Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-06-17
2002-04-30
Wang, Andrew (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S101000, C435S069100, C435S069800, C435S410000, C435S419000, C800S284000, C800S287000, C800S263000
Reexamination Certificate
active
06379968
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a transgenic organism.
In particular, the present invention relates to a transgenic starch producing organism having an increased ability to synthesize starch and one that is capable of producing starch in high yields. More particularly the present invention relates to a transgenic organism comprising a nucleotide sequence coding for exogenous ADP glucose pyrophosphorylase (AGP).
In a preferred embodiment the present invention relates to a transgenic plant or plant cell capable of expressing exogenous AGP in the starch producing centres in the plant, namely the chloroplasts and the amyloplasts. The invention also relates to a recombinant DNA construct for use in the transformation of a plants or plant cell showing enhanced starch production, and plants and plant cells transformed with the recombinant DNA construct.
ADP glucose pyrophosphorylase (E.C.2.7.7.27) (AGP) is one of the primary enzymes involved in the biosynthesis of starch and glycogen in organisms such as plants, algae, fungi and bacteria, particularly plants.
AGP catalyses the reaction:
&agr;-glucose-1-P+ATP⇄ADP-glucose+PP
1
the product ADP-glucose being the major donor of glucose in the biosynthesis of starch in plants. Moreover, that reaction has been shown to be the rate limiting factor in the synthesis of starch in organisms such as plants, the rate of that reaction in turn being critically dependent upon the AGP concentration. Because of that, AGP has been the subject of intensive investigation and for a general review of recent studies on plant AGP, reference should be made to Kleczkowski et al: Z. Naturforsch. 46c, 605-612 (1991).
As reported by Kleczkowski et al (ibid) and elsewhere, AGP is widely distributed throughout the plant kingdom and is found in some starch producing bacteria, such as
E. coli.
Plant AGP exists as a tetramer (210 to 240 kDa) composed of two small sub-units (50 to 55 kDa) and two large sub-units (51 to 60 kDa) in contrast to bacterial AGP which appears to consist of four units of equal size. AGP has also been shown to be produced in cyanobacteria and in algae, where its tetrameric structure is similar to that in plants, i.e. two large and two small sub-units, rather than the homotetrameric structure found in ordinary bacteria.
Because of the commercial importance of starch, primarily as a foodstuff but also as an important industrial chemical, AGP itself and recombinant DNA constructs containing DNA sequences encoding AGP for the transfection of plants and plant cells as a means of increasing plant AGP concentration and hence increased biosynthesis of starch in plants and increased starch yields, have formed the subject matter of several recently published patent applications.
For example, in EP-A-0368506 a method of extracting AGP from wheat leaf and wheat endosperm is disclosed. Also disclosed are the cDNA sequences encoding wheat leaf and wheat endosperm AGP, and various plasmids containing those sequences for subsequent insertion into plants to provide plants having an increased ability to synthesise starch, although that latter step is not described in detail, nor are any examples given of transgenic plants containing those constructs.
WO 91/19806 discloses transformed plant cells and plants having elevated levels of starch and starch biosynthesis achieved by incorporating into the plant genome a DNA construct comprising in sequence a plant promoter, a DNA sequence encoding a fusion polypeptide consisting of a plastid transit peptide and a bacterial (
E. coli
) AGP, and a 3′-non-translated region which functions in the plant cell to cause transcriptional termination and the addition of a polyadenylated tail to the 3′-end of the corresponding DNA sequence. The DNA sequence encoding
E. coli
AGP is given, as well as the deduced amino acid sequence. Transgenic potato and tomato plants transformed with the
E. coli
AGP gene are shown to produce increased starch yields. It is suggested that other bacterial sources besides
E. coli,
and also algae, may be used as a source for the AGP gene to be used in the transformation of the plants and plant cells to provide increased starch yields. However, there is no mention of the isolation of nucleotide sequences coding for AGP enzymes from those other sources or their expression in such transgenic systems.
A similar disclosure is contained in WO 92/11382 which likewise discloses the transformation of plants, especially potato plants, with bacterial (
E. coli
) DNA encoding bacterial AGP, with the objective of increasing starch biosynthesis and starch yield in such plants.
A slightly different objective is set out in EP-A-0455316. There the objective is to increase sugar and protein concentrations in plant-based foodstuffs at the expense of starch formation. That is achieved by incorporating into the plant genome a DNA sequence encoding AGP, but in an inverted orientation in the transformation vector. Transcription of the reversed sequence results in an anti-sense mRNA which inhibits the production of AGP in the plant cell leading to reduced AGP activity and reduced starch production.
All plant AGPs investigated so far have been reported to be strongly activated by 3-phospho-glycerate (PGA) and inhibited by inorganic phosphate (P
i
). Also, the PGA/P
i
ratio in the chloroplasts and amyloplasts where biosynthetic starch production is concentrated is believed to play a key regulatory role in starch synthesis. It is known, for example, that chloroplast PGA/P
i
ratios are at the highest activity during the daylight hours, i.e. during photosynthesis, which period coincides with the peak period of starch production in the chloroplasts. The regulation of the AGP formation in non-photosynthetic tissues is less well understood, but the activatory and inhibitory roles of PGA and P
i
, respectively, i.e. the PGA/P
i
ratio, is believed still to play an important part.
SUMMARY OF THE INVENTION
The present invention addresses the problem of how to increase AGPase levels and/or starch levels in starch producing organisms.
According to a first aspect of the present invention there is provided a transgenic starch producing organism comprising a nucleotide sequence coding for an exogenous ADP glucose pyrophosphorylase (AGP) enzyme or a sub-unit thereof which retains the enzymatic activity of the AGP enzyme, wherein the nucleotide sequence is capable of being expressed in the organism; characterised in that the activity of the enzyme or sub-unit thereof is substantially independent of any level of in vivo 3-phospho-glycerate and/or any in vivo level of inorganic phosphate; and further characterised in that the activity of the enzyme or sub-unit thereof is not stimulated by fructose-1,6bisP and/or is not inhibited by AMP.
According to a second aspect of the present invention there is provided a transgenic starch producing organism comprising exogenous ADP glucose pyrophosphorylase (AGP) enzyme or a sub-unit thereof which retains the enzymatic activity of the AGP enzyme, wherein the activity of the enzyme or sub-unit thereof is substantially independent of any level of in vivo 3-phospho-glycerate and/or any in vivo level of inorganic phosphate and wherein the activity of the enzyme or sub-unit thereof is not stimulated by fructose-1,6-bisP and/or is not inhibited by AMP.
According to a third aspect of the present invention there is provided a potato tuber containing an enhanced starch content.
According to a fourth aspect of the present invention there is provided a method of increasing the rate and/or yield of starch production in an organism, especially a plant or a plant cell, which method comprises introducing into an organism a nucleotide sequence according to the present invention to form a transgenic organism according to the present invention and expressing the nucleotide sequence.
According to a fifth aspect of the present invention there is provided a method of increasing the rate and/or yield of starch production in an organism, especially a plant or a plant cell
Danisco A/S
Katcheves Konstantina
Knobbe Martens Olson & Bear LLP
Wang Andrew
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