PLANTS WITH REDUCED ASPARAGINE CONTENT

• US 20190169628A1
• Filed: 09/06/2016
• Published: 06/06/2019
• Est. Priority Date: 09/09/2015
• Status: Active Grant

• Current Assignee: Philip Morris Products SA (Philip Morris Limited)
• Original Assignee: Philip Morris Products SA (Philip Morris Limited)
• Inventors: BOVET, Lucien
• Granted Patent: US 10,883,114 B2
• Time in Patent Office: Days
• CPC Class Codes: A24B 15/20 Biochemical treatment
• CPC Class Codes: A24B 3/12 Steaming, curing, or flavouring tobacco
• 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
• CPC Class Codes: C12N 15/8251 Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
• CPC Class Codes: C12N 9/93 Ligases (6)

Abstract

There is described herein a mutant, non-naturally occurring or transgenic plant or part thereof having reduced expression or activity of asparagine synthetase, said asparagine synthetase comprising, consisting or consisting essentially of: (i) a polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 90% sequence identity to SEQ ID NO:1 or at least 72% sequence identity to SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7; or (ii) a polypeptide encoded by the polynucleotide set forth in (i); or (iii) a polypeptide having at least 78% sequence identity to SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8; wherein the expression or activity of the asparagine synthetase set forth in (i), (ii) or (iii) is reduced as compared to a control plant.

Claims

1. A mutant, non-naturally occurring or transgenic plant or part thereof having reduced expression or activity of asparagine synthetase, said asparagine synthetase comprising:

(i) a polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 90% sequence identity to SEQ ID NO;

1 or at least 72% sequence identity to SEQ ID NO;

3 or SEQ ID NO;

5 or SEQ ID NO;

7;

or

(ii) a polypeptide encoded by the polynucleotide set forth in (i);

or

(iii) a polypeptide having at least 78% sequence identity to SEQ ID NO;

2 or SEQ ID NO;

4 or SEQ ID NO;

6 or SEQ ID NO;

8;

wherein the expression or activity of the asparagine synthetase set forth in (i), (ii) or (iii) is reduced as compared to a control plant.

2. The mutant, non-naturally occurring or transgenic plant or part thereof according to claim 1, wherein the plant comprises at least one genetic alteration in a regulatory region or in the coding sequence of the polynucleotide encoding the asparagine synthetase.

3. The mutant, non-naturally occurring or transgenic plant or part thereof according to claim 1, wherein the reduced expression or activity of the asparagine synthetase confers a reduction in the level of asparagine in cured or dried leaf derived from the mutant, non-naturally occurring or transgenic plant or part thereof as compared to the level of asparagine in cured or dried leaf derived from the control plant, suitably wherein the level of asparagine is reduced by at least about 17% as compared to the control plant;

and/or

wherein the reduced expression or activity of the asparagine synthetase confers a reduction in the level of acrylamide in aerosol produced upon heating or combusting the cured or dried leaf derived from the mutant, non-naturally occurring or transgenic plant or part thereof as compared to cured or dried leaf derived from the control plant, suitably, wherein the level of acrylamide in aerosol is reduced by at least 20% as compared to cured or dried leaf of the control plant; and

/or

wherein the level of asparagine in cured or dried leaf derived from the mutant, non-naturally occurring or transgenic plant or part thereof is at least about 22% lower and the amount of acrylamide in aerosol produced upon heating or combusting the cured or dried leaf derived from the mutant, non-naturally occurring or transgenic plant or part thereof is at least 24% lower than cured or dried leaf and aerosol of the control plant;

suitably, wherein the level of asparagine in cured or dried leaf derived from the mutant, non-naturally occurring or transgenic plant or part thereof is at least about 44% lower and the amount of acrylamide in aerosol produced upon heating or combusting the cured or dried leaf derived from the mutant, non-naturally occurring or transgenic plant or part thereof is at least 66% lower as compared to cured or dried leaf and aerosol of the control plant;

suitably, wherein the level of asparagine in cured or dried leaf derived from the mutant, non-naturally occurring or transgenic plant or part thereof is at least about 70% lower and the amount of acrylamide in aerosol produced upon heating or combusting the cured or dried leaf derived from the mutant, non-naturally occurring or transgenic plant or part thereof is at least about 88% lower as compared to cured or dried leaf and aerosol of the control plant.

4. The mutant, non-naturally occurring or transgenic plant or part thereof according to claim 1, wherein the level of nicotine in cured or dried leaf from the mutant, non-naturally occurring or transgenic plant or part thereof is substantially the same as the level of nicotine in cured or dried leaf of the control plant;

and/or

wherein the formation of glutamine, aspartic acid and glutamic acid is increased in cured or dried leaf derived from the mutant, non-naturally occurring or transgenic plant or part thereof as compared to cured or dried leaf of the control plant; and

/or

wherein the plant is selected form the group consisting of tobacco or tea, preferably,

wherein the tobacco plant is a Nicotiana tabacum tobacco plant, preferably, a Burley type Nicotiana tabacum tobacco plant.

5. The mutant, non-naturally occurring or transgenic plant or part thereof according to claim 1, wherein the plant comprises at least one mutation in each copy of the polynucleotide sequence encoding the asparagine synthetase set forth in (i), (ii) or (iii), suitably a stop mutation(s) and/or a gene fragment(s) that interferes with the translation of an RNA transcript encoding the asparagine synthetase.

