Acyltransferase regulation to increase the percent of polyunsaturated fatty acids in total lipids and oils of oleaginous organisms

• US 20060094088A1
• Filed: 07/27/2005
• Published: 05/04/2006
• Est. Priority Date: 11/04/2004
• Status: Active Grant

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Yadav, Narendra S., Zhang, Hongxiang, Picataggio, Stephen K.
• Granted Patent: US 8,685,679 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

Methods to increase the percent of polyunsaturated fatty acids (PUFAs) within the total lipids and oils of PUFA-producing oleaginous organisms are provided herein, by regulating the activity of specific acyltransferases. Specifically, since oil biosynthesis is expected to compete with polyunsaturation during oleaginy, it is possible to reduce or inactivate the activity of an organism'"'"'s DAG ATs (e.g., phospholipid:diacylglycerol acyltransferase (PDAT) and/or diacylglycerol acyltransferase 1 (DGAT1) and/or diacylglycerol acyltransferase 2 (DGAT2)) to thereby reduce the overall rate of oil biosynthesis while concomitantly increasing the percent of PUFAs that are incorporated into the lipid and oil fractions. The teachings herein will thereby enable one to engineer a wide variety of oleaginous organisms to produce oils with very specific fatty acid compositions.

Claims

1. A method for increasing the percent of polyunsaturated fatty acids in the total lipid or oil of an oleaginous organism, comprising:

c) providing an oleaginous organism, comprising;

(iii) native genes encoding at least one acyltransferase selected from the group consisting of;

a phospholipid;

diacylglycerol acyltransferase, a diacylglycerol acyltransferase 1 and a diacylglycerol acyltransferase 2; and

(iv) genes encoding a functional polyunsaturated fatty acid biosynthetic pathway;

d) reducing the activity of at least one native acyltransferase enzyme, wherein the organism'"'"'s overall rate of oil biosynthesis is reduced and the percent of polyunsaturated fatty acids in the total lipid or oil is increased as compared with the oleaginous organism where the activity of at least one native acyltransferase enzyme is not reduced.

2. A method according to claim 1, wherein the activity of the at least one native acyltransferase is reduced by a means selected from the group consisting of:

a) gene disruption;

b) antisense or iRNA technology;

c) use of a mutant host having diminished DAG AT activity;

d) over-expression of a mutagenized hereosubunit; and

,

e) manipulation of the regulatory sequences controlling expression of the protein.

3. A method according to claim 1, wherein the oleaginous organism comprises a total of “

n”

native acyltransferases and wherein the activity of a total of “

n-1”

acyltransferases are reduced while the remaining acyltransferase retains its wildtype activity.

4. A method according to claim 3, wherein the activity of a total of “

n-1”

acyltransferases are eliminated by gene knock-out.

5. A method according to claim 1, wherein the diacylglycerol acyltransferase 1 enzyme is selected from the group consisting of:

a) a diacylglycerol acyltransferase 1 enzyme comprising all of the amino acid motifs as set forth in;

SEQ ID NO;

151;

SEQ ID NO;

152;

SEQ ID NO;

153;

SEQ ID NO;

154;

SEQ ID NO;

155;

SEQ ID NO;

156; and

SEQ ID NO;

157;

b) a diacylglycerol acyltransferase 1 enzyme comprising all of the amino acid motifs as set forth in;

SEQ ID NO;

143;

SEQ ID NO;

144;

SEQ ID NO;

145;

SEQ ID NO;

146;

SEQ ID NO;

147;

SEQ ID NO;

148;

SEQ ID NO;

149; and

SEQ ID NO;

150;

c) a diacylglycerol acyltransferase 1 enzyme selected from the group consisting of;

SEQ ID NOs;

84, 132, 133, 134, 135, 136, 137, 138, 139, 140,141 and 142; and

,

d) a diacylglycerol acyltransferase 1 enzyme having the amino acid sequence as described by the GenBank Accession Nos selected from the group consisting of;

AY445635, AF384160, NM_—

053437, NM_—

174693, AY116586, AY327327, AY327326, AF298815, AF164434 and NM_—

010046.

