Methods of producing triacylglyceride compositions comprising tailored oils

• US 9,279,136 B2
• Filed: 09/28/2012
• Issued: 03/08/2016
• Est. Priority Date: 05/28/2010
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Franklin, Scott, Somanchi, Aravind, Wee, Janice, Rudenko, George, Moseley, Jeffrey L., Rakitsky, Walt
• Primary Examiner(s): Mondesi, Robert
• Assistant Examiner(s): Epstein, Todd M
• Application Number:
  US13/630,757
• Publication Number:
  US 20130102039A1
• Time in Patent Office: 1,257 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A23D 7/00 Edible oil or fat compositions containing an aqueous phase, e.g. margarines
• CPC Class Codes: A23D 9/00 Other edible oils or fats, e.g. shortenings, cooking oils
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: C11B 1/00 Production of fats or fatty oils from raw materials
• CPC Class Codes: C11C 3/00 Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom sulfonated fats or oils C07C309/62; factice C08H; drying oils C09F
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/2431 Beta-fructofuranosidase (3.2.1.26), i.e. invertase
• CPC Class Codes: C12N 9/2465 acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
• 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 7/6445 Glycerides
• CPC Class Codes: C12P 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: C12Y 114/19001 Stearoyl-CoA 9-desaturase (1.14.19.1), i.e. DELTA9-desaturase
• CPC Class Codes: C12Y 301/02 Thioester hydrolases (3.1.2)
• CPC Class Codes: C12Y 301/02014 Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase
• CPC Class Codes: C12Y 302/01022 Alpha-galactosidase (3.2.1.22)
• 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 30/20 : using bio-feedstock
• CPC Class Codes: Y02T 50/678 : Aviation using fuels of non-fossil origin

Abstract

Methods and compositions for the production of food compositions, oils, fuels, oleochemicals, and other compounds in recombinant microorganisms are provided, including oil-bearing microorganisms and methods of low cost cultivation of such microorganisms. Microalgal cells containing exogenous genes encoding, for example, a lipase, a sucrose transporter, a sucrose invertase, a fructokinase, a polysaccharide-degrading enzyme, a keto acyl-ACP synthase enzyme, a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, and/or an acyl carrier protein are useful in manufacturing food compositions, and transportation fuels such as renewable diesel, biodiesel, and renewable jet fuel, as well as oleochemicals such as functional fluids, surfactants, soaps and lubricants.

Claims

1. A method for producing a triacylglyceride composition comprising:

(a) cultivating at a first pH, in a seed production phase, a seed culture of a recombinant oleaginous microalgal cell comprising at least one acyl-ACP thioesterase gene under the control of a promoter of a Prototheca ammonium transporter gene; and

(b) seeding a lipid production phase with the seed culture to produce triacylglyceride, the lipid production phase comprising cultivation at a second pH.

2. A method according to claim 1, wherein the acyl-ACP thioesterase gene encodes an acyl-ACP thioesterase that preferentially liberates C10, C12, C14, or C16 acyl groups.

3. A method according to claim 1, wherein the acyl-ACP thioesterase gene is from Cinnamonum camphora, Umbellularia californica, Ulmus Americana, or Cuphea hookeriana.

4. A method according to claim 1, wherein the triacylglyceride composition is altered in its fatty acid profile as compared to a comparable organism lacking the gene.

5. A method according to claim 4, wherein the composition is enriched in C10, C12, C14, or C16 fatty acids.

6. A method according to claim 4, wherein the composition is enriched in saturated fatty acids.

7. A method according to claim 6, wherein the saturated fatty acids comprise at least 50% of the fatty acids of the triacylglyceride.

8. A method according to claim 6, wherein the saturated fatty acids comprise at least 80% of the fatty acids of the triacylglyceride.

9. A method according to claim 4, wherein the composition is reduced in unsaturated fatty acids.

10. A method according to claim 9, wherein the C18:

1 fatty acids are reduced by over 8 fold.

11. A method according to claim 1, wherein the gene further comprises a plastid transit peptide.

12. A method according to claim 1, wherein the gene is codon optimized.

13. A method according to claim 1, wherein the cell further comprises a transgene that encodes an active sucrose invertase so as to allow cultivation using sucrose as a sole carbon source.

14. A method according to claim 1, wherein the microalgal cell is of the genus Prototheca.

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

16. The method of claim 14, wherein the promoter of the endogenous ammonium transporter protein is selected from the group consisting of amt01, amt02 and amt03.

17. The method of claim 16, wherein the promoter is amt02.

18. The method of claim 16, wherein the promoter is amt03.

19. A method for producing a triacylglyceride composition comprising:

(a) cultivating at a first pH, in a seed production phase, a seed culture of a recombinant oleaginous microalgal cell comprising at least one acyl-ACP thioesterase gene under the control of a promoter of a Prototheca ammonium transporter gene; and

(b) seeding a lipid production phase with the seed culture to produce triacylglyceride, the lipid production phase comprising cultivation at a second pH, wherein the first pH is lower than the second pH.

20. The method of claim 19, wherein the first pH is 5.0 and/or the second pH is 7.0.

21. The method of claim 19, wherein the recombinant microalgal cell is of the genus Prototheca.

22. The method of claim 21, wherein the microalgal cell is Prototheca moriformis.