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
1999-12-22
2002-12-31
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
C800S288000, C800S298000, C435S419000, C435S430000, C435S468000
Reexamination Certificate
active
06501005
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to DNA sequences which lead to the formation of polyfructans (levans), as well as a process for preparing transgenic plants using plasmids on which these DNA sequences are located.
High molecular weight, water soluble, linear polymers, for example those based on polyacrylates or polymethacrylates, are products of mineral oils and have many important uses. In particular their properties in increasing the viscosity of aqueous systems, in suspending or sedimentation acceleration and complexing are especially valuable from the technical viewpoint. These products are also used in exceptionally large amounts in super absorbers for water binding and in water dilutable lacquers. In spite of the outstanding positive properties, because such products are difficult to dispose of, their use is increasingly coming under criticism because they are not biodegradable.
Alternatives based on recyclable raw materials, especially starches and cellulose, because of the macromolecular structure of these polysaccharides, have been shown to have limited value. As a replacement for non-biodegradable chemically derived polymers, a number of derivatised high polymeric polysaccharides have been considered. Until now, such polysaccharides could only be obtained biotechnologically via suitable fermentation and transglycosidation processes. The products obtained in this way, such as dextrans and polyfructans (levans) are not competitive as raw materials for mass production.
Polyfructans are found in a number of monocotyledonous and dicotyledonous higher plants, in green algae as well as in a number of gram positive and gram negative bacteria (Meier and Reid, (1982) Encyclopedia of Plant Physiology, New Series, 13A 418-471). The role of fructans for the plant development and plant growth is not fully understood. Functions of the fructans that have been proposed are as a protectant against freezing at low temperatures, as alternative carbohydrate stores which limit starch biosynthesis, as well as applied intermediary stores for photoassimilates which are situated in the stems of grasses shortly before their transfer into the seeds.
All fructans contain, as starter molecule for the polymerisation reaction, a molecule of sucrose (glucose-fructose) to which fructose polymers are added.
Depending on the coupling of the fructose molecule, fructans of-plant origin can be classified into four classes (Meier and Reid (1982), Encyclopedia of Plant Physiology, New Series, 13A, 418-471):
a) (2-1) coupled &bgr; D-fructans (inulin type)
b) (2-6) coupled &bgr;-D-fructans (phlein or levan type)
c) highly branched fructans with a mixture of 2-1 and 2-6 couplings.
d) (2-1) coupled &bgr;-D-fructans, which in contrast to the types under a-c, are added completely from fructose residues of polymerisation both from glucose and also from fructose residues from polyfructose residues (neokestose type).
Fructans of bacterial origin correspond either to the levan or to the inulin type (Carlsson (1970) Caries Research 4, 97-113) and Dedonder (1966) Methods Enzymology 8, 500-505).
Experiments on the biosynthesis of fructans in plants and bacteria lead one to conclude that the biosynthesis proceeds by various routes. Bacterial and plant fructans are further distinguished, not particularly in their primary structure but mainly in their molecular weight. Thus, fructans isolated from plants have been shown to have molecular weights of between 5000 and 50,000 d (Pollock and Chatterton (1988) in: The Biochemistry of Plants 14, 109-140), while fructans isolated from bacteria, molecular weights of up to 2,000,000 d have been described (Clarke et al (1991) in: Carbohydrates as Organic Raw Materials, VCH Weinheim, 169-182).
Various microorganisms from the group of Bacillus spp as well as Streptococcus spp produce polyfructoses in which both fructans of the levan type and fructans of the inulin type have been described (Carlsson (1 970) Caries Research 4, 97-113 and Dedonder (1966) Methods Enzymology 8, 500-505).
Experiments on biosynthesis pathways have made it clear that, in comparison to biosynthesis pathways in higher plants, there is a simpler pattern and a sharing of only one enzyme. This enzyme with the trivial name levan sucrase is a transfructosylase (sucrose:.&bgr;-D-fructosyl transferase, E.C.2.4.1.10.), which catalyzes the following reaction:
sucrose+acceptor
glucose+fructosyl acceptor
Representative acceptors are water, alcohol, sugar or polyfructoses. The hypothesis that only one enzyme catalyses this reaction, depends on the one hand on the examination of the protein chemically purified enzyme, and on the other, to the fact that the gene for levan sucrase has been isolated from various Bacillus spp. as well as from a Streptococcus spp. and after transfer into
E. coli
leads to the formation of levan in
E. coli
(Gay et al (1983) J. Bacteriology 153, 1424-1431 and Sato et al. (1986) Infection and Immunity 52, 166-170).
Until now, genes for levan sucrase from
Bacillus amyloliquefaciens
(Tang et al. (1990) Gene 96, 89-93) and
Bacillus subtilis
(Steinmetz et al. (1985) Mol. Gen. Genetics 200, 220-228), have been described, and demonstrate relatively high homology with each other and both of which catalyze the synthesis of fructans of the levan type. Further, a fructosyl transferase from
Streptococcus mutans
(Shiroza et al. (1988) J. Bacteriology 170, 810-816) has been described. This shows little homology to either levan sucrases from Bacillus spp.. The fructan formed in
Streptococcus mutans
is of the inulin type.
In WO 89/12386, there is described the possibility of producing carbohydrate polymers such as dextran or levan in transgenic plants, especially in the fruit of transgenic plants. To prepare these plants, the use of levan sucrases from
Aerobacter levanicum, Streptococcus salivarius
and
Bacillus subtilis
and the use of dextran sucrases from
Leuconostoc mesenteroides
have been described.
Further, the construction of chimeric genes is described which may be suitable for the expression of the levan sucrase from
Bacillus subtilis
as well as the dextran sucrase fom
Leuconostoc mesenteroides
in transgenic plants. Also described is the preparation of transgenic plants containing these constructs. Further, the preparation of transgenic plants that contain these constructs are described. Whether polyfructans can actually be produced by the described process is not known.
There is also described a series of processes for modifying the carbohydrate concentration and/or concentrating carbohydrates in transgenic plants by means of biotechnological methods. Thus, in view of the fact that increasing of the starch concentration and the modification of the starch in physical and chemical respects is already known, then a modification of the carbohydrate content of potato plants by raising or lowering the ADP-glucose-pyrophosphorylase activity can be achieved (EP 455 316).
From EP 442 592 it is further known that a modification of the distribution of photoassimilates by means of cytosolic and apoplastic invertase is possible and that the yield as well as the drought and frost resistance of potato plants can be modified through the expression of a heterologous pyrophosphatase gene in potato plants.
In order to adapt the physico-chemical parameters of raw materials which are increasingly being used, such as polysaccharides, to the requirements of the chemical industry, as well as to minimize the costs of obtaining these products, processes for the preparation of transgenic plants have to be developed which lead in comparison with known processes to better, higher yielding plants.
SUMMARY OF THE INVENTION
It has now been surprisingly found that the DNA sequence of the levan sucrase from a gram-negative bacterium of the species Erwinia amylovora with the nucleotide sequence (Seq-ID NO 1):
GGATCCCCCG GGCTGCAGCG ATCATGGTTA TTTATAAGGG ATTGTTATGT
50
CCTGAAAACC ACACAACAGA ACCAGAGTGA TTTCAAAAAA TAAAAAGCTA
100
TTAATATACA GACCTTCAGC AAGAAGGTAT TCGAAATAAC CTGTGAGGAT
150
A
Geider Klaus
Geier Gebhard
Röber Manuela
Willmitzer Lothar
Fox David T.
Frommer & Lawrence & Haug LLP
Hoechst Schering AgrEvo
Kubelik Anne
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