GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND PHOSPHOGLYCERATE MUTASE PROMOTERS FOR GENE EXPRESSION IN OLEAGINOUS YEAST

• US 20110059496A1
• Filed: 11/17/2010
• Published: 03/10/2011
• Est. Priority Date: 06/25/2003
• Status: Abandoned Application

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: ZHU, QUINN QUN
• Application Number:
  US12/948,330
• Publication Number:
  US 20110059496A1
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone

Abstract

Promoter regions associated with the Yarrowia lipolytica glyceraldehyde-3-phosphate dehydrogenase (gpd) gene have been found to be particularly effective for the expression of heterologous genes in yeast. Promoter regions of a Yarrowia gpd gene shown to drive high-level expression of genes involved in the production of omega-3 and omega-6 fatty acids are disclosed.

Claims

1. A method for the expression of a coding region of interest in a transformed yeast cell comprising:

a) providing the transformed yeast cell having a chimeric gene, wherein the chimeric gene comprises;

(1) a promoter region of a gpd Yarrowia gene; and

,

(2) the 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 chimeric gene of step (a) is expressed.

2. The method according to claim 1 wherein the promoter region of a gpd Yarrowia gene comprises SEQ ID NO:

16,

3. The method according to claim 1 wherein the promoter region of a gpd Yarrowia gene is set forth in SEQ ID NO:

15, wherein said promoter 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259 or 260 consecutive nucleotides, wherein the first nucleotide deleted is the thymine nucleotide [‘

T’

] at position 1 of SEQ ID NO;

15;

(b) insertion of any two nucleotides [‘

NN’

] after the adenine [‘

A’

] nucleotide at position +160 and before the guanine [‘

G’

] nucleotide at position +161 of SEQ ID NO;

15;

(c) insertion of a cytosine [‘

C’

] nucleotide at the 3′

end of SEQ ID NO;

15 after the cytosine [‘

C’

] nucleotide at position +1068; and

,

(d) any combination of part (a), part (b) and part (c) above.

4. The method according to claim 1 wherein the promoter region of a gpd Yarrowia gene is set forth in SEQ ID NO:

14, wherein said promoter 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 or 60 consecutive nucleotides, wherein the first nucleotide deleted is the guanine nucleotide [‘

G’

] at position 1 of SEQ ID NO;

14;

(b) insertion of a thymine nucleotide and a cytosine nucleotide [‘

TC’

] after the adenine [‘

A’

] nucleotide at position +60 and before the guanine [‘

G’

] nucleotide at position +61 of SEQ ID NO;

14;

(c) insertion of any two nucleotides [‘

NN’

] after the adenine [‘

A’

] nucleotide at position +60 and before the guanine [‘

G’

] nucleotide at position +61 of SEQ ID NO;

14;

(d) insertion of a cytosine [‘

C’

] nucleotide at the 3′

end of SEQ ID NO;

14 after the cytosine [‘

C’

] nucleotide at position +968;

(e) any combination of part (a), part (b), part (c) and part (d) above.

5. The method according to claim 4 wherein the promoter region of a gpd Yarrowia gene is selected from the group consisting of SEQ ID NO:

3, SEQ ID NO;

5, SEQ ID NO;

6 and SEQ ID NO;

7.

6. The method according to claim 1 wherein the transformed yeast cell is an oleaginous yeast.

7. The method of claim 6, wherein the oleaginous yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

8. 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, catalyases, 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, phosphotases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminases and xylanases.

9. A method for the production of an omega-3 fatty acid or omega-6 fatty acid comprising:

a) providing a transformed oleaginous yeast comprising a chimeric gene, wherein the chimeric gene comprises;

i) a promoter region of a gpd Yarrowia gene; and

,

ii) a coding region encoding at least one omega-3 fatty acid or omega-6 fatty acid biosynthetic pathway enzyme;

wherein the promoter region and the coding region are operably linked; and

,

b) growing the transformed oleaginous yeast of step (a) under conditions whereby the at least one omega-3 fatty acid or omega-6 fatty acid biosynthetic pathway enzyme is expressed and the omega-3 fatty acid or the omega-6 fatty acid is produced; and

,

c) optionally recovering the omega-3 fatty acid or the omega-6 fatty acid.

