Insecticidal polypeptides having improved activity spectrum and uses thereof

Abstract

The disclosure provides nucleic acids, and variants and fragments thereof, derived from strains of Bacillus thuringiensis encoding variant polypeptides having increased pesticidal activity against insect pests, including Lepidoptera and Coleopteran. Particular embodiments of the disclosure provide isolated nucleic acids encoding pesticidal proteins, pesticidal compositions, DNA constructs, and transformed microorganisms and plants comprising a nucleic acid of the embodiments. These compositions find use in methods for controlling pests, especially plant pests.

1 . A variant Cry1B polypeptide comprising an amino acid sequence having at least one amino acid substitution compared to the corresponding reference Cry1B polypeptide, wherein the variant Cry1B polypeptide has increased insecticidal activity against corn earworm and/or fall armyworm compared to the corresponding reference Cry1B polypeptide. 
     
     
         2 . The variant Cry1B polypeptide of  claim 1 , wherein the corresponding reference Cry1B polypeptide is selected from:
 a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1;   b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 47;   c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 52; and   d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 54.   
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The variant Cry1B polypeptide of  claim 1 , wherein the corresponding reference Cry1B polypeptide comprises the amino sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45. 
     
     
         7 . The variant Cry1B polypeptide of  claim 1 , wherein the corresponding reference Cry1B polypeptide comprises a Cry1Be type Domain I and a Cry1Ah type Domain III. 
     
     
         8 . The variant Cry1B polypeptide of  claim 7 , wherein Cry1Be type Domain I is selected from;
 a) a Cry1Be type Domain I having at least 90% identity to amino acids 36-276 of SEQ ID NO: 58; and   b) a Cry1Be type Domain I having at least 90% identity to amino acids 35-276 of SEQ ID NO: 47.   
     
     
         9 . (canceled) 
     
     
         10 . The variant Cry1B polypeptide of  claim 7  or  8 , wherein the Cry1Ah type Domain III is selected from:
 a) a Cry1Ah type Domain III having at least 80% sequence identity to amino acids 483 to 643 of SEQ ID NO: 61; and 
 b) a Cry1Ah type Domain III having at least 80% sequence identity to amino acids 494 to 655 of SEQ ID NO: 47. 
 
     
     
         11 . (canceled) 
     
     
         12 . The variant Cry1B polypeptide of  claim 1 , wherein the corresponding reference Cry1B polypeptide comprises a Cry1Ba type Domain I and Domain II selected from:
 a) a Cry1Ba type Domain I and Domain II having at least 80% sequence identity to amino acids 30 to 489 of SEQ ID NO: 55;   b) a Cry1Be type Domain I and Domain II having at least 80% sequence identity to amino acids 35 to 494 of SEQ ID NO: 58; and   c) a Cry1Be type Domain I and Domain II having at least 80% sequence identity to amino acids 35 to 493 of SEQ ID NO: 47.   
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The variant Cry1B polypeptide of  claim 12 , wherein the corresponding reference Cry1B polypeptide further comprises a Cry1Ah type Domain III selected from:
 a) a Cry1Ah type Domain III having at least 80% sequence identity to amino acids 483 to 643 of SEQ ID NO: 61; and   b) a Cry1Ah type Domain III having at least 80% sequence identity to amino acids 494 to 655 of SEQ ID NO: 47.   
     
     
         16 . (canceled) 
     
