ZA200102854B - Nucleic acid molecules which code a branching enzyme from bacteria of the genus neisseria, and a method for producing alpha-1,6-branched alpha-1,4-glucans. - Google Patents

Description

de ’

PCT application PCT/EP99/07562

PlantTec Biotechnology GmbH et al.

Our Ref.: C 1434 PCT

English translation of the PCT application PCT/EP99/07562

Nucleic acid molecules encoding a branching enzyme from bacteria of the genus Neisseria as well as methods for the production of a-1,6-branched a-1,4-glucans

The present invention relates to nucleic acid molecules encoding a branching enzyme from bacteria of the genus Neisseria, vectors, host cells, plant cells and plants containing such nucleic acid molecules as well as starch obtainable from the plants described.

Furthermore, the present invention relates to in-vitro methods for the production of a-1,6- : branched a-1,4-glucans on the basis of sucrose and a combination of enzymes of an amylosucrase and a branching enzyme. Moreover, the invention relates to glucans that are obtainable by the method described.

In many respects, a-1,6-branched a-1,4-glucans are of enormous interest since they are suitable, for instance, as regards the production of products in the pharmaceutical and cosmetic industry. They can be used, e.g. as binding agent for tablets, as carrier substances for pharmaceutical agents, as packaging material, as carrier substance for powder additives, as UV-absorbing additive in sun creme and as carrier substance of flavourings and scents.

In plants, a-1,6-branched «-1,4-glucans can mainly be found as amylopectin, a component of starch. In animals and in bacteria, glucans mainly occur in form of glycogen.

The polysaccharide starch is formed of chemically uniform basic building blocks, i.e. the glucose molecules, it is, however, a complex mixture of different forms of molecules which differ with regard to the degree of polymerization and branching and which, thus, differ strongly in their physico-chemical properties. It has to be differentiated between amylose starch, which is an essentially non-branched polymer i

P noe of a-1,4-glycosidically linked glucose units, and the amylopectin starch, which is a branched polymer in which the branchings are formed due to the presence of additional «-1,6-glycosidical linkings. According to textbooks (Voet and Voet,

Biochemistry, John Wiley & Sons, 1990), the a-1,6-branchings occur after every 24 to 30 glucose residues on average, which corresponds to a branching degree of approximately 3% to 4%. The indications as to the branching degree vary and depend on the origin of the respective starch (e.g. plant species, plant variety). In plants that are typically used for the industrial production of starch the share of amylose in the overall share of starch varies between 10% and 25%. Various approaches for the production of «-1,6-branched a-1,4-glucans with different branching degrees have already been described, with these approaches comprising the use of (transgenic) plants.

The heterologous expression of a bacterial glycogen synthase in potato plants, for instance, leads to a slight decrease of the amylose content, to an increase in the branching degree and to a modification of the branching pattern of the amylopectin when compared to wild type plants (Shewmaker et al., Plant. Physiol. 104 (1994), 1159-1166). Furthermore, it was observed that the heterologous expression of the branching enzyme from E. coli (9igB) in amylose-free potato mutants (amf) (Jacobsen et al., Euphytica 44 (1989), 43-48) leads to amylopectin molecules which have 25% more branching points (Kortstee et al., Plant J. 10 (1996), 83-90) than the control (amf). For isolating the glucans with different branching degrees, which were produced in transgenic plants, it is necessary to carry out additional purification steps in order to remove, for example, the amylose component. These purification steps are laborious and, therefore, time-consuming and cost-intensive. Furthermore, it is not possible to achieve a particular branching degree by means of these approaches.

What is more, due to varying experimental conditions (environmental factors, location), such in-vivo methods vary considerably with regard to the quality of the product. i

Glycogen has a higher branching degree than the amylopectin. This polysaccharide, too, contains «-1,6-branched a-1,4-glucans. Glycogen also differs from starch in the average length of the side-chains and in the degree of polymerization. According to textbooks (Voet and Voet, Biochemistry, John Wiley & Sons, 1990), glycogen contains, on average, an q-1 ,8-branching point after every 8 to 12 glucose residues.

This corresponds to a branching degree of approximately 8% to 12%. There are varying indications as to the molecular weight of glycogen, which range from 1 million to more than 1000 millions (D. J. Manners in: Advances in Carbohydrate Chemistry,

Ed. M. L. Wolfrom, Academic Press, New York (1957), 261-298: Geddes et al.,

Carbohydr. Res. 261 (1994), 79-89). These indications, too, strongly depend on the respective organism of origin, its state of nutrition and the kind of isolation of the

“

Ww glycogen. Glycogen is usually recovered from mussels (e.g. Mytillus edulis), from mammalian liver or muscles (e.g. rabbit, rat) (Bell et al., Biochem. J. 28 (1934), 882;

Bueding and Orrell, J. Biol. Chem. 236 (1961), 2854). This renders the production on an industrial scale very time-consuming and cost-intensive.

The naturally-occurring «-1,6-branched a-1,4-glucans described, starch and glycogen, are very different depending on their content of 1,6-glycosidic branchings.

This holds true, amongst others, with regard to solubility, transparency, enzymatic hydrolysis, rheology, gel formation and retrogradation properties. For many industrial applications, such variations in the properties, however, cannot always be tolerated.

