Docosahexaenoic acid producing strains of Yarrowia lipolytica

• US 20060110806A1
• Filed: 11/01/2005
• Published: 05/25/2006
• Est. Priority Date: 11/04/2004
• Status: Active Grant

• Current Assignee: Dupont US Holding, LLC (International Flavors & Fragrances Incorporated)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Damude, Howard Glenn, Xue, Zhixiong, Yadav, Narendra S., Zhang, Hongxiang, Zhu, Quinn Qun, Seip, John E., Ragghianti, James John, Macool, Daniel Joseph, Picataggio, Stephen K., Gillies, Peter John
• Granted Patent: US 7,550,286 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: A23D 9/00 Other edible oils or fats, e.g. shortenings, cooking oils
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A23L 33/12 Fatty acids or derivatives thereof
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A23V 2200/30 Foods, ingredients or supplements having a functional effect on health
• CPC Class Codes: A23V 2250/187 Eicosapentaenoic acid
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/14 Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/20 Hypnotics; Sedatives
• CPC Class Codes: A61P 25/24 Antidepressants
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 25/32 Alcohol-abuse
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 : Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/06 : Antihyperlipidemics
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: C12N 1/16 : Yeasts; Culture media therefor
• CPC Class Codes: C12N 15/52 : Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/746 : for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 15/815 : for yeasts other than Saccharomyces
• CPC Class Codes: C12N 9/0083 : Miscellaneous (1.14.99)
• CPC Class Codes: C12N 9/1029 : transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/6434 : Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6472 : containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

An engineered strain of the oleaginous yeast Yarrowia lipolytica capable of producing greater than 5.6% docosahexaenoic acid acid (DHA, an w-3 polyunsaturated fatty acid) in the total oil fraction is described. This strain comprises various chimeric genes expressing heterologous desaturases, elongases and acyltransferases and optionally comprises various native desaturase and acyltransferase knockouts to enable synthesis and high accumulation of DHA. Production host cells are claimed, as are methods for producing DHA within said host cells.

Claims

1. A recombinant production host cell for the production of docosahexaenoic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω

-3/ω

-6 fatty acid biosynthetic pathway;

a) at least one gene encoding Δ

6 desaturase;

b) at least one gene encoding C_18/20 elongase;

c) at least one gene encoding Δ

5 desaturase;

d) at least one gene encoding Δ

17 desaturase;

e) at least one gene encoding C_20/22 elongase; and

,

f) at least one gene encoding Δ

4 desaturase.

5. A recombinant production host according to any one of claims 1, 2, 3 or 4 wherein the gene pool optionally comprises at least one gene encoding a Δ

12 desaturase.

6. A recombinant production host according to claim 5 wherein the background Yarrowia sp. is devoid of any native gene encoding a polypeptide having Δ

12 desaturase activity

7. A recombinant production host according to any one of claims 1, 2, 3 or 4, wherein at least one of said ω

-3/ω

-6 fatty acid biosynthetic pathway genes is under the control of a promoter sequence having the nucleic acid sequence selected from the group consisting of SEQ ID NOs;

210-221.

8. A recombinant production host according to claim 5 wherein said Δ

12 desaturase has the amino acid sequence selected from the group consisting of SEQ ID NOs;

29, 31, 33, 35, 36, 37, 39, 41, 43, 45, 46, 48-50.

9. A recombinant production host cell according to either of claims 1 or 2 wherein said Δ

6 desaturase has an amino acid sequence selected from the group consisting of SEQ ID NOs;

2 and 5; and

wherein said C_18/20 elongase has an amino acid sequence selected from the group consisting of SEQ ID NOs;

23 and 26

11. A recombinant production host according to any one of claims 1, 2, 3, 4 wherein said at least one gene encoding a Δ

5 desaturase encodes a bifunctional Δ

5/Δ

6 desaturase polypeptide which binds at least two fatty acids as an enzymatic substrate, selected from the group consisting of;

a) linolenic acid and dihomo-γ

-linolenic acid;

b) α

-linolenic acid and eicosatetraenoic acid; and

,

c) linolenic acid, dihomo-γ

-linolenic acid, α

-linolenic acid; and

eicosatetraenoic acid.

12. A recombinant production host according to claim 11 wherein said bifunctional Δ

5/Δ

6 desaturase polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs;

15 and 18.

