Patent
Promoter regions associated with the Yarrowia lipolytica peroxisomal 2,4-dienoyl-CoA reductase (SPS19) gene are disclosed and have been found to be particularly effective for the expression of heterologous genes in yeast. These promoter regions will be useful for driving high-level expression of genes involved in the production of omega-3 and omega-6 fatty acids.
1. A method for the expression of a coding region of interest in a transformed yeast cell comprising:
a) providing a transformed yeast cell having a recombinant construct, wherein the recombinant construct comprises:
(1) a promoter region comprising a sequence selected from the group consisting of SEQ ID NOs: 10, 36, 37, 38 and 39; and
(2) a coding region of interest which is expressible in the yeast cell;
wherein the promoter region is operably linked to the coding region of interest; and
b) growing the transformed yeast cell of step (a) under conditions whereby the recombinant construct of step (a) is expressed.
2. The method according to claim 1 , wherein the promoter region comprises SEQ ID NO:38, and wherein said promoter region optionally comprises at least one modification selected from the group consisting of:
a) a deletion at the 5′-terminus of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 consecutive nucleotides, wherein the first nucleotide deleted is the adenine nucleotide [A] at position 1 of SEQ ID NO:38; and
b) a deletion of part (a) in combination with a substitution of a ‘CAA’ nucleotide sequence for an ‘ACC’ nucleotide sequence at positions 173 to 175 of SEQ ID NO:38.
3. The method according to claim 1 , wherein the transformed yeast cell is an oleaginous yeast cell.
4. The method of claim 3 , wherein the oleaginous yeast cell is a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
5. The method according to claim 1 , wherein the coding region of interest encodes a polypeptide, wherein the polypeptide is selected from the group consisting of: desaturases, elongases, acyltransferases, aminopeptidases, amylases, carbohydrases, carboxypeptidases, catalases, cellulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, alpha-galactosidases, beta-galactosidases, glucoamylases, alpha-glucosidases, beta-glucanases, beta-glucosidases, invertases, laccases, lipases, mannosidases, mutanases, oxidases, pectinolytic enzymes, peroxidases, phospholipases, phosphatases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminases and xylanases.
6. The method according to claim 1 , wherein the coding region encodes at least one omega-3 fatty acid or omega-6 fatty acid biosynthetic pathway enzyme, and wherein an omega-3 fatty acid or omega-6 fatty acid is produced in step b).
7. The method according to claim 6 , wherein the omega-3 fatty acid or omega-6 fatty acid biosynthetic pathway enzyme is selected from the group consisting of desaturases and elongases.
8. The method according to claim 6 , wherein the oleaginous yeast cell is a member of a genus selected from the group of consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
9. The method according to claim 6 , wherein the omega-3 fatty acid or the omega-6 fatty acid is selected from the group consisting of: linoleic acid, gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, alpha-linoleic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosatetraenoic acid, omega-6 docosapentaenoic acid, omega-3 docosapentaenoic acid and docosahexaenoic acid.