In-vitro approaches are an alternative to the recovery of a-1,6-branched o-14- glucans from plants or animal organisms. Compared to in-vivo methods, in-vitro methods are generally better to control and are reproducible to a greater extent since the reaction conditions in vitro can be exactly adjusted in comparison with the conditions in a living organism. This usually allows the production of invariable products with a high degree of uniformity and purity and, thus, of high quality, which is very important for any further industrial application. The preparation of products of a steady quality leads to a reduction of costs since the procedural parameter that are necessary for the preparation do not have to be optimised for every preparation set- up. Another advantage of certain in-vitro methods is the fact that the products are free of the organisms used in the in-vivo method. This is absolutely necessary for particular applications in the food and pharmaceutical industries.

In general, in-vitro methods can be divided into two different groups.

In the first group of methods, various substrates, such as amylose, amylopectin and glycogen, are subjected to the activity of a branching enzyme.

Borovsky et al. (Eur. J. Biochem. 59 (1975), 615-625) were able to prove that using the branching enzyme from potato in connection with the substrate amylose leads to products that are similar to amylopectin, but that differ from it in their structure.

Boyer and Preiss (Biochemistry 16 (1977), 3693-3699) showed, in addition, that a purified branching enzyme (a-1,4-glucan: «-1,4-glucan 6-glycosyltransferase) from

E. coli may be used to increase the branching degree of amylose or amylopectin.

If, however, glycogen from E. coli or rabbit liver is incubated with the branching enzyme from E. coli, only a slight increase in the branching degree can be achieved (Boyer and Preiss, loc. cit.).

Rumbak et al. (J. Bacteriol. 173 (1991), 6732-6741), too, could subsequently increase the branching degree of amylose, amylopectin and glycogen by incubating these substrates with the branching enzyme from Butyrivibrio fibrisolvens.

Okada et al. made a similar approach (patent no. US 4454161) to improve the properties of starch-containing foodstuffs. They incubated substances, such as amylose, amylopectin, starch or dextrin with a branching enzyme. This had

’ advantageous effects on the durability of foodstuffs containing substances that were modified correspondingly. Furthermore, the patent application EP-A1 0 690 170 describes the reaction of jellied starch in an aqueous solution using a branching enzyme. This results in starches having advantageous properties in the production of paper.

However, the aforementioned in-vitro methods have the disadvantage that they, due to the varying branching degree of the educts (e.g. starch, amylopectin, etc.), make it impossible to produce uniform products. In addition, it is not possible to intentionally control the branching degree and, what is more, the substrates used are quite expensive.

The other group of in-vitro methods comprises the de-novo synthesis of o-1,6- branched o-1 .4-glucans starting from various Substrates (glucose-1-phosphate, ADP glucose, UDP glucose) using a combination of enzymes that consists of a 1,4-glucan- chain-forming enzyme (phosphorylase, starch synthase, glycogen synthase) and a branching enzyme. llingwort et al. (Proc. Nat. Acad. Sci. USA 47 (1961), 469-478) were able to show for an in-vitro method using a phosphorylase A from muscles (organism unknown) in combination with a branching enzyme (organism unknown) that the de-novo synthesis of molecules similar to glycogen using the substrate glucose-1-phosphate was possible. Boyer and Preiss (loc. cit.) combined the enzymatic activity of a phosphorylase from rabbit muscles or a glycogen synthase from E. coli with the activity of a branching enzyme from E. coli using the substrate glucose-1-phosphate or UDP glucose and in this way generated branched a-glucans. Borovsky et al. (Eur.

J. Biochem. 59 (1975), 615-625), too, analysed the de-novo synthesis of c-1,6- branched «-1,4-glucans from glucose-1-phosphate using a branching enzyme from potato in combination with a phosphorylase (1,4-a-D-glucan: orthophosphate - glycosyitransferase [EC 2.4.1.1]) from maize. Doi (Biochimica et Biophysica Acta 184 (1969), 477-485) showed that the enzyme combination of a starch synthase (ADP-D- glucose: «-1,4-glucan a-4-glucosyltransferase) from spinach and a branching enzyme from potato using the substrate ADP glucose resulted in products similar to amylopectin. Parodi et al. (Arch. Biochem. Biophys. 132 (1969), 11-117) used 3 glycogen synthase from rat liver combined with a branching enzyme from rat liver for the de-novo synthesis of branched glucans from UDP glucose. They obtained a polymer which was similar to native glycogen and which differs from the polymers that are based on glucose-1-phosphate.

This second group of in-vitro methods, too, has the disadvantage that the substrates, e.g. glucose-1-phosphate, UDP glucose and ADP glucose, are very expensive.

. v : Furthermore, it does not seem to be possible either to intentionally control the branching degree.

Bittcher et al. (J. Bacteriol. 179 (1997), 3324-3330) describe an in-vitro method for the production of water-insoluble o-1 4-glucans using an amylosucrase and sucrose as substrates. However, only linear c-1 ,4-glucans without branchings are synthesized.

Thus, the technical problem underlying the present invention is to provide a method allowing the cheap production of a-1,6-branched o-1 ,4-glucans for industrial purposes, as well as nucleic acid molecules encoding the enzymes that may be used in said methods, in particular branching enzymes.

This technical problem has been solved by providing the embodiments characterised in the claims.

