Recombinant microbial cells that produce at least 28% eicosapentaenoic acid as dry cell weight

Patent metadata

Field	Value
Publication number	US-9701989-B2
Application number	US-201314132022-A
Country	US
Kind code	B2
Filing date	Dec 18, 2013
Priority date	Dec 21, 2012
Publication date	Jul 11, 2017
Grant date	Jul 11, 2017

Abstract

Recombinant microbial cells are disclosed herein that produce an oil comprising at least 28 percent eicosapentaenoic acid (EPA) measured as a weight percent of dry cell weight. These cells may comprise a polynucleotide sequence encoding an active acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) comprising at least one amino acid mutation in a membrane-bound O-acyltransferase motif. In addition, the cells may comprise a down-regulation of an endogenous polynucleotide sequence encoding Sou2 sorbitol utilization protein, and/or one or more polynucleotides encoding phospholipid:diacylglycerol acyltransferase (PDAT), delta-12 desaturase, a dihomo-gamma-linolenic acid (DGLA) synthase multizyme, delta-8 desaturase, malonyl-CoA synthetase (MCS), or acyl-CoA:lysophosphatidic acid acyltransferase (LPAAT). Also disclosed are methods of using the recombinant microbial cells to produce oil containing omega-3 polyunsaturated fatty acids such as EPA.

First claim

What is claimed is:

1. A recombinant Yarrowia cell that produces an oil comprising at least 28 percent eicosapentaenoic acid (EPA) measured as a weight percent of dry cell weight, wherein the cell comprises:
(a) at least one polynucleotide sequence encoding an acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 40 (YILPCAT), and
(b) the LPCAT comprises mutations at (i) amino acid position 136 changing methionine to a different amino acid, and (ii) amino acid position 389 changing threonine to a different amino acid,
wherein said LPCAT has LPCAT activity, and wherein the polynucleotide encoding LPCAT is operably linked to at least one regulatory sequence.

2. The recombinant Yarrowia cell of claim 1 , wherein the LPCAT is a Yarrowia lipolytica LPCAT corresponding to the sequence set forth in SEQ ID NO:40 and the LPCAT comprises mutations at (i) amino acid position 136 changing methionine to a different amino acid, and (ii) amino acid position 389 changing threonine to a different amino acid.

3. The recombinant microbial Yarrowia cell of claim 1 or claim 2 , wherein the cell further comprises a down-regulation of an endogenous polynucleotide sequence encoding Sou2 sorbitol utilization protein.

4. The recombinant Yarrowia cell of claim 3 , wherein said down-regulation is due to a mutation of said endogenous polynucleotide sequence, and wherein the mutation is selected from the group consisting of a substitution, deletion and insertion.

5. The recombinant Yarrowia cell of claim 4 , wherein the Sou2 sorbitol utilization protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:10.

6. The recombinant Yarrowia cell of claim 3 , wherein said down-regulation decreases the total amount of sugar alcohols produced by said cell.

7. The recombinant Yarrowia cell of claim 1 or claim 2 , wherein the cell further comprises:
(a) at least one polynucleotide sequence encoding phospholipid:diacylglycerol acyltransferase (PDAT),
(b) at least one polynucleotide sequence encoding delta-12 desaturase, and
(c) at least one polynucleotide sequence encoding a dihomo-gamma-linolenic acid (DGLA) synthase multizyme;
wherein each of the polynucleotide sequences of (a)-(c) is operably linked to at least one regulatory sequence.

8. The recombinant Yarrowia cell of claim 7 , wherein the DGLA synthase multizyme comprises a delta-9 elongase linked to a delta-8 desaturase.

9. The recombinant Yarrowia cell of claim 1 or claim 2 , wherein the cell further comprises:
(a) at least one polynucleotide sequence encoding delta-8 desaturase,
(b) at least one polynucleotide sequence encoding malonyl-CoA synthetase (MCS), and
(c) at least one polynucleotide sequence encoding acyl-CoA:lysophosphatidic acid acyltransferase (LPAAT)
wherein each of the polynucleotide sequences of (a)-(c) is operably linked to at least one regulatory sequence.

10. The recombinant Yarrowia cell of claim 1 or claim 2 , wherein the oil comprises at least 30 percent EPA measured as a weight percent of the dry cell weight of the cell.

11. A method of producing a microbial oil comprising eicosapentaenoic acid, said method comprising:
a) culturing the recombinant Yarrowia cell of claim 1 , wherein a microbial oil comprising eicosapentaenoic acid is produced; and
b) optionally recovering the microbial oil of step (a).

Assignees

• Du Pont

Inventors

• Zhu Quinn Qun
• Hong Seung-Pyo
• Sharpe Pamela L
• Yadav Narendra S
• Fan Xiaochun
• Dauner Michael

Classifications

• C12N1/16

  Yeasts; Culture media therefor · CPC title

• C12P7/6427 Primary

  Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone · CPC title

• C12P7/6472

  containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone · CPC title

• C12P7/6463

  obtained from glyceride producing microorganisms, e.g. single cell oil · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title