Novel plant acyltransferases specific for long-chained, multiply unsaturated fatty acids

• US 20060174376A1
• Filed: 03/26/2004
• Published: 08/03/2006
• Est. Priority Date: 03/31/2003
• Status: Active Grant

• Current Assignee: University of Bristol
• Original Assignee: University of Bristol
• Inventors: Sozer, Nursen, Renz, Andreas, Keith, Stobart, Bauer, Jorg, Frentzen, Margit
• Granted Patent: US 7,855,321 B2
• Time in Patent Office: Days
• US Class Current: 800/281
• 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 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6458 by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

The present invention relates to a process for the production of long-chain polyunsaturated fatty acids in an organism by introducing, into the organism, nucleic acids which code for polypeptides with acyl transferase activity. These nucleic acid sequences, if appropriate together with further nucleic acid sequences which code for polypeptides of the fatty acid or lipid metabolism biosynthesis, can advantageously be expressed in the organism. Furthermore, the invention relates to a method for the production of oils and/or triacylglycerides with an elevated content of long-chain polyunsaturated fatty acids. The invention furthermore relates to the nucleic acid sequences, nucleic acid constructs, vectors and organisms comprising the nucleic acid sequences according to the invention, vectors comprising the nucleic acid sequences and/or the nucleic acid constructs and to transgenic organisms comprising the abovementioned nucleic acid sequences, nucleic acid constructs and/or vectors. A further part of the invention relates to oils, lipids and/or fatty acids produced by the process according to the invention and to their use.

Claims

1. A process for the production of polyunsaturated fatty acids in an organism, which comprises the following steps:

(a) introducing, into the organism, at least one nucleic acid sequence with the sequence shown in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

11, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18 or SEQ ID NO;

20, which codes for a polypeptide with lysophosphatidic acid acyltransferase activity;

or

(b) introducing, into the organism, at least one nucleic acid sequence with the sequence shown in SEQ ID NO;

22, SEQ ID NO;

24 or SEQ ID NO;

26, which codes for a polypeptide with glycerol-3-phosphate acyltransferase activity;

or

(c) introducing, into the organism, at least one nucleic acid sequence with the sequence shown in SEQ ID NO;

28, SEQ ID NO;

30 or SEQ ID NO;

32 which codes for a polypeptide with diacylglycerol acyltransferase activity;

or

(d) introducing, into the organism, at least one nucleic acid sequence with the sequence shown in SEQ ID NO;

34 or SEQ ID NO;

36, which codes for a polypeptide with lecithin cholesterol acyltransferase activity;

or

(e) introducing, into the organism, at least one nucleic acid sequence which can be derived from the coding sequence in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

1, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

20, SEQ ID NO;

22, SEQ ID NO;

24, SEQ ID NO;

26, SEQ ID NO;

28, SEQ ID NO;

30, SEQ ID NO;

32, SEQ ID NO;

34 or SEQ ID NO;

36 as the result of the degeneracy of the genetic code, or

(f) introducing, into the organism, at least one derivative of the nucleic acid sequence shown in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

1, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

20, SEQ ID NO;

22, SEQ ID NO;

24, SEQ ID NO;

26, SEQ ID NO;

28, SEQ ID NO;

30, SEQ ID NO;

32, SEQ ID NO;

34 or SEQ ID NO;

36, which code for polypeptides with the amino acid sequence shown in SEQ ID NO;

2, SEQ ID NO;

5, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

15, SEQ ID NO;

17, SEQ ID NO;

19, SEQ ID NO;

21, SEQ ID NO;

23, SEQ ID NO;

25, SEQ ID NO;

27, SEQ ID NO;

29, SEQ ID NO;

31, SEQ ID NO;

33, SEQ ID NO;

35 or SEQ ID NO;

37 and which have at least 40% homology at the amino acid level with SEQ ID NO;

2, SEQ ID NO;

5, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

15, SEQ ID NO;

17, SEQ ID NO;

19, SEQ ID NO;

21, SEQ ID NO;

23, SEQ ID NO;

25, SEQ ID NO;

27, SEQ ID NO;

29, SEQ ID NO;

31, SEQ ID NO;

33, SEQ ID NO;

35 or SEQ ID NO;

37 and have an equivalent lysophosphatidic acid acyltransferase activity, glycerol-3-phosphate acyltransferase activity, diacylglycerol acyltransferase activity or lecithin cholesterol acyltransferase activity, and

(g) culturing and harvesting the organism.

