Tailored oils produced from recombinant heterotrophic microorganisms

• US 8,765,424 B2
• Filed: 07/06/2012
• Issued: 07/01/2014
• 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): KAM, CHIH MIN
• Application Number:
  US13/543,666
• Publication Number:
  US 20120324784A1
• Time in Patent Office: 725 Days
• Field of Search: 435/134, 435/41, 435/252.3, 435/254.2, 435/257.2, 554/227, 443/08
• US Class Current: 435/134
• 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 7/6458 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: 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 oil, 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 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 microbial oil obtained from a recombinant microalga, wherein the fatty acid profile of the oil comprises at least 40% saturated fatty acid having C10, C12 and C14, and the oil further comprises sterols of the recombinant microalga, wherein the recombinant microalga has been genetically engineered to express at least one lipid pathway enzyme at an altered level compared to a non-engineered microalga, the lipid pathway enzyme being selected from the group consisting of a desaturase, a ketoacyl synthase, and an acyl-ACP thioesterase.

2. The microbial oil of claim 1, wherein the at least one lipid pathway enzyme is an endogenous lipid pathway enzyme that is expressed at a lower level compared to the non-engineered microalga.

3. The microbial oil of claim 2, wherein the at least one endogenous lipid pathway enzyme is a desaturase.

4. The microbial oil of claim 3, wherein the at least one endogenous lipid pathway enzyme is fatty acyl desaturase enzyme.

5. The microbial oil of claim 3, wherein the at least one endogenous lipid pathway enzyme is a stearoyl ACP desaturase-enzyme.

6. The microbial oil of claim 2, wherein the at least oneendogenous lipid pathway enzyme is an acyl-ACP thioesterase.

7. The microbial oil of claim 2, wherein the at least one endogenous lipid pathway enzyme is a ketoacyl synthase.

8. The microbial oil of claim 7, wherein the at least one endogenous lipid pathway enzyme is a ketoacyl synthase II enzyme.

9. The microbial oil of claim 7, wherein the at least one endogenous lipid pathway enzyme is a ketoacyl synthase I enzyme.

10. The microbial oil of claim 7, wherein the at least one endogenous lipid pathway enzyme is a ketoacyl synthase IV enzyme.

11. The microbial oil of claim 1, wherein the at least one lipid pathway enzyme is expressed at a higher level compared to the non-engineered microalga.

12. The microbial oil of claim 11, wherein the at least one lipid pathway enzyme is a ketoacyl synthase.

13. The microbial oil of claim 1, wherein the fatty acid profile of the oil has not been altered by further processing the microbial oil by one or more of esterification, distillation, fractionation, crystallization, and precipitation.

14. The microbial oil of claim 1, wherein the fatty acid profile of the oil comprises at least 70% saturated fatty acid.

15. The microbial oil of claim 1, wherein the fatty acid profile of the oil comprises at least 4% C10.

16. The microbial oil of claim 1, wherein the fatty acid profile of the oil comprises at least 10% C12.

17. The microbial oil of claim 1, wherein the fatty acid profile of the oil comprises at least 10% C14.

18. The microbial oil of claim 1, wherein the fatty acid profile of the oil comprises at least 40% C14.

19. The microbial oil of claim 1, wherein the fatty acid profile of the comprises at least 50% total combined amounts of C10:

0, C12;

0 and C14;

0.

20. The microbial oil of claim 1, wherein the microbial oil is Prototheca oil or Chlorella oil.

21. The microbial oil of claim 20, wherein the microbial oil is Prototheca moriformis oil or Chlorella protothecoides oil.

22. A blended composition comprising a microbial oil of claim 1 and an oil from soy, rapeseed, canola, palm, palm kernel, coconut, corn, waste vegetable, Chinese tallow, olive, sunflower, cottonseed, chicken fat, beef tallow, porcine tallow, microalgae, macroalgae, microbes, Cuphea, flax, peanut, choice white grease, lard, Camelina sativa, mustard seed, cashew nut, oats, lupine, kenaf, calendula, help, coffee, linseed (flax), hazelnut, euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice, tung tree, cocoa, copra, opium poppy, castor beans, pecan, jojoba, macadamia, Brazil nuts, avocado, petroleum, or a distillate fraction of any of the preceding oils.

23. The oil of claim 1, wherein the oil has a C12:

C14 fatty acid ratio of at least 5;

1.

24. The oil of claim 1, wherein the oil has a C12-C14 level of over 49%.

25. The microbial oil of claim 1, wherein the fatty acid profile of the oil comprises at least 70% total combined amounts of C10:

0, C12;

0 and C14;

0.