     
         17 . The variant Cry1B polypeptide of  claim 1 , wherein the variant Cry1B polypeptide comprises an amino acid substitution at position 50, 57, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 87, 91, 92, 93, 94, 95, 106, 108, 109, 110, 111, 112, 113, 114, 115, 118, 119, 122, 123, 125, 129, 136, 140, 143, 144, 145, 146, 147, 148, 149, 158, 159, 160, 166, 167, 173, 177, 178, 179, 180, 201, 206, 209, 210, 211, 213, 214, 218, 219, 221, 222, 225, 226, 230, 233, 234, 236, 240, 241, 242, 243, 245, 246, 247, 248, 252, 277, 280, 281, 303, 306, 360, 362, 367, 406, 407, 418, 425, 427, 429, 431, 435, 439, 447, 473, 476, 477, 478, 479, 490, 495, 499, 502, 509, 512, 513, 515, 517, 518, 520, 521, 526, 532, 534, 535, 537, 538, 541, 545, 547, 551, 552, 553, 554, 555, 556, 557, 558, 559, 563, 564, 565, 568, 569, 570, 571, 572, 573, 574, 577, 581, 582, 583, 584, 585, 586, 587, 590, 591, 592, 593, 595, 596, 598, 599, 601, 602, 603, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 618, 624, 626, 628, 629, 636, 641, 643, 645, or 646 corresponding to the amino acid sequence of SEQ ID NO: 47. 
     
     
         18 . The variant Cry1B polypeptide of  claim 17 , wherein the amino acid substitution
 at position 50 is selected from R, I, D, A, H, V, S, F, V, K, and N;   at position 57 is selected from V, R, L, N, G, and D;   at position 65 is selected from Q, A, S, and G;   at position 67 is selected from M, F, and I;   at position 68 is selected from A, R, and F;   at position 70 is selected from E, W, and H;   at position 71 is S;   at position 73 is selected from S and G;   at position 74 is selected from I, E, S, R, V, and D;   at position 76 is selected from T, S, Y, V, D, and R;   at position 77 is selected from N, D, G, L, I, H, P, A, T, M, C, and S;   at position 79 is selected from S, V, T, L, R, I, P, N, Q, and K;   at position 80 is selected from Q, K, G, E, R, M, N, C, W, Y, and D;   at position 82 is F;   at position 83 is selected from E, D, G, A, K, H, R, Y, and L;   at position 87 is selected from D, K, N, C, W, and H;   at position 91 is selected from S, Y, T, and D;   at position 92 is selected from E, G, F, V, L, and T;   at position 93 is selected from H, D, and I;   at position 94 is selected from L, H, T, and S;   at position 95 is selected from G, Q, V, and F;   at position 106 is selected from I, A, F, G, H, C, K, V, R, and S;   at position 108 is selected from L, M, and T;   at position 109 is selected from S, V, and N;   at position 110 is selected from T, R, V, F, and H;   at position 111 is selected from H, L, S, M, R, G, A, Q, N, K, Y, and E;   at position 112 is L;   at position 113 is selected from L, V, S, N, and K;   at position 114 is selected from L, T, M, H, F, I, Y, A, S, V, E, and D;   at position 115 is P;   at position 118 is selected from V, T, E, D, F, V, and G;   at position 119 is selected from A, M, S, K, H, E, R, and V;   at position 122 is selected from R, I, F, N, G, and T;   at position 123 is K;   at position 125 is selected from N, R, and E;   at position 129 is selected from K, W, L, P, and V;   at position 136 is selected from I, F, and I;   at position 140 is E;   at position 143 is selected from S, R, G, Y, M, Q, L, W, T, A, N, and P;   at position 144 is selected from M, A, and T;   at position 145 is selected from N, P, A, L, and S;   at position 146 is selected from W, T, H, and V;   at position 147 is selected from V, R, D, and S;   at position 148 is selected from F, W, P, N, and L;   at position 149 is selected from V, A, S, and L;   at position 158 is F;   at position 159 is V;   at position 160 is V;   at position 166 is selected from V, E, C, I, and T;   at position 167 is selected from T, M, Q, L, and A;   at position 173 is selected from F, and T;   at position 177 is selected from C, S, T, and P;   at position 178 is K;   at position 179 is selected from I, and L;   at position 180 is selected from A, S, and L, M;   at position 201 is V;   at position 206 is selected from L, I, T, and W;   at position 209 is selected from E, R, D, L, V, and C;   at position 210 is selected from P, T, I, and R;   at position 211 is selected from I, R, G, T, P, and L;   at position 213 is selected from V, T, L, M, Q, N, and G;   at position 214 is W;   at position 218 is selected from T, A, H, S, I, V, Y, W, and D;   at position 219 is N;   at position 221 is selected from L, Y, V, K, I, D, G, H, W, R, T, and F;   at position 222 is selected from G, M, K, T, D, and I;   at position 225 is selected from V, Q, M, F, L, G, I, Y, C, N, and T;   at position 226 is selected from D, S, V, C, Y, R, and A;   at position 230 is selected from A, L, and S;   at position 233 is selected from K, D, Q, G, I, A, and Y;   at position 234 is selected from V, M, L, I, A, R, F, Y, and S;   at position 236 is selected from E, K, S, T, and L;   at position 240 is selected from Y, A, M, S, T, G, K, F, L, R, W, and C;   at position 241 is selected from S, I, W, M, K, Y, V, L, and C;   at position 242 is selected from P, and V;   at position 243 is selected from M, V, T, C, K, I, S, and Q;   at position 245 is selected from Q, Y, K, G, A, I, W, H, S, M, D, N, V, R, and F;   at position 246 is selected from T, S, G, and Q;   at position 247 is selected from E, S, G, and P;   at position 248 is selected from S, N, T, L, Y, V, R, and F;   at position 252 is selected from N, A, and F;   at position 277 is selected from Q, G, and V;   at position 280 is selected from H, C, and T;   at position 281 is selected from Q, M, R, K, S, H, and A;   at position 303 is selected from N, and P;   at position 306 is G;   at position 360 is selected from S, N, T, Y, and M;   at position 362 is selected from Y, H, W, K, I, D, V, A, L, G, and E;   at position 367 is selected from H, Q, N, W, T, L, Y, I, and A;   at position 406 is M;   at position 407 is W;   at position 418 is selected from K, and T;   at position 425 is selected from P, and G;   at position 427 is Y;   at position 429 is I;   at position 431 is selected from L, H, G, and A;   at position 435 is selected from Y, H, and L;   at position 439 is selected from M, and Q;   at position 447 is selected from N, V, I, S, L, A, E, and M;   at position 473 is selected from T, G, A, S, M, N, K, D, and Y;   at position 476 is selected from Y, H, G, L, S, F, and M;   at position 477 is selected from S, and A;   at position 478 is selected from G, and K;   at position 479 is V;   at position 490 is Q;   at position 495 is N;   at position 499 is selected from R, S, G, M, C, V, P, and W;   at position 502 is selected from K, V, A, T, N, E, L, Q, P, H, R, F, S, and Y;   at position 509 is T;   at position 512 is selected from A, Y, P, M, R, K, G, S, Q, I, and W;   at position 513 is selected from G, V, P, L, H, and C;   at position 515 is H;   at position 517 is selected from A, H, and S;   at position 518 is selected from D, A, Y, K, V, L, G, H, E, R, T, and C;   at position 520 is selected from V, R, Y, C, K, M, E, L, F, S, and A;   at position 521 is selected from G, L, V, A, D, I, Q, F, P, N, and M;   at position 526 is L;   at position 532 is selected from K, C, W, S, L, V, H, and G;   at position 534 is selected from S, Y, Q, W, E, H, D, and L;   at position 535 is selected from M, Q, E, K, F, D, R, L, A, P, S, and I;   at position 537 is selected from W, E, F, A, K, S, Q, Y, R, C, D, V, N, H, and T;   at position 538 is selected from M, G, I, C, H, T, S, F, A, Y, V, W, L, Q, I, D, E, and K;   at position 541 is selected from Y, N, W, and F;   at position 545 is selected from F, and L;   at position 547 is selected from A, S, G, I, M, and Q;   at position 551 is selected from H, C, R, A, S, D, and Y;   at position 552 is selected from T, V, and W;   at position 553 is selected from Q, D, R, E, A, F, L, P, G, W, S, and T;   at position 554 is selected from Y, R, D, H, N, and G;   at position 555 is selected from V, M, I, and W;   at position 556 is selected from A, W, G, D, C, and P;   at position 557 is selected from I, R, G, S, Q, M, V, A, and C;   at position 558 is selected from Y, K, T, L, N, G, S, E, I, D, F, P, V, M, and H;   at position 559 is W;   at position 563 is selected from N, L, I, and A;   at position 564 is selected from H, V, W, I, K, C, S, and A;   at position 565 is F;   at position 568 is selected from C, A, E, F, R, G, L, S, W, and N;   at position 569 is selected from I, M, G, and S;   at position 570 is selected from M, F, W, and T;   at position 571 is selected from G, V, T, C, and L;   at position 572 is selected from H, P, R, I, K, F, S, A, V, W, and M;   at position 573 is selected from A, T, and G;   at position 574 is R;   at position 577 is selected from R, F, K, M, V, A, T, H, G, and I;   at position 581 is selected from S, and K;   at position 582 is V;   at position 583 is S;   at position 584 is R;   at position 585 is selected from R, T, K, H, Q, L, W, N, M, F, and I;   at position 586 is selected from M, Y, P, A, S, K, R, F, G, V, Q, N, L, W, and T;   at position 587 is selected from H, C, N, S, D, R, A, T, K, E, W, L, Y, F, and Q;   at position 590 is selected from A, D, F, S, and G;   at position 591 is selected from H, V, N, T, D, R, S, K, C, E, W, L, Y, F, P, and Q;   at position 592 is selected from Q, M, A, Y, N, K, P, S, D, I, G, F, V, and W;   at position 593 is selected from Y, G, R, and V;   at position 595 is selected from R, G, H, N, V, F, K, T, Y, I, M, A, and P;   at position 596 is selected from V, T, I, S, G, L, W, Y, H, P, and D;   at position 598 is selected from V, G, D, and I;   at position 599 is selected from C, Q, L, Y, T, V, A, and P;   at position 601 is selected from Y, F, V, G, M, and E;   at position 602 is M;   at position 603 is selected from M, A, Y, R, S, L, W, D, and T;   at position 605 is selected from S, W, R, M, A, I, C, V, K, D, Y, N, Q, G, E, and P;   at position 606 is selected from R, H, K, F, Q, W, G, Y, M, T, A, I, L, and N;   at position 607 is selected from R, C, T, I, Q, G, D, E, and V;   at position 608 is selected from R, S, V, L, F, G, Y, A, K, W, and Q;   at position 609 is selected from G, P, L, R, S, V, F, and I;   at position 610 is selected from G, F, P, and L;   at position 611 is selected from L, K, G, W, and V;   at position 612 is selected from F, H, G, E, N, D, and P;   at position 613 is selected from M, T, W, V, N, R, and Y;   at position 614 is selected from M, S, L, H, V, R, G, Y, and D;   at position 615 is selected from V, Q, G, K, M, R, C, and L;   at position 618 is selected from N, H, W, R, G, L, D, and T;   at position 624 is selected from A, and M;   at position 626 is selected from K, G, R, T, H, A, N, I, Y, Q, P, and S;   at position 628 is selected from F, K, Q, S, R, G, L, I, and D;   at position 629 is selected from L, V, K, H, M, S, R, C, Q, E, T, P, and A;   at position 636 is selected from A, and C;   at position 641 is selected from P, H, A, L, Q, Y, E, I, S, V, D, and G;   at position 643 is selected from L, A, Q, H, S, D, M, C, and R;   at position 645 is selected from T, M, L, Y, A, N, V, P, I, W, C, S; and   at position 646 is selected from S, Y, D, E, M, F, H, V, W, and I.   
     