13. A recombinant production host cell according to any one of claims 1, 2, 3, or 4 wherein said Δ

5 desaturase has an amino acid sequence selected from the group consisting of SEQ ID NOs;

7, 9, 12, 15 and 18;

wherein said C_20/22 elongase has an amino acid sequence selected from the group consisting of SEQ ID NOs;

101 and 103; and

wherein said Δ

4 desaturase has the amino acid sequence selected from the group consisting of SEQ ID NOs;

105 and 107.

14. A recombinant production host cell according to either of claims 1 or 3 wherein said Δ

17 desaturase has the amino acid sequence as set forth in SEQ ID NO;

20.

15. A recombinant production host cell according to either of claims 1 or 3 wherein the gene pool optionally comprises at least one gene encoding a Δ

15 desaturase.

19. A recombinant production host according to any one of claims 1, 2, 3, or 4 wherein the gene pool optionally comprises a ω

-3/ω

-6 fatty acid biosynthetic pathway gene selected from the group consisting of;

a) at least one gene encoding Δ

9 desaturase;

b) at least one gene encoding C_16/18 elongase; and

,

c) at least one gene encoding C_14/16 elongase.

20. A recombinant production host according to claim 19 wherein said C_16/18 elongase has the amino acid sequence selected from the group consisting of:

SEQ ID NOs;

84, 87 and 95; and

wherein said C_14/16 elongase has the amino acid sequence as set forth in SEQ ID NO;

98.

21. A recombinant production host according to any one of claims 1, 2, 3, or 4 wherein the gene pool optionally comprises at least one gene encoding an acyltransferase selected from the group consisting of:

a) diacylglycerol acyltransferase (DGAT1);

b) diacylglycerol acyltransferase (DGAT2);

c) phospholipid;

diacylglycerol acyltransferase (PDAT);

d) acyl-CoA;

1-acyl lysophosphatidylcholine acyltransferase (LPCAT);

e) glycerol-3-phosphate acyltransferase (GPAT); and

,

f) lysophosphatidic acid acyltransferase (LPAAT).

22. A recombinant production host according to claim 21 wherein said diacylglycerol acyltransferase (DGAT1) has an amino acid sequence selected from the group consisting of SEQ ID NOs:

123 and 125-129;

wherein said diacylglycerol acyltransferase (DGAT2) has an amino acid sequence selected from the group consisting SEQ ID NOs;

131, 133, 135 and 137;

wherein said phospholipid;

diacylglycerol acyltransferase (PDAT) has an amino acid sequence as set forth in SEQ ID NO;

118;

wherein said a glycerol-3-phosphate acyltransferase (GPAT) has an amino acid sequence as set forth in SEQ ID NO;

139;

wherein said lysophosphatidic acid acyltransferase (LPAAT) has the amino acid sequence selected from the group consisting of SEQ ID NOs;

109, 111, 113 and 116; and

wherein said acyl-CoA;

1-acyl lysophosphatidylcholine acyltransferase (LPCAT) has an amino acid sequence as set forth in SEQ ID NO;

121.

25. A recombinant production host according to any one of claims 1, 2, 3, or 4 wherein the host produces a microbial oil comprising at least about 5% docosahexaenoic acid as a percent of the total fatty acids.

26. A recombinant production host according to claim 25 wherein the host produces a microbial oil comprising docosahexaenoic acid and wherein the microbial oil is devoid of any γ

-linoleic acid.

27. A method for the production of a microbial oil comprising docosahexaenoic acid comprising:

a) culturing the production host of any one of claims 1, 2, 3, or 4 wherein a microbial oil comprising docosahexaenoic acid is produced; and

b) optionally recovering the microbial oil of step (a).

28. A microbial oil produced by the method of claim 27.

29. The microbial oil of claim 28 wherein the oil contains at least about 5% docosahexaenoic acid.

30. The microbial oil of claim 28 wherein the oil is devoid of any γ

-linoleic acid.

31. A blended oil according to claim 28, wherein the oil comprises a fatty acid selected from the group consisting of linoleic acid, γ

-linolenic acid, eicosadienoic acid, dihomo-γ

-linoleic acid, arachidonic acid, α

-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, docosapentaenoic acid and docosahexaenoic acid.

