Gene encoding oxalate decarboxylase from aspergillus phoenices

Details Gene encoding oxalate decarboxylase from aspergillus phoenices Gene encoding oxalate decarboxylase from aspergillus phoenices

1999-04-12

2001-10-16

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C800S288000, C800S301000, C800S306000, C800S322000, C800S312000, C800S313000, C435S468000, C435S419000, C435S418000, C435S416000, C435S415000

Reexamination Certificate

active

06303846

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a novel nucleic acid sequence encoding oxalate decarboylyase isolated from
Aspergillus phoenices
and to use of the nucleic acid sequence to produce its encoded protein.
BACKGROUND OF THE INVENTION
Oxalic acid (oxalate) is a diffusable toxin associated with various plant diseases, particularly those caused by fungi. Some leafy green vegetables, including spinach and rhubarb, produce oxalate as a nutritional stress factor. When plants containing oxalate are consumed in large amounts, they can be toxic to humans.
Oxalate is used by pathogens to gain access into and subsequently throughout an infected plant. See for example, Mehta and Datta,
The Journal of Biological Chemistry,
266:23548-23553, 1991; and published PCT Application WO92/14824.
Field crops such as sunflower, bean, canola, alfalfa, soybean, flax, safflower, peanut, clover, as well as numerous vegetable crops, flowers, and trees are susceptible to oxalate-secreting pathogens. For example, fungal species including Sclerotinia and Sclerotium use oxalic acid to provide an opportunistic route of entry into plants, causing serious damage to crops such as sunflower.
Because of the role of oxalate in plant disease and toxicity, compounds that inhibit oxalate mediated disease, and particularly genes encoding such inhibitory degrading molecules, are greatly needed.
Enzymes that utilize oxalate as a substrate have been identified. These include oxalate oxidase and oxalate decarboxylase. Oxalate oxidase catalyzes the conversion of oxalate to CO
2
and H
2
O
2
. A gene encoding barley oxalate oxidase has been cloned from a barley root cDNA library and sequenced (See PCT publication No. WO92/14824). A gene encoding wheat oxalate oxidase activity (Germin) has been isolated and sequenced, (PCT publication No. WO 94/13790) and the gene has been introduced into a canola variety. Canola plants harboring the gene appeared to show some resistance to
Sclerotinia sclerotiorum,
in vitro (Dumas, et al., 1994, Abstracts: 4th Int'l.
Congress of Plant Molecular Biology,
#1906).
Oxalate decarboxylase converts oxalate to CO
2
and formic acid. A gene encoding oxalate decarboxylase has been isolated from
Collybia velutipes
(now termed
Flammulina velutipes
) and the cDNA clone has been sequenced (WO94/12622, published Jun. 9, 1994). Oxalate decarboxylase activities have also been described in
Aspergillus niger
and
Aspergillus phoenices
(Emiliani et al., 1964,
ARCH. Biochem. Biophys.
105:488-493), however the amino acid sequence and nucleic acid sequence encoding these enzyme activities have not been isolated or characterized.
Enzymatic assays for clinical analysis of urinary oxalate provide significant advantages in sensitivity and qualification Obzansky, et al., 1983,
Clinical Chem.
29:1815-1819. For many reasons, including reactivity with interfering analytes and the high cost of available oxalate oxidase used in this diagnostic assay, alternative enzymes are needed. (Lathika et al., 1995,
Analytical Letters
28: 425-442).
In this application, we disclose the isolation, cloning, and sequencing of a unique gene encoding an oxalate decarboxylase enzyme from
Aspergillus phoenices.
The gene is useful in producing highly purified
Aspergillus phoenices
oxalate decarboxylase enzyme, in producing transonic plant cells and plants expressing the enzyme in vivo, and in diagnostic assays of oxalate.
SUMMARY OF THE INVENTION
The present invention provides a nucleic acid sequence encoding oxalate decarboxylase isolated from
Aspergillus phoenices
(APOXD). The gene sequence [Seq ID No:1], the recombinant protein produced therefrom [Seq ID No:2], and vectors, transformed cells, and plants containing the gene sequence are provided as individual embodiments of the invention, as well as methods using the gene or its encoded protein. The nucleic acid is useful for producing oxalate decarboxylase for commercial applications, including degradation of oxalic acid, protection against oxalic acid toxicity, and diagnostic assays to quantify oxalate.
The nucleic acid of the invention is also useful as a selectable marker. Growth of plant cells in the presence of oxalic acid favors survival of plant cells transformed with the coding sequence of the gene.
The present invention also includes compositions and methods for degrading oxalic acid, in providing protection against oxalic acid toxicity, and in combating and providing protection against plant pathogens that utilize oxalate to gain access to plant tissue or otherwise in the course of the pathogenesis of the disease. Oxalate decarboxylase from
Aspergillis phoenices
(APOXD) of the present invention is combined with an appropriate carrier for delivery to the soil or plants. Alternatively, plant cells are transformed with the nucleic acid sequence of the invention for expression of APOXD in vivo.


REFERENCES:
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patent: WO 92/14824 (1992-09-01), None
patent: WO 94/12622 (1994-06-01), None
patent: WO 94/13790 (1994-06-01), None
patent: WO 96/30530 (1996-10-01), None
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