DNA molecules coding for debranching enzymes derived from plants

Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase

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4352523, 4353201, 435410, 435412, 435417, 536 232, 536102, 800278, 800295, C12N 944, C12N 120, C12N 1500, C07H 2104

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061176656

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BRIEF SUMMARY
The present invention relates to DNA molecules coding for proteins from plants having the enzymatic activity of a debranching enzyme (R enzyme). The invention furthermore relates to a process for modifying the branching degree of amylopectin synthesized in plants, and to plants and plant cells in which an amylopectin having a modified branching degree is synthesized due to the expression of an additional debranching enzyme activity or the inhibition of an endogenous debranching enzyme activity, as well as to the starch obtainable from said plant cells and plants.
Starch plays an important role both as storage substance in a variety of plants and as reproductive, commercially useful raw material and is gaining significance. For the industrial application of starch it is necessary that the starch meets the requirements of the manufacturing industry in terms of its structure, form and/or other physico-chemical parameters. For the starch to be useful in as many fields of application as possible it is furthermore necessary that it is obtainable in as many forms as possible. While the polysaccharide starch is composed of chemically uniform components, the glucose molecules, it is a complex mixture of different molecule forms that exhibit differences as regards their polymerization degree and the presence of branches. One distinguishes the amylose starch, an essentially unbranched polymer of .alpha.-1,4 glycosidically linked glucose molecules from the amylopectin starch, a branched polymer, the branches of which are the result of additional .alpha.-1,6 glycosidic bonds.
In plants typically used for starch production, such as maize or potato, both starch forms are present in a ratio of about 25 parts of amylose to 75 parts of amylopectin. In addition to amylopectin, maize, for example, exhibits; another branched polysaccharide, the so-called phytoglycogen which differs from the amylopectin by a higher branching degree and a differing solubility (see, e.g., Lee et al., Arch. Biochem. Biophys. 143 (1971), 365-374; Pan and Nelson, Plant Physiol. 74 (1984), 324-328). In the present application the term amylopectin is intended to comprises phytoglycogen.
With a view to the uniformity of the basic compound starch for its industrial application starch-producing plants are required that contain, e.g., either only the component amylopectin or only the component amylose. For other applications plants are required that synthesize forms of amylopectin of different degrees of branching. Such plants can be generated, e.g., by breeding or mutagenesis techniques. It is known of certain plant species, e.g., maize, that mutagenesis can be used to generate varieties producing only amylopectin. For potato, a genotype was generated by chemical mutagenesis for a haploid line that does not produce amylose (Hovenkamp-Hermelink, Theor. Appl. Genet. 75 (1987), 217-221). Haploid lines, however, or the homozygous diploid or tetraploid lines derived thereof are not useful for agricultural purposes. Mutagenesis techniques, however, cannot be applied to the tetraploid lines that are interesting for agriculture since due to the presence of four different genotypes inactivation of all copies of a gene is not technically feasible. Therefore, in the case of potato, one must fall back on other techniques, e.g., the specific genetically engineered modification of plants.
For example it is known from Visser et al. (Mol. Gen. Genet.. 225 (1991), 289) and WO 92/11376 that varieties can be generated by anti-sense inhibition of the gene for the starch granule-bound starch synthase in potato that synthesize substantially pure amylopectin starch.
WO 92/14827 discloses DNA sequences coding for a branching enzyme (Q enzyme) that introduces .alpha.-1,6 branches into amylopectin starch. With these DNA sequences it should be possible to generate transgenic plants which exhibit a modified amylose/amylopectin ratio of the starch.
In order to furthermore specifically modify the branching degree of starch synthesized in plants by using genetic engineering it is stil

REFERENCES:
patent: 4454161 (1984-06-01), Okada et al.
R.C. Black et al., "Genetic Interactions Affecting Maize Phytoglycogen and the Phytoglycogen-Forming Branching Enzyme", Genetics, 53, pp. 661-668 (Apr. 1966).
L. Curtis Hannah et al. "Biotechnological Modification of Carbohydrates for Sweet Corn and Maize Improvement", Scientia Horticulturae, 55, pp. 177-197 (1993).
Yukuo Ishizaki et al., "Debranching Enzymes of Potato Tubers (Solanum tuberosum L.). I. Purification and Some Properties of Potato Isoamylase", Agric. Biol. Chem., 47(4), pp. 771-779 (1983).
Nobuhiro Katsuragi et al., "Entire Nucleotide Sequence of the Pullulanese Gene of Klebsiella aerogenes W70", Journal of Bacteriology, 169, pp. 2301-2306 (May 1987).
Bin Li et al., "Characterization and Subcellular Localization of Debranching Enzyme and Endoamylase from Leaves of Sugar Beet", Plant Physiol., 98, pp. 1277-1284 (1992).
Isabella Ludwig et al., "Purification and Properties of Spinach Leaf Debranching Enzyme", Plant Physiol., 74, pp. 856-861 (1984).
D.J. Manners et al., "Studies on Carbohydrate-Metabolising Enzymes: Part XX. Sweet Corn Debranching Enzymes", Carbohyd. Res., 9, pp. 107-121 (1969).
David Pan et al., "A Debranching Enzyme Deficiency in Endosperms of the Sugary-1 Mutants of Maize", Plant Physiol., 74, pp. 324-328 (1984).
A. Renz et al., "S.oleracea L. mRNA for Pullulanase", EMBL Sequence Database, Accession No. X83969 (Jan. 19, 1995).

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