2. The process for the production of polyunsaturated fatty acids according to claim 1, wherein, additionally to the nucleic acid sequences mentioned under (a) to (f), further nucleic acid sequences which code for polypeptides of the fatty acid metabolism or lipid metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP[=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA:

lysophospholipid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenase(s), lipoxygenase(s), triacylglycerol lipase(s), allene oxide synthase(s), hydroperoxide lyase(s) or fatty acid elongase(s) were introduced into the organism.

3. The process for the production of polyunsaturated fatty acids according to claim 1, wherein, additionally to the nucleic acid sequences mentioned under (a) to (f), further nucleic acid sequences which code for polypeptides selected from the group consisting of acyl-CoA:

lysophospholipid acyltransferase, Δ

-4-desaturase, Δ

-5-desaturase, Δ

-6-desaturase, Δ

-8-desaturase, Δ

-9-desaturase, Δ

-12-desaturase, Δ

-5-elongase, Δ

-6-elongase or Δ

-9-elongase, were introduced into the organism.

4. The process for the production of polyunsaturated fatty acids according to claim 1, wherein the polyunsaturated fatty acids produced are C₁₈-, C₂₀-, C₂₂- or C₂₄-fatty acids.

5. The process for the production of polyunsaturated fatty acids according to claim 1, wherein the polyunsaturated fatty acids are isolated from the organism in the form of an oil, a lipid or a free fatty acid.

6. The process for the production of polyunsaturated fatty acids according to claim 1, wherein the polyunsaturated fatty acids produced in the process are C₁₈-, C₂₀-, C₂₂- or C₂₄-fatty acids with at least two double bonds in the molecule.

7. The process for the production of polyunsaturated fatty acids according to claim 1, wherein a polyunsaturated fatty acid selected from the group consisting of dihomo-γ

-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid is produced in the process.

8. The process for the production of polyunsaturated fatty acids according to claim 1, wherein the organism is a microorganism, a nonhuman animal or a plant.

9. The process for the production of polyunsaturated fatty acids according to claim 1, wherein the organism is a transgenic plant.

10. The process for the production of polyunsaturated fatty acids according to claim 1, wherein the transgenic plant is an oil crop plant.

24. An oil, a lipid or a fatty acid or a fraction of these, produced by the process according to claim 1.

25. An oil, a lipid or a fatty acid composition comprising polyunsaturated fatty acids prepared by a process according to claim 1 and originating from transgenic plants.

29. A method of making feed, foodstuffs, cosmetics or pharmaceuticals comprising incorporating the polyunsaturated fatty acids prepared according to claim 1 in said feed, foodstuffs, cosmetics or pharmaceuticals.

30. A method of making feed, foodstuffs, cosmetics or pharmaceuticals comprising incorporating the oil, lipid or fatty acid composition according to claim 25 in said feed, foodstuffs, cosmetics or pharmaceuticals.

11. An isolated nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence with the sequence shown in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

11, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18 or SEQ ID NO;

20,

(b) nucleic acid sequences which, as the result of the degeneracy of the genetic code, can be derived from the coding sequence in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

1, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18 or SEQ ID NO;

20,

(c) derivatives of the nucleic acid sequence shown in SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

11, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18 or SEQ ID NO;

20, which code for polypeptides with the amino acid sequence shown in SEQ ID NO;

2, SEQ ID NO;

5, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

15, SEQ ID NO;

17, SEQ ID NO;

19 or SEQ ID NO;

21 and which have at least 40% homology at the amino acid level with SEQ ID NO;

2, SEQ ID NO;

5, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

15, SEQ ID NO;

17, SEQ ID NO;

19 or SEQ ID NO;

21 and have lysophosphatidic acid acyltransferase activity.

15. The isolated nucleic acid sequence according to claim 11, which sequence originates from a eukaryote.

16. An amino acid sequence which is encoded by an isolated nucleic acid sequence according to claim 11.

17. A gene construct comprising an isolated nucleic acid according to claim 11, where the nucleic acid is linked functionally to one or more regulatory signals.

18. The gene construct according to claim 17, wherein the nucleic acid construct comprises additional biosynthesis genes of the fatty acid metabolism or lipid metabolism selected from the group consisting of fatty acid metabolism or lipid metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP[=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), acyl-CoA:

lysophospholipid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenase(s), lipoxygenase(s), triacylglycerol lipase(s), allene oxide synthase(s), hydroperoxide lyase(s) or fatty acid elongase(s).

19. The gene construct according to claim 17, wherein the nucleic acid construct comprises additional biosynthesis genes of the fatty acid metabolism or lipid metabolism selected from the group consisting of acyl-CoA:

lysophospholipid acyltransferase, Δ

-4-desaturase, Δ

-5-desaturase, Δ

-6-desaturase, Δ

-8-desaturase, Δ

-9-desaturase, Δ

-12-desaturase, Δ

-5-elongase, Δ

-6-elongase or Δ

-9-elongase.

20. A vector comprising a nucleic acid according to or a gene construct according to claim 17.

21. A transgenic nonhuman organism comprising at least one nucleic acid according to claim 11.

22. The transgenic nonhuman organism according to claim 21, wherein the organism is a microorganism, a nonhuman animal or a plant.

23. The A transgenic nonhuman organism according to claim 21, wherein the organism is a plant.

27. A transgenic nonhuman organism comprising at least a gene construct according to claim 17.

28. A transgenic nonhuman organism comprising at least a vector according to claim 20.

12. An isolated nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence with the sequence shown in SEQ ID NO;

22, SEQ ID NO;

24 or SEQ ID NO;

26,

(b) nucleic acid sequences which, as the result of the degeneracy of the genetic code, can be derived from the coding sequence in SEQ ID NO;

22, SEQ ID NO;

24 or SEQ ID NO;

26,

(c) derivatives of the nucleic acid sequence shown in SEQ ID NO;

22, SEQ ID NO;

24 or SEQ ID NO;

26, which code for polypeptides with the amino acid sequence shown in SEQ ID NO;

23, SEQ ID NO;

25 or SEQ ID NO;

27 and have at least 40% homology at the amino acid level with SEQ ID NO;

23, SEQ ID NO;

25 or SEQ ID NO;

27 and have glycerol-3-phosphate acyltransferase activity.

13. An isolated nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence with the sequence shown in SEQ ID NO;

28, SEQ ID NO;

30 or SEQ ID NO;

32,

(b) nucleic acid sequences which, as the result of the degeneracy of the genetic code, can be derived from the coding sequence in SEQ ID NO;

28, SEQ ID NO;

30 or SEQ ID NO;

32,

(c) derivatives of the nucleic acid sequence shown in SEQ ID NO;

28, SEQ ID NO;

30 or SEQ ID NO;

32, which code for polypeptides with the amino acid sequence shown in SEQ ID NO;

29, SEQ ID NO;

31 or SEQ ID NO;

33 and have at least 40% homology at the amino acid level with SEQ ID NO;

29, SEQ ID NO;

31 or SEQ ID NO;

33 and which have diacylglycerol acyltransferase activity.

14. An isolated nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence with the sequence shown in SEQ ID NO;

34 or SEQ ID NO;

36,

(b) nucleic acid sequences which, as the result of the degeneracy of the genetic code, can be derived from the coding sequence in SEQ ID NO;

34 or SEQ ID NO;

36,

(c) derivatives of the nucleic acid sequence shown in SEQ ID NO;

34 or SEQ ID NO;

36, which code for polypeptides with the amino acid sequence shown in SEQ ID NO;

35 or SEQ ID NO;

37 and which have at least 40% homology at the amino acid level with SEQ ID NO;

35 or SEQ ID NO;

37 and have lecithin cholesterol acyltransferase activity.

26. (canceled)