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
1999-02-05
2002-04-02
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
C800S287000, C800S320000, C800S320100, C536S023600, C435S101000, C435S193000, C435S419000
Reexamination Certificate
active
06365800
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns methods for synthesis and accumulation of fructose polymers in transgenic maize (
Zea mays
L.) by selective expression of plant-derived fructosyltransferase genes.
TECHNICAL BACKGROUND
Higher plants accumulate various commercially useful carbohydrate polymers such as cellulose, starch and fructan. Starch and cellulose are currently used in numerous food and non-food applications in their native form, but are more likely to be enzymatically or chemically modified, which greatly expands their usefulness.
Fructans are linear or branched polymers of repeating fructose residues. The number of residues contained in an individual polymer, also known as the degree of polymerization (DP), varies greatly depending on the source from which it is isolated. For example, fructan synthesized by fungal species, such as in
Aspergillus syndowi
may contain only two or three fructose residues. By contrast, polymers with a DP of 5000 or greater are synthesized by several bacterial lines, including
Bacillus amyloliquefaciens
and
Streptococcus mutans
. Intermediate sized fructan, with a DP of 3 to 60, are found in over 40,000 plant species (
Science and Technology of Fructans
, (1993) M. Suzuki and N. Chatterton, eds. CRC Press Inc., Boca Raton, Fla., pp. 169-190).
Regardless of size, fructose polymers are not metabolized by humans. Because of this, and due to their relative sweetness, small fructans with a DP of 3-4 are used in a wide variety of low calorie food products. Polymer size is critical to its commercial use. High DP polymers are not sweet, however, they do provide texture to food products very similar to that of fat. High DP fructan used as a fat replacer also contributes very little to the caloric value of the product.
Fructans are also considered to be an excellent source of fructose for the production of high fructose syrup (Fuchs, A. (1993) in
Science and Technology of Fructans
, M. Suzuki and N. Chatterton, eds. CRC Press Inc., Boca Raton, Fla., pp. 319-352). Simple hydrolysis of fructan into individual fructose residues has a tremendous advantage over the current, technically demanding process of enzymatically converting starch into high fructose syrup. Using fructan as the starting material would, therefore, significantly reduce production costs.
The commercial potential for fructan is extremely high, however, its use is severely limited due mainly to the high cost of production. Fructan used in low-calorie foods is currently produced by fermentation culture. Larger polymers synthesized by bacteria are not currently produced on a commercial scale. Isolation from plants would reduce the production costs, but fructan is not found in many crops of agricultural importance. Traditional crops, adapted to wide growing regions, such as oat, wheat and barley accumulate fructan, but only at extremely low levels. Fructan is currently harvested from plants on a relatively small commercial scale and only from a single plant species,
Cichorium intybus.
Transgenic crops accumulating fructan through expression of chimeric fructosyltransferase (FTF) genes would have a significant advantage over native fructan-storing plants by making use of established breeding programs, pest resistance and adaptation to a variety of growing regions throughout the world. Examples of fructan synthesis in transgenic plants containing genes from bacterial species, such as Bacillus, Streptococcus and Erwinia have been reported (Caimi et al., (1996)
Plant Physiol.
110:355-363; Ebskamp et al., (1994)
Biotechnol.
12:272-275; Rober et al., (1996)
Planta
199:528-536). Synthesis of fructan in these non-fructan-storing plants was demonstrated, but accumulation was often very low and in tissues where high levels of fructan were reported to have a detrimental effect on plant development.
Several important differences between transgenic plants expressing chimeric bacterial FTF genes and native fructan-storing plants were reported. The most obvious difference was in the size of the polymers synthesized. Transgenic lines containing bacterial FTF genes accumulate fructan with a DP of greater than 5000 (Ebskamp et al., (1994)
Biotechnol.
12:272-275; Caimi et al., (1996)
Plant Physiol.
110:355-363). Polymers synthesized in transgenic plants are, therefore, several thousand times larger than fructans which accumulate in plants such as chicory (
Cichorium intibus
L.) and Jerusalem artichoke (
Helinathus tuberosus
L.).
Differences in the specificity for donor and acceptor molecules have also been reported for bacterial and plant FTFs. The bacterial enzymes are known to release significant amounts of fructose to water as an acceptor (invertase activity), whereas the plant enzymes do not have invertase activity (Chambert, R. and Petit-Glatron, M. (1993) in
Inulin and Inulin Containing Crops
, A. Fuchs ed. Elsevier Press, Amsterdam. pp. 259-266). Fructose, liberated from sucrose by invertase activity, can not be used to increase the length of a polymer. Bacterial FTFs, therefore, convert sucrose to fructan less efficiently than do the plant enzymes.
The two classes of FTFs also differ in their affinity for sucrose, the sole substrate. Jerusalem artichoke sucrose-sucrose-fructosyltransferase (SST) has a Km for sucrose reported to be approximately 100 mM (Koops, A. and Jonker, H., (1994)
J. Exp. Bot.
45:1623-1631). By contrast, the bacterial enzyme has a much lower Km of approximately 20 mM (Chambert, R., and Petit-Glatron, M. (1991)
Biochem. J.
279:35-41). This difference may have a critical effect on fructan synthesis, resulting in higher or lower levels of accumulation, depending on the concentration of sucrose in the cell. The fundamental differences between FTF enzymes prevents meaningful predictions regarding the outcome of expression of plant genes in transgenic tissue, based on expression of bacterial FTF genes.
Predicting whether or not fructan would accumulate in a transgenic line containing the plant-derived FTF genes could be significantly enhanced if a greater understanding of the fructan metabolic pathway in native fructan-storing plants existed. The currently accepted model for fructan synthesis in plants suggests that synthesis is a two step reaction. The initial reaction involves the enzyme sucrose-sucrose-fructosyltransferase (SST). SST catalyzes the synthesis of a trisaccharide from two sucrose residues. The second step, chain elongation, is carried out by the enzyme fructan-fructan-fructosyltransferase (FFT), (Edelman J., and Jefford T. (1968)
New Phytol.
67:517-531. The model has been applied to all fructan-storing plants (ca 45000 species). However, it is based largely on data from a single species,
Helianthus tuberosus
, and has undergone several revisions. A recent study demonstrates that the SST can act alone in producing long chain fructan (Van der Ende, W. and Van Laere, A., (1996)
J. Exp. Bot.
47:1797-1803). Thus, additional revisions in the model are necessary and suggests that there is only a rudimentary knowledge of fructan synthesis in plants.
Examples of fructan synthesis in transgenic plants containing microbial or plant-derived FTF genes has been reported (Vijn, et al., (1997)
The Plant J.
11:387-398; Smeekens et al., WO 96/01904; Van Tunen et al., WO 96/21023; Sevenier et al., (1998)
Nature Biotechnology
16:843-846). This previous work involves expression of microbial or plant-derived SST genes only in transgenic dicotyledenous (dicots) plants. The present invention describes a method of increasing the level of fructan synthesis in transgenic monocotyledonous plants containing plant-derived SST genes or plant-derived SST and FFT genes.
Numerous differences between monocotyledonous (moncots) plants and dicots exist which inhibit useful extrapolation of events occurring in one plant based on data from another. These differences include, but are not limited to, the competition for sucrose as an energy source among biosynthetic pathways in various plant organs and among biosynthetic pathways in different plant species.
D
E. I. du Pont Nemours & Company
Fox David T.
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