     
         19 . The variant Cry1B polypeptide of  claim 1 , wherein the variant Cry1B polypeptide is selected from:
 a) a variant Cry1B polypeptide having has at least 80% identity to SEQ ID NO: 1;   b) a variant Cry1B polypeptide having at least 80% identity to SEQ ID NO: 47;   c) a variant Cry1B polypeptide having at least 80% identity to SEQ ID NO: 52; and   d) a variant Cry1B polypeptide having at least 80% identity to SEQ ID NO: 54.   
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . The variant Cry1B polypeptide of  claim 1 , wherein the variant Cry1B polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45. 
     
     
         24 . The variant Cry1B polypeptide of  claim 1 , wherein the variant Cry1B polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45. 
     
     
         25 . The variant Cry1B polypeptide of  claim 1 , wherein the variant Cry1B polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45. 
     
     
         26 . The variant Cry1B polypeptide of  claim 1  wherein the increased insecticidal activity is against corn earworm. 
     
     
         27 . The variant Cry1B polypeptide of  claim 26 , wherein the increased insecticidal activity is quantified as a Mean FAE index. 
     
     
         28 . The variant Cry1B polypeptide of  claim 1 , wherein the increased insecticidal activity is against fall armyworm. 
     
     
         29 . The variant Cry1B polypeptide of  claim 28 , wherein the variant Cry1B polypeptide comprises the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29. 
     
     
         30 . A recombinant polynucleotide encoding the variant Cry1B polypeptide of  claim 1 . 
     
     
         31 . The recombinant polynucleotide of  claim 30 , wherein the nucleic acid sequence has been optimized for expression in a plant. 
     
     
         32 . The recombinant polynucleotide of  claim 31 , wherein the nucleic acid sequence is a synthetic nucleotide sequence having plant preferred codons that have been designed for expression in a plant. 
     
     
         33 . The recombinant polynucleotide of  claim 32 , wherein the nucleic acid sequence has been optimized for expression in maize or soybean. 
     
     
         34 . The recombinant polynucleotide of  claim 30 , wherein the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46. 
     
     
         35 . A DNA construct comprising a polynucleotide of  claim 30  operably linked to a heterologous regulatory element. 
     
     
         36 . A host cell comprising the DNA construct of  claim 35 . 
     
     
         37 . The host cell of  claim 36 , wherein the host cell is a bacterial cell. 
     
     
         38 . The host cell of  claim 37 , wherein the host cell is a plant cell. 
     
     
         39 . The host cell of  claim 38 , wherein the host cell is a soybean or maize cell. 
     
     
         40 . A transgenic plant comprising the DNA construct of  claim 30 . 
     
     
         41 . The transgenic plant of  claim 40 , wherein said plant is selected from the group consisting of maize,  sorghum , wheat, cabbage, sunflower, tomato, a crucifer species, a pepper species, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape. 
     
     
         42 . A seed comprising the DNA construct of  claim 35 . 
     
     
         43 . A composition comprising the variant Cry1B polypeptide of  claim 1 . 
     
     
         44 . The composition of  claim 43 , wherein the composition comprises from 1% to 99% by weight of the variant Cry1B polypeptide. 
     
     
         45 . A method for controlling a Lepidopteran pest population comprising contacting said population with a pesticidally-effective amount of the variant Cry1B polypeptide of  claim 1 . 
     
     
         46 . A method for killing a Lepidopteran pest comprising contacting said pest with, or feeding to said pest, a pesticidally-effective amount of the variant Cry1B polypeptide of  claim 1 . 
     
     
         47 . A method for producing a polypeptide with pesticidal activity, comprising culturing the host cell of  claim 36  under conditions in which the polynucleotide encoding the polypeptide is expressed. 
     
     
         48 . A plant or plant cell having stably incorporated into its genome the DNA construct of  claim 35 . 
     
     
         49 . A method of protecting a plant from an insect pest, comprising introducing into said plant the DNA construct of  claim 35 . 
     
     
         50 . The method of  claim 49 , wherein the variant Cry1B polypeptide has increased insecticidal activity against corn earworm and/or fall armyworm compared to the polypeptide of SEQ ID NO: 47. 
     
     
         51 . The method of  claim 49  or  50 , wherein the insect pest is resistant to a non-Cry1B insecticidal polypeptide. 
     
     
         52 . The method of  claim 49 , wherein the insect pest is corn earworm, fall armyworm or European corn borer.