32. A food product comprising an effective amount of a microbial oil produced by the method of claim 27.

33. A food product according to claim 32 selected from the group consisting of a food analogue, a meat product, a cereal product, a baked food, a snack food, and a dairy product.

34. A product selected from the group consisting of a medical food, a dietary supplement;

an infant formula and a pharmaceutical comprising an effective amount of a microbial oil produced by the method of claim 27.

35. An animal feed comprising an effective amount of the microbial oil produced by the method of claim 27.

36. An animal feed according to claim 35 wherein the animal feed is selected from the group consisting of a pet feed, a ruminant feed, a poultry feed, and and aquacultural feed.

37. An animial feed comprising an effective amount of microbial oil and optionally comprising a yeast biomass comprising the recombinant host of any of claims 1, 2, 3 or 4.

38. An animal feed according to claim 37 wherein the yeast biomass comprises feed nutrients selected from the group consisting of proteins, lipids, carbohydrates, vitamins, minerals, and nucleic acids.

39. A method of making a food product supplemented with docosahexaenoic acid comprising combining a microbial oil produced by the method of claim 27 with a food product.

40. A method of making a product selected from the group consisting of medical food;

a dietary supplement, an infant formula and a pharmaceutical wherein the product is supplemented with docosahexaenoic acid comprising combining a microbial oil produced by the method of claim 27 with the product.

41. A method of making an animal feed supplemented with docosahexaenoic acid comprising combining a microbial oil produced by the method of claim 27 with an animal feed.

42. A method of supplementing an animal feed comprising docosahexaenoic acid with feed nutrients comprising combining the animal feed of claim 37 with a yeast biomass comprising feed nutrients.

43. A method according to claim 42 wherein the feed nuturients are selected from the group consisting of proteins, lipids, carbohydrates, vitamins, minerals, and nucleic acids.

44. A method for providing a human, animal or aquaculture organism diet supplement enriched with eicosapentaenoic acid comprising providing a microbial oil produced by the method of claim 27 containing docosahexaenoic acid in a form consumable or usable by humans or animals.

45. A method for treating a deficiency in docosahexaenoic acid in animals or humans comprising providing a microbial oil produced by the method of claim 27 containing docosahexaenoic acid in a form consumable or usable by humans or animals to treat said deficiency

2. A recombinant production host cell for the production of docosahexaenoic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω

-3/ω

-6 fatty acid biosynthetic pathway;

a) at least one gene encoding Δ

15 desaturase;

b) at least one gene encoding Δ

6 desaturase;

c) at least one gene encoding C_18/20 elongase;

d) at least one gene encoding Δ

5 desaturase;

e) at least one gene encoding C_20/22 elongase; and

,

f) at least one gene encoding Δ

4 desaturase.

16. A recombinant production host according to any one of claims 2, or 4 wherein said at least one gene encoding a Δ

15 desaturase encodes a bifunctional Δ

15/Δ

12 desaturase polypeptide which binds both oleic acid and linoleic acid as an enzymatic substrate.

17. A recombinant production host according to claim 16 wherein said bifunctional Δ

15/Δ

12 desaturase polypeptide has an amino acid sequence as set forth in SEQ ID NO;

52.

18. A recombinant production host according to any one of claims 2, or 4 wherein said Δ

15 desaturase has the amino acid sequence selected from the group consisting of SEQ ID NOs;

52, 54, 56, 58, 60, 62 and 64-68.

3. A recombinant production host cell for the production of docosahexaenoic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω

-3/ω

-6 fatty acid biosynthetic pathway;

a) at least one gene encoding Δ

9 elongase;

b) at least one gene encoding Δ

8 desaturase;

c) at least one gene encoding Δ

5 desaturase;

d) at least one gene encoding Δ

17 desaturase;

e) at least one gene encoding C_20/22 elongase; and

f) at least one gene encoding Δ

4 desaturase.

10. A recombinant production host cell according to either of claims 3 or 4 wherein said Δ

9 elongase has the amino acid sequence selected from the group consisting of SEQ ID NOs;

70 and 23;

and wherein said Δ

8 desaturase has an amino acid sequence selected from the group consisting of SEQ ID NOs;

78, 80 and 82.

