Tailored oils produced from recombinant oleaginous microorganisms

• US 8,633,012 B2
• Filed: 10/11/2012
• Issued: 01/21/2014
• Est. Priority Date: 02/02/2011
• 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, Zhao, Xinhua, Bhat, Riyaz
• Primary Examiner(s): Fox, David T
• Assistant Examiner(s): KEOGH, MATTHEW R
• Application Number:
  US13/650,024
• Publication Number:
  US 20130034887A1
• Time in Patent Office: 467 Days
• Field of Search: None
• US Class Current: 435/257.2
• CPC Class Codes: C07C 69/52 Esters of acyclic unsaturated carboxylic acids having the esterified carboxyl group bound to an acyclic carbon atom
• CPC Class Codes: C10L 1/02 essentially based on components consisting of carbon, hydrogen, and oxygen only
• CPC Class Codes: C10L 2200/0476 Biodiesel, i.e. defined lower alkyl esters of fatty acids first generation biodiesel
• CPC Class Codes: C10L 2290/26 Composting, fermenting or anaerobic digestion fuel components or materials from which fuels are prepared
• CPC Class Codes: C10L 2290/30 Pressing, compressing or compacting
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• 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: C12P 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

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 acid hydroxylase, a desaturase enzyme, 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 method for producing an oil, the method comprising:

(a) cultivating a microalga of the genus Prototheca, the microalga comprising a gene knockout of an endogenous FATA acyl-ACP thioesterase gene and wherein the microalga produces an oil enriched in oleic acid due to the gene knockout; and

(b) obtaining the oil.

2. The method of claim 1, wherein the microalga is cultivated so as to comprise 50 to 90% triglyceride by dry cell weight.

3. The method of claim 1, wherein the oil produced is characterized by a fatty acid profile elevated in C18:

1 fatty acids and reduced in C16 fatty acids as a result of the gene knockout.

4. The method of claim 1, wherein the microalga is of the species Prototheca moriformis.

5. The method of claim 1, wherein the microalga expresses an exogenous sucrose invertase so as to be cultivatable on sucrose as a sole carbon source.

6. A method for producing an oil, the method comprising:

cultivating a microalga of the genus Prototheca, the microalga comprising a gene knockout of an endogenous FATA acyl-ACP thioesterase gene,

wherein the microalga produces an oil enriched in oleic acid due to the gene knockout and the microalga comprises 50 to 90% triglyceride by dry cell weight, and

wherein the oil produced is characterized by a fatty acid profile elevated in C18;

1 fatty acids and reduced in C16 fatty acids as a result of the gene knockout; and

obtaining the oil.

7. The method of claim 6, wherein the microalga further comprises recombinant nucleic acids encoding an active sucrose invertase so that the microalga can be cultivated on sucrose as a sole carbon source.

8. A method for producing an oil, the method comprising:

(a) cultivating a microalga of the genus Prototheca, the microalga comprising an RNAi suppression construct, which targets and decreases production of an acyl-ACP thioesterase encoded by a FATA acyl-ACP thioesterase gene, and wherein the microalga produces an oil enriched in oleic acid due to the RNAi suppression construct; and

(b) obtaining the oil.

9. The method of claim 8, wherein the microalga is cultivated so as to comprise 50 to 90% triglyceride by dry cell weight.

10. The method of claim 8, wherein expression of the RNAi suppression construct leads to formation of a hairpin RNA.

11. The method of claim 10, wherein the oil produced is characterized by a fatty acid profile elevated in C18:

1 fatty acids and reduced in C16 fatty acids as a result of the RNAi suppression construct.

12. The method of claim 8, wherein the microalga is of the species Prototheca moriformis.

13. The method of claim 8, wherein the microalga expresses an exogenous sucrose invertase so as to be cultivatable on sucrose as a sole carbon source.

14. A method for producing an oil, the method comprising:

(a) cultivating a microalga of the genus Prototheca, the microalga comprising a dsRNA suppression construct, which targets and decreases production of an acyl-ACP thioesterase encoded by a FATA acyl-ACP thioesterase gene, and wherein the microalga produces an oil enriched in oleic acid due to the dsRNA suppression construct; and

(b) obtaining the oil.

