# US 9,752,158 B2 · AXMI115 variant insecticidal gene and methods for its use

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- Number: US 9,752,158 B2
- Document ID: US9752158B2
- Status: Granted
- Kind: B2
- Application number: 14/851,442
- Filed: 2015-09-11
- Published: 2016-06-30
- Granted: 2017-09-05
- Earliest claimed: 2011-04-05
- Assignees: Athenix Corporation
- Applicants: Athenix Corp.
- Inventors: Nalini Manoj Desai; Volker Heinrichs; Duane Lehtinen
- Web page: https://patentgrep.com/patent/US9752158B2

## CPC

- C12N 15/8286: {for insect resistance}
- C07K 14/325: Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
- C07K 2319/00: Fusion polypeptide
- C07K 2319/24: containing a MBP (maltose binding protein)-tag
- 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)
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. The toxin coding sequences can be used in DNA constructs or expression cassettes for expression in plants and bacteria. Compositions include transformed bacteria, plants, plant cells, tissues, and seeds. In particular, polynucleotide sequences and the toxin proteins encoded thereby are provided. Also provided are antibodies specifically binding to those amino acid sequences. In particular, the invention encompasses nucleotide sequences encoding fusion proteins, as well as biologically active variants and fragments thereof, wherein the fusion protein contains the C-terminal portion of SEQ ID NO:43. The fusion protein may also contain the N-terminal portion of SEQ ID NO:45. The invention also includes the nucleotide sequence of SEQ ID NO:47 and 1-14, or a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:48 and 15-31, including biologically active variants and fragments thereof.

## Claims

### Claim 1

A recombinant nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6;
b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20: and
c) a nucleotide sequence that encodes a polypeptide comprising from one to five amino acid substitutions, deletions, or insertions relative to the amino acid sequence of SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO:19 or SEQ ID NO:20, wherein the pesticidal activity of the polypeptide is improved relative to the pesticidal activity of SEQ ID NO:43.

### Claim 2 (depends on 1)

The recombinant nucleic acid molecule of claim 1, wherein said nucleotide sequence is selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; and
b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

### Claim 3 (depends on 1)

The recombinant nucleic acid molecule of claim 1, wherein said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant.

### Claim 4 (depends on 1)

The recombinant nucleic acid molecule of claim 1, wherein said nucleotide sequence is operably linked to a promoter capable of directing expression of said nucleotide sequence in a plant cell.

### Claim 5 (depends on 1)

A vector comprising the recombinant nucleic acid molecule of claim 1.

### Claim 6 (depends on 5)

The vector of claim 5, further comprising a nucleic acid molecule encoding a heterologous polypeptide.

### Claim 7 (depends on 1)

A host cell that contains the recombinant nucleic acid of claim 1.

### Claim 8 (depends on 7)

The host cell of claim 7 that is a bacterial host cell.

### Claim 9 (depends on 7)

The host cell of claim 7 that is a plant cell.

### Claim 10 (depends on 9)

A transgenic plant comprising the host cell of claim 9.

### Claim 11 (depends on 10)

The transgenic plant of claim 10, wherein said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape.

### Claim 12 (depends on 1)

A transgenic seed comprising the nucleic acid molecule of claim 1.

### Claim 13

A recombinant polypeptide having pesticidal activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:
a) the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; and
b) an amino acid sequence comprising from one to five amino acid substitutions, deletions, or insertions relative to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20, wherein the pesticidal activity of the polypeptide is improved relative to the pesticidal activity of SEQ ID NO:43.

### Claim 14 (depends on 13)

The recombinant polypeptide of claim 13, wherein said polypeptide comprises SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

### Claim 15 (depends on 13)

The polypeptide of claim 13 further comprising heterologous amino acid sequences.

### Claim 16 (depends on 13)

A composition comprising the polypeptide of claim 13.

### Claim 17 (depends on 16)

The composition of claim 16, wherein said composition is selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution.

### Claim 18 (depends on 16)

The composition of claim 16, wherein said composition is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of bacterial cells.

### Claim 19 (depends on 16)

The composition of claim 16, comprising from about 1% to about 99% by weight of said polypeptide.

### Claim 20 (depends on 13)

A method for controlling a lepidopteran, hemipteran, coleopteran, nematode, or dipteran pest population, said method comprising contacting said population with a pesticidally-effective amount of the polypeptide of claim 13.

### Claim 21 (depends on 13)

A method for killing a lepidopteran, hemipteran, coleopteran, nematode, or dipteran pest, said method comprising contacting said pest with, or feeding to said pest, a pesticidally-effective amount of the polypeptide of claim 13.

### Claim 22 (depends on 7)

A method for producing a polypeptide with pesticidal activity, said method comprising culturing the host cell of claim 7 under conditions in which the nucleic acid molecule encoding the polypeptide is expressed.

### Claim 23

A plant having stably incorporated into its genome a DNA construct comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6;
b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; and
c) a nucleotide sequence that encodes a polypeptide comprising from one to five amino acid substitutions, deletions, or insertions relative to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20, wherein the pesticidal activity of the polypeptide is improved relative to the pesticidal activity of SEQ ID NO:43.

### Claim 24 (depends on 23)

The plant of claim 23, wherein said nucleotide sequence is selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; and
b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

### Claim 25 (depends on 24)

The plant of claim 24, wherein said plant is a plant cell.

### Claim 26

A method for protecting a plant from a pest, said method comprising expressing in a plant or cell thereof a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6;
b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; and
c) a nucleotide sequence that encodes a polypeptide comprising from one to five amino acid substitutions, deletions, or insertions relative to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20, wherein the pesticidal activity of the polypeptide is improved relative to the pesticidal activity of SEQ ID NO:43.

### Claim 27 (depends on 26)

The method of claim 26, wherein said nucleotide sequence is selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; and
b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

### Claim 28 (depends on 26)

The method of claim 26, wherein said plant produces a pesticidal polypeptide having pesticidal activity against a lepidopteran, hemipteran, coleopteran, nematode, or dipteran pest.

### Claim 29

A method for increasing yield in a plant, said method comprising growing in a field a plant of or a seed thereof having stably incorporated into its genome a DNA construct comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6;
b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; and
c) a nucleotide sequence that encodes a polypeptide comprising from one to five amino acid substitutions, deletions, or insertions relative to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20, wherein the pesticidal activity of the polypeptide is improved relative to the pesticidal activity of SEQ ID NO:43.

### Claim 30 (depends on 29)

The method of claim 29, wherein said nucleotide sequence is selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; and
b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.