Back to results
US 5,928,937 AGranted
Structure-based designed herbicide resistant products
AssigneeAmerican Cyanamid Company
InventorsGenichi Kakefuda, Karl-Heinz Ott, Jae-Gyu Kwagh, Gerald W. Stockton
Life of this patent
Estimating the term…
Abstract
Disclosed herein are structure-based modelling methods for the preparation of acetohydroxy acid synthase (AHAS) variants, including those that exhibit selectively increased resistance to herbicides such as imidazoline herbicides and AHAS inhibiting herbicides. The invention encompasses isolated DNAs encoding such variants, vectors that include the DNAs, and methods for producing the variant polypeptides and herbicide resistant plants containing specific AHAS gene mutations. Methods for weed control in crops are also provided.
Claims30
An isolated DNA encoding an acetohydroxy acid synthase (AHAS) variant protein, said variant protein comprising an AHAS protein modified by
- (i) substitution of at least one different amino acid residue at an amino acid residue of the sequence of FIG. 1, SEQ ID NO:1, selected from the group consisting of S52, M53, E54, A84, A95, T96, S97, G98, P99, G100, A101, R127, R128, M129, I187, T259, T260, L261, M262, G263, R276, T283, V284, G300, V301, T308, G309, K310, I311, E312, A313, F314, A315, S316, R317, A318, K319, I320, E329, I330, K332, N333, K334, Q335, T404, G413, V414, G415, Q416, H417, Q418, M419, W420, A421, A422, L434, S435, S436, A437, G438, L439, G440, A441, M442, G443, D467, G468, S469, L471, N473, L477, M479, Q495, H496, L497, Y508, K509, A510, N511, R512, A513, H514, T515, S524, H572, Q573, E574, H575, V576, L577, P578, M579, I580, P581, G583, G584, functional equivalents of any of the foregoing, and any combination of any of the foregoing;
- (ii) deletion of up to 5 amino acid residues preceding, or up to 5 amino acid residues following at least one amino acid residue of the sequence of FIG. 1, SEQ ID NO:1, selected from the group consisting of S52, M53, E54, A84, A95, T96, S97, G98, P99, G100, A101, R127, R128, M129, I187, T259, T260, L261, M262, G263, R276, T283, V284, G300, V301, T308, G309, K310, I311, E312, A313, F314, A315, S316, R317, A318, K319, I320, E329, I330, K332, N333, K334, Q335, T404, G413, V414, G415, Q416, H417, Q418, M419, W420, A421, A422, L434, S435, S436, A437, G438, L439, G440, A441, M442, G443, D467, G468, S469, L471, N473, L477, M479, Q495, H496, L497, Y508, K509, A510, N511, R512, A513, H514, T515, S524, H572, Q573, E574, H575, V576, L577, P578, M579, I580, P581, G583, G584, functional equivalents of any of the foregoing, and any combination of any of the foregoing;
- (iii) deletion of at least one amino acid residue or a functional equivalent thereof between Q124 and H150 of the sequence of FIG. 1, SEQ ID NO:1,;
- (iv) addition of at least one amino acid residue or a functional equivalent thereof between Q124 and H150 of the sequence of FIG. 1, SEQ ID NO:1,;
- (v) deletion of at least one amino acid residue or a functional equivalent thereof between G300 and D324 of the sequence of FIG. 1, SEQ ID NO:1,;
- (vi) addition of at least one amino acid residue or a functional equivalent thereof between G300 and D324 of the sequence of FIG. 1, SEQ ID NO:1,;
- or
- (vii) any combination of any of the foregoing, or functional equivalents thereof,
- said DNA having the property of conferring herbicide resistance.
6 more elements
A method for production of herbicide-resistant AHAS protein, said method comprising:
- (a) selecting an amino acid position in an AHAS protein as a target for mutation;
- (b) mutating DNA encoding AHAS to produce a mutated DNA encoding a mutation at said position;
- (c) expressing said mutated DNA in a first cell, under conditions in which a variant AHAS is produced containing said mutation at said position;
- (d) expressing wild-type AHAS protein in parallel in a second cell;
- (e) purifying said wild-type and said variant AHAS proteins, from said cells;
- (f) assaying said wild-type and said variant AHAS proteins, for catalytic activity in conversion of pyruvate to acetolactate, in the absence and in the presence of imidazolinone or sulfonylurea herbicides;
- and
- (g) repeating steps (a)-(g), wherein said mutated DNA is used as the AHAS-encoding DNA in step (b) until an herbicide resistant AHAS protein having:
- (i) catalytic activity in the absence of herbicides of more than about 20% of the catalytic activity of said wild-type AHAS;
- (ii) catalytic activity that is relatively more resistant to the presence of imidazolinone herbicides compared to wild type AHAS;
- and
- (iii) catalytic activity that is relatively more sensitive to the presence of sulfonylurea herbicides compared to imidazolinone herbicides is identified.
