# US 8,785,164 B2 · Expression of caleosin in recombinant oleaginous microorganisms to increase oil content therein

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- Number: US 8,785,164 B2
- Document ID: US8785164B2
- Status: Granted
- Kind: B2
- Application number: 13/478,522
- Filed: 2012-05-23
- Published: 2012-11-29
- Granted: 2014-07-22
- Earliest claimed: 2011-05-26
- Assignees: E. I. Du Pont de Nemours and Company
- Applicants: Seung-Pyo Hong; Quinn Qun Zhu
- Inventors: Seung-Pyo Hong; Quinn Qun Zhu
- Web page: https://patentgrep.com/patent/US8785164B2

## CPC

- C07K 14/37: from fungi
- C12P 7/6458: by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
- C12P 7/6463: obtained from glyceride producing microorganisms, e.g. single cell oil
- C12P 7/6472: containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Recombinant oleaginous microorganisms having increased oil content due to the expression of a caleosin polypeptide are described. A recombinant oleaginous microorganism of the disclosed invention produces at least 25% of its dry cell weight as oil, and comprises a functional polyunsaturated fatty acid (PUFA) biosynthetic pathway and at least one genetic construct encoding a caleosin polypeptide. A method for increasing the amount of oil in a recombinant oleaginous microorganism is also described.

## Claims

### Claim 1

A recombinant oleaginous Yarrowia lipolytica that produces at least 25% of its dry cell weight as oil, wherein said recombinant oleaginous Yarrowia lipolytica comprises a functional polyunsaturated fatty acid biosynthetic pathway and at least one genetic construct encoding a caleosin polypeptide comprising the amino acid sequence of SEQ ID NO:2;
further wherein said recombinant oleaginous Yarrowia lipolytica comprises at least 35% eicosapentaenoic acid as a weight percentage of total fatty acids of the Yarrowia lipolytica.

### Claim 2 (depends on 1)

The recombinant oleaginous microorganism of claim 1, wherein the caleosin polypeptide is linked to an enzyme that catalyzes acylation of diacylglycerol.

### Claim 3 (depends on 2)

The recombinant oleaginous microorganism of claim 2, wherein said enzyme is a phospholipid:diacylglycerol acyltransferase (PDAT).

### Claim 4 (depends on 1)

A method for increasing the amount of oil contained in a recombinant oleaginous Yarrowia lipolytica, said method comprising the steps of:
a) providing a recombinant oleaginous Yarrowia lipolytica according to claim 1;
b) growing the recombinant oleaginous Yarrowia lipolytica of step (a) under conditions whereby said oil is produced; and
c) optionally, recovering the oil of step (b).

### Claim 5 (depends on 1)

The recombinant oleaginous Yarrowia lipolytica of claim 1, wherein the Yarrowia lipolytica comprises at least 45% eicosapentaenoic acid as a weight percentage of the total fatty acids.

### Claim 6 (depends on 3)

The recombinant oleaginous Yarrowia lipolytica of claim 3, wherein the PDAT is Yarrowia lipolytica PDAT.

### Claim 7 (depends on 4)

The method of claim 4, wherein the Yarrowia lipolytica comprises at least 45% eicosapentaenoic acid as a weight percentage of the total fatty acids.