Therefore, the present invention relates to nucleic acid molecules encoding a branching enzyme (EC 2.4.1.18) from bacteria of the genus Neisseria selected from the group consisting of : (@ nucleic acid molecules encoding a protein which comprises the amino acid sequence depicted in SEQ ID NO. 2; (b) nucleic acid molecules comprising the nucleotide sequence of the coding region which is depicted in SEQ ID NO. 1: (¢) nucleic acid molecules encoding a protein which comprises the amino acid sequence that is encoded by the insert of the plasmid DSM 12425; (d) nucleic acid molecules comprising the region of the insert of the plasmid DSM 12425, which encodes a branching enzyme from Neisseria denitrificans: (e) nucleic acid molecules encoding a protein the sequence of which has within the first 100 amino acids a homology of at least 65% with regard to the sequence depicted in SEQ ID NO. 2; (f) nucleic acid molecules the complementary strand of which hybridizes to a nucleic acid molecule according to (a), (b), (¢), (d) and/or (e) and which encode a branching enzyme from a bacterium of the genus Neisseria; and (9) nucleic acid molecules the nucleic acid sequence of which differs from the sequence of a nucleic acid molecule according to (f) due to the degeneracy of the genetic code.

The nucleic acid sequence depicted in SEQ ID NO. 1 is a genomic sequence which comprises a coding region for a branching enzyme from Neisseria denitrificans. A plasmid containing said DNA sequence has been deposited as DSM 12425. By means of

® . o said sequence or said molecule, the person skilled in the art can now isolate homologous sequences from other Neisseria species or Neisseria strains. He/she may do so using conventional methods, like screening of cDNA or genomic libraries with suitable hybridization probes. The homologous sequences may also be isolated as described in

Example 1. Thus, it is possible, for example, to identify and isolate nucleic acid molecules that hybridize to the sequence depicted in SEQ ID NO. 1 and that encode a branching enzyme.

The nucleic acid molecules of the invention may, in principle, encode a branching enzyme from any bacterium of the genus Neisseria, they preferably encode a branching enzyme from Neisseria denitrificans. ~ According to the present invention, the term "hybridization" means hybridization under conventional hybridization conditions, preferably under stringent conditions as have been described, e.g. in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2™ edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The term "hybridization" is particularly preferred to mean a hybridization under the following conditions: hybridization buffer: 2xSSC; 10x Denhardt solution (Fikoll 400+PEG+BSA:; at a ratio of 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na,HPO,; 250 pg/ml herring sperm DNA; 50 pg/ml tRNA; or

M sodium phosphate buffer, pH 7.2; 1 mM EDTA: 7%

SDS hybridization temperature: T = 65 to 68°C washing buffer: 0.2xSSC; 0.1% SDS washing temperature: T =651t0 68°C.

Nucleic acid molecules hybridizing to the nucleic acid molecules of the invention may, in principle, be derived from any bacterium of the genus Neisseria which expresses a corresponding protein, preferably they are derived from Neisseria denitrificans. Nucleic acid molecules hybridizing to the molecules of the invention, may, for instance, be isolated from genomic or from cDNA libraries. Such nucleic acid molecules can be identified and isolated using the nucleic acid molecules of the invention or parts of said molecules or the reverse complements of said molecules, e.g. by hybridizing according to standard techniques (cf. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2™ edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or by amplification by means of PCR.

As hybridization probe nucleic acid molecules can be used which have exactly or essentially the nucleotide sequence depicted in SEQ ID NO. 1 or parts thereof. The fragments used as hybridization probes may also be synthetic fragments which have been produced by means of conventional synthesis techniques and the sequence of which is essentially identical to the one of a nucleic acid molecule of the invention. If genes have been identified and isolated to which the nucleic acid sequences of the invention hybridize, the sequence should be determined and the properties of the proteins encoded by said sequence should be analysed to find out whether they are branching enzymes. For this purpose, it is particularly suitable to compare the homology on the nucleic acid and amino acid sequence level and to determine the enzymatic activity.

The molecules hybridizing to the nucleic acid molecules of the invention comprise, in particular, fragments, derivatives and allelic variants of the above-described nucleic acid molecules encoding a branching enzyme from bacteria of the genus Neisseria, preferably from Neisseria denitrificans. In this context, the term "derivative" means that the sequences of said molecules differ from the sequences of the aforementioned nucleic acid molecules in one of more positions and have a high degree of homology to said sequences. Homology, in this context, means that there is, over the entire length, a sequence identity of at least 60%, in particular an identity of at least 70%, preferably of more than 80%, more preferably of more than 90% and most preferably of at least 95%.

The deviations from the above-described nucleic acid molecules may be caused by, e.g. deletion, addition, substitution, insertion or recombination.

Furthermore, homology means that there is a functional and/or structural equivalence between the respective nucleic acid molecules or the proteins encoded by these. The nucleic acid molecules which are homologous to the aforementioned molecules and which are derivatives of said molecules are usually variations of said molecules which are modifications that have the same biological functions. These may be both naturally- occurring variations, e.g. sequences from other Neisseria species or Neisseria strains and mutations with these mutations occurring naturally or being introduced by directed mutagenesis. Furthermore, the variations may be sequences produced synthetically. The allelic variants may be both naturally-occurring variants and variants that have been produced synthetically or by recombinant DNA techniques.

The proteins encoded by the different variants of the nucleic acid molecules of the invention have certain characteristics in common. These may include, for instance, biological activity, molecular weight, immunological reactivity, conformation, etc., as well as physical properties, such as the migration behaviour in gel electrophoreses, chromatographic behaviour, sedimentation coefficients, solubility, spectroscopic properties, stability; pH optimum, temperature optimum, etc.

