Method to produce acetyldiacylglycerols (ac-TAGs) by expression of an acetyltransferase gene isolated from;(burning bush)

• US 9,328,335 B2
• Filed: 12/29/2010
• Issued: 05/03/2016
• Est. Priority Date: 12/30/2009
• 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: Durrett, Timothy, Ohlrogge, John, Pollard, Michael
• Primary Examiner(s): Ibrahim, Medina A
• Assistant Examiner(s): Zhong, Wayne
• Application Number:
  US13/519,660
• Publication Number:
  US 20130116462A1
• Time in Patent Office: 1,952 Days
• Field of Search: None
• US Class Current: 1/1
• 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 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: Y02T 50/678 Aviation using fuels of non-fossil origin

Abstract

The present invention relates to novel diacylglycerol acyltransferase genes and proteins, and methods of their use. In particular, the invention describes genes encoding proteins having diacylglycerol acetyltransferase activity, specifically for transferring an acetyl group to a diacylglycerol substrate to form acetyl-Triacylglycerols (ac-TAGS), for example, a 3-acetyl-1,2-diacyl-sn-glycerol. The present invention encompasses both native and recombinant wild-type forms of the transferase, as well as mutants and variant forms. The present invention also relates to methods of using novel diacylglycerol acyltransferase genes and proteins, including their expression in transgenic organisms at commercially viable levels, for increasing production of 3-acetyl-1,2-diacyl-sn-glycerols in plant oils and altering the composition of oils produced by microorganisms, such as yeast, by increasing ac-TAG production. Additionally, oils produced by methods of the present inventions comprising genes and proteins are contemplated for use as biodiesel fuel, in polymer production and as naturally produced food oils with reduced calories.

Claims

1. An isolated nucleic acid encoding a protein operably linked to a heterologous promoter, wherein said protein is at least 95% identical to SEQ ID NO:

01.

2. The nucleic acid of claim 1, wherein said protein is capable of acetylating a diacylglycerol substrate comprising a fatty acid to form an acetyltriacylglycerol.

3. The nucleic acid claim 2, wherein said fatty acid is Oleate.

4. The nucleic acid of claim 2, wherein said diacylglycerol substrate is 1,2-dioleoyl-glycerol.

5. The nucleic acid of claim 2, wherein said protein is capable of acylating said diacylglycerol substrate with an acyl-coenzyme A substrate.

6. The nucleic acid of claim 5, wherein said acyl-coenzyme A substrate is a short chain acyl-coenzyme A.

7. The nucleic acid of claim 5, wherein said acyl-coenzyme A substrate is propionyl-coenzyme A.

8. The nucleic acid claim 1, wherein said protein is SEQ ID NO:

01.

9. The nucleic acid of claim 1, wherein said protein is from a plant selected from the group of Celastraceae, Lardizabalaceae, Ranunculaceae, Rosaceae, and Vitaceae.

10. The nucleic acid of claim 1, wherein said protein is from a plant selected from the group of Euonymus, Maytenus, Akebia, Adonis, Sorbus and Vitis species.

11. The nucleic acid of claim 1, wherein said protein is from an Euonymus alata plant.

12. The nucleic acid sequence of claim 1, wherein said heterologous promoter is a seed specific promoter.

13. A vector comprising the nucleic acid of claim 1.

14. A host cell comprising the vector of claim 13.

15. The host cell of claim 14, wherein said host cell is selected from the group consisting of a plant cell and a microorganism.

16. The host cell of claim 15, wherein said plant cell is selected from the group consisting of an edible crop plant cell, an oil seed crop plant cell, a seed cell, a pollen cell, an ovule cell, mesenchymal cell, meristem cell, an endosperm cell, a male reproductive cell, a female reproductive cell, and an embryo cell.

17. The host cell of claim 15, wherein said plant cell is selected from the group consisting of a Jatropha plant, an oil crop plant, a palm oil plant, and an alga.

18. The host cell of claim 15, wherein said plant cell is selected from the group consisting of Brassica plants and Brassicaceae plants.

19. The host cell of claim 15, wherein said plant cell is selected from the group consisting of Arabidopsis plants, Camelina plants, and crambe plants.

20. The host cell of claim 15, wherein said microorganism is a fungus cell.

21. The host cell of claim 20, wherein said fungus cell is a yeast cell.

22. The host cell of claim 14, wherein said host cell has lower long chain-triacylglycerol production.

23. The host cell of claim 22, wherein said host cell further comprises at least one mutant fatty acid elongase I gene, wherein said mutation results in lowered long chain-triacylglycerol production in host cell.

24. The host cell of claim 22, wherein said host cell further comprises at least one silenced triacylglycerol synthesis gene, wherein said gene is silenced due to a mutation which results in lowered expression of said gene.

25. The host cell of claim 24, wherein said silenced triacylglycerol synthesis gene is diacylglycerol acyltransferase 1.

26. A seed, wherein said seed comprises a heterologous plant nucleic acid encoding a protein that is at least 95% identical to SEQ ID NO:

01.

27. A composition comprising a host cell, wherein said host cell comprises a heterologous plant nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO:

01.

28. The composition of claim 27, wherein said host cell is an oil seed plant cell, a mesenchymal cell, meristem cell, an endosperm cell, a pollen cell, a male reproductive cell, a female reproductive cell, and an embryo cell.

29. The composition of claim 27, further comprising acetyltriacylglycerol.

30. The composition of claim 29, wherein said acetyltriacylglycerol comprises a 3-acetyl-1, 2-diacyl-sn-glycerol.