AU777455B2 - Nucleic acid molecules from artichoke (cynara scolymus) encoding enzymes having fructosyl polymerase activity - Google Patents

Description

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN E.V.

Invention Title: NUCLEIC ACID MOLECULES FROM ARTICHOKE (CYNARA SCOLYMUS) ENCODING ENZYMES HAVING FRUCTOSYL POLYMERASE ACTIVITY.

The following statement is a full description of this invention, including the best method of performing it known to us: tI 11 la Max-Planck-Gesellschaft zur Forderung Der Wissenschaften e.V.

Nucleic acid molecules encoding enzymes having fructosyl polymerase activity All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

The present invention relates to nucleic acid molecules encoding sucrose dependent sucrose fructosyltransferases (SST). Furthermore, this invention relates to vectors and hosts containing such nucleic acid molecules, as well as plant cells and plants transformed with the described nucleic acid molecules. Furthermore, methods for the production of transgenic plants are described that synthesize short-chain fructosyl polymers due to the introduction of DNA molecules encoding an SST from artichoke. The present invention also relates to methods for the production of SST for producing shortchain fructosyl polymers in various host organisms as well as to the SST with the help of which short-chain fructosyl polymers can be produced using various methods, for example fermentative or other biotechnological methods.

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Ib Water-soluble, linear polymers have many various applications, for example for increasing the viscosity of aqueous systems, as detergents, as suspending agents or for accelerating the sedimentation process and for complexing but also for binding water. Polymers on the basis of saccharides, for example fructosyl polysaccharides, are especially interesting raw materials since they are biodegradable.

Apart from their application as regenerative raw materials for industrial production and processing, fructosyl polymers are also interesting as food additives, for example as artificial sweeteners. Polymers having a low polymerization level are particularly suitable for this purpose.

Up to now only processes for the production of long-chain fructane polysaccharides in plants by expression of enzymes of bacterial origin as well as a process for the production of transgenic plants expressing fructosyltransferases from Helianthus tuberosus have been described. Processes for the production of enzymes for producing short-chain fructosyl polymers are not known.

In the spefcification of PCT/USA89/02729 the possibility to produce carbohydrate polymers, in particular H.\Pcabral\Xeep\speci\682S4.98.doc 28/05/02 dextrane or polyfructose, in transgenic plants, in particular in the fruits of transgenic plants, is described. For the production of such modified plants the use of levane sucrases from microorganisms, in particular from Aerobacter levanicum, Streptococcus salivarius and Bacillus subtilis, or from dextrane sucrases from Leuconostoc mesenteroides are suggested. The production of neither the active enzymes nor of levane or dextrane nor of transgenic plants is described. The specification of PCT/EP93/02110 discloses a process for the production of transgenic plants expressing the Isc gene of levane sucrase from the gram-negative bacterium Erwinia amylovora. In the specification of PCT/NL93/00279 the transformation of plants having chimeric genes that contain the sacB gene from Bacillus subtilis or the ftf gene from Streptococcus mutans is described. In the case of the sacB gene a modification in the region of the gene is recommended in order to increase the expression level in transgenic plants. The specification of PCT/NL96/00012 discloses DNA sequences encoding the enzymes synthesizing carbohydrate polymers and the production of transgenic plants with the help of these DNA sequences. The disclosed sequences originate from Helianthus tuberosus. According to PCTL/NL96/00012 the disclosed sequences are not only suitable to modify the fructane profile of, for example, petunia and potato but also of Helianthus tuberosus itself. Therefore, the specification of PCT/NL96/00012 describes inter alia transgenic potato plants expressing an SST from Helianthus tuberosus. Even though the enzymatic activity of the SST expressed in the transgenic plants could be detected, only a low level of conversion of the substrate sucrose to short-chain fructosyl polymers could be achieved. This may be related to various factors, such as a low affinity of the enzyme to its substrate or a possible inhibition of the enzyme by the produced product.

Therefore, the problem of the present invention is to provide nucleic acid molecules encoding a sucrose dependent sucrose fructosyltransferase (SST) with the help of which it is possible to produce organisms modified by genetic engineering that are able to form short-chain fructosyl polymers.

This problem is solved by providing the embodiments described in the claims.

3 Therefore, the present invention relates to nucleic acid molecules encoding the proteins having the biological activity of an SST and being selected from the group consisting of nucleic acid molecules encoding a protein that comprises the amino acid sequence depicted in SEQ ID No. 2 and SEQ ID No. 4; nucleic acid molecules comprising the nucleotide sequence depicted in SEQ ID No. 1 or a corresponding ribonucleotide sequence; nucleic acid molecules comprising the nucleotide sequence depicted in SEQ ID No. 3 or a corresponding ribonucleotide sequence; nucleic acid molecules hybridizing to the nucleic acid molecules mentioned in or and encoding an SST the amino acid of which is to at least 90 identical to the amino acid sequence depicted in SEQ ID No. 2; and nucleic acid molecules the nucleotide sequence of which deviates from the sequence mentioned in or due to the degeneration of the genetic code.

In the context of the present invention an enzyme having the fructosyl polymerase activity is understood to be a protein that is able to catalyze the linking of p-2,1 glycosidic or p-2,6 glycosidic bonds between fructose units. Hereby, a fructosyl residue to be transferred can originate from sucrose or a fructan polymer.

A short-chain fructosyl polymer is understood to be a molecule containing at least two but not more than 100 fructosyl residues that are linked either 0-2,1 glycosidically or 3- 2,6 glycosidically. The fructosyl polymer can carry a glucose residue at its terminal that is linked via the C-1 OH-group of the glucose and the C-2 OH-group of a fructosyl. In this case a molecule of sucrose is contained in the fructosyl polymer.

In a preferred embodiment the nucleic acid sequences of the invention are derived from artichoke.

It was surprisingly found that during the expression of the nucleic acid molecules of the invention large amounts of fructosyl polymers were produced.

4 In contrast to the potatoes described in the specification of PCT/NL96/00012 a large amount of oligofructan is obtained that is even larger than the cellular content of the substrate sucrose when the nucleic acid molecules of the invention are used.

The nucleic acid molecules of the invention can be both DNA and RNA molecules.

Suitable DNA molecules are, for example, genomic or cDNA molecules. The nucleic acid molecules of the invention can be isolated from natural sources, preferably artichoke, or can be synthesized according to known methods.

By means of conventional molecular biological processes it is possible (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2 d edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) to introduce different mutations into the nucleic acid molecules of the invention. As a result proteins with possibly modified biological properties are synthesized. One possibility is the production of deletion mutants in which nucleic acid molecules are produced by continuous deletions from the or 3'-terminal of the coding DNA sequence and that lead to the synthesis of proteins that are shortened accordingly. By such deletions at the 5'-terminal of the nucleotide sequence it is, for example, possible to identify amino acid sequences that are responsible for the translocation of the enzyme in the plastids (transition peptides). This allows the specific production of enzymes that are, due to the removal of the corresponding sequences, no longer located in the vacuole but in the cytosol or that are, due to the addition of other signal sequences, located in other compartments.

Another possibility is the introduction of single-point mutation at positions where a modification of the amino acid sequence influences, the enzyme activity or the regulation of the enzyme. By this method mutants can be produced, for example, that possess a modified Km-value or that are no longer subject to the regulation mechanisms that normally exist in the cell with regard to allosteric regulation or covalent modification.

Furthermore, mutants can be produced showing a modified substrate or product specificity. Also mutants can be produced showing a modified activity-temperature profile.

For the manipulation in prokaryotic cells by means of genetic engineering the nucleic acid molecules of the invention or parts of these molecules can be introduced into plasmids allowing a mutagenesis or a modification of a sequence by recombination of DNA sequences. By means of conventional methods (cf. Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2 d edition, Cold Spring Harbor Laboratory Press, NY, USA) bases can be exchanged and natural or synthetic sequences can be added. In order to link the DNA fragments with each other adapters or linkers can be added to the fragments. Furthermore, manipulations can be performed that provide suitable cleavage sites or that remove superfluous DNA or cleavage sites. If insertions, deletions or substitutions are possible, in vitro mutagenesis, primer repair, restriction or ligation can be performed. As analysis method usually sequence analysis, restriction analysis and other biochemical or molecular biological methods are used.

The term "hybridization" in the context of this invention has the meaning of hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 d edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Nucleic acid molecules that hybridize to the molecules of the invention can be isolated, from genomic or cDNA libraries that were produced from artichoke.

