Nucleic acid molecules coding for debranching enzymes from...

Details Nucleic acid molecules coding for debranching enzymes from... Nucleic acid molecules coding for debranching enzymes from...

2001-05-08

2004-07-13

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, C800S286000, C800S320100, C435S320100, C435S101000, C435S412000, C435S419000, C536S023600, C536S024500

Reexamination Certificate

active

06762346

ABSTRACT:

The present invention relates to nucleic acid molecules encoding proteins from
maize
with the enzymatic activity of a debranching enzyme (R enzyme). The invention further relates to transgenic plants and plant cells, in which an amylopectin with an altered degree of branching is synthesized due to the expression of an additional debranching enzyme activity from
maize
or due to the inhibition of an endogeneous debranching enzyme activity. The invention also relates to the starch obtainable from said transgenic plant cells and plants.
Starch plays an important role as storage substance in a multitude of plants and also as a regenerative, industrially usable raw material and has gained increasing significance. For the industrial use of starch it is necessary that it meets the demands of the processing industry with respect to its structure, form and/or other physico-chemical parameters. In order to enable the use in as many areas as possible it is furthermore necessary to achieve a large variety of substances.
The polysaccharide starch is made up of chemically homogeneous basic components, namely the glucose molecules. However, it constitutes a highly complex mixture of various types of molecules which differ from each other in their degree of polymerization and in the degree of branching. One differentiates between amylose-starch, a basically non-branched polymer made up of &agr;-1,4-glycosidically branched glucose molecules, and amylopectin-starch, a branched polymer, in which the branching results from additional &agr;-1,6-glycosidic interlinkings.
In plants used typically for the production of starch, such as
maize
or potato, the synthesized starch consists of approximately 25% amylose-starch and of about 75% amylopectin-starch. In the case of
maize
, for example, a further branched polysaccharide, apart from amylopectin, occurs, namely the so-called phytoglycogen which differs from amylopectin by exhibiting a higher degree of branching and different 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 scope of the present application the term amylopectin is used in such a way as to comprise the phytoglycogen.
With respect to the homogeneity of the basic component starch for its use in the industrial area, starch-producing plants are needed which contain, for example, only the component amylopectin or only the component amylose. For a number of other uses plants are needed that synthesize amylopectin types with different degrees of branchings.
Such plants may for example be obtained by breeding or by means of mutagenesis techniques. It is known for various plant species, such as for
maize
, that by means of mutagenesis varieties may be produced in which only amylopectin is formed. Also in the case of potato a genotype was produced from a haploid line by means of chemical mutagenesis. Said genotype does not form amylose (Hovenkamp-Hermelink, Theor. Appl. Genet. 75 (1987), 217-221).
From Visser et al. (Mol. Gen. Genet. 225 (1991), 289) and WO 92/11376 it is furthermore known that by means of an antisense-inhibition of the gene of the granule-bound starch synthase in potato varieties may be produced that mainly synthesize pure amylopectin. Moreover, DNA sequences are known from WO 92/14827 that encode a branching enzyme (Q enzyme), that introduces &agr;-1,6 branchings into amylopectin starch. By means of these DNA sequences it should be possible to produce transgenic plants in which the amylose/amylopectin ratio of the starch is altered.
For a further targeted modification of the degree of branching of starch synthesized in plants by means of recombinant DNA techniques, it is still necessary to identify DNA sequences that encode enzymes participating in the starch metabolism, particularly in the branching of starch molecules.
Apart from the Q enzymes that introduce branchings into starch molecules, enzymes occur in plants which are capable of dissolving branchings. These enzymes are called debranching enzymes and are classified as three groups according to their substrate specifity:
(a) Pullulanases which also, apart from pullulan, use amylopectin as a substrate, occur in microorganisms, e.g. Klebsiella, and in plants. In plants these enzymes are also called R enzymes.
(b) Isoamylases, which do not use pullulan, but glycogen and amylopectin as a substrate, also occur in microorganisms and plants. Isoamylases were described for example in the case of
maize
(Manners & Rowe, Carbohydr. Res. 9 (1969), 107) and potato (Ishizaki et al., Agric. Biol. Chem. 47 (1983), 771-779).
(c) Amylo-1,6-glucosidases were described in the case of mammals and yeast; as a substrate, they make use of dextrines.
In the case of sugar beet, Li et al. (Plant Physiol. 98 (1992), 1277-1284) could only prove the occurrence of one debranching enzyme of the pullulanase type, apart from five endo- and two exoamylases. This enzyme having a size of approximately 100 kD and an optimum pH value of 5.5 is located within the chloroplasts. A debranching enzyme using pullulan as a substrate was also described for spinach. The debranching enzyme from spinach as well as that from sugar beet exhibit a fivefold lower activity in a reaction with amylopectin as substrate when compared to a reaction with pullulan as a substrate (Ludwig et al., Plant Physiol. 74 (1984), 856-861; Li et al., Plant Physiol. 98 (1992), 1277-1284).
In the case of the agriculturally significant starch-storing cultured plant potato, the activity of a debranching enzyme was examined by Hobson et al. (J. Chem. Soc., (1951), 1451). It was proven that the respective enzyme, contrary to the Q enzyme, does not exhibit any activities leading to an elongation of the polysaccharide chain, but merely hydrolyses &agr;-1,6-glycosidic bonds. So far, however, the enzyme could not be characterized in more detail.
In the case of potato, methods for the purification of the debranching enzyme as well as partial peptide sequences of the purified protein have already been proposed (WO 95/04826).
The purification of a debranching enzyme and the isolation of a corresponding cDNA has by now been described for spinach (Renz et al., Plant Physiol. 108 (1995), 1342).
For the most significant starch-delivering plant, namely
maize
, so far only the existence of one debranching enzyme was described in the prior art. Due to its substrate specificity, this debranching enzyme is classified as an isoamylase (see e.g. Hannah et al., Scientia Horticulturae 55 (1993), 177-197 or Garwood (1984) in Starch Chemistry and Technology, Whistler, R. L., BeMiller, J. N., Puschall, E. F. (eds.), Academic Press San Diego, New York, Boston, 25-86). The corresponding mutant was designated su (sugary). The gene of the sugary locus was cloned recently (see James et al., Plant Cell 7 (1995), 417-429). So far no other gene locus apart from the sugary locus is known for
maize
, which encodes a protein with debranching enzyme activity. Thus, there is so far no indication as to the existence of further types of debranching enzymes from
maize
. If transgenic
maize
plants are to be produced which no longer exhibit any debranching enzyme activity, e.g. in order to achieve a modification of the degree of branching of the amylopectin starch, it is necessary to identify all debranching enzymes occurring in
maize
and to isolate the corresponding genes or cDNA sequences.
Therefore, the technical problem underlying the present invention is to identify further debranching enzymes possibly occurring in
maize
and to isolate corresponding nucleic acid molecules encoding these enzymes.
This problem is solved by the provision of the embodiments as defined in the claims.
Thus, the present invention relates to nucleic acid molecules encoding proteins with the biological activity of a debranching enzyme from
maize
or a biologically active fragment thereof, wherein such nucleic acid molecules preferably encode a debranching enzyme from
maize
that exhibits the amino acid sequence depicted in SEQ ID No. 2. In a particula

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