresponding non-genetically modified plant cells.

Description

DESCRIPTION

Improved procedures and means to produce hyaluronan

The present invention relates to plant and plant cells that synthesize an increased amount of hyaluronan and to the methods for preparing said plants, and also to the methods for preparing hyaluronan with the help of these plant or plant cells. Here, the genetically modified plant or plant cells according to the invention have hyaluronan synthase activity and additionally an increased glutamine activity: fructose 6-phosphate amidotransferase (GFAT) and an increased activity of UDP glucose dehydrogenase (UDP-Glc-DH), compared to natural plant cells or natural plants. The present invention further relates to the use of plants that have increased hyaluronan synthesis to prepare hyaluronan and food or
food products containing hyaluronan.

Hyaluronan is a natural, unbranched linear mucopolysaccharide (glucosaminoglycan) that is constructed of alternating molecules of glucuronic acid and N-acetyl-glucosamine. The basic hyaluronan building block consists of the glucuronic beta-1,3-N-acetyl-glucosamine acid disaccharide. In hyaluronan, these repeat units are linked together by beta-1,4 bonds. In pharmacy, the term hyaluronic acid is often used. Because hyaluronan is present in most cases as a polyanion and not as free acid, the term hyaluronan is preferably used herein, but it should be understood that each term encompasses both molecular forms.

Hyaluronanne has unusual physicochemical properties, such as, for example, polyelectrolyte properties, viscoelastic properties, a high capacity to bind to water, gel formation properties, which, in addition, additional properties of hyaluronan are described in a review article Lapcik et al., (1998, Chemical Reviews 98 (8), 2663-2684).

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

Recently, hyaluronan has also been found in non-vertebrate animal organisms (molluscs) (Volpi and Maccari, 2003, Biochimie 85, 619-625).

In addition, some gram-positive pathogenic bacteria (Streptococcus group A and C) and gram-negative bacteria (Pasteurella) synthesize hyaluronan as exopolysaccharides that protect these bacteria against attack by their host's immune system, because hyaluronan is a non-immunogenic substance.

Viruses that infect unicellular green algae of the genus Chlorella, some of which are present in endosymbiotic species of Paramecium, grant unicellular green algae the ability to synthesize hyaluronan after virus infection (Graves et al., 1999, Virology 257 , 15-23). However, the ability to synthesize hyaluronan is not a characteristic that characterizes the algae in question. The ability of algae to synthesize hyaluronan is mediated by an infection with a virus whose genome has a coding sequence for hyaluronan synthase (DeAngelis, 1997, Science 278, 1800-1803). In addition, the virus genome contains coding sequences for UDP-glucose dehydrogenase (UDP-Glc-DH) and a glutamine: fructose 6-phosphate amidotransferase (GFAT). UDP-Glc-DH catalyzes the synthesis of UDP-glucuronic acid used as a substrate by hyaluronan synthase. GFAT converts fructose 6-phosphate and glutamine into glucosamine 6. phosphate, which is an important metabolite in the metabolic pathway for the synthesis of hyaluronan in, for example, bacteria. Both algae genes encode active proteins that, in the same way as the hyaluronan synthase of the virus, are transcribed simultaneously in the early phase of viral infection (DeAngelis et al., 1997, Science 278, 1800-1803, Graves et al. , 1999, Virology 257, 15-23). The activity of a protein that has glutamine: fructose activity 6-phosphate amidotransferase (GFAT) would never be detected in extracts from cells not infected by a virus or in cells infected with viruses (Landstein et al., 1998, Virology 250, 388- 396). Therefore, the role of UDP-Glc-DH and GFAT expression in Chlorella cells infected with virus for hyaluronan synthesis, and whatever is required for hyaluronan synthesis is not known.

Plants of natural origin by themselves do not have any nucleic acid, which encodes the proteins that catalyze the synthesis of hyaluronan and, although a large number of vegetable carbohydrates have been described and characterized, it has not been possible until now to detect hyaluronan or Hyaluronan-related molecules in non-infected, naturally occurring plants (Graves et al., 1999, Virology 257, 15-23).

The catalysis of hyaluronan synthesis is carried out by means of a membrane-associated enzyme or integrated to a single membrane, hyaluronan synthase. 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).

Hyaluronan synthases of vertebrates are further distinguished by the isoenzymes identified. The different isoenzymes are named for identification using Arabic numerals (for example, hsHAS1, hsHAS2, hsHAS3).

The mechanism of transferring synthesized hyaluronan molecules through the cytoplasmic membrane in the environment surrounding the cell has not yet been completely elucidated. The initial hypotheses have meant that transport through the cell membrane has been carried out using hyaluronan synthase itself. However, more recent results indicate that the transport of hyaluronan molecules through the cytoplasmic membrane takes place by energy-dependent transport through transport proteins responsible for this action. Therefore, Streptococcus strains have been generated by mutation in which the synthesis of an active transport protein has been inhibited. These strains synthesized less hyaluronan than the corresponding natural bacterial strains (Ouskova et al., 2004, Glycobiology 14 (10), 931-938). In human fibroblasts, it has been possible to demonstrate, with the help of agents that specifically inhibit known transport proteins, that it is possible to reduce the amount of hyaluronan produced and the activity of hyaluronan synthases (Prehm and Schumacher, 2004, Biochemical Pharmacology 68, 1401-1410). In which it is not known what amount, in any case, of transport proteins capable of transporting hyaluronan are present in plants.

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

In the medical field, products containing hyaluronan are currently used for the intra-articular treatment of osteoarthritis and in ophthalmic treatments used for eye surgery. Hyaluronan is also used to treat joint disorders in racehorses. In addition, hyaluronic acid is a component of some rhinology that, for example, in the form of eye and nasal drops, serves to moisten dry mucous membranes. Solutions containing hyaluronan for injection are used as analgesics and anti-rheumatic. Patches comprising hyaluronan or derivatized hyaluronan are used in wound healing. As dermal, gel implants containing hyaluronan are used to correct skin deformations 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 skin fillers. Injecting hyaluronan, for a limited period of time, it is possible to smooth wrinkles or increase the volume of the lips.

In cosmetic products, in particular in creams and lotions for the skin, hyaluronan is frequently used as a humectant because of its high water binding capacity.

In addition, preparations containing hyaluronan are sold as the so-called nutraceuticals (food supplements) that can also be used in animals (eg, dogs, horses) for the prophylaxis and relief of osteoarthritis.

The hyaluronan used for commercial purposes is currently isolated from animal tissues (cocks crests) or fermentatively prepared using bacterial cultures.

US 4,141,973 describes a method for isolating hyaluronan from rooster crests or alternatively from umbilical cords. In addition to hyaluronan, animal tissues (eg, cockscomb, umbilical cords) also contain additional mucopolysaccharides related to hyaluronan, such as chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate and heparin. In addition, animal organisms contain proteins (hialadherins) that specifically bind to hyaluronan and are required for the most different functions in the body, such as, for example, the degradation of hyaluronin in the liver, the function of hyaluronan as a conductive structure for cell migration, regulation of endocytosis, anchoring of hyaluronan on cell structure or formation of hyaluronan networks (Turley, 1991, Adv Drug Delivery Rev 7, 257 FF; Laurent and Fraser, 1992, FASEB J. 6 , 183 FF; Stamenkovic and Aruffo, 1993, Methods Enzymol. 245, 195 FF; Knudson and Knudson, 1993, FASEB 7, 1233 et seq.).

Streptococcus strains used for the production of hyaluronan are exclusively pathogenic bacteria. During the culture, also, these bacteria produce exotoxins (pyrogens) and hemolysins (streptolysin, in particular, alpha and beta hemolysin (Kilian, M., Streptococcus and Enterococcus. In: Medical Microbiology. Greenwood, D .; Slack, RCA; Peutherer , JF (Eds.) Chapter 16. Churchill Livingstone, Edinburgh, United Kingdom: pp. 174-188, 2002, ISBN 0443070776) that are released into the culture medium.This makes purification and isolation of prepared hyaluronan more difficult. With the help of Streptococcus strains, in particular for pharmaceutical applications, the presence of exotoxins and hemolysins in preparations is a problem.

US 4,801,539 describes the preparation of hyaluronan by fermentation of a strain of mutagenized bacteria (Streptococcus zooedemicus). The strain of mutagenized bacteria used does not synthesize large amounts of beta-hemolysin. The yield achieved was 3.6 g of hyaluronan per liter of culture.

EP 0694616 describes a method for cultivating Streptococcus zooedemicus or Streptococcus equi, in which, under the culture conditions employed, streptolysin is not synthesized, but increased amounts of hyaluronan are synthesized. The yield achieved was 3.5 g of hyaluronan per liter of culture.

During cultivation, Streptococcus strains release the hyaluronidase enzyme in the culture medium, as a consequence of which, in this production system, also, the molecular weight is reduced during purification. The use of negative Streptococcus hyaluronidase strains or of the processes for the production of hyaluronan in which the production of hyaluronidase has been inhibited during cultivation is described in US 4,782,046. The yield achieved was up to 2.5 g of hyaluronan per liter of culture, and the maximum average molecular weight achieved was 3.8 x 106 Da, at a molecular weight distribution between 2.4 x 106 to 4.0 x

6

10.

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

WO 05 012529 describes the preparation of transgenic tobacco plants using nucleic acid molecules encoding the hyaluronan synthases of viruses that infect Chlorella. In WO 05/012529, on the other hand, nucleic acid sequences encoding the hyaluronan synthase of the viral strain CVHI1 of Chlorella and, on the other hand, of the viral strain CVKA1 of Chlorella have been used to transform plants of tobacco. The synthesis of hyaluronan could only be demonstrated for a plant transformed with a nucleic acid sequence encoding the hyaluronan synthase isolated from the viral strain CVKA1 of Chlorella. For tobacco plants transformed with a nucleic acid sequence encoding hyaluronan synthase from the viral strain CVHI1 of Chlorella, it has not been possible to detect hyaluronan synthesis in the corresponding transgenic plants. The amount of hyaluronan synthesized only by the transgenic tobacco plant producing hyaluronan in WO 05 012529 indicates that it is approximately 4.2 µg of hyaluronan per ml of measured volume which, taking into account the description to perform the experiment in question, corresponds approximately to an amount of at least 12 µg of hyaluronan produced per gram of fresh weight of plant material.

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

In plant cells, UDP-glucuronic acid serves as a metabolite for a plurality of possible routes to synthesize ascorbic acid (Lorence et al., 2004, Plant Physiol 134, 1200-1205) and as a fundamental metabolite in the synthesis of pectin components and hemicellulose of cell walls that are synthesized in the endoplasmic reticulum of the plant cell (Reiter, 1998, Plant Physiol Biochem 36 (1), 167-176). The most important and most frequently produced pectin monomer is D-galacturonic acid (2004, H. W. Heldt in "Plant Biochemistry", 3rd Edition, Academic Press, ISBN 0120883910) which is synthesized using UDP-glucuronic acid. In addition, it is also possible, among other things, to synthesize UDP-xylose, UDP-arabinose, UDP galacturonic acid and UDP-apiosa, the metabolites for the synthesis of hemicellulose and pectin, using UDP-glucuronic acid (Seitz et al., 2000, Plant Journal, 21 (6), 537-546). In plant cells, UDP-glucuronic acid can be synthesized either by the metabolism of hexose phosphate which comprises, among other things, the conversion of UDP-glucose into UDP-glucuronic acid by UDP-Glc-DH or by the oxidative metabolism of mine -inositol comprising the conversion of glucuronate 1-phosphate uridylyl transferase. Both metabolic pathways to synthesize glucuronic acid appear to exist independently of each other and alternatively at different stages of tissue / development in Arabidopsis plants (Seitz et al., 2000, Plant Journal 21 (6), 537-546). The respective contribution of the two mentioned metabolic pathways (from hexose phosphate or from oxidative metabolism of myo-inositol) towards the synthesis of UDP-glucuronic acid has not yet been elucidated (Karkonen, 2005, Plant Biosystems 139 (1), 46- 49).

The UDP-Glc-DH enzyme catalyzes the conversion of UDP-glucose into UDP-glucuronic acid. Samac et al. (2004, Applied Biochemistry and Biotechnology 113-116, Humana Press, Editor Ashok Mulehandani, 1167-1182) describes the tissue-specific excess expression of a soy UDP-Glc-DH in Alfalfa phloem cells in order to increase the pectin content in the stems of these plants. The activity of UDP-Glc-DH was increased, in comparison with the corresponding natural plants, 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 natural plants. The amount of xylose and rhamnose monomers in the cell fraction wall of the transgenic plants increased while the amount of mannose monomers in the cell wall fraction was reduced.

The constitutive excess expression of UDP-Glc-DH in Arabidosis plants resulted in an abnormal growth of the plants in question compared to the corresponding natural plants and a dwarf phenotype. The fraction of the cell wall of the corresponding plants has an increased amount of mannose and galactose and a reduced amount of xylose, arabinose and uronic acids compared to the corresponding natural plants (Roman, 2004, "Studies on The Role of UDP-Glc -DH in Polysaccharide Biosynthesis ", PhD Thesis, Acta Universitatis Upsaliensis, ISBN 91-554-6088-7, ISSN 0282-7476). Therefore, these results contradict at least in part the results of Samac et al. (2004, Applied Biochemistry and Biotechnology 113116, Humana Press, Editor Ashok Mulehandani, 1167-1182) that detected a reduced amount of mannose and an increased amount of xylose in the cell wall fraction of the corresponding transgenic plants.

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

WO 00 11192 describes the specific excess expression of endosperm of a corn nucleic acid molecule that encodes a protein that has the enzymatic activity of a GFAT in transgenic corn plants in order to synthesize a cationic starch in plants that have 2-aminoanhydroglucose molecules. The metabolic pathway describes that, According to the description of WO 00 11192, 2-amino-anhydroglucose that is incorporated into the starch must result, including, among other things, the incorporation of UDP-glucosamine by starch synthases and / or glycogen starch synthases. It is indicated that increased amounts of UDP-glucosamine could be detected in endosperm flour of transgenic corn plants that express in excess in question a nucleic acid molecule that encodes a protein that has the (enzymatic) activity of GFAT translationally fused with a plastid signaling peptide. When the protein having the (enzymatic) activity of a GFAT was expressed without signaling peptide, it has been possible to detect an increased amount of glucosamine 1-phosphate in the corresponding flours from the corn endosperm tissue. It has not been possible to detect cationic starch in transgenic plants.

The production of hyaluronan by fermentation of bacterial strains is associated with high costs, because the bacteria have to be fermented in sterile hermetically sealed containers under conditions of expensive controlled culture (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 developed for excessively large volumes of culture. Particular mention should be made here that only the homogeneous mixture of substances fed from abroad (for example, sources of essential nutrients for bacteria, reagents for regulating pH, oxygen) with the culture medium required for production can be ensured effective, which, in large fermenters, only in any case, with a significant technical expense.

The purification of hyaluronan from animal organisms is complicated due to the presence, in animal tissues, of other mucopolysaccharides and proteins that specifically bind hyaluronan. In patients, the use of hyaluronan containing medical preparations contaminated by animal proteins may result in unwanted immune reactions of the body (US 4,141,973), particularly if the patient is allergic to animal proteins (eg egg white of chicken). In addition, the amounts (yields) of hyaluronan that can be obtained from animal tissues in satisfactory quality and purity are low (rooster crest: 0.079% in w / w, EP 0144019, US 4,782,046), which needs the processing large quantities of animal tissues. An additional problem in the isolation of hyaluronan from animal tissues is that the molecular weight of the hyaluronan during purification is reduced because the animal tissues also contain a hyaluronan degrading enzyme (hyaluronidase).

In addition to the hyaluronidases and exotoxins mentioned, Streptococcus strains also produce endotoxins that, when present in pharmacological products, pose risks to the patient's health. In a scientific study, it has been shown that medical products containing hyaluronan in the market contain detectable amounts of bacterial endotoxins (Dick et al., 2003, Eur J Pharmacol. 13 (2), 176-184). An additional disadvantage of the hyaluronan produced with the help of Streptococcus strains is the fact that the isolated hyaluronan has a lower molecular weight than the isolated hyaluronan of rooster crests (Lapcik et al.

1998, Chemical Reviews 98 (8), 2663-2684). US 20030134393 describes the use of a Streptococcus strain to produce hyaluronan that synthesizes a particularly pronounced (supercapsulated) hyaluronan capsule. The hyaluronan isolated after fermentation had a molecular weight of 9.1 x 106 Da. However, the yield was only 350 mg per liter.

Some of the disadvantages of producing hyaluronan by bacterial fermentation or by isolating animal tissues by producing hyaluronan using transgenic plants can be avoided; however, the amounts of hyaluronan currently obtained can be produced using transgenic plants that would require large areas of cultivation to produce relatively large amounts of hyaluronan. In addition, the isolation or purification of hyaluronan from plants that have a lower hyaluronan content is considerably more complicated and expensive than the isolation or purification of plants that have a higher hyaluronan content.

Although hyaluronan has unusual properties, it is, due to its scarcity and the high price, rarely used, in any case, for industrial applications.

Accordingly, it is an object of the present invention to provide means and processes that allow the provision of hyaluronan in sufficient quantities and quality and that make it possible to provide hyaluronan even for industrial applications and applications in the field of food and feed.

This object is achieved by the embodiments outlined in the claims.

Therefore, the present invention relates to a genetically modified plant cell having a nucleic acid molecule encoding a hyaluronan synthase linked to regulatory sequences that ensure transcription in plant cells stably integrated into its genome, in which said cell The plant additionally has a plurality of foreign nucleic acid molecules stably integrated into its genome, in which a foreign nucleic acid molecule encodes a protein that has the activity of a glutamine: fructose 6-phosphate amidotransferase-2 (GFAT- 2) that originates from animals or a glutamine: fructose 6-phosphate amidotransferase (GFAT) that originates from bacteria and a second molecule of a foreign nucleic acid that encodes a protein that has the activity of a UDP- glucose dehydrogenase (UDP-Glc-DH) and in which said genetically modified plant cell has an increased activity a of a protein that has the activity of a glutamine fructose 6-phosphate amidotransferase (GFAT) and an increased activity of a protein that has the activity of a UDP-glucose dehydrogenase (UDP-Glc-DH) compared to non-plant cells genetically modified.

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

Here, the genetic modification of genetically modified plant cells according to the invention or of genetically modified plants according to the invention can be any genetic modification that results in a stable integration of a nucleic acid molecule that encodes a hyaluronan synthase in a plant cell or plant.

In the context of the present invention, the term "natural plant cell" should be understood as meaning plant cells that have served as starting material for the preparation of genetically modified plant cells according to the invention, that is, their genetic information, apart from the genetic modifications introduced and which result in a stable integration of a nucleic acid molecule that encodes a hyaluronan synthase and increasing the activity of a protein that has the activity of a GFAT, and increasing the activity of a protein that has the activity of a UDP-Glc-DH corresponds to that of a genetically modified plant cell according to the invention.

In the context of the present invention, the term "natural plant" should be understood as meaning plants that have served as starting material for the preparation of genetically modified plants according to the invention, that is, their genetic information, apart from genetic modifications introduced and resulting in a stable integration of a nucleic acid molecule that encodes a hyaluronan synthase and increasing the activity of a protein that has the activity of a GFAT, and increasing the activity of a protein that has the activity of a UDP-Glc-DH 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 comparing a plurality of objects, the objects in question that are compared with each other must be maintained in the same conditions. In the context of the present invention, the term "corresponding" in the context of natural plant cells or natural plants means that the plant cells or plants compared to each other have been grown under the same growing conditions and have the same age (of the crop).