The molecular weight of the branching enzyme from Neisseria denitrificans is 86.3 kDa, with the molecular weight being deduced from the amino acid sequence. Hence, the i deduced molecular weight of a protein of the invention preferably ranges from 70 kDa to 100 kDa, more preferably from 77 kDa to 95 kDa and most preferably it is about 86 kDa.

The present invention also relates to nucleic acid molecules encoding a protein having the enzymatic activity of a branching enzyme with the encoding protein having a homology of at least 65%, preferably of at least 80% and most preferably of at least 95% in the region of the N-terminus, preferably in the first 100 amino acids, more preferably in the first 110 amino acids and most preferably in the first 120 amino acids to the amino acid sequence depicted in SEQ ID NO. 2.

In another embodiment, the present application relates to nucleic acid molecules encoding a protein having activity of a branching enzyme, the protein comprising at least one, preferably at least 5, more preferably at least 10 and most preferably at least 20 of the following peptide motifs: (a) MNRNRHI (SEQ ID NO. 8), (b) RPDAHH (SEQ ID NO. 9), (c) HAPDYAL (SEQ ID NO. 10), (d) EGEAA (SEQ ID NO. 11), (e) DDYRF (SEQ ID NO. 12), () SALQH (SEQ ID NO. 13), (9) YETLG (SEQ ID NO. 14), (h) VSGVR (SEQ ID NO. 15), (i) VSVIG (SEQ ID NO. 16), () FNGWD (SEQ ID NO. 17), (k} LYKFS (SEQ ID NO. 18), (I) PYAFG (SEQ ID NO. 19), (m) RPTTAS (SEQ ID NO. 20), (n) FRRRA (SEQ ID NO. 21), (0) DELVNY (SEQID NO. 22), (p) LPLSEY (SEQ ID NO. 23), (@) YQATGL (SEQ ID NO. 24), () DDHGL (SEQ ID NO. 25), (s) HQDWN (SEQ ID NO. 26), (ty) DGIRV (SEQ ID NO. 27), (u) YGGSEN (SEQ ID NO. 28), (v) SFAEES (SEQ ID NO. 29), (w) DPVHR (SEQ ID NO. 30), : (x) WQQFAN (SEQ ID NO. 31),

i) ’ (y) EILNS (SEQ ID NO. 32), (z) ATEIQTAL (SEQ ID NO. 33), (aa) VKDKQAKAK (SEQ ID NO. 34).

The nucleic acid molecules of the invention may be any nucleic acid molecules, in particular DNA or RNA molecules, e.g. cDNA, genomic DNA, mRNA, etc. They may be naturally-occurring molecules or molecules produced by means of genetic or chemical synthesis techniques. They may be single-stranded molecules which either contain the coding or the non-coding strand, or they may also be double-stranded molecules.

Furthermore, the present invention relates to nucleic acid molecules which are at least 15, preferably more than 50 and most preferably more than 200 nucleotides in length, these nucleic acid molecules specifically hybridizing to at least one nucleic acid molecule of the invention. In this context, the term "specifically hybridizing" means that said molecules hybridize to nucleic acid molecules encoding a protein of the invention, however, not to nucleic acid molecules encoding other proteins. The term "hybridizing" means preferably hybridizing under stringent conditions (see above). In particular, the invention relates to nucleic acid molecules which hybridize to transcripts of nucleic acid molecules of the invention and which, thus, can prevent the translation thereof. Such nucleic acid molecules which specifically hybridize to the nucleic acid molecules of the invention may, for instance, be components of anti-sense constructs or ribozymes or may be used as primers for amplification by means of PCR.

Moreover, the invention relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors that are usually used in genetic engineering and that contain the above-described nucleic acid molecules of the invention.

In a preferred embodiment, the nucleic acid molecules contained in the vectors are linked in sense-orientation to regulatory elements guaranteeing expression in prokaryotic or eukaryotic cells. In this context, the term "expression" means both transcription or transcription and translation.

The expression of the nucleic acid molecules of the invention in prokaryotic cells, e.g. in

Escherichia coli, allows, for instance, a more exact characterisation of the enzymatic activities of the proteins encoded. In addition, it is possible to introduce various mutations into the nucleic acid molecules of the invention by means of conventional techniques of molecular biology (cf. e.g. Sambrook et al., loc. cit). This leads to the synthesis of proteins the properties of which have optionally been modified. It is also possible to produce deletion mutants by continued deletion of the 5' or 3' end of the encoding DNA sequence, which results in the generation of nucleic acid molecules leading to the synthesis of correspondingly shortened proteins. Moreover, it is possible to introduce point mutations at positions that influence, for instance, the enzyme activity or the regulation of the enzyme. In this way, mutants may be generated that have a modified Ky, value or that are no longer subjected to the usual regulation mechanisms in the cells via allosteric regulation or covalent modification. In addition, mutants may be produced which have a modified substrate or product specificity. Furthermore, mutants may be produced which have a modified activity-temperature profile. The genetic manipulation in prokaryotic cells may be carried out according to methods known to the skilled person (cf.

Sambrook et al., loc. cit.).

Regulatory sequences for the expression in prokaryotic organisms, e.g. E. coli, and in eukaryotic organisms have been sufficiently described in the literature, in particular sequences for the expression in yeast, such as Saccharomyces cerevisiae. Methods in

Enzymology 153 (1987), 383-516 and Bitter et al. (Methods in Enzymology 153 (1987), 516-544) give an overview of various systems for the expression for proteins in various host organisms.

