Regulatory nucleic acid molecules for enhancing seed-specific gene expression in plants promoting enhanced polyunsaturated fatty acid synthesis

Patent metadata

Field	Value
Publication number	US-9428757-B2
Application number	US-201013393063-A
Country	US
Kind code	B2
Filing date	Aug 27, 2010
Priority date	Aug 31, 2009
Publication date	Aug 30, 2016
Grant date	Aug 30, 2016

Abstract

The invention in principle pertains to the field of recombinant manufacture of fatty acids. It provides novel nucleic acid molecules comprising nucleic acid sequences encoding fatty acid desaturases, elongases, acyltransferases, terminator sequences and high expressing seed-specific promoters operatively linked to the said nucleic acid sequences wherein nucleic acid expression enhancing nucleic acids (NEENAs) are functionally linked to said promoters.

First claim

The invention claimed is:

1. A polynucleotide comprising:
a) at least one nucleic acid sequence encoding a polypeptide having desaturase or elongase activity;
b) at least one seed-specific plant promoter operatively linked to the nucleic acid sequence of a);
c) at least one terminator sequence operatively linked to the nucleic acid sequence of a); and
d) at least one nucleic acid expression enhancing nucleic acid (NEENA) molecule functionally linked to the promoter of b),
wherein said at least one NEENA molecule is heterologous to the promoter of b) and to the polypeptide of a),
wherein said at least one NEENA molecule comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24,
wherein said at least one NEENA molecule is not able to drive expression of the nucleic acid sequence of a) but is able to enhance expression of said nucleic acid sequence when functionally linked to the promoter of b),
and wherein said at least one NEENA molecule enhances seed-specific expression of said nucleic acid sequence of a) in the plant or part thereof as compared to a corresponding control plant or part thereof.

2. The polynucleotide of claim 1 , comprising:
e) at least one nucleic acid sequence encoding a polypeptide having beta-ketoacyl reductase activity;
f) at least one nucleic acid sequence encoding a polypeptide having dehydratase activity; and/or
g) at least one nucleic acid sequence encoding a polypeptide having enoyl-CoA reductase activity,
wherein the nucleic acid sequences of e) to g) are heterologous to said polypeptide having desaturase or elongase activity.

3. The polynucleotide of claim 2 , comprising at least one nucleic acid sequence encoding a polypeptide having acyltransferase activity, wherein the nucleic acid sequence is heterologous to said polypeptide having desaturase, elongase, beta-ketoacyl reductase, dehydratase, or enoyl-CoA reductase activity.

4. A vector comprising the polynucleotide of claim 1 .

5. A host cell comprising:
a) the polynucleotide of claim 1 ; or
b) a vector comprising said polynucleotide.

6. A method for the manufacture of a polypeptide encoded by the polynucleotide of claim 1 , comprising:
a) cultivating a host cell comprising the polynucleotide of claim 1 or a vector comprising said polynucleotide under conditions which allow for the production of the polypeptide; and
b) obtaining the polypeptide from the host cell.

7. A non-human transgenic organism comprising the polynucleotide of claim 1 or a vector comprising said polynucleotide.

8. The non-human transgenic organism of claim 7 , which is a plant or a plant part.

9. A method for the manufacture of polyunsaturated fatty acids comprising:
a) cultivating the host cell of claim 5 under conditions which allow for the production of polyunsaturated fatty acids in said host cell; and
b) obtaining said polyunsaturated fatty acids from the host cell.

10. A method for the manufacture of polyunsaturated fatty acids comprising:
a) cultivating the non-human transgenic organism of claim 7 under conditions which allow for the production of a polyunsaturated fatty acid in said non-human transgenic organism; and
b) obtaining said polyunsaturated fatty acid from the non-human transgenic organism.

11. The method of claim 9 , wherein said polyunsaturated fatty acid is arachidonic acid (ARA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA).

12. A method for the manufacture of an oil, lipid, or fatty acid composition comprising:
a) cultivating the host cell of claim 5 under conditions which allow for the production of a polyunsaturated fatty acid in said host cell;
b) obtaining said polyunsaturated fatty acid from said host cell; and
c) formulating said polyunsaturated fatty acid as an oil, lipid, or fatty acid composition.

13. A method for the production of foodstuffs, animal feed, a seed, a pharmaceutical, or a fine chemical, comprising:
a) providing the host cell of claim 5 , or a host cell culture, non-human transgenic organism, transgenic plant, plant part, or plant seed derived from a transgenic non-human organism or plant comprising said host cell; and
b) preparing foodstuffs, animal feed, a seed, a pharmaceutical, or a fine chemical.

14. A method for enhancing expression of at least one enzyme of the polyunsaturated fatty acid biosynthetic pathway in a plant or part thereof, comprising transforming a plant or part thereof with a polynucleotide comprising:
i) at least one nucleic acid sequence encoding a polypeptide selected from the group consisting of:
a) a polypeptide having desaturase or elongase activity;
b) a polypeptide having beta-ketoacyl reductase activity;
c) a polypeptide having dehydratase activity; and
d) a polypeptide having enoyl-CoA reductase activity;

ii) at least one seed-specific plant promoter operatively linked to the nucleic acid sequence of i);
iii) at least one terminator sequence operatively linked to the nucleic acid sequence of i); and
iv) at least one nucleic acid expression enhancing nucleic acid (NEENA) molecule functionally linked to the promoter of ii),
wherein said at least one NEENA molecule is heterologous to the promoter of ii) and to the nucleic acid sequence of i),
wherein said at least one NEENA molecule comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24,
wherein said at least one NEENA molecule is not able to drive expression of the nucleic acid sequence of i) but is able to enhance expression of said nucleic acid sequence when functionally linked to the promoter of ii),
and wherein said at least one NEENA molecule enhances seed-specific expression of said nucleic acid sequence of i) in the plant or part thereof as compared to a corresponding control plant or part thereof.

15. The method of claim 14 , wherein said at least one NEENA molecule comprises the nucleotide sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

16. The polynucleotide of claim 1 , wherein said at least one NEENA molecule comprises the nucleotide sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

Assignees

• Senger Toralf
• Bauer Jörg
• Kuhn Josef Martin
• Basf Plant Science Co Gmbh

Inventors

• Senger Toralf
• Bauer Jörg
• Kuhn Josef Martin

Classifications

• C12N15/8216 Primary

  Methods for controlling, regulating or enhancing expression of transgenes in plant cells · CPC title

• C12N15/8247

  involving modified lipid metabolism, e.g. seed oil composition · CPC title

• C12N15/8234

  Seed-specific, e.g. embryo, endosperm · CPC title