Modulating sugar and amino acid content in a plant (SULTR3)

Patent metadata

Field	Value
Publication number	US-12419264-B2
Application number	US-202017765746-A
Country	US
Kind code	B2
Filing date	Sep 28, 2020
Priority date	Oct 1, 2019
Publication date	Sep 23, 2025
Grant date	Sep 23, 2025

Abstract

There is described herein a plant cell (i) a polynucleotide comprising, consisting or consisting essentially of a sequence having at least 60% sequence identity to SEQ ID NO: 1 (NtSULTR3;1A-S), SEQ ID NO: 3 (NtSULTR3;1A-T), SEQ ID NO: 5 (NtSULTR3;1B-S), SEQ ID NO: 7 (NtSULTR3;1B-T), SEQ ID NO: 15 (NtSULTR3;3-T), SEQ ID NO: 17 (NtSULTR3;4A-S), SEQ ID NO: 19 (NtSULTR3;4A-T) or SEQ ID NO: 23 (NtSULTR3;4B-T); (ii) a polypeptide encoded by the polynucleotide set forth in (i); (iii) a polypeptide comprising, consisting or consisting essentially of a sequence having at least 87% sequence identity to SEQ ID NO: 2 (NtSULTR3;1A-S) or at least 87% sequence identity to SEQ ID NO: 4 (NtSULTR3;1A-T) or at least 87% sequence identity to SEQ ID NO: 6 (NtSULTR3;1B-S), or at least 88% sequence identity to SEQ ID NO: 8 (NtSULTR3;1B-T), or at least 70% sequence identity to SEQ ID NO: 16 (NtSULTR3;3-T), or at least 84% sequence identity to SEQ ID NO: 18 (NtSULTR3;4A-S) or at least 79% sequence identity to SEQ ID NO: 20 (NtSULTR3;4A-T); or at least 87% sequence identity to SEQ ID NO: 24 (NtSULTR3;4B-T); or (iv) a construct, vector or expression vector comprising the isolated polynucleotide set forth in (i), wherein said plant cell comprises at least one modification which modulates (a) the expression or activity of the polynucleotide or (b) the expression or activity of the polynucleotide the polypeptide, as compared to a control plant cell in which the expression or activity of the polynucleotide or polypeptide has not been modified.

First claim

The invention claimed is:

1. A plant cell comprising:
(i) a polynucleotide comprising a sequence having at least 90 sequence identity to SEQ ID NO: 1 (NtSULTR3;1A-S) or SEQ ID NO: 3 (NtSULTR3;1A-T);
(ii) a polypeptide encoded by the polynucleotide set forth in (i);
(iii) a polypeptide comprising a sequence having at least 90% sequence identity to SEQ ID NO: 2 (NtSULTR3;1A-S) or at least 90% sequence identity to SEQ ID NO: 4 (NtSULTR3;1A-T); or
(iv) a construct, vector or expression vector comprising the isolated polynucleotide set forth in (i),
wherein said plant cell comprises at least one modification which reduces (a) the expression or activity of the polynucleotide or (b) the expression or activity of the polypeptide, as compared to a control plant cell in which the expression or activity of the polynucleotide or polypeptide has not been modified.

2. The plant cell according to claim 1 , wherein the reduced expression or reduced activity reduced the levels of glucose, fructose and sucrose in cured leaf of a plant comprising the plant cell as compared to the levels of glucose, fructose and sucrose in a control cured leaf.

3. The plant cell according to claim 2 , wherein the level of glucose is reduced at least about 60% as compared to a control cured leaf; or
wherein the level of fructose is reduced at least about 60% as compared to a control cured leaf; or
wherein the level of sucrose is reduced at least about 60% as compared to a control cured leaf; or
wherein the levels of glucose, fructose and sucrose are reduced at least about 60% as compared to a control cured leaf.

4. The plant cell according to claim 1 , wherein the reduced expression or reduced activity increases the levels of free amino acids, glutamine, glutamate and aspartate in cured leaf of a plant comprising the plant cell as compared to the levels of free amino acids, glutamine, glutamate and aspartate in a control cured leaf.

5. The plant cell according to claim 4 , wherein the level of free amino acids is increased by at least about 1.5 times as compared to a control cured leaf; or
wherein the level of glutamine is increased by at least about 1.5 times as compared to a control cured leaf; or
wherein the level of glutamate is increased by at least about 1.5 times as compared to a control cured leaf; or
wherein the level of aspartate is increased by at least about 1.5 times as compared to a control cured leaf; or
wherein the levels of free amino acids, glutamine, glutamate and aspartate are increased by at least about 1.5 times as compared to a control cured leaf.

6. The plant cell according to claim 1 , wherein the at least one modification is a genetic mutation in the polynucleotide and the plant is Nicotiana tabacum.