6. A method according to claim 1, wherein the diacylglycerol acyltransferase 2 enzyme is selected from the group consisting of:

a) a diacylglycerol acyltransferase 2 enzyme comprising an amino acid motif selected from the group consisting of;

SEQ ID NO;

158 and SEQ ID NO;

159;

b) a diacylglycerol acyltransferase 2 enzyme selected from the group consisting of;

SEQ ID NO;

30, 44, 74 and 76; and

,

c) a diacylglycerol acyltransferase 2 enzyme selected from the group consisting of GenBank Accession Nos;

NC_—

001147 (locus NP_—

014888), NM_—

012079, NM_—

127503, AF051849, AJ238008, NM_—

026384, AF384160, AB057816, AY093657, AB062762, AF221132, AF391089, AF391090, AF129003, AF251794 and AF164434.

7. A method according to claim 1, wherein the phospholipid:

diacylglycerol acyltransferase enzyme is selected from the group consisting of;

SEQ ID NO;

59 and GenBank Accession Nos. P40345, O94680, NP_—

596330, NP_—

190069 and AB006704 [gi;

2351069].

8. A method according to claim 1, wherein the oleaginous organism is selected from the group consisting of:

oleaginous algae, oleaginous moss, oleaginous bacteria, oleaginous fungi, oleaginous yeast and oleaginous plants.

9. A method according to claim 8, wherein the oleaginous yeast is selected from the group consisting of:

Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

10. A method according to claim 9, wherein the oleaginous yeast is Yarrowia lipolytica.

11. A method according to claim 10, wherein the Yarrowia lipolytica is a strain selected from the group consisting of:

Yarrowia lipolytica ATCC #20362, Yarrowia lipolytica ATCC #8862, Yarrowia lipolytica ATCC #18944, Yarrowia lipolytica ATCG #76982, Yarrowia lipolytica ATCC #90812 and Yarrowia lipolytica LGAM S(7)1.

12. A method according to claim 8, wherein the oleaginous alga is selected from the group consisting of:

Cyclotella sp., Nitzschia sp., Thraustochytrium sp. and Schizochytrium sp.

13. A method according to claim 8, wherein the oleaginous fungus is Mortierella.

14. A method according to claim 8, wherein the oleaginous plant is selected from the group consisting of:

cacao bean, colza, borage, sunflower, peanut, soya, rapeseed, copra, tobacco, oat, olive, oil palm, rye, barley, wheat, pepper, primrose, marigolds, linseed, canola, hemp, flax, coconut palm, kenaf, corn, maize, sesame, castor, safflower, soybean, cotton, tamarind, spurges, meadowfoam, Arabidopsis thaliana, Acrocomia, Aiphanes, Apeiba, Attalea, Beilschmiedia, Bertholettia, Ceiba, Corozo, Dialyanthera, Elaeia, Jessenia, Lecythis, Lepidium, Licania, Moringa, Papaver, Pentaclethra, Scheelia, Simarouba, Terminalia, Virola, Ximenia, Cuphea, Limnanthes, Crambe, Lesquerella, Vemonia and Simmondsia.

15. A method according to claim 14, wherein the oleaginous plant is Glycine max.

16. A method according to claim 1, wherein the genes encoding a functional polyunsaturated fatty acid biosynthetic pathway are selected from the group consisting of:

desaturases and elongases.

17. A method according to claim 16, wherein the desaturase is selected from the group consisting of:

Δ

9 desaturase, Δ

12 desaturase, Δ

6 desaturase, Δ

5 desaturase, Δ

17 desaturase, Δ

8 desaturase, Δ

15 desaturase and Δ

4 desaturase.

18. A method according to claim 16, wherein the functional polyunsaturated fatty acid biosynthetic pathway is either native to the oleaginous organism or genetically engineered.

19. A method according to claim 1, wherein the oleic acid is either produced endogenously or introduced exogenously.

20. A method according to claim 1, wherein the polyunsaturated fatty acid produced in step (d) is selected from the group consisting of:

linoleic acid, conjugated linoleic acid, γ

-linoleic acid, dihomo- γ

-linoleic acid, arachidonic acid, α

-linoleic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid, eicosadienoic acid, eicosatrienoic acid, and hydroxylated or expoxy fatty acids thereof.

21. Oil or lipids obtained from the oleaginous organism of claim 1.

22. Use of the oil or lipids of claim 21 in food, animal feed or an industrial application.

23. An oilseed plant made by the method of claim 1.

24. Seeds obtained from the oilseed plant of claim 23.

25. A microbe made by the method of claim 1.