Recombinant microalgae including keto-acyl ACP synthase

• US 10,006,034 B2
• Filed: 08/05/2015
• Issued: 06/26/2018
• Est. Priority Date: 05/28/2010
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Corbion Biotech, Inc. (Corbion NV)
• Inventors: Franklin, Scott, Somanchi, Aravind, Wee, Janice, Rudenko, George, Moseley, Jeffrey L., Rakitsky, Walt
• Primary Examiner(s): Holland, Paul J
• Application Number:
  US14/819,117
• Publication Number:
  US 20160024538A1
• Time in Patent Office: 1,056 Days
• Field of Search: None
• 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 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 process for producing a triglyceride oil from a microalgae, the process comprising:

(i) cultivating a recombinant oleaginous microalgal cell comprising exogenous nucleic acids encoding a KAS (keto-acyl ACP synthase) or wherein nucleic acids encoding an endogenous KAS are disrupted by homologous recombination to produce said triglyceride oil; and

(ii) isolating said triglyceride oil from said recombinant oleaginous microalgal cell,

wherein said triglyceride oil has a fatty acid profile that is at least 40% saturated fatty acids, and said fatty acid profile of said triglyceride oil comprises at least;

10% C8;

0, 20% C10;

0, 50% C12;

0, 40% C14;

0, 60% C16;

0, or 30% C18;

0.

2. The process of claim 1, wherein said exogenous nucleic acids encoding said KAS encode a KAS I enzyme, a KAS II enzyme, or a KAS IV enzyme.

3. The process of claim 1, wherein said recombinant oleaginous microalgal cell further comprises nucleic acids encoding a FatB acyl-ACP thioesterase.

4. The process of claim 3, wherein said exogenous nucleic acids encoding said FatB acyl-ACP thioesterase are from a plant of the genus Cuphea, Ulmus, or Cinnamomum.

5. The process of claim 1, wherein nucleic acids encoding an endogenous desaturase of said recombinant oleaginous microalgal cell have been disrupted.

6. The process of claim 5, wherein exogenous nucleic acids encoding a FatB acyl-ACP thioesterase disrupt nucleic acids encoding an endogenous desaturase.

7. The process of claim 6, wherein said exogenous nucleic acids encoding said FatB acyl-ACP thioesterase are from a plant of the genus Cuphea.

8. The process of claim 1, wherein said triglyceride oil has a fatty acid profile that is at least 55% saturated fatty acids.

9. The process of claim 1, wherein said triglyceride oil has a fatty acid profile that is at least 65% saturated fatty acids.

10. The process of claim 1, wherein said triglyceride oil has a fatty acid profile that is at least 80% saturated fatty acids.

11. The process of claim 1, wherein said triglyceride oil is a cocoa butter mimetic having percentages of C16:

0, C18;

0 and C18;

1 fatty acid constituents within 10% of the percentages, respectively, of cocoa butter.

12. The process of claim 1, wherein said recombinant oleaginous microalgal cell is of the genus Prototheca.

13. The process of claim 12, wherein said recombinant oleaginous microalgal cell is a species of Prototheca selected from Prototheca moriformis, Prototheca krugani, Prototheca stagnora, Prototheca wickerhamii, or Prototheca zopfii.

14. The process of claim 13, wherein said recombinant oleaginous microalgal cell is Prototheca moriformis.

15. The process of claim 1, wherein said recombinant oleaginous microalgal cell is of the genus Chlorella.

16. The process of claim 15, wherein said recombinant oleaginous microalgal cell is Chlorella prothecoides.

17. The process of claim 2, wherein said exogenous nucleic acids encoding said KAS encodes KAS II.

18. A process for producing a triglyceride oil from a microalgal cell of the genus Prototheca, the process comprising:

(i) cultivating a recombinant oleaginous Prototheca cell comprising exogenous nucleic acids encoding a KAS (keto-acyl ACP synthase) or wherein nucleic acids encoding an endogenous KAS are disrupted by homologous recombination to produce said triglyceride oil; and

(ii) isolating said triglyceride oil from said recombinant oleaginous Prototheca cell,

wherein said triglyceride oil has a fatty acid profile that is at least 40% saturated fatty acids, and said fatty acid profile of said triglyceride oil comprises at least;

10% C8;

0, 20% C10;

0, 50% C12;

0, 40% C14;

0, 60% C16;

0, or 30% C18;

0.

19. The process of claim 18, wherein said exogenous nucleic acids encoding said KAS encode a KAS I enzyme, a KAS II enzyme, or a KAS IV enzyme.

20. The process of claim 18, wherein nucleic acids encoding an endogenous desaturase of said recombinant oleaginous Prototheca cell have been disrupted.

21. The process of claim 18, wherein said triglyceride oil has a fatty acid profile that is at least 50% saturated fatty acids.

22. The process of claim 18, wherein said triglyceride oil is a cocoa butter mimetic having percentages of C16:

0, C18;

0 and C18;

1 fatty acid constituents within 10% of the percentages, respectively, of cocoa butter.

23. The process of claim 18, wherein said recombinant oleaginous Prototheca cell is selected from Prototheca moriformis, Prototheca krugani, Prototheca stagnora, Prototheca wickerhamii, or Prototheca zopfii.

24. The process of claim 23, wherein said recombinant oleaginous Prototheca cell is Prototheca moriformis.

30. The process of claim 18, wherein said triglyceride oil has a fatty acid profile of at least 60% saturated fatty acids.

31. The process of claim 18, wherein said triglyceride oil has a fatty acid profile of at least 65% saturated fatty acids.

25. A process for producing a triglyceride oil from a Prototheca moriformis cell, the process comprising:

(i) cultivating a recombinant Prototheca moriformis cell comprising exogenous nucleic acids encoding a KAS (keto-acyl ACP synthase) or wherein nucleic acids encoding an endogenous KAS are disrupted by homologous recombination to produce said triglyceride oil; and

(ii) isolating said triglyceride oil from said Prototheca moriformis cell, wherein said triglyceride oil has a fatty acid profile that is at least 40% saturated fatty acids, and said fatty acid profile of said triglyceride oil comprises at least;

10% C8;

0, 20% C10;

0, 50% C12;

0, 40% C14;

0, 60% C16;

0, or 30% C18;

0.

26. The process of claim 25, wherein said exogenous nucleic acids encoding said KAS encode a KAS I enzyme, a KAS II enzyme, or a KAS IV enzyme.

27. The process of claim 25, wherein nucleic acids encoding an endogenous desaturase of said Prototheca moriformis cell have been disrupted.

28. The process of claim 25, wherein said triglyceride oil has a fatty acid profile that is at least 50% saturated fatty acids.

29. The process of claim 25, wherein said triglyceride oil is a cocoa butter mimetic having percentages of C16:

0, C18;

0 and C18;

1 fatty acid constituents within 10% of the percentages, respectively, of cocoa butter.

32. The process of claim 25, wherein said triglyceride oil has a fatty acid profile of at least 55% saturated fatty acids.

33. The process of claim 25, wherein said triglyceride oil has a fatty acid profile of at least 60% saturated fatty acids.

34. The process of claim 25, wherein said triglyceride oil has a fatty acid profile that is at least 65% saturated fatty acids.