Enzyme directed oil biosynthesis in microalgae

• US 9,771,605 B2
• Filed: 06/22/2015
• Issued: 09/26/2017
• Est. Priority Date: 12/18/2008
• Status: Active Grant

• Current Assignee: The Board of Trustees of Michigan State University (Michigan State University)
• Original Assignee: The Board of Trustees of Michigan State University (Michigan State University)
• Inventors: Benning, Christoph, Miller, Rachel, Moellering, Eric R.
• Primary Examiner(s): O HARA, EILEEN B
• Application Number:
  US14/746,680
• Publication Number:
  US 20150376660A1
• Time in Patent Office: 827 Days
• Field of Search: None
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/6409 Fatty acids
• CPC Class Codes: C12P 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: C12Y 203/0102 Diacylglycerol O-acyltransferase (2.3.1.20)

Abstract

The present invention is related to biosynthetic oil compositions and methods of making thereof. In some embodiments, the invention relates to the use of endogenous enzymes in plants capable of synthesizing oil. In preferred embodiments, said plants are algae. In further embodiments, said algae are from the family Chlamydomonas, Nannochloropsis, Dunaliella, Chiarella and Scenedesmus. In still further embodiments, said endogenous enzymes are diacylglycerol acyltransferases.

Claims

1. A method comprising:

a) providing;

i) a host cell, and

ii) a vector comprising a nucleic acid encoding a diacylglycerol acyltransferase operably linked to a promoter, wherein said nucleic acid has greater than 90% sequence identity to a sequence is selected from SEQ ID) NO;

2 (DGTT1 cDNA), SEQ ID NO;

5 (DGTT2 cDNA), SEQ ID NO;

8 (DGTT3 cDNA), SEQ ID NO;

11 (DGTT4 cDNA), and SEQ ID NO;

14 (DGTT5 cDNA);

b) transfecting said host with said vector under conditions such that said diacylglycerol acyltransferase is expressed.

2. The method of claim 1, wherein said expression is under conditions such that triacylglycerol (TAG), free fatty acid and/or diacylglyerol (DAG) is produced.

3. The method of claim 2, further comprising c) recovering said TAG, said free fatty acid, and/or said DAG from said host and/or oil from said host.

4. The method of claim 1, wherein said expression is ectopic.

5. The method of claim 1, wherein said expression of said nucleic acid is upregulated between 1.5-3 told relative to wild type expression of said nucleic acid.

6. The method of claim 1, wherein said host is Lipomyces, Candida, Rhodotorula, Rhodosporidium, or Cryptococcus.

7. The method of claim 1, wherein said host is soybean, rutabaga, rapeseed, canola, sunflower, cotton, corn, cocoa, safflower, oil palm, coconut palm, flax, castor, or peanut.

8. A host transfected with a nucleic acid encoding a diacylglycerol acyltransferase operably linked to a promoter, wherein said nucleic acid has greater than 90% sequence identity to a sequence selected from SEQ ID NO:

2 (DGTT1 cDNA), SEQ ID NO;

5 (DCTT2 cDNA), SEQ ID NO;

8 (DGTT3 cDNA), SEQ ID NO;

11 (DGTT4 cDNA), and SEQ ID NO;

14 (DGIT5 cDNA).

9. The host of claim 8, wherein said promoter is a heterologous promoter.

10. The host of claim 8, wherein said promoter is an inducible promoter.

11. A tissue from the host of claim 8, wherein the tissue comprises roots, shoots, leaves, pollen, seeds, tumors, or cells.

12. The host of claim 8, wherein said host is soybean, rutabaga, rapeseed, canola, sunflower, cotton, corn, cocoa, safflower, oil palm, coconut palm, flax, castor, or peanut.

13. A non-algae plant tissue comprising a nucleic acid encoding an algae diacylglycerol acyltransferase operably linked to a promoter, wherein said nucleic acid has greater than 90% sequence identity to a sequence selected from SEQ ID NO:

2 (DGTT1 cDNA) or SEQ ID NO;

14 (DGTT5 cDNA).

14. The non-algae plant tissue of claim 13, selected from the group consisting of soybean (Glycine max), rutabaga (Brassica napohrassica), rapeseed (Brassica napus), canola, Brassica campestris, sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linum ushatissimum), castor (Ricinus communis) and peanut (Arachis hypogaea) plant tissue.

15. The non-algae plant tissue of claim 13, selected from the group consisting of roots, shoots, leaves, pollen, seeds, tumors, and cells.

16. A vector, comprising a nucleic acid encoding an algae diacylglycerol acyltransferase, wherein said nucleic acid is operably linked to a promoter, and wherein said nucleic acid has greater than 90% sequence identity to a sequence selected from SEQ ID NO:

2 (DGTT1 cDNA) or SEQ I) NO;

14 (DGTT5 cDNA).

17. The vector of claim 16, wherein said vector is a yeast shuttle vector.