Production of Oil in Microorganisms

• US 20110015417A1
• Filed: 04/30/2010
• Published: 01/20/2011
• Est. Priority Date: 06/01/2007
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Franklin, Scott, Day, Anthony G., Dillon, Harrison F., Trimbur, Donald E., Im, Chung-Soon, Coragliotti, Anna
• Granted Patent: US 8,512,999 B2
• Time in Patent Office: Days
• US Class Current: 554/127
• 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 cultivating oil-bearing microbes using xylose alone or in combination with other depolymerized cellulosic material. Also provided are microorganisms comprising an exogenous gene encoding a polysaccharide degrading enzyme, such as a cellulase, a hemicellulase, a pectinase, or a driselase. Some methods of microbial fermentation are provided that comprise the use of xylose and depolymerized cellulosic materials for the production of oil-bearing microorgansims.

Claims

1. A microalgae containing an exogenous gene encoding a polysaccharide degrading enzyme.

2. The microalgae of claim 1, wherein the enzyme is a cellulase, a hemicellulase, a pectinase, or a driselase.

3. The microalgae of claim 1, wherein the microalgae is selected from Table 1.

4. The microalgae of claim 1 that is an obligate heterotroph.

5. The microalgae of claim 1 that is a species of the genus Prototheca.

6. The microalgae of claim 5, wherein the microalgae is selected from the group consisting of Prototheca stagnora, Prototheca portoricensis, Prototheca wickerhamii, Prototheca moriformis, and Prototheca zopfii.

7. The microalgae of claim 1 that contains a second exogenous gene that encodes a a lipid modification enzyme.

8. The microalgae of claim 7, wherein the lipid modification enzyme is a thioesterase.

9. The microalgae of claim 8, wherein the thioesterase is selected from the group consisting of Umbellularia californica fatty acyl-ACP thioesterase (GenBank #AAC49001), Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank #Q39473), Umbellularia californica fatty acyl-ACP thioesterase (GenBank #Q41635), Myristica fragrans fatty acyl-ACP thioesterase (GenBank #AAB71729), Myristica fragrans fatty acyl-ACP thioesterase (GenBank #AAB71730), Elaeis guineensis fatty acyl-ACP thioesterase (GenBank #ABD83939), Elaeis guineensis fatty acyl-ACP thioesterase (GenBank #AAD42220), Populus tomentosa fatty acyl-ACP thioesterase (GenBank #ABC47311), Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank #NP_—

172327), Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank #CAA85387), Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank #CAA85388), Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank #Q9SQI3), Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank #CAA54060), Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank #AAC72882), Cuphea calophylla subsp. mesostemon fatty acyl-ACP thioesterase (GenBank #ABB71581), Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank #CAC19933), Elaeis guineensis fatty acyl-ACP thioesterase (GenBank #AAL15645), Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank #Q39513), Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank #AAD01982), Vitis vinifera fatty acyl-ACP thioesterase (GenBank #CAN81819), Garcinia mangostana fatty acyl-ACP thioesterase (GenBank #AAB51525), Brassica juncea fatty acyl-ACP thioