# US 11,441,151 B2 · Increased protein expression in plants

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- Number: US 11,441,151 B2
- Document ID: US11441151B2
- Status: Granted
- Kind: B2
- Application number: 17/021,710
- Filed: 2020-09-15
- Published: 2020-12-31
- Granted: 2022-09-13
- Earliest claimed: 2014-01-17
- Assignees: Dow AgroSciences LLC
- Applicants: Dow AgroSciences LLC
- Inventors: Aaron Woosley; Sarah Worden
- Web page: https://patentgrep.com/patent/US11441151B2

## CPC

- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- A01N 37/46: N-acyl derivatives
- A23L 7/198: {Dry unshaped finely divided cereal products, not provided for in groups A23L7/117 - A23L7/196 and A23L29/00, e.g. meal, flour, powder, dried cereal creams or extracts}
- C12N 15/8286: {for insect resistance}
- 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
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

This disclosure concerns synthetic polynucleotides encoding a polypeptide of interest that are particularly well-suited for expression in target plants.

## Claims

### Claim 1

A polynucleotide comprising:
a plant promoter that is functional in maize; and
a synthetic nucleotide sequence encoding a polypeptide encoded by a native nucleotide sequence in the genome of an organism other than a maize plant,
wherein the synthetic nucleotide sequence has been codon-optimized to remove codons selected from the group consisting of TTA, CTA, GTA, CGT, AGT, and CGA, from the native nucleotide sequence,
wherein the synthetic nucleotide sequence comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the native nucleotide sequence,
wherein the synthetic nucleotide sequence does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the native nucleotide sequence, and
wherein the synthetic nucleotide sequence does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA.

### Claim 2 (depends on 1)

The polynucleotide of claim 1, wherein the nucleotide sequence encoding the polypeptide has been codon-optimized to remove from the reference nucleotide sequence all codons selected from the group consisting of TTA, CTA, GTA, CGT, AGT, and CGA.

### Claim 3 (depends on 1)

The polynucleotide of claim 1, wherein the polypeptide is selected from the group consisting of insecticidal polypeptides, herbicide tolerance polypeptides, stress tolerance-related polypeptides, and oil profile modification polypeptides.

### Claim 4 (depends on 3)

The polynucleotide of claim 3, wherein the polypeptide is an insecticidal polypeptide or herbicide tolerance polypeptide.

### Claim 5 (depends on 4)

The polynucleotide of claim 4, wherein the polypeptide is an insecticidal polypeptide.

### Claim 6 (depends on 1)

A plant expression construct comprising the polynucleotide of claim 1.

### Claim 7 (depends on 6)

A vector comprising the plant expression construct of claim 6.

### Claim 8

A method for producing a synthetic polynucleotide, the method comprising:
removing codons selected from the group consisting of TTA, CTA, GTA, CGT, AGT, and CGA, from a nucleotide sequence encoding a polypeptide that is native in an organism other than a maize plant, to produce a codon-optimized nucleotide sequence;
engineering the codon-optimized nucleotide sequence such that:
the codon-optimized nucleotide sequence comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the nucleotide sequence encoding the polypeptide that is native in the organism other than a maize plant,
the codon-optimized nucleotide sequence does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the nucleotide sequence encoding the polypeptide that is native in the organism other than a maize plant, and
the codon-optimized nucleotide sequence does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA; and
constructing the synthetic polynucleotide.

### Claim 9 (depends on 8)

The method according to claim 8, wherein constructing the synthetic polynucleotide comprises concatenation of smaller oligonucleotides, or restriction digestion and ligation.

### Claim 10 (depends on 8)

The method according to claim 8, further comprising ligating the synthetic polynucleotide into an expression vector comprising a plant promoter that is functional in maize.

### Claim 11 (depends on 7)

A method for producing transgenic maize, the method comprising:
transforming a maize cell with the vector of claim 7, thereby producing a transgenic maize cell.

### Claim 12 (depends on 11)

The method according to claim 11, further comprising regenerating a transgenic maize plant from the transgenic maize cell.

### Claim 13

A polynucleotide comprising:
a plant promoter that is functional in soybean; and
a synthetic nucleotide sequence encoding a polypeptide encoded by a native nucleotide sequence in the genome of an organism other than a soybean plant,
wherein the synthetic nucleotide sequence has been codon-optimized to remove codons selected from the group consisting of TTA, CTA, TCG, CCG, ACG, GCG, CGA, and CGG, from the native nucleotide sequence,
wherein the synthetic nucleotide sequence comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the native nucleotide sequence,
wherein the synthetic nucleotide sequence does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the native nucleotide sequence, and
wherein the synthetic nucleotide sequence does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA.

### Claim 14 (depends on 13)

The polynucleotide of claim 13, wherein the nucleotide sequence encoding the polypeptide has been codon-optimized to remove from the reference nucleotide sequence all codons selected from the group consisting of TTA, CTA, TCG, CCG, ACG, GCG, CGA, and CGG.

### Claim 15 (depends on 13)

The polynucleotide of claim 13, wherein the polypeptide is selected from the group consisting of insecticidal polypeptides, herbicide tolerance polypeptides, stress tolerance-related polypeptides, and oil profile modification polypeptides.

### Claim 16 (depends on 15)

The polynucleotide of claim 15, wherein the polypeptide is an insecticidal polypeptide or herbicide tolerance polypeptide.

### Claim 17 (depends on 16)

The polynucleotide of claim 16, wherein the polypeptide is an insecticidal polypeptide.

### Claim 18 (depends on 13)

A plant expression construct comprising the polynucleotide of claim 13.

### Claim 19 (depends on 18)

A vector comprising the plant expression construct of claim 18.

### Claim 20

A method for producing a synthetic polynucleotide, the method comprising:
removing codons selected from the group consisting of TTA, CTA, TCG, CCG, ACG, GCG, CGA, and CGG, from a nucleotide sequence encoding a polypeptide that is native in an organism other than a soybean plant, to produce a codon-optimized nucleotide sequence;
engineering the codon-optimized nucleotide sequence such that:
the codon-optimized nucleotide sequence comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the nucleotide sequence encoding the polypeptide that is native in the organism other than a maize plant,
the codon-optimized nucleotide sequence does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the nucleotide sequence encoding the polypeptide that is native in the organism other than a maize plant, and
the codon-optimized nucleotide sequence does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA; and
constructing the synthetic polynucleotide.

### Claim 21 (depends on 20)

The method according to claim 20, wherein constructing the synthetic polynucleotide comprises concatenation of smaller oligonucleotides, or restriction digestion and ligation.

### Claim 22 (depends on 20)

The method according to claim 20, further comprising ligating the synthetic polynucleotide into an expression vector comprising a plant promoter that is functional in soybean.

### Claim 23 (depends on 19)

A method for producing transgenic soybean, the method comprising:
transforming a soybean cell or protoplast with the vector of claim 19, thereby producing a transgenic soybean cell.

### Claim 24 (depends on 23)

The method according to claim 23, further comprising regenerating a transgenic soybean plant from the transgenic soybean cell.