# US 9,752,156 B2 · Nucleic acid sequences encoding transcription factors regulating alkaloid biosynthesis and their use in modifying plant metabolism

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- Number: US 9,752,156 B2
- Document ID: US9752156B2
- Status: Granted
- Kind: B2
- Application number: 14/840,019
- Filed: 2015-08-30
- Published: 2016-01-07
- Granted: 2017-09-05
- Earliest claimed: 2007-05-25
- Assignees: 22nd Century Limited, LLC
- Applicants: 22nd Century Limited, LLC
- Inventors: Jonathan Page; Andrea T. Todd
- Web page: https://patentgrep.com/patent/US9752156B2

## CPC

- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- A24B 13/00: Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff (reconstituted tobacco products A24B3/14)
- A24B 15/10: Chemical features of tobacco products or tobacco substitutes
- A24B 15/12: of reconstituted tobacco
- A24D 1/00: Cigars; Cigarettes
- A24D 1/10: with extinguishers
- C07K 14/415: from plants
- C07K 14/4702: {Regulators; Modulating activity}
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A24B: MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24D: CIGARS; CIGARETTES; TOBACCO SMOKE FILTERS; MOUTHPIECES OF CIGARS OR CIGARETTES; MANUFACTURE OF TOBACCO SMOKE FILTERS OR MOUTHPIECES
- 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)

## Abstract

Plant metabolism and alkaloid levels can be regulated by transcription factors that regulate the nicotinic alkaloid biosynthetic pathway. In one embodiment, the disclosure provides a transcription factor that negatively regulates alkaloid biosynthesis, such as nicotine biosynthesis.

## Claims

### Claim 1

An isolated cDNA molecule comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9;
(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10; and
(c) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (a) or (b);
wherein (a), (b), and (c) encode a transcription factor that positively regulates nicotinic alkaloid biosynthesis.

### Claim 2 (depends on 1)

A Nicotiana cell comprising the cDNA molecule of claim 1.

### Claim 3 (depends on 2)

The Nicotiana cell of claim 2, wherein the level of the transcription factor in the Nicotiana cell is higher than in a Nicotiana cell that does not contain the nucleic acid molecule.

### Claim 4 (depends on 1)

An expression vector comprising the nucleic acid molecule of claim 1, operably linked to one or more control sequences suitable for directing expression in a Nicotiana cell.

### Claim 5 (depends on 4)

A Nicotiana cell comprising the expression vector of claim 4.

### Claim 6 (depends on 5)

The Nicotiana cell of claim 5, wherein the level of the transcription factor in the Nicotiana cell is higher than in a Nicotiana cell that does not contain the expression vector.

### Claim 7 (depends on 1)

The isolated cDNA molecule of claim 1, wherein the nucleotide sequence is set forth in SEQ ID NO: 8.

### Claim 8 (depends on 1)

The isolated cDNA molecule of claim 1, wherein the nucleotide sequence is set forth in SEQ ID NO: 9.

### Claim 9 (depends on 1)

The isolated cDNA molecule of claim 1, wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO: 8, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.

### Claim 10 (depends on 1)

The isolated cDNA molecule of claim 1, wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO: 9, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.

### Claim 11 (depends on 1)

The isolated cDNA molecule of claim 1, wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.

### Claim 12

A method for reducing a nicotinic alkaloid in a Nicotiana plant, comprising down-regulating a transcription factor that positively regulates nicotinic alkaloid biosynthesis, wherein the transcription factor is down-regulated by:
(a) genetically engineering a population of plant cells to eliminate production of a protein encoded by the nucleotide sequence set forth in SEQ ID NO: 8, comprising introducing one or more mutations into the region of SEQ ID NO: 8 that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10; and
(b) detecting and selecting a target mutated plant cell or a plant derived from such a cell, wherein the target mutated plant cell or plant has a mutation in a gene encoding a transcription factor that positively regulates nicotinic alkaloid biosynthesis and wherein the targeted plant cell or plant has a decreased nicotinic alkaloid content relative to a control plant cell or plant.

### Claim 13 (depends on 12)

The method of claim 12, wherein the genetic engineering further comprises introducing into the population of cells a reagent comprising a recombinagenic oligonucleobase.

### Claim 14 (depends on 12)

The method of claim 12, wherein the genetic engineering further comprises introducing into the population of cells a reagent comprising a targeted nuclease.

### Claim 15 (depends on 12)

The method of claim 12, wherein the genetic engineering further comprises suppressing within the Nicotiana plant at least one of NBB1, A622, quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO).

### Claim 16 (depends on 12)

The method of claim 12, wherein the genetic engineering further comprises suppressing within the Nicotiana plant at least one additional transcription factor that positively regulates nicotinic alkaloid biosynthesis.

### Claim 17 (depends on 12)

The method of claim 12, wherein the genetic engineering further comprises overexpressing within the Nicotiana plant at least one transcription factor that negatively regulates nicotinic alkaloid biosynthesis.

### Claim 18 (depends on 12)

The method of claim 12, wherein the genetic engineering further comprises introducing one or more mutations into regions of SEQ ID NO: 8 resulting in a complete deletion of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 10.

### Claim 19 (depends on 12)

A mutated Nicotiana plant produced by the method of claim 12, wherein the plant has reduced expression of a transcription factor that positively regulates nicotinic alkaloid biosynthesis and reduced nicotinic alkaloid content, as compared to a control plant.

### Claim 20 (depends on 19)

The mutated plant of claim 19, wherein the plant is a Nicotiana tabacum plant.

### Claim 21 (depends on 19)

A product having a reduced level of a nicotinic alkaloid comprising the mutated plant of claim 19, or portions thereof, relative to a product produced from a control plant.

### Claim 22 (depends on 21)

The product of claim 21, wherein the product is selected from the group consisting of a cigarette, cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, lozenges, and reconstituted tobacco.

### Claim 23 (depends on 19)

Seeds from the mutated plant of claim 19.