Synthesis of long-chain polyunsaturated fatty acids by recombinant cell
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.
1. A process for producing a composition, the process comprising
(i) obtaining a yeast cell comprising
eicosapentaenoic acid and docosapentaenoic acid in an esterified form as part of triacylglycerols in the yeast cell;
a polynucleotide which encodes a Δ5 desaturase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;
a polynucleotide which encodes a Δ8 desaturase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;
a polynucleotide which encodes a Δ9 elongase operably linked to a promoter which directs expression of the polynucleotide in the yeast cell;
an exogenous desaturase which desaturates an acyl-CoA substrate in the yeast cell; and
a total fatty acid content in the yeast cell which includes 2.5% (w/w) C20 ω3 long-chain polyunsaturated fatty acids (LC-PUFAs) and 0.1% (w/w) docosapentaenoic acid,
(ii) extracting oil comprising eicosapentaenoic acid from the cell, and
(iii) processing the oil comprising eicosapentaenoic acid, thereby producing the composition comprising eicosapentaenoic acid.
2. The process of claim 1 , wherein 50% of the docosapentaenoic acid in the yeast cell is incorporated into triacylglycerols.
3. The process of claim 1 , wherein 50% of the eicosapentaenoic acid in the yeast cell is incorporated into triacylglycerols.
4. The process of claim 1 , wherein the total fatty acid content in the yeast cell comprises 0.13% (w/w) docosapentaenoic acid.
5. The process of claim 1 , wherein the total fatty acid content in the yeast cell comprises at least 0.5% (w/w) docosapentaenoic acid.
6. The process of claim 1 , wherein the desaturase which desaturates an acyl-CoA substrate is the Δ5 desaturase.
7. The process of claim 1 , wherein the Δ8 desaturase does not also have Δ6 desaturase activity.
8. The process of claim 1 , wherein the Δ8 desaturase is able to catalyse the desaturation of both 18:3ω3 and 20:3ω3 fatty acids.
9. The process of claim 1 , wherein at least one of said desaturases comprises a motif having the amino acid sequence QIEHHLF (SEQ ID NO: 45).
10. The process of claim 1 , wherein the Δ8 desaturase comprises amino acids having the sequence set forth as SEQ ID NO: 36 or a sequence at least 90% identical thereto.
11. The process of claim 1 , wherein the yeast cell is of the species Yarrowia lipolytica.
12. The process of claim 1 , wherein the composition is a pharmaceutical composition.
13. The process of claim 12 , wherein the pharmaceutical composition is in the form of a capsule.
14. The process of claim 1 , wherein in step (iii) processing the oil comprises converting the eicosapentaenoic acid to an ester.
15. The process of claim 14 , wherein the ester is a methyl ester.