Nova Patents
CA2425956C

Novel glyphosate n-acetyltransferase (gat) genes

Abstract

Novel proteins are provided herein, including proteins capable of catalyzing the acetylation of glyphosate and other structurally related proteins. Also provided are novel polynucleotides capable of encoding these proteins, compositions that include one or more of these novel proteins and/or polynucleotides, recombinant cells and transgenic plants comprising these novel compounds, diversification methods involving the novel compounds, and methods of using the compounds. Some of the novel methods and compounds provided herein can be used to render an organism, such as a plant, resistant to glyphosate.

CA2425956C, drawing sheet 1
Sheet 1 of 15

Term

Term ended

Expired 29 October 2021, 4.9 years ago.

  1. Priority
  2. Filed
  3. Granted
  4. Expired
  5. Today

34 claims: 13 independent, 21 dependent

  1. 1
    Claims:1. An isolated or recombinant polynucleotide that encodes a polypeptide that has glyphosate N-acetyl transferase activity comprising: (a) a nucleotide sequence encoding an amino acid sequence which when optimally aligned using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1 with a sequence selected from the group consisting of SEQ. ID. No.: 300, SEQ. ID. No.: 445 and SEQ. ID. No.: 457 generates a similarity score of at least 460;or, in the alternative, (b) a nucleotide sequence encoding at least 20 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ. ID. No.: 300, SEQ. ID. No.: 445 and SEQ. ID. No.: 457 wherein said encoded polypeptide has glyphosate Nacetyl transferase activity;or, in the alternative, (c) a nucleotide sequence that encodes the amino acid sequence of SEQ. ID. Nos.: 6-10 and 263-514, or, in the alternative, (d) a nucleotide sequence of claim 1 (a), wherein said sequence encodes a polypeptide wherein at least 80% of the positions conform to the following restrictions: (a) at position 2 the amino acid residue is I or L;(b) at position 3 the amino acid residue is E or D;(c) at position 4 the amino acid residue is V, A or I;(d) at position 5 the amino acid residue is K, R or N;(e) at position 6 the amino acid residue is P or L;(f) at position 8 the amino acid residue is N, S or T;(g) at position 10 the amino acid residue is E or G;(h) at position 11 the amino acid residue is D or E;(i) at position 12 the amino acid residue is T or A;(j) at position 14 the amino acid residue is E or K;(k) at position 15 the amino acid residue is I or L;(l) at position 17 the amino acid residue is H or Q;(m) at position 18 the amino acid residue is R, C or K;198 CA 02425956 2013-03-06 (n) at position 19 the amino acid residue is I or V;(o) at position 24 the amino acid residue is Q or R;(p) at position 26 the amino acid residue is L or I;(q) at position 27 the amino acid residue is E or D;(r) at position 28 the amino acid residue is A or V;(s) at position 30 the amino acid residue is K, M or R;(t) at position 31 the amino acid residue is Y or F;(u) at position 32 the amino acid residue is E or G;(v) at position 33 the amino acid residue is T, A or S;(w) at position 35 the amino acid residue is L, S or M;(x) at position 37 the amino acid residue is R, G, E or Q;(y) at position 38 the amino acid residue is G or S;(z) at position 39 the amino acid residue is T, A or S;(aa) at position 40 the amino acid residue is F, L or S;(ab) at position 45 the amino acid residue is Y or F;(ac) at position 47 the amino acid residue is R, Q or G;(ad) at position 48 the amino acid residue is G or D;(ae) at position 49 the amino acid residue is K, R, E or Q;(af) at position 51 the amino acid residue is I or V;(ag) at position 52 the amino acid residue is S, C or G;(ah) at position 53 the amino acid residue is I or T;(ai) at position 54 the amino acid residue is A or V;(aj) at position 57 the amino acid residue is H or N;(ak) at position 58 the amino acid residue is Q, K, N or P;(al) at position 59 the amino acid residue is A or S;(am) at position 60 the amino acid residue is E, K, G, V or D;(an) at position 61 the amino acid residue is H or Q;(ao) at position 62 the amino acid residue is P, S or T;(ap) at position 63 the amino acid residue is E, G or D;(aq) at position 65 the amino acid residue is E, D, V or Q;199 CA 02425956 2013-03-06 (ar) at position 67 the amino acid residue is Q, E, R, L, H or K;(as) at position 68 the amino acid residue is K, R, E, or N;(at) at position 69 the amino acid residue is Q or P;(au) at position 79 the amino acid residue is E or D;(av) at position 80 the amino acid residue is G or E;(aw) at position 81 the amino acid residue is Y, N or F;(ax) at position 82 the amino acid