Preferably, the nucleic acid molecule of the invention which has been inserted in a vector of the invention is madified in such a way that it is easier to isolate the encoded protein from the culture medium after it had been expressed in a suitable host organism. There is, for instance, the possibility of expressing the encoded branching enzyme as a fusion protein together with a further polypeptide sequence the specific binding properties of which allow the isolation of the fusion protein by means of affinity chromatography (cf.

Chong et al., Gene 192 (1997), 271-281; Hopp et al., Bio/Technology 6 (1988), 1204- 1210; Sassenfeld, Trends Biotechnol. 8 (1990), 88-93).

Furthermore, the nucleic acid molecule contained in vector of the invention is preferred to comprise nucleotide sequences which allow the secretion of the branching enzyme into the culture medium. Preferably, a sequence is used which codes for the signal peptide of the a-CGTase from Klebsiella oxytoca M5A1 (Fiedler et al., J. Mol. Biol. 256 (1996), 279- 291; Genebank acc. no. X86014, CDS 11529-11618). The recovery and the purification is made easier by the secretion of the enzyme into the culture medium. A disruption of the cells is avoided and the enzyme can be recovered from the culture medium with conventional methods, such as dialysis, osmosis, chromatographic methods, etc. being used for removing residuary components of the culture medium.

Furthermore, the vectors of the invention may comprise other functional units which may bring about a stabilisation of the vector in a host organism, such as a bacterial replication origin or the 2u-DNA for the stabilisation in S. cerevisiae.

In another embodiment, the invention relates to host cells, in particular to prokaryotic or eukaryotic cells which have been transformed with a nucleic acid molecule or a vector as described above, as well as to cells which are derived from said host cells and which contain the described nucleic acid molecules or vectors. The host cells may be bacterial cells (e.g. E. coli) or fungal cells (e.g. yeast, in particular S. cerevisiae), as well as plant or animal cells. The term "transformed" means that the cells of the invention have been genetically modified with a nucleic acid molecule of the invention in so far as they contain at least one nucleic acid molecule of the invention in addition to their natural genome.

Said nucleic acid molecule may be present free in the cell, optionally as self-replicating molecule, or it may be stably integrated into the genome of the host cell.

The host cells are preferred to be microorganisms. Within the present invention, such microorganisms may be all bacteria and all protista (e.g. fungi, in particular yeasts and algae) as have been defined, for instance, in Schlegel "Allgemeine Mikrobiologie" (Georg

Thieme Verlag (1985), 1-2).

The host cells of the invention are particularly preferred to be plant cells. In principle, these may include plant cells from any plant species, i.e. both from monocotyledonous and dicotyledonous plants. Preferably, said cells are plant cells from agricultural useful plants, i.e. plants that people cultivate for nutritional or technical purposes, in particular, for industrial purposes. The invention preferably relates to plants cells from fibre-forming plants (e.g. flax, hemp, cotton), oil-storing plants (e.g. rape, sunflower, soy bean), sugar- storing plants (e.g. sugar beat, sugar cane, sugar millet, banana) and protein-storing plants (e.g. leguminoses).

In another embodiment, the invention relates to plant cells from forage plants (e.g. forage grass and pasture grass (alfalfa, clover, etc.)), vegetable plants (e.g. tomato, lettuce, chicory).

In a preferred embodiment, the invention relates to plant cells from starch-storing plants (e.g. wheat, barley, oat, rye, potato, maize, rice, pea, cassava, mung bean). Plant cells from maize, rice, wheat and potato plants are particularly preferred.

Moreover, the present invention relates to a method for producing a branching enzyme from bacteria of the genus Neisseria. In said method, the host cells of the invention are cultivated under conditions allowing the protein to be expressed and the protein is recovered from the culture, i.e. from the cells and/or the culture medium. Preferably, a host organism that secretes the branching enzyme is used.

Furthermore, the present invention relates to a method for producing a branching enzyme from bacteria of the genus Neisseria with the protein being produced in an in-vitro i transcription and translation system using a nucleic acid molecule of the invention. The person skilled in the art is familiar with such systems.

The invention also relates to proteins which are encoded by the nucleic acid molecules of the invention or which are obtainable by a method of the invention.

Furthermore, the present invention relates to antibodies which specifically recognise a protein of the invention. These antibodies may be, for instance, monoclonal or polyclonal antibodies. They may also be fragments of antibodies which recognise the proteins of the invention. The person skilled in the art is familiar with methods for producing said antibodies or fragments.

Furthermore, the present invention relates to the use of a branching enzyme of the invention for the production of a-1,6-branched o-1 ,4-glucans in in-vitro systems.

In particular, the present invention also relates to transgenic plant cells which contain the nucleic acid molecules or vectors of the invention. Preferably, the cells of the invention are characterised in that the nucleic acid molecule of the invention which has been introduced is stably integrated into the genome and is controlled by a promoter active in plant cells.

There is a plurality of promoters or regulatory elements at disposal for expressing a nucleic acid molecule of the invention in plant cells. In principle, all promoters, enhancers, terminators, etc. that are active in plants are regulatory elements for the expression in plant cells. Basically any promoter which is functional in the plants selected for the transformation can be used. With regard to the plant species used, the promoter can be homologous or heterologous. Said promoter may be selected in such a way that the expression takes place in a constitutive manner or only in a particular tissue, at a certain time in the development of the plant or at a time that is determined by external influence.