In the context of the present invention, the term "hyaluronan synthase" (EC 2.4.1.212) should be understood as meaning a protein that synthesizes hyaluronan from substrates of UDP-glucuronic acid (UDP-GlcA) and N-acetyl- glucosamine (UDP-GlcNAc). Hyaluronan synthesis is catalyzed according to the following reaction schemes:

nUDP-GlcA + nUDP-GlcNAc → beta-1,4- [GlcA-beta-1,3-GlcNAc] n + 2 nUDP

Nucleic acid molecules and corresponding protein sequences encoding hyaluronan synthases have been described, among other things, for the following organisms: rabbit (Oryctolagus cuniculus) ocHas2 (EMBL AB055978.1, US 20030235893), ocHas3 (EMBL AB055979. 1, US 20030235893); baboon (Papio anubis) paHas1 (EMBL AY463695.1); frog (Xenopus laevis) xlHas1 (EMBL M22249.1, US 20030235893), xlHas2 (DG42) (EMBL AF168465.1), xlHas3 (EMBL AY302252.1); human being (Homo sapiens) hsHAS1 (EMBL D84424.1, US 20030235893), hsHAS2 (EMBL U54804.1, US 20030235893), hsHAS3 (EMBL AF232772.1, US 20030235893); mouse (Mus musculus), mmHas1 (EMBL D82964.1, US 20030235893), mmHAS2 (EMBL U52524.2, US 20030235893), mmHas3 (EMBL U86408.2, US 20030235893); cow (Bos taurus) btHas2 (EMBL AJ004951.1, US 20030235893); chicken (Gallus gallus) ggHas2 (EMBL AF106940.1, US 20030235893); rat (Rattus norvegicus) 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, GI: 23428486, pig (Sus scrofa ) sscHAS2 (NCBI NM_214053.1, GI: 47522921, sscHas 3 (EMBLAB159675), zebrafish (Danio rerio) brHas1 (EMBL AY437407), brHas2 (EMBL AF190742.1) brHas3 (EMBL AF190743.1m); PasteHure1 (cult) AF036004.2); Streptococcus pyogenes spHas (EMBL, L20853.1, L21187.1, documents US 6,455,304, document US 20030235893); Streptococcus equis seHas (EMBL AF347022.1, AY173078.1), Streptococcus uberis AHAS29 (EM24) .2, document US 20030235893), Streptococcus equisimilis seqHas (EMBL AF023876.1, document US 20030235893); Sulfolobus solfataricus ssHAS (US 20030235893), Sulfolobus tokodaii stHas (AP000988.1), Paramevium cursary (EM000 AFR) 1 (B000). 3, PB42580, US 20030235893).

In the context of the present invention, the term "UDP-glucose dehydrogenase (UDP-Glc-DH)" (EC 1.1.1.22) should be understood as meaning a protein that synthesizes, from UDP-glucose (UDP-Glc) and NAD +, UDPglucuronic acid (UDP-GlcA) and NADH. This catalysis proceeds according to the following reaction scheme:

UDP-Glc + 2 NAD + → UDP-GlcA + 2 NADH

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

Glutamine + Fruc-6-P → GlcN-6-P + Glutamate

In particular in animal organisms, it has been possible to demonstrate two different isoforms of proteins that have the (enzymatic) activity of a GFAT (called GFAT-1 and GFAT-2, respectively, in the literature). Hu et al. (2004), J. Biol. Chem. 279 (29), 29988-29993 describes the differences of the respective proteins from the mouse: in addition to the differences in the specific tissue expression of the proteins in question that have the activity (enzymatic) ) of a glutamine: fructose 6-phosphate amidotransferase 1 (GFAT-1) and a glutamine: fructose 6-phosphate amidotransferase 2 (GFAT-2), it has been possible to show that both isoforms are regulated by phosphorylation by a protein kinase dependent on CAMP. the activity of a protein that has the (enzymatic) activity of a GFAT-1 is inhibited by the phosphorylation of a conserved serine residue (serine 205 in the GFAT-1 from the mouse, GenBank accession number: AF334736.1) of the amino acid sequence in question, while the activity of a protein having the activity of a GFAT-2 is increased by the phosphorylation of a conserved serine residue (serine 202 in the GFAT-2 from the mouse, Accession No. to GenBank: NM_013529) of the amino acid sequence in question. Both proteins that have the activity of a GFAT-1 and the proteins that have the activity of a GFAT-2 are inhibited in a concentration-dependent manner by UDP-N-acetylglucosamine; however, for a protein that has the activity of a GFAT2, the inhibition by UDP-N-acetylglucosamine is lower (maximum reduction of activity by UDP-Nacetylglucosamine is approximately 15%) compared to a protein that has the activity of a GFAT-1 (the maximum reduction in activity by UDP-N-acetylglucosamine is approximately 51% or 80%). There are indications that the inhibition of a protein that has the activity of a GFAT-1 in animal organisms is based on the fact that at high concentrations of UDP-N-acetylglucosamine there is a glycosylation of the O-glucose-N-acetylglucosamine of The proteins in question. It is not yet fully understood whether regulation of protein activity by O-glycosylation in plant cells also takes place (Huber and Hardin, 2004, Current Opinion in Plant Biotechnology 7, 318-322).

In the context of the present invention, it should be understood that the term "glutamine: fructose 6-phosphate amidotransferase-1 (GFAT 1)" means a protein that has the activity of a GFAT and whose activity is inhibited by phosphorylation of a protein cAMP dependent kinase.

In the context of the present invention, the term "glutamine: fructose 6-phosphateamidotransferase-2 (GFAT-2)" should be understood to mean a protein that has the activity of a GFAT and that is activated by phosphorylation by a protein kinase dependent cAMP.

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

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

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

The genetically modified plant cells according to the invention or the genetically modified plants according to the invention fulfill in each case at least one of the conditions mentioned above, which means an increased enzymatic activity of a protein for the proteins having the activity ( enzymatic) of a GFAT and for proteins that have the (enzymatic) activity of a UDP-Glc-DH.

An increased expression can be determined, for example, by measuring the amount of transcripts encoding a protein that has the activity of a GFAT or that encodes a protein that has the activity of a UDP-Glc-DH, for example, by analyzing Northern transfer or by RT-PCR. Here, an increase preferably means an increase in the amount of transcripts compared to the corresponding non-genetically modified natural plant cells or non-genetically modified natural plants by at least 50%, in particular at least 70%, preferably at at least 85% and particularly preferably at least 100%. An increase in the amount of transcripts encoding a protein that has the activity of a GFAT or that encodes a protein that has the activity of a UDP-Glc-DH also means that plants

or plant cells that do not have detectable amounts of transcripts encode a protein that has the activity of a GFAT and / or encode a protein that has the activity of a UDP-Glc-DH that has, after genetic modification according to the invention, amounts detectable transcripts encoding a protein that has the activity of a GFAT or that encodes a protein that has the activity of a UDP-Glc-DH.

The increase in the amount of protein that has the activity of a GFAT or of proteins that have the activity of a UDP-Glc-DH can be determined results in an increased activity of these proteins in the plant cells in question, for example, by immunological procedures, such as Western blot analysis, ELISA (Adsorption Enzyme Immunoassay) or RIA (Radioimmune Assay). Those skilled in the art are aware of methods for preparing antibodies that react specifically with a specific protein, that is, specifically bind to said protein (see, for example, Lottspeich and Zorbas (Eds.), 1998, Bioanalytik [Bioanalysis], Spektrum Akad Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4). Some companies (for example, Eurogentec, Belgium) offer the preparation of such antibodies as an à la carte service. Here, an increase in the amount of protein preferably means an increase in the amount of protein that has the activity of a GFAT and / or proteins that have the activity of a UDP-Glc-DH compared to non-genetically modified plant cells. corresponding at least 50%, in particular at least 70%, preferably at least 85% and particularly preferably at least 100%. An increase in the amount of protein that has the activity of a GFAT and / or protein that has the activity of a UDP-Glc-DH also means that plants or plant cells that do not have detectable amounts of a protein that has the activity of a GFAT and / or having no detectable activity of a protein that has the activity of a UDP-Glc-DH having, after genetic modification according to the invention, a detectable amount of a protein encoding a protein that has the activity of a GFAT and / or a detectable amount of a protein that has the activity of a UDP-Glc-DH protein.

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

The increased activity of a protein having the activity of a UDP-Glc-DH in plant extracts can be described using the methods known to those skilled in the art, as described, for example, in WO 00/11192 provides preferred method for determining the amount of activity of a protein that has the activity of a UDP-Glc-DH in General Methods, article 7.

An increased amount of (enzymatic) activity of proteins that have the activity of one or of proteins that have the activity of a UDP-Glc-DH preferably means an increase in the activity of said proteins by at least 50%, preferably at at least 70%, especially preferably at least 85%, and especially preferably at least 100% compared to the corresponding non-genetically modified natural plant cells or non-genetically modified natural plants. An increase in the amount of (enzymatic) activity of proteins that has the activity of a GFAT and / or of a protein that has the activity of a UDP-Glc-DH also means that plants or plant cells that do not have detectable amounts of a protein that has the activity of a GFAT and / or that has no detectable activity of a protein that has the activity of a UDP-Glc-DH that has, after genetic modification according to the invention, a detectable amount of a protein that it has the activity of a GFAT and / or a detectable amount of a protein that has the activity of a UDP-Glc-DH.

In the context of the present invention, it should be understood that the term "genome" means the complete genetic material present in a plant cell. A person skilled in the art knows that, in addition to the nucleus, other compartments (for example, plastids, mitochondria) also contain genetic material.

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

A large number of techniques are available to stably integrate nucleic acid molecules into a host plant cell. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a means of transformation, fusion of protoplasts, injection, DNA electroporation, introduction of DNA through the biolistic solution and also additional options (review in "Transgenic Plants ", Leandro ed., Humana Press 2004, ISBN 1-59259-827-7).

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

5,565,347.

The transformation of monocot plants using Agrobacterium-based vectors, (Chan et al., Plant Mol. Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6, (1994) 271 has also been described. -282; Deng et al., Science in China 33, (1990), 28-34; Wilmink et al., Plant Cell Reports 11, (1992), 76-80; May et al., Bio / Technology 13, (1995 ), 486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al, Transgenic Res. 2, (1993), 252-265). An alternative system for transforming monocot plants is transformation using the biolistic solution (Wan and Lamaux, Plant Physiol. 104, (1994), 37-48; Vasil et al., Bio / Technology 11 (1993), 15531558; Ritala et al. ., Plant Mol. Biol. 24, (1994), 317-325; Spencer et al., Theor. Appl. Genet. 79, (1990), 625-631), protoplast transformation, electroporation of partially permeabilized cells , the introduction of DNA using glass fibers. In particular, corn transformation has been described sometimes in the literature (see, for example, WO95 / 06128, EP0513849, EP0465875, EP0292435; Fromm et al., Biotechnology 8, (1990), 833-844; Gordon -Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appl. Genet. 80, (1990), 721-726). the transformation of other herbs has also been described, such as, for example, rod grass (Panicum virgatum) (Richards et al., 2001, Plant Cell Reporters 20, 48-54).

The satisfactory transformation of other cereal species has also been described, for example, for barley (Wan and Lemaux, see above; Ritala et al., See above; Krens et al., Nature 296, (1982), 72-74 ) and for wheat (Nehra et al., Plant J: 5, (1994), 285-297; Becker et al., 1994, Plant Journal 5, 299-307). All the above procedures 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 greater amounts of hyaluronan than plants that have only the activity of hyaluronan synthase. This allows cheap hyaluronan to be produced due to the isolation of hyaluronan from plants that have a higher hyaluronan content that is less complicated and cheaper. Furthermore, in comparison to the plants described in the prior art, smaller cultivation areas are required to produce hyaluronan using the genetically modified plants according to the invention. This leads to the possibility of providing hyaluronan in sufficient quantities even for industrial applications in which it is not currently used due to its shortage and its high price. Plant organisms infected with Chlorella genus virus are inadequate to produce large amounts of hyaluronan. In hyaluronan production, 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), in this way, to produce hyaluronan, the viral infection must be repeated. Therefore, it is not possible to isolate individual Chlorella cells that continuously synthesize the desired quality and quantity of hyaluronan. In addition, in Chlorella algae infected with virus, hyaluronan occurs only for a limited period of time, and as a result of lysis produced by the virus, algae die only approximately 8 hours after infection (Van Etten et al., 2002 , Arch Virol 147, 1479-1516). On the contrary, 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 vegetative or sexually unlimited way and that produce hyaluronan continuously.

The transgenic plants described in WO 05/012529, which have a nucleic acid molecule encoding a hyaluronan synthase, synthesize a relatively small amount of hyaluronan. On the contrary, the present invention offers the advantage of genetically modified plant cells according to the invention and genetically modified plants according to the invention synthesize considerably 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 that synthesize hyaluronan. the genetically modified plant cells according to the invention or the 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 µg of hyaluronan per g of fresh weight (PF) of plant material.

Preferably, the plant cells according to the invention or the plants according to the invention synthesize almost 25,000 µg of hyaluronan per gram of fresh weight, preferably almost 20,000 µg of hyaluronan per gram of fresh weight, particularly preferably almost 15,000 µg of hyaluronan per gram of fresh weight, especially preferably almost 10,000 µg of hyaluronan per gram of fresh weight and most preferably almost 6500 µg of hyaluronan per gram of fresh weight.

To determine the content of hyaluronan with respect to fresh weight in genetically modified plant cells according to the invention or genetically modified plants according to the invention, preference is given to using the working method of the plant material described in the General Procedures, article 2 and the procedure for determining the amount of hyaluronan described in the General Procedures, article 4.

The present invention also provides genetically modified plant cells according to the invention or the genetically modified plants according to the invention preferably synthesize at least 1000, preferably at least 2000, particularly preferably at least 4000, especially preferably at least 5000 µg of hyaluronan per g dry weight (PS) 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 working method of the plant material described in Example 13k ) and the procedure to determine the amount of hyaluronan described in the General Procedures, article 4.

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

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

Because the plant tissue has the advantage that it does not contain hyaluronidases, a simple and rapid isolation procedure 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. . To this end, water is added to the plant tissue to be examined and then the plant tissue is mechanically crushed (with the help of, for example, a pearl mill, a crusher wheel mill, a Warring mixer, an extractor of juice, etc.). If necessary, more water can then be added to the suspension, and then cell debris and water insoluble components are removed by centrifugation or sieving. The presence of hyaluronan in the supernatant obtained can then be demonstrated using, for example, a protein that specifically binds to hyaluronan. A method for detecting hyaluronan is described with the help of a protein that specifically binds hyaluronan, for example, in US 5,019,498. Test kits for performing the procedure described in US 5,019,498 are commercially available (for example, the hyaluronic acid (AH) test kit from Corgenix, Inc., Colorado, USA, Prod. No. 029- 001; see also General Procedures article 4). In parallel, it is possible to initially digest an aliquot of the centrifugal supernatant obtained with a hyaluronidase and then confirm the presence of hyaluronan with the help of the protein that specifically binds to the hyaluronan, as described above. Through the action of hyaluronidase in the parallel batch, the hyaluronan present in the previous one is degraded, so that after complete digestion it is not possible to detect significant amounts of hyaluronan for longer.

The presence of hyaluronan in the centrifugal supernatant can also be confirmed using other analysis procedures such as, for example, IR, NMR or mass spectrometry.

As discussed above, it is not clear that the metabolic pathway (hexose phosphate pathway or myo-inositol oxidative metabolic pathway) is primarily used in plant cells to synthesize UDPglucuronic acid, and either of both metabolic pathways can make different contributions. quantitative to the synthesis of UDP-glucuronic acid, depending on the tissue and / or the stage of development of the plant. In addition, excess expression of a UDP-Glc-DH in transgenic plants does not lead to consistent results, and it has not been possible to achieve the objective of increasing the pectin content of the cell wall by adopting said solution. Additionally, the regulation of the activity of proteins that have the activity of a UDP-Glc-DH was inhibited by UDP-xylose. This has been demonstrated for relevant proteins originated from prokaryotes (Campbell et al., 1997, J. Biol. Chem. 272 (6), 3416-3422; Schiller et al., 1973, Biochim. Biophys Acta 293 (1), 1 -10), from animal organisms (Balduini et al., 1970, Biochem. J. 120 (4), 719-724) and from plants (Hinterberg, 2002, Plant Physiol. Biochem. 40, 1011-1017). In addition, the reaction products of glucuronic acid and NADH originating from the reaction catalyzed by a protein that has the activity of a UDP-Glc-DH are inhibitors that regulate the activity of a protein that has the activity of a GFAT (Campbell and col., 1997, J. Biol. Chem. 272 (6), 3416-3422, Ordman and Kirkwood, 1977, Biochim Biophys Acta 482 (1) 25-32; Turner and Botha, 2002, Archives of Biochem. Biophys. 407 , 209-216).

Expression in excess, in corn, of a protein that has the (enzymatic) activity of a GFAT translationally fused with a plastid signaling peptide resulted in an increased UDP-glucosamine content, and cytosolic excess expression in corn, of a protein that has the (enzymatic) activity of a GFAT resulted in an increased glucosamine 1-phosphate content in underground endosperm tissue. However, UDP-glucosamine and glucosamine 1-phosphate are not starting materials for the synthesis of hyaluronan by hyaluronan synthase. In addition, it is known that glucosamine has a cytotoxic effect on plant cells (Roberts et al., 1971, Plant Physiol. 48, 36-42) and that, if relatively high concentrations are present in plant cells, it is converted into glucosamine 6- phosphate. Glucosamine 6-phosphate is probably toxic to plant cells (WO 98 35047, US 6,444,878). Furthermore, it is known that proteins that have the activity of a GFAT can be regulated in an inhibitory manner by metabolites that are formed in the additional metabolic pathway for the synthesis of UDP-N-acetyl-glucosamine. Proteins that have the activity of a GFAT are inhibited isolated from eukaryotes (with animal and plant organisms), for example, by UDPN-acetyl-glucosamine, which is one of two substrates for hyaluronan synthase (Konrfeld, 1967, J. Biol. Chem. 242 (13), 3135-3141; Graack et al., 2001, Biochem. J. 360, 401-412; Mayer et al., 1968, Plant Physiol. 43, 10971107). Bacterial proteins that have the activity of a GFAT are inhibited by glucosamine 6-phosphate, a direct reaction product of the reaction catalyzed with GFAT (Deng et al., 2005, Metabolic Engineering 7, 201214). There are no indications in the literature that can limit the amount of hyaluronan synthesized in plant cells.