residue is R or H;(ay) at position 83 the amino acid residue is E, G or D;(az) at position 84 the amino acid residue is Q, R or L;(ba) at position 86 the amino acid residue is A or V;(bb) at position 89 the amino acid residue is T or S;(be) at position 90 the amino acid residue is L or I;(bd) at position 91 the amino acid residue is I or V;(be) at position 92 the amino acid residue is R or K;(bf) at position 93 the amino acid residue is H, Y or Q;(bg) at position 96 the amino acid residue is E, A or Q;(bh) at position 97 the amino acid residue is L or I;(bi) at position 100 the amino acid residue is K, R, N or E;(bj) at position 101 the amino acidi residue is K or R;(bk) at position 103 the amino acid residue is A or V;(bl) at position 104 the amino acid residue is D or N;(bm) at position 105 the amino acid residue is L or M;(bn) at position 106 the amino acid residue is L or I;(bo) at position 112 the amino acid residue is T or I;(bp) at position 113 the amino acid residue is S, T or F;(bq) at position 114 the amino acid residue is A or V;(br) at position 115 the amino acid residue is S, R or A;(bs) at position 119 the amino acid residue is K, E or R;(bt) at position 120 the amino acid residue is K or R;(bu) at position 123 the amino acid residue is F or L;200 CA 02425956 2013-03-06 (bv) at position 124 the amino acid residue is S or R;(bw) at position 125 the amino acid residue is E, K, G or D;(bx) at position 126 the amino acid residue is Q or H;(by) at position 128 the amino acid residue is E, G or K;(bz) at position 129 the amino acid residue is V, I or A;(ca) at position 130 the amino acid residue is Y, H, F or C;(cb) at position 131 the amino acid residue is D, G, N or E;(cc) at position 132 the amino acid residue is I, T, A, Μ, V or L;(cd) at position 135 the amino acid residue is V, T, A or I;(ce) at position 138 the amino acid residue is H or Y;(cf) at position 139 the amino acid residue is I or V;(eg) at position 140 the amino acid residue is L or S;(ch) at position 142 the amino acid residue is Y or H;(ci) at position 143 the amino acid residue is K, T or E;(cj) at position 144 the amino acid residue is K, E or R;(ck) at position 145 the amino acid residue is L or I;and (cl) at position 146 the amino acid residue is T or A;(cm) at position 9, 76, 94 and 110 the amino acid residue is A;(cn) at position 29 and 108 the amino acid residue is C;(co) at position 34 the amino acid residueis D;(cp) at position 95 the amino acid tesidueis E;(cq) at position 56 the amino acid residueis F;(cr) at position 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 the amino acid residue is G;(cs) at position 41 the amino acid residue is H;(ct) at position 7 the amino acid residue is I;(cu) at position 85 the amino acid residue is K;(cv) at position 20, 36, 42, 50, 72, 78, 98 and 121 the amino acid residue is L;(cw) at position 1, 75 and 141 the amino acid residue is M;201 CA 02425956 2013-03-06 (ex) at position 23, 64 and 109 the amino acid residue is N;(cy) at position 22, 25,133, 134 and 137 the amino acid residue is P;(cz) at position 71 the amino acid residue is Q;(da) at position 16, 21, 73, 99 and 111 the amino acid residue is R;(db) at position 55 and 88 the amino acid residue is S;(de) at position 77 the amino acid residue is T;(dd) at position 107 the amino acid residue is W;and (de) at position 13,46, 70,117 and 118 the amino acid residue is Y.
  2. 5
    The isolated or recombinant polynucleotide of any one of claims 1-3, wherein (a) a parental codon has been replaced by a synonymous codon that is preferentially used in plants relative to the parental codon;or, in the alternative, (b) said polynucleotide further comprises a nucleotide sequence encoding an N-terminal chloroplast transit peptide.
  3. 7
    A nucleic acid construct comprising the polynucleotide of any one of claims 1-6, said construct comprising a promoter operably linked to said polynucleotide wherein the 203 CA 02425956 2013-12-04 promoter is heterologous with respect to the polynucleotide and effective to cause expression of the encoded polypeptide.
  4. 13
