Tailored oils

• US 9,102,973 B2
• Filed: 07/12/2013
• Issued: 08/11/2015
• 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): Prouty, Rebecca
• Assistant Examiner(s): EPSTEIN, TODD MATTHEW
• Application Number:
  US13/941,357
• Publication Number:
  US 20130317240A1
• Time in Patent Office: 760 Days
• Field of Search: None
• US Class Current: 1/1
• 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 comprising:

(a) providing a recombinant oleaginous microalgal cell of the class Trebouxiophyte, the cell comprising an exogenous delta-12 fatty acid desaturase gene under control of an amt03 promoter and an allelic disruption of an endogenous delta-12 fatty acid desaturase gene;

(b) cultivating the cell under a first environmental condition wherein the exogenous delta-12 fatty acid desaturase gene is expressed, linoleic acid is produced and the cell divides, increasing the number of cells;

(c) cultivating the cells under a second environmental condition of reduced pH relative to the first environmental condition wherein expression of the exogenous delta-12 fatty acid desaturase gene and consequently production of linoleic acid is reduced and oil accumulates in the cells; and

(d) extracting the oil from the cells, wherein the oil is characterized by a polyunsaturated fatty acid profile with 5% linoleic acid or less.

2. The method of claim 1, wherein the cell comprises 23S rRNA having at least 65% nucleotide sequence identity to SEQ ID NO:

76.

3. The method of claim 1, wherein the cell is an obligately heterotrophic microalga.

4. The method of claim 3, wherein the cell is of the genus Prototheca.

5. The method of claim 1, wherein the linoleic acid is reduced in the oil by at least 50%.

6. The method of claim 1, wherein the cell is cultivated heterotrophically.

7. The method of claim 1, wherein the cell produces at least 40% oil by dry cell weight.

8. The method of claim 1, wherein, when extracted from the cell, the oil is stable at 110°

C. so that the inflection point in conductance is not yet reached by 20 hours under conditions of the AOCS Cd 12b-92 Rancimat test.

9. The method of claim 1, wherein, when extracted from the cell, the oil is stable at 110°

C. so that the inflection point in conductance is not yet reached by 5 days under conditions of the AOCS Cd 12b-92 Rancimat test, when 1050 ppm of tocopherol and 500 pm of ascorbyl palmitate are added to the oil.

10. The method of claim 1, wherein the cell comprises an exogenous gene encoding a KAS II enzyme.

11. The method of claim 10, wherein the cell further comprises a knockout or knockdown of a FatA gene.

12. The method of claim 1, wherein the oil is characterized by a fatty acid profile with greater than 60% oleic acid and less than 3% polyunsaturates.

13. The method of claim 1, wherein the oil is characterized by a fatty acid profile with greater than 70% oleic acid and less than 2% polyunsaturates.

14. The method of claim 1, wherein the oil is characterized by a fatty acid profile with greater than 80% oleic acid and less than 1% polyunsaturates.

15. The method of claim 1, wherein the oil is characterized by a fatty acid profile with less 3% polyunsaturates or less.

16. The method of claim 1, wherein the oil is characterized by a fatty acid profile with less than 2% polyunsaturates or less.

17. The method of claim 1, wherein the oil is characterized by a fatty acid profile with less than 1% polyunsaturates or less.

18. The method of claim 1, further comprising frying a food in the oil.

19. The method of claim 1, wherein the cell division rate is decreased by more than 40% under the second environmental condition.