In order to identify and isolate such nucleic acid molecules the molecules of the invention or parts of these molecules or the reverse complements of these molecules can be used, for example by means of hybridization according to conventional methods (see, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

As a hybridization probe nucleic acid molecules can be used, for example, that have exactly or basically the nucleotide sequence depicted in Seq ID No. 1 or parts of these sequences. The fragments used as hybridization probe can be synthetic fragments that were produced by means of conventional synthesis methods and the sequence of which basically corresponds to the sequence of a nucleic acid molecule of the invention.

The molecules hybridizing to the nucleic acid molecules of the invention also comprise fragments, derivatives and allelic variants of the nucleic acid molecules described above encoding a protein of the invention. "Fragments" are understood to be parts of 6 the nucleic acid molecules that are long enough to encode one of the described proteins. The term "derivative" in this context means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above at one or several positions but have a high level of homology to these sequences.

Homology hereby means a sequence identity of at least 40 in particular an identity of at least 60 preferably of more than 80 and particularly preferred of more than These proteins encoded by the nucleic acid molecules have a sequence identity to the amino acid sequence depicted in SEQ ID No. 2 of at least 80 preferably of and particularly preferred of more than 90 95 97 and 99 The deviations to the above-described nucleic acid molecules may have been produced by deletion, substitution, insertion or recombination.

The nucleic acid molecules that are homologous to the above-described molecules and that represent derivatives of these molecules usually are variations of these molecules that represent modifications having the same biological function. They can be naturally occurring variations, for example sequences from other organisms, or mutations that can either occur naturally or that have been introduced by specific mutagenesis. Furthermore, the variations can be synthetically produced sequences.

The allelic variants can be either naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA processes.

The proteins encoded by the various variants of the nucleic acid molecules of the invention show certain common characteristics, such as enzyme activity, molecular weight, immunological reactivity or conformation or physical properties like the electorphoretical mobility, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability; pH optimum, temperature optimum.

In another preferred embodiment the invention relates to nucleic acid molecules specifically hybridizing to transcripts of the nucleic acid molecules. These nucleic acid molecules preferably are oligonucleotides having a length of at least 10, in particular of at least 15 and particularly preferred of at least 50 nucleotides. The nucleic acid molecules and oligonucleotides of the invention can be used, for example, as primers for a PCR reaction. They can also be components of antisense constructs or of DNA molecules encoding suitable ribozymes.

The invention furthermore relates to vectors containing nucleic acid molecules of the invention. Preferably, they are plasmids, cosmids, viruses, bacteriophages and other vectors usually used in the field of genetic engineering.

Preferably, the nucleic acid sequence of the invention is operatively linked to the regulatory elements in the vector of the invention that guarantee the transcription and synthesis of an RNA in prokaryotic and/or eukaryotic cells that can be translated.

The expression vectors of the invention allow the production of enzymes synthesizing short-chain fructosyl polymers in various host organisms.

The encoded enzymes can be used also outside the host organisms for the production of short-chain fructosyl polymers. Thereby, fermentative and other biotechnological methods for the production of short-chain fructosyl polymers can be used. For example, it is also imaginable to produce fructosyl polymers by means of immobilized enzymes.

According to the invention regulatory elements of the patatin B33 promoter are preferred. Other preferred promoters are the 35S CaMV promoter and the promoter of the alcohol dehydrogenase gene from Saccharomyces cerevisiae.

The vectors of the invention can possess further functional units effecting the stabilization of the vector in the host organism, such as a bacterial replication origin or the 2 -t DNA for the purpose of stabilization in Saccharomyces cerevisiae.

Furthermore, "left border" and "right border" sequences of agrobacterial T-DNA can be contained, whereby a stable integration into the genome of plants is made possible.

Furthermore, the vectors of the invention can contain functional terminators, such as the terminator of the octopine synthase gene from agrobacteria.

In another embodiment the nucleic acid molecule of the invention is linked to the vector of the invention by a nucleic acid molecule encoding a functional signal 8 sequence in order to transport the enzyme to various cell compartments. This modification can be, for example, the addition of an N-terminal signal sequence for secretion into the cell membrane space of higher plants but also any other modification that leads to the fusion of a signal sequence to the encoded fructosyltransferase can be the subject matter of the invention.

In a particularly preferred embodiment the invention relates to the plasmid pB33cySST the construction of which is described in the examples (Fig. 1).

The expression of the nucleic acid molecules of the invention in prokaryotic cells, for example in Escherichia coli, is interesting because this way a closer characterization of the enzymatic activities of the enzymes encoding these molecules is possible.

In a further embodiment the invention relates to host cells transiently or stably containing the nucleic acid molecules or vectors of the invention. A host cell is understood to be an organism that is capable to take up in vitro recombinant DNA and, if the case may be, to synthesize the proteins encoded by the nucleic acid molecules of the invention.

Preferably, these cells are prokaryotic or eukaryotic cells. In particular, the invention relates to plant cells containing the vector systems of the invention or derivatives or parts therof. Preferably, they are able to synthesize enzymes for the production of short-chain fructosyl polymers due to the fact that they have taken up the vector systems of the invention, derivatives or parts thereof. The cells of the invention are preferably characterized by the fact that the introduced nucleic acid molecule of the invention either is heterologous with regard to the transformed cell, i.e. that it does not naturally occur in these cells, or is localized at a place in the genome different from that of the corresponding naturally occurring sequence.

A further embodiment of the invention relates to proteins being encoded by the nucleic acid molecules of the invention, as well as to methods for their production, whereby a host cell of the invention is cultivated under conditions allowing the synthesis of the protein and the protein is subsequently isolated from the cultivated cells and/or the culture medium. Furthermore, the invention relates to the SSTs that can be produced with the plants of the invention.

By providing the nucleic acid molecules of the invention it is now possible to produce short-chain fructosyl polymers in any organisms by means of genetic engineering, whereas up to now it had not been possible to modify plants by conventional methods, for example breeding methods, so that they are able to synthesize fructosyl polymers.

By increasing the activity of the proteins of the invention, for example by overexpressing suitable nucleic acid molecules or by providing mutants that are no longer subject to the cell-specific regulation mechanisms and/or that have altered temperature dependencies with respect to their activity, it is possible to increase the yield in plants modified by genetic engineering.

Therefore, the expression of the nucleic acid molecules of the invention in plant cells in order to increase the activity of the corresponding SST or the introduction into cells normally not expressing this enzyme is now possible. Furthermore, it is possible to modify the nucleic acid molecules of the invention according to the methods known to the person skilled in the art in order to obtain SSTs of the invention that are no longer subject to the cell-specific regulation mechanisms or that have modified temperature dependencies or substrate or product specificities.

When the nucleic acid molecules are expressed in plants, the synthesized protein may be located in any compartment of the plant cell. In order to achieve the localization in a specific compartment, the sequence guaranteeing the localization in vacuole has to be deleted and, if necessary, the remaining coding region has to be linked to DNA sequences guaranteeing the localization in the specific compartment. Such sequences are known (see, Braun et al., EMBO J. 11 (1992), 3219-3227; Wolter et al., Proc.

Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al., Plant J. 1 (1991), 95-106).

The present invention therefore also relates to transgenic plant cells that were transformed with one or several nucleotide molecule(s) of the invention as well as to transgenic plant cells originating from such transformed cells. Such cells contain one or several nucleic acid molecule(s) of the invention with it/them preferably being linked to regulatory DNA elements guaranteeing the transcription in plant cells, in particular with a promoter. Such plants can be distinguished from naturally occurring plant cells by the fact that they contain at least one nucleic acid molecule according to the invention which does not naturally occur in these cells or by the fact that such a molecule is integrated into the genome of the cell where it does not naturally occur, i.e.

in another genomic region.

The transgenic plant cells can be regenerated to whole plants using methods known to the person skilled in the art. The subject matter of the present invention relates to the plants obtainable by regeneration of the transgenic plant cells of the invention.

Furthermore, the subject matter of the invention relates to plants containing the transgenic plant cells described above. The transgenic plants can basically be plants of any plant species, i.e. both monocotyledonous and dikotyledonous plants.

Preferably they are crops, in particular plants that synthesize and/or store starch, such as wheat, barley, rice, maize, sugar beet, sugar cane or potato. Particularly preferred are sucrose storing plants.

The invention also relates to propagation material and harvest products of the plants of the invention, for example fruits, seeds, tubers, root stocks, seedlings, cuttings etc.

The transgenic plant cells and plants of the invention synthesize short-chain fructosyl polymers due to the expression or additional expression of at least one nucleic acid molecule of the invention.

The subject matter of the invention therefore also relates to the short-chain fructosyl polymers obtainable from the transgenic plant cells and plants of the invention as well as from the propagation material and harvest products.