    An isolated or recombinant polypeptide that has glyphosate N-acetyl transferase activity wherein (a) said polypeptide comprises an amino acid sequence which when optimally aligned using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1 with a sequence selected from the group consisting of SEQ. ID. No.:300, SEQ. ID. No.: 445 and SEQ. ID. No.: 457 generate a similarity score of 204 CA 02425956 2013-03-06 at least 460, or, in the alternative, (b) said polypeptide comprises at least 20 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ. ID. No.: 300, SEQ. ID. No.: 445 and SEQ. ID. No.: 457, wherein said polypeptide has glyphosate N-acetyl transferase activity, or, in the alternative, (c) said polypeptide of claim 13(a) has a Km for glyphosate of at least 2 mM or less;a Km for acetyl CoA of at least 200 μΜ or less;and a Kcat equal to at least 6/minute;or, in the alternative, (d) the polypeptide of claim 13(a) wherein said sequence comprises at least 80% of the positions of the polypeptide conform to the following restrictions: (a) at position 2 the amino acid residue is I or L;(b) at position 3 the amino acid residue is E or D;(c) at position 4 the amino acid residue is V, A or I;(d) at position 5 the amino acid residue is K, R or N;(e) at position 6 the amino acid residue is P or L;(f) at position 8 the amino acid residue is N, S or T;(g) at position 10 the amino acid residue is E or G;(h) at position 11 the amino acid residue is D or E;(i) at position 12 the amino acid residue is T or A;(j) at position 14 the amino acid residue is E or K;(k) at position 15 the amino acid residue is I or L;(l) at position 17 the amino acid residue is H or Q;(m) at position 18 the amino acid residue is R, C or K;(n) at position 19 the amino acid residue is I or V;(o) at position 24 the amino acid residue is Q or R;(p) at position 26 the amino acid residue is L or I;(q) at position 27 the amino acid residue is E or D;(r) at position 28 the amino acid residue is A or V;(s) at position 30 the amino acid residue is K, M or R;(t) at position 31 the amino acid residue is Y or F;205 CA 02425956 2013-03-06 (u) at position 32 the amino acid residue is E or G;(v) at position 33 the amino acid residue is T, A or S;(w) at position 35 the amino acid residue is L, S or M;(x) at position 37 the amino acid residue is R, G, E or Q;(y) at position 38 the amino acid residue is G or S;(z) at position 39 the amino acid residue is T, A or S;(aa) at position 40 the amino acid residue is F, L or S;(ab) at position 45 the amino acid residue is Y or F;(ac) at position 47 the amino acid residue is R, Q or G;(ad) at position 48 the amino acid residue is G or D;(ae) at position 49 the amino acid residue is K, R, E or Q;(af) at position 51 the amino acid residue is I or V;(ag) at position 52 the amino acid residue is S, C or G;(ah) at position 53 the amino acid residue is I or T;(ai) at position 54 the amino acid residue is A or V;(aj) at position 57 the amino acid residue is H or N;(ak) at position 58 the amino acid residue is Q, K, N or P;(al) at position 59 the amino acid residue is A or S;(am) at position 60 the amino acid residue is E, K, G, V or D;(an) at position 61 the amino acid residue is H or Q;(ao) at position 62 the amino acid residue is P, S or T;(ap) at position 63 the amino acid residue is E, G or D;(aq) at position 65 the amino acid residue is E, D, V or Q;(ar) at position 67 the amino acid residue is Q, E, R, L, H or K;(as) at position 68 the amino acid residue is K, R, E, or N;(at) at position 69 the amino acid residue is Q or P;(au) at position 79 the amino acid residue is E or D;(av) at position 80 the amino acid residue is G or E;(aw) at position 81 the amino acid residue is Y, N or F;(ax) at position 82 the amino acid residue is R or H;206 CA 02425956 2013-03-06 (ay) at position 83 the amino acid residue is E, G or D;(az) at position 84 the amino acid residue is Q, R or L;(ba) at position 86 the amino acid residue is A or V;(bb) at position 89 the amino acid residue is T or S;(be) at position 90 the amino acid residue is L or I;(bd) at position 91 the amino acid residue is I or V;(be) at position 92 the amino acid residue is R or K;(bf) at position 93 the amino acid residue is H, Y or Q;(bg) at position 96 the amino acid residue is E, A or Q;(bh) at position 97 the amino acid residue is L or I;(bi) at position 100 the amino acid residue is K, R, N or E;(bj) at position 101 the amino acid residue is K or R;(bk) at position 103 the amino acid residue is A or V;(bl) at position 104 the amino acid residue is D or N;(bm) at position 105 the amino acid residue is L or M;(bn) at position 106 the amino acid residue is L orI;(bo) at position 112 the amino acid residue is T orI;(bp) at position 113 the amino acid residue is S, T orF;(bq) at position 114 the amino acid residue is A orV;(br) at position 115 the amino acid residue is S, R or A;(bs) at position 119 the amino acid residue is K, E or R;(bt) at position 120 the amino acid residue is K or R;(bu) at position 123 the amino acid residue is F or L;(bv) at position 124 the amino acid residue is S or R;(bw) at position 125 the amino acid residue is E, K, G or D;(bx) at position 126 the amino acid residue is Q or H;(by) at