# US 12,180,523 B2 · Southern green stink bug pheromone synthesis enzymes and uses thereof

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- Number: US 12,180,523 B2
- Document ID: US12180523B2
- Status: Granted
- Kind: B2
- Application number: 17/291,604
- Filed: 2019-09-28
- Published: 2021-12-16
- Granted: 2024-12-31
- Earliest claimed: 2018-11-06
- Assignees: Virginia Tech Intellectual Properties, Inc.
- Applicants: Virginia Tech Intellectual Properties, Inc.
- Inventors: Dorothea Tholl; Jason Lancaster
- Web page: https://patentgrep.com/patent/US12180523B2

## CPC

- C12N 9/88: Lyases (4.)
- C12N 15/8286: {for insect resistance}
- A01N 63/60: Isolated nucleic acids
- C12N 9/1085: {transferring alkyl or aryl groups other than methyl groups (2.5)}
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- C12P 5/007: {containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00)}
- C12P 17/02: Oxygen as only ring hetero atoms
- C12Q 1/68: involving nucleic acids
- 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
- 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)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Described herein are engineered polynucleotides and vectors capable of encoding one or more engineered southern green stink bug pheromone synthesis enzymes. Also described herein are engineered southern green stink bug pheromone synthesis enzymes. Also described herein are methods of making modified plants capable of expressing one or more southern green stink bug pheromone synthesis enzymes.

## Claims

### Claim 1

An engineered polynucleotide comprising:
one or more polynucleotides, wherein the one or more polynucleotides
(a) have a sequence that is 95% to 100% identical to SEQ ID NO: 1 or 3 and encode a terpene synthase (TPS) protein comprising an amino acid sequence that is about 95% to 100% identical to SEQ ID NO: 4;
(b) have a sequence that is 95% to 100% identical to SEQ ID NO: 2 and
encode an isoprenyl diphosphate synthase (IDS) protein comprising an amino acid sequence that is about 95% to 100% identical to SEQ ID NO: 5; or
(c) have both (a) and (b); and one or more heterologous regulatory polynucleotides operably coupled to the one or more polynucleotides.

### Claim 2 (depends on 1)

A vector comprising:
one or more engineered polynucleotides of claim 1.

### Claim 3 (depends on 1)

A cell comprising:
the engineered polynucleotide of claim 1, or a vector comprising the engineered polynucleotide, wherein the engineered polynucleotide encodes a terpene synthase (TPS) protein comprising an amino acid sequence that is about 95% to 100% identical to SEQ ID NO: 4 or encodes an isoprenyl diphosphate synthase (IDS) protein comprising an amino acid sequence that is about 95% to 100% identical to SEQ ID NO: 5, or a combination thereof.

### Claim 4 (depends on 3)

The cell of claim 3, wherein the cell is a plant cell.

### Claim 5 (depends on 3)

The cell of claim 3, wherein the plant cell is from a plant species suitable for use as a trap crop for management of the southern green stink bug.

### Claim 6 (depends on 3)

The cell of claim 3, wherein the cell is capable of producing a southern green stink bug pheromone, a southern green stink bug pheromone intermediate, or both.

### Claim 7 (depends on 3)

A genetically modified plant comprising: the cell of claim 3 or a population thereof.

### Claim 8 (depends on 7)

The genetically modified plant of claim 7, wherein the genetically modified plant is of a species suitable for use as a trap crop for management of the southern green stink bug.

### Claim 9 (depends on 8)

A method of managing southern green stink bug infestation of a desired crop, the method comprising:
planting the genetically modified plant of claim 8.

### Claim 10 (depends on 9)

The method of claim 9, wherein the genetically modified plant is planted in a location next to or near the desired crop.

### Claim 11 (depends on 6)

The cell of claim 6, wherein the cell is a sunflower plant cell, a squash plant cell, a zucchini plant cell, a pumpkin plant cell, a hollyhock cell, a buckwheat cell, a triticale cell, a crimson clover cell, a vetch sorghum cell, or a millet cell.

### Claim 12 (depends on 8)

The genetically modified plant of claim 8, wherein the genetically modified plant is a sunflower plant, a squash plant, a zucchini plant, a pumpkin plant, hollyhock, buckwheat, triticale, crimson clover, vetch sorghum, or millet.

### Claim 13 (depends on 10)

The method of claim 10, wherein the desired crop is a cash crop.

### Claim 14 (depends on 10)

The method of claim 10, further comprising positioning a visual attractant near the genetically modified plant, positioning semiochemical attractants near the genetically modified plant, or both.

### Claim 15 (depends on 10)

The method of claim 10, wherein the genetically modified plant is effective as a trap crop for the southern green stink bug.

### Claim 16 (depends on 1)

The engineered polynucleotide of claim 1, further comprising a polynucleotide that encodes a cytochrome P450 protein comprising an amino acid sequence that is about 100% identical to any one of SEQ ID NOs: 6-79.

### Claim 17 (depends on 1)

The engineered polynucleotide of claim 1, wherein the TPS protein comprises a first aspartate rich motif (FARM).

### Claim 18 (depends on 1)

The engineered polynucleotide of claim 1, wherein the IDS protein comprises a second aspartate rich motif SARM.

### Claim 19 (depends on 3)

The cell of claim 3, further comprising a cytochrome P450 protein comprising an amino acid sequence that is about 100% identical to any one of SEQ ID NOs: 6-79.

### Claim 20 (depends on 7)

The genetically modified plant of claim 7, wherein the cell further comprises a cytochrome P450 protein comprising an amino acid sequence that is about 100% identical to any one of SEQ ID NOs: 6-79.