The transgenic plant cells of the invention can be regenerated to whole plants according to methods known to the person skilled in the art. Therefore, the subject matter of the invention also relates to plants containing the transgenic plant cells of the invention. These plants preferably are crops, in particular plants that synthesize and/or store sucrose and/or starch. Particularly preferred is potato. The invention also relates to the propagation material of the plants of the invention, in particular tubers.

In order to express the nucleic acid molecules of the invention in sense or antisense orientation in plant cells, they are linked to regulatory DNA elements guaranteeing the transcription in plant cells. These are particularly promoters. Basically, any promoter active in plant cells is suitable for the expression.

11 The promoter can be selected such that the expression takes place constitutively or only in a certain tissue, at a certain stage of the plant development or at a point of time determined by external stimuli. With regard to the plant the promoter can be homologous or heterologous. Suitable promoters are, for example, the promoter of the RNA of the cauliflower mosaic virus and the ubiquitin promoter from maize for a constitutive expression, particularly preferred the patatin gen promoter B33 (Rocha- Sosa et al., EMBO J. 8 (1989), 23-29) for a tuber specific expression in potato or a promoter only guaranteeing the expression in photosynthetically active tissue, 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 an endosperm specific expression the HMG promoters from wheat, the USP promoter, the Phaseolin promoter or promoters from zein genes from maize.

Furthermore, there can be a termination sequence serving for the correct termination of the transcription as well as the addition of a poly-A tail to the transcript which is regarded as having a function for the stabilization of the transcripts. Such elements are described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged arbitrarily.

In order to prepare the introduction of foreign genes into higher plants there is a great number of cloning vectors available containing a replication signal for E.coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence can be introduced into the vector at a suitable cleavage site. The plasmid obtained is suitable for the transformation of E.coli cells. Transformed E.coli cells are cultivated in a suitable medium, then harvested and lysed. The plasmid is regenerated. Usually, restriction analyses, gel electrophoreses and other biochemical or molecular biological methods are used as analysis methods for the characterization of the regenerated plasmid DNA. After every manipulation the plasmid DNA can be cleaved and the regenerated DNA fragments linked to other DNA sequences. Every plasmid DNA sequence can be cloned into the same or other plasmids.

For the introduction of DNA into a plant host cell a great number of methods are available. These methods comprise the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as means for transformation, 12 the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA by means of the biolistic methods as well as further possibilities.

For the injection and electroporation of DNA in plant cells there are no specific requirements for the plasmids used. Simple plasmids such as pUC derivatives can be used. If whole plants are to be regenerated from such transformed cells, there should be a selectable marker.

Depending on the method for the introduction of desired genes into the plant cell further DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right border, often, however, the right and left border of the Ti and Ri plasmid T-DNA have to be linked as flanking region to the genes to be introduced.

If agrobacteria are used for the transformation, the DNA to be introduced has to be cloned into specific plasmids, either into an intermediary vector or into a binary vector.

The intermediary vectors can be integrated into the Ti or Ri plasmid of the agrobacteria due to sequences that are homologous to sequences in the T-DNA by homologous recombination. The Ti or Ri plasmid furthermore contains the vir region necessary for the transfer of the T-DNA. Intermediary vectors cannot replicate in agrobacteria. By means of a helper plasmid the intermediary vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate both in E.coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al., Mol. Gen. Genet. 163 (1978), 181-187). The agrobacterium serving as a host cell should contain a plasmid carrying a vir region.

The vir region is necessary for the transfer of the T-DNA into the plant cell. There may be additional T-DNA. The agrobacterium transformed such is used for the transformation of plant cells. The use of T-DNA for the transformation of plant cells has extensively been examined and described in EP-A-120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters Alblasserdam (1985), Chapter V, Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and An et al., EMBO J. 4 (1985), 277-287.

For the transfer of the DNA into the plant cell plant explants can be co-cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material pieces of leaf, stem segments, roots, but also protoplasts or plant cells cultivated by suspension) whole plants can be regenerated in a suitable medium, 13 which may contain antibiotics or biozides for the selection of transformed cells. The plants obtained this way can be examined for the presence of the introduced DNA.

Other possibilities of introducing foreign DNA using the biolistic methods or by protoplast transformation are known Willmitzer, 1993 Transgenic plants.

In: Biotechnology, A Multi-Volume Comprehensive Treatise Rehm, G. Reed, A.

Puhler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel- Cambridge).

Alternative systems for the transformation of monocotyledonous plants are the transformation by means of the biolistic approach, the electrically or chemically induced introduction of DNA into protoplasts, the electroporation of partially permeabilized cells, the macroinjection of DNA into flowers, the microinjection of DNA into microspores and pro-embryos, the introduction of DNA into germinating pollen and the introduction of DNA into embryos by swelling (for review: Potrykus, Physiol. Plant (1990), 269-273).

While the transformation of dicotyledonous plants via Ti plasmid vector systems with the help of Agrobacterium tumefaciens is well-established, more recent research work indicates that also monocotyledonous plants are accessible for transformation by means of vectors based on Agrobacterium (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994), 271-282; Bytebier et al., Proc. Natl. Acad. Sci.

USA 84 (1987), 5345-5349; Raineri et al., Bio/Technology 8 (1990), 33-38; Gould et al., Plant Physiol. 95 (1991), 426-434; Mooney et al., Plant, Cell Tiss. Org. Cult. (1991), 209-218; Li et al., Plant Mol. Biol. 20 (1992), 1037-1048).

Three of the above-mentioned transformation systems could be established for various cereals: the electroporation of tissues, the transformation of protoplasts and the DNA transfer by particle bombardment in regenerative tissue and cells (for review: Jahne et al., Euphytica 85 (1995), 35-44).

The transformation of wheat has been frequently described in the literature (for review: Maheshwari et al., Critical Reviews in Plant Science 14 (1995), 149-178).

The invention also relates to plants containing at least one, preferably a number of cells containing the vector systems of the invention or derivatives or parts thereof and being able to synthesize enzymes for the production of short-chain fructosyl polymers due to the introduction of the vector systems, derivatives or parts of the vector systems 14 of the invention. The invention also provides plants of many species, genuses, families, orders and classes that are able to synthesize enzymes for the production of short-chain fructosyl polymers due to the introduced vector systems or derivatives or parts thereof. Since the known plants are not able to only produce short-chain fructosyl polymers, it is easy to check whether the method has been successfully performed, for example by chromatographic analysis of the sugars containing fructose. They are advantageous vis-&-vis the few plants containing fructosyl polymers since there is a defined molecular size, i.e. the size of the short-chain fructosyl polymer. Furthermore, a localization in the various cell compartments and various organs as well as an increase of the expression ratio and therefore of the yield is possible.

In another embodiment the invention relates to methods for the production of short-chain fructosyl polymers comprising: contacting sucrose or an equivalent substrate with an SST of the invention under conditions allowing the conversion to short-chain fructosyl polymers; and obtaining the fructosyl polymers produced this way.

The nature of the produced fructosyl polymers depends on the enzymatic specificity of the fructosyl transferase. When an SST of the invention is used, preferably kestose but also nystose and fructosylnystose are produced.

Furthermore, the invention relates to the fructosyl polymers produced from a plant cell or plant of the invention or from the propagation material or harvest product of plants or plant cells of the invention or obtained according to the aboveLdescribed method of the H-\Pcabral\KeeP\Bpeci\68254.98.doc 28/05/02 14a invention. These fructosyl polymers can preferably be used for the production of food such as baked goods or pasta. Preferably, these fructosyl polymers can be used for increasing the viscosity in aqueous systems, as detergents, as suspending agents or for accelerating the sedimentation process and complexing but also for binding water.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

H.\Pcabral\Keep\speci\68254.98.dc 28/05/02 The figures show: Figure 1 shows the construction of the plasmid pB33-cySST.

Vector: pBinB33 (derivative of pBin19; Bevan, 1984, Nucl Acids Res 12: 8711) promoter: B33 promoter (Rocha-Sosa et al., 1989, EMBO J 8: 23- 29) donor: Solanum tuberosum coding region: orientation: terminator: donator: resistance: SST gene from Cynars scolymus sense Polyadenylation signal of the octopin synthase gene from A. tumefaciens plasmid pTiACH5 (Gielen et al., 1984, EMBO J 3: 835-846) Agrobacterium tumefaciens kanamycin Figure 2 shows the analysis of the soluble sugars in the tubers of transgenic plants that were produced using the vector system pB33-cySST. The short-chain fructosyl polymers (in particular 1-kestose) produced due to the genetic modification have been labeled.

Figure 3 shows the analysis of the soluble sugars in transgenic plants that were produced using the vector system pB33-cySST and respectively, compared to wildtype plants.

