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
1997-02-27
2001-07-24
Bui, Phuong 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
C536S023600, C536S023100, C536S023740, C435S419000, C435S410000, C435S243000, C435S252300, C435S320100, C435S468000, C435S254100, C435S254110, C800S278000, C800S290000
Reexamination Certificate
active
06265635
ABSTRACT:
The present invention relates to recombinant DNA techniques for producing plants and microorganisms capable of intra- or extracellularly expressing a protein exhibiting amylosucrase activity and catalyzing the synthesis of linear &agr;-1,4 glucans from sucrose. The present invention further relates to new DNA sequences and plasmids containing said DNA sequences which, after integration into a plant genome or after transformation in microorganisms, particularly bacteria or fungi, result in the expression of an enzyme catalyzing the synthesis of linear &agr;-1,4 glucans from sucrose, as well as to transgenic organisms (i.e., plants, fungi and microorganisms) containing the above-mentioned DNA sequences.
Linear &agr;-1,4 glucans are polysaccharides consisting of glucose monomers, the latter being exclusively linked to each other by &agr;-1,4 glycosidic bonds. The most frequently occurring natural &agr;-1,4 glucan is the amylose, a component of plant starch. Recently, more and more importance has been attached to the commercial use of linear &agr;-1,4 glucans. Due to its physico-chemical properties amylose can be used to produce films that are colorless, odorless and flavorless, non-toxic and biologically degradable. Already today, there are various possibilities of application, e.g., in the food industry, the textile industry, the glass fiber industry and in the production of paper.
One has also succeeded in producing fibers from amylose whose properties are similar to those of natural cellulose fibers and which allow to partially or even completely replace them in the production of paper. Being the most important representative of the linear &agr;-1,4 glucans, amylose is particularly used as binder for the production of tablets, as thickener of puddings and creams, as gelatin substitute, as binder in the production of sound-insulating wall panels and to improve the flow properties of waxy oils. Another property of the &agr;-1,4 glucans, which recently has gained increasing attention, is the capability of these molecules to form inclusion compounds with organic complexers due to their helical structure. This property allows to use the &agr;-1,4 glucans for a wide variety of applications. Present considerations relate to their use for the molecular encapsulation of vitamins, pharmaceutical compounds and aromatic substances, as well as their use for the chromatographic separation of mixtures of substances over immobilized linear &agr;-1,4 glucans. Amylose also serves as starting material for the production of so-called cyclodextrins (also referred to as cycloamyloses, cyclomaltoses) which in turn are widely used in the pharmaceutical industry, food processing technology, cosmetic industry and analytic separation technology. These cyclodextrins are cyclic maltooligosaccharides from 6-8 monosaccharide units, which are freely soluble in water but have a hydrophobic cavity which can be utilized to form inclusion compounds.
Today, linear &agr;-1,4 glucans are obtained in the form of amylose from starch. Starch itself consists of two components. One component forms the amylose as an unbranched chain of &agr;-1,4 linked glucose units. The other component forms the amylopectin, a highly branched polymer from glucose units in which in addition to the &agr;-1,4 links the glucose chains can also be branched via &agr;-1,6 links. Due to their different structure and the resulting physico-chemical properties, the two components are also used for different fields of application. In order to be able to directly utilize the properties of the individual components, it is necessary to obtain them in pure form. Both components can be obtained from starch, the process, however, requiring several purification steps and involving considerable time and money. Therefore, there is an urgent need to find possibilities of obtaining both components of the starch in a uniform manner. To this end, so far starch-producing plants have been altered by breeding or by genetic manipulation to produce starch with an altered amylose/amolypectin proportion. While the normal amylopectin percentage of corn starch is e.g., 70%, one succeeded in establishing a maize variety (waxy maize) by breeding whose starch consists of almost 100% of amylopectin (Akatsuka and Nelson, 1966, J. Biol. Chem. 241:2280-2285). Furthermore, several maize varieties having an increased amylose content (60-70%) have been produced by breeding, e.g., the amylose extender and dull varieties (Wolf et al., 1955, J. Am. Chem. Soc. 77:1654-1659; Boyer et al., 1976, Die Stärke: 28:405-410). Other plant species were used to obtain varieties that synthesize uniform starches in form of amylopectin, e.g., rice (Sano, 1984, Theor. Appl. Genet. 68:467-473) and barley (Shannon and Garwood, 1984, in: Whistler, Bemiller, Paschall, Starch: Chemistry and Technology, Academic Press, Orlando, 2nd Edition: 25-86) or that synthesize highly amylose-containing starch (e.g., peas). In addition to the above approaches of classical breeding, approaches based on the genetic manipulation of starch-producing plants have been reported.
Visser et al. (1991, Mol. Gen. Genet. 255:289-296), for example, describe that potato varieties synthesizing substantially pure amylopectin starch can be obtained by anti-sense inhibition of the gene that codes for the starch-granule bound starch synthetase. WO 92/14827 discloses the production of potato plants which due to the anti-sense inhibition of the expression of the branching enzyme produce a starch having an increased amylose/amylopectin proportion. However, the plants described in WO 92/14827 do not produce a highly amylose-containing starch.
Despite numerous attempts and varied approaches, one has so far not succeeded in obtaining plants producing a pure amylose starch. Also, so far no possibility has been described to produce highly pure amylose or pure linear &agr;-1,4 glucans by using other processes, e.g., genetically engineered microorganisms.
Furthermore, so far no DNA sequences have been found that encode enzymes that would be capable of catalyzing the synthesis of linear &agr;-1,4 glucans in plants, fungi, microorganisms or in vitro.
It is therefore the object of the present invention to provide DNA sequences and processes that are capable of allowing the production of plants, fungi and microorganisms capable of synthesizing linear &agr;-1,4 glucans.
The object of the present invention is achieved by the provision of the embodiments characterized by the patent claims.
The invention therefore relates to DNA sequences coding for proteins having the enzymatic activity of an amylosucrase.
Particularly, it relates to DNA sequences coding for a protein having the amino acid sequence indicated under Seq ID No. 1, and to the encoding DNA sequence indicated under Seq ID No. 1. The present invention furthermore relates to DNA sequences hybridizing to the above-mentioned sequences of the invention and coding for a protein having the enzymatic activity of an amylosucrase, as well as to DNA sequences which, due to the genetic code, are degenerate in comparison to the above-mentioned DNA sequences of the invention.
The term “hybridization” in this context means hybridization under conventional hybridization conditions, preferably under stringent conditions such as described by, e.g., Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
In another embodiment, the invention relates to DNA sequences coding for a protein having the enzymatic activity of an amylosucrase which are obtainable by a process comprising the following steps:
(a) preparing a genomic or a cDNA library on the basis of the genomic DNA or the mRNA of cells of an organism;
(b) transforming a suitable host with the library constructed in step (a);
(c) subjecting the transformed cells to iodine vapor;
(d) identifying the cells that are stained blue;
(e) isolating and cultivating the cells identified in step
(d); and
(f) isolating the genomic DNA insert or the cDNA insert from the transformed c
Buttcher Volker
Kossmann Jens
Welsh Thomas
Bui Phuong T.
Fish & Neave
Haley Jr. James F.
Plantec Biotechnologie GmbH Forschung & Entwicklung
Shin Elinor K.
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