Compositions and methods for minimizing nornicotine synthesis in tobacco

• US 10,681,883 B2
• Filed: 06/29/2017
• Issued: 06/16/2020
• Est. Priority Date: 01/15/2010
• Status: Active Grant

• Current Assignee: North Carolina State University (University of North Carolina System)
• Original Assignee: North Carolina State University (University of North Carolina System)
• Inventors: Dewey, Ralph E., Lewis, Ramsey S.
• Primary Examiner(s): Fan, Weihua
• Application Number:
  US15/637,865
• Publication Number:
  US 20170359994A1
• Time in Patent Office: 1,083 Days
• CPC Class Codes: A01H 5/12 Leaves
• CPC Class Codes: A01H 6/823 Nicotiana, e.g. tobacco
• 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: C12N 15/8223 Vegetative tissue-specific promoters
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12N 9/0073 with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2600/13 Plant traits
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) in tobacco plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for a root-specific nicotine demethylases, CYP82E10, and variants thereof, that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Compositions of the invention also include tobacco plants, or plant parts thereof, comprising a mutation in a gene encoding a CYP82E10 nicotine demethylase, wherein the mutation results in reduced expression or function of the CYP82E10 nicotine demethylase. Seed of these tobacco plants, or progeny thereof, and tobacco products prepared from the tobacco plants of the invention, or from plant parts or progeny thereof, are also provided. Methods for reducing the level of nornicotine, or reducing the rate of conversion of nicotine to nornicotine, in a tobacco plant, or plant part thereof are also provided. The methods comprise introducing into the genome of a tobacco plant a mutation within at least one allele of each of at least three nicotine demethylase genes, wherein the mutation reduces expression of the nicotine demethylase gene, and wherein a first of these nicotine demethylase genes encodes a root-specific nicotine demethylase involved in the metabolic conversion of nicotine to nornicotine in a tobacco plant or a plant part thereof. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products.

Claims

1. Cured tobacco material or a tobacco product comprising said cured tobacco material, wherein said cured tobacco material is made from a tobacco plant comprising a mutant CYP82E10 gene encoding a truncated CYP82E10 polypeptide comprising a mutation compared to SEQ ID NO:

2, wherein said mutant CYP82E10 gene results in reduced expression or function of said CYP82E10 polypeptide when compared to the activity of a wild-type CYP82E10 polypeptide under the same test conditions.

2. The cured tobacco material or tobacco product of claim 1, wherein said truncated CYP82E10 polypeptide comprises at least 75 contiguous amino acids of SEQ ID NO:

2.

3. The cured tobacco material or tobacco product of claim 1, wherein said truncated CYP82E10 polypeptide is encoded by a polynucleotide comprising a mutation as compared to SEQ ID NO:

4.

4. The cured tobacco material or tobacco product of claim 3, wherein said mutation is selected from the group consisting of a substitution, an insertion, or a deletion.

5. The cured tobacco material or tobacco product of claim 1, wherein said truncated CYP82E10 polypeptide has nicotine demethylase activity that is inhibited by at least 25% when compared to the activity of a wild-type CYP82E10 polypeptide under the same test conditions.

6. The cured tobacco material or tobacco product of claim 1, wherein said truncated CYP82E10 polypeptide has nicotine demethylase activity that is inhibited by at least 50% when compared to the activity of a wild-type CYP82E10 polypeptide under the same test conditions.

7. The cured tobacco material or tobacco product of claim 3, wherein said polynucleotide mutation is positioned between two sequences having the sequences of SEQ ID NOs:

35 and 36.

8. The tobacco product of claim 1, wherein said tobacco product is selected from the group consisting of a cigarette, a cigarillo, a non-ventilated filter cigarette, a vented recess filter cigarette, a cigar, pipe tobacco, snuff, chewing tobacco, gum, a lozenge, and a dissolving strip.

9. Cured tobacco material or a tobacco product comprising said cured tobacco material, wherein said cured tobacco material comprises a polynucleotide comprising a mutation in a CYP82E10 exon 1 sequence as compared to SEQ ID NO:

4, wherein said mutation results in reduced expression or function of a CYP82E10 polypeptide encoded by said polynucleotide when compared to the activity of a wild-type CYP82E10 polypeptide under the same test conditions.

10. The cured tobacco material or tobacco product of claim 9, wherein said CYP82E10 exon 1 sequence comprises nucleotides 115 to 1053 of SEQ ID NO:

4.

11. The cured tobacco material or tobacco product of claim 9, wherein said mutation is selected from the group consisting of a substitution, an insertion, or a deletion.

12. The cured tobacco material or tobacco product of claim 9, wherein a CYP82E10 polypeptide encoded by said polynucleotide comprises nicotine demethylase activity that is inhibited by at least 25% when compared to the activity of a wild-type CYP82E10 polypeptide under the same test conditions.

13. The cured tobacco material or tobacco product of claim 9, wherein a CYP82E10 polypeptide encoded by said polynucleotide comprises nicotine demethylase activity that is inhibited by at least 50% when compared to the activity of a wild-type CYP82E10 polypeptide under the same test conditions.

14. The tobacco product of claim 9, wherein said tobacco product is selected from the group consisting of a cigarette, a cigarillo, a non-ventilated filter cigarette, a vented recess filter cigarette, a cigar, pipe tobacco, snuff, chewing tobacco, gum, a lozenge, and a dissolving strip.

15. The cured tobacco material or tobacco product of claim 9, wherein said mutation is positioned between two sequences identical or complementary to SEQ ID NOs:

35 and 36.

16. Cured tobacco material or a tobacco product comprising said cured tobacco material, wherein said cured tobacco material comprises a null mutation in a polynucleotide encoding a CYP82E10 polypeptide comprising a mutation compared to SEQ ID NO:

2.

17. The cured tobacco material or tobacco product of claim 16, wherein said null mutation is located in a CYP82E10 exon 1 sequence as compared to SEQ ID NO:

4.

18. The cured tobacco material or tobacco product of claim 16, wherein said null mutation is positioned between two sequences identical or complementary to SEQ ID NOs:

35 and 36.

19. The cured tobacco material or tobacco product of claim 16, wherein said mutation is selected from the group consisting of a substitution, an insertion, or a deletion.

20. The tobacco product comprising said cured tobacco material of claim 16, wherein said tobacco product is selected from the group consisting of a cigarette, a cigarillo, a non-ventilated filter cigarette, a vented recess filter cigarette, a cigar, pipe tobacco, snuff, chewing tobacco, gum, a lozenge, and a dissolving strip.