Examples of suitable promoters are the 35S promoter of the cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812 or US 5 352 605), which ensures a constitutive expression in all tissues of a plant, and the promoter construct described in WO/9401571.

The ubiquitin promoter (cf. e.g. US 5 614 399) and the promoters of the polyubiquitin genes from maize (Christensen et al., loc. cit.) are further examples. However, also promoters which are only activated at a time determined by external influence (cf. e.g.

WOQO/9307279) can be used. Promoters of heat shock proteins allowing a simple induction may be of particular interest. Furthermore, promoters can be used which lead to the expression of downstream sequences in a certain tissue of the plant, e.g. in photosynthetically active tissue. Examples thereof are 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), the Calb promoter (cf. e.g. US 5 656 496, US 5 639 952, Bansal et al., Proc. Natl. Acad. Sci. USA 89 (1992), 3654-3658) and the Rubisco SSU promoter (cf. e.g. US 5034 322 and US 4 962 028). In addition, promoters that are active in the starch- storing organs of plants to be transformed are to be mentioned. It is, for instance, the maize kernels in maize, whereas in potatoes, it is the tubers. For over-expressing the nucleic acid molecules of the invention in potato, the tuber-specific patatin gene promoter ‘B33 (Rocha-Sosa et al, EMBO J. 8 (1989), 23-29) can, for example, be used. Seed- specific promoters have already been described for various plant species. The USP promoter from Vicia faba, which guarantees a seed-specific expression in V. faba and other plants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Baumlein et al., Mol. Gen.

Genet. 225 (1991), 459-467) is an example thereof.

Moreover, fruit-specific promoters as described in WO 91/01373 can also be used.

Promoters for an endosperm-specific expression, such as the glutelin promoter (Leisy et al., Plant Mol. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993), 357-366), the HMG promoter from wheat, the USP promoter, the phaseolin promoter or promoters of zein genes from maize (Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., Plant

Mol. Biol. 15 (1990), 81-93) are particularly preferred. By means of endosperm-specific promoters it is possible to increase the amounts of transcripts of the nucleic acid molecules of the invention in the endosperm in comparison with the endosperm of corresponding wild type plants.

The shrunken-1-promoter (sh-1) from maize (Werr et al., EMBO J. 4 (1985), 1373-1380) is particularly preferred.

In addition, there may be a terminator sequence which is responsible for the correct termination of the transcription and the addition of a poly-A tail to the transcript having the function of stabilising the transcripts. Such elements have been described in the literature (cf. e.g. Gielen et al., EMBO J. 8 (1989), 23-29) and may be exchanged at will.

Therefore, it is possible to express the nucleic acid molecules of the invention in plant cells.

Thus, the present invention also relates to a method for producing transgenic plant cells comprising introducing a nucleic acid molecule or a vector of the invention into plant cells.

The person skilled in the art has various plant transformation systems at disposal, e.g. the use of T-DNA for transforming plant cells has been examined extensively and has been described in EP-A-120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij

Kanters B.V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46 and

An, EMBO J. 4 (1985), 277-287.

For transferring the DNA in the plant cells, plant explants may suitably be co-cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants may then be

) regenerated from the infected plant material (e.g. parts of leaves, stem segments, roots and protoplasts or plant cells cultivated in suspensions) in a suitable medium which can contain antibiotics or biocides for selecting transformed cells. The plants obtained in that way can then be examined for the presence of the DNA introduced. Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation are known (cf. Willmitzer, L. 1993 Transgenic plants. In: Biotechnology, A Multi-Volume

Comprehensive Treatise (H. J. Rehm, G. Reed, A. Pihler, P. Stadler, eds.), Vol. 2, 627- 659, VCH Weinheim-New York-Basel-Cambridge).

Alternative systems for transforming monocotyledonous plants are the transformation by means of the biolistic method, the electrically or chemically induced DNA absorption in protoplasts, the electroporation of partially permeabilised cells, the microinjection of DNA in the inflorescence, the microinjection of DNA in microspores and pro-embryos, the DNA absorption through germinating pollens and the DNA absorption in embryos by swelling (cf. e.g. Lusardi, Plant J. 5 (1994), 571-582; Paszowski, Biotechnology 24 (1992), 387- 392).

While the transformation of dicotyledonous plants via Ti-plasmid vector systems by means of Agrobacterium tumefaciens is well established, more recent studies point to the : fact that monocotyledonous plants, too, can indeed be transformed by means of vectors based on Agrobacterium (Chan, Plant Mol. Biol. 22 (1993), 491-506; Hiei, Plant J. 6 (1994), 271-282; Bytebier, Proc. Natl. Acad. Sci. USA 84 (1987), 5345-5349; Rainer,

Bio/Technology 8 (1990), 33-38: Gould, Plant Physiol. 95 (1991), 426-434; Mooney,

Plant, Cell Tiss. & Org. Cult. 25 (1991), 209-218; Li, Plant Mol. Biol. 20 (1992), 1037- 1048).

In the past, three of the above transformation systems could be established for various cereals: the electroporation of tissue, the transformation of protoplasts and the DNA transfer by particle bombardment in regenerable tissue and cells (for an overview see

Jahne, Euphytica 85 (1995), 35-44). The transformation of wheat has been described several times in the literature (for an overview see Maheshwari, Critical Reviews in Plant

Science 14 (2) (1995), 149-178).