Example 1: Identification, isolation and characterization of a cDNA encoding a sucrose dependent sucrose-fructosyltransferase from artichoke (Cynare scolymus) Total RNA was isolated from blossom discs of artichoke (Sambrook et al., see supra).

Poly(A)' mRNA was isolated using the mRNA isolation system PolyATtract (Promega Corporation, Madison, WI, USA). Complementary DNA (cDNA) was produced from 16 pg of this RNA by means of the ZAp-cDNA synthesis kit of Stratagene according to the manufacturer's instructions. 2x10 6 independent recombinant phages were obtained.

The amplified cDNA library was screened by conventional methods with a DNA fragment labeled with 32 P and corresponding to the 3'-terminal of the 6-SFT cDNA (Sprenger et al., Proc. Natl. Acad. Sci. USA 92 (1995), 11652) having a length of 392 bp. This fragment was obtained from the complete RNA by RT-PCR (RT-PCR Kit, Stratagene, Heidelberg, Germany) as matrix from light-induced (72 hours) primary leaves from barley. Positive clones were further examined.

Example 2: Sequence analysis of the cDNA insertion of the plasmid pCy21 The plasmid DNA was isolated from the clone pCy21. The sequence of the cDNA insertion was determined by conventional methods by means of the dideoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci USA 74 (1977), 5463-5467).

The insertion of the clone pCy21 is a DNA of 2055 bp. The nucleotide sequence is depicted in Seq ID No. 1. The corresponding amino acid sequence is depicted in Seq ID No. 2.

A sequence analysis and a comparison with already published sequences showed that the sequence depicted in Seq ID No. 1 is novel and comprises a coding region showing homologies to SSTs from other organisms.

Example 3: Production of the plasmid pB33-cySST and introduction of the plasmid into the genome of potato The plasmid pB33-cySST contains three fragments A, B and C in the binary vector pBin19 (Bevan, 1984, Nucl Acids Res 12: 8711, modified according to Becker, 1990, Nucl Acids Res 18: 203) (cf. Fig. Fragment A contains the B33 promoter of the patatin gene b33 of potato. It contains a Dral fragment (position 1512 to position +14) of the patatin gene B33 (Rocha-Sosa et al., 1989, EMBO J 8:23-29), which is inserted between the EcoRI and the Sad cleavage site of the polylinker of pBinl9-Hyg.

Fragment B contains the coding region of the sequence depicted in SEQ ID No. 1.

Fragment B was obtained as Notl fragment with blunt ends from the vector pBluescript 17 SK, in which it is inserted into the EcoRI cleavage site via an EcoRI/Not I linker sequence. Fragment C contains the polyadenylation signal of the gene 3 of the T-DNA of the Ti plasmid pTi ACH 5 (Gielen et al (1984); EMBO J. 3, 835-846) nucleotides 11749 11939, which was isolated as Pvu II-Hind III fragment from the plasmid pAGV (Herrera-Estrella et al (1983) Nature 303, 209 213) and cloned between the Sphl and the Hind III cleavage site of the polylinker of pBinl9-Hyg after the addition of Sph I linkers to the Pvu II cleavage site. The plasmid pB33-cySST has a size of approx. 14 kb. The plasmid was introduced into agrobacteria (H6fgen and Willmitzer, Nucleic Acids Res. 16 (1988), 9877).

The plasmid pB33-cySST was introduced into potato plants via the gene transfer induced by Agrobacterium according to the above-described conventional methods.

Intact plants were regenerated from transformed cells. From regenerated plants enzyme extracts were obtained and examined for the presence of fructosyl polymers.

The analysis was carried out as described in Rober (Planta 199, 528-536). The analysis of the tubers of a number of transformed plants transformed with this vector clearly showed the presence of short-chain fructosyl polymers, in particular 1-kestose, which can be put down to the expression of the SST gene of the invention (cf. Fig. 2).

Example 4 Analysis of soluble sugar in wildtype and SST containing transgenic plants Transgenic plants containing vectors pB33-cySST and 35S-cySST (having the coding region of SEQ ID No. 1 under the control of the 35S promoter) were generated as described in Example 3. Extracts were obtained from transgenic plants and wildtype plants and examined for the presence of fructosyl polymers; see Example 3. HPAECanalysis shown in Figure 3 demonstrates the production of oligofructanes. The results are summarized in Table 1, below.

18 Table 1 Soluble sugars (sucrose and oligofructane) in wildtype and transgenic plants line sucrose 1-kestose nystose F-nystose WT 1 (D6siree) 2,09-- WT 2 (D6siree) 1,67 B33-cySST 6 2,26 3,58 1,60 B33-cySST 54 5,13 3,06 2,90 0,23 18 4,08 4,05 1,51 0,12 22 4,80 4,14 2,19 0,1 Values in g carbohydrate per kg fresh weight As is evident from Figure 3 and Table 1, supra, the content of fructosyl polymers, in particular 1-kestose exceeds the content of sucrose. Thus, the experiments performed in accordance with the present invention demonstrate the usefulness of the nucleic acid molecules of the invention for the production of fructosyl polymers in transgenic plants.

The entire disclosure in the complete specification of our Australian Patent Application No. 68254/98 is by this cross-reference incorporated into the present specification.

Page(s) -are claims pages They appear after the sequence listing(s) 19 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V.

STREET: none CITY: Berlin COUNTRY: DE ZIP CODE: NONE (ii) TITLE OF THE INVENTION: Nucleic acid molecules encoding enzymes having fructosyl polymerase activity (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPA) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 2226 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) IMMEDIATE SOURCE: ORGANISM: Cynara Scolymus (ix) FEATURE: NAME/KEY: CDS LOCATION: 8..1918 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CCACCAC ATG GCT TCC TCT ACC ACC ACC CCA CTC CTC CCT CAC CAC CAC 49 Met Ala Ser Ser Thr Thr Thr Pro Leu Leu Pro His His His 1 5 CTT CAG AAC CCG CAA CAA CTC GCC GGA TCT CCG GCA GCT CAT CGT CTA 97 Leu Gln Asn Pro Gln Gin Leu Ala Gly Ser Pro Ala Ala His Arg Leu 20 25 TCC CGA CCC ACA CTC CTT TCT GGG ATC CTT GTT TCG GTC CTA GTC ATC 145 Ser Arg Pro Thr Leu Leu Ser Gly Ile Leu Val Ser Val Leu Val Ile 40 TGT GOT CTC GTT GCT GTA ATC CAC Cys Ala Leu Val Ala Val Ile His

AAC

Asn 55 CAA TCA CAG CAA Gin Ser Gin Gin CCC TAC CAT Pro Tyr His ACC TTC CCA Thr Phe Pro GAC GGC GGA Asp Giy Gly GCT AAA CCC TCC Ala Lys Pro Ser

TOO

Ser 70 TOO GCC GCT ACC Ser Ala Ala Thr

ACC

Thr ACA GCG Thr Ala TCG CCA GAA GCT Ser Pro Giu Ala TTG AAA CGG TTT Leu Lys Arg Phe CCC ATT GAG TTG AAA Pro Ile Glu Leu Lys TAC CAT TTT CAG CCC Tyr His Phe Gin Pro

ACG

Thr AAT GCT GAG GTT Asn Ala Glu Val

GAG

Glu 100 TGG CAA CGC TOG Trp Gin Arg Ser

GCT

Ala 105 GAT AAG AAC TAC Asp Lys Aen Tyr

ATT

Ile 115 AGO GAT OCT GAT Ser Asp Pro Asp

GGC

Gly 120 OCA ATG TAT CAC Pro Met Tyr His ATG GGG Met Gly 125 TGG TAT CAT Trp Tyr His AAC ATC ACA Asn Ile Thr 145

CTO

Leu 130 TTC TAT CAG TAO Phe Tyr Gin Tyr OCA GAG TOT GCC Pro Giu Ser Ala ATC TGG GGG Ile Trp Gly 140 AAC TGG TTC Asn Trp Phe TGG GGC CAC TOO Trp Gly His Ser

GTA

Val 150 TCC AAA GAO ATG Ser Lys Asp Met

ATO

Ile 155 CAT CTC His Leu 160 CCC TTC GOC ATG Pro Phe Ala Met

GTO

Val1 165 COT GAC CAA TGG Pro Asp Gin Trp

TAO

Tyr 170 GAT ATO GAA GGT Asp Ile Glu Gly

GTC

Val 175 ATG ACC GGO TOO Met Thr Gly Ser ACC GTC CTC OCT Thr Val Leu Pro GGT CAG ATO ATC Gly Gin Ile Ile CTC TAO ACC GGO Leu Tyr Thr Gly

