DNA sequences encoding carbohydrate polymer synthesizing enzymes

Details DNA sequences encoding carbohydrate polymer synthesizing enzymes DNA sequences encoding carbohydrate polymer synthesizing enzymes

1997-08-25

2000-05-02

Fox, David T.

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters carbohydrate production in the plant

800286, 800298, 435101, 435419, 435468, 435471, 435193, 536 236, 536 245, C12N 1582, C12N 1529, C12N 1554, C12P 1904, A01H 500

Patent

active

060574941

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

This invention relates to nucleotide sequences encoding fructan synthesizing enzymes, a recombinant DNA sequence comprising one or more of said nucleotide sequences, a method for producing a genetically transformed host organism showing a modified fructan profile, and transformed plants or plant parts showing said modified fructan profile.
Fructans refer to a group of carbohydrate compounds in which one or more fructosyl-fructose linkages constitute a majority of the linkages. Fructans are fructosepolymers with usually, but not necessarily, one terminal glucosylunit [G-(F).sub.n, G=optional, n.gtoreq.2]. The fructosyl-fructose linkages in fructans are of the .beta.-2,6 or .beta.-2,1 linkage type. Fructans with predominantly .beta.-2,6 fructosyl-fructose linkages are usually called levan(s). Fructans with predominantly .beta.-2,1 fructosyl-fructose linkages are usually called inulin(s).
Fructan biosynthesis is common in several bacterial, fungal and algal families and also in specific plant families, such as the Liliaceae (e.g. Allium cepa), Poaceae (e.g. Lolium perenne) and Asteraceae (e.g. Helianthus tuberosus). The function of fructans in bacteria and fungi is poorly understood. It has been suggested that fructans act as extracellular stores of carbohydrate that can be mobilized during periods of carbohydrate stress (Jacques, 1993). In plants, fructans can function as reserve carbohydrates which serve as a source of carbon for (re)growth (Meier and Reid, 1982). In fructan-storing crops, tructan synthesis is restricted not only to specific organs (e.g. the stems or tubers of H. tuberosus, the bulbs of Allium spp, the leaf bases and stems of grasses) but also specific cell types within these organs (usually the parenchyma cells). In these specific cell types, the vacuole is probably the location of both fructan biosynthesis and storage (Darwen and John, 1989; Wagner et al., 1983).
In bacteria, examples of fructan synthesizing bacteria are Streptococcus mutans and Bacillus subtilus, the biosynthesis of fructans from sucrose is catalysed by only one enzyme: levansucrase (EC 2.4.1.10) in B. subtilus (Dedonder 1966) and levansucrase, but also called fructosyltransferase, (FTF, EC 2.4.1.10) in S. mutans (Carlsson, 1970). Bacterial fructan synthesis proceeds via the direct transfer of fructose from a donor-sucrose (G-F) to sucrose or other acceptor molecules according to the following reversible reaction:
Water, hexoses, sucrose, oligosaccharides and levan may act as acceptor molecules for fructosyl units from sucrose (fructosyl donor).
Bacterial DNA sequences encoding FTF in S. mutans and levansucrase in B. subtilus are already described in the literature (Sato and Kuramitsu, 1986; Steinmetz et al. 1985). Bacterial genes from several sources were used to transform specific host plants which normally cannot synthesize fructans, thereby inducing fructan synthesis (see for example: Van der Meer et al., 1994; Ebskamp et al., 1994). A method to enhance the solid content of tomato fruits, using the levansucrase gene from B. subtilus and the dextransucrase gene from Leuconostoc mesenretoides is described in application WO 89/12386. A method to modify the fructan pattern in plants which normally cannot synthesize fructans, using the levansucrase-encoding ftf gene from S. mutans and the levansucrase-encoding SacB gene from B. subtilus is described in applications NL A 9300646 and WO 94/14970. The use of a levansucrase-encoding DNA sequence from Erwinia amylovora, which after integration in the host plant genome leads to the synthesis of levans, is described in DE 4227061 A1 and WO A 9404692. In all said applications, transgenic plants are described which are transformed with levansucrase genes from bacteria. Accordingly, these transgenic plants synthesize and accumulate fructans structurally comparable to those synthesized by the donor bacteria (Van der Meer et al., 1994; Ebskamp et al., 1994).
The present application differs from said applications in that it is related to fructosyltransfera

REFERENCES:
Stratagene Instruction Manual: UniZAP.TM. Library, Jan. 19, 1994, pp. 1-10.
Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Lab. Press, pp. 8.46-8.47, 1989.
Hellwege et al. Plant Journal 12(5):1057-1065, 1997.
Bournay et al. Nucl. Acids Res. 24(12):2347-2351, 1996.
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Angenent et al. "Purification and Properties of Sucrose Fructosyltransferases from Barley Leaves and Onion Seeds" by Angenent, G.C., et al., Insulin and Insulin-containing Crops, 1993, pp. 173-184, Fuchs, A., ed.
Praznik, W., et al., "Isolation and Characterization of Sucrose:Sucrose 1.sup.F -.beta.-D- Fructosyltransferase from Tubers of Helianthus tuberosus L." Agricultural Biol. Chem., vol. 54, No. 9, 1990, pp. 2429-2431.
Luscher, M., et al., "Purification and characterization of fructan: fructan fructosyltransferase from Jerusalem artichoke (Helianthus tuberosus L.)", New Phytol, vol. 123, 1993, pp. 717-724.
Koops, A., et al., "Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus "Colombia" I. Fructan:fructan fructosyltransferase" Journal of Experimental Botany, vol. 45, No. 280, Nov. 1994, pp. 1623-1631.

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