In particular, the transformation of maize has been described several times in the literature (cf. e.g. WO 95/06128, EP 0513849, EO 0465875, EP 292435; 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 successful transformation of other kinds of cereals has also been described, e.g. for barley (Wan and Lemausx, loc. cit.: Ritala et al., loc. cit.; Krens et al., Nature 296 (1982), 72-74) and for wheat (Nehra et al, Plant J. 5 (1994), 285-297).

For expressing the nucleic acid molecules of the invention in plants it is, in principle, possible for the synthesized protein to be located in any compartment of the plant cell.

~The coding region must optionally be linked to DNA sequences which guarantee the localisation in the respective compartment in order to achieve localisation in a particular compartment. Such sequences are known (cf. e.g. Braun, EMBO J. 11 (1 992), 3219- 3227; Sonnewald, Plant J. 1 (1991), 95-106; Rocha-Sosa, EMBO J. 8 (1989), 23-29).

As plastidial signal sequence, for instance, the one of ferrodoxin:NADP+ oxidoreductase (FNR) from spinach can be used. Said sequence contains the 5' non-translated region and the flanking transit peptide sequence of the cDNA of the plastidial protein ferrodoxin:NADP+ oxidoreductase from spinach (nucleotide ~171 to +165; Jansen et al.,

Current Genetics 13 (1988), 517-522).

Furthermore, the transit peptide of the waxy protein from maize plus the first 34 amino acids of the mature waxy protein (Klosgen et al., Mol. Gen. Genet. 217 (1989), 155-161) may also be used as plastidial signal sequence. In addition, the transit peptide of the waxy protein from maize (cf. above) may also be used without the 34 amino acids of the mature waxy protein.

Moreover, it is also thinkable to use to following plastidial signal sequences: the signal sequence of the ribulose biphosphate carboxylase small subunit (Wolter et al., Proc. Natl.

Acad. Sci. USA 85 (1988), 846-850; Nawrath et al., Proc. Natl. Acad. Sci. USA 91 (1994), 12760-12764); the signal sequence of the NADP malate dehydrogenase (Gallardo et al.,

Planta 197 (1995), 324-332); the signal sequence of the glutathione reductase (Creissen etal, Plant J. 8 (1995), 167-175).

Therefore, the present invention also relates to transgenic plant cells that were transformed with one or more of the nucleic acid molecule(s) of the invention, as well as to transgenic plant cells that are derived from cells transformed in such a way. Such cells contain one or more nucleic acid molecule(s) of the invention with said molecule(s) preferably being linked to regulatory DNA elements which guarantee the transcription in plant cells, in particular with a promoter. Such cells can be differentiated from naturally- occurring plant cells in that they contain at least one nucleic acid molecule of the invention.

The transgenic plant cells may be regenerated to whole plants using techniques well- known to the person skilled in the art. The plants obtainable by means of regeneration of the transgenic plant cells of the invention are also a subject matter of the present invention.

Moreover, plants containing the aforementioned plant cells are a subject matter of the present invention. The plants of the invention may, in principle, be plants of any plant species, i.e. both monocotyledonous and dicotyledonous plants. They are preferred to be useful plants, i.e. plants which are cultivated for nutritional or technical, in particular, industrial purposes. Preferably, the invention relates to plant cells from fibre-forming

Claims (48)

1. Claims

   ) 1. A nucleic acid molecule encoding a branching enzyme from a bacterium of the genus Neisseria selected from the group consisting of (@) nucleic acid molecules encoding a protein which comprises the amino acid sequence depicted in SEQ ID NO. 2; (b) nucleic acid molecules comprising the coding region depicted in SEQ ID

   NO. 1; (c) nucleic acid molecules encoding a protein which comprises the amino acid sequence encoded by the insert in plasmid DSM 12425: (d) nucleic acid molecules comprising the coding region for a branching enzyme, which is contained in the insert of the plasmid DSM 12425; (8) nucleic acid molecules encoding a protein the sequence of which has, in the first 100 amino acids, a homology of at least 65% to the amino acid sequence depicted in SEQ ID NO. 2; (f) nucleic acid molecules the complementary strand of which hybridizes to a nucleic acid molecule of (a), (b), (c), (d) and/or (e) and which encode a branching enzyme from a bacterium of the genus Neisseria; and (@) nucleic acid molecules the sequence of which deviates from the sequence of a nucleic acid molecule of (f) due to the degeneracy of the genetic code.
2. 2. A vector containing a nucleic acid molecule according to claim 1.
3. 3. The vector according to claim 2, wherein the nucleic acid molecule is linked in sense-orientation to regulatory sequences guaranteeing the transcription in prokaryotic or eukaryotic cells.
4. 4. A host cell which is genetically modified with a nucleic acid molecule according to claim 1 or with a vector according to claim 2 or 2.
5. 3. A method for producing a branching enzyme from a bacterium of the genus Neisseria, wherein a host cell according to claim 4 is cultivated under conditions allowing the expression of the protein, and wherein the protein is isolated from the cultivated cells and/or the culture medium.
6. 6. A method for producing a branching enzyme from a bacterium of the genus Neisseria, wherein the protein is produced in an in-vitro transcription and translation system using a nucleic acid molecule according to claim 1.
7. " 7. A protein encoded by a nucleic acid molecule according to claim 1 or obtainable by a method according to claim 5 or 6.
8. 8. An antibody which specifically recognises a protein according to claim 7.
9. 9. Use of a protein according to claim 7 for producing «-1,6-branched «-1,4- glucans in in-vitro systems.
10. 10. A transgenic plant cell containing a nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is linked to regulatory sequences guaranteeing the transcription in plant cells.
11. 11. The transgenic plant cell according to claim 10, wherein the nucleic acid molecule is linked to a sequence encoding a signal sequence which guarantees the localisation of the encoded protein in the plastids of the cells.
12. 12. Atransgenic plant containing plant cells according to claim 10 or 11.
13. 13. A method for producing a transgenic plant, wherein (@) a plant cell is genetically modified by introducing a nucleic acid molecule according to claim 1 or a vector according to claim 2 or 3: (b) a plant is regenerated from the cell produced according to step (a); and (c) optionally further plants are produced from the plant produced according to step (b).
14. 14. Harvestable parts of plants according to claim 12 or of plants obtainable by a method according to claim 13, wherein said parts of plants contain transgenic niant ralle anArArdinm $a Alama 10 Ar 14 . MivAtIL Wii hd dR ALE] WJ wi@nil iw wi [I I
15. 15. Starch obtainable form transgenic plant cells according to claim 10 or 11, from transgenic plants according to claim 12, from transgenic plants obtainable by a method according to claim 13 or from parts of plants according to claim 14.
16. 16. The starch according to claim 15, wherein the composition of the starch is modified in such a way that it has an increased gel texture and/or a reduced phosphate content and/or a reduced peak viscosity and/or a reduced pastification temperature and/or a reduced size of the starch granules and/or a