Accordingly, it has been surprisingly found that genetically modified plant cells or genetically modified plants that have a nucleic acid molecule encoding a hyaluronan synthase and that additionally has an increased GFAT activity and an increased UDP-Glc-DH activity in Comparison with genetically modified plant cells or genetically modified plants that have (only) hyaluronan synthase activity produce significantly larger 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, in which they produce an increased amount of hyaluronan compared to genetically modified plant cells that have ( only) the activity of a hyaluronan synthase or compared to genetically modified plant cells that have the activity of a hyaluronan synthase and an unimproved activity of a protein that has the activity of a GFAT and an unimproved activity of a protein that has the activity of a UDPGlc-DH. Preferably, the amount of hyaluronan produced with respect to the fresh weight of the plant material in the genetically modified plant cells according to the invention or in the genetically modified plants according to the invention is at least 1.5 times, preferably at least 5 times, particularly preferably at least 7.5 times and especially preferably at least 10 times more, compared to genetically modified plant cells or compared to genetically modified plants that have (only) the activity of hyaluronan synthase . In order to determine the increase of the hyaluronan content with respect to the fresh weight of the plant material in the genetically modified plant cells according to the invention or 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 plant cells or corresponding plants that have (only) the activity of hyaluronan synthase, in which the equivalent material (e.g., leaf, tuber) of plant or plant cells should be compared, plant or plant cells from which this material must have been grown under the same conditions and in which the hyaluronan content of the plant material having a comparable age (development stage) must be compared. You should not, for example, compare young leaves of one plant with old leaves of another plant or plants.

In the context of the present invention, it should be understood that the term "plant cell or plant that has (only) the activity of a hyaluronanosyntase" means a genetically modified plant cell or a genetically modified plant in which the genetic modification is that it comprises a nucleic acid molecule encoding a hyaluronan synthase, as compared to the corresponding non-genetically modified natural plant cells or with the non-genetically modified natural plants.

In particular, "plant cells or plants that have (only) the activity of a hyaluronan synthase" are characterized in that they synthesize hyaluronan and in that they have no additional genetic modifications different from those of the introduction of a nucleic acid molecule encoding a hyaluronan synthase in non-genetically modified natural plant cells or in non-genetically modified natural plants. Preferably, said plants do not have an increased activity of a protein that has the activity of a GFAT and an unintended activity of a protein that has the activity of a UDP-Glc-DH.

The amount of hyaluronan produced by plant cells or plants can be determined with the help of the procedures described above, using, for example, a commercial test kit (for example, the hyualuronic acid (AH) test kit of Corgenix, Inc., Colorado, USA, Prod. No. 029-001). A preferred method is described in the context of the present invention for determining the content of hyaluronan in plant or plant cells in the General Procedures, article 4.

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

In the context of the present invention, the term "green land plant (Embryophyte) should be understood as defined in Strasburger," Lehrbuch der Botanik "[Textbook of Botany], 34th ed., Spektrum Akad. Verl., 1999, ISBN 38274 -0779-6).

A preferred embodiment of the present invention relates to genetically modified plant cells according to the invention or multicellular plants or genetically modified plants according to the invention which are multicellular organisms. Therefore, this embodiment refers to plant cells or plants that do not originate single-celled (protist) plants or that are not protist.

The genetically modified plant cells according to the invention or the genetically modified plants according to the invention may, in principle, plant cells and plants, respectively, or any species of plant, that is, monocotyledonous and dicotyledonous plants. there are preferably crop plants, that is, man-grown plants for the purpose of feeding man and animals or for producing biomass and / or for preparing substances for technical purposes, industrial purposes (eg, corn, rice, wheat, alfalfa , rye, oats, barley, cassava, potato, tomato, grass stick (Panicum virgatum), sago, mung beans, peas, sorghum, carrots, eggplant, radish, oilseed rape, soybeans, peanuts, cucumbers, pumpkins, melons, leeks, garlic, cabbage, spinach, sweet potatoes, asparagus, zucchini, lettuce, artichokes, sweet corn, parsnips, black salsify, patacas, banana, sugar beet, sugar cane, beet, broccoli, cabbage, onion, yellow beet, dandelion, strawberry, apple, apricot, plum, peach, vines, cauliflower, celery, peppers, Swedish 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 in which the nucleic acid molecule encoding hyaluronan synthase is characterized in that it encodes a viral hyaluronan synthase . The nucleic acid molecule encoding hyaluronan synthase preferably encodes a hyaluronan synthase of a virus that infects algae.

With respect to a virus that infects algae, the nucleic acid molecule that encodes hyaluronan synthase preferably encodes a hyaluronan synthase of a virus that infects Chlorella, particularly preferably, a hyaluronan synthase of a Paramecium bursaria Chlorella virus 1 and especially Preferably a hyaluronan synthase of an H1 strain of a Paramecium bursaria Chlorella virus.

In a further preferred embodiment, the present invention relates to genetically modified plant cells according to the invention or to genetically modified plants according to the invention in which the nucleic acid molecules encoding hyaluronan synthase are characterized in that the codons of The nucleic acid molecules encoding a hyaluronan synthase are modified compared to the codons of the nucleic acid molecules encoding the organism's hyaluronan synthase that the hyaluronan synthase originated from. With particular preference, the codons of the hyaluronan synthase have been modified in such a way that they adapt to the frequency of the use of the codons of the plant or plant cell in whose genome they are integrated or will be integrated.

Due to the degeneracy of the genetic code, amino acids can be encoded by one or more codons. In different organisms, codons that encode an amino acid are used at different frequencies. Adapting the codons of a nucleic acid coding sequence to the frequency of their use in the plant cell or in the plant in whose genome the sequence to be integrated is expressed can contribute to an increased amount of translated protein and / or to the Stability of the mRNA in question in plant cells or specific plants. The person skilled in the art can determine the frequency of codon use in the plant or plant cells in question by examining how many nucleic acid coding sequences are possible for the frequency with which certain codons are used to encode a given amino acid. The person skilled in the art knows the frequency of codon usage of certain organisms and can be determined simply and quickly using computer programs. The appropriate computer programs are publicly accessible and are provided among other things for free on the Internet (for example
http: //gcua.schoedl
.from/;
http://www.kazusa.or.jp/codon/;

http://www.entelechon.com/eng/cutanalysis.html). Adapting the codons of a nucleic acid coding sequence to the frequency of use in the plant cell or in the plant in whose genome the sequence to be expressed is integrated can be performed by in vitro mutagenesis or, preferably, by synthesis of de novo of the gene sequence. Those skilled in the art are aware of the procedures for de novo synthesis of nucleic acid sequences. De novo synthesis can be performed, for example, by initially synthesizing individual nucleic acid oligonucleotides, hybridizing these with oligonucleotides complementary to the above, so that they form a double stranded DNA, and then joining the double stranded individual oligonucleotides in such a way that it is obtained the desired nucleic acid sequence. The de novo synthesis of nucleic acid sequences can be outsourced including the adaptation of the frequency with which the codons of a given target organism are used to companies offering this service (for example, Entelechon GmbH, Regensburg, Germany).

The nucleic acid molecule encoding the hyaluronan synthase is preferably characterized in 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 95% and most preferably at least 98% identical to the amino acid sequence shown in SEQ ID NO. 2. More preferably, the nucleic acid molecule encoding hyaluronan synthase is characterized in that it encodes a hyaluronan synthase having the sequence. of amino acids shown in SEQ ID NO. 2.

The nucleic acid molecule encoding a hyaluronan synthase may be 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 nucleic acid sequence shown in SEQ ID No. 1 or in SEQ ID No. 3. The nucleic acid molecule encoding hyaluronan synthase is characterized in that it encodes a hyaluronan synthase having the amino acid sequence shown in SEC ID No. 3.

On 25 June 2004, plasmid IC 341-222, which comprises a synthetic nucleic acid molecule that encodes the hyalurosan synthase of Paramecium bursaria Chlorella virus was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Brunswick, Germany, with the number DSM16664, according to the Budapest treaty. The amino acid sequence shown in SEQ ID No. 2 of the coding region of the nucleic acid sequence integrated in plasmid 341-222 for the hyaluronan synthase of Paramecium bursaria Chlorella virus can be derived.

Accordingly, the present description also refers to genetically modified plant cells or genetically modified plants in which the nucleic acid molecules encoding hyaluronan synthase are characterized in that they encode a protein whose amino acid sequence can be derived from the coding region of the nucleic acid sequence inserted into plasmid DSM16664 or that 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 is derived from the coding region of the nucleic acid sequence inserted in plasmid DSM 16664.

The present description also refers to genetically modified plant cells or genetically modified plants in which the nucleic acid molecule encoding the hyaluronan synthase is characterized in that it is the nucleic acid sequence encoding the hyaluronan synthase integrated in the plasmid DSM16664 or which 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 nucleic acid sequence integrated in plasmid DSM16664.

The present invention further describes genetically modified plant cells according to the invention or genetically modified plants according to the invention that are characterized in that they have a foreign nucleic acid molecule stably integrated in their genome or a plurality of foreign nucleic acid molecules. stably integrated into its genome, said foreign nucleic acid molecule or said foreign nucleic acid molecules increase the activity of a protein that has the activity of a GFAT and increase the activity of a UDP-Glc-DH compared to cells non-genetically modified vegetables or corresponding to non-genetically modified natural plants.

It can be a single foreign nucleic acid molecule that, by integrating into the genome of genetically modified plant cells according to the invention or genetically modified plants according to the invention, increases the activity of the protein having the activity of a GFAT and simultaneously increases the activity of a protein that has the activity of a UDP-Glc-DH in comparison with the corresponding natural plant cells or with the corresponding natural plants. However, it can also be a plurality of foreign nucleic acid molecules, a foreign nucleic acid molecule that increases the activity of a protein that has the activity of a UDP-Glc-DH and another foreign nucleic acid molecule that increases the activity of a protein that has the activity of a UDP-Glc-DH in comparison with the corresponding plant cells or with the corresponding natural plants. if a plurality of foreign nucleic acid molecules are integrated into the genome of a genetically modified plant cell according to the invention or of a genetically modified plant according to the invention, both foreign nucleic acid molecules together may be at a site where the genome of the plant or plant cell, or they can be located at different sites in the genome of the plant or plant cell (for example, on different chromosomes

or in different sections of chromosomes). Accordingly, foreign nucleic acid molecules can be inherited either as a binding locus or as loci coupled according to Mendel's laws, or they can be inherited as separate loci independently from each other according to Mendel's laws.

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

In the context of the present invention, it should be understood that the term "recombinant nucleic acid molecule" means a nucleic acid molecule that has various nucleic acid molecules that are not naturally present in a combination similar to that present in an acid molecule. recombinant nucleic Thus, recombinant nucleic acid molecules can, in addition to nucleic acid molecules encoding a hyaluronan synthase and / or a protein that has the activity of a GFAT or a protein that has the activity of a UDP-Glc-DH, additionally they have nucleic acid sequences that are not naturally present in combination with the aforementioned nucleic acid molecules. the aforementioned 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 has the activity of a GFAT and / or a protein that has the activity of a UDP-Glc-DH can be any sequences. For example, they can be genomic sequences of plant nucleic acids. The aforementioned additional nucleic acid sequences are preferably regulatory sequences (promoters, and termination signals, enhancers), particularly preferable regulatory sequences that are active in plant tissues, especially tissue specific regulatory sequences that are active in a plant. Plant tissue. The person skilled in the art knows the procedures for generating recombinant nucleic acid molecules and understands genetic engineering procedures, such as, for example, the binding of nucleic acid molecules by ligation, genetic recombination or de novo synthesis of molecules of molecules. nucleic acids (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 Biology, John Wiley &Sons; 5th edition (2002), ISBN: 0471250929).

Genetically modified plant cells and genetically modified plants that have a foreign nucleic acid molecule integrated in their genome or a plurality of foreign nucleic acid molecules stably integrated in their genome that encode hyaluronan synthase and that increase the activity of a protein that has the activity of a GFAT and increases the activity of a protein that has the activity of a UDP-Glc-DH compared to non-genetically modified natural plant cells or non-genetically modified natural plants can be distinguished from said plant cells natural and of said plant cells, respectively, among other things by the fact that they comprise a foreign nucleic acid molecule that does not occur naturally in natural plant cells and in natural plants, respectively, or that said molecule is integrated into a site in the veget cell genome to the genetically modified according to the invention or in the genome of the genetically modified plant according to the invention in which it is not produced in natural plant cells and in natural plants, respectively, that is, in a different genomic environment. Furthermore, said genetically modified plant cells according to the invention and genetically modified plants according to the invention can be distinguished from genetically modified natural plant cells and non-genetically modified natural plants, respectively, in that it comprises at least one copy of the molecule of foreign nucleic acid stably integrated into its genome, if appropriate in addition to copies of said molecule naturally present in natural plant cells or natural plants. if the molecule (s) introduced into genetically modified plant cells according to the invention or genetically modified plants according to the invention are additional copies of molecules already naturally present in natural plant cells or natural plants, genetically modified plant cells according to the invention and genetically modified plants according to the invention can be distinguished from natural plant cells and natural plants, respectively, in particular by the fact that this additional copy / these additional copies are located / n at sites in the genome where it is / are / are not present in natural plant cells and natural plants, respectively.

The stable integration of a nucleic acid molecule into the genome of a plant cell or a plant can be demonstrated by genetic procedures and / or molecular biology procedures. A stable integration of a nucleic acid molecule into the genome of a plant cell or the genome of a plant is characterized in that the progeny that has inherited said nucleic acid molecule, the stably integrated nucleic acid molecule is present in it Genomic environment than in the parent generation. The presence of a stable integration of a nucleic acid sequence in the genome of a plant cell or in the genome of a plant can be demonstrated using procedures known to the person skilled in the art, among other things with the help of transfer analysis. Southern RFLP (Restriction Fragment Length Polymorphism) analysis (Nam et al., 1989, The Plant Cell 1, 699-705; Leister and Dean, 1993, The Plant Journal 4 (4), 745-750), with PCR-based procedures, such as, for example, the analysis of length differences in the amplified fragment (Amplified Fragment Length Polymorphism, AFLP) (Castiglioni et al., 1998, Genetics 149, 2039-2056; Meksem and col., 2001, Molecular Genetics and Genomics 265, 207-214; Meyer et al., 1998, Molecular and General Genetics 259, 150-160) or using amplified fragments cleaved with the aid of restriction endonucleases (Amplified Polymorphic Sequences s Split, CAPS) (Konieczny and Ausubel, 1993, The Plant Journal 4, 403-410; Jarvis et al., 1994, Plant Molecular Biology 24, 685-687; Bachem et al., 1996, The Plant Journal 9 (5), 745-753).

The present invention describes genetically modified plant cells or genetically modified plants that are characterized in that at least one foreign nucleic acid molecule encodes a protein that has the activity (enzymatic) of a GFAT or the at least one foreign nucleic acid molecule encodes a protein which has the (enzymatic) activity of a UDP-Glc-DH.

The present invention describes genetically modified plant cells according to the invention or genetically modified plants according to the invention that are characterized in that a first foreign nucleic acid molecule encodes a protein that has the (enzymatic) activity of a GFAT and a second molecule of foreign nucleic acid encodes a protein that has the (enzymatic) activity of a UDP-Glc-DH.

The foreign nucleic acid molecule that encodes a protein that has the (enzymatic) activity of a GFAT can originate from bacteria, or, animals, particularly preferably from mammals or bacteria and especially preferably from mouse. or Escherichia coli.

With respect to a foreign nucleic acid molecule that encodes a protein that has the (enzymatic) activity of a GFAT that originates from animal organisms, preferably a nucleic acid molecule that encodes a protein that has the (enzymatic) activity of a GFAT-2; with particular preference, the protein that has the (enzymatic) activity of a GFAT-2 originates from the mouse.

Instead of a naturally occurring nucleic acid molecule that encodes a protein that has the activity (enzymatic) of a GFAT, it is also possible to introduce a nucleic acid molecule generated by mutagenesis into genetically modified plant cells according to the invention. or genetically modified plants according to the invention, wherein said mutagenized foreign nucleic acid molecule is characterized in that it encodes a protein that has the (enzymatic) activity of a GFAT with a reduced inhibition by metabolites (for example, of metabolism of glucosamine). The preparation of said mutagenized nucleic acid molecules is described in an illustrative manner for a protein that has the (enzymatic) activity of an Escherichia coli GFAT in Deng et al. (2005, Metabolic Engineering 7, 201-214; WO 04/003175). Mutants of a protein having 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 has the (enzymatic) activity of a UDP-Glc-DH can originate from any organism; preferably, said nucleic acid molecule originates from bacteria, fungi, animals, plants or viruses, particularly preferably from bacteria, plants or viruses, especially preferably from viruses.

With respect to viruses, the foreign nucleic acid molecule that encodes a protein that has the (enzymatic) activity of a UDP-Glc-DH can preferably originate from a virus that infects algae, preferably from a virus that It infects algae of the genus Chlorella, particularly preferably from a Paramecium bursaria Chlorella virus and especially preferably from a Paramecium bursaria Chlorella virus from an H1 strain.

Instead of a naturally occurring nucleic acid molecule that encodes a protein that has the (enzymatic) activity of a UDP-Glc-DH, it is also possible to introduce a nucleic acid molecule generated by mutagenesis into genetically modified plant cells of according to the invention or genetically modified plants according to the invention, wherein said mutagenized foreign nucleic acid molecule is characterized in that it encodes a protein that has the (enzymatic) activity of a UDP-Glc-DH with reduced inhibition by metabolites (for example, of glucuronic acid metabolism).

Those skilled in the art know the nucleic acid molecules that encode a protein that has the activity of a GFAT and are described in the literature. Thus, nucleic acid molecules that encode a protein that has the activity of a GFAT are described from viruses, for example, for Chlorella virus k2 (EMBL Accession No. AB107976.1), from bacteria, for example Escherichia coli (Dutka-Malen, 1988, Biochemie 70 (2), 287-290; EMBL Accession No.: L10328.1), from fungi, for example Saccharomyces cerevisiae (EMBL Accession No. AF334737 .1, Watzele et al., 1989, J. Biol. Chem. 264, 8753-8758), Aspergillus niger (EMBL Accession No. AY594332.1), Candida albicans (EMBL Accession No. X94753.1), from of insects, for example Aedes aegyti (Kato et al., 2002, insects. Biol. 11 (3), 207, 216; EMBL accession number AF399922.1), Drosophila melanogaster (GFAT-1: Accession number EMBL Y18627.1, GFAT-2: NCBI accession number NM_143360.2), from algae of Volvariella volvacea (EMBL accession number AY661466.1), from vertebrates, for example of Homo sapiens (GFAT- 1: No. of access of the EMBL AF334737.1; GFAT-2: NCBI accession number BC000012.2, Oki et al., 1999, Genomics 57 (2), 227-34), Mus musculus (GFAT-1: EMBL accession number AF334736.1; GFAT-2 : EMBL access number AB016780.1), or from plants, for example from Arabidopsis thaliana (EMBL access number AP001297.1; CDBI accession number NCBI BAB03027.1).

The present description refers to genetically modified plant cells according to the invention and genetically modified plants in which the foreign nucleic acid molecule encoding a protein having the activity of a GFAT is selected from the group consisting of

a) nucleic acid molecules encoding a protein that has the sequence provided in SEQ ID No. 10 or a protein that has the amino acid sequence provided in SEQ ID No. 12; b) nucleic acid molecules that encode a protein whose sequence is at least 60%, preferably at least 80%, preferably at least 90%, especially preferably at least 95% and most preferably at least 98% identical to the amino acid sequence of SEQ ID No. 10 or SEQ ID No. 12; c) the nucleic acid molecules comprising the nucleotide sequences shown in the nucleotide sequence shown in SEQ ID No. 9 or a sequence complementary to the above, the nucleotide sequence of SEQ ID No. 11 or a sequence complementary to the previous one or the nucleotide sequence shown in SEQ ID No. 13 or a sequence complementary to the previous one; d) nucleic acid molecules that are 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 acid sequence nucleic described in a) or c); e) nucleic acid molecules that hybridize under restrictive conditions with at least one strand of the nucleic acid sequences described in a) or c); f) nucleic acid molecules whose nucleotide sequences differ from the sequence of nucleic acid molecules mentioned in a) or c) due to the degeneracy of the genetic code; and g) nucleic acid molecules that are fragments, variants and / or allelic derivatives of the nucleic acid molecules mentioned in a), b), c), d), e) or f).

