Recombinant microalgae cells producing novel oils

• US 8,697,427 B2
• Filed: 05/23/2012
• Issued: 04/15/2014
• 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:
  US13/479,200
• Publication Number:
  US 20120277453A1
• Time in Patent Office: 692 Days
• Field of Search: 554/225, 435/134, 435/257.1, 435/69.1, 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 method of producing an oil, the method comprising:

heterotrophically cultivating a microalgal cell of the genus Prototheca, the cell comprising an exogenous gene encoding a fatty acid desaturase, wherein the exogenous gene is integrated into the nuclear chromosome of the microalgal cell and is expressed, so as to alter the proportion of unsaturated to saturated fatty acids in an oil produced by the cell; and

extracting the oil produced by the cell.

2. The method of claim 1, wherein the fatty acid desaturase is a ω

-6 fatty acid desaturase.

3. The method of claim 1, wherein the fatty acid desaturase is a ω

-3 fatty acid desaturase.

4. The method of claim 1, wherein the fatty acid desaturase is a stearoyl-ACP desaturase.

5. The method 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.

6. The method of claim 1, wherein the chromosome comprises a sucrose invertase gene.

7. The method of claim 1, wherein the chromosome comprises a fatty acyl-ACP thioesterase gene.

8. The method of claim 7, wherein the fatty acyl-ACP thioesterase gene 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 method of claim 8, wherein the oil has a fatty acid profile of at least 30% C8-C14.

10. The method of claim 1, wherein the chromosome comprises one or more of a sucrose invertase gene and a fatty acyl-ACP thioesterase gene.

11. The method of claim 10, wherein the fatty acyl-ACP thioesterase gene 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.

12. The method of claim 11, wherein the oil has a fatty acid profile of at least 30% C8-C14.

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

14. The method of claim 1, wherein the microalgal cell is of Prototheca moriformis.

15. The method of claim 1, wherein the microalgal cell has a 23s rRNA sequence with at least 75% nucleotide identity to SEQ ID NO:

15.

16. A method of producing an oil, the method comprising:

heterotrophically cultivating a microalgal cell of a species having 23S rRNA with at least 75% nucleotide identity to SEQ ID NO;

15, the cell comprising an exogenous gene, wherein the integration of the exogenous gene ablates the expression of an endogenous fatty acid desaturase gene so as to alter the proportion of unsaturated to saturated fatty acids in an oil produced by the cell; and

extracting the oil produced by the cell.

17. The method of claim 16, wherein the microalgal cell is of a species having 23S rRNA with at least 85% nucleotide identity to SEQ ID NO:

15.

18. The method of claim 16, wherein the microalgal cell is of a species having 23S rRNA with at least 95% nucleotide identity to SEQ ID NO:

15.

19. The method of claim 16, wherein the cell is a cell of the genus Prototheca.

20. A method of producing an oil, the method comprising:

heterotrophically cultivating a microalgal cell of a species having 23S rRNA with at least 75% nucleotide identity to SEQ ID NO;

15, the cell comprising an exogenous gene, wherein the integration of the exogenous gene ablates the expression of an endogenous stearoyl ACP desaturase gene so as to cause the cell to produce an oil that is reduced in unsaturated fatty acids.