# US 2023/0272018 A1 · Insecticidal polypeptides having improved activity spectrum and uses thereof

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- Number: US 2023/0272018 A1
- Document ID: US20230272018A1
- Status: Published application
- Kind: A1
- Application number: 18/047,163
- Filed: 2022-10-17
- Published: 2023-08-31
- Earliest claimed: 2017-05-26
- Assignees: Pioneer Hi-Bred International, Inc.
- Applicants: Pioneer Hi-Bred International, Inc.
- Inventors: ALBERT L. LU; MARK EDWARD NELSON; GUSUI WU; TAKASHI YAMAMOTO
- Web page: https://patentgrep.com/patent/US20230272018A1

## CPC

- C07K 14/325: Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
- A01N 63/50: Isolated enzymes; Isolated proteins (peptides A01N37/46)
- C12N 15/8286: {for insect resistance}
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- 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)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## 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.

## Claims

### Claim 1

A DNA construct comprising a polynucleotide encoding a one Cry1B variant polypeptide with a second polynucleotide encoding a different second Cry1B variant polypeptide, wherein the first Cry1B variant polypeptide and the second Cry1B variant polypeptide, wherein the first and second Cry1B variant polypeptides each have insecticidal activity.

### Claim 2 (depends on 1)

The DNA construct of claim 1, wherein the polynucleotides encoding the first and second Cry1B variant polypeptides are each operably linked to a heterologous regulatory element.

### Claim 3 (depends on 1)

The DNA construct of claim 1, wherein the first Cry 1B variant polypeptide and the different second Cry1B variant polypeptide each comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B21 (SEQ ID NO: 5), IP1B-B22 (SEQ ID NO: 7), IP1B-B23 (SEQ ID NO: 9), IP1B-B24 (SEQ ID NO: 11), IP1B-B25 (SEQ ID NO: 13), IP1B-B26 (SEQ ID NO: 15), IP1B-B27 (SEQ ID NO: 17), IP1B-B28 (SEQ ID NO: 19), IP1B-B29 (SEQ ID NO: 21), IP1B-B40 (SEQ ID NO: 31), IP1B-B41 (SEQ ID NO: 33), IP1B-B42 (SEQ ID NO: 35), IP1B-B43 (SEQ ID NO: 37), IP1B-B44 (SEQ ID NO: 39), IP1B-B45 (SEQ ID NO: 41), IP1B-B46 (SEQ ID NO: 43), IP1B-B47 (SEQ ID NO: 45), IP1B-B60 (SEQ ID NO: 62), IP1B-B61 (SEQ ID NO: 63), IP1B-B62 (SEQ ID NO: 64), IP1B-B63 (SEQ ID NO: 65), IP1B-B64 (SEQ ID NO: 66), IP1B-B65 (SEQ ID NO: 67), IP1B-B66 (SEQ ID NO: 68), IP1B-B67 (SEQ ID NO: 69),), IP1B-B68 (SEQ ID NO: 70), IP1B-B69 (SEQ ID NO: 71), IP1B-B80 (SEQ ID NO: 72), IP1B-B81 (SEQ ID NO: 73), IP1B-B82 (SEQ ID NO: 74), IP1B-B83 (SEQ ID NO: 75), IP1B-B100 (SEQ ID NO: 76), and IP1B-B101 (SEQ ID NO: 77), IP1B-B102 (SEQ ID NO: 78), SL8-01 (SEQ ID NO: 143), SL8-02 (SEQ ID NO: 144), IP1B-B31 (SEQ ID NO: 23), IP1B-B32 (SEQ ID NO: 25), IP1B-B33 (SEQ ID NO: 27), and IP1B-B34 (SEQ ID NO: 29).

### Claim 4 (depends on 1)

The DNA construct of claim 1, wherein the first Cry 1B variant polypeptide and the different second Cry1B variant polypeptide each have a different site of action, mode of action, or second Cry1B variant polypeptide has activity on insect resistant to the activity of the first Cry1B variant polypeptide.

