Tailored oils

• US 8,945,908 B2
• Filed: 04/18/2013
• Issued: 02/03/2015
• Est. Priority Date: 04/18/2012
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Franklin, Scott, Somanchi, Aravind, Rudenko, George, Bhat, Riyaz, Zhao, Xinhua, Bond, Risha, Rakitsky, Walter, Marangoni, Alejandro, Braksmayer, Diza
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US13/865,974
• Publication Number:
  US 20130316410A1
• Time in Patent Office: 656 Days
• Field of Search: 435/134, 435/257.2
• US Class Current: 435/257.2
• CPC Class Codes: A23D 9/00 Other edible oils or fats, e.g. shortenings, cooking oils
• CPC Class Codes: A23D 9/013 Other fatty acid esters, e.g. phosphatides
• CPC Class Codes: C07C 57/03 Monocarboxylic acids
• CPC Class Codes: C07J 9/00 Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
• CPC Class Codes: C11B 1/00 Production of fats or fatty oils from raw materials
• CPC Class Codes: C12N 1/125 Unicellular algae isolates
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• 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 33/00 Preparation of steroids
• 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 7/6445 Glycerides
• 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 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12R 2001/89 : Algae ; Processes using algae
• CPC Class Codes: C12Y 203/01015 : Glycerol-3-phosphate O-acyltransferase (2.3.1.15)
• CPC Class Codes: C12Y 203/0102 : Diacylglycerol O-acyltransferase (2.3.1.20)
• CPC Class Codes: C12Y 203/01041 : Beta-ketoacyl-acyl-carrier-protein synthase I (2.3.1.41)
• CPC Class Codes: C12Y 203/01051 : 1-Acylglycerol-3-phosphate O-acyltransferase (2.3.1.51)
• CPC Class Codes: C12Y 203/01179 : Beta-ketoacyl-acyl-carrier-protein synthase II (2.3.1.179)
• CPC Class Codes: C12Y 203/0118 : Beta-ketoacyl-acyl-carrier-protein synthase III (2.3.1.180)
• CPC Class Codes: C12Y 302/01014 : Chitinase (3.2.1.14)
• 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

Abstract

Recombinant DNA techniques are used to produce oleaginous recombinant cells that produce triglyceride oils having desired fatty acid profiles and regiospecific or stereospecific profiles. Genes manipulated include those encoding stearoyl-ACP desturase, delta 12 fatty acid desaturase, acyl-ACP thioesterase, ketoacyl-ACP synthase, and lysophosphatidic acid acyltransferase. The oil produced can have enhanced oxidative or thermal stability, or can be useful as a frying oil, shortening, roll-in shortening, tempering fat, cocoa butter replacement, as a lubricant, or as a feedstock for various chemical processes. The fatty acid profile can be enriched in midchain profiles or the oil can be enriched in triglycerides of the saturated-unsaturated-saturated type.

Claims

1. An oleaginous microalgal cell of genus Prototheca, the cell comprising an active exogenous KASII (ketoacyl ACP synthase) gene encoding a protein having at least 95% sequence identity to the protein encoded by nucleotides 4039 to 5328 of SEQ ID NO:

95 and producing an oil comprising triglycerides, wherein the oil is enriched in stearic-oleic-stearic.

2. The cell of claim 1, further comprising an exogenous gene that encodes an active fatty acyl-ACP thioesterase.

3. The cell of claim 1, wherein the cell further comprises nucleic acids operable to reduce the expression of a stearoyl-ACP desaturase.

4. The cell of claim 3, wherein the oil is enriched in triglycerides of the saturated-unsaturated-saturated type, wherein the sum of palmitic-oleic-palmitic, stearic-oleic-stearic and palmitic-oleic-stearic is at least 30% of the triglycerides.

5. The cell of claim 4, wherein the sum of palmitic-oleic-palmitic, stearic-oleic-stearic and palmitic-oleic-stearic is at least 50% of the triglycerides.

6. The cell of claim 5, wherein the oil comprises triglycerides comprising at least 50% stearic-oleic-stearic, and optionally less than 10% stearic-stearic-stearic.

7. The cell of claim 1, further comprising a knockout or knockdown of a stearoyl-ACP desaturase gene, a FatA gene, or both.

8. The cell of claim 4, wherein the oil comprises POP at 16%±

20%, POS at 38%±

20%, and SOS at 23%±

20%.

9. The cell of claim 7, wherein the cell further comprises nucleic acids operable to reduce the expression of a delta 12 fatty acid desaturase, optionally via a regulatable promoter, so as to produce an oil with linoleic and linolenic acids totaling 5 area percent or less.

10. The cell of claim 4, wherein the oil has greater than 65% stearic-oleic-stearic, less than 45% unsaturated fatty acid, less than 5% polyunsaturated fatty acids, less than 1% lauric acid, and less than 2% trans fatty acids.

11. A method for producing an oil comprising providing or cultivating a cell according to claim 1, and extracting the oil.

12. The cell of claim 1, wherein the cell is an obligately heterotrophic cell.

13. The cell of claim 1, comprising an exogenous sucrose invertase gene so that the cell can grow on sucrose as a sole carbon source.

14. The cell of claim 3, wherein the nucleic acids operable to reduce the expression of a stearoyl-ACP desaturase disrupt one or both alleles of the endogenous stearoyl-ACP desaturase coding sequences.

15. The cell of claim 3, wherein the nucleic acids operable to reduce the expression of a stearoyl-ACP desaturase is a nucleic acid that encodes an RNA construct that reduces the activity of stearoyl-ACP desaturase.

16. An oleaginous microalgal cell of genus Prototheca, the cell comprising an exogenous gene encoding an active KAS II (ketoacyl ACP synthase) enzyme that has at least 95% sequence identity to the protein encoded by nucleotides 4039 to 5328 of SEQ ID NO:

95 and nucleic acids operable to reduce the expression of a stearoyl-ACP desaturase, the cell producing an oil comprising triglycerides, wherein the oil is enriched in triglycerides of the saturated-unsaturated-saturated type.

17. The cell of claim 16, wherein the palmitic-oleic-palmitic, stearic-oleic-stearic and palmitic-oleic-stearic in the oil comprise at least 30% in sum.

18. The cell of claim 16, wherein the palmitic-oleic-palmitic, stearic-oleic-stearic and palmitic-oleic-stearic in the oil comprise at least 50% in sum.

19. The cell of claim 16, wherein the stearic-oleic-stearic in the oil comprise at least 40%.

20. The cell of claim 16, wherein the stearic-oleic-stearic in the oil comprise at least 50%.