Synthesis of long-chain polyunsaturated fatty acids by recombinant cell

• US 9,926,579 B2
• Filed: 07/07/2017
• Issued: 03/27/2018
• Est. Priority Date: 04/22/2004
• Status: Active Grant

• Current Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Original Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Inventors: Singh, Surinder Pal, Robert, Stanley Suresh, Nichols, Peter David, Blackburn, Susan Irene Ellis, Zhou, Xue-Rong, Petrie, James Robertson, Green, Allan Graham
• Primary Examiner(s): O Hara, Eileen B
• Application Number:
  US15/644,647
• Publication Number:
  US 20170327852A1
• Time in Patent Office: 263 Days
• Field of Search: None
• CPC Class Codes: A01H 5/10 Seeds
• CPC Class Codes: A23K 10/12 by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
• CPC Class Codes: A23K 10/30 from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms obtained by microbiological or biochemical processes, e.g. using yeasts or enzymes, A23K10/10
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: C11B 1/10 by extracting
• CPC Class Codes: C11C 3/12 by hydrogenation
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• 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 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12Y 114/19004 DELTA8-fatty-acid desaturase (1.14.19.4)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present invention relates to methods of synthesizing long-chain polyunsaturated fatty acids, especially eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, in recombinant cells such as yeast or plant cells. Also provided are recombinant cells or plants which produce long-chain polyunsaturated fatty acids. Furthermore, the present invention relates to a group of new enzymes which possess desatorase or elongase activity that can be used in methods of synthesizing long-chain polyunsaturated fatty acids.

Claims

1. A Brassica plant cell for producing docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in an esterified form as part of triacylglycerols in the plant cell, comprising

a polynucleotide which encodes a Δ

5 elongase operably linked to a promoter which directs expression of the polynucleotide in the plant cell;

a polynucleotide which encodes a Δ

4 desaturase whose amino acid sequence is at least 99.5% identical to the sequence set forth as SEQ ID NO;

33, operably linked to a promoter which directs expression of the polynucleotide in the plant cell; and

an exogenous desaturase which desaturates an acyl-CoA substrate.

2. The Brassica plant cell of claim 1 which is a Brassica napus plant cell.

3. The Brassica napus plant cell of claim 2 which is a Brassica napus seed cell.

4. A Brassica napus seed comprising the Brassica napus seed cell of claim 3.

5. A process for producing DPA and DHA in an esterified form as part of triacylglycerols comprising

i) obtaining the Brassica napus seed of claim 4, and

ii) extracting triacylglycerols from the seed.

6. The process of claim 5 which further comprises purifying the extracted triacylglycerols.

7. A Brassica plant cell comprising

a polynucleotide which encodes a Δ

5 elongase operably linked to a promoter which directs expression of the polynucleotide in the plant cell;

a polynucleotide which encodes a Δ

6 elongase which has the amino acid sequence set forth as SEQ ID NO;

31, operably linked to a promoter which directs expression of the polynucleotide in the plant cell;

a polynucleotide which encodes a Δ

4 desaturase whose amino acid sequence is at least 99.5% identical to the sequence set forth as SEQ ID NO;

33, operably linked to a promoter which directs expression of the polynucleotide in the plant cell;

a polynucleotide which encodes a Δ

5 desaturase which has the amino acid sequence set forth as SEQ ID NO;

18, operably linked to a promoter which directs expression of the polynucleotide in the plant cell;

a polynucleotide which encodes a Δ

6 desaturase operably linked to a promoter which directs expression of the polynucleotide in the plant cell; and

an exogenous desaturase which desaturates an acyl-CoA substrate.

8. The Brassica plant cell of claim 7, wherein the Δ

6 desaturase is a Δ

5/Δ

6 bifunctional desaturase.

9. The Brassica plant cell of claim 7 which is a Brassica napus plant cell.

10. The Brassica napus plant cell of claim 9 which is a Brassica napus seed cell.

11. A Brassica napus seed comprising the Brassica napus seed cell of claim 10.

12. The Brassica napus seed of claim 11 comprising docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in an esterified form as part of triacylglycerols in the seed.

13. A process for producing DPA and DHA in an esterified form as part of triacylglycerols comprising

i) obtaining the Brassica napus seed of claim 12, and

ii) extracting triacylglycerols from the seed.

14. The process of claim 13 which further comprises purifying the extracted triacylglycerols.