Nucleic acid molecules that encode a protein that has the activity of a UDP-Glc-DH are described in the literature and known to those skilled in the art. Thus, nucleic acid molecules that encode a protein that has the activity of a UDP-Glc-DH from virus are described, for example for Chlorella virus 1 (NCBI Accession No. NC_000852.3), from bacteria, for example Escherichia coli (EMBL accession number: AF176356.1), from fungi, for example Aspergillus niger (EMBL accession number AY594332.1), Cryptococcus neoformans (EMBL accession number AF405548. 1), from insects, for example from Drosophila melanogaster (EMBL Accession No. AF001310.1), from vertebrates, for example from Homo sapiens (EMBL Accession No. AF061016.1), Mus musculus (No. of EMBL access AF061017.1), Bos taurus (EMBL access no. AF095792.1), Xenopus laevis (EMBL access no. AY762616.1) or from plants, for example poplars (EMBL access no. AF053973.1 ), Colocasia esculenta (EMBL accession number AY222335.1), Dunaliella salina (EMBL accession number AY795899.1), Glycine max (EMBL access number U53418.1).

The present description refers to genetically modified plant cells and genetically modified plants in which the foreign nucleic acid molecule encoding a protein having the activity of a UDP-Glc-DH is selected from the group consisting of

a) nucleic acid molecules encoding a protein that has the amino acid sequence provided in 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 most preferably at least 98% identical to the amino acid sequence provided in SEQ ID No. 5; c) nucleic acid molecules comprising the nucleotide sequence of SEQ ID No. 4 or a complementary sequence of the above or the nucleotide sequence shown in SEQ ID No. 6 or a complementary sequence of the above; d) nucleic acid molecules that are 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 nucleic acid sequences described in a) or c); e) nucleic acid molecules that hybridize under restrictive conditions with at least one strand of the nucleic acid sequences described in a) or c); f) nucleic acid molecules whose nucleotide sequences differ from the sequence of nucleic acid molecules mentioned in a) or c) due to the degeneracy of the genetic code; and g) nucleic acid molecules that are fragments, variants and / or allelic derivatives of the nucleic acid molecules mentioned in 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 restrictive conditions, as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. . (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). With particular preference, "hybridization" means a hybridization under the following conditions:

Hybridization Buffer:

2xSSC; 10 x Denhardt solution (Fikoll 400 + PEG + BSA; 1: 1: 1 ratio); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 µg / ml herring sperm DNA; 50 mg / ml tRNA; or 25 mM sodium phosphate buffer, pH 7.2; 1 mM EDTA;

7% SDS

Hybridization temperature:

T = 65 at 68 ° C

Wash buffer: 0.1xSSC; 0.1% SDS Washing temperature: T = 65 to 68 ° C.

Nucleic acid molecules that hybridize with nucleic acid molecules that encode a protein that has the activity of a UDP-Glc-DH can originate from any organism; accordingly, they can originate from bacteria, fungi, animals, plants or viruses.

Nucleic acid molecules that hybridize with nucleic acid molecules that encode a protein that has the activity of a UDP-Glc-DH preferably originate from a virus that infects algae, preferably a virus that infects algae of the genus Chlorella , particularly preferably from a Paramecium bursaria Chlorella virus and especially preferably from a Paramecium bursaria Chlorella virus from an H1 strain.

Nucleic acid molecules that hybridize with nucleic acid molecules that encode a protein that has the activity of a GFAT can originate from mammals, or bacteria and especially preferably from mouse or Escherichia coli.

Nucleic acid molecules that hybridize with nucleic acid molecules that encode a protein that has the activity of a GFAT -2 can originate from an animal organism, preferably from a mouse.

Nucleic acid molecules that hybridize with the mentioned molecules can be isolated, for example, from genomic libraries or cDNAs. Such nucleic acid molecules can be identified and isolated using the aforementioned nucleic acid molecules or parts of these molecules or the inverse complements of these molecules, for example, by hybridization according to standard procedures (see, for example, Sambrook et al. ., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or by amplification using PCR.

As a hybridization sample to isolate a nucleic acid sequence encoding a protein that has the activity of a UDP-Glc-DH, it is possible to use, for example, nucleic acid molecules that have exactly or essentially the nucleotide sequences provided in the SEQ ID No. 4 or in SEQ ID No. 6, or parts of these sequences.

As a hybridization sample to isolate a nucleic acid sequence encoding a protein that has the activity of a GFAT, it is possible to use, for example, nucleic acid molecules that have exactly or essentially the nucleotide sequences provided in SEQ ID NO: 9 or in SEQ ID No. 11 or in SEQ ID No. 13, or parts of these sequences.

The fragments used as hybridization samples may also be synthetic fragments or oligonucleotides prepared using custom synthesis techniques, the sequence of which is essentially identical to the nucleic acid molecule described in the context of the present invention. Once the genes that hybridize with 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 by this sequence must be analyzed to determine if they are proteins that have the activity of a GFAT, a GFAT-1 or a GFAT-2 or the activity of a UDP-Glc-DH. The procedures of how to determine if a protein has the activity of a protein that has the activity of a GFAT (for example, Mayer et al., 1968, Plant Physiol. 43, 1097-1107; Deng et al., 2005, Metabolic Engineering 7 , 201-214), a GFAT-1 or a GFAT-2 (for example Hu et al., 2004, J. Biol. Chem. 279 (29), 2998829993) or a UDP-Glc-DH (for example De Luca et al., 1976, Connective Tissue Research 4, 247-254; Bar-Peled et al., 2004, Biochem. J. 381, 131-136; Turner and Botha, 2002, Archives Biochem. Biophys. 407, 209-216 ) are known by persons skilled in the art and are described, among other things, in the literature described.

The molecules that hybridize with the nucleic acid molecules described in the context of the present invention comprise in particular fragment derivatives, and allelic variants of the aforementioned nucleic acid molecules. In the context of the present invention, the term "derivative" means that the sequences of these molecules differ in one or more positions of the nucleic acid molecule sequences described above and are very identical to these sequences. The differences with the nucleic acid molecules described above may, for example, be due to deletion, additions, substitution, insertion or recombination.

In the context of the present invention, the term "identity" means a sequence identity over the entire length of the coding region of a nucleic acid molecule or the full length of an amino acid sequence encoding a protein of at least 60 %, in particular 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, it should be understood that the term "identity" means the number of identical amino acids / nucleotides (identity) with other proteins / nucleic acids, expressed as a percentage. Preferably, the identity with respect to a protein having the activity of a UDP-Glc-DH is determined by comparison with the amino acid sequence provided in SEQ ID NO. 5, the identity with respect to a nucleic acid molecule encoding a Protein that has the activity of a UDP-Glc-DH is determined by comparison with the nucleic acid sequence provided in SEQ ID No. 4 or in SEQ ID No. 6, the identity with respect to a protein that has the activity of a GFAT is determined by comparison with the amino acid sequence provided in SEQ ID No. 10 or in SEQ ID No. 12, and the identity with respect to a nucleic acid molecule encoding a protein that has the activity of a GFAT is determined by comparison with the nucleic acid sequence provided in SEQ ID No. 9 or SEQ ID No. 11 or SEQ ID No. 13 with other proteins / nucleic acids with the aid of computer programs. If the sequences to be compared with each other are of different lengths, the identity will be determined by determining the percentage identity of the number of amino acids that the shortest sequence shares with the longest sequence. Preferably, identity is determined using the known and publicly available ClustaIW program (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalW is publicly available from Julie Thompson

(Thompson@EMBL-Heidelberg.DE) and Toby Gibson (
Gibson @ EMBL-Heidelberg
.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can also be downloaded from various websites, among others from the IGBMC (Institut de Genetique et de Biologie Moleculaire et Cellulaire, BP163, 67404 Illkirch Cedex, France); ftp://ftp-igbmc.u-strasbg.fr/pub/) and the EBI (ftp://ftp.ebi.ac.uk/pub/software/) and the duplicate Internet pages of the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom).

Preferably, use is made of the ClustalW software of version 1.8 to determine the identity between the proteins described in the context of the present invention and other proteins. Here, the parameters must be configured as follows: KTUPLE = 1, TOPDIAG = 5, WINDOW = 5, PAIRGAP = 3, GAPOPEN = 10, GAPEXTEND = 0.05, GAPDIST = 8, MAX-DIV = 40, MATRIX = GONNET, ENDGAPS (OFF), NOPGAP, NOHGAP.

Preferably, use is made of the ClustaIW software of version 1.8 to determine the identity of the example between the nucleotide sequence of the nucleic acid molecules described in the context of the present invention and the nucleotide sequence of other nucleic acid molecules. Here, the parameters must be configured as follows:

KTUPLE = 2, TOPDIAGS = 4, PAIRGAP = 5, DNAMATRIX: IUB, GAPOPEN = 10, GAPEXT = 5, MAXDIV = 40, TRANSITIONS: unweighted.

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

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

These can be, for example, biological or enzymatic activity, substrate specificity, molecular weight, immunological reactivity, conformation, etc., and also physical properties, such as, for example, the properties of advance in gel electrophoresis, chromatographic behavior , sedimentation coefficients, solubility, spectroscopic properties, stability, optimum pH, optimum temperature, etc. Those skilled in the art know the preferred properties of proteins that have the activity of a GFAT or a UDPGlc-DH, 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 in which the nucleic acid molecules encoding a protein having the (enzymatic) activity of a GFAT and / or encoding a protein that has the (enzymatic) activity of a UDP-Glc-DH are characterized in that the codons of said nucleic acid molecules are different from the codons of the nucleic acid molecules encoding said protein that it has the (enzymatic) activity of a GFAT or they encode said protein that has the (enzymatic) activity of a UDP-Glc-DH of the parent organism. Particularly preferably, the codons of nucleic acid molecules that encode a protein that has the (enzymatic) activity of a GFAT or that encode a protein that has the (enzymatic) activity of a UDP-Glc-DH are changed in such a way. so that they adapt to the frequency of use of the codons of the plant or plant cell in whose genome they are integrated or will be integrated.

The present invention further provides genetically modified plant cells according to the invention or genetically modified plants according to the invention in which the foreign nucleic acid molecules stably integrated into the genome of the plant or plant cell encoding a hyaluronan synthase and / or that encode a protein that has the (enzymatic) activity of a GFAT and / or that encode a protein that has the (enzymatic) activity of a UDP-Glc-DH bind to regulatory elements that initiate transcription in plant cells (promoters). These may be homologous or heterologous promoters. The promoters can be constitutive, tissue specific, developmental specific or regulated by external factors (for example, after the application of chemical substances, through the action of abiotic factors, such as heat and / or cold, drought, disease, etc. ). Here, nucleic acid molecules encoding a hyaluronan synthase or a protein that has the activity (enzymatic) of a GFAT or a protein that has the activity of a UDP-Glc-DH, whose nucleic acid molecules are integrated into the genome of A genetically modified plant cell according to the invention or of a genetically modified plant according to the invention can, in each case, bind to the same promoter, or the individual sequences can bind to different promoters. Here, two or three promoters in any combination can in each case be linked to a relevant foreign nucleic acid molecule that encodes a hyaluronan synthase or a protein that has the (enzymatic) activity of a GFAT or a protein that has the (enzymatic) activity of a UDP-Glc-DH in a genetically modified plant cell according to the invention or in a genetically modified plant according to the invention.

The present description refers to genetically modified plant cells according to the invention or to genetically modified plants according to the invention in which at least one foreign nucleic acid molecule, particularly preferably at least two foreign nucleic acid molecules, of especially preferably three foreign nucleic acid molecules selected from the group consisting of nucleic acid molecules encoding a hyaluronan synthase or a protein that has the (enzymatic) activity of a GFAT or a protein that has the (enzymatic) activity of a UDP-Glc-DH binds to a tissue specific promoter. Preferred tissue specific promoters are promoters that initiate transcription specifically in plant tubers, fruits or seed or leaf cells.

To express nucleic acid molecules encoding a hyaluronan synthase or a protein that has the activity (enzymatic) of a GFAT or a protein that has the activity of a UDP-Glc-DH, they bind to regulatory DNA sequences that ensure the transcription in plant cells. These include specific promoters. In general, any active promoter in plant cells is suitable for expression.

Here, the promoter can be chosen in such a way that the expression is constitutive or only in a certain tissue, at a certain point of plant development or at a time point determined by external factors. With respect to the plant and with respect to the nucleic acid molecule to be expressed, the promoter can be homologous or heterologous.

Suitable promoters are, for example, the 35S RNA promoter of cauliflower mosaic virus or the ubiquitin promoter from corn or Cestrum YLCV (Yellow Curly Leaf Virus; WO 01 73087; Stavolone et al. ., 2003, Plant Mol. Biol. 53, 703-713) for a constitutive expression, the B33 promoter of patatingen (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) for a specific expression of potato tuber

or a specific tomato fruit promoter, such as, for example, the tomato polygalacturonase promoter (Montgomery et al., 1993, Plant Cell 5, 1049-1062) or the E8 tomato promoter (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 ensures expression only in photosynthetically active tissues, for example, the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989) , 2445-2451) or for a specific expression of endosperm, the wheat HMWG promoter, the USP promoter, the phaseolin promoter, the promoters of the maize corin genes (Pedersen et al., Cell 29 (1982) , 1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93), the glutelin promoter (Leisy et al., Plant Mol. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993), 357-366; Yoshihara et al., FEBS Lett., 383 (1996), 213218) or the shrunken-1 promoter (Werr et al., EMBO J. 4 (1985), 1373-1380), a globulin promoter (Nakase et al. , 1996, Gene 170 (2), 223-226) or a prolamine promoter (Qu and Takaiwa, 2004, Plant Biotechnology Journal 2 (2), 113-125). However, it is also possible to use promoters that are only active at a time point determined by external factors (see, for example, WO 9307279). Of particular interest here may be heat shock protein promoters that allow for simple induction. Seed-specific promoters are also possible, such as, for example, the Vicia taba USP promoter that ensures a specific expression of seed in Vicia taba and other plants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669 -679; Baumlein et al., Mol. Gen. Genet. 225 (1991), 459-467).

Also suitable is the use of promoters present in the genome of viruses that infect to express nucleic acid sequences in plants (Mitra et al., 1994, Biochem. Biophys Res Commun 204 (1), 187-194; Mitra and Higgins, 1994, Plant Mol Biol 26 (1), 85-93, Van Etten et al., 2002, Arch Virol 147, 1479-1516).

In the context of the present invention, it should be understood that the term "tissue specific" means the substantial limitation of a manifestation (eg, onset of transcription) to a particular tissue.

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

In the context of the present invention, it should be understood that the term "homologous promoter" means a promoter that is naturally present in plant cells or plants used for the preparation of genetically modified plant cells according to the invention or genetically modified plants according to the invention (homologous with respect to the plant cell or plant) or means a promoter that regulates the regulation of the expression of a gene in an organism from which the sequence has been isolated (homologous with respect to the nucleic acid molecule that will be expressed).

In the context of the present invention, it should be understood that the term "heterologous promoter" means 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 genetically modified plants according to with the invention (heterologous with respect to the plant cell or the plant) or means a promoter that is, in the organism from which a nucleic acid molecule to be expressed has been isolated, not naturally present to regulate the expression of said nucleic acid sequence (heterologous with respect to the nucleic acid molecule to be expressed).

A termination sequence (polyadenylation signal) may also be present that serves to add a poly-A tail to the transcript. The poly-A tail is thought to act in the stabilization of the transcripts. Such elements are described in the literature (see Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged as desired.

It is also possible that intronic sequences are present between the promoter and the coding region. Such intronic sequences can lead to expression stability and increased expression in plants (Callis et al., 1987, Genes Devel. 1, 1183-1200; Luehrsen, and Walbot, 1991, Mol. Gen. Genet. 225, 81 -93; Rethmeier et al., 1997; Plant Journal 12 (4), 895-899; Rose and Beliakoff, 2000, Plant Physiol. 122 (2), 535-542; Vasil et al., 1989, Plant Physiol. 91 , 1575-1579; XU et al., 2003, Science in China Series C Vol. 46 No. 6, 561-569). Suitable intronic sequences are, for example, the first intron of the corn sh1 gene, the first intron of the corn polyubiquitin gene 1, the first intron of the rice EPSPS gene or one 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. Said plants can be produced by regeneration of 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, it should be understood that the term "processable parts" means parts of plants that are used to prepare edible foods and feeds, which are used as a source of raw materials in industrial processes, as a source of raw materials for the preparation of pharmaceutical products or as a source of raw materials for the preparation of cosmetic products.

In the context of the present invention, it should be understood that the term "consumable parts" means parts of plants that serve as food for man or that are used as food for animals.

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

Here, the term "propagation material" comprises two plant components that are suitable for generating progeny through the vegetative or generative path. Suitable for vegetative propagation are, for example, cuttings, callus cultures, rhizomes or tubers. Other propagation material includes, for example, fruits, seeds, seedlings, protoplasts, cell cultures, etc. The propagation material preferably takes the form of tubers, fruits or seeds.

In a further embodiment, the present invention relates to harvested plant parts of genetically modified plants according to the invention, such as fruits, for storage and other roots, flowers, flower buds, buds, leaves or stems, preferably seeds, fruits or tubers, these harvested parts comprise genetically modified plant cells according to the invention.

Preferably, the present invention relates to the propagation material according to the invention or to harvested parts of plants according to the invention comprising hyaluronan. Particularly preferred is the propagation material according to the invention or the harvested parts of plants according to the invention that synthesize hyaluronan.

In the context of the present invention, it should be understood that the term "potato plant" or "potato" means species of plants of the genus Solanum, particularly species producing tubers of the genus Solanum and in particular Solanum tuberosum.

In the context of the present invention, it should be understood that the term "tomato plant" or "tomato" means species of plants of the genus Lycopersicon, in particular Lycopersicum esculentum.

A further advantage of the present invention is that the harvested parts, propagation material, processable parts or consumable plants of genetically modified plants according to the invention comprise more hyaluronan than the transgenic hyaluronan synthesizing plants described in the literature. Accordingly, the genetically modified plants according to the invention are not only particularly suitable for use as raw material from which hyaluronan can be isolated but can also be used directly as edible food / feedstuffs or to prepare edible food / feedstuffs. which have a prophylactic or therapeutic character (for example, for the prophylaxis of osteoarthritis, US 6,607,745). Because the genetically modified plants according to the invention have a hyaluronan content greater than the plants described in the literature, the preparation of said edible foods / feeds requires smaller amounts of harvested parts, propagation material, processable parts or parts consumables of genetically modified plants according to the invention. If the consumable parts of genetically modified plants according to the invention are consumed, for example, directly as they are called "nutraceuticals" in this way, it is possible to achieve a positive effect by ingesting even relatively small amounts of substances. This can be of particular significance among others in the production of animal foods, because animal foods have a very high content of plant components, are inadequate as feed for various animal species.