### Claim 5 (depends on 1)

The DNA construct of claim 1, wherein first Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in SEQ IP1B-B21 (SEQ ID NO: 5), IP1B-B22 (SEQ ID NO: 7), IP1B-B23 (SEQ ID NO: 9), IP1B-B24 (SEQ ID NO: 11), IP1B-B25 (SEQ ID NO: 13), IP1B-B26 (SEQ ID NO: 15), IP1B-B27 (SEQ ID NO: 17), IP1B-B28 (SEQ ID NO: 19), IP1B-B29 (SEQ ID NO: 21), IP1B-B40 (SEQ ID NO: 31), IP1B-B41 (SEQ ID NO: 33), IP1B-B42 (SEQ ID NO: 35), IP1B-B43 (SEQ ID NO: 37), IP1B-B44 (SEQ ID NO: 39), IP1B-B45 (SEQ ID NO: 41), IP1B-B46 (SEQ ID NO: 43), IP1B-B47 (SEQ ID NO: 45), IP1B-B60 (SEQ ID NO: 62), IP1B-B61 (SEQ ID NO: 63), IP1B-B62 (SEQ ID NO: 64), IP1B-B63 (SEQ ID NO: 65), IP1B-B64 (SEQ ID NO: 66), IP1B-B65 (SEQ ID NO: 67), IP1B-B66 (SEQ ID NO: 68), IP1B-B67 (SEQ ID NO: 69),), IP1B-B68 (SEQ ID NO: 70), IP1B-B69 (SEQ ID NO: 71), IP1B-B80 (SEQ ID NO: 72), IP1B-B81 (SEQ ID NO: 73), IP1B-B82 (SEQ ID NO: 74), IP1B-B83 (SEQ ID NO: 75), IP1B-B100 (SEQ ID NO: 76), and IP1B-B101 (SEQ ID NO: 77), and IP1B-B102 (SEQ ID NO: 78), SL8-01 (SEQ ID NO: 143), and SL8-02 (SEQ ID NO: 144), and wherein the second Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B31 (SEQ ID NO: 23), IP1B-B32 (SEQ ID NO: 25), IP1B-B33 (SEQ ID NO: 27), and IP1B-B34 (SEQ ID NO: 29).

### Claim 6 (depends on 1)

The DNA construct of claim 1, wherein first Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B60 (SEQ ID NO: 62), IP1B-B61 (SEQ ID NO: 63), IP1B-B62 (SEQ ID NO: 64), IP1B-B63 (SEQ ID NO: 65), IP1B-B64 (SEQ ID NO: 66), IP1B-B65 (SEQ ID NO: 67), and IP1B-B66 (SEQ ID NO: 68), and wherein the second Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B100 (SEQ ID NO: 76), and IP1B-B101 (SEQ ID NO: 77), IP1B-B102 (SEQ ID NO: 78), SL8-01 (SEQ ID NO: 143), and SL8-02 (SEQ ID NO: 144).

### Claim 7

A transgenic plant comprising a molecular stack comprising a polynucleotide encoding a one Cry1B variant polypeptide with a second polynucleotide encoding a different second Cry1B variant polypeptide, wherein the first Cry1B variant polypeptide and the second Cry1B variant polypeptide, wherein the first and second Cry1B variant polypeptides each have insecticidal activity.

### Claim 8 (depends on 7)

The transgenic plant of claim 7, wherein the polynucleotides encoding the first and second Cry1B variant polypeptides are each operably linked to a heterologous regulatory element.

### Claim 9 (depends on 7)

The transgenic plant of claim 7, wherein the first Cry 1B variant polypeptide and the different second Cry1B variant polypeptide each comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B21 (SEQ ID NO: 5), IP1B-B22 (SEQ ID NO: 7), IP1B-B23 (SEQ ID NO: 9), IP1B-B24 (SEQ ID NO: 11), IP1B-B25 (SEQ ID NO: 13), IP1B-B26 (SEQ ID NO: 15), IP1B-B27 (SEQ ID NO: 17), IP1B-B28 (SEQ ID NO: 19), IP1B-B29 (SEQ ID NO: 21), IP1B-B40 (SEQ ID NO: 31), IP1B-B41 (SEQ ID NO: 33), IP1B-B42 (SEQ ID NO: 35), IP1B-B43 (SEQ ID NO: 37), IP1B-B44 (SEQ ID NO: 39), IP1B-B45 (SEQ ID NO: 41), IP1B-B46 (SEQ ID NO: 43), IP1B-B47 (SEQ ID NO: 45), IP1B-B60 (SEQ ID NO: 62), IP1B-B61 (SEQ ID NO: 63), IP1B-B62 (SEQ ID NO: 64), IP1B-B63 (SEQ ID NO: 65), IP1B-B64 (SEQ ID NO: 66), IP1B-B65 (SEQ ID NO: 67), IP1B-B66 (SEQ ID NO: 68), IP1B-B67 (SEQ ID NO: 69),), IP1B-B68 (SEQ ID NO: 70), IP1B-B69 (SEQ ID NO: 71), IP1B-B80 (SEQ ID NO: 72), IP1B-B81 (SEQ ID NO: 73), IP1B-B82 (SEQ ID NO: 74), IP1B-B83 (SEQ ID NO: 75), IP1B-B100 (SEQ ID NO: 76), and IP1B-B101 (SEQ ID NO: 77), IP1B-B102 (SEQ ID NO: 78), SL8-01 (SEQ ID NO: 143), SL8-02 (SEQ ID NO: 144), IP1B-B31 (SEQ ID NO: 23), IP1B-B32 (SEQ ID NO: 25), IP1B-B33 (SEQ ID NO: 27), and IP1B-B34 (SEQ ID NO: 29).

### Claim 10 (depends on 7)

The transgenic plant of claim 7, wherein the first Cry 1B variant polypeptide and the different second Cry1B variant polypeptide each have a different site of action, mode of action, or second Cry1B variant polypeptide has activity on insect resistant to the activity of the first Cry1B variant polypeptide.

### Claim 11 (depends on 7)

The transgenic plant of claim 7, wherein first Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in SEQ IP1B-B21 (SEQ ID NO: 5), IP1B-B22 (SEQ ID NO: 7), IP1B-B23 (SEQ ID NO: 9), IP1B-B24 (SEQ ID NO: 11), IP1B-B25 (SEQ ID NO: 13), IP1B-B26 (SEQ ID NO: 15), IP1B-B27 (SEQ ID NO: 17), IP1B-B28 (SEQ ID NO: 19), IP1B-B29 (SEQ ID NO: 21), IP1B-B40 (SEQ ID NO: 31), IP1B-B41 (SEQ ID NO: 33), IP1B-B42 (SEQ ID NO: 35), IP1B-B43 (SEQ ID NO: 37), IP1B-B44 (SEQ ID NO: 39), IP1B-B45 (SEQ ID NO: 41), IP1B-B46 (SEQ ID NO: 43), IP1B-B47 (SEQ ID NO: 45), IP1B-B60 (SEQ ID NO: 62), IP1B-B61 (SEQ ID NO: 63), IP1B-B62 (SEQ ID NO: 64), IP1B-B63 (SEQ ID NO: 65), IP1B-B64 (SEQ ID NO: 66), IP1B-B65 (SEQ ID NO: 67), IP1B-B66 (SEQ ID NO: 68), IP1B-B67 (SEQ ID NO: 69),), IP1B-B68 (SEQ ID NO: 70), IP1B-B69 (SEQ ID NO: 71), IP1B-B80 (SEQ ID NO: 72), IP1B-B81 (SEQ ID NO: 73), IP1B-B82 (SEQ ID NO: 74), IP1B-B83 (SEQ ID NO: 75), IP1B-B100 (SEQ ID NO: 76), and IP1B-B101 (SEQ ID NO: 77), and IP1B-B102 (SEQ ID NO: 78), SL8-01 (SEQ ID NO: 143), and SL8-02 (SEQ ID NO: 144), and wherein the second Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B31 (SEQ ID NO: 23), IP1B-B32 (SEQ ID NO: 25), IP1B-B33 (SEQ ID NO: 27), and IP1B-B34 (SEQ ID NO: 29).

### Claim 12 (depends on 7)

The transgenic plant of claim 7, wherein first Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B60 (SEQ ID NO: 62), IP1B-B61 (SEQ ID NO: 63), IP1B-B62 (SEQ ID NO: 64), IP1B-B63 (SEQ ID NO: 65), IP1B-B64 (SEQ ID NO: 66), IP1B-B65 (SEQ ID NO: 67), and IP1B-B66 (SEQ ID NO: 68), and wherein the second Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B100 (SEQ ID NO: 76), and IP1B-B101 (SEQ ID NO: 77), IP1B-B102 (SEQ ID NO: 78), SL8-01 (SEQ ID NO: 143), and SL8-02 (SEQ ID NO: 144).

### Claim 13

A transgenic plant comprising a breeding stack of a polynucleotide encoding a one Cry1B variant polypeptide with a second polynucleotide encoding a different second Cry1B variant polypeptide, wherein the first Cry1B variant polypeptide and the second Cry1B variant polypeptide, wherein the first and second Cry1B variant polypeptides each have insecticidal activity.

### Claim 14 (depends on 5)

The transgenic plant of claim 5, wherein the polynucleotides encoding the first and second Cry1B variant polypeptides are each operably linked to a heterologous regulatory element.

### Claim 15 (depends on 13)

The transgenic plant of claim 13, wherein the first Cry 1B variant polypeptide and the different second Cry1B variant polypeptide each comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B21 (SEQ ID NO: 5), IP1B-B22 (SEQ ID NO: 7), IP1B-B23 (SEQ ID NO: 9), IP1B-B24 (SEQ ID NO: 11), IP1B-B25 (SEQ ID NO: 13), IP1B-B26 (SEQ ID NO: 15), IP1B-B27 (SEQ ID NO: 17), IP1B-B28 (SEQ ID NO: 19), IP1B-B29 (SEQ ID NO: 21), IP1B-B40 (SEQ ID NO: 31), IP1B-B41 (SEQ ID NO: 33), IP1B-B42 (SEQ ID NO: 35), IP1B-B43 (SEQ ID NO: 37), IP1B-B44 (SEQ ID NO: 39), IP1B-B45 (SEQ ID NO: 41), IP1B-B46 (SEQ ID NO: 43), IP1B-B47 (SEQ ID NO: 45), IP1B-B60 (SEQ ID NO: 62), IP1B-B61 (SEQ ID NO: 63), IP1B-B62 (SEQ ID NO: 64), IP1B-B63 (SEQ ID NO: 65), IP1B-B64 (SEQ ID NO: 66), IP1B-B65 (SEQ ID NO: 67), IP1B-B66 (SEQ ID NO: 68), IP1B-B67 (SEQ ID NO: 69),), IP1B-B68 (SEQ ID NO: 70), IP1B-B69 (SEQ ID NO: 71), IP1B-B80 (SEQ ID NO: 72), IP1B-B81 (SEQ ID NO: 73), IP1B-B82 (SEQ ID NO: 74), IP1B-B83 (SEQ ID NO: 75), IP1B-B100 (SEQ ID NO: 76), and IP1B-B101 (SEQ ID NO: 77), IP1B-B102 (SEQ ID NO: 78), SL8-01 (SEQ ID NO: 143), SL8-02 (SEQ ID NO: 144), IP1B-B31 (SEQ ID NO: 23), IP1B-B32 (SEQ ID NO: 25), IP1B-B33 (SEQ ID NO: 27), and IP1B-B34 (SEQ ID NO: 29).

### Claim 16 (depends on 13)

The transgenic plant of claim 13, wherein the first Cry 1B variant polypeptide and the different second Cry1B variant polypeptide each have a different site of action, mode of action, or second Cry1B variant polypeptide has activity on insect resistant to the activity of the first Cry1B variant polypeptide.

