# US 10,480,008 B2 · Insecticidal polypeptides having broad spectrum activity and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,480,008 B2 - Document ID: US10480008B2 - Status: Granted - Kind: B2 - Application number: 15/518,721 - Filed: 2015-10-09 - Published: 2017-08-17 - Granted: 2019-11-19 - Earliest claimed: 2014-10-16 - Assignees: Poineer Hi-Bred International, Inc. - Applicants: Pioneer Hi-Bred International, Inc. - Inventors: Andre R. Abad; Andrew C. Crow; Brad Poland; Xiaomei Shi; Thomas C. Wolfe - Web page: https://patentgrep.com/patent/US10480008B2 ## CPC - C12N 15/8286: {for insect resistance} - A01N 63/50: Isolated enzymes; Isolated proteins (peptides A01N37/46) - C07K 14/32: from Bacillus (G) - C12N 5/04: Plant cells or tissues {(culture media C12N5/0025)} - C12N 15/8279: {for biotic stress resistance, pathogen resistance, disease resistance} - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C12N 2510/02: Cells for production - Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract The invention provides nucleic acids, and variants and fragments thereof, obtained from strains of Bacillus thuringiensis encoding polypeptides having pesticidal activity against insect pests, including Lepidoptera and Coleoptera. Particular embodiments of the invention 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. ## Claims ### Claim 1 An isolated nucleic acid molecule operably linked to a heterologous regulatory element, wherein said isolated nucleic acid is selected from the group consisting of: (a) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3; (b) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; (c) a nucleic acid molecule comprising a nucleotide sequence encoding a protein, having insecticidal activity against soybean looper, comprising an amino acid sequence that differs by 1 to 3 amino acid resides compared to the amino acid sequence of SEQ ID NO: 4; and (d) a nucleic acid molecule comprising a nucleotide sequence encoding a protein, having insecticidal activity against soybean looper, comprising an amino acid sequence that differs by 1 to 3 amino acid resides compared to the amino acid sequence of SEQ ID NO: 2. ### Claim 2 (depends on 1) The isolated nucleic acid molecule of claim 1, wherein said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant. ### Claim 3 (depends on 1) A DNA construct comprising a heterologous regulatory element operably linked to the nucleic acid molecule of claim 1. ### Claim 4 (depends on 3) A host cell that contains the DNA construct of claim 3. ### Claim 5 (depends on 4) The host cell of claim 4 that is a bacterial cell. ### Claim 6 (depends on 4) The host cell of claim 4 that is a plant cell. ### Claim 7 (depends on 6) A transgenic plant comprising the host cell of claim 6. ### Claim 8 (depends on 7) The transgenic plant of claim 7, wherein said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape. ### Claim 9 (depends on 8) Transformed seed of the plant of claim 8, wherein the seed comprise the DNA construct. ### Claim 10 An isolated polypeptide operably linked to a heterologous signal peptide, wherein the isolated polypeptide is selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; (b) a polypeptide that is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3; (c) a polypeptide sequence that differs by 1 to 3 amino acid resides compared to the polypeptide sequence of SEQ ID NO: 2, wherein the polypeptide has insecticidal activity; and (d) a polypeptide sequence that differs by 1 to 3 amino acid resides compared to the polypeptide sequence of SEQ ID NO: 2, wherein the polypeptide of (a), (b), (c) or (d) has insecticidal activity against soybean looper. ### Claim 11 A composition comprising an agriculturally acceptable carrier and a polypeptide operably linked to a heterologous signal peptide, wherein the polypeptide is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; (b) a polypeptide that is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3; (c) a polypeptide sequence that differs by 1 to 3 amino acid resides compared to the polypeptide sequence of SEQ ID NO: 4, wherein the polypeptide has insecticidal activity against soybean looper; and (d) a polypeptide sequence that differs by 1 to 3 amino acid resides compared to the polypeptide sequence of SEQ ID NO: 2, wherein the polypeptide has insecticidal activity against soybean looper. ### Claim 12 (depends on 11) The composition of claim 11, wherein said composition is selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution. ### Claim 13 (depends on 12) The composition of claim 12, wherein said composition is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of Bacillus thuringiensis cells. ### Claim 14 (depends on 12) The composition of claim 12, comprising from 1% to 99% by weight of said polypeptide. ### Claim 15 (depends on 11) A method for controlling a Lepidopteran or Coleopteran pest population comprising contacting said population with a pesticidally-effective amount of the composition of claim 11. ### Claim 16 (depends on 11) A method for killing a lepidopteran pest comprising contacting said pest with, or feeding to said pest, a pesticidally-effective amount of the composition of claim 11. ### Claim 17 (depends on 4) A method for producing a polypeptide with pesticidal activity, comprising culturing the host cell of claim 4 under conditions in which a nucleic acid molecule encoding the polypeptide is expressed, said polypeptide being selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; (b) a polypeptide that is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3; (c) a polypeptide sequence that differs by 1 to 3 amino acid resides compared to the polypeptide of SEQ ID NO: 4; and (d) a polypeptide sequence that differs by 1 to 3 amino acid resides compared to the polypeptide sequence of SEQ ID NO: 2. ### Claim 18 A plant having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a protein having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of: (a) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3; (b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; (c) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that differs by 1 to 3 amino acid resides compared to the amino acid sequence of-SEQ ID NO: 4, wherein the polypeptide has insecticidal activity; and (d) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that differs by 1 to 3 amino acid resides compared to the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide has insecticidal activity; wherein said nucleotide sequence is operably linked to a promoter that drives expression of a coding sequence in a plant cell. ### Claim 19 (depends on 18) The plant of claim 18, wherein said plant is a plant cell. ### Claim 20 A method for protecting a plant from a pest, comprising introducing into said plant or cell thereof at least one expression vector comprising a nucleotide sequence that encodes a pesticidal polypeptide, wherein said nucleotide sequence is selected from the group consisting of: (a) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3; (b) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; (c) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that differs by 1 to 3 amino acid resides compared to the amino acid sequence of SEQ ID NO: 4, wherein the polypeptide has insecticidal activity; and (d) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that differs by 1 to 3 amino acid resides compared to amino acid sequence of SEQ ID NO: 2, wherein the polypeptide has insecticidal activity. ### Claim 21 (depends on 20) The method of claim 20, wherein said plant produces a pesticidal polypeptide having insecticidal activity against a Lepidopteran pest.