By virtue of the high capacity of hyaluronan to bind to water, harvested parts, propagation material, processable parts or consumable parts of genetically modified plants according to the invention have the advantage that less thickeners are required when produced edible food / solidified feed. Therefore, for example, the production of jellies requires less sugar which is associated with an additional positive effect on health. in the production of edible food / feedstuffs in which the dehydration of the raw material of the plant is required, the advantage of using harvested parts, propagation material, processable parts or consumable parts of genetically modified plants in accordance with the The invention consists in the fact that less water has to be removed from the material of the plant in question, resulting in lower production costs and, due to milder preparation processes (for example, a lower heat input and / or shorter), a high nutritional value of the edible food / feed in question. Therefore, for example, in the production of tomato sauce, less energy has to be introduced in order to achieve the desired consistency.

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

a) genetically modify a plant cell, in which the genetic modification comprises the following stages i to iii

i) the introduction of a foreign nucleic acid molecule encoding a hyaluronan synthase linked to regulatory sequences that ensure transcription in plant cells in the plant cell ii) introduction of a foreign nucleic acid molecule in the plant cell, the acid molecule A foreign nucleic comprising a coding sequence of a protein that has the activity of a glutamine: fructose 6-phosphate amidotransferase-2 (GFAT-2) that originates from animals or a glutamine: fructose 6-phosphate amidotransferase (GFAT) originating from bacteria iii) introduction of a foreign nucleic acid molecule into the plant cell, the foreign nucleic acid molecule comprising a coding sequence of a protein that has the activity of a glutamine: fructose 6-phosphate UDP-glucose dehydrogenase

where steps i to iii can be performed in any order, individually, or any combination of stages i to iii can be done simultaneously b) regenerating a plant from plant cells of stage a); c) generating, if appropriate, additional plants using the plants according to stage b), in which, if appropriate, plant cells were isolated from plants according to stages b) or c) and the process steps a ) ac) were repeated until the plant was generated that had a foreign nucleic acid molecule encoding a hyaluronan synthase and had an increased activity of a protein that had the (enzymatic) activity of a GFAT compared to non-natural plant cells corresponding genetically modified and an increased activity of a protein that had the (enzymatic) activity of a UDP-Glc-DH compared to the corresponding non-genetically modified natural plant cells.

The present description preferably refers to processes for preparing a plant that synthesizes hyaluronan which comprises

a) genetically modify a plant cell, in which the genetic modification comprises the following stages i to iii in any order, or any combination of stages i to iii can be performed individually or simultaneously,

i) introduction of a foreign nucleic acid molecule encoding a hyaluronan synthase linked to regulatory sequences that ensure transcription in plant cells in the plant cell ii) the introduction of a foreign nucleic acid molecule in the plant cell, the acid molecule A foreign nucleic comprising a coding sequence of a protein that has the activity of a glutamine: fructose 6-phosphate amidotransferase-2 (GFAT-2) that originates from animals or a glutamine: fructose 6-phosphate amidotransferase (GFAT) which originates from bacteria iii) the introduction of a foreign nucleic acid molecule into the plant cell, the foreign nucleic acid molecule comprising a coding sequence of a protein that has the activity of a glutamine: fructose 6-phosphate UDP-glucose dehydrogenase

b) regenerating a plant from plant cells that comprises genetic modification according to the stages

i)

a) i

ii)

a) ii

iii)

a) iii

iv)

a) i and a) ii,

v)

a) i and a) iii,

saw)

a) ii and a) iii, or

vii)

a) i and a) ii and a) iii

c) introduce into plant plant cells according to the stage

i) b) i a genetic modification according to stage a) ii, ii) b) i a genetic modification according to stage a) iii, iii) b) i a genetic modification according to stage ) ii and simultaneously a genetic modification according to stage a) iii, iv) b) ii a genetic modification according to stage a) i, v) b) ii a genetic modification according to stage a) iii, vi) b) ii a genetic modification according to stage a) i and simultaneously a genetic modification according to stage a) iii, vii) b) iii a genetic modification according to stage a) i, viii) b) iii a genetic modification according to stage a) ii, ix) b) iii a genetic modification according to stage a) i and simultaneously a genetic modification according to stage a) ii, x) b) iv a genetic modification according to stage a) iii, xi) b) v a genetic modification according to et apa a) ii, or xii) b) I saw a genetic modification according to stage a) i

and regenerate a plant

d) introduce into plant plant cells according to the stage

i)

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

ii)

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

iii)

c) iv a genetic modification according to stage a) iii,

iv)

c) v a genetic modification according to stage a) ii,

v)

c) vii a genetic modification according to stage a) ii,

saw)

c) vii a genetic modification according to stage a) i, or

vii)

c) ix a genetic modification according to stage a) ii

and regenerate a plant e) generating, if appropriate, additional plants with the help of the plants according to any of the stages b) vii, c) iii, c) vi, c) x, c) xi, c) xii or according to any of the stages d) iad) vii.

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

The generation of additional plants can be performed (depending on the procedure according to stage c) or stage e)) for example, by vegetative propagation (for example, by means of skeletons, tubers or by callus culture and regeneration of intact plants) or through generative propagation. In this context, generative propagation generally takes place under controlled conditions, that is, selected plants with specific characteristics hybridize to each other and multiply. The selection preferably takes place in such a way that the additional plants (depending on the procedure generated according to stage c) or stage e)) comprise the modifications introduced in the preceding stages.

In the methods according to the invention to prepare plants that synthesize hyaluronan, genetic modifications can be made to generate the genetically modified plant cells according to the invention in simultaneous or successive stages and in any combination. Natural plants and natural plant cells can be used as a starting point in which a foreign nucleic acid molecule that encodes a hyaluronan synthase has not been introduced and in which a genetic modification that increases the activity of a plant has not been introduced protein that has the (enzymatic) activity of a GFAT compared to non-genetically modified plant cells and in which a genetic modification that increases the activity of a protein that has the (enzymatic) activity of a UDP has not yet been introduced. Glc-DH compared to the corresponding genetically unmodified plant cells, Plant cells or plants that have already been genetically modified in which a nucleic acid molecule encoding a hyaluronan synthase has not been introduced and into which it has already been introduced a modification to increase the activity of a protein that has the (enzymatic) activity of a GFAT compared to the corresponding non-genetically modified natural plant cells and in which they have already been introduced and / or in which a genetic modification has already been introduced to increase the activity of a protein that has the (enzymatic) activity of a GFAT compared to the corresponding genetically unmodified plant cells. Here, it is immaterial if the same procedure as for the genetic modification resulting in an increased activity of a protein that has the (enzymatic) activity of a UDP-Glc-DH is used for successive genetic modifications that result in an increased activity of a protein that has the (enzymatic) activity of a GFAT, provided that both genetic modifications together result in an increased activity of a protein that has the (enzymatic) activity of a GFAT and a protein that has the (enzymatic) activity of a UDP -Glc-DH in the same plant cell. It is also immaterial, whose procedure is used to introduce a foreign nucleic acid molecule that encodes a hyaluronan synthase in the plant cell.

The procedures for preparing a plant that synthesizes hyaluronan, consist of the introduction of at least one foreign nucleic acid molecule or a plurality of foreign nucleic acid molecules into the genome of the plant cell, in which the molecule (s) s) of foreign nucleic acids comprises / comprises a hyaluronan synthase and a coding sequence of a protein that has the (enzymatic) activity of a GFAT and a coding sequence of a protein that has the (enzymatic) activity of a UDP-Glc -DH.

As already described above for the foreign nucleic acid molecules introduced for genetic modification in the plant cell or plant, which is introduced in step a) of the methods according to the invention to prepare a plant that synthesizes hyaluronan can be an individual nucleic acid molecule or a plurality of nucleic acid molecules. Therefore, foreign nucleic acid molecules that encode a hyaluronan synthase and / or that encode a protein that has the (enzymatic) activity of a GFAT and / or that encode a protein that has the (enzymatic) activity of a UDP-Glc -DH may be present together in a single nucleic acid molecule, or two of the aforementioned 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 molecule of Nucleic acid, in any possible combination, or the three foreign nucleic acid molecules mentioned may in each case be present in individual separate nucleic acid molecules.

Furthermore, to introduce a foreign nucleic acid molecule in the practice of the methods according to the invention to prepare a plant that synthesizes hyaluronan, it is possible to use, instead of natural plant cells or natural plants, mutant or mutant cells that are distinguished because they already have an increased activity of a protein that has the (enzymatic) activity of a GFAT and / or an increased activity of a protein that has the (enzymatic) activity of a UDP-Glc-DH. If the mutant cell already has an increased activity of a protein that has the (enzymatic) activity of a GFAT or an increased activity of a protein that has the (enzymatic) activity of a UDP-Glc-DH compared to natural plant cells corresponding or with the corresponding natural plants, it is sufficient to perform a process according to the invention to prepare a plant that synthesizes hyaluronan than a foreign nucleic acid molecule that encodes a hyaluronan synthase and a genetic modification that results in an increase in activity of a protein that has the (enzymatic) activity of a UDP-Glc-DH or an increase in the activity of a protein that has the (enzymatic) activity of a GFAT, compared to non-genetically corresponding natural plant cells, is introduced in said mutant or mutant cells. If the mutant cell already has an increased activity of a protein that has the (enzymatic) activity of a GFAT and an increased activity of a protein that has the (enzymatic) activity of a UDP-Glc-DH compared to natural plant cells corresponding to a corresponding natural plant, a foreign nucleic acid molecule encoding a hyaluronan synthase can be introduced into said mutant or mutant cell to perform a method according to the invention to prepare a plant that synthesizes hyaluronan.

Everything mentioned above that refers to the use of mutants for the preparation of genetically modified plant cells according to the invention or of genetically modified plants according to the invention is applied here analogously.

The present description relates to methods according to the invention for preparing a plant that synthesizes hyaluronan, in which the nucleic acid molecule encoding a hyaluronan synthase in step a) is selected from the group consisting of:

a) nucleic acid molecules characterized in that they encode a viral hyaluronan synthase, b) synthetic acid molecules characterized in that they encode a hyaluronan synthase of a virus that infects Chlorella, c) nucleic acid molecules characterized in that they encode a hyaluronan synthase of a Paramecium bursaria Chlorella virus 1, d) nucleic acid molecules characterized in that they encode a hyaluronan synthase of a Paramecium bursaria Chlorella virus 1 of strain H1, e) nucleic acid molecules characterized in that the codons of the nucleic acid molecules encoding a hyaluronanosyntase are modified in comparison with the codons of the nucleic acid molecules that encode the hyaluronan synthase in the parent organism of the hyaluronan synthase, f) nucleic acid molecules characterized in that the codons of the hyaluronan synthase have been modified in such a way that they adapt to the frequency from l use of the codons of the plant or plant cell in whose genome they are to be integrated or integrated, g) nucleic acid molecules characterized by a hyaluronan synthase having the amino acid sequence shown in SEQ ID NO: 2 or encoding 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 identical to the amino acid sequence shown in SEQ ID NO: 2, h) nucleic acid molecules characterized in that they encode a protein whose amino acid sequence can be derived from the nucleic acid coding sequence inserted in plasmid DSM16664 or encoding a protein whose amino acid sequence is at least 70%, preferably at least 80%, particularly preferably at least 90%, specifically at least 95% and most preferably at least 98% identical to the amino acid sequence that is derived from the coding region of the nucleic acid sequence inserted in plasmid DSM16664, i) nucleic acid molecules comprising a nucleic acid sequence shown in SEQ ID No. 1 or in SEQ ID No. 3 or that 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 nucleic acid sequence shown in SEQ ID No. 1 or in SEQ ID No. 3, j) nucleic acid molecules comprising the nucleic acid sequence inserted into the plasmid DSM16664 or that 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 inserted in plasmid DSM16664, k) molecules of nucleic acids encoding a hyaluronan synthase, in which the nucleic acid sequences encoding hyaluronan synthase bind to regulatory elements (promoters) that initiate transcription in plant cells ol) nucleic acid molecules according to k) in which the promoters are tissue specific promoters, particularly preferable promoters that initiate the initiation of transcription specifically in plant tuber cells, fruits or plant seeds.

Preferably, the nucleic acid molecule encoding a protein that has the activity of a GFAT is selected from the group consisting of:

a) nucleic acid molecules characterized in that they encode a protein that has the activity of a GFAT that originates from bacteria or animals, preferably from Escherichia coli or mouse, b) nucleic acid molecules characterized in that the codons of the molecule of nucleic acid encoding a protein that has the activity of a GFAT are modified compared to the codons of the nucleic acid molecule encoding the corresponding protein that has the activity of a GFAT of the parent organism, c) nucleic acid molecules characterized because the codons of the protein that has the activity of a GFAT synthase have been modified in such a way that they adapt to the frequency of the use of the codons of the plant or plant cell in whose genome they are to be integrated or integrated, d ) nucleic acid molecules that encode a protein that has the sequence shown in SEQ ID No. 10 or for a protein that has the amino acid sequence shown in SEQ ID NO: 12; e) 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 amino acid sequence of SEQ ID No. 10 or SEQ ID No. 12; f) the nucleic acid molecules comprising the nucleic acid sequence shown in SEQ ID No. 9 or a complementary sequence of the above or the nucleic acid sequence shown in SEQ ID No. 11 or a complementary sequence of the above or the nucleic acid sequence shown in SEQ ID No. 13 or a complementary sequence of the above; g) nucleic acid molecules that are 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 sequences of nucleic acids described in h); h) nucleic acid molecules that hybridize under restrictive conditions with at least one strand of the nucleic acid sequences described in f) or h); i) nucleic acid molecules whose nucleotide sequences differ from the sequence of nucleic acid molecules mentioned in f) or h) due to the degeneracy of the genetic code; and j) nucleic acid molecules that are fragments, variants and / or allelic derivatives of the nucleic acid molecules mentioned in a), b), c), d), e, f) oh), k) nucleic acid molecules that encode a protein that has the activity of a GFAT, in which the nucleic acid sequences that encode a protein that has the activity of a GFAT bind to regulatory elements (promoters) that initiate transcription in plant cells ol) acid molecules nuclei according to m), in which the promoters are tissue specific, particularly preferable promoters that initiate the initiation of transcription specifically in plant tuber cells, leaves, fruits or plant seeds.

The present description relates to methods according to the invention for preparing a plant that synthesizes hyaluronan, in which the nucleic acid molecule encoding a protein that has the activity of a UDP-Glc-DH is selected from the group consisting of :

a) nucleic acid molecules characterized in that they encode a protein that has the activity of a UDP-Glc-DH that originates from viruses, bacteria, animals or plants, b) nucleic acid molecules characterized in that they encode a protein that has the activity of a UDP-Glc-DH of a virus that infects Chlorella, c) nucleic acid molecules characterized in that they encode a protein that has the activity of a UDP-Glc-DH of a Paramecium bursaria Chlorella virus, d) acid molecules Nuclei characterized in that the codons of the nucleic acid molecule encode a protein that has the activity of a UDP-Glc-DH are modified compared to the codons of the nucleic acid molecule that encodes the corresponding protein that has the activity of a UDP -Glc-DH of the parent organism, e) nucleic acid molecules characterized by the codons of the protein having the activity of a UDP-Glc- DH have been modified in such a way that they adapt to the frequency of the use of the codons of the plant or plant cell in whose genome they are to be integrated or integrated, f) nucleic acid molecules that encode a protein that has the amino acid sequence shown in 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 amino acid sequence shown in SEQ ID NO: 5; h) nucleic acid molecules comprising the nucleotide sequence of SEQ ID No. 4 or a complementary sequence of the above or the nucleotide sequence shown in SEQ ID No. 6 or a complementary sequence of the above; i) nucleic acid molecules that are at least 70%, preferably at least 80%, preferably at least 90%, especially preferably 95% and most preferably at least 98% identical to the nucleic acid sequences described in h); j) nucleic acid molecules that hybridize under restrictive conditions with at least one strand of the nucleic acid molecules described in f) or h); k) nucleic acid molecules whose nucleotide sequences differ from the sequence of nucleic acid molecules mentioned in f) or h) due to the degeneracy of the genetic code; and l) nucleic acid molecules that are fragments, variants and / or allelic derivatives of the nucleic acid molecules mentioned in a), b), c), d), e, f) oh), m) nucleic acid molecules that encode a protein that has the activity of a UDP-Glc-DH in which the nucleic acid sequences encoding a protein that has the activity of a UDP-Glc-DH bind to regulatory elements (promoters) that initiate transcription in plant cells on) nucleic acid molecules according to m), in which the promoters are tissue specific, particularly preferable promoters that initiate the initiation of transcription specifically in plant tuber cells, leaves, fruits or plant seeds.

In a preferred embodiment of the present invention, the methods for preparing a plant that synthesizes hyaluronan refers to procedures for preparing a plant that synthesizes at least 100, preferably at least 600, particularly preferably at least 1000, especially preferably the less 1500, µg of hyaluronan per g of fresh weight (PF) of plant material.

In a further preferred embodiment, the methods according to the invention to prepare a plant that synthesizes hyaluronan are used to prepare genetically modified plants according to the invention.

The present invention also provides plants obtainable by a process according to the invention to prepare a plant that synthesizes hyaluronan.

The present invention further relates to a process for preparing hyaluronan which comprises the step of extracting hyaluronan from genetically modified plant cells according to the invention, from genetically modified plants according to the invention, from propagation material according to the invention, from parts of harvested plants according to the invention or from plants or parts of these plants obtainable by a process according to the invention to prepare plants that synthesize hyaluronan.

Preferably, said method also comprises the step of harvesting the genetically modified plant cells according to the invention, the genetically modified plants according to the invention, the propagation material according to the invention, the parts of harvested plants according to the invention. invention, the parts of processable plants according to the invention before the hyaluronan is extracted, and particularly preferably also the step of cultivating genetically modified plant cells according to the invention or genetically modified plants according to the invention before harvesting .

Unlike bacteria or animal tissues, plant tissues do not have hyaluronidases and do not contain any hialaadherin. Therefore, as described above, the extraction of hyaluronan from plant tissues using relatively simple procedures. If necessary, the aqueous extracts, described above, of plant cells or tissues containing hyaluronan can be purified using methods known to those skilled in the art, such as, for example, repeated precipitation with ethanol. A preferred procedure for purifying hyaluronan is described in the General Procedures, article 3.

The methods already described for extracting hyaluronan from genetically modified plant cells according to the invention or from genetically modified plants according to the invention are also suitable for isolating hyaluronan from propagation material according to the invention, from parts of plants. harvested according to the invention or from plants or parts of these plants obtainable by a process according to the invention to prepare plants that synthesize hyaluronan.

The present invention also provides the use of genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, parts of harvested plants according to the invention, parts of plants processable according to the invention or plants obtainable by a process according to the invention to prepare hyaluronan.

The present invention further describes compositions comprising plant cells according to the invention. Here, it does not matter if the plant cells are intact or are no longer intact because they have been destroyed, for example, due to processing. The compositions are edible food or feed, pharmaceutical or cosmetic products.

The present invention further describes compositions comprising plant cells according to the invention, of genetically modified plants according to the invention, of propagation material according to the invention, of parts of harvested plants according to the invention or of obtainable plants by a method according to the invention and comprising recombinant nucleic acid molecules, in which the recombinant nucleic acid molecules are characterized in that they comprise nucleic acid molecules encoding a hyaluronan synthase and proteins that have the (enzymatic) activity of a GFAT and proteins that have the (enzymatic) activity of a UDP-Glc-DH.

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 plant cell genome by genomic sequences of plant nucleic acids. The compositions described herein are characterized in that the recombinant nucleic acid molecules present in the composition are flanked by genomic sequences of plant nucleic acids.

