HIGH EICOSAPENTAENOIC ACID OILS FROM IMPROVED OPTIMIZED STRAINS OF YARROWIA LIPOLYTICA

• US 20100317735A1
• Filed: 06/14/2010
• Published: 12/16/2010
• Est. Priority Date: 06/16/2009
• 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: Sharpe, Pamela L., Xue, Zhixiong, Yadav, Narendra S., Zhu, Quinn Qun, Hong, Seung-Pyo
• Granted Patent: US 8,399,226 B2
• Time in Patent Office: Days
• US Class Current: 514/560
• CPC Class Codes: A21D 2/165 Triglycerides
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• 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 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 9/12 Antihypertensives
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/13 transferring sulfur containing groups (2.8)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6481 Phosphoglycerides phosphoglycerides having carboxylic acids with less than seven carbon atoms C12P7/62

Abstract

Described are engineered strains of the oleaginous yeast Yarrowia lipolytica capable of producing an oil comprising greater than 50 weight percent of eicosapentaenoic acid [“EPA”], an ω-3 polyunsaturated fatty acid, measured as a weight percent of total fatty acids [“% TFAs”] and having a ratio of at least 3.1 of EPA % TFAs, to linoleic acid, measured as % TFAs. These strains over-express at least one Δ9 elongase/Δ8 desaturase multizyme, in addition to other heterologous Δ9 elongases, Δ8 desaturases, Δ5 desaturases, Δ17 desaturases, Δ12 desaturases, C_16/18 elongases, and optionally over-express diacylglycerol cholinephosphotransferases, malonyl CoA synthetases and/or acyl-CoA lysophospholipid acyltransferases. The strains possess at least one peroxisome biogenesis factor protein knockout. Methods for producing EPA within said host cells, oils obtained from the cells, and products therefrom are claimed.

Claims

1. An extracted oil comprising:

(a) at least 50 weight percent of eicosapentaenoic acid measured as a weight percent of total fatty acids; and

,

(b) having a ratio of at least 3.1 of eicosapentaenoic acid, measured as a weight percent of total fatty acids, to linoleic acid, measured as a weight percent of total fatty acids.

2. The extracted oil of claim 1 wherein said oil is a microbial oil.

3. The extracted oil of claim 2 wherein said oil is extracted from fermented recombinant Yarrowia sp. cells, engineered for the production of eicosapentaenoic acid wherein said cells comprise:

a) at least one multizyme which comprises a polypeptide having at least one Δ

9 elongase linked to at least one Δ

8 desaturase;

(b) at least one peroxisome biogenesis factor protein whose expression has been down-regulated; and

,

(c) at least one recombinant construct comprising a nucleotide sequence encoding an enzyme selected from the group consisting of malonyl CoA synthetase and acyl-CoA lysophospholipid acyltransferase.

4. The extracted oil of claim 3 wherein said oil is extracted from fermented recombinant Yarrowia sp. cells and wherein said cells comprise at least one recombinant construct comprising a nucleotide sequence encoding malonyl CoA synthetase, wherein the malonyl CoA synthetase consists essentially of a sequence selected from the group consisting of SEQ ID NO:

40 and SEQ ID NO;

42.

5. The extracted oil of claim 3 wherein said oil is extracted from fermented recombinant Yarrowia sp. cells and wherein said cells comprise at least one recombinant construct comprising a nucleotide sequence encoding an acyl-CoA lysophospholipid acyltransferase, wherein the acyl-CoA lysophospholipid acyltransferase consists essentially of the sequence selected from the group consisting of SEQ ID NO:

15, SEQ ID NO;

17, SEQ ID NO;

25, SEQ ID NO;

29, SEQ ID NO;

31 and SEQ ID NO;

32.

6. The extracted oil of claim 3 wherein said oil is extracted from fermented recombinant Yarrowia sp. cells and wherein said cells comprise at least at least one multizyme which comprises a polypeptide having at least one Δ

9 elongase linked to at least one Δ

8 desaturase, wherein the multizyme linker is selected from the group consisting of;

SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6 and SEQ ID NO;

7.

7. The extracted oil of claim 3 wherein said oil is extracted from fermented recombinant Yarrowia sp. cells and wherein said cells comprise at least at least one multizyme, wherein the multizyme consists essentially of the sequence selected from the group consisting of:

SEQ ID NO;

9, SEQ ID NO;

11 and SEQ ID NO;

13.

8. A blended oil comprising the oil of any of claims 1-7 or a derivative thereof.

9. A blended oil comprising the oil of claim 8 and an additional quantity of a fatty acid selected from the group consisting of:

linoleic acid, γ

-linolenic, eicosadienoic acid, dihomo-γ

-linolenic acid, arachidonic acid, docosatetraenoic acid, ω

-6 docosapentaenoic acid, α

-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, ω

-3 docosapentaenoic acid and docosahexaenoic acid.

10. Food or feed comprising the oil of claim 8 or a derivative thereof or a blend of the oil or derivative thereof.

11. Food or feed comprising the oil of claim 9 or a derivative thereof or a blend of the oil or derivative thereof.

12. The food of claim 10 wherein said food is selected from the group consisting of a food analog, a functional food, a medical food and a medical nutritional.

13. The food of claim 11 wherein said food is selected from the group consisting of a food analog, a functional food, a medical food and a medical nutritional.

14. A product comprising the oil any of claims 1-7 or a derivative thereof or a blend of the oil or derivative thereof, wherein said product is selected from the group consisting of a pharmaceutical product, an infant formula, a dietary supplement, and an animal feed.

15. A microbial biomass comprising the oil of any of claims 1-7.

16. An animal feed comprising the microbial biomass of claim 15.

17. The animal feed of claim 16 wherein the feed is an aquaculture feed.

18. The oil of any of claims 1-7 wherein said oil is extracted from a recombinant Yarrowia sp. host cell using a process selected from the group consisting of:

extraction with organic solvents, sonication and supercritical fluid extraction.