Acyltransferases for alteration of polyunsaturated fatty acids and oil content in oleaginous yeasts
View Patent ↗Two acyltransferases are provided, suitable for use in the manufacture of microbial oils enriched in omega fatty acids in oleaginous yeast (e.g., Yarrowia lipolytica ). Specifically, the genes encoding phophatidylcholine-diacylglycerol acyltransferase (PDAT) and diacylglycerol acyltransferase (DGAT2) have been isolated from Y. lipolytica . These genes encode enzymes that participate in the terminal step in oil biosynthesis in yeast. Each is expected to play a key role in altering the quantity of polyunsaturated fatty acids produced in oils of oleaginous yeasts.
1. An isolated nucleic acid molecule encoding a phospholipid:diacylglycerol acyltransferase 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:46;
(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;
(c) an isolated nucleic acid molecule that is completely complementary to (a) or (b); and, (d) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a phospholipid:diacylglycerol acyltransferase enzyme of at least 648 amino acids that has at least 95% identity based on the Clustal W method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:46 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
2. The isolated nucleic acid molecule of claim 1 having the nucleotide sequence as set forth in SEQ ID NO 45.
3. A chimeric gene comprising the isolated nucleic acid molecule of claim 1 operably linked to suitable regulatory sequences.
4. A transformed host cell comprising the chimeric gene of claim 3 .
5. The transformed host cell of claim 4 , selected from the group consisting of algae, bacteria, molds, fungi and yeasts.
6. The transformed host cell of claim 5 , wherein the yeast is an oleaginous yeast.
7. The transformed host cell of claim 6 , wherein the oleaginous yeast cell is selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
8. The transformed host cell of claim 7 , wherein the host cell is Yarrowia lipolytica.
9. A method of increasing triacylglycerol content in a transformed host cell comprising:
(a) providing a transformed host cell comprising:
(i) at least one gene encoding an acyltransferase enzyme having the amino acid sequence as set forth in SEQ ID NO:46 under the control of suitable regulatory sequences; and
(ii) a source of fatty acids;
(b) growing the cell of step (a) under conditions whereby the at least one gene encoding an acyltransferase enzyme is expressed, resulting in the transfer of the fatty acids to triacylglycerol; and
(c) optionally recovering the triacylglycerol of step (b).
10. The method according to claim 9 , wherein growing the cell of step (a) under conditions whereby the genes of (i) and (ii) are expressed results in the production of at least one ω-3 or ω-6 fatty acid and its transfer to triacylglycerol.
11. The method according to claim 10 , wherein the at least one gene encoding at least one enzyme of the ω-3/ω-6 fatty acid biosynthetic pathway is selected from the group consisting of desaturases and elongases.
12. The method according to claim 11 , wherein the desaturase is selected from the group consisting of: Δ9 desaturase, Δ12 desaturase, Δ6 desaturase, Δ5 desaturase, Δ17 desaturase, Δ8 desaturase, Δ15 desaturase and Δ4 desaturase.
13. The method according to claim 9 , wherein the host cell is selected from the group consisting of algae, bacteria, molds, fungi and yeasts.
14. The method according to claim 13 , wherein the host cell is an oleaginous yeast.
15. The method according to claim 14 wherein the oleaginous yeast is a member of a genus selected from the group of consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
16. The method according to claim 15 , wherein the oleaginous yeast is Yarrowia lipolytica.
17. The method according to claim 9 wherein the fatty acid is selected from the group consisting of: stearate, oleic acid, linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid, eicosadienoic acid and eicosatrienoic acid.