Here, the genomic sequences of plant nucleic acids can be any sequences naturally present in the genome of the plant or plant cell used to prepare the composition.

The recombinant nucleic acid molecules present in the compositions described herein may be individual nucleic acid molecules or various nucleic acid molecules in which the nucleic acid molecules encoding a hyaluronan synthase and the proteins that have the activity (enzymatic ) of a GFAT and proteins that have the (enzymatic) activity of a UDP-Glc-DH are present in a nucleic acid molecule, or those in which the aforementioned nucleic acid molecules may be present in recombinant nucleic acid molecules separated. Nucleic acid molecules that encode a hyaluronan synthase or encode a protein that has the (enzymatic) activity of a GFAT or that encode a protein that has the (enzymatic) activity of a UDP-Glc-DH can be present together in a single recombinant nucleic acid molecule, or two of the aforementioned 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 The three mentioned nucleic acid molecules can in each case be present in individual separated nucleic acid molecules. Depending on how the nucleic acid molecules encode a hyaluronan synthase or encode a protein that has the (enzymatic) activity of a GFAT or encode a protein that has the (enzymatic) activity of a UDP-Glc-DH are present in a composition, they may be flanked by genomic sequences of nucleic acids from identical or different plants.

These compositions described herein comprising recombinant nucleic acid molecules can be demonstrated using methods known to those skilled in the art, such as, for example, hybridization-based procedures or, preferably, using PCR-based (reaction) procedures. in polymerase chain).

Preferably, the compositions described herein 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, the compositions described herein comprise almost 5%, preferably almost 2%, particularly preferably almost 1% and especially preferably at least 0.5% hyaluronan.

As already mentioned above, it is possible to use genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, parts of harvested plants according to the invention, parts of processable plants according to the invention, parts of consumable plants according to the invention or plants obtainable by a process according to the invention to prepare edible food or feed. However, it is also possible to use as raw materials for industrial applications, without having to isolate hyaluronan. Therefore, for example, genetically modified plants according to the invention or parts of genetically modified plants according to the invention can be applied to areas under agricultural cultivation to achieve an increase in the binding of water to soil. In addition, genetically modified plants according to the invention or genetically modified plant cells according to the invention can be used to prepare drying agents (for example for use with moisture sensitive articles during shipment) or as absorbents of liquids (for example, in diapers or to absorb spills of aqueous liquids). For such applications, it is possible to use genetically modified whole plants according to the invention, parts of genetically modified plants according to the invention or genetically modified crushed plants (for example, ground) according to the invention or parts of plants according to the invention, as necessary. Suitable for applications in which plants or parts of ground plants are used are, in particular, parts of plants containing hyaluronan, but only a low proportion of water. These are preferably grains of cereal plants (corn, rice, wheat, rye, oats, barley, sago or sorghum). Because the genetically modified plant cells according to the invention and the genetically modified plants according to the invention have a higher hyaluronan content than the transgenic plants described in the literature, in comparison with this less material should be used for industrial applications when use is made of genetically modified plant cells according to the invention or genetically modified plants according to the invention.

The present invention also provides methods for preparing a composition according to the invention, in which genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, are used. parts of harvested plants according to the invention, parts of processable plants according to the invention, parts of consumable plants according to the invention or plants obtainable by a process according to the invention to prepare a plant that synthesizes hyaluronan. The processes for preparing a composition according to the invention are preferably procedures for preparing edible foods or feeds, procedures for preparing a pharmaceutical product or procedures for preparing a cosmetic product.

5 Persons skilled in the art know the procedures for preparing edible foods or feed. Those skilled in the art also know the methods for using genetically modified plants according to the invention or parts of plants according to the invention in industrial areas and include among others the crushing or grinding of genetically modified plants according to the invention. or parts of plants according to the invention; however, they are not limited exclusively to the foregoing. They have

10 described above some of the advantages resulting from the use of subject materials according to the invention for preparing edible food / feedstuffs or for use in industrial areas.

A process according to the invention is particularly preferable for preparing a composition comprising hyaluronan.

Compositions obtainable by a method 15 for preparing a composition according to the invention are also provided herein.

The present invention also relates to the use of genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, parts of harvested plants according to the invention, parts of processable plants according to the invention, parts of consumable plants according to the invention or plants obtainable by means of a process according to the invention to prepare a plant that synthesizes hyaluronan to prepare a composition according to the invention. Preference is given to the use of genetically modified plant cells according to the invention, genetically modified plants according to the invention, propagation material according to the invention, parts of harvested plants according to the invention, parts of processable plants according to the invention, parts of consumable plants according to the invention or

25 of plants obtainable by a process according to the invention to prepare a plant that synthesizes hyaluronan to prepare edible food or feed, to prepare a pharmaceutical product or to prepare a cosmetic product.

Description of the sequences

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

SEQ ID No. 2: Amino acid sequence of a hyaluronan synthase of Paramecium bursaria Chlorella virus 1. The amino acid sequence shown, which can be derived from SEQ ID No. 1.

SEQ ID NO. 3: The synthetic nucleic acid sequence encoding a hyaluronan synthase of a Paramecium bursaria Chlorella virus 1. 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 in SEQ ID NO: 2.

SEQ ID NO: 4 A nucleic acid sequence encoding a protein that has the activity of a UDP-Glc-DH of Paramecium bursaria Chlorella virus 1.

SEQ ID No. 5: The amino acid sequence of a protein that has the activity of a UDP-Glc-DH of Paramecium bursaria Chlorella virus 1. The amino acid sequence shown, which can be derived from SEQ ID No. 4.

SEQ ID NO: 6 A synthetic nucleic acid sequence encoding a protein that has the activity of a UDP-Glc-DH of Paramecium bursaria Chlorella virus 1. The synthesis of the codons 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 in SEQ ID NO: 5.

SEQ ID NO. 7: The nucleic acid sequence of a protein that has the activity of a mouse GFAT-1.

SEQ ID NO. 8: The amino acid sequence of a protein that has the activity of a mouse GFAT-1. The amino acid sequence shown, which can be derived from SEQ ID NO. 7.

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

5

10

fifteen

twenty

25

30

35

40

SEQ ID No. 10:

The amino acid sequence of a protein that has the activity of a mouse GFAT-2. The amino acid sequence shown, which can be derived from SEQ ID NO. 9.

SEQ ID No. 11:

The nucleic acid sequence encoding a protein that has the activity of a GFAT from Escherichia coli.

SEQ ID No. 12:

The amino acid sequence of a protein that has the activity of an Escherichia coli GFAT. The amino acid sequence shown, which can be derived from SEQ ID NO: 11.

SEQ ID No. 13:

The synthetic nucleic acid sequence encoding a protein that has the activity of a GFAT from Escherichia coli. 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 in SEQ ID NO: 12.

SEQ ID No. 14: Synthetic oligonucleotide used as a primer in Example 1

SEQ ID No. 15: Synthetic oligonucleotide used as a primer in Example 1

Description of the figures

Figure 1: Shows a calibration curve and the corresponding regression line equation used to calculate the hyaluronan content in the plant tissue. The calibration curve was established with the help of the commercial test kit (hyaluronic acid (AH) test kit from Corgenix, Inc., Colorado, USA, product no. 029-001) and the standard solutions supplied to the previous one .

General procedures

The procedures that can be used in conjunction with the present invention are described below.

one.

Potato plant transformation

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

2.

Hyaluronan isolation of plant tissue

To detect the presence of hyaluronan and determine the content of hyaluronan in the plant tissue, the plant material was worked as follows: approximately 200 µl of water (demineralized, conductivity = 18 MΩ) was added to approximately 0.3 g of material of tuber, and the mixture was crushed in a laboratory oscillating ball mill (MM200, from Retsch, Germany) (30 s to 30 Hz). Next, approximately 800 µl of water (demineralized, conductivity = 18 MΩ) was added, and the mixture was mixed well (using, for example, a Vortex mixer). Cellular debris and insoluble components were separated from the supernatant by centrifugation at 16,000 xg for 5 minutes.

3.

Hyaluronan purification

Approximately 100 grams of tubers were stripped, cut into pieces of a size of approximately 1 cm3 and, after adding 100 ml of water (demineralized, conductivity = 18 MΩ), crushed in a Warring mixer at maximum speed for approximately 30 seconds. Cellular debris was then removed using a tea sleeve. The cellular debris that had been removed was resuspended in 300 ml of water (demineralized, conductivity = 18 MΩ) and removed again using a tea sleeve. The two suspensions obtained (100 ml + 300 ml) were combined and centrifuged at 13,000 xg for 15 minutes. NaCl was added to the centrifugal supernatant obtained until reaching a final concentration of 1%. Once the NaCl had dissolved, the precipitation was performed by adding twice the volume of ethanol followed by the complete mixing and incubation at -20 ° C overnight. The mixture was then centrifuged at 13,000 for 15 minutes. The sedimented precipitate obtained after this centrifugation was dissolved in 100 ml of buffer (50 mM TrisHCl, pH 8, 1 mM CaCl2) and then proteinase K was added to a final concentration of 100 mg / ml and the solution was incubated at 42 ° C for 2 hours This was followed by 10 minutes of incubation at 95 ° C. Again, NaCl was added to this solution until a final concentration of 1% was reached. Once the NaCl had dissolved, another 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 x g. The sedimented precipitate obtained after this centrifugation was dissolved in 30 ml of water (demineralized, conductivity = 18 MΩ), 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 occurred. The precipitate obtained after the subsequent centrifugation at 13,000 xg for 15 minutes was dissolved in 20 ml of water (demineralized, conductivity = 18 MΩ).

Another purification was performed by centrifugal filtration. To this end, in each case 5 ml of the dissolved precipitate is

applied to a membrane filter (CentriconAmicon, pore width 10,000 NMWL, Prod. No. UCF8 01096), and the sample was centrifuged at 2200 xg until only about 3 ml of the solution remained above the filter. Twice more, after adding 3 ml of water (demineralized, conductivity = 18 MΩ) to the solution above the membrane and, in each case, they were centrifuged again in identical conditions until, finally, only 5 remained approximately 3 ml of the solution above the filter. The solutions still present above the membrane after centrifugal filtration were removed, and the membrane was repeatedly washed (three to five times) with approximately 1.5 ml of water (demineralized, conductivity = 18MΩ). All the solutions remaining above the membrane and the solutions obtained from the washing were combined, NaCl was added to a final concentration of 1%, once the NaCl had dissolved, the solution was added twice.

10 volume of ethanol, the sample was mixed and a precipitate was obtained by storage at -20 ° C overnight. The precipitate obtained after the subsequent centrifugation at 13,000 xg for 15 minutes was dissolved in 4 ml of water (demineralized, conductivity = 18 MΩ) and then it was dried (24 hours at a pressure of 0.37 mbar, Christ Alpha cryo-drying equipment 1-4 of Christ, Osterode, Germany).

4. Hyaluronan detection and hyaluronan content determination

15 Hyaluronan was detected using a commercial test (hyaluronic acid (AH) test kit from Corgenix, Inc., Colorado, USA, Prod. No. 029-001) in accordance with the manufacturer's instructions incorporated herein. document in its specification as a reference. The principle of the test is based on the availability of a protein that specifically binds to hyaluronan (HABP) and is carried out similarly to an ELISA, in which a color reaction indicates the content of hyaluronan in the examined sample . Therefore, for the

In the quantitative determination of hyaluronan, the samples to be measured should be used in a concentration that is within the limits indicated (for example, dilution of the sample in question or use of less water to extract the hyaluronan from the plant tissue, depending on if the limit has been exceeded or not reached).

In parallel batches, aliquots of the samples to be determined were initially digested with hyaluronidase and then measured using the commercial test (hyaluronic acid (AH) test kit from Corgenix, Inc., Colorado,

25 USA, Prod. No. 029-001). Digestion with hyaluronidase was performed using 400 µl of the potato tuber extracted in hyaluronidase buffer (0.1 M potassium phosphate buffer, pH 5.3; NaCl 150) by adding 5 µg (approximately 3 units) of hyaluronidase ((hyaluronidase) Sigma type III, Prod. No. H 2251) and incubation at 37 ° C for 30 min.

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

5. The standard deviations cited were calculated according to the following formula:

image 1

where x is the value of the individual measured values and n is the sum of all the measured values to determine the standard deviation in question.

35 6. Determination of the activity of a GFAT

The activity of a protein that has the activity of a GFAT was determined as described in Rachel et al. (1996, J. Bacteriol. 178 (8), 2320-2327).

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

40 7. Determination of the activity of a UDP-Glc-DH

The activity of a protein that has the activity of a UDP-Glc-DH was determined as described in Rachel et al. (1998, J. Bacteriol. 273 (39), 25117-25124).

8. Transformation of tomato plants

Tomato plants were transformed with the help of Agrobacterium using the procedure described in US 5,565,347.

Examples

1. Preparation of the plant expression vector IR 47-71 Plasmid pBinAR is a derivative of the binary vector plasmid pBin19 (Bevan, 1984, Nucl Acids Res 12: 8711-8721)

It was built as follows:

A 529 bp length fragment comprising nucleotides 6909-7437 of the 35S promoter of cauliflower mosaic virus was isolated as an EcoR IIKpn I fragment from plasmid pDH51 (Pietrzak et al, 1986 Nucleic Acids Res 14, 5858) and linked between the EcoR I and Kpn I restriction sites of

5 polylinker of pUC18. In this way, plasmid pUC18-35S was formed. Using the restriction endonucleases Hind III and Pvu II, a fragment with a length of 192 bp that included the polyadenylation signal (3 'end) of the Octopin synthase gene (gene 3) of the Ti-T plasmid pTiACH5 (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 the addition of the Sph I linkers to the Pvu II restriction site, the fragment was ligated between the Sph I and Hind III restriction sites of pUC18-35S. This produced plasmid pA7. Here, the complete polylinker comprising the 35S promoter and the OCS terminator was removed with EcoR I and Hind III and ligated into the properly cleaved vector pBin19. This produced the plant expression vector pBinAR (Höfgen and Willmitzer, 1990, Plant Science 66, 221-230).

The promoter of the Patatin B33 gene of Solanum tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) was, at

The same as the Ora I fragment (nucleotides -1512 - +14), ligated into the pUC19 vector cleaved with Sst I whose ends had been enromed using the T4 DNA polymerase. This produced plasmid pUC19-B33. From this plasmid the B33 promoter was removed using EcoR I and Sma I and ligated into the suitably restricted pBinAr vector. This produced the plant expression vector pBinB33.

To facilitate additional cloning stages, the MCS (multiple cloning site) was extended. To this end, two complementary oligonucleotides were synthesized, heated at 95 ° C for 5 minutes, slowly cooled to room temperature to allow good fixation (hybridization) and cloned into the restriction sites Salt I and Kpn I of pBinB33. The oligonucleotides used for this purpose had the following sequence:

5'-TCg ACA ggC CTg gAT CCT TAA TTA AAC TAg TCT CgA ggA gCT Cgg TAC-3 '25 5'-CgA gCT CCT CgA gAC TAg TTT AAT TAA ggA TCC Agg CCT g-3'

The plasmid obtained was called IR 47-71.

2.

Preparation of the plant expression vector pBinARHyg

The fragment comprising the 35S promoter, the Ocs terminator and the complete multiple cloning site was removed from pA7 using the restriction endonucleases EcoR I and Hind III and cloned into the vector pBIBHyg (Becker, 1990, Nucleic Acids Res. 18 , 203) that had been cut with the same restriction endonucleases. The plasmid obtained was called pBinARHyg.

3.

Preparation of cloning vector IC 317-204

Nucleic acid fragments comprising the OCS terminator were isolated from IR 47-71 plasmid using restriction endonucleases Xho I and Hind III 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 IR 47-71 plasmid using Bam HI and Eco RI restriction endonucleases and cloned into the pBlueScript KS vector (from Stratagene, Prod. No. 212207) that 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 Bam HI restriction endonuclease and was cloned into plasmid IC 314-204 that had been cut with the same restriction endonuclease. The plasmid obtained was named IC 317-204.

4. Synthesis of nucleic acid molecules

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

The nucleic acid sequence encoding a hyaluronan synthase (HAS) from Paramecium bursaria Chlorella virus 1 was synthesized by Medigenomix GmbH (Munich, Germany) and was cloned into the pCR2.1 vector of Invitrogen (Prod. No. K2000-01). The plasmid obtained was named IC 323-215. The synthetic nucleic acid sequence encoding the HAS protein of Paramecium bursaria Chlorella virus 1 is shown in SEQ ID No. 3. The corresponding nucleic acid sequence originally isolated from Paramecium bursaria Chlorella virus 1 is shown in SEQ ID No. 1.

b) Synthesis of nucleic acid molecules that encode a protein that has the activity of a UDP-Glc-DH of Paramecium bursaria Chlorella virus 1

The nucleic acid sequence encoding a protein that has the activity of a UDP-Glc-DH of Paramecium bursaria Chlorella virus 1, was synthesized by Entelechon GmbH and was cloned into the vector pCR4 Topo of Invitrogen (Prod. No. K4510-20). The plasmid obtained was named IC 339-222. The synthetic nucleic acid sequence encoding the UDP-Glc-DH protein of Paramecium bursaria Chlorella virus 1, is shown in SEQ ID No. 6. The corresponding nucleic acid sequence originally isolated from Paramecium bursaria Chlorella virus 1 is shown in SEC. ID No. 4.

c) Synthesis of nucleic acid molecules that encode a protein that has the activity of a GFAT of

Escherichia coli

The nucleic acid sequence encoding a protein that has the activity of an Escherichia coli GFAT was synthesized by Entelechon GmbH and was cloned into the pCR4 Topo vector of Invitrogen (Prod. No. K4510-20). The plasmid obtained was named IC 373-256. The synthetic nucleic acid sequence encoding a protein that has the activity of an Escherichia coli GFAT is shown in SEQ ID No. 13. The corresponding nucleic acid sequence originally isolated from Escherichia coli is shown in SEQ ID No. 11.

5. Origin of additional nucleic acid molecules

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

The nucleic acid sequence encoding a protein that has the activity of a GFAT-1 was purchased from BioCat GmbH, Heidelberg (Art. No. MMM1013-65346, cDNA clone MGC: 58262, IMAGE: 6742987). It is a clone produced by IMAGE Konsortium

(http://image.llnl.gov) and distributed by BioCat GmbH. Here, the cDNA encoding a protein that has the activity of a GFAT-1 was cloned into the pCMV Sport 6 vector of Invitrogen. The plasmid obtained was named IC 365-256. The nucleic acid sequence, inserted in IC 365-256, which encodes a protein that has the activity of a Mus musculus GFAT-1, compared to the nucleic acid sequence shown in SEQ ID NO: 7, a change of basis of T to C at position 1090 and a base change from G to at position 2027. These base changes do not result in amino acid changes of the amino acid sequences encoded by the two different nucleic acid molecules.

The nucleic acid sequence encoding the protein that has the activity of a mouse GFAT-1 is shown in SEQ ID NO: 8.

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

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

Nucleic acid molecules encoding a protein that has the activity of a mouse GFAT-2 were purchased from (Clone ID 4167189, cDNA clone MGC: 18324, IMAGE: 4167189). It is a clone produced by

IMAGE
Konsortium (http://image.llnl.gov) and distributed by Invitrogen. Here, the cDNA encoding a protein that has the activity of a GFAT-2 was cloned into the pCMV Sport 6 vector of Invitrogen. The plasmid was named IC 369

256. The nucleic acid sequence encoding the protein that has the activity of a Mus musculus GFAT-2 is shown in SEQ ID NO: 9.

