Compositions and methods for minimizing nornicotine synthesis in tobacco

• US 9,247,706 B2
• Filed: 01/13/2011
• Issued: 02/02/2016
• 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): Kallis, Russell
• Assistant Examiner(s): FAN, WEIHUA
• Application Number:
  US13/521,766
• Publication Number:
  US 20130037040A1
• Time in Patent Office: 1,846 Days
• US Class Current: 1/1
• 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 first mutation in a CYP82E10 nicotine demethylase, wherein said first mutation results in reduced expression or function of said CYP82E10 nicotine demethylase, and wherein said tobacco plant is a Nicotiana tabacum plant.

2. The cured tobacco material or a tobacco product comprising said cured tobacco material, according to claim 1, wherein said tobacco plant further comprises a second mutation in a CYP82E4 nicotine demethylase, wherein said second mutation results in reduced expression or function of said CYP82E4 nicotine demethylase.

3. The cured tobacco material or a tobacco product comprising said cured tobacco material, according to claim 1, wherein said tobacco plant further comprises a second mutation in a CYP82E5v2 nicotine demethylase, wherein said second mutation results in reduced expression or function of said CYP82E5v2 nicotine demethylase.

4. The cured tobacco material or a tobacco product comprising said cured tobacco material, according to claim 2, wherein said tobacco plant further comprises a third mutation in a CYP82E5v2 nicotine demethylase, wherein said third mutation results in reduced expression or function of said CYP82E5v2 nicotine demethylase.

5. The tobacco product according to claim 4.

6. The tobacco product according to claim 4, wherein said first mutation results in a Proline to Serine substitution at amino acid position 381 of CYP82E10 and said CYP82E10 numbering is according to SEQ ID NO:

2;

wherein said second mutation results in a Tryptophan to stop codon substitution at amino acid position 329 of CYP82E4 and said CYP82E4 numbering is according to SEQ ID NO;

14; and

wherein said third mutation results in a Tryptophan to stop codon substitution at amino acid position 422 of CYP82E5v2 and said CYP82E5v2 numbering is according to SEQ ID NO;

26.

7. The tobacco product according to claim 4, wherein each of said mutations is homozygous.

8. The tobacco product according to claim 4, wherein said tobacco material is made by a curing process selected from the group consisting of flue curing, air curing, and fire curing.

9. The tobacco product according to claim 4, wherein said tobacco product is selected from the group consisting of a cigar, a cigarette, pipe tobacco, a cigarillo, a non-ventilated or vented recess filter cigarette, a dissolving strip, a gum, a tablet, snuff, and chewing tobacco.

10. The tobacco product according to claim 4, wherein said cured tobacco material comprises a lower level of nornicotine or N′

-nitrosonornicotine compared to cured tobacco material made from a tobacco plant comprising wild type alleles CYP82E4, CYP82E5v2, and CYP82E10.

11. The tobacco product according to claim 4, wherein the conversion of nicotine to nornicotine in said cured tobacco material is selected from the group consisting of less than 0.3%, less than 0.5%, less than 0.7%, between 0.1%-0.5%, between 0.1%-0.4%, between 0.1%-0.7%, and between 0.1%-1.0%.

12. The tobacco product according to claim 4, wherein said tobacco plant has a non-converter phenotype and reduced nicotine demethylase activity.

13. The tobacco product according to claim 4, wherein said tobacco plant is a hybrid.

14. The tobacco product according to claim 4, wherein said tobacco plant is selected from the group consisting of a Burley type, a dark type, a flue-cured type, and an Oriental type.

15. The tobacco product according to claim 4, wherein said first, second, or third mutation is selected from the group consisting of a point mutation, a deletion, an insertion, and an inversion.

16. The tobacco product according to claim 4, wherein said CYP82E10 mutation comprises a Cytosine to Thymine substitution at nucleic acid position 1141.

17. The tobacco product according to claim 4, wherein said CYP82E10 mutation comprises a Cytosine to Thymine substitution at nucleic acid position 1141;

wherein said nucleic acid position numbering is according to SEQ ID NO;

1;

wherein said second mutation results in a Tryptophan to stop codon substitution at amino acid position 329 of CYP82E4 and said CYP82E4 numbering is according to SEQ ID NO;

14; and

wherein said third mutation results in a Tryptophan to stop codon substitution at amino acid position 422 of CYP82E5v2 and said CYP82E5v2 numbering is according to SEQ ID NO;

26.

18. A tobacco seed, tobacco plant developed therefrom, or plant part thereof, said tobacco plant comprising a CYP82E10 nicotine demethylase having a Cytosine to Thymine mutation at nucleic acid position 1141, and said CYP82E10 position numbering is according to SEQ ID NO:

1;

said tobacco plant also comprising a CYP82E4 nicotine demethylase having a mutation at amino acid position 329, and said CYP82E4 numbering is according to SEQ ID NO;

14; and

said tobacco plant further comprising a CYP82E5v2 nicotine demethylase having a mutation at amino acid position 422, wherein said CYP82E5v2 numbering is according to SEQ ID NO;

26; and

wherein said tobacco plant is a Nicotiana tabacum plant.

19. The tobacco seed, tobacco plant developed therefrom, or plant part thereof, according to claim 18, wherein said CYP82E4 mutation at amino acid position 329 and said CYP82E5 mutation at amino acid position 422 both result in a stop codon.