Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
1999-09-03
2003-02-04
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C800S278000, C800S287000, C800S317200, C800S320000, C800S320100, C800S320200, C800S320300, C435S069100, C435S101000, C435S193000, C435S320100, C435S419000, C435S468000, C536S023600
Reexamination Certificate
active
06515203
ABSTRACT:
FIELD OF THE INVENTION
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 short-chain 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.
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 specification of PCT/USA89/02729 the possibility to produce carbohydrate polymers, in particular 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 levan sucrases from microorganisms, in particular from
Aerobacter levanicum, Streptococcus salivarius
and
Bacillus subtilis
, or from dextran sucrases from
Leuconostoc mesenteroides
are suggested. The production of neither the active enzymes nor of levan 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 levan 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 5′-untranslated 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/NL96/100012 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.
SUMMARY OF THE INVENTION
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.
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
(a) nucleic acid molecules encoding a protein that comprises the amino acid sequence depicted in SEQ ID No. 2 and SEQ ID No. 4;
(b) nucleic acid molecules comprising the nucleotide sequence depicted in SEQ ID No. 1 or a corresponding ribonucleotide sequence;
(c) nucleic acid molecules comprising the nucleotide sequence depicted in SEQ ID No. 3 or a corresponding ribonucleotide sequence;
(d) nucleic acid molecules hybridizing to the nucleic acid molecules mentioned in (a) or (b) 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
(e) nucleic acid molecules the nucleotide sequence of which deviates from the sequence mentioned in (a), (b) or (c) 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 &bgr;-2,1 glycosidic or &bgr;-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 &bgr;-2,1 glycosidically or &bgr;-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.
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
nd
edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) 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 5′- 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
Gritscher Dominique
Hellwege Elke
Heyer Arnd G.
Brown Karen E.
Fox David T.
Haley Jr. James F.
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.
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