6.

Preparation of the plant expression vector IC 341-222 comprising a nucleic acid sequence encoding a hyaluronan synthase of Paramecium bursaria Chlorella virus 1

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

7.

Preparation of plant expression vectors IC 370-256 and IC 376-256 comprising nucleic acid sequences encoding a protein that has the activity of a mouse GFAT-1 and a protein that has the activity of a UDP-Glc -DH of Paramecium bursaria Chlorella virus 1

Using restriction digestion with BamH I and Kpn I, nucleic acid molecules comprising the coding sequence of a protein having the activity of a UDP-Glc-DH of Paramecium bursaria Chlorella virus 1 were isolated from plasmid IC 339 -222 and were cloned into plasmid pA7 that 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 having the activity of a UDP-Glc-DH of Paramecium bursaria Chlorella virus 1 were isolated from plasmid IC 342 -222 and were cloned into the pBinAR Hyg expression vector that had been restricted with Xba I and Kpn I. The plasmid obtained was designated IC 349-222.

In the next step, a nucleic acid fragment comprising the B33 promoter and the OCS terminator, fragment that had been isolated from IC 317-204 by restriction digestion with Eco IR, was cloned into the Eco IR restriction site of IC 349-222. Here, it was guaranteed that the promoters (35S and 833) were oriented in parallel. The vector obtained was named IC 354-222.

In an additional cloning step, a nucleic acid fragment comprising the coding sequence of the protein that has the activity of a mouse GFAT-1 was isolated by restriction digestion with Xho I and Eco RV from IC 367- 256 and was cloned into plasmid IC 354-222, which had been restricted with Xho I and Ecl136 II. The plant expression vector obtained was named IC 370-256.

After a sequence analysis of plasmid IC 370-256, it was discovered that the nucleic acid sequence encoding the protein having the activity of a mouse GFAT-1 had modifications in two positions compared to the inserted nucleic acid sequence in plasmid IC 365-256. Compared to the nucleic acid sequence shown in SEQ ID No. 7, the nucleic acid sequence encoding the protein that has the activity of a mouse GFAT-1 contained in plasmid IC 370-256 has a base change of G a A at position 1160, a base change from T to C at position 1190, a base change from T to C at position 1245 and from G to A at position 2027. This modified nucleic acid sequence encodes a protein which, with respect to the amino acid sequence shown in SEQ ID NO. 8, has a modification in amino acids at position 304 from R to Q and at position 366 from C to R.

To obtain a plant expression vector comprising the nucleic acid sequence encoding a protein that has the activity of a correct GFAT-1 from mouse, the protein coding sequence that has the activity of a GFAT-1 of the mouse was again isolated from IC 365-256 by restriction digestion with Xho I and Eco RV and cloned into plasmid IC 354-222, restricted with Xho I and Ecl136 II. The plant expression vector obtained was named IC 376-256.

The nucleic acid sequence encoding the protein that has the activity of a mouse GFAT-1 that was included in plasmid IC 376-256 is identical to the coding sequence of a protein that has the activity of a GFAT-1 of the mouse inserted into plasmid 365-256. The amino acid sequence encoded by this nucleic acid molecule is shown in SEQ ID NO. 8.

8.

Preparation of the plant expression vector IC 372-256 comprising nucleic acid sequences encoding a protein that has the activity of a mouse GFAT-2 and a protein that has the activity of a UDP-Glc-DH of Paramecium bursaria Chlorella virus 1

A nucleic acid fragment comprising the protein coding sequence that has 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 the plasmid IC 354-222, restricted with Xho I and Ecl136 II. The plant expression vector obtained was named IC 372-256.

9.

Preparation of the plant expression vector IC 375-271 comprising nucleic acid sequences encoding a protein that has the activity of an Escherichia coli GFAT and a protein that has the activity of a UDP-Glc-DH of Paramecium bursaria Chlorella virus one

A nucleic acid fragment comprising the protein coding sequence that has the activity of an Escherichia coli GFAT was isolated from IC 373-256 by restriction digestion with Xho I and Eco RV and was cloned into plasmid IC 354-222, restricted with Xho I and Ecl136 II. The plant expression vector obtained was named IC 375-271.

10.

Transformation of potato plants with plant expression vectors comprising nucleic acid molecules encoding a hyaluronan synthase

Potato plants were transformed using the plant expression vector IC 341-222, which comprises a nucleic acid sequence encoding a hyaluronan synthase of Paramecium bursaria Chlorella virus 1 under the control of the promoter of the B33 patatin gene of Solanum tuberosum ( Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) using the procedure provided in section 1 of the General Procedures. The obtained transgenic potato plants, which were transformed with plasmid IC 341-222, were called 365 ES.

eleven.

Analysis of the transgenic plants transformed with plant expression vectors comprising nucleic acid molecules encoding a hyaluronan synthase

a) Construction of a calibration curve

A calibration curve was constructed using standard solutions supplied with the commercial test kit (hyaluronic acid (AH) from Corgenix, Inc., Colorado, USA, Prod. No. 029-001), in accordance with the procedures described by the maker. To determine the extinction at 1600 ng / ml of hyaluronan, double the

5 quantity, depending on the quantity of standard supplied indicated by the manufacturer, comprising 800 ng / ml of hyaluronan. In each case, three series of independent measurements were made, and the corresponding average was determined. This produced the following calibration curve:

Table 1: Values to construct a calibration curve for the quantitative determination of hyaluronan content in plant tissue. With the help of a computer program (Microsoft Office Excel 2002, SP2),

10 measured values obtained were entered in a diagram, and the equation of the trend line function was determined (see Fig. 1). E450nm refers to extinction at a wavelength of 450 nm, s.d. is the standard deviation of the calculated mean of the individual values.

Hyaluronan concentration

Individual independent measurements Average s.d.

E450 nm

E450 nm

E450 nm

0 ng / ml

0.100 0.096 0.096 0.097 0.002

50 ng / ml

0.224 0.183 0.222 0.210 0.023

100 ng / ml

0.396 0.263 0.377 0.345 0.072

200 ng / ml

0.554 0.443 0.653 0.550 0.105

500 ng / ml

1,231 0.850 1,221 1,101 0.217

800 ng / ml

1,465 1,265 1,795 1,508 0.268

1600 ng / ml

2,089 2,487 3,170 2,582 0.547

b) Analysis of potato tubers of 365 ES lines

15 In a greenhouse, individual plants of the 365 ES line were grown in the soil in 6 cm pots. In each case, approximately 0.3 g of potato tuber material from the individual plants were processed according to the procedure described in section 2 of the General Procedures.

Using the procedure described in point 4 of the General Procedures, the amount of hyaluronan present in the respective plant extracts was determined by the calibration curve shown in Example 10a) and Fig. 1. Here, the supernatant obtained after the Centrifugation was used in a dilution of 1:10 to determine the content of hyaluronan. The following results were obtained for the selected plants:

Table 2: Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced by independent transgenic plants selected from line 365 ES. Column 1 refers to the plant from which the tuber material has been obtained (here, "natural type" refers to the non-transformed plants that, however, have the genotype used as the starting material for the transformation) . Column 2 indicates the amount of tuber material of the plant in question used to determine the hyaluronan content. Column 3 contains the measured extinction of a 1:10 dilution of the respective plant extract. Column 4 was calculated using the linear regression equation (see Fig. 1) taking into account the dilution factor, as set out below: ((column value 3 - 0.149) / 0.00185) x 10. The column 5 indicates the amount of hyaluronan based on the

30 fresh weight used and calculated as follows: (column value 4 / column value 2) / 1000. "n.d." means not detectable.

Plant name

the Weight of plant material used [g] E450 Extinction Amount of hyaluronan [ng / ml] Fresh Hyaluronan from [g / g] based on plant material weight

365 EP 13

0.297 2,746 14038 47

365 EP 74

0.306 4,000 20816 68

Natural type

0.305 0.111 n.d. n.d.

12. Transformation of plants that synthesize hyaluronan with plant expression vectors comprising nucleic acid sequences encoding a protein that has the activity of a GFAT and a protein that has the activity of a UDP-Glc-DH. In each case, the potato plants of lines 365 ES 13 and 365 ES 74 were transformed with the plant expression vectors IC 370-256, RO 376-256, IC 372-256 and IC 375-271 using the procedure provided in point 1 of the General Procedures.

The transgenic potato plants obtained from line 365 ES 13 that had been transformed with plasmid IC370-256 were called 393 ES.

The transgenic potato plants obtained from line 365 ES 74 that had been transformed with plasmid IC370-256 were called 394 ES.

The transgenic potato plants obtained from line 365 ES 13 that had been transformed with plasmid IC372-256 were called 395 ES.

The transgenic potato plants obtained from line 365 ES 74 that had been transformed with plasmid IC372-256 were called 396 ES.

The transgenic potato plants obtained from line 365 ES 13 that had been transformed with plasmid IC375-271 were named 403 ES.

The transgenic potato plants obtained from line 365 ES 74 that had been transformed with plasmid IC375-271 were designated 404 ES.

The transgenic potato plants obtained from line 365 ES 13 that had been transformed with plasmid IC376-256 were designated 408 ES.

The transgenic potato plants obtained from line 365 ES 74 that had been transformed with plasmid IC376-256 were designated 409 ES.

13. Analysis of transgenic potato plants that synthesize hyaluronan additionally transformed with plant expression vectors comprising nucleic acid sequences encoding a protein that has the activity of a GFAT and a protein that has the activity of a UDP-Glc-DH

In a greenhouse, individual plants of lines 393 ES, 394 ES, 395 ES, 396 ES, 403 ES, 404 ES and 409 ES were grown in the soil in 6 cm pots. In each case, approximately 0.3 g of tuber material or potato leaves of the individual plants were processed according to the procedure described in section 2 of the General Procedures. Using the procedure described in point 4 of the General Procedures, the amount of hyaluronan contained in the respective plant extracts was determined by a calibration curve generated according to Example 10a), said calibration curve was generated new for each series of individual measurements Here, to determine the content of hyaluronan, the supernatant obtained after centrifugation was diluted in each case with water (demineralized, conductivity = 18 Mn) in such a way that the measured extinction values for the individual samples were in the linear range. of the calibration curve. The results of plants originating from linear transformations with several plasmids are shown below.

a) Analysis of the tubers of line 393 EN

For tuber tasting of the individual transgenic plants of the lines called 393 ES, two independent samples were taken, and the hyaluronan content, in each case, was determined separately. The mean and standard deviation of the values obtained from the individual measurements were then calculated using the formula provided in section 5 of the General Procedures. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES 13 plants were calculated by calculating the amount of hyaluronan in tubers of, in each case, ten different plants that were vegetative progeny of the natural plant (Solanum tuberosum cv. Desiree ) and line 365 ES 13, respectively. The following results were obtained for the selected plants:

Table 3: Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in tubers of independent transgenic plants selected from line 393 ES. Column 1 contains the name of the plant from which the tuber material was obtained (here, "natural" refers to plants that have not been transformed; 365 ES 13 refers to plants that express a hyaluronan synthase and were used as Starting material for transformation with the plant expression vector IC 370-256 to generate lines 393 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.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

393 EP 6

126.42 12.7

393 EP 18

113.10 26.1

393 EP 23

112.83 18.7

393 EP 38

102.81 19.2

393 EP 36

99.71 16.7

393 EP 52

90.78 3.5

393 EP 50

90.31 8.7

393 EP 49

88.63 14.4

393 EP 32

87.82 15.2

393 EP 16

86.09 17.8

393 EP 33

80.47 16.3

Natural type

0.52 0.8

365 EP 13

72.62 16.4

b) Analysis of the tubers of line 394 EN

For tuber tasting of the individual transgenic plants of the lines called 394 ES, two were taken

5 independent samples, and the hyaluronan content, in each case, was determined separately. The mean and standard deviation of the values obtained from the individual measurements were then calculated using the formula provided in section 5 of the General Procedures. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating the amount of hyaluronan in tubers of, in each case, 18 different natural plants and 26 plants of line 365

10 ES 74 which were the vegetative progeny of the natural plant (Solanum tuberosum cv. Desiree) and of the line 365 ES 74, respectively. The following results were obtained for the selected plants:

Table 4 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in tubers of independent transgenic plants selected from line 394 ES. Column 1 contains the name of the plant from which the tuber material was obtained (here, "natural" refers to plants that have not been transformed; 365 EN 74

15 refers to plants expressing a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 370-256 to generate lines 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.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

394 EP 47

242.24 12.7

394 EP 37

227.66 15.2

394 EP 45

185.90 12.8

394 EP 56

176.82 25.1

394 EP 43

172.83 15.3

394 EP 14

168.80 27.1

394 EP 52

157.81 16.1

394 EP 28

145.20 8.5

394 EP 5

131.11 17.9

394 EP 26

127.56 13.5

(continuation)

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

394 EP 1

126.22 15.4

394 EP 15

125.46 9.9

Natural type

1.29 0.8

365 EP 74

104.34 26.1

c) Analysis of the tubers of line 395 EN

For tuber tasting of individual transgenic plants of the lines called 395 ES, if possible,

5 took two independent samples, and the hyaluronan content, in each case, was determined separately. The mean and standard deviation of the values obtained from the individual measurements were then calculated using the formula provided in section 5 of the General Procedures. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating the amount of hyaluronan in tubers of, in each case, 10 different natural plants and 18 plants

10 of line 365 ES 13 that were vegetative progeny of the natural plant (Solanum tuberosum cv. Desiree) and of line 365 ES 13, respectively. The following results were obtained for the selected plants:

Table 5 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in tubers of independent transgenic plants selected from line 395 ES. Column 1 contains the name of the plant from which the tuber material was obtained (here, "natural" refers to plants that have not been transformed; 365 EN 13

15 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 lines 395 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 for which the standard deviation is not indicated, the hyaluronan content of a single tuber sample was determined.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [mg / g] Standard deviation

395 EP 17

1321.75

395 EP 29

1145.32

395 EP 60

999.66 484.50

395 EP 16

791.64

395 EP 53

770.93 57.35

395 EP 10

651.30

395 EP 26

299.37 58.78

395 EP 3

288.21

395 EP 13

228.22

395 EP 38

96.10 12.30

Natural type

0.31 0.22

365 EP 13

77.16 16.56

twenty

d) Analysis of the sheets of line 395 EN

The hyaluronan content of the individual leaves of the plants of the lines having the name 395 ES was determined. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating in each case the amount of hyaluronan in leaves of 4 different natural plants 25 and 9 plants of the line 365 ES 13, which were vegetative progeny of the natural plant (Solanum tuberosum

cv. Desiree) and line 365 ES 13, respectively. The following results were obtained for the selected plants:

Table 6 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in leaves of independent transgenic plants selected from line 395 ES. Column 1 contains the name of the plant from which the tuber material was obtained (here, "natural" refers to plants that have not been transformed; 365 ES 13 refers to plants that express a hyaluronan synthase and were used as Starting material for transformation with the plant expression vector IC 372-256 to generate lines 395 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 for which the standard deviation is not indicated, the hyaluronan content of a single leaf sample was determined.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

395 EP 34

619.17

395 EP 45

589.52

395 EP 51

420.81

395 EP 46

405.81

395 EP 24

401.68

395 EP 12

392.90

395 EP 43

381.78

395 EP 21

368.04

395 EP 33

352.25

395 EP 25

350.90

395 EP 22

344.44

395 EP 48

338.52

395 EP 4

300.86

395 EP 28

298.30

395 EP 36

291.51

395 EP 2

274.05

395 EP 14

219.96

395 EP 56

158.39

395 EP 57

94.17

Natural type

0.18 0.14

365 EP 13

44.76 18.71

10 e) Analysis of the tubers of line 396 EN

For tuber tasting of the individual transgenic plants of the lines named 396, two independent samples were taken, and the hyaluronan content, in each case, was determined separately. The mean and standard deviation of the values obtained from the individual measurements were then calculated using the formula provided in section 5 of the General Procedures. The means and deviations were calculated

15 standard indicated for the so-called natural plants and the so-called 365 ES plants by calculating the amount of hyaluronan in tubers of, in each case, 12 different natural plants and 14 plants of line 365 ES 74 that were vegetative progeny of the natural plant (Solanum tuberosum cv. Desiree) and line 365 ES 74, respectively. The following results were obtained for the selected plants:

Table 7 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in tubers of independent transgenic plants selected from line 396 ES. Column 1 contains the name of the plant from which the tuber material was obtained (here, "natural" refers to plants that have not been transformed; 365 EN 74 refers to plants that express a hyaluronan synthase and were used as Starting material for transformation with the plant expression vector IC 372-256 to generate lines 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.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

396 EP 42

1283.02 229.8

396 ES44

1146.29 235.1

396 EP 33

804.90 500.1

396 EP 9

670.56 91.7

396 EP 2

389.61 67.2

396 EP 15

380.60 18.4

396 EP 28

371.66 159.9

396 EP 30

204.20 13.5

396 EP 8

186.69 55.5

396 EP 4

161.61 25.8

Natural type

0.95 0.6

365 EP 74

142.70 57.5

f) Analysis of the sheets of line 396 EN

10 The hyaluronan content of the individual leaves of the plants of the lines having the name 396 ES was determined. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating in each case the amount of hyaluronan in leaves of 4 different natural plants and 6 plants of line 365 ES 13, which were vegetative progeny of the natural plant (Solanum tuberosum

cv. Desiree) and line 365 ES 13, respectively. The following results were obtained for the selected plants:

Table 8 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in leaves of independent transgenic plants selected from line 396 ES. Column 1 contains the name of the plant from which the leaf material was obtained (here, "natural" refers to plants that have not been transformed; 365 EN 74 refers to plants that express a hyaluronan synthase and were used as a starting material for the transformation

20 with the plant expression vector IC 372-256 to generate lines 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. In the case of plants for which the standard deviation is not indicated, the hyaluronan content of a single leaf sample was determined.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

396 EP 51

1160.57

396 EP 32

941.89

396 EP 11

938.33

396 EP 36

860.54

396 EP 57

807.97

(continuation)

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

396 EP 25

801.58

396 EP 34

796.79

396 EP 50

619.23

396 EP 49

538.75

396 EP 48

461.05

396 EP 24

443.57

396 EP 17

426.79

396 EP 16

416.43

396 EP 23

271.85

396 EP 43

258.47

396 EP 14

186.78

Natural type

0.15 0.08

365 EP 74

106.35 56.77

g) Analysis of the tubers of line 403 EN

For each tuber of the individual transgenic plants of the lines called 403 ES, two were taken

5 independent samples, if possible, and the hyaluronan content, in each case, was determined separately. The mean and standard deviation of the values obtained from the individual measurements were then calculated using the formula provided in section 5 of the General Procedures. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating the amount of hyaluronan in tubers of, in each case, 10 different natural plants and 10 plants

10 of line 365 ES 13 that were vegetative progeny of the natural plant (Solanum tuberosum cv. Desiree) and of line 365 ES 13, respectively. The following results were obtained for the selected plants:

Table 9 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in tubers of independent transgenic plants selected from line 403 ES. Column 1 contains the name of the plant from which the tuber material was obtained (here, "natural" refers to plants that have not been transformed; 365 EN 13 15 refers to plants that express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 375-271 to generate lines 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." It means that it was not possible to detect the hyaluronan in the tubers. In the case of plants for which the standard deviation is not indicated, the content of

20 hyaluronan of a single tuber sample.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

