Δ6 desaturases and their use in making polyunsaturated fatty acids
View Patent ↗The present invention relates to Δ6 desaturases, which have the ability to convert linoleic acid [“LA”; 18:2 ω-6] to γ-linolenic acid [“GLA”; 18:3 ω-6] and/or α-linolenic acid [“ALA”; 18:3 ω-3] to stearidonic acid [“STA”; 18:4 ω-3]. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding Δ6 desaturases, along with methods of making long-chain polyunsaturated fatty acids [“PUFAs”] using these Δ6 desaturases in oleaginous yeast, are disclosed.
1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a Δ6 desaturase enzyme, selected from the group consisting of:
(a) an isolated nucleic acid molecule encoding the amino acid sequence as set forth in SEQ ID NO:2;
(b) an isolated nucleic acid molecule that hybridizes with (a) under the following 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; or,
an isolated nucleic acid molecule that is completely complementary to (a) or (b).
2. The isolated nucleic acid molecule of claim 1 , wherein at least 227 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:46.
4. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a Δ6 desaturase enzyme of at least 471 amino acids that has at least 95% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2;
or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
5. A chimeric gene comprising the isolated nucleic acid molecule of any one of claim 1 or 4 operably linked to at least one regulatory sequence.
6. A microbial host cell comprising the isolated nucleic acid sequence of claim 1 or claim 4 .
7. The microbial host cell of claim 6 wherein the microbial host cell is selected from the group consisting of yeast, algae, bacteria, euglenoids, stramenopiles, oomycetes and fungi.
8. The microbial host cell of claim 7 wherein the cell is a member of a genus selected from the group consisting of Mortierella, Thraustochytrium , and Schizochytrium.
9. The microbial host cell of claim 7 wherein the cell is a yeast that is oleaginous.
10. The microbial host cell of claim 9 wherein the oleaginous yeast is member of a genus selected from the group consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
11. A method for the production of γ-linolenic acid comprising:
a) providing a microbial host cell of claim 6 ; and
b) growing the host cell of (a) in the presence of a source of linoleic acid under conditions wherein γ-linolenic acid is produced.
12. A method for the production of stearidonic acid comprising:
a) providing a microbial host cell of claim 6 ; and
b) growing the host cell of (a) in the presence of a source of α-linolenic acid under conditions wherein stearidonic acid is produced.