Delta-4 desaturase and its use in making polyunsaturated fatty acids
View Patent ↗Described here are Δ4 desaturases that convert all-cis-7,10,13,16,19-docosapentaenoic acid [“DPA”; 22:5 ω-3] to docosahexaenoic acid [“DHA”; 22:6 ω-3], with secondary activity in converting docosatetraenoic acid [“DTA”; 22:4 ω-6] to all-cis-4,7,10,13,16-docosapentaenoic acid [“DPAn-6”; 22:5 ω-6]. Also, described here are isolated nuclei acid fragments and recombinant constructs comprising such fragments encoding Δ4 desaturases as well as methods of making long chain polyunsaturated fatty acids [“PUFAs”] using this Δ4 desaturase in oleaginous yeast.
1. An isolated nucleic acid molecule selected from the group consisting of:
(a) an isolated nucleotide sequence encoding a delta-4 desaturase enzyme selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4;
(b) an isolated nucleotide sequence encoding a delta-4 desaturase enzyme that hybridizes with (a) under the following stringent hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
(c) an isolated nucleotide sequence encoding a delta-4 desaturase enzyme having at least 90% identity when compared to a polypeptide haying the amino acid sequence as set forth in SEQ ID NO:2, based on the Clustal W method of alignment; and,
(d) an isolated nucleotide sequence that is completely complementary to
(a), (b) or (c).
2. The isolated nucleic acid molecule of claim 1 , wherein at least 191 codons are codon-optimized for expression in Yarrowia.
3. The isolated nucleic acid molecule of claim 1 selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3.
4. The isolated nucleic acid molecule of claim 1 , wherein the nucleotide sequence encoding a delta-4 desaturase enzyme comprises at least 514 amino acids.
5. A chimeric gene comprising the isolated nucleic acid molecule of claim 1 operably linked to at least one regulatory sequence.
6. A host cell comprising the isolated nucleic acid sequence of claim 1 .
7. The host cell of claim 6 , wherein the host cell is selected from the group consisting of yeast; oleaginous yeast preferably selected from the group consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces ; algae; bacteria; euglenoids; stramenopiles, fungi; Mortierella; Thraustochytrium ; and Schizochytrium.
8. The host cell of claim 6 , wherein the cell is from a plant selected from the group consisting of soybean, corn, flax, rapeseed, primrose, canola, maize, cotton, safflower and sunflower.
9. A host cell comprising the isolated nucleic acid sequence of claim 1 .
10. The host cell of claim 9 , wherein the host cell is selected from the group consisting of yeast; oleaginous yeast preferably selected from the group consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces ; algae; bacteria; euglenoids; stramenopiles, fungi; Mortierella; Thraustochytrium ; and Schizochytrium.
11. The host cell of claim 9 , wherein the cell is from a plant selected from the group consisting of soybean, corn, flax, rapeseed, primrose, canola, maize, cotton, safflower and sunflower.
12. A transformed Yarrowia comprising the isolated nucleic acid molecule of claim 1 .
13. A method of making a polyunsaturated fatty acid selected from the group consisting of docosahexaenoic acid and all-cis-4,7,10,13,16-docosapentaenoic acid (22:5, omega-6), comprising:
(a) providing a host cell comprising:
(i) an isolated nucleotide molecule encoding a delta-4 desaturase polypeptide having at least 90% identity when compared to a polypeptide having the amino acid sequence as set forth in SEQ ID NO:2, based on the Clustal W method of alignment; and,
(ii) a source fatty acid selected from the group consisting of all-cis-7,10,13,16,19-docosapentaenoic acid (22:5, omega-3) and docosatetraenoic acid;
(b) growing the host cell of step (a) under conditions to express the nucleic acid fragment encoding the delta-4 desaturase polypeptide and to convert the source fatty acid to a polyunsaturated fatty acid selected from the group consisting of all-cis-7,10,13,16,19-docosapentaenoic acid (22:5, omega-3) and docosatetraenoic acid, such that when all-cis-7,10,13,16,19-docosapentaenoic acid (22:5, omega-3) is the source fatty acid, then docosahexaenoic acid is the polyunsaturated fatty acid produced; and when docosatetraenoic acid is the source fatty acid, then all-cis-4,7,10,13,16-docosapentaenoic acid (22:5, omega-6) is the polyunsaturated fatty acid produced; and,
(c) optionally recovering the polyunsaturated fatty acid produced in step (b).
14. The method of claim 13 , wherein the isolated nucleic acid molecule encodes a delta-4 desaturase polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
15. The method of claim 13 , wherein the isolated nucleic acid molecule has a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3 and the host cell is selected from the group consisting of: algae; bacteria; yeast; oleaginous yeast preferably selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces ; stramenopiles; euglenoids; fungi; plant cells; and animal cells.