403 EP 2

687.90

403 EP 5

457.56

403 EP 4

366.34

403 EP 15

295.00

403 EP 30

241.03

403 EP 8

140.51

403 EP 41

107.65

Natural type

n.d. -

365 EP 13

89.42 24.87

5

10

fifteen

twenty

25

30

h) Analysis of the sheets of line 403 ES

The hyaluronan content of the individual leaves of the plants of the lines having the name 403 ES was determined. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating in each case the amount of hyaluronan in leaves of 5 different natural plants and 5 plants of line 365 ES 13, which were vegetative progeny of the natural plant (Solanum tuberosum

cv. Desiree) and line 365 ES 13, respectively. The following results were obtained for the selected plants:

Table 10 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in leaves of independent transgenic plants selected from line 403 ES. Column 1 contains the name of the plant from which the leaf material was obtained (here, "natural" refers to plants that have not been transformed; 365 EN 13 refers to plants that express a hyaluronan synthase and were used as starting material for the transformation with the plant expression vector IC 375-271 to generate lines 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. In the case of plants for which the standard deviation is not indicated, the hyaluronan content of a single leaf sample was determined.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

403 EP 10

1186.32

403 EP 50

1141.75

403 EP 9

1017.62

403 EP 42

959.01

403 EP 40

930.39

403 EP 33

904.65

403 EP 6

884.45

403 EP 47

841.92

403 EP 37

725.39

403 EP 2

653.23

403 EP 48

579.14

403 EP 27

510.98

Natural type

3.93 2.87

365 EP 13

85.46 17.3

i) Analysis of the tubers of line 404 ES

For tuber tasting of the individual transgenic plants of the lines called 404 ES, two independent samples were taken, if possible, and the hyaluronan content, in each case, was determined separately. The mean and standard deviation of the values obtained from the individual measurements were then calculated using the formula provided in section 5 of the General Procedures. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating the amount of hyaluronan in tubers of, in each case, 10 different natural plants and 12 plants of line 365 ES 74 that were vegetative progeny of the natural plant (Solanum tuberosum cv. Desiree) and line 365 ES 74, respectively. The following results were obtained for the selected plants:

Table 11 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in tubers of independent transgenic plants selected from line 404 ES. Column 1 contains the name of the plant from which the tuber material was obtained (here, "natural" refers to plants that have not been transformed; 365 EN 74 refers to plants that express a hyaluronan synthase and were used as Starting material for transformation with the plant expression vector IC 375-271 to generate lines 404 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." It means that it was not possible to detect the hyaluronan in the tubers. In the case of plants for which the standard deviation is not indicated, the hyaluronan content of a single tuber sample was determined.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

404 EP 16

633.53

404 EP 47

188.42 7.80

404 EP 45

174.21 1.03

404 EP 17

155.09

404 EP 2

138.33 4.91

404 EP 30

124.38

404 EP 18

116.10 14.98

Natural type

n.d. -

365 EP 74

110.23 15.94

j) Analysis of the 404 ES line sheets

The hyaluronan content of the individual leaves of the plants of the lines having the name 404 ES was determined. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating in each case the amount of hyaluronan in leaves of 7 different 5 natural plants and 9 plants of the line 365 ES 74, which were vegetative progeny of the natural plant (Solanum tuberosum

cv. Desiree) and line 365 ES 74, respectively. The following results were obtained for the selected plants:

Table 12 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in leaves of independent transgenic plants selected from line 404 ES. Column 1 contains the name of the plant from which the leaf material has been obtained (here, "natural" refers to plants that have not been transformed; 365 EN 74 refers to plants that express a hyaluronan synthase and is used as starting material for the transformation with the plant expression vector IC 375-271 to generate lines 404 ES). Column 2 shows the average amount of hyaluronan determined for the leaves in question. Column 3 shows the standard deviation of the determined means. In the case of plants for which the standard deviation is not indicated, the

15 hyaluronan content of a single leaf sample.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

404 EP 13

1547.12

404 EP 35

1388.51

404 EP 29

1146.68

404 EP 36

1095.11

404 EP 44

921.11

404 EP 42

849.43

404 EP 46

846.81

404 EP 15

832.32

404 EP 23

817.91

404 EP 1

801.14

404 EP 38

651.12

404 EP 14

616.79

404 EP 16

615.92

404 EP 20

581.11

404 EP 37

533.89

404 EP 8

521.92

(continuation)

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

404 EP 21

489.73

404 EP 43

479.34

404 EP 24

434.06

404 EP 40

371.88

404 EP 9

366.46

404 EP 6

365.15

404 EP 28

359.96

404 EP 39

353.74

404 EP 34

310.76

404 EP 48

302.54

404 EP 11

231.39

404 EP 10

226.83

404 EP 7

218.42

404 EP 26

205.00

Natural type

0.25 0.09

365 EP 74

83.24 44.73

k) Analysis of the tubers of line 409 ES

For tuber tasting of the individual transgenic plants of the lines called 409 ES, were taken

5 samples, and the hyaluronan content was determined in each case. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating the amount of hyaluronan in tubers of, in each case, 4 different natural plants and 6 plants of line 365 ES 74 that were vegetative progeny of the natural plant (Solanum tuberosum cv. Desiree) and line 365 ES 74, respectively. The mean and standard deviation of the values obtained from the measurements were calculated

10 individuals using the formula provided in point 5 of the General Procedures. The following results were obtained for the selected plants:

Table 13 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in tubers of independent transgenic plants selected from line 395 ES. Column 1 contains the name of the plant from which the tuber material was obtained (here, "natural" refers to plants that have not been transformed, 365 EN 74 15 refers to plants that express a hyaluronan synthase and were used as a starting material for the transformation with the plant expression vector IC 375-271 to generate lines 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." It means that it was not possible to detect the hyaluronan in the tubers. In the case of plants for which the standard deviation is not indicated, the content of

20 hyaluronan of a single tuber sample.

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

409 EP 3

68.75

409 EP 4

59.80

409 EP 13

55.87

409 EP 16

60.28

409 EP 22

69.47

409 EP 23

108.67

5

10

fifteen

twenty

25

30 (continued)

Plant name

Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

409 EP 28

66.95

409 EP 29

79.58

Natural type

n.d. -

365 EP 74

40.53 16.75

l) Analysis of the sheets of line 409 EN

The hyaluronan content of the individual leaves of the plants of the lines having the name 409 ES was determined. The means and standard deviations indicated for the so-called natural plants and the so-called 365 ES plants were calculated by calculating in each case the amount of hyaluronan in leaves of 4 different natural plants and 6 plants of line 365 ES 74, which were vegetative progeny of the natural plant (Solanum tuberosum

cv. Desiree) and line 365 ES 74, respectively. The following results were obtained for the selected plants:

Table 14 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in leaves of independent transgenic plants selected from line 409 ES. Column 1 contains the name of the plant from which the leaf material was obtained ("natural" refers to plants that have not been transformed); 365 ES 74 refers to plants that express a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 376-256 to generate lines 409 ES). Column 2 shows the amount of hyaluronan determined for the leaves in question. Column 3 shows the standard deviation of the determined means. "n.d." means that the hyaluronan could not be detected in the tubers. In the case of plants for which the standard deviation is not indicated, the hyaluronan content of a single leaf sample was determined.

Plant name

Amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

409 EP 3

68.75

409 EP 4

59.80

409 EP 13

55.87

409 EP 16

60.28

409 EP 22

69.47

409 EP 23

108.67

409 EP 28

66.95

409 EP 29

79.58

Natural type

n.d. -

365 EP 74

40.53 16.75

m) Determination of hyaluronan content with respect to fresh weight and with respect to dry weight

The individual leaves of the plants of lines 395 ES and 396 ES, before collecting the tubers of the plants in question, were removed from the plants and divided in half. One half of each individual leaf, in each case, was frozen with liquid nitrogen, the corresponding other half was dried during the night.

Approximately 0.3 g of freeze sheet material or about 0.02 g of the samples of cryo-dried leaves were crushed in a laboratory oscillating ball mill (MM200, from Retsch, Germany) (30 s at 30 HZ). Approximately 300 ml of water (demineralized, conductivity = 18 Mn) was then added to each individual crushed sample, which was then mixed with a vortex mixer, and cell debris and insoluble components were then separated from the supernatant by centrifugation. (5 minutes at 16,000 xg). The supernatant was removed, and each sample was filled up to 500 with water (demineralized, conductivity = 18 Mn). Aliquots of the samples prepared in this way were used to determine the hyaluronan content using the procedure described in section 4 of the General Procedures 4. The mean and standard deviation were calculated using the formula provided in section 5 of the General Procedures . The following results were obtained for the selected plants:

Table 15 Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in leaves of independent transgenic plants selected from lines 395 ES and 396 ES. Column 1 cites the plant from which the sheet material was obtained. 365 ES 13 refers to plants that express a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 372-256 to generate lines 395 ES. 365 ES 74 refers to plants that express a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 372-256 to generate lines 396 ES. Column 2 indicates the amount of hyaluronan determined for the different leaves of the plants in question. Column 3 indicates the average of the amounts of hyaluronan measured in different leaves of a plant. Column 4 shows the standard deviation of the determined means.

Plant name

Amount of hyaluronan based on fresh weight of plant material [g / g] Average amount of hyaluronan based on fresh weight of plant material [g / g] Standard deviation

395ES 16 I

570.87 491.04 146.80

395ES 16 II

321.62

395ES 16 III

580.64

395ES 17 I

414.39 532.55 120.37

395ES 17 II

655.02

395EN 17 III

528.23

396ES 9 I

316.64 241.21 88.31

396ES 9 II

144.08

396EN 9 III

262.92

396ES 16 I

462.80 622.99 139.45

396ES 16 II

688.92

396ES 16 III

717.24

365EN 13 I

43.23 52.77 16.04

365EN 13 II

71.28

365EN 13 III

43.80

365ES 74 I

169.75 158.00 12.52

365ES 74 II

144.83

365EN 74 III

159.42

10

Table 16 Amount of hyaluronan (in mg of hyaluronan per g of dry weight) produced in leaves of independent transgenic plants selected from lines 395 ES and 396 ES. Column 1 cites the plant from which the sheet material was obtained. 365 ES 13 refers to plants that express a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 372-256 to generate the

15 lines 395 ES. 365 ES 74 refers to plants that express a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 372-256 to generate lines 396 ES. Column 2 indicates the amount of hyaluronan determined for the different leaves of the plants in question. Column 3 indicates the average of the amounts of hyaluronan measured in different leaves of a plant. Column 4 shows the standard deviation of the determined means.

Plant name

Amount of hyaluronan based on dry weight of plant material [g / g] Average amount of hyaluronan based on dry weight of plant material [g / g] Standard deviation

395ES 16 I

5212.63 4633.54 636.00

395ES 16 II

3952.86

395ES 16 III

4735.12

(continuation)

Plant name

Amount of hyaluronan based on dry weight of plant material [g / g] Average amount of hyaluronan based on dry weight of plant material [g / g] Standard deviation

395ES 17 I

4402.04 4313.57 77.25

395ES 17 II

4,259.45

395EN 17 III

4279.23

396ES 9 I

3918.45 2543.02 1383.15

396ES 9 II

1152.27

396EN 9 III

2558.35

396ES 16 I

4428.93 5077.92 932.12

396ES 16 II

6146.03

396ES 16 III

4658.81

365EN 13 I

373.90 398.74 104.57

365EN 13 II

513.49

365EN 13 III

308.82

365ES 74 I

1403.83 1207.71 170.52

365ES 74 II

1094.43

365EN 74 III

1124.87

14. Transformation of tomato plants with plant expression vectors comprising nucleic acid molecules encoding a hyaluronan synthase

5 Tomato plants were initially transformed using the plant expression vector IC 341-222 comprising a nucleic acid sequence encoding a hyaluronan synthase of Paramecium bursaria Chlorella virus 1 under the control of the Solanum B33 patatin gene promoter tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) using the procedure provided in item 8 of the General Procedures. The obtained transgenic tomato plants, which had been transformed with plasmid IC 341-222, were named

10 367 ES.

The tomato plants of lines 367 ES 25 and 367 ES 42 were then transformed with the plant expression vector IC 341-222 using the procedure provided in item 8 of the General Procedures. The transgenic tomato plants obtained from line 367 ES 25, which had been transformed with plasmid IC 341-222, were designated 399 ES. The transgenic tomato plants obtained from line 367 ES 42,

15 that had been transformed with plasmid IC 341-222, were designated 400 ES.

The tomato plants of lines 367 ES 25 were then transformed with the plant expression vector IC 375-271 using the procedure provided in item 8 of the General Procedures. The transgenic tomato plants obtained from line 367 ES 25, which had been transformed with plasmid IC 375-271, were designated 405 ES.

20 15. Analysis of transgenic tomato plants that synthesize hyaluronan additionally transformed with plant expression vectors comprising nucleic acid sequences encoding a protein that has the activity of a GFAT and a protein that has the activity of a UDP-Glc- DH

a) Tomato plant leaves of lines 399 ES and 400 ES

From different tomato plants selected from lines 399 ES and 400 ES, which had been grown in the soil

25 in a greenhouse, in each case a leaf was collected that was frozen in liquid nitrogen. Further elaboration and determination of the hyaluronan content were performed as described in Example 11b) for the leaves of the potato plants. The following results were obtained:

Table 17: Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in leaves of independent transgenic plants selected from lines 395 ES and 396 ES. Column 1 refers to the plant from which the sheet material was obtained. 367 ES 25 and 367 ES 42 refer to different plants that express a hyaluronan synthase and were used as starting material for transformation with the plant expression vector IC 372-256 to generate lines 399 ES and 400 ES, respectively. Column 2 indicates the value of the amount of hyaluronan determined for the different leaves of the plants in question. Natural refers to plants that have not been transformed.

Plant name

Amount of hyaluronan based on fresh weight of plant material [g / g]

Natural type

0.06

367 EP 25

57.19

399 EP 1

260.24

399 EP 11

835.69

367 EP 42

88.99

400 EP 3

513.27

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

10 From different tomato plants selected from lines 399 ES and 400 ES, which had been grown in the soil in a greenhouse, ripe fruits were picked in each case, crushed, centrifuged, and the supernatant, after centrifugation, filter. Further elaboration of the filtrate and the determination of the hyaluronan content were performed as described in Example 11b) for the leaves of the potato plants. The following results were obtained:

15 Table 18: Amount of hyaluronan (in mg of hyaluronan per g of fresh weight) produced in mature fruits of independent transgenic plants selected from lines 395 ES and 396 ES. Column 1 refers to the plant from which the sheet material was obtained. 367 ES 25-1 and 267 ES-2 refer to different clonal progeny of the plant 367 ES 25, and 367 ES 42-1 and 267 ES 42-2 refer to different clonal progeny of the plant 367 ES 42. The column 2 indicates the average amount of hyaluronan determined in the fruits of the plants in question.

20 For this purpose, the hyaluronan content of, in each case, 3 (lines 399 ES-1, 400 ES 3), 5 (lines 367 ES-25-1, 325 ES-2, 367 ES 42-1) was determined , 367 ES 42-2) or 6 (line 399 ES-11) different fruits of the lines in question. Column 3 shows the standard deviation of the determined means.

Plant name

Amount of hyaluronan based on fresh weight of plant material [µg / g] Standard deviation

Natural type

0.01 0.01

367 EN 25 -1

12.04 3.9

367 EN 25 -2

8.51 1.8

399 EP 1

87.02 20.6

399 EP 11

292.79 51.3

367 EN 42-1

12.20 2.4

367 EN 42-2

10.35 2.9

400 EP 3

31.59 13.7

c) Fruits of tomato plants of lines 405 EN

25 From different tomato plants selected from the lines and 405 ES, which had been grown in the soil in a greenhouse, ripe fruits were collected in each case, crushed, centrifuged, and the supernatant, after centrifugation, was filtered. Further elaboration of the filtrate and the determination of the hyaluronan content were performed as described in Example 11b) for the leaves of the potato plants. The following results were obtained:

30

Claims (10)

image 1

one.

A genetically modified plant cell that has a nucleic acid molecule that encodes a hyaluronan synthase linked to regulatory sequences that ensure transcription in plant cells stably integrated into its genome, wherein said plant cell additionally has a plurality of acid molecules foreign nucleic stably integrated into its genome, in which a first foreign nucleic acid molecule encodes a protein that has the activity of a glutamine: fructose 6-phosphate amidotransferase-2 (GFAT-2) that originates from animals or a glutamine: fructose 6-phosphate amidotransferase (GFAT) that originates from bacteria and a second molecule of a foreign nucleic acid that encodes a protein that has the activity of a UDP-glucose dehydrogenase (UDP-Glc-DH) and wherein said genetically modified plant cell has an increased activity of a protein that has the activity of a to glutamine: fructose 6-phosphate amidotransferase (GFAT) and an increased activity of a protein that has the activity of a UDP-glucose dehydrogenase (UDP-Glc-DH) compared to the corresponding genetically unmodified plant cells.

2.

The genetically modified plant cell according to claim 1 which synthesizes an increased amount of hyaluronan compared to plant cells that have the activity of a hyaluronan synthase and an unincorporated activity of a glutamine: fructose 6-phosphate amidotransferase (GFAT) and an unintended activity of a UDP-glucose dehydrogenase.

3.

A cell comprising genetically modified plant cells according to any one of claims 1 or 2.

Four.

Plant propagation material according to claim 3, comprising genetically modified plant cells according to any one of claims 1 or 2.

5.

A harvested plant part of a plant according to claim 3, comprising genetically modified plant cells according to any one of claims 1 or 2.

6.

 A process for preparing a plant that synthesizes hyaluronan, which comprises

a) genetically modify a plant cell, in which the genetic modification comprises the following stages i to iii i) introduction of a foreign nucleic acid molecule encoding a hyaluronan synthase linked to regulatory sequences that ensure transcription in plant cells in the plant cell ii) introduction of a foreign nucleic acid molecule in the plant cell, comprising the acid molecule Nucleic strains a coding sequence of a protein that has the activity of a glutamine: fructose 6-phosphate amidotransferase-2 (GFAT-2) that originates from animals or a glutamine: fructose 6-phosphate amidotransferase (GFAT) that originates from bacteria iii) introduction of a foreign nucleic acid molecule into the plant cell, the foreign nucleic acid molecule comprising a coding sequence of a protein having the activity of a glutamine: fructose 6-phosphate UDP-glucose dehydrogenase wherein steps i to iii can be performed in any order, individually, or any combination of stages i to iii can be done simultaneously b) regenerating a plant from the plant cells of stage a).

7.

A hyaluronan preparation process comprising the step of extracting hyaluronan from genetically modified plant cells according to any one of claims 1 or 2, from plants according to claim 3, from the propagation material according to with claim 4, or from harvestable parts of plants according to claim 5.

8.

The use of a genetically modified plant cell according to any one of claims 1 or 2, a plant according to claim 3, propagation material according to claim 4 or the harvested parts of the plant according to claim 5 for the preparation of hyaluronan.

9.

A method of preparing a composition comprising hyaluronan in which the genetically modified plant cells according to any one of claims 1 or 2 are used, the plants according to claim 3, propagation material according to claim 4, or the harvested parts of the plant according to claim 5.

10.

 The use of genetically modified plant cells according to any of claims 1 or 2, of plants according to claim 3, of propagation material according to claim 4 or of the harvestable parts of the plant for the preparation of a composition according to claim 5 to prepare a composition.

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