position 128 the amino acid residue is E, G or K;(bz) at position 129 the amino acid residue is V, I or A;(ca) at position 130 the amino acid residue is Y, H, F or C;(cb) at position 131 the amino acid residue is D, G, N or E;207 CA 02425956 2013-03-06 (cc) at position 132 the amino acid residue is I, T, A, Μ, V or L;(cd) at position 135 the amino acid residue is V, T, A or I;(ce) at position 138 the amino acid residue is H or Y;(cf) at position 139 the amino acid residue is I or V;(eg) at position 140 the amino acid residue is L or S;(ch) at position 142 the amino acid residue is Y or H;(ci) at position 143 the amino acid residue is K, T or E;(cj) at position 144 the amino acid residue is K, E or R;(ck) at position 145 the amino acid residue is L or I;and (cl) at position 146 the amino acid residue is T or A.
  5. 18
    A method of producing a glyphosate resistant transgenic plant seed thereof, or plant cell comprising:transforming a plant or plant cell with a polynucleotide or a nucleic acid construct encoding a polypeptide having glyphosate N-acetyltransferase activity, wherein said polynucleotide is a polynucleotide as defined in any one of claims 1 to 6 and said construct is a construct as defined in claim 7 or 8.
  6. 22
    The method of any one of claims 18 to 21, wherein the plant is or the plant cell is of, a crop plant that is:Eleusine, Lollium, Bambusa, Brassica, Dactylis, Sorghum, Pennisetum, Zea, Oryza, Triticum, Secale, Avena, Hordeum, Saccharum, Coix, Glycine or Gossypium.
  7. 23
    A method for selectively controlling weeds in a field containing a crop comprising:(a) planting the field with crop seeds or plants which are glyphosate-tolerant as a result of being transformed with a polynucleotide encoding a polypeptide having glyphosate N-acetyltransferase activity and wherein said encoded polypeptide acetylates the secondary amine of glyphosate;and (b) applying to the crop and weeds in the field an amount of glyphosate to control the weeds without significantly affecting the crop;wherein the polypeptide with glyphosate N-acetyltransferase activity is expressed from a polynucleotide as defined in any one of claims 1 to 6.
  8. 24
    A transgenic plant cell having an enhanced tolerance to glyphosate, wherein the plant cell expresses:a polypeptide with glyphosate N-acetyltransferase activity and which acetylates the secondary amine of glyphosate;and, (a) at least one polypeptide imparting glyphosate tolerance by an additional mechanism or, in the alternative, (b) at least one polypeptide imparting tolerance to an additional herbicide;and wherein the polypeptide with glyphosate N-acetyltransferase activity is expressed from a polynucleotide as defined in any one of claims 1 to 6. 210 CA 02425956 2013-12-04
  9. 27
    A method for controlling weeds in a field containing a crop comprising:(a) planting the field with at least one of a crop seed or a plant comprising plant cells as defined in claim 24 or 25, and (b) applying to the crop and weeds in the field an application of glyphosate sufficient to inhibit growth of weeds in the field without significantly affecting the crop.
  10. 31
    The method of any one of claims 27 to 30, wherein said plant is or said crop is of a crop plant that is of the genera:Eleusine, Lollium, Bambusa, Brassica, Dactylis, Sorghum, Pennisetum, Zea, Oryza, Triticum, Secale, Avena, Hordeum, Saccharum, Coix, Glycine or Gossypium.
  11. 32
    A plant cell expressing a heterologous polypeptide having glyphosate N-acetyl transferase activity and acetylates the secondary amine of glyphosate, wherein said plant cell produces N-acetylglyphosate when treated with glyphosate and wherein said polypeptide having glyphosate N-acetyl transferase activity comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:300,445 or 457.
  12. 33
    A method for detectins the nresence or evaluatine the activity of a nolvnentide CA 02425956 2013-12-04 secondary amine of glyphosate comprising:assaying plant tissue using PCR amplification to detect a polynucleotide that encodes said polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 300,445 or 457.
  13. 34
    37. A method for determining whether a polypeptide having glyphosate N-acetyl transferase activity confers resistance to glyphosate in transgenic plants comprising the steps of:transforming a plant with a polynucleotide that encodes a glyphosate N-acetyl transferase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 300,445 or 457;treating the transformed plant with glyphosate;and determining whether the plant is damaged or killed by the glyphosate treatment. 213 CA 02425956 2003-04-14
Independent claims13