AAO

Asn 195 GOG TAO GAT CTC Ala Tyr Asp Leu

TOG

Ser 200 CAA OTG CAA TGO Gin Leu Gin Cys TTA GOA Leu Ala 205 625 TAT GCC GTC Tyr Ala Val GAG GGA AAT Glu Gly Asn 225

AAC

Asn 210 TOG TCT GAT COO Ser Ser Asp Pro

OTO

Leu 215 OTC CTO GAT TGG Leu Leu Asp Trp AAA AAG TAC Lys Lye Tyr 220 TAO AAG GAT Tyr Lys Asp 673 COO ATO TTG TTO Pro Ile Leu Phe

OCA

Pro 230 COT OCT GGG GTG Pro Pro Gly Val

GGA

Gly 235 TTT CGG Phe Arg 240

GAO

Asp CCA TOT ACA Pro Ser Thr TOG TTG GGT COO Trp Leu Gly Pro

GAT

Asp 250 GOT GAA TAO AGA Gly Giu Tyr Arg

ATG

Met 255 GTA ATG GGG TOO Val Met Gly Ser

AAG

Lys 260 OAT AAO GAG ACC His Asn Glu Thr

ATO

Ile 265 GOT TGT GOC TTG Gly Cys Ala Leu

ATT

Ile 270 817 TAO CAT ACC ACT Tyr His Thr Thr

AAT

Aen 275 TTT ACG CAT TTO Phe Thr His Phe CTO AAG GAA GAG Leu Lys Giu Glu GTG OTT Val Leu 285 865 CAC GCC GTT His Ala Val GTA TCC ACC Val Ser Thr 305

CCC

Pro 290 CAC ACG GGT ATG His Thr Gly Met

TGG

Trp 295 GAA TGT GTG GAT Giu Cys Val Asp CTT TAT CCG Leu Tyr Pro 300 AAC GGG CCG Asn Gly Pro 913 ACG CAC ACA AAC Thr His Thr Asn

GGG

Gly 310 TTG GAC ATG GTG Leu Asp Met Val

GAT

Asp 315 961 AAT GTG Asn Val 320 AAG CAT GTG TTG Lys His Val. Leu

AAA

Lys 325 CAA AGT GGG GAT Gin Ser Gly Asp GAT CGA CAT GAT Asp Arg His Asp

TGG

Trp 335 TAT GCG CTC GGG Tyr Ala Leu Gly

ACT

Thr 340 TAT GAC GTC GTG Tyr Asp Val Val

AAT

Asn 345 GAT AAG TGG TAT Asp Lys Trp Tyr

CCA

Pro 350 1009 1057 1105 GAT GAC CCT GAA Asp Asp Pro Giu

AAC

Asn 355 GAT GTG GGT ATC Asp Val Gly Ile TTA AGA TAC GAT Leu Arg Tyr Asp TTC GGA Phe Gly 365 AAG TTT TAT Lys Phe Tyr GTC CTT TGG Val Leu Trp 385 TCA AAG ACG TTC Ser Lys Thr Phe

TAC

Tyr 375 GAC CAA CAT AAG Asp Gin His Lys AAG AGA CGG Lys Arg Arg 380 TAC GAC GTT Tyr Asp Val 1153 1201 GGT TAC GTT GGA Gly Tyr Val Gly

GAA

Giu 390 ACC GAT CCC CCT Thr Asp Pro Pro

AAA

Lys 395 TAC AAG Tyr Lys 400 GGA TGG GCT AAC Gly Trp Ala Asn

ATT

Ile 405 TTG AAC ATT CCA Leu Asn Ile Pro ACC ATA GTT TTG Thr Ile Val Leu 1249 1297

GAC

Asp 415

GAA

Glu ACG AAA ACG AAT Thr Lys Thr Asn AAC TTG AGA TCG Asn Leu Arg Ser 435

ACC

Thr 420 AAT TTG ATT CAA Asn Leu Ile Gin

TGG

Trp 425 CCA ATT GCG GAA Pro Ilie Ala Glu

GTC

Val 430 AAT AAA TAC AAT Asn Lys Tyr Asn

GAA

Glu 440 TTC AAA GAC GTG Phe Lys Asp Val GAG CTG Giu Leu 445 1345 AAA CCG GGA Lys Pro Gly GAT ATA ACT Asp Ile Thr 465

TCA

Ser 450 CTG ATT CCG CTC Leu Ile Pro Leu

GAG

G lu 455 ATA GGC ACA GCA Ile Gly Thr Ala ACA CAG TTG Thr Gin Leu 460 GAA TCG ACG Glu Ser Thr 1393 1441 GCG ACA TTC GAA Aia Thr Phe Glu GTT GAT Val Asp 470 CAA ACG ATG Gin Thr Met

TTG

Leu 475 CTT GAA Leu Giu 480 GCC GAT GTT TTG Ala Asp Val Leu

TTC

Phe 485 AAT TGT ACG ACC Asn Cys Thr Thr

AGT

Ser 490 GAA GGT TCA GCC Giu Gly Ser Ala 1489 1537

GGG

G ly 495 AGA GGG GTG TTG Arg Gly Val. Leu

GGG

Gly 500 CCA TTT GGA CTG Pro Phe Gly Leu GTT CTA GCT GAT Val Leu Ala Asp

GCC

Ala 510 GAA CGA TCT GAG Glu Arg Ser Glu

CAA

Gin 515 CTT CCT GTG TAT Leu Pro Val Tyr

TTC

Phe 520 TAT ATA GCA AAA Tyr Ile Ala Lys GAC ACC Asp Thr 525 1585 GAT GGA TCC Asp Gly Ser AAC GAT GTA Asn Asp Val 545

TCA

Ser 530 AAA ACT TAC TTC Lys Thr Tyr Phe

TGT

Cys 535 GCC GAT GAA TCA Ala Asp Glu Ser AGA TCA TCG Arg Ser Ser 540 GTT CCT GTT Val Pro Val 1633 1681 GAC ATA GGG AAA Asp Ile Gly Lys

TGG

Trp 550 GTG TAC GGA AGC Val Tyr Gly Ser CTA GAA Leu Glu 560 GGC GAA AAA TTC Gly Glu Lys Phe

AAC

Aen 565 ATG AGG TTG CTG Met Arg Leu Leu

GTG

Val 570 GAT CAT TCA ATT Asp His Ser Ile

GTC

Val 575 GAA GGC TTC GCA Glu Gly Phe Ala

CAA

Gin 580 GGA GGC AGA ACG Gly Gly Arg Thr

GTG

Val 585 GTG ACA TCA AGA Val Thr Ser Arg 1729 1777 1825 TAT CCG GCG AAG Tyr Pro Ala Lys

GCG

Ala 595 ATC TAC GGC GCT Ile Tyr Gly Ala

GCA

Ala 600 AAG TTA TTT TTG Lys Leu Phe Leu TTC AAC Phe Asn 605 AAC GCC ACC Asn Ala Thr AAG GAA GCA Lys Glu Ala 625

GGA

Gly 610 ATC AGC GTG AAG Ile Ser Val Lys

GCA

Ala 615 TCT CTC AAG ATC Ser Leu Lys Ile TGG AAA ATG Trp Lys Met 620 1873 CAA CTG GAT CCA Gin Leu Asp Pro

TTC

Phe 630 CCT CTT TCT GGA Pro Leu Ser Gly TGG AGT TCT Trp Ser Ser 635 1918 TGATGATGAT GATGATTAAG AACTCATTTC ATGAAGATGA TGATTAAGAA CTCATTTCAT GATGATGATG ATGATTCCAG TTTATATGCG TACCCTGTTC CCTTTACCTG TATGTGGTGG TGGTGGTGAA ATATGGTTAG CATGATTCCG GGTTGGCGAG GGCAATATGG TAATTTACTA TCGCTGTAGT AGTACTCCAC TTGTGAGATT ATATTTCATA AATTCAATTA TTATTCCTGT TTACAACCTT TTTCATTGTA TCATACCACC CATTGAATCC CATCATGTTC AATTAGTGTT