    AMENDED SHEET modified distribution of the side-chains in comparison with the starch from corresponding wild type plants.
17. 17. A regulatory region which naturally controls the transcription of a nucleic acid molecule according to claim 1 in bacterial cells.
18. 18. The regulatory region according to claim 17 containing a nucleotide sequence selected from the group consisting of: (a) nucleotide sequences comprising the nucleotides 1 to 169 of the nucleotide sequence depicted in SEQ ID NO. 1; (b) the nuclectide sequence of the regulatory region which is contained in the insert of the plasmid DSM 12425, or parts thereof, (c) nucleotide sequences hybridizing to the sequences of (a) or (b) under stringent conditions.
19. 19. An in-vitro method for producing «-1,6-branched «-1,4-glucans using the substrate sucrose and a combination of enzymes of an amylosucrase and a branching enzyme.
20. 20. The method according to claim 19, wherein the branching enzyme is encoded by a nucleic acid molecule according to claim 1.
21. 21. A nucleic acid molecule as claimed in claim 1, substantially as hereinbefore described or exemplified.
22. 22. A nucleic acid molecule including any new and inventive integer or combination of integers, substantially as herein described.
23. 23. A vector as claimed in either of claims 2 or 3, substantially as hereinbefore described or exemplified.
24. 24. A vector including any new and inventive integer or combination of integers, substantially as herein described.
25. 25. A host cell as claimed in claim 4, substantially as hereinbefore described or exemplified.
26. 26. A host cell including any new and inventive integer or combination of integers, substantially as herein described.

    “ AMENDED SHEET N
27. 27. A method according to the invention for producing a branching enzyme, substantially as hereinbefore described or exemplified.
28. 28. A method for producing a branching enzyme including any new and inventive integer or combination of integers, substantially as herein described.
29. 29. A protein as claimed in claim 7, substantially as hereinbefore described or exemplified.
30. 30. A protein including any new and inventive integer or combination of integers, substantially as herein described.
31. 31. An antibody as claimed in claim 8, substantially as hereinbefore described or exemplified.
32. 32. An antibody including any new and inventive integer or combination of integers, substantially as herein described.
33. 33. The use of a protein as claimed in claim 9, substantially as hereinbefore described or exemplified.
34. 34. The use of a protein including any new and inventive integer or combination of integers, substantially as herein described.
35. 35. A transgenic plant cell as claimed in either of claims 10 or 11, substantially as hereinbefore described or exemplified.
36. 36. A transgenic plant cell including any new and inventive integer or combination of integers, substantially as herein described.
37. 37. A transgenic plant as claimed in claim 12, substantially as hereinbefore described or exemplified.
38. 38. A transgenic plant including any new and inventive integer or combination of integers, substantially as herein described.
39. 39. A method according to the invention for producing a transgenic plant, substantially as hereinbefore described or exemplified.
40. 40. A method for producing a transgenic plant including any new and inventive integer or combination of integers, substantially as herein described.

    : AMENDED SHEET "
41. 41. Harvestable parts of plants as claimed in claim 14, substantially as hereinbefore described or exemplified.
42. 42. Harvestable parts of plants including any new and inventive integer or combination of integers, substantially as herein described.
43. 43. Starch obtainable from transgenic plant cells as claimed in either of claims 15 or 16, substantially as hereinbefore described or exemplified.
44. 44. Starch obtainable from transgenic plant cells including any new and inventive integer or combination of integers, substantially as herein described.
45. 45. A regulatory region as claimed in either of claims 17 or 18, substantially as hereinbefore described or exemplified.
46. 46. A regulatory region including any new and inventive integer or combination of integers, substantially as herein described.
47. 47. An in-vitro method according to the invention for producing a-1,6-branched a-1,4- glucan, substantially as hereinbefore described or exemplified.
48. 48. An in-vitro method for producing a-1,6-branched a-1,4-glucan including any new and inventive integer or combination of integers, substantially as herein described.