Recombinant microalgae cells producing novel oils

• US 8,187,860 B2
• Filed: 03/28/2011
• Issued: 05/29/2012
• Est. Priority Date: 11/28/2008
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Franklin, Scott, Somanchi, Aravind, Espina, Karen, Rudenko, George, Chua, Penelope
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US13/073,757
• Publication Number:
  US 20110250658A1
• Time in Patent Office: 428 Days
• Field of Search: 435/257.1, 435/196, 435/197, 435/134, 435/69.1, 536/23.1, 536/23.2
• US Class Current: 435/257.1
• CPC Class Codes: C07C 1/2078 by a transformation in which at least one -C(=O)-O- moiety is eliminated
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C10L 1/02 essentially based on components consisting of carbon, hydrogen, and oxygen only
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• 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 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12P 7/20 Glycerol
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• CPC Class Codes: C12P 7/6409 Fatty acids
• CPC Class Codes: C12P 7/6418 by hydrolysis of fatty acid esters
• 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 7/6458 by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
• CPC Class Codes: C12P 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: C12P 7/649 Biodiesel, i.e. fatty acid alkyl esters
• CPC Class Codes: C12Y 301/02014 Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase
• CPC Class Codes: C12Y 302/01026 Beta-fructofuranosidase (3.2.1.26), i.e. invertase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts
• CPC Class Codes: Y02P 60/87 Re-use of by-products of food processing for fodder production
• CPC Class Codes: Y02T 50/678 : Aviation using fuels of non-fossil origin

Abstract

Disclosed herein are obligate heterotrophic microalgae cells containing an exogenous gene. In some embodiments the gene is a sucrose utilization gene, and further disclosed are methods of manufacturing triglyceride oils using sugar cane or sugar beets as a feedstock in a heterotrophic fermentation. In other embodiments the feedstock is depolymerized cellulosic material. Also disclosed are cells that produce medium chain fatty acids at levels not produced in non-recombinant cells of the same species and genus.

Claims

1. A cell of the genus Prototheca comprising an exogenous gene encoding a fatty acid desaturase, wherein the exogenous gene is integrated into the nuclear genome of the cell and is expressed thereby modifying the saturation of fatty acids in the cell.

2. The cell of claim 1, wherein the fatty acid desaturase is selected from the group consisting of ω

-6 fatty acid desaturase, ω

-3 fatty acid desaturase, ω

-6 oleate desaturase and stearoyl ACP desaturase.

3. The cell of claim 1, wherein the coding sequence of the exogenous gene is in operable linkage with a promoter from a gene endogenous to a species of the genus Prototheca.

4. The cell of claim 1, further comprising one or more additional exogenous genes, wherein the one or more additional exogenous genes are selected from the group consisting of a sucrose invertase gene, a fatty acyl-ACP thioesterase gene, a fatty acyl-CoA/aldehyde reductase gene, a fatty acyl-CoA reductase gene and a fatty aldehyde decarbonylase gene and integrated into a nuclear genome of the cell.

5. The cell of claim 4 wherein the cell is Prototheca moriformis, Prototheca krugani, Prototheca stagnora, or Prototheca zopfii.

6. The cell of claim 4, wherein the one or more additional exogenous genes are selected from the group consisting of a sucrose invertase gene and a fatty acyl-ACP thioesterase gene.

7. The cell of claim 6, wherein the additional exogenous gene is a fatty acyl-ACP thioesterase gene that encodes a fatty acyl-ACP thioesterase that has hydrolysis activity towards one or more fatty acyl-ACP substrates of chain length C8, C10, C12 or C14.

8. The cell of claim 4, wherein the additional exogenous gene is a fatty acyl-ACP thioesterase gene that encodes a fatty acyl-ACP thioesterase that has hydrolysis activity towards one or more fatty acyl-ACP substrates of chain length C8, C10, C12 or C14.

9. The cell of claim 4, wherein the cell is Prototheca moriformis.

10. The cell of claim 1 wherein the cell is Prototheca moriformis, Prototheca krugani, Prototheca stagnora, or Prototheca zopfii.

11. A method of making a triglyceride composition comprising cultivating the cell of claim 1 under heterotrophic conditions suitable for triglyceride production, thereby producing triglyceride in the cell.

12. The cell of claim 1, wherein the cell is Prototheca moriformis.

13. A cell of the genus Prototheca comprising an exogenous gene that is integrated into a nuclear chromosome of the cell, wherein the integration of the exogenous gene ablates the expression of an endogenous fatty acid desaturase gene thereby modifying the saturation of fatty acids in the cell.

14. The cell of claim 13, wherein the endogenous fatty acid desaturase is a stearoyl ACP desaturase.

15. The cell of claim 13, wherein the exogenous gene encodes a fatty acid desaturase selected from the group consisting of ω

-6 fatty acid desaturase, ω

-3 fatty acid desaturase, ω

-6 oleate desaturase and stearoyl ACP desaturase, and wherein the expressed exogenous gene modifies the saturation of fatty acids in the cell.

16. The cell of claim 13, wherein the exogenous gene encodes for a selectable marker.

17. The cell of claim 16, wherein the selectable marker is a sucrose invertase.

18. The cell of claim 13 further comprising one or more additional exogenous gene wherein the one or more additional exogenous gene is selected from the group consisting of a sucrose invertase gene, a fatty acyl-ACP thioesterase gene, a fatty acyl-CoA/aldehyde reductase gene, a fatty acyl-CoA reductase gene or a fatty aldehyde decarbonylase gene and is integrated into a nuclear genome of the cell.

19. The cell of claim 18, wherein the one or more exogenous gene is a fatty acyl-ACP thioesterase gene that encodes a fatty acyl-ACP thioesterase that has hydrolysis activity towards one or more fatty acyl-ACP substrates of chain length C8, C10, C12 or C14.