GCAAAAAA

INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH:: 637 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Ala Ser Ser Thr Thr Thr Pro Leu Leu Pro His His His Leu Gin 1 5 10 1978 2038 2098 2158 2218 2226 23 Asn Pro Gin Gin Leu Ala Gly Ser Pro Ala Ala His Arg Leu Ser Arg 25 Pro Thr Leu Leu Ser Gly Ile Leu Val Ser Val Leu Val Ile Cys Ala 40 Leu Val Ala Val Ile His Asn Gin Ser Gin Gin Pro Tyr His Asp Gly 55 Gly Ala Lys Pro Ser Ser Ser Ala Ala Thr Thr Thr Phe Pro Thr Ala 70 75 Ser Pro Glu Ala Gly Leu Lys Arg Phe Pro Ile Glu Leu Lys Thr Asn 90 Ala Glu Val Glu Trp Gln Arg Ser Ala Tyr His Phe Gin Pro Asp Lys 100 105 110 Asn Tyr Ile Ser Asp Pro Asp Gly Pro Met Tyr His Met Gly Trp Tyr 115 120 125 His Leu Phe Tyr Gln Tyr Asn Pro Glu Ser Ala Ile Trp Gly Asn Ile 130 135 140 Thr Trp Gly His Ser Val Ser Lys Asp Met Ile Asn Trp Phe His Leu 145 150 155 160 Pro Phe Ala Met Val Pro Asp Gin Trp Tyr Asp Ile Glu Gly Val Met 165 170 175 Thr Gly Ser Ala Thr Val Leu Pro Asp Gly Gin Ile Ile Met Leu Tyr 180 185 190 Thr Gly Asn Ala Tyr Asp Leu Ser Gin Leu Gin Cys Leu Ala Tyr Ala 195 200 205 Val Asn Ser Ser Asp Pro Leu Leu Leu Asp Trp Lys Lys Tyr Glu Gly 210 215 220 Asn Pro Ile Leu Phe Pro Pro Pro Gly Val Gly Tyr Lys Asp Phe Arg 225 230 235 240 Asp Pro Ser Thr Leu Trp Leu Gly Pro Asp Gly Glu Tyr Arg Met Val 245 250 255 Met Gly Ser Lys His Asn Glu Thr Ile Gly Cys Ala Leu Ile Tyr His 260 265 270 Thr Thr Asn Phe Thr His Phe Glu Leu Lys Glu Glu Val Leu His Ala 275 280 285 Val Pro His Thr Gly Met Trp Glu Cys Val Asp Leu Tyr Pro Val Ser 290 295 300 Thr Thr His Thr Asn Gly Leu Asp Met Val Asp Asn Gly Pro Asn Val 305 310 315 320 Lys His Val Leu Lys Gin Ser Gly Asp Glu Asp Arg His Asp Trp Tyr 325 330 335 24 Ala Leu Gly Thr Tyr Asp Val Val Asn Asp Lys Trp Tyr Pro Asp Asp Pro Tyr Trp 385 Gly Lys Leu Gly Thr 465 Ala Gly Ser Ser Val 545 Gly Gly Ala Thr Glu Ala 370 Gly Trp Thr Arg Ser 450 Ala Asp Val Glu Ser 530 Asp Glu Phe Lys Gly 610 Aen 355 Ser Tyr Ala Asn Ser 435 Leu Thr Val Leu Gin 515 Lys Ile Lys Ala Ala 595 Ile 340 Asp Lys Val Asn Thr 420 Asn Ile Phe Leu Gly 500 Leu Thr Giy Phe Gin 580 Ile Ser Val Thr Gly Ile 405 Asn Lys Pro Giu Phe 485 Pro Pro Tyr Lys Asn 565 Gly Tyr Val Gly Phe Glu 390 Leu Leu Tyr Leu Val 470 Asn Phe Val Phe Trp 550 Met Gly Giy Lys Ile Tyr 375 Thr Asn Ile Asn Giu 455 Asp cy s Gly Tyr cys 535 Val Arg Arg Ala Ala 615 Gly 360 Asp Asp Ile Gin Giu 440 Ile Gin Thr Leu Phe 520 Al a Tyr Leu Thr Ala 600 Ser 345 Leu Gin Pro Pro Trp 425 Phe Gly Thr Thr Val1 505 Tyr Asp Gly Leu Val 585 Lys Leu Arg His Pro Arg 410 Pro Lye Thr Met Ser 490 Val1 Ile Giu Ser Val1 570 Val1 Leu Lys Tyr Lys Lys 395 Thr Ile Asp Ala Leu 475 Giu Leu Ala Ser Ser 555 Asp Thr Phe Ile Asp Lys 380 Tyr Ile Ala Val1 Thr 460 Giu Gly Ala Lys Arg 540 Val1 His Ser Leu Trp 620 Phe 365 Arg Asp Val Giu Glu 445 Gin Ser Ser Asp Asp 525 Ser Pro Ser Arg Phe 605 Lys 350 Gly Arg Val Leu Val 430 Leu Leu Thr Ala Ala 510 Thr Ser Val Ile Val 590 Asn Met Lys Val Tyr Asp 415 Giu Lys Asp Leu Gly 495 Giu Asp Asn Leu Val 575 Tyr Asn Lys Phe Leu Lys 400 Thr Asn Pro Ile Giu 480 Arg Arg Gly Asp Giu 560 Giu Pro Ala Glu Ala Gin Leu Asp Pro Phe Pro Leu Ser Gly Trp Ser Ser 630 635 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 1911 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic DNA" (ix) FEATURE: NAME/KEY: CDS LOCATION:1..1911 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATG GCA AGC Met Ala Ser 640 TCT ACG ACT ACA Ser Thr Thr Thr TTG TTA CCG CAC Leu Leu Pro His

CAC

His 650 CAT TTG CAG His Leu Gin AAT CCT Asn Pro 655 CAG CAG TTG GCT Gin Gin Leu Ala

GGA

Gly 660 AGT CCA GCT GCA Ser Pro Ala Ala

CAC

His 665 AGG TTG AGT CGT Arg Leu Ser Arg

CCT

Pro 670 ACT CTT TTG AGT Thr Leu Leu Ser

GGT

Gly 675 ATA TTG GTA AGT Ile Leu Val Ser

GTA

Val 680 CTG GTC ATC TGC Leu Val Ile Cys

GCA

Ala 685 144 TTG GTC GCA GTT Leu Val Ala Val

ATA

Ile 690 CAT AAT CAG TCT His Asn Gin Ser

CAA

Gin 695 CAG CCA TAC CAT Gin Pro Tyr His GAT GGT Asp Gly 700 192 GGT GCC AAG Gly Ala Lys AGC CCT GAA Ser Pro Glu 720

CCT

Pro 705 AGC TCT AGC GCT Ser Ser Ser Ala

GCC

Ala 710 ACG ACT ACT TTT Thr Thr Thr Phe CCT ACA GCC Pro Thr Ala 715 AAG ACC AAC Lys Thr Asn 240 GCA GGA TTG AAA Ala Gly Leu Lys TTC CCT ATC GAA Phe Pro Ile Glu

CTC

Leu 730 288 GCA GAA Ala Glu 735 GTC GAG TGG CAG Val Glu Trp Gin

AGA

Arg 740 AGT GCA TAC CAC Ser Ala Tyr His

TTC

Phe 745 CAG CCA GAT AAG Gin Pro Asp Lys 336

AAC

Asn 750 TAT ATC TCA GAC Tyr Ile Ser Asp

CCA

Pro 755 GAC GGG CCT ATG Asp Gly Pro Met CAT ATG GGT TGG His Met Gly Trp

TAC

Tyr 765 384 CAC TTA TTC TAC His Leu Phe Tyr

CAA

Gin 770 TAT AAT CCA GAG Tyr Asn Pro Glu

AGT

Ser 775 GCA ATA TGG GGA Ala Ile Trp Gly AAT ATA Asn Ile 780 432 ACT TGG GGT Thr Trp Gly

CAT

His 785 AGC GTT AGC AAG Ser Val Ser Lys

GAT

Asp 790 ATG ATT AAT TGG Met Ile Asn Trp TTT CAC TTG Phe His Leu 795 480 CCA TTT GCG ATG GTC CCA GAT CAA TGG TAT GAT ATT GAG GGC GTT ATG 528 Pro Phe Ala 800 Met Val Pro Asp Gin 805 Trp Tyr Asp Ile Gly Val Met ACT GGA Thr Gly 815 AGC GCA ACT GTT Ser Ala Thr Val

TTG

Leu 820 CCA GAC GGA CAG Pro Asp Gly Gin

ATC

Ile 825 ATT ATG TTG TAT Ile Met Leu Tyr

ACC

Thr 830 GGT AAT GCA TAC Gly Asn Ala Tyr

GAC

Asp 835 TTG AGT CAG TTG Leu Ser Gin Leu

CAG

Gin 840 TGT CTC GCC TAT Cys Leu Ala Tyr

GCC

Ala 845 GTT AAT AGC AGC Val Asn Ser Ser

GAC

Asp 850 CCC TTG TTG CTC Pro Leu Leu Leu

GAT

Asp 855 TGG AAG AAG TAC Trp Lys Lys Tyr GAG GGC Glu Gly 860 AAT CCG ATT Asn Pro Ile GAT CCC AGT Asp Pro Ser 880

