Structuring fats and methods of producing structuring fats

• US 10,287,613 B2
• Filed: 01/19/2018
• Issued: 05/14/2019
• Est. Priority Date: 04/18/2012
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Corbion Biotech, Inc. (Corbion NV)
• Inventors: Franklin, Scott, Somanchi, Aravind, Rudenko, George N., Bhat, Riyaz, Zhao, Xinhua, Bond, Risha, Rakitsky, Walter, Marangoni, Alejandro, Braksmayer, Diza
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US15/875,984
• Publication Number:
  US 20180237811A1
• Time in Patent Office: 480 Days
• Field of Search: 435189, 435193, 4352573, 435134, 435195, 435232
• 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. A method of producing a triglyceride oil, the method comprising:

cultivating a recombinant microalgal cell expressing an exogenous polynucleotide encoding a ketoacyl-ACP synthase; and

extracting the oil produced by the cell;

wherein the triglyceride oil is enriched in stearic-oleic-stearic triglyceride as compared to a non-recombinant microalgal cell.

2. The method of claim 1, wherein the microalgal cell is a cell of the genus Prototheca or Chlorella.

3. The method of claim 2, wherein the microalgal cell is a Prototheca moriformis cell.

4. The method of claim 1, wherein the recombinant microalgal cell further comprises an exogenous polynucleotide that encodes a fatty acyl-ACP thioesterase.

5. The method claim 1, wherein the recombinant microalgal cell further comprises nucleic acids operable to reduce the expression of stearoyl-ACP desaturase or a fatty acyl desaturase.

6. The method of claim 5, wherein the nucleic acids operable to reduce the expression of a stearoyl-ACP desaturase or a fatty acyl desaturase is a nucleic acid that encodes an RNA construct that reduces the activity of stearoyl-ACP desaturase or the fatty acyl desaturase.

7. The method of claim 1, 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.

8. The method of claim 7, wherein the sum of palmitic-oleic-palmitic, stearic-oleic-stearic and palmitic-oleic-stearic is at least 50% of the triglycerides.

9. The method of claim 8, wherein the oil comprises triglycerides comprising at least 50% stearic-oleic-stearic, and optionally less than 10% stearic-stearic-stearic.

10. The method of claim 1, wherein the recombinant microalgal cell further comprises an exogenous sucrose invertase gene so that the cell can grow on sucrose as a sole carbon source.

11. A method of producing a triglyceride oil, the method comprising:

cultivating a recombinant microalgal cell expressing an exogenous polynucleotide encoding a ketoacyl-ACP synthase and a fatty acyl-ACP thioesterase; and

extracting the oil produced by the cell;

wherein the triglyceride oil is enriched in stearic-oleic-stearic triglyceride as compared to a non-recombinant microalgal cell.

12. The method of claim 11, wherein the microalgal cell is a cell of the genus Prototheca or Chlorella.

13. The method of claim 12, wherein the microalgal cell is a cell of the genus Prototheca.

14. The method of claim 13, wherein the microalgal cell is a Prototheca moriformis cell.

15. The method claim 11, wherein the recombinant microalgal cell further comprises nucleic acids operable to reduce the expression of stearoyl-ACP desaturase or a fatty acyl desaturase.

16. The method of claim 11, wherein the nucleic acids operable to reduce the expression of a stearoyl-ACP desaturase or a fatty acyl desaturase is a nucleic acid that encodes an RNA construct that reduces the activity of stearoyl-ACP desaturase or the fatty acyl desaturase.

17. The method of claim 11, 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.

18. The method of claim 17, wherein the sum of palmitic-oleic-palmitic, stearic-oleic-stearic and palmitic-oleic-stearic is at least 50% of the triglycerides.

19. The method of claim 18, wherein the oil comprises triglycerides comprising at least 50% stearic-oleic-stearic, and optionally less than 10% stearic-stearic-stearic.

20. The method of claim 11, wherein the recombinant microalgal cell further comprises an exogenous sucrose invertase gene so that the cell can grow on sucrose as a sole carbon source.