### Claim 17 (depends on 13)

The transgenic plant of claim 13, wherein first Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in SEQ IP1B-B21 (SEQ ID NO: 5), IP1B-B22 (SEQ ID NO: 7), IP1B-B23 (SEQ ID NO: 9), IP1B-B24 (SEQ ID NO: 11), IP1B-B25 (SEQ ID NO: 13), IP1B-B26 (SEQ ID NO: 15), IP1B-B27 (SEQ ID NO: 17), IP1B-B28 (SEQ ID NO: 19), IP1B-B29 (SEQ ID NO: 21), IP1B-B40 (SEQ ID NO: 31), IP1B-B41 (SEQ ID NO: 33), IP1B-B42 (SEQ ID NO: 35), IP1B-B43 (SEQ ID NO: 37), IP1B-B44 (SEQ ID NO: 39), IP1B-B45 (SEQ ID NO: 41), IP1B-B46 (SEQ ID NO: 43), IP1B-B47 (SEQ ID NO: 45), IP1B-B60 (SEQ ID NO: 62), IP1B-B61 (SEQ ID NO: 63), IP1B-B62 (SEQ ID NO: 64), IP1B-B63 (SEQ ID NO: 65), IP1B-B64 (SEQ ID NO: 66), IP1B-B65 (SEQ ID NO: 67), IP1B-B66 (SEQ ID NO: 68), IP1B-B67 (SEQ ID NO: 69),), IP1B-B68 (SEQ ID NO: 70), IP1B-B69 (SEQ ID NO: 71), IP1B-B80 (SEQ ID NO: 72), IP1B-B81 (SEQ ID NO: 73), IP1B-B82 (SEQ ID NO: 74), IP1B-B83 (SEQ ID NO: 75), IP1B-B100 (SEQ ID NO: 76), and IP1B-B101 (SEQ ID NO: 77), and IP1B-B102 (SEQ ID NO: 78), SL8-01 (SEQ ID NO: 143), and SL8-02 (SEQ ID NO: 144), and wherein the second Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B31 (SEQ ID NO: 23), IP1B-B32 (SEQ ID NO: 25), IP1B-B33 (SEQ ID NO: 27), and IP1B-B34 (SEQ ID NO: 29).

### Claim 18 (depends on 13)

The transgenic plant of claim 13, wherein first Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B60 (SEQ ID NO: 62), IP1B-B61 (SEQ ID NO: 63), IP1B-B62 (SEQ ID NO: 64), IP1B-B63 (SEQ ID NO: 65), IP1B-B64 (SEQ ID NO: 66), IP1B-B65 (SEQ ID NO: 67), and IP1B-B66 (SEQ ID NO: 68), and wherein the second Cry 1B variant polypeptide comprises a sequence having at least 95% identity to an one of the sequences as set forth in IP1B-B100 (SEQ ID NO: 76), and IP1B-B101 (SEQ ID NO: 77), IP1B-B102 (SEQ ID NO: 78), SL8-01 (SEQ ID NO: 143), and SL8-02 (SEQ ID NO: 144).

### Claim 19 (depends on 1)

The transgenic plant or progeny thereof comprising the DNA construct of claim 1, wherein said transgenic plant is corn or soy.

### Claim 20 (depends on 7)

The transgenic plant or progeny thereof comprising the molecular stack of claim 7, wherein said transgenic plant is corn or soy.

### Claim 21 (depends on 13)

The transgenic plant or progeny thereof comprising the breeding stack of claim 13, wherein said transgenic plant is corn or soy.

### Claim 22

A composition comprising a polynucleotide encoding a one Cry1B variant polypeptide with a second polynucleotide encoding a different second Cry1B variant polypeptide, wherein the first Cry1B variant polypeptide and the second Cry1B variant polypeptide, wherein the first and second Cry1B variant polypeptides each have insecticidal activity.

### Claim 23 (depends on 7)

A method for controlling an insect pest population comprising contacting the insect pest population with the transgenic plant of claim 7.