15. The method of claim 14, wherein the microalga is cultivated so as to comprise 50 to 90% triglyceride by dry cell weight.

16. The method of claim 14, wherein the oil produced is characterized by a fatty acid profile elevated in C18:

1 fatty acids and reduced in C16 fatty acids as a result of the dsRNA suppression construct.

17. The method of claim 14, wherein the microalga is of the species Prototheca moriformis.

18. The method of claim 14, wherein the microalga expresses an exogenous sucrose invertase so as to be cultivatable on sucrose as a sole carbon source.

19. A method for producing an oil, the method comprising:

(a) cultivating a microalga of the genus Prototheca, the microalga comprising an anti-sense RNA suppression construct, which targets and decreases production of an acyl-ACP thioesterase encoded by a FATA acyl-ACP thioesterase gene, and wherein the microalga produces an oil enriched in oleic acid due to the anti-sense RNA suppression construct; and

(b) obtaining the oil.

20. The method of claim 19, wherein the microalga is cultivated so as to comprise 50 to 90% triglyceride by dry cell weight.

21. The method of claim 19, wherein the oil produced is characterized by a fatty acid profile elevated in C18:

1 fatty acids and reduced in C16 fatty acids as a result of the anti-sense RNA suppression construct.

22. The method of claim 19, wherein the microalga is of the species Prototheca moriformis.

23. The method of claim 19, wherein the microalga expresses an exogenous sucrose invertase so as to be cultivatable on sucrose as a sole carbon source.

24. A method for producing an oil, the method comprising:

cultivating a microalga of the genus Prototheca, the microalga comprising an RNAi suppression construct, which targets and decreases production of an acyl-ACP thioesterase encoded by a FATA acyl-ACP thioesterase gene,

wherein the microalga produces an oil enriched in oleic acid due to the RNAi suppression construct and the microalga comprises 50 to 90% triglyceride by dry cell weight, and

wherein the oil produced is characterized by a fatty acid profile elevated in C18;

1 fatty acids and reduced in C16 fatty acids as a result of the RNAi suppression construct; and

obtaining the oil.

25. The method of claim 24, wherein the microalga further comprises recombinant nucleic acids encoding an active sucrose invertase so that the microalga can be cultivated on sucrose as a sole carbon source.

26. The method of claim 24, wherein expression of the RNAi suppression construct leads to formation of a hairpin RNA.

27. A method for producing an oil, the method comprising:

cultivating a microalga of the genus Prototheca, the microalga comprising a dsRNA suppression construct, which targets and decreases production of an acyl-ACP thioesterase encoded by a FATA acyl-ACP thioesterase gene,

wherein the microalga produces an oil enriched in oleic acid due to the dsRNA suppression construct and the microalga comprises 50 to 90% triglyceride by dry cell weight, and

wherein the oil produced is characterized by a fatty acid profile elevated in C18;

1 fatty acids and reduced in C16 fatty acids as a result of the dsRNA suppression construct; and

obtaining the oil.

28. The method of claim 27, wherein the microalga further comprises recombinant nucleic acids encoding an active sucrose invertase so that the microalga can be cultivated on sucrose as a sole carbon source.

29. A method for producing an oil, the method comprising:

cultivating a microalga of the genus Prototheca, the microalga comprising an anti-sense RNA suppression construct, which targets and decreases production of an acyl-ACP thioesterase encoded by a FATA acyl-ACP thioesterase gene,

wherein the microalga produces an oil enriched in oleic acid due to the anti-sense RNA suppression construct and the microalga comprises 50 to 90% triglyceride by dry cell weight, and

wherein the oil produced is characterized by a fatty acid profile elevated in C18;

1 fatty acids and reduced in C16 fatty acids as a result of the anti-sense RNA suppression construct; and

obtaining the oil.

30. The method of claim 29, wherein the microalga further comprises recombinant nucleic acids encoding an active sucrose invertase so that the microalga can be cultivated on sucrose as a sole carbon source.