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
2000-05-05
2003-05-06
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, C800S306000, C800S312000, C800S317200, C800S317400, C800S320000, C800S320100, C800S320200, C800S320300, C435S069100, C435S101000, C435S193000, C435S320100, C435S419000, C536S023600
Reexamination Certificate
active
06559356
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to nucleic acid molecules encoding proteins with the enzymatic activity of a fructosyl transferase (FFT). The invention also relates to vectors containing such nucleic acid molecules as well as to host cells transformed with said nucleic acid molecules, in particular plant cells, plant tissue and plants. Moreover, methods for the production of transgenic plants are described which synthesize long-chain inulin due to the introduction of nucleic acid molecules encoding an FFT. The present invention also relates to methods of producing FFT and to the production of long-chain inulin in various host organisms, in particular plants, as well as to in vitro methods for producing long-chain inulin by means of the FFT of the invention. The present invention further relates to the host cells of the invention and to the inulin obtainable by the processes of the present invention.
Water-soluble, linear polymers allow for a variety of applications, for example for increasing the viscosity in aqueous systems, as detergents, as suspending agents or for speeding up sedimentation, for complexing and, however, also for binding water. Polymers which are based on saccharides, such as fructosyl polysaccharides, are particularly interesting raw materials as they are biodegradable. Apart from their application as regenerable raw materials for the industrial production and processing, fructosyl polymers are also to be considered as additives in foodstuffs, for example as sweeteners. For various uses, polymers with varying chain-lengths are needed. Whereas short- and medium-chain polymers are particularly preferred in the food processing industry, polymers with a high degree of polymerization (DP) are needed for technical uses, such as the production of surfactants.
So far only methods for producing long-chain fructan polysaccharides in plants have been described in which fructosyl transferases of bacterial origin are expressed. Most bacterial fructosyl transferases synthesize levan, a &bgr;-2,6 linked fructosyl polymer which has numerous &bgr;-2,1-branchings. Due to its numerous branchings levan has decisive disadvantages when it comes to technical processing and is therefore considerably less significant as a technical raw material then inulin. Up to now, only one bacterial gene is known, the gene product of which is involved in the synthesis of inulin, namely the ftf gene from
Streptococcus mutans.
It is in principle possible to express the gene in plants if the gene has previously been genetically engineered. However, the inulin yield obtained from transgenic plants is so low that the economic utilization of the transgenic plants is out of question.
Furthermore, a method for producing transgenic plants expressing fructosyl transferases from
Helianthus tuberosus
is known. The expression of these genes in transgenic plants leads to the production of inulin with an average degree of polymerization of DP=6 to DP=10. Polymers with this degree of polymerization may not be referred to as long-chain inulin. Inulin with an average DP=6 to DP=10 is unsuitable for most technical uses.
Methods for an economic production of long-chain inulin in plants or for synthesizing enzymes for the production of long-chain inulin are not known.
PCT/US89/02729 describes the possibility of synthesizing carbohydrate polymers, in particular dextran or polyfructose, in transgenic plant cells, specifically in the fruits of transgenic plants. In order to produce plants modified in such a way, the use of levan sucrases from microorganisms, in particular from
Aerobacter levanicum, Streptococcus salivarius
and
Bacillus subtilis,
or of dextran sucrases from
Leuconostoc mesenteroides
is proposed. Neither the formation of the active enzymes nor that of levan or dextran or the production of transgenic plants is described. PCT/EP93/02110 discloses a method for producing transgenic plants expressing the Isc gene of the levan sucrase from the gram-negative bacterium
Erwinia amylovora.
The plants produce a high-molecular, strongly branched levan. PCT/NL93/00279 describes the transformation of plants with chimeric genes containing the sacB gene from
Bacillus subtilis
or the ftf gene from
Streptococcus mutans.
Transgenic plants expressing the sacB gene produce a branched levan. Plants expressing the ftf gene synthesize high-molecular inulin; the yield, however, is so low that an economic utilization is out of question. PCT/NL96/00012 discloses DNA sequences encoding enzymes synthesizing carbohydrate polymers as well as the production of transgenic plants by means of these DNA sequences. The disclosed sequences are derived from
Helianthus tuberosus.
According to PCT/NL96/00012, the disclosed sequences may be used in order to modify the fructan profile of petunia and potato, but also of
Helianthus tuberosus
itself. When expressing the SST and the FFT gene in transgenic plants, it is possible to produce inulin. The average degree of polymerization of inulin, however, ranges between DP=6 and DP=10. The production of high-molecular inulin is not possible by means of the method described in PCT/NL96/00012. PCT/EP97/02195 describes a method for producing transgenic, inulin-producing plants by means of the ftf gene from
Streptococcus mutans.
The yield of high-molecular inulin is low, as is the case with the plants described in PCT/NL9300279. DE 197 08 774.4 describes the production of short-chain inulin by means of enzymes exhibiting fructosyl polymerase activity. The short-chain inulin may be produced in transgenic plants. The yield of short-chain inuin is high and in potato it corresponds to the cellular content of sucrose. The production of long-chain inulin, however, is not described.
The synthesis of inulin in plants has been thoroughly examined (Pollock & Chafterton, Fructans, The Biochemistry of Plants Vol. 14 (1988), Academic Press, pp.109-140). However, the inulin occurring naturally in plants is short-chain fructan with a maximum degree of polymerization of approximately DP=35 (Pollock & Chatterton, 1988, loc.cit.). Synthesis and metabolism of fructans in plants are based on the activity of at least three enzymes: a sucrose-dependent sucrose-fructosyl transferase (SST) forming the tri-saccharide kestose, a fructan-dependent fructan-fructosyl transferase (FFT) which transfers fructosyl residues from fructan molecules with a minimum degree of polymerization of DP=3 (kestose) to sucrose and higher fructans, and a fructan exohydrolase (FEH) which removes fructose residues from fructan molecules. It is not known whether differences in the average molecular weight of the inulin in various plant species, for example about 2×10
3
in the case of
Allium cepa
and 5×10
3
in the case of
Helianthus tuberosus,
are based on the different properties of their SST, FFT or FEH.
For this reason it is not possible in view of the present knowledge relating to the inulin synthesis in plants to identify suitable DNA sequences by means of which high-molecular inulin might be synthesized in plants in economically interesting amounts.
Thus, the technical problem underlying the present invention is to provide nucleic acid molecules and methods which allow for the production of genetically modified organisms, in particular plants, capable of forming long-chain inulin.
This problem is solved by the provision of the embodiments characterized in the claims.
SUMMARY OF THE INVENTION
Therefore, the present invention relates to nucleic acid molecules encoding proteins with the enzymatic activity of an FFT, selected from the group consisting of
(a) nucleic acid molecules encoding a protein comprising the amino acid sequence indicated under SEQ ID NO: 2 or SEQ ID NO: 4;
(b) nucleic acid molecules comprising the nucleotide sequence indicated under SEQ ID NO: 1 or SEQ ID NO: 3 or a corresponding ribonucleotide sequence;
(c) nucleic acid molecules which hybridize to a complementary strand of the nucleic acid molecules mentioned
Gritscher Dominique
Hellwege Elke
Heyer Arnd G.
Brown Karen E.
Fish & Neave
Fox David T.
Haley Jr. James F.
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V
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