Multizymes comprising delta-9 elongase and delta-8 desaturase and their use in making polyunsaturated fatty acids
Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding multizymes (i.e., single polypeptides having at least two independent and separable enzymatic activities) along with a method of making long-chain polyunsaturated fatty acids (PUFAs) using these multizymes in plants and oleaginous yeast are disclosed.
1. A method for the conversion of linoleic acid to dihomo gamma-linolenic acid, comprising:
a) providing a recombinant microbial host cell comprising:
i) a DGLA synthase comprising an amino acid sequence selected from the group consisting of SEQ ID NO:441, SEQ ID NO:447, SEQ ID NO:454, SEQ ID NO:461, SEQ ID NO:464, SEQ ID NO:471, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, and SEQ ID NO:519; and
ii) a source of linoleic acid; and
b) growing the host cell of (a) under conditions whereby dihomo gamma-linolenic acid is produced.
2. A method for the conversion of alpha-linolenic acid to eicosatetraenoic acid, comprising:
a) providing a recombinant microbial host cell comprising:
i) a DGLA synthase comprising an amino acid sequence selected from the group consisting of SEQ ID NO:441, SEQ ID NO:447, SEQ ID NO:454, SEQ ID NO:461, SEQ ID NO:464, SEQ ID NO:471, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:518, and SEQ ID NO:519; and
ii) a source of alpha-linolenic acid; and
b) growing the host cell of (a) under conditions whereby eicosatetraenoic acid is produced.
3. A method for the conversion of linoleic acid to dihomo-gamma-linolenic acid comprising:
a) providing a recombinant microbial host cell comprising:
i) a DGLA synthase comprising:
1) at least one delta-9 elongase having an amino acid sequence selected from the group consisting of SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:319, SEQ ID NO:359, SEQ ID NO:420, SEQ ID NO:422, and SEQ ID NO:513;
2) at least one delta-8 desaturase; and
3) a linker selected from the group consisting of a polypeptide bond, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:235, SEQ ID NO:438, SEQ ID NO:445, SEQ ID NO:472, and SEQ ID NO:504; wherein the linker is interposed between the delta-9 elongase and the delta-8 desaturase such that the delta-9 elongase is located at the N-terminus of the DGLA synthase; and
ii) a source of linoleic acid; and
b) growing the host cell of (a) under conditions whereby dihomo-gamma-linolenic acid is produced.
4. A method for the conversion of linoleic acid to dihomo-gamma-linolenic acid comprising:
a) providing a recombinant microbial host cell comprising:
i) a DGLA synthase comprising:
1) at least one delta-9 elongase;
2) at least one delta-8 desaturase having an amino acid sequence selected from the group consisting of SEQ ID NO:328, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, and SEQ ID NO:514; and
3) a linker selected from the group consisting of a polypeptide bond, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:235, SEQ ID NO:438, SEQ ID NO:445, SEQ ID NO:472, and SEQ ID NO:504;
wherein the linker is interposed between the delta-9 elongase and the delta-8 desaturase such that the delta-9 elongase is located at the N-terminus of the DGLA synthase; and
ii) a source of linoleic acid; and
b) growing the host cell of (a) under conditions whereby dihomo-gamma-linolenic acid is produced.
5. A host cell comprising in its genome the recombinant construct of claim 4 .
6. The host cell of claim 5 , wherein said cell is selected from the group consisting of plants and yeast.
7. A transformed Yarrowia comprising the recombinant construct of claim 4 .
8. A method for transforming a cell, comprising transforming a cell with the recombinant construct of claim 4 and selecting those cells transformed with said recombinant construct.
9. A method for producing a transformed plant comprising transforming a plant cell with the recombinant construct of claim 4 and regenerating a plant from the transformed plant cell.
10. The method of claim 9 , wherein the plant is a soybean plant.
11. A method for producing yeast, comprising transforming a yeast cell with the recombinant construct of claim 4 and growing yeast from the transformed yeast cell.
12. A plant comprising in its genome the recombinant construct of claim 4 .
13. The plant of claim 12 , wherein the plant is an oilseed plant.
14. The plant of either of claim 12 or 13 , wherein the plant is soybean.
15. Seed obtained from the plant of either of claim 12 or 13 , wherein the seed comprises the recombinant construct.
16. Seed obtained from the plant of claim 14 , wherein the seed comprises the recombinant construct.
17. A method for the conversion of alpha-linolenic acid to eicosatetraenoic acid, comprising:
a) providing a recombinant microbial host cell comprising:
i) a DGLA synthase comprising:
1) at least one delta-9 elongase having an amino acid sequence selected from the group consisting of SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:319, SEQ ID NO:359, SEQ ID NO:420, SEQ ID NO:422, and SEQ ID NO:513;
2) at least one delta-8 desaturase; and
3) a linker selected from the group consisting of a polypeptide bond, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:235, SEQ ID NO:438, SEQ ID NO:445, SEQ ID NO:472, and SEQ ID NO:504;
wherein the linker is interposed between the delta-9 elongase and the delta-8 desaturase such that the delta-9 elongase is located at the N-terminus of the DGLA synthase; and
ii) a source of alpha-linolenic acid; and
b) growing the host cell of (a) under conditions whereby eicosatetraenoic acid is produced.
18. A method for the conversion of alpha-linolenic acid to eicosatetraenoic acid, comprising:
a) providing a recombinant microbial host cell comprising:
i) a DGLA synthase comprising:
1) at least one delta-9 elongase;
2) at least one delta-8 desaturase having an amino acid sequence selected from the group consisting of SEQ ID NO:328, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, and SEQ ID NO:514; and
3) a linker selected from the group consisting of a polypeptide bond, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:235, SEQ ID NO:438, SEQ ID NO:445, SEQ ID NO:472, and SEQ ID NO:504;
wherein the linker is interposed between the delta-9 elongase and the delta-8 desaturase such that the delta-9 elongase is located at the N-terminus of the DGLA synthase; and
ii) a source of alpha-linolenic acid; and
b) growing the host cell of (a) under conditions whereby eicosatetraenoic acid is produced.
19. A DGLA synthase comprising:
(i) at least one delta-9 elongase having an amino acid sequence selected from the group consisting of SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:319, SEQ ID NO:359, SEQ ID NO:420, SEQ ID NO:422, and SEQ ID NO:513;
(ii) at least one delta-8 desaturase; and
(iii) a linker selected from the group consisting of a polypeptide bond, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:235, SEQ ID NO:438, SEQ ID NO:445, SEQ ID NO:472, and SEQ ID NO:504;
wherein the linker is interposed between the delta-9 elongase and the delta-8 desaturase such that the delta-9 elongase is located at the N-terminus of the DGLA synthase.
20. A DGLA synthase comprising:
(i) at least one delta-9 elongase;
(ii) at least one delta-8 desaturase having an amino acid sequence selected from the group consisting of SEQ ID NO:328, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, and SEQ ID NO:514; and
(iii) a linker selected from the group consisting of a polypeptide bond, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:235, SEQ ID NO:438, SEQ ID NO:445, SEQ ID NO:472, and SEQ ID NO:504;
wherein the linker is interposed between the delta-9 elongase and the delta-8 desaturase such that the delta-9 elongase is located at the N-terminus of the DGLA synthase.