9 more elements
An isolated DNA encoding an herbicide-resistant acetohydroxy acid synthase (AHAS) variant protein, said variant protein comprising an AHAS protein modified by
- (i) substitution of at least one different amino acid residue at an amino acid residue of the sequence of FIG. 1, SEQ ID NO:1, selected from the group consisting of P48, G49, S52, M53, E54, A84, A95, T96, S97, G98, P99, G100, A101, V125, R127, R128, M129, I130, G131, T132, D133, F135, Q136, D186, I187, T259, T260, L261, M262, G263, R276, M277, L278, G279, H281, G282, T283, V284, G300, V301, R302, F303, D304, R306, V307, T308, G309, K310, I311, E312, A313, F314, A315, S316, R317, A318, K319, I320, E329, I330, K332, N333, K334, Q335, T404, G413, V414, G415, Q416, H417, Q418, M419, W420, A421, A422, L434, S435, S436, A437, G438, L439, G440, A441, M442, G443, D467, G468, S469, L471, N473, L477, M479, Q495, H496, L497, G498, M499, V501, Q502, Q504, D505, R506, Y508, K509, A510, N511, R512, A513, H514, T515, S524, H572, Q573, E574, H575, V576, L577, P578, M579, I580, P581, G583, G584, functional equivalents of any of the foregoing, and any combination of any of the foregoing;
- (ii) deletion of up to 5 amino acid residues preceding, or up to 5 amino acid residues following at least one amino acid residue of the sequence of FIG. 1, SEQ ID NO:1, selected from the group consisting of P48, G49, S52, M53, E54, A84, A95, T96, S97, G98, P99, G100, A101, V125, R127, R128, M129, I130, G131, T132, D133, F135, Q136, D186, I187, T259, T260, L261, M262, G263, R276, M277, L278, G279, H281, G282, T283, V284, G300, V301, R302, F303, D304, R306, V307, T308, G309, K310, I311, E312, A313, F314, A315, S316, R317, A318, K319, I320, E329, I330, K332, N333, K334, Q335, T404, G413, V414, G415, Q416, H417, Q418, M419, W420, A421, A422, L434, S435, S436, A437, G438, L439, G440, A441, M442, G443, D467, G468, S469, L471, N473, L477, M479, Q495, H496, L497, G498, M499, V501, Q502, Q504, D505, R506, Y508, K509, A510, N511, R512, A513, H514, T515, S524, H572, Q573, E574, H575, V576, L577, P578, M579, I580, P581, G583, G584, functional equivalents of any of the foregoing, and any combination of any of the foregoing;
- (iii) deletion of at least one amino acid residue or a functional equivalent thereof between Q124 and H150 of the sequence of FIG. 1, SEQ ID NO:1;
- (iv) addition of at least one amino acid residue or a functional equivalent thereof between Q124 and H150 of the sequence of FIG. 1, SEQ ID NO:1;
- (v) deletion of at least one amino acid residue or a functional equivalent thereof between G300 and D324 of the sequence of FIG. 1, SEQ ID NO:1;
- (vi) addition of at least one amino acid residue or a functional equivalent thereof between G300 and D324 of the sequence of FIG. 1, SEQ ID NO:1;
- and
- (vii) any combination of any of the foregoing,
- wherein said herbicide resistant AHAS variant has:
- (i) catalytic activity in the absence of herbicides of more than about 20% of the catalytic activity of wild-type AHAS;
- (ii) catalytic activity that is relatively more resistant to the presence of imidazolinone herbicides compared to wild-type AHAS;
- and
- (iii) catalytic activity that is relatively more sensitive to the presence of sulfonylurea herbicides compared to imidazolinone herbicides.
10 more elements
Dependencies are inferred from the claim text.