CTC

Leu 865 TTT CCG CCT CCT Phe Pro Pro Pro

GGC

Gly 870 GTC GGA TAT AAA Val Gly Tyr Lys GAT TTC AGA Asp Phe Arg 875 CGT ATG GTC Arg Met Val ACT CTC TGG CTC Thr Leu Trp Leu

GGT

Gly 885 CCA GAC GGA GAG Pro Asp Gly Glu

TAC

Tyr 890 ATG GGC Met Gly 895 AGC AAA CAC AAT Ser Lys His Asn

GAA

Glu 900 ACA ATC GGG TGC Thr Ile Gly Cys

GCA

Ala 905 CTC ATC TAT CAC Leu Ile Tyr His 816

ACG

Thr 910 ACA AAC TTC ACG Thr Asn Phe Thr

CAC

His 915 TTC GAG CTC AAG Phe Glu Leu Lys

GAA

Glu 920 GAA GTC TTA CAC Glu Val Leu His

GCT

Ala 925 864 GTT CCT CAC ACA Val Pro His Thr

GGA

Gly 930 ATG TGG GAG TGC Met Trp Glu Cys

GTC

Val 935 GAC TTA TAT CCC Asp Leu Tyr Pro GTC AGT Val Ser 940 912 ACT ACT CAT Thr Thr His AAA CAT GTC Lys His Val 960

ACG

Thr 945 AAT GGC TTG GAT Asn Gly Leu Asp

ATG

Met 950 GTC GAC AAT GGT Val Asp Asn Gly CCC AAC GTC Pro Asn Val 955 GAC TGG TAC Asp Trp Tyr 960 CTC AAG CAG TCC Leu Lys Gin Ser

GGC

Gly 965 GAC GAG GAC AGG Asp Glu Asp Arg

CAC

His 970 1008 GCT TTA Ala Leu 975 GGT ACA TAT GAC Gly Thr Tyr Asp

GTC

Val 980 GTC AAC GAC AAA Val Asn Asp Lys

TGG

Trp 985 TAT CCC GAC GAT Tyr Pro Asp Asp 1056 1104 CCC Pro 990 GAG AAC GAC GTC Glu Asn Asp Val

GGA

Gly 995 ATT GGC CTT CGT Ile Gly Leu Arg TAC GAC TTC GGC AAG Tyr Asp Phe Gly Lys 1000

TTC

Phe 1005 TAC GCC AGT AAA Tyr Ala Ser Lys ACA TTC TAC GAT CAG Thr Phe Tyr Asp Gin 1010 CAC AAA AAA His Lys Lys 1015 CCC AAA TAC Pro Lys Tyr CGT CGT GTT TTA Arg Arg Val Leu 1020 GAT GTC TAC AAA Asp Val Tyr Lys 1035 1152 1200 TGG GGA TAC Trp Gly Tyr GTC GGC GAG ACG GAC Val Gly Glu Thr Asp 1025

CCG

Pro 1030 GGT TGG GCA AAT ATC CTC AAC ATA CCT CGC ACT ATT GTC CTC GAT ACG 1248 Gly Trp Ala Asn Ile Leu Asn Ile Pro Arg Thr Ile Val Leu Asp Thr 1040 1045 1050 AAG ACA AAC Lys Thr Asn 1055 ACG AAC CTC ATA CAG Thr Asn Leu Ile Gin 1060 TGG CCT ATT GCC GAG Trp Pro Ile Ala Glu 1065 GTG GAG AAT Val Glu Asn 1296 TTA CGT Leu Arg 1070 AGC AAC AAA Ser Asn Lys TAC AAC Tyr Asn 1075 GAG TTC AAG Glu Phe Lys GAT GTG Asp Val 1080 GAA TTG AAG Glu Leu Lys

CCT

Pro 1085 1344 1392 GGA AGT TTG ATT Gly Ser Leu Ile CCG TTA Pro Leu 1090 GAA ATC GGT Glu Ile Gly ACT GCT Thr Ala 1095 ACT CAA CTC Thr Gin Leu GAC ATC Asp Ile 1100 ACC GCT ACT Thr Ala Thr TTT GAG GTC Phe Glu Val 1105 GAT CAG ACC ATG CTC Asp Gln Thr Met Leu 1110 GAG AGT ACC TTA GAA Glu Ser Thr Leu Glu 1115 1440 GCG GAC GTA TTA TTT AAC TGT ACC ACA TCC GAG GGG AGC GCA GGT CGC Ala Asp Val Leu Phe Asn Cys Thr Thr Ser Glu Gly Ser Ala Gly Arg 1120 1125 1130 GGA GTC CTT GGT CCA TTC GGA CTT GTC GTC TTA GCG GAC GCA GAA AGA Gly Val Leu Gly Pro Phe Gly Leu Val Val Leu Ala Asp Ala Glu Arg 1135 1140 1145 1488 1536 AGC GAG Ser Glu 1150 CAG TTG CCC GTC TAT Gin Leu Pro Val Tyr 1155 TTT TAC ATT GCC AAG Phe Tyr Ile Ala Lys 1160 GAC ACC GAC GGT Asp Thr Asp Gly 1165 1584 TCC AGC AAG ACA Ser Ser Lys Thr TAC TTC Tyr Phe 1170 TGC GCA GAT GAG TCC CGC AGC AGC Cys Ala Asp Glu Ser Arg Ser Ser 1175 AAC GAC Asn Asp 1180 1632 GTC GAT ATC Val Asp Ile GGC AAG TGG Gly Lys Trp 1185 GTC TAT GGT TCG TCA Val Tyr Gly Ser Ser 1190 GTC CCA GTG TTG GAG Val Pro Val Leu Glu 1195 1680 GGA GAG AAA TTT AAC ATG CGC CTG CTT GTC GAC CAC AGC ATC GTC GAA Gly Glu Lys Phe Asn Met Arg Leu Leu Val Asp His Ser Ile Val Glu 1200 1205 1210 GGC TTC GCT CAG GGT GGC CGT ACT GTC GTA ACC AGT CGT GTC TAC CCT Gly Phe Ala Gln Gly Gly Arg Thr Val Val Thr Ser Arg Val Tyr Pro 1215 1220 1225 GCT AAA GCC ATA TAT GGG GCA GCC AAA CTC TTC CTC TTT AAT AAT GCC Ala Lys Ala Ile Tyr Gly Ala Ala Lys Leu Phe Leu Phe Asn Asn Ala 1230 1235 1240 1245 ACA GGC ATA TCA GTC AAA GCC AGC TTA AAA ATT TGG AAA ATG AAA GAG Thr Gly Ile Ser Val Lys Ala Ser Leu Lys Ile Trp Lys Met Lys Glu 1250 1255 1260 1728 1776 1824 1872 28 GCT CAG TTG GAC CCG TTT CCA TTA AGC GGC TGG TCT AGC 1911 Ala Gin Leu Asp Pro Phe Pro Leu Ser Gly Trp Ser Ser 1265 1270 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 637 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Ala Ser Ser Thr Thr Thr Pro Leu Leu Pro His His His Leu Gin 1 5 10 Asn Pro Gin Gln Leu Ala Gly Ser Pro Ala Ala His Arg Leu Ser Arg 25 Pro Thr Leu Leu Ser Gly Ile Leu Val Ser Val Leu Val Ile Cys Ala 40 Leu Val Ala Val Ile His Asn Gin Ser Gin Gin Pro Tyr His Asp Gly 55 Gly Ala Lys Pro Ser Ser Ser Ala Ala Thr Thr Thr Phe Pro Thr Ala 70 75 Ser Pro Glu Ala Gly Leu Lys Arg Phe Pro Ile Glu Leu Lys Thr Asn 90 Ala Glu Val Glu Trp Gln Arg Ser Ala Tyr His Phe Gin Pro Asp Lys 100 105 110 Asn Tyr Ile Ser Asp Pro Asp Gly Pro Met Tyr His Met Gly Trp Tyr 115 120 125 His Leu Phe Tyr Gin Tyr Asn Pro Glu Ser Ala Ile Trp Gly Asn Ile 130 135 140 Thr Trp Gly His Ser Val Ser Lys Asp Met Ile Asn Trp Phe His Leu 145 150 155 160 Pro Phe Ala Met Val Pro Asp Gin Trp Tyr Asp Ile Glu Gly Val Met 165 170 175 Thr Gly Set Ala Thr Val Leu Pro Asp Gly Gin Ile Ile Met Leu Tyr 180 185 190 Thr Gly Asn Ala Tyr Asp Leu Ser Gin Leu Gln Cys Leu Ala Tyr Ala 195 200 205 Val Asn Ser Ser Asp Pro Leu Leu Leu Asp Trp Lys Lys Tyr Glu Gly 210 215 220 29 Aen Pro Ile Leu Phe Pro Pro Pro Gly Val Gly Tyr Lys Asp Phe Arg 225 230 235 240 Asp Pro Met Gly Thr Thr Val Pro 290 Thr Thr 305 Lys His Ala Leu Pro Glu Tyr Ala 370 Trp Gly 385 Gly Trp Lys Thr Leu Arg Gly Ser 450 Thr Ala 465 Ala Asp Gly Val Ser Glu Ser Thr Ser Lys 260 Aen Phe 275 His Thr His Thr Val Leu Gly Thr 340 Asn Asp 355 Ser Lys Tyr Val Ala Asn Asn Thr 420 Ser Asn 435 Leu Ile Thr Phe Val Leu Leu Gly 500 Gin Leu 515 Leu 245 His Thr Gly Aen Lye 325 Tyr Val1 Thr Gly Ile 405 Asn Lys Pro Giu Phe 485 Pro Pro Trp Leu Aen Glu His Phe Met Trp 295 Gly Leu 310 Gin Ser Asp Val Gly Ile Phe Tyr 375 Giu Thr 390 Leu Asn Leu Ile Tyr Asn Leu Glu 455 Vai Asp 470 Asn Cys Phe Gly Val Tyr Gly Thr Giu 280 Giu Asp Gly Val Gly 360 Asp Asp Ile Gin Giu 440 Ile Gin Thr Leu Phe 520 Pro Ile 265 Leu Cys Met Asp Asn 345 Leu Gin Pro Pro Trp 425 Phe Giy Thr Thr Val1 505 Tyr Asp 250 Gly Lye Vai Vai Giu 330 Asp Arg His Pro Arg 410 Pro Lye Thr Met Ser 490 Vali Ile Gly Cys G iu Asp Asp 315 Asp Lye Tyr Lye Lys 395 Thr Ile Asp Aia Leu 475 Giu Leu Ala Giu Ala G iu Leu 300 Asn Arg Trp Asp Lye 380 Tyr Ile Ala Val Thr 460 Giu Gly Ala Lys Tyr Leu Vali 285 Tyr Gly His Tyr Phe 365 Arg Asp Val Giu Giu 445 Gin Ser Ser Asp Asp 525 Arg Ile 270 Leu Pro Pro Asp Pro 350 G ly Arg Val Leu Val 430 Leu Leu Thr Ala Ala 510 Thr Met 255 Tyr His Val Asn Trp 335 Asp Lye Val1 Tyr Asp 415 Giu Lye Asp Leu Gly 495 Giu Asp Val His Ala Ser Val 320 Tyr Asp Phe Leu Lys 400 Thr Asn Pro Ile Giu 480 Arg Arg G ly Ser Ser Lye Thr Tyr Phe Cys Ala Asp Giu Ser Arg Ser Ser Asn Asp 530 535 540 Val 545 Gly Gly Ala Thr Ala 625 Asp Glu Phe Lys Gly 610 Gin Ile Lys Ala Ala 595 Ile Leu Giy Phe Gin 580 Ile Ser Asp Lys Asn 565 Giy Tyr Val Pro Trp 550 Met Gly Gly Lye Phe 630 Vai Arg Arg Ala Ala 615 Pro Tyr Leu Thr Ala 600 Ser Leu Ser Ser 555 Vai Asp 570 Val Thr Leu Phe Lys Ile Gly Trp 635 Val His Ser Leu Trp 620 Ser Pro Ser Arg Phe 605 Lys Ser Leu Val 575 Tyr Asn Lys Giu 560 Giu Pro Ala Glu