10. The method according to claim 9 wherein the promoter region of a gpd Yarrowia gene is set forth in SEQ ID NO:

14, wherein said promoter 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 or 60 consecutive nucleotides, wherein the first nucleotide deleted is the guanine nucleotide [‘

G’

] at position 1 of SEQ ID NO;

14;

b) insertion of a thymine nucleotide and a cytosine nucleotide [‘

TC’

] after the adenine [‘

A’

] nucleotide at position +60 and before the guanine [‘

G’

] nucleotide at position +61 of SEQ ID NO;

14;

c) insertion of any two nucleotides [‘

NN’

] after the adenine [‘

A’

] nucleotide at position +60 and before the guanine [‘

G’

] nucleotide at position +61 of SEQ ID NO;

14;

d) insertion of a cytosine [‘

C’

] nucleotide at the 3′

end of SEQ ID NO;

14 after the cytosine [‘

C’

] nucleotide at position +968;

e) any combination of part (a), part (b), part (c) and part (d) above.

11. The method according to claim 10 wherein the promoter region of a gpd Yarrowia gene is selected from the group consisting of SEQ ID NO:

3, SEQ ID NO;

5, SEQ ID NO;

6 and SEQ ID NO;

7.

12. The method according to claim 9 wherein the coding region encoding at least one omega-3 fatty acid or omega-6 fatty acid biosynthetic pathway enzyme is selected from the group consisting of:

desaturases and elongases.

13. The method according to claim 12 wherein the desaturase is selected from the group consisting of:

delta-9 desaturase, delta-8 desaturase, delta-12 desaturase, delta-6 desaturase, delta-5 desaturase, delta-17 desaturase, delta-15 desaturase and delta-4 desaturase and the elongase is selected from the group consisting of;

a delta-9 elongase, a C_14/16 elongase, a C_16/18 elongase, a C_18/20 elongase and a C_20/22 elongase.

14. The method according to claim 9 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.

15. The method according to claim 14 wherein the oleaginous yeast is Yarrowia lipolytica.

16. The method according to claim 9 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.

17. An isolated nucleic acid molecule comprising a promoter region of a gpd Yarrowia gene as set forth in SEQ ID NO:

15, wherein said promoter 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259 or 260 consecutive nucleotides, wherein the first nucleotide deleted is the thymine nucleotide [‘

T’

] at position 1 of SEQ ID NO;

15;

(b) insertion of any two nucleotides [‘

NN’

] after the adenine [‘

A’

] nucleotide at position +160 and before the guanine [‘

G’

] nucleotide at position +161 of SEQ ID NO;

15;

(c) insertion of a cytosine [‘

C’

] nucleotide at the 3′

end of SEQ ID NO;

15 after the cytosine [‘

C’

] nucleotide at position +1068; and

,

(d) any combination of part (a), part (b) and part (c) above.

18. An isolated nucleic acid molecule comprising a promoter region of a gpd Yarrowia gene as set forth in SEQ ID NO:

14, wherein said promoter 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 or 60 consecutive nucleotides, wherein the first nucleotide deleted is the guanine nucleotide [‘

G’

] at position 1 of SEQ ID NO;

14;

(b) insertion of a thymine nucleotide and a cytosine nucleotide [‘

TC’

] after the adenine [‘

A’

] nucleotide at position +60 and before the guanine [‘

G’

] nucleotide at position +61 of SEQ ID NO;

14;

(c) insertion of any two nucleotides [‘

NN’

] after the adenine [‘

A’

] nucleotide at position +60 and before the guanine [‘

G’

] nucleotide at position +61 of SEQ ID NO;

14;

(d) insertion of a cytosine [‘

C’

] nucleotide at the 3′

end of SEQ ID NO;

14 after the cytosine [‘

C’

] nucleotide at position +968;

(e) any combination of part (a), part (b), part (c) and part (d) above.

19. The isolated nucleic acid molecule of claim 18 selected from the group consisting of SEQ ID NO:

3, SEQ ID NO;

5, SEQ ID NO;

6 and SEQ ID NO;

7.

20. An isolated nucleic acid molecule comprising a promoter region of a gpd Yarrowia gene comprising SEQ ID NO:

16.