Plant acyltransferases specific for long-chained, multiply unsaturated fatty acids

• US 7,855,321 B2
• Filed: 03/26/2004
• Issued: 12/21/2010
• Est. Priority Date: 03/31/2003
• Status: Expired due to Fees

• Current Assignee: University of Bristol
• Original Assignee: University of Bristol
• Inventors: Abbadi, Amine, Keith, Stobart, Fraser, Thomas, Sözer, Nursen, Frentzen, Margit, Renz, Andreas, Bauer, Jörg, Heinz, Ernst, Lazarus, Colin M., Qi, Baoxiu
• Primary Examiner(s): McElwain; Elizabeth F
• Application Number:
  US10/552,013
• Publication Number:
  US 20060174376A1
• Time in Patent Office: 2,461 Days
• Field of Search: None
• 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 comprising

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

16 which codes for a polypeptide with lysophosphatidic acid acyltransferase activity;

or

(b) introducing, into the organism, at least one nucleic acid comprising a nucleic acid sequence having at least 95% identity with the sequence of SEQ ID NO;

16, wherein the nucleic acid codes for a polypeptide with lysophosphatidic acid acyltransferase activity, or

(c) introducing, into the organism, at least one nucleic acid which codes for a polypeptide comprising the amino acid sequence shown in SEQ ID NO;

17 or comprising an amino acid sequence having at least 95% identity with SEQ ID NO;

17 and has lysophosphatidic acid acyltransferase activity, and

(d) culturing and harvesting the organism

wherein the organism is a microorganism, yeast, or a plant.

2. The process for the production of polyunsaturated fatty acids according to claim 1, wherein, additionally to the nucleic acid sequence mentioned under (a) to (c), at least one further nucleic acid sequence which codes for a polypeptide 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) and fatty acid elongase(s) is introduced into the organism.

3. The process for the production of polyunsaturated fatty acids according to claim 1, wherein, additionally to the nucleic acid sequence mentioned under (a) to (c), at least one further nucleic acid sequence which codes for a polypeptide 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 and Δ

-9-elongase, is 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 three double bonds.

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 transgenic plant.

9. The process for the production of polyunsaturated fatty acids according to claim 8, wherein the transgenic plant is an oil crop plant.

10. An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of:

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

16,

(b) a nucleic acid sequence having at least 95% identity with the sequence of SEQ ID NO;

16 and coding for a polypeptide having lysophosphatidic acid acyltransferase activity,

(c) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO;

17, and

(d) a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence having at least 95% identity with the sequence of SEQ ID NO;

17 and having lysophosphatidic acid acyltransferase activity.

11. The isolated nucleic acid of claim 10, wherein the nucleic acid sequence originates from a eukaryote.

12. A gene construct comprising the isolated nucleic acid of claim 10, where the nucleic acid is linked functionally to one or more regulatory signals.

13. The gene construct of claim 12, wherein the gene construct comprises an additional biosynthesis gene 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) and fatty acid elongase(s).

14. The gene construct of claim 12, wherein the gene construct comprises an additional biosynthesis gene 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 and Δ

-9-elongase.

15. A vector comprising the nucleic acid of claim 10 or a gene construct comprising said nucleic acid.

16. A transgenic organism comprising the nucleic acid of claim 10, wherein the organism is a microorganism, yeast, or a plant.

17. The transgenic organism of claim 16, wherein the organism is a plant.

18. A transgenic organism comprising the gene construct of claim 12, wherein the transgenic organism is a microorganism, yeast, or a plant.

19. A transgenic organism comprising the vector of claim 15, wherein the transgenic organism is a microorganism, yeast, or a plant.

20. The nucleic acid of claim 10, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO:

16.

21. The nucleic acid of claim 10, wherein the nucleic acid sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:

17.