Claims (21)

1. A nucleic acid molecule encoding a sucrose dependent sucrose fructosyltransferase (SST), selected from the group consisting of: a nucleic acid molecule encoding a protein comprising the amino acid sequence of SEQ ID No: 2 and SEQ ID No: 4; a nucleic acid molecule comprising the nucleotide sequence of SEQ ID No: 1 or a corresponding ribonucleotide sequence; a nucleic acid molecule comprising the nucleotide sequence of SEQ ID No: 3 or a corresponding ribonucleotide sequence; a nucleic acid molecule encoding a protein having a sequence identity of at least 85% when compared to the amino acid sequence of SEQ ID No: 2 or SEQ ID No: 4; a nucleic acid molecule having a sequence identity of at least 60% when compared with the nucleotide sequence of SEQ ID No: 3; and a nucleic acid molecule comprising a fragment of a nucleic acid molecule mentioned in to said fragment encoding a protein that catalyzes the linking of P-2, 1-glycosidic or 3-2, 6- glycosidic bonds between fructose units.

2. A nucleic acid molecule according to claim 1, which is a DNA molecule.

3. A nucleic acid molecule according to claim 2, wherein the DNA molecule is a cDNA molecule.

4. A nucleic acid molecule according to claim 1, which is 35 an RNA molecule. 0: 0 5. A vector comprising a nucleic acid molecule according S to any one of claims 1 to 4. Sclam 27 Kirstie/keep/retype/P46049 claims 20/07/04 32

6. A vector according to claim 5, wherein the nucleic acid molecule is operatively linked to regulatory elements allowing the transcription and synthesis of a translatable RNA in prokaryotic and/or eukaryotic cells.

7. A vector according to claim 6, wherein the regulatory elements are derived from the patatin B33 promoter.

8. A host cell transformed with a nucleic acid molecule according to any one of claims 1 to 4 or a vector according to claim 6 or claim 7.

9. A cell derived from a host cell according to claim 8, wherein said cell comprises a nucleic acid molecule according to any one of claims 1 to 4 or a vector according to claim 6 or claim 7. A method for the production of an SST protein, comprising the step of cultivation of a host cell according to claim 8 under conditions allowing the synthesis of the SST and isolating said SST from the cultivated cells and/or the culture medium.

11. An SST encoded by a nucleic acid molecule according to any one of claims 1 to 4 or produced by a method according to claim

12. A transgenic plant cell transformed with a nucleic acid 30 molecule according to any one of claims 1 to 4 or a vector according to claim 6 or claim 7, wherein the nucleic acid molecule encoding an SST from artichoke is controlled by regulatory elements allowing the transcription of a translatable mRNA in plant cells.

13. A cell derived from a transgenic plant cell according to claim 12, wherein said cell comprises a nucleic acid molecule according to any one of claims 1 to 4 or a vector Kirstie/keep/retype/P46049 claims 20/07/04 33 according to claim 6 or claim 7.

14. A plant containing a cell according to claim 12 or claim 13. A plant according to claim 14, which is a useful plant. A plant according to claim 14, which is a useful plant.or

16. A plant according to claim 15, which is a sucrose or starch-storing plant.

17. A plant according to claim 16, which is a potato plant.

18. Propagation material of a plant according to any one of claims 14 to 17, containing a plant cell according to claim 12 or claim 13.

19. A harvest product of a plant according to any one of claims 14 to 17, containing a plant cell according to claim 12 or claim 13. A method for the production of short-chain fructosyl polymers comprising the steps of: cultivating a host cell according to claim 8 or a plant cell according to claim 12 or claim 13 under conditions allowing the production of an SST protein and conversion of, if necessary, externally added sucrose or of an equivalent substrate to short-chain fructosyl polymers; and e 30 obtaining the fructosyl polymers produced ~this way from the cultivated cells or from the medium.

21. A method for the production of short-chain fructosyl 35 polymers comprising the steps of: contacting sucrose or an equivalent substrate with an SST protein according to claim 11 under conditions allowing the conversion to .c Kirstie/keep/retype/P46049 claims 20/07/04 34 short-chain fructosyl polymers; and obtaining the fructosyl polymers so produced.

22. A method for the production of short-chain fructosyl polymers comprising the steps of: cultivating a plant according to any one of claims 14 to 17; and obtaining the fructosyl polymers from these plants or their propagation material according to claim 14 or the harvest products according to claim 19.

23. A nucleic acid molecule according to claim 1, substantially as hereinbefore described with reference to any one of the examples.

24. A vector according to claim 5, substantially as hereinbefore described with reference to any one of the examples. A method according to any one of claims 9 or 20 to 22 substantially as hereinbefore described with reference to any one of the examples.

26. A transgenic plant cell according to claim 12, substantially as hereinbefore described with reference to any one of the examples. 30 Dated this 20th day of July 2004 MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN V. By their Patent Attorneys GRIFFITH HACK 35 Fellows Institute of Patent and Trade Mark Attorneys of Australia *o *o* o o Kirstie/keep/retype/P46049 claims 20/07/04