# US 8,569,583 B2 · Secreted insecticidal protein and gene compositions from Bacillus thuringiensis and uses therefor

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- Number: US 8,569,583 B2
- Document ID: US8569583B2
- Status: Granted
- Kind: B2
- Application number: 13/171,519
- Filed: 2011-06-29
- Published: 2011-11-17
- Granted: 2013-10-29
- Earliest claimed: 2003-12-16
- Assignees: Monsanto Technology LLC
- Applicants: Judith Donovan; William Donovan; James Engleman; Thomas Malvar; John Pitkin
- Inventors: Judith Donovan; William Donovan; James Engleman; Thomas Malvar; John Pitkin
- Web page: https://patentgrep.com/patent/US8569583B2

## CPC

- C07K 14/32: from Bacillus (G)
- C07K 14/325: Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
- 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)
- 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 present invention relates to the isolation and characterization of nucleotide sequences encoding novel insecticidal proteins secreted into the extracellular space from Bacillus thuringiensis and related strains. The proteins are isolated from culture supernatants of Bacillus thuringiensis and related strains and display insecticidal activity against lepidopteran insects including European corn borer (ECB), tobacco budworm (TBW) and diamondback moth (DBM). Insecticidal proteins encoded by nucleotide sequences that hybridize under stringent conditions to the isolated and characterized nucleotide sequences are disclosed. Methods are disclosed for making and using transgenic cells and plants comprising the novel nucleotide sequence of the invention.

## Claims

### Claim 1

A DNA construct comprising a polynucleotide operably linked to a heterologous promoter, wherein said polynucleotide encodes a Bacillus thuringiensis insecticidal toxin protein or insecticidal fragment thereof, active against a lepidopteran insect pest, wherein said insecticidal toxin protein comprises the polypeptide sequence-as set forth in SEQ ID NO:4 (TIC900), SEQ ID NO:8 (TIC403), SEQ ID NO:10 (TIC404), SEQ ID NO:30 (TIC434), SEQ ID NO:12 (TIC961), SEQ ID NO:14 (TIC962), SEQ ID NO:16 (TIC963), SEQ ID NO:18 (TIC965) or SEQ ID NO:20 (TIC966).

### Claim 2 (depends on 1)

The DNA construct of claim 1, wherein said lepidopteran insect pest is selected from the group consisting of a Noctuidae, a Tortricidae, Epinotia aporema, Anticarsia gemmatalis, Pseudoplusia includens, European Corn Borer (ECB), a Tobacco Budworm (TBW), Black Cutworm (BCW), and a Diamondback Moth (DBM).

### Claim 3 (depends on 1)

The DNA construct of claim 1, wherein said insecticidal toxin protein has a molecular weight between approximately 65 kDa and approximately 70 kDa, and wherein said insecticidal toxin protein is selected from the group consisting of SEQ ID NO:4 (TIC900), SEQ ID NO:8 (TIC403), SEQ ID NO:10 (TIC404), SEQ ID NO:30 (TIC434), SEQ ID NO:12 (TIC961), SEQ ID NO:14 (TIC962), SEQ ID NO:16 (TIC963), SEQ ID NO:18 (TIC965) and SEQ ID NO:20 (TIC966).

### Claim 4 (depends on 1)

The DNA construct of claim 1, wherein the sequence of said polynucleotide has been optimized for expression in plants.

### Claim 5 (depends on 4)

The DNA construct of claim 4, wherein said sequence of said polynucleotide has been optimized for
(a) expression in a monocot plant, said optimization comprising one or more of the steps selected from the group consisting of
(i) removing polyadenylation sequences,
(ii) adjusting the A and T content of the nucleotide sequence to be from about 40% to about 49% without modifying the amino acid sequence of the protein, and
(iii) modifying codons in the coding sequence to be consistent with the steps (i) and (ii), or
(b) expression in a dicot plant, said optimization comprising one or more of the steps selected from the group consisting of
(i) removing polyadenylation sequences,
(ii) adjusting the A and T content of the nucleotide sequence to be from about 40% to about 49% without modifying the amino acid sequence of the protein, and
(iii) modifying codons in the coding sequence to be consistent with the steps (i) and (ii).

### Claim 6

An insecticidal protein active against Lepidopteran insects, said protein comprising the amino acid sequence as set forth in SEQ ID NO:4 (TIC900), SEQ ID NO:8 (TIC403), SEQ ID NO:10 (TIC404), SEQ ID NO:30 (TIC434), SEQ ID NO:12 (TIC961), SEQ ID NO:14 (TIC962), SEQ ID NO:16 (TIC963), SEQ ID NO:18 (TIC965) or SEQ ID NO:20 (TIC966).

### Claim 7 (depends on 1)

A host cell comprising the DNA construct of claim 1.

### Claim 8 (depends on 7)

The host cell of claim 7, wherein said host cell is a plant cell.

### Claim 9 (depends on 6)

A method for controlling a lepidopteran insect pest, said method comprising contacting said pest with a pesticidal amount of the insecticidal protein of claim 6 or an insecticidal fragment thereof.

### Claim 10 (depends on 8)

The host cell of claim 8, said plant cell selected from the group consisting of a corn plant cell, a wheat plant cell, a rice plant cell, an oat plant cell, an onion plant cell, and a grass plant cell; and wherein said dicot plant cell comprises a cotton plant cell, a canola plant cell, a soybean plant cell, a tobacco plant cell, a fruit tree plant cell, a cruciferous plant cell, a pepper plant cell, an ornamental plant cell, a sunflower plant cell, a cucurbit plant cell, and a melon plant cell.

### Claim 11

A method for expressing a lepidopteran-active toxin protein in a plant, comprising the steps of:
(a) inserting into the genome of a plant cell a nucleic acid sequence comprising in the 5′ to 3′ direction an operably linked recombinant, double-stranded DNA molecule, wherein the recombinant, double-stranded DNA molecule comprises:
(i) a promoter that functions in the plant cell;
(ii) a nucleotide sequence encoding the insecticidal amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:30, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20; and
(iii) a 3′ non-translated nucleotide sequence that functions in the cells of the plant to cause termination of transcription;
(b) obtaining a transformed plant cell containing the nucleic acid sequence of step (a); and
(d) generating from said transformed plant cell a plant that expresses the lepidopteran-active toxin protein in the transformed plant.

### Claim 12 (depends on 1)

A plasmid vector comprising the DNA construct of claim 1.

### Claim 13 (depends on 1)

A transformed plant comprising the DNA construct of claim 1.

### Claim 14 (depends on 13)

A seed from the transformed plant of claim 13, wherein said seed comprises said DNA construct.

### Claim 15 (depends on 14)

Progeny of the seed of claim 14, wherein said progeny comprise said DNA construct.

### Claim 16 (depends on 13)

A biological sample derived from tissues or seed of the transformed plant of claim 13, said sample comprising a detectable amount of said DNA construct.