Production of Oil in Microorganisms

• US 20100323414A1
• Filed: 04/30/2010
• Published: 12/23/2010
• Est. Priority Date: 06/01/2007
• Status: Abandoned Application

• Current Assignee: Terravia Holdings, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Franklin, Scott, Day, Anthony G., Dillon, Harrison F., Trimbur, Donald E., Im, Chung-Soon, Coragliotti, Anna
• Application Number:
  US12/772,164
• Publication Number:
  US 20100323414A1
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C10L 1/026 for compression ignition
• CPC Class Codes: C10L 1/06 for spark ignition
• CPC Class Codes: C10L 2200/0484 Vegetable or animal oils
• CPC Class Codes: C10L 2270/026 for diesel engines, e.g. automobiles, stationary, marine
• CPC Class Codes: C10L 2270/04 for turbines, planes, power generation
• 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 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 1/32 Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12N 9/2408 acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2431 Beta-fructofuranosidase (3.2.1.26), i.e. invertase
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12P 5/02 : acyclic C12P5/007 takes precedence
• CPC Class Codes: C12P 7/6409 : Fatty acids
• 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 7/649 : Biodiesel, i.e. fatty acid alkyl esters
• 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: Y02E 50/30 : Fuel from waste, e.g. synthetic alcohol or diesel
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts
• CPC Class Codes: Y02T 50/678 : Aviation using fuels of non-fossil origin

Abstract

The invention provides methods of producing lipids by oleaginous microorganisms. The invention also provides genetically engineered oleaginous microorganisms and methods of cultivation for lipid production. Also provided are oils, fuels, oleochemicals, chemical precursors, and other compounds manufactured such microorganisms. Exemplary oleaginous microorganisms include oleaginous fungi and such oleaginous fungi that have been genetically modified.

Claims

1. A fungus containing an exogenous gene encoding a polysaccharide degradation enzyme.

2. The fungus of claim 1 that is an oleaginous yeast.

3. The fungus of claim 1 that is a non-yeast fungus.

4. The fungus of claim 1, wherein the fungus contains a second exogenous gene encoding a protein selected from the group consisting of a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, a lipid modification enzyme, a hydrocarbon modification enzyme and a fructokinase.

5. The fungus of claim 1 that is selected from Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, Yarrowia lipolytica, a species of the genus Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, a species of the genus Hensenulo, a species of the genus Chaetomium, a species of the genus Cladosporium, a species of the genus Malbranchea, a species of the genus Rhizopus, and a species of the genus Pythium.

6. The fungus of claim 1 that contains at least 10% lipid by dry weight.

7. A fungi culture containing:

(a) a population of oleaginous fungi; and

(b) a culture medium comprising a carbon source selected from depolymerized cellulosic material, sorghum, sugar cane juice, sugar beet or molasses.

8. The fungi culture of claim 7, wherein the fungi are selected from Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, Yarrowia lipolytica, a species of the genus Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, a species of the genus Hensenulo, a species of the genus Chaetomium, a species of the genus Cladosporium, a species of the genus Malbranchea, a species of the genus Rhizopus, and a species of the genus Pythium.

9. The fungi culture of claim 7, wherein sucrose is provided by the carbon source.

10. The fungi culture of claim 7, wherein the oleaginous fungi are oleaginous yeast.

11. The fungi culture of claim 7, wherein the oleaginous fungi are non-yeast fungi.

12. The fungi culture of claim 7, wherein the culture medium is essentially free of carbon sources other than sucrose, glucose and fructose.

13. The fungi culture of claim 7, wherein the culture is axenic.

14. The fungi culture of claim 7, wherein the oleaginous fungi are at least 50% lipid by dry weight.

15. A method of cultivating fungi, comprising:

(a) providing a fungus;

(b) placing the fungus in culture medium comprising sucrose as a carbon source; and

(c) culturing the fungus under growth conditions in the presence of a sucrose invertase enzyme until the fungus accumulates at least 10% of its dry cell weight as lipid.

16. The method of claim 15, wherein the fungus is an oleaginous yeast.

17. The method of claim 16, wherein the oleaginous yeast is selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, Yarrowia lipolytica.

18. The method of claim 15, wherein the fungus is a non-yeast fungus.

19. The method of claim 18, wherein the non-yeast fungus is selected from the group consisting of a species of the genus Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, a species of the genus Hensenulo, a species of the genus Chaetomium, a species of the genus Cladosporium, a species of the genus Malbranchea, a species of the genus Rhizopus, and a species of the genus Pythium.

20. The method of claim 15, wherein the culture medium is axenic.

21. The method of claim 15, wherein the culture medium is essentially free of carbon sources other than sucrose, glucose and fructose.

22. The method of claim 15, wherein the sucrose is provided by a material selected from the group consisting of sorghum, sugar cane juice, sugar beet and molasses.

23. The method of claim 15, wherein the culture medium further comprises at least one additional carbon source selected from glucose, glycerol, xylose, fructose, arabinose, mannose, galactose, acetate, depolymerized cellulosic material, sugarcane bagasse, rice hulls, corn stover, wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels, hardwood thinnings, softwood thinnings, wood chips, sawdust, pulp mill waste, urban paper waste, grass clippings, switchgrass, hybrid poplar wood, miscanthus, fiber cane or fiber sorghum.

24. The method of claim 15, wherein the lipid comprises at least 50% C18:

1.

25. The method of claim 15, wherein the fungus is at least 50% lipid by dry weight.

26. The method of claim 15, where the culture medium comprises at least 1% sucrose.

27. The method of claim 15, wherein the culturing step occurs primarily in an absence of light.

28. The method of claim 15, wherein the fungus has a lipid profile comprising at least 3% C:

18;

0 and at least 8% C;

16;

0.

29. The method of claim 15, further comprising isolating the lipid from the fungus.

30. The method of claim 29, further comprising subjecting the lipid to a chemical reaction that changes the chemical structure of the lipid.

31. The method of claim 30, wherein the chemical reaction is selected from cracking, transesterification, hydrogenation, deoxygenation, and isomerization.

32. The method of claim 31, wherein the lipid is subjected to hydrogenation, deoxygenation and isomerization to produce a renewable diesel fuel that meets ASTM D975 specifications.

33. The method of claim 31, wherein the lipid is subjected to transesterification to produce a biodiesel fuel that meets ASTM D6751 specifications.

34. The method of claim 15, wherein the culturing is continued until the fungus accumulates at least 60% of its dry cell weight as lipid.

35. A method of cultivating fungi, comprising culturing a fungus (a) in a culture medium containing sucrose as a primary carbon source, (b) under heterotrophic growth and limited nitrogen conditions, and (c) until the fungus accumulates at least 10% of its dry cell weight as lipid, wherein the sucrose is provided to the culture medium in a feedstock selected from the group consisting of sorghum, sugar beets, molasses or sugar cane juice.

36. The method of claim 35, wherein the sucrose is provided to the culture medium as sugar cane juice.

37. The method of claim 35, wherein the fungus accumulates at least 40% of its dry cell weight as lipid .

38. The method of claim 35, wherein the culturing is continued until the fungus accumulates at least 60% of its dry cell weight as lipid.

39. The method of claim 36, wherein the culturing is continued until the fungus accumulates at least 60% of its dry cell weight as lipid.

40. The method of claim 37, wherein the culturing is continued until the fungus accumulates at least 60% of its dry cell weight as lipid.