7. The plant cell according to claim 1 , further comprising:
(i) at least one modification in a NtSUS polynucleotide or polypeptide encoded thereby,
suitably, wherein the NtSUS polynucleotide or polypeptide encoded thereby is selected from the group consisting of NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T or a combination of two or more thereof,
more suitably, wherein the NtSUS polynucleotide or polypeptide encoded thereby is selected from the group consisting of NtSUS2-S, NtSUS3-S, NtSUS3-T and NtSUS4-S or a combination of two or more thereof; or
(ii) further comprising at least one modification in a CLC-Nt2 polynucleotide or polypeptide encoded thereby or a NtCLCe polynucleotide or polypeptide encoded thereby, or a combination thereof; or
(iii) a combination of (i) and (ii).

8. A plant or part thereof comprising the plant cell according to claim 1 .

9. Plant material, cured plant material, or homogenized plant material, derived or obtained from the plant or part thereof of claim 8 ; suitably,
wherein the plant material is selected from the group consisting of biomass, seed, stem, flowers, or leaves or a combination of two or more thereof; optionally,
wherein the cured plant material is selected from the group consisting of flue-cured plant material, sun-cured plant material or air-cured plant material or a combination of two or more thereof.

10. A tobacco product comprising the plant cell of claim 1 .

11. A method for producing the plant of claim 8 , comprising the steps of:
(a) providing the plant cell comprising at least one modification; and
(b) propagating the plant cell into a plant.

12. The method according to claim 11 , wherein in step (a) the at least one modification is introduced by genome editing; suitably,
wherein the genome editing is selected from CRISPR-mediated genome editing, zinc finger nuclease-mediated mutagenesis, chemical or radiation mutagenesis, homologous recombination, oligonucleotide-directed mutagenesis and meganuclease-mediated mutagenesis; or
wherein in step (a) the at least one modification is introduced using an interference polynucleotide or by introducing at least one mutation or a combination thereof.

13. A method for producing cured plant material with altered levels of glucose, fructose and sucrose and altered levels of free amino acids, glutamine, glutamate and aspartate as compared to control plant material, comprising the steps of:
(a) providing a plant or part thereof according to claim 8 ;
(b) harvesting plant material therefrom; and
(c) curing the plant material.

14. A method of producing a liquid tobacco extract, the method comprising the steps of:
(a) preparing a first tobacco starting material from a plant or part thereof containing a plant cell according to claim 1 ;
(b) preparing a second tobacco starting material from a plant or part thereof containing a plant cell comprising:
(i) at least one modification in a NtSUS polynucleotide or polypeptide encoded thereby, suitably, wherein the NtSUS polynucleotide or polypeptide encoded thereby is selected from the group consisting of NtSUS2-T, NtSUS3-S, NtSUS3-T, NtSUS4-S, NtSUS4-T or a combination of two or more thereof, more suitably, wherein the NtSUS polynucleotide or polypeptide encoded thereby is selected from the group consisting of NtSUS2-S, NtSUS3-S, NtSUS3-T and NtSUS4-S or a combination of two or more thereof; or
(ii) comprising at least one modification in a CLC-Nt2 polynucleotide or polypeptide encoded thereby or a NtCLCe polynucleotide or polypeptide encoded thereby, or a combination thereof; or
(iii) a combination of (i) and (ii);
(c) heating the first tobacco starting material at a first extraction temperature;
(d) heating the second tobacco starting material at a second extraction temperature;
(e) collecting the volatile compounds released from the first tobacco starting materials and second tobacco starting materials during heating; and
(f) combining the collected volatile compounds released from the first and second tobacco starting materials and forming a liquid tobacco extract from the combined volatile compounds.

15. The plant cell according to claim 1 wherein the plant cell is a tobacco plant cell.

16. The plant cell according to claim 1 wherein comprising a polynucleotide comprising a sequence having at least 95% sequence identity to SEQ ID NO: 1 (NtSULTR3;1A-S) or SEQ ID NO: 3 (NtSULTR3;1A-T) and a polypeptide comprising a sequence having at least 95% sequence identity to SEQ ID NO: 2 (NtSULTR3;1A-S) or at least 95% sequence identity to SEQ ID NO: 4 (NtSULTR3;1A-T).

17. The plant cell according to claim 1 wherein comprising a polynucleotide comprising a sequence having at least 99% sequence identity to SEQ ID NO: 1 (NtSULTR3;1A-S) or SEQ ID NO: 3 (NtSULTR3;1A-T) and a polypeptide comprising a sequence having at le

Assignees

• Philip Morris Products Sa

Inventors

• Bovet Lucien

Classifications

• C12N15/8251 Primary

  Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis · CPC title

• C12N15/8245

  involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis · CPC title

• A24B3/10

  Roasting or cooling tobacco · CPC title

• A24B15/24

  by extraction; Tobacco extracts · CPC title

• A01H6/823 Primary

  Nicotiana, e.g. tobacco · CPC title