Nucleic acid sequences encoding transcription factors regulating alkaloid biosynthesis and their use in modifying plant metabolism

• US 10,030,249 B2
• Filed: 09/11/2015
• Issued: 07/24/2018
• Est. Priority Date: 05/25/2007
• Status: Active Grant

• Current Assignee: 22nd Century Limited, LLC (22nd Century Group, Inc.)
• Original Assignee: 22nd Century Limited, LLC (22nd Century Group, Inc.)
• Inventors: Page, Jonathan, Todd, Andrea T.
• Primary Examiner(s): COLLINS, CYNTHIA E
• Application Number:
  US14/852,158
• Publication Number:
  US 20160130601A1
• Time in Patent Office: 1,047 Days
• Field of Search: None
• CPC Class Codes: A24B 13/00 Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff reconstituted tobacco products A24B3/14
• CPC Class Codes: A24B 15/10 Chemical features of tobacco products or tobacco substitutes
• CPC Class Codes: A24B 15/12 of reconstituted tobacco
• CPC Class Codes: A24D 1/00 Cigars; Cigarettes
• CPC Class Codes: A24D 1/10 with extinguishers
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

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

1. A method for increasing a nicotinic alkaloid in a Nicotiana plant, comprising up-regulating a transcription factor that positively regulates nicotinic alkaloid biosynthesis, wherein the transcription factor is up-regulated by:

(a) introducing into the Nicotiana plant an expression construct comprising a nucleic acid molecule selected from the group consisting of;

(i) the nucleotide sequence set forth in SEQ ID NO;

1 or SEQ ID NO;

2;

(ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO;

3; and

(iii) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (i) or (ii), and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis; and

(b) growing the Nicotiana plant under conditions whereby the expression construct increases levels of the transcription factor in the Nicotiana plant as compared to a control Nicotiana plant grown under similar conditions;

wherein the nucleic acid molecule is operably linked to one or more heterologous control sequences suitable for directing expression in a Nicotiana plant.

2. The method of claim 1, further comprising overexpressing at least one additional transcription factor that positively regulates nicotinic alkaloid biosynthesis.

3. The method of claim 2, further comprising suppressing the expression of at least one transcription factor that negatively regulates nicotinic alkaloid biosynthesis.

4. A genetically engineered nicotinic alkaloid-producing Nicotiana plant produced by the method of claim 2, wherein the plant comprises a chimeric nucleic acid construct comprising a nucleic acid molecule encoded by a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence set forth in SEQ ID NO;

1 or SEQ ID NO;

2;

(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO;

3; and

(c) a nucleotide sequence at least 90% identical to the nucleotide sequences of (a) or (b), and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis;

wherein the nucleic acid molecule is linked to a heterologous nucleic acid to form a chimeric nucleic acid construct.

5. The engineered Nicotiana plant of claim 4, wherein the plant is a Nicotiana tabacum plant.

6. Seeds from the engineered Nicotiana plant of claim 4, wherein the seeds comprise the chimeric nucleic acid construct.

7. A product comprising the engineered Nicotiana plant of claim 4, wherein the product has an increased level of nicotinic alkaloid relative to a product produced from a non-transformed control Nicotiana plant.