Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1998-07-30
2002-04-23
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S300000, C435S410000, C435S418000, C435S419000, C435S255200, C435S320100, C536S063000, C536S023100, C536S023200, C536S023600, C536S023700
Reexamination Certificate
active
06376753
ABSTRACT:
BACKGROUND OF THE INVENTION
The gene CYP76B1 of a cytochrome P450 has been isolated from
Helianthus tuberosus
tuber. The expression of this gene in yeast has shown that it encoded an enzyme very actively catalyzing the O-dealkylation of various exogenous molecules with a high efficiency. It could, for this reason, be used as oxygenase, as biocatalyst either for the degradation of environmental pollutants or for the biosynthesis of organic compounds such as medicaments, perfumes or pigments.
The expression of CYP76B1 is strongly induced in plants brought into contact with certain exogenous metals or organic compounds. This induction may be exploited for the detection of environmental pollutants.
CYP76B1 metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresofurins and several herbicides of the class of phenylureas. CYP76B1 also catalyzes the mono- and didealkylation of phenylureas such as the two herbicides chlortoluron and isoproturon, with turnover rates comparable to those reported for physiological substrates and produces non-phytotoxic compounds. CYP76B1 can therefore be used to alter the resistance of plants sensitive to this family of herbicides and for soil and groundwater bioremediation.
1. Field of Invention
The present invention relates to purified polypeptides and DNA sequences of CYP76B1, a cytochrome P450 molecule, which has been isolated from
Helianthus tuberosus
tuber, and a method for preparing the same. It further relates to methods for detecting environmental chemical pollution comprising determining the presence of CYP76B1 polypeptide. It furthermore relates to methods for producing transgenic plants incorporating a gene or a gene fragment CYP76B1 in a bioremediation perspective and for an agricultural use.
2. Description of the Related Art
Plants respond to environmental stress with a wide range of adaptative changes, including the induction of defense mechanisms. Plants defense against the attack from pathogens and predators, or against changes in environmental conditions like drought, salinity, increased UV light or extreme temperatures have been largely documented. Much less is known about plant response to chemical agression. Increasing amounts of pesticides and other industrial chemicals are introduced in the environment without real knowledge of their impact on plant metabolism, development and organoleptic properties. Evidence has been obtained that plant can accumulate, transform and store exogenous chemicals as conjugated, compartmented or bound residues (2, 3). The enzymatic equipment allowing plants to cope with the toxicity of xenobiotics is very similar to the enzymes involved in the metabolism of drugs in the liver of mammals (4, 5, 6, 7). It includes reductive, oxidative and hydrolytic enzymes, various transferases for the formation of conjugated metabolites, and systems for the regeneration of antioxidants like glutathione. The cytochrome P450s constitute one of the principal classes of enzymes responsible for this metabolism (12, 7). A characteristic common to both animal and plant enzymes is their inducibility by drugs and other exogenous toxins.
As in animals, cytochromes P450 form, in plants, the main class of oxidases involved in the metabolism of exogenous molecules, including herbicides and pollutants (8, 9, 10, 11, 12). They have been shown to catalyze hydroxylation, S-oxidation, N- or O-dealkylation of environmental chemicals, thereby increasing their hydrophylicity and allowing their subsequent conjugation or immobilization. Increased P450 content and enzymatic activities following treatment with metals or organic molecules including drugs, herbicides, herbicide safeners, solvents and industrial pollutants have been observed in many plant species (1, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). The induction of certain cytochrome P450s and of their enzymatic activities is currently used as an early marker of chemical pollution in marine environments in particular (46, 47, 26). Studies, which are very preliminary, have suggested that an induction of P450s may be detected in a polluted environment (47). In the case of the cinnamate 4-hydroxylase (C4H), increase in catalytic activity induced by chemicals was paralleled by an increase in the steady-state level of CYP73A1 transcripts (22).
7-Ethoxycoumarin is a synthetic fluorescent molecule, very similar to natural coumarins, widely used to assay the catalytic activity of cytochromes P450 involved in the metabolism of foreign compounds (23, 24). Increase in 7-10 ethoxycoumarin O-deethylase (ECOD) activity, fast and easy to measure, has often been taken as an index of the induction of these P450s in animals following environmental chemical stress (25, 26). P450-dependent ECOD activity was detected in several plant species (27, 28, 29). In both dicots and monocots, the activity was found strongly induced in response to exogenous chemicals (1, 27, 29). A more detailed analysis, performed on
Helianthus tuberosus
tuber, showed that the induction of ECOD activity was often several fold higher than that of a physiological activity like C4H (22). ECOD thus appeared as a good potential marker of chemical contamination. Fluorescence assay of ECOD activity is impossible in extracts from green plants (interference with chlorophyll).
Because of the enormous losses caused by indesirable plant growth, the problem of weed control is a major one in the agricultural economy. Herbicides are broadly used for controlling the growth of weeds in crops. Among herbicides, compounds show broad spectrum control, making them useful where complete eradication of vegetation is needed, and others show selective control with tolerance to agronomic crops. New methods for producing transgenic plants resistant to herbicides have been developed (EP 730030). These transgenic plants are transformed and generally express or overexpress an enzyme capable of detoxifying the chosen herbicide, making them resistant to that herbicide.
Among the herbicides or pesticides which have been developed, many of them are substitued urea or thiourea compounds, represented by the formula: R1R2N—CO—NR3R4 or R1R2N—CS—NR3R4, and are used for destruction and prevention of weeds (WO 90/06680; WO 95/22547; U.S. Pat. Nos. 2,655,444; 2,655,447; 2,857,430; 3,912,496; 5,393,733; 5,512,535 FR 69 03 235; FR 71 06 517).
Most of these substituted urea or thiourea compounds have cyclic groups and amine radicals, which can be substituted with alkoxy radicals and/or alkyl radicals. Among them, the phenylurea class which can be represented by the formula: (substituted-phenyl)—NH—CO—NR1R2, in which the phenyl group and the secondary amine radical can be substituted with alkoxy and/or alkyl radicals. This phenylurea class can be illustrated by chlortoluron (N-(3-chloro-p-tolyl)-N′,N′-dimethylurea) and by isoproturon (N-(4-isopropylphenyl)-N′,N′-dimethylurea).
The first coding sequence of plant P450 (CYP73A1) capable of metabolizing an herbicide (i.e. capable of hydroxylating chlortoluron) has been isolated in the laboratory (33). The reaction turnover is however too low (0.014 min
−1
) for the enzyme to be capable of substantially increasing the resistance of plants to chlortoluron.
It has, moreover, been demonstrated that the resistance of plants to herbicides (chlortoluron* and chlorsulfuron) could be manipulated (increased or decreased) by genetic transformation using animal or bacterial P450 genes (48, 49, 50). No experiment of this type has been carried out up until now with plant genes. The work carried out with bacterial and animal genes strongly suggests, however, that it is possible to transfer the tolerance to herbicides from one plant to another.
The advantage of using plant genes is two-fold: i) the efficiency of the biotransformation with the plant P450 appears to be substantially greater than that obtained with the animal P450s tested, ii) the transformation of plants with plant genes is accepted better by public opinion.
The present invention is directed to purifie
Batard Yannick
Didierjean Luc
Durst Francis
Robineau Tiburce
Werck-Reichhart Daniele
Centre National de la Recherche Scientifique
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Nelson Amy J.
Zaghmout Ousama M. F.
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