Synthesis of long-chain polyunsaturated fatty acids by recombinant cell

• US 10,443,079 B2
• Filed: 07/05/2017
• Issued: 10/15/2019
• 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, Zhong, Xue-Rong, Petrie, James Robertson, Green, Allan Graham
• Primary Examiner(s): Fronda, Christian L
• Application Number:
  US15/642,207
• Publication Number:
  US 20180119184A1
• Time in Patent Office: 832 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 process for increasing the levels of long chain polyunsaturated fatty acid(s) (LC-PUFA) in fish comprising:

a) obtaining feedstuff which comprises oil extracted from transgenic Brassica seeds which comprise eicosapentaenoic acid (EPA) and heterologous polynucleotides which encode enzymes that comprise;

i) a Δ

6 desaturase, a Δ

6 elongase and a Δ

5 desaturase;

or

ii) a Δ

5/Δ

6 bifunctional desaturase and a Δ

5/Δ

6 bifunctional elongase;

wherein the heterologous polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica seed cells, wherein the enzymes encoded by said polynucleotides comprise at least one desaturase which is able to act on an acyl-CoA substrate; and

b) administering the feedstuff to fish,

thereby increasing the levels of LC-PUFA in the fish.

2. The process of claim 1, wherein the enzymes encoded by the polynucleotides comprise at least one Δ

5 elongase which catalyses the conversion of EPA to docosapentaenoic acid (DPA) in the Brassica seed cells.

3. The process of claim 1, wherein at least one promoter is a seed specific promoter.

4. The process of claim 1, wherein the Brassica seeds comprise docosapentaenoic (DPA).

5. The process of claim 1, wherein the Brassica seeds comprise docosahexaenoic acid (DHA).

6. The process of claim 1, wherein the Brassica seeds comprise

a) DHA which is produced from a fatty acid which is ALA, LA, GLA, arachidonic acid (ARA), SDA, ETA, EPA, DPA, or any combination or mixture of these, and/or

b) DPA which is produced from a fatty acid which is ALA, LA, GLA, ARA, SDA, ETA, EPA, or any combination or mixture of these.

7. The process of claim 1, wherein the Brassica seeds have a germination rate which is substantially the same as that of isogenic non-transgenic seed.

8. The process of claim 1, wherein the Brassica seeds are homozygous for the heterologous polynucleotides.

9. The process of claim 1, wherein the heterologous polynucleotides encode a Δ

6 desaturase, a Δ

6 elongase and a Δ

5 desaturase.

10. The process of claim 9, wherein the heterologous polynucleotides encode a Δ

5 elongase which catalyses the conversion of EPA to DPA in the Brassica seed cells.

11. The process of claim 10, wherein the Brassica seeds comprise docosahexaenoic acid (DHA).

12. The process of claim 1, wherein the heterologous polynucleotides encode a Δ

5/Δ

6 bifunctional desaturase and a Δ

5/Δ

6 bifunctional elongase.

13. A process for increasing the levels of long chain polyunsaturated fatty acid(s) (LC-PUFA) in fish comprising:

a) obtaining feedstuff which comprises oil extracted from transgenic Brassica seeds which comprise docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in an esterified form as part of triacylglycerols in the seeds, and which comprise

i) a polynucleotide which encodes a Δ

5 elongase operably linked to a promoter which directs expression of the polynucleotide in Brassica seed cells;

ii) a polynucleotide which encodes a Δ

4 desaturase whose amino acid sequence is at least 99% identical to the sequence set forth as SEQ ID NO;

33, operably linked to a promoter which directs expression of the polynucleotide in the Brassica seed cells; and

iii) an exogenous desaturase which desaturates an acyl-CoA substrate;

and

b) administering the feedstuff to fish,

thereby increasing the levels of LC-PUFA in the fish.

14. A process for increasing the levels of long chain polyunsaturated fatty acid(s) (LC-PUFA) in fish comprising:

a) obtaining feedstuff which comprises oil extracted from transgenic Brassica seeds which comprise docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in an esterified form as part of triacylglycerols in the seeds, and heterologous polynucleotides which encode enzymes that comprise;

i) a Δ

5 elongase;

ii) a Δ

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

31;

iii) a Δ

4 desaturase whose amino acid sequence is at least 99% identical to the sequence set forth as SEQ ID NO;

33;

iv) a Δ

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

18; and

v) a Δ

6 desaturase;

wherein the heterologous polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica seed cells, wherein the Brassica seeds comprise an exogenous desaturase which desaturates an acyl-CoA substrate;

and

b) administering the feedstuff to fish,

thereby increasing the levels of LC-PUFA in the fish.

15. A process for producing a feedstuff which comprises docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) comprising:

a) obtaining transgenic Brassica seeds which comprise docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in an esterified form as part of triacylglycerols in the seeds, and heterologous polynucleotides which encode enzymes that comprise;

i) a Δ

5 elongase;

ii) a Δ

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

31;

iii) a Δ

4 desaturase whose amino acid sequence is at least 99% identical to the sequence set forth as SEQ ID NO;

33;

iv) a Δ

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

18; and

v) a Δ

6 desaturase;

wherein the heterologous polynucleotides are operably linked to one or more promoters that are capable of directing expression of said polynucleotides in Brassica seed cells, wherein the Brassica seeds comprise an exogenous desaturase which desaturates an acyl-CoA substrate;

b) extracting oil from the transgenic Brassica seeds; and

c) admixing the extracted oil with a suitable carrier

thereby producing a feedstuff which comprises docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA).

16. The process of claim 15, wherein the carrier has nutritional value.