6. The mutant, non-naturally occurring or transgenic plant or part thereof according to claim 1, wherein the asparagine synthetase nucleotide sequence comprises a nucleotide sequence encoding a stop mutation at a position equivalent to the location of the nucleotide sequence encoding the stop mutation in SEQ ID Nos. 19, 21 and 23, or wherein the asparagine synthetase comprises a nucleotide sequence encoding a stop codon at a position equivalent to the location of the stop codon in SEQ ID Nos. 20, 22 and 24.

7. The mutant, non-naturally occurring or transgenic plant or part thereof according to claim 1, wherein the asparagine synthetase comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos. 19, 21 and 23, or is encoded by the nucleotide sequence selected from the group consisting of SEQ ID Nos. 19, 21 and 23, or comprises, consists or consists essentially of the polypeptide sequence set forth in SEQ ID Nos. 20, 22 and 24;

and/or

wherein the amount of leaf biomass from the mutant, non-naturally occurring or transgenic plant or part thereof plant is substantially the same as the amount of leaf biomass from the control plant.

8. The mutant, non-naturally occurring or transgenic plant or part thereof according to claim 3, wherein the leaf is air cured, suitably, wherein the air cured leaf is sun cured or fire cured;

or wherein the leaf is air dried, suitably, wherein the air dried leaf is sun dried or fire dried.

9. Plant material or cured or dried plant material from the plant of claim 1.

10. A plant product comprising at least a part of the plant of claim 1.

14. A method for producing plant material with reduced levels of asparagine and reduced levels of acrylamide in aerosol derived from cured or dried plant material as compared to a control plant, said method comprising the steps of:

(a) providing a plant according to claim 8;

(b) harvesting plant material from the plant;

(c) curing or drying the plant material for a period of time; and

(d) obtaining cured or dried plant material that has reduced levels of asparagine and reduced levels of acrylamide in aerosol derived from the cured or dried plant material, suitably, wherein the level of nicotine is substantially the same as the level of nicotine in the control plant;

suitably, wherein the formation of glutamine, aspartic acid and glutamic acid is increased as compared to the control plant.

15. A tobacco product or a smoking article comprising the plant material of claim 9.

17. A plant product comprising at least a part of the plant of claim 9.

18. A method for producing plant material with reduced levels of asparagine and reduced levels of acrylamide in aerosol derived from cured or dried plant material as compared to a control plant, said method comprising the steps of:

(a) providing the plant material according to claim 9;

(b) harvesting plant material from the plant;

(c) curing or drying the plant material for a period of time; and

(d) obtaining cured or dried plant material that has reduced levels of asparagine and reduced levels of acrylamide in aerosol derived from the cured or dried plant material, suitably, wherein the level of nicotine is substantially the same as the level of nicotine in the control plant;

suitably, wherein the formation of glutamine, aspartic acid and glutamic acid is increased as compared to the control plant.

11. A method of preparing plant material with reduced levels of asparagine and reduced levels of acrylamide in aerosol derived from said plant material as compared to plant material from a control plant, said method comprising the steps of:

(a) providing a plant or part thereof comprising a polynucleotide comprising a sequence encoding an asparagine synthetase and having at least 90% sequence identity to SEQ ID NO;

1 or at least 72% sequence identity to SEQ ID NO;

3 or SEQ ID NO;

5 or SEQ ID NO;

7;

(b) reducing the expression of the polynucleotide or the activity of the protein encoded thereby in the plant or part thereof;

(c) harvesting plant material from the plant or part thereof;

(d) drying or curing the plant material;

(e) optionally, measuring the levels of asparagine in the plant or part thereof and/or measuring the levels of acrylamide in aerosol derived from the plant or part thereof; and

(f) obtaining cured or dried plant material with reduced levels of asparagine and reduced levels of acrylamide in aerosol derived from said plant material, suitably, wherein the level of nicotine is substantially the same as the level of nicotine in the control plant;

suitably, wherein the formation of glutamine, aspartic acid and glutamic acid is increased as compared to cured or dried leaf of the control plant.

12. The method according to claim 11, wherein the plant material is cured or dried for at least about 3 days after harvesting;

and/or

wherein the plant material is air cured, suitably, wherein the air cured leaf is sun cured or fire cured, or wherein the plant material is air dried or sun dried or fire dried.

13. Plant material obtained or obtainable by the method of claim 11.

16. A tobacco product or a smoking article comprising the plant material of claim 13.

19. A mutant, non-naturally occurring, or transgenic plant, or part thereof having asparagine synthetase comprising:

(i) a polynucleotide sequence comprising a sequence having at least 90% sequence identity to SEQ ID NO;

1 or at least 72% sequence identity to SEQ ID NO;

3 or SEQ ID NO;

5 or SEQ ID NO;

7;

or

(ii) a polypeptide encoded by the polynucleotide set forth in (i);

or

(iii) a polypeptide having at least 78% sequence identity to SEQ ID NO;

2 or SEQ ID NO;

4 or SEQ ID NO;

6 or SEQ ID NO;

8.

20. A method comprising altering the plant to comprise a polynucleotide comprising a sequence encoding an asparagine synthetase having at least 90% sequence identity to SEQ ID NO:

1 or at least 72% sequence identity to SEQ ID NO;

3 or SEQ ID NO;

5 or SEQ ID NO;

7.