4. A recombinant production host cell for the production of docosahexaenoic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω

-3/ω

-6 fatty acid biosynthetic pathway;

a) at least one gene encoding Δ

15 desaturase;

b) at least one gene encoding Δ

9 elongase;

c) at least one gene encoding Δ

8 desaturase;

d) at least one gene encoding Δ

5 desaturase;

e) at least one gene encoding C_20/22 elongase; and

f) at least one gene encoding Δ

4 desaturase.

23. A recombinant production host cell for the production of docosahexaenoic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω

-3/ω

-6 fatty acid biosynthetic pathway;

a) at least one gene encoding Δ

6 desaturase;

b) at least one gene encoding C_18/20 elongase;

c) at least one gene encoding Δ

5 desaturase;

d) at least one gene encoding Δ

17 desaturase;

e) at least one gene encoding C_20/22 elongase;

f) at least one gene encoding Δ

4 desaturase;

g) at least one gene encoding C_16/18 elongase; and

,

h) at least one gene encoding Δ

12 desaturase; and

wherein the background Yarrowia sp. is devoid of any native gene encoding an enzyme selected from the group consisting of;

orotidine-5′

-phosphate decarboxylase (Ura3), isopropyl malate dehydrogenase (Leu2), acyl-coA oxidase (Pox3), acyl-CoA oxidase (Pox2), Δ

12 desaurase and lipase 1 (Lip1).

24. A recombinant production host cell for the production of docosahexaenoic acid comprising a background Yarrowia sp. comprising a gene pool comprising the following genes of the ω

-3/ω

-6 fatty acid biosynthetic pathway;

a) at least one set of genes selected from the group consisting of;

i. at least one gene encoding Δ

6 desaturase; and

at least one gene encoding C_18/20 elongase; and

at least one gene encoding Δ

5 desaturase; and

at least one gene encoding Δ

17 desaturase; and

at least one gene encoding C_20/22 elongase; and

at least one gene encoding Δ

4 desaturase;

ii. at least one gene encoding Δ

9 elongase; and

at least one gene encoding Δ

8 desaturase; and

at least one gene encoding Δ

5 desaturase; and

at least one gene encoding Δ

17 desaturase; and

at least one gene encoding C_20/22 elongase; and

at least one gene encoding Δ

4 desaturase;

iii. at least one gene encoding Δ

15 desaturase;

at least one gene encoding Δ

6 desaturase; and

at least one gene encoding C_18/20 elongase; and

at least one gene encoding Δ

5 desaturase; and

at least one gene encoding C_20/22 elongase; and

at least one gene encoding Δ

4 desaturase;

iv. at least one gene encoding Δ

15 desaturase;

at least one gene encoding Δ

9 elongase; and

at least one gene encoding Δ

8 desaturase; and

at least one gene encoding Δ

5 desaturase; and

at least one gene encoding C_20/22 elongase; and

at least one gene encoding Δ

4 desaturase; and

,

b) at least one gene encoding an enzyme selected from the group consisting of;

i. Δ

12 desaturase;

ii. Δ

9 desaturase;

iii. C_14/16 elongase;

iv. C_16/18 elongase; and

,

c) at least one gene encoding an enzyme selected from the group consisting of;

(i) diacylglycerol acyltransferase selected from the group consisting of DGAT1, DGAT2, and PDAT;

(ii) acyl-CoA;

1-acyl lysophosphatidylcholine acyltransferase (LPCAT);

(iii) glycerol-3-phosphate acyltransferase (GPAT);

(iv) lysophosphatidic acid acyltransferase (LPAAT);

(v) phospholipase C; and

,

(vi) phospholipase A₂;

wherein;

(1) the background Yarrowia sp. is devoid of any native gene encoding a polypeptide having Δ

12 desaturase activity; and

,

(2) the background Yarrowia sp. is devoid of any native gene encoding an enzyme selected from the group consisting of lipase 1 (Lip1-), peroxisomal acyl CoA oxidase ACO3 (Pox3-), acyl-CoA oxidase 2 (Pox2-), orotidine-5′

-phosphate decarboxylase (Ura3-), saccharopine dehydrogenase (Lys5-), lipase 2 (Lip2-) and isopropyl malate dehydrogenase (Leu2-).

46. The recombinant production host Yarrowia lipolytica Y3000, useful for the production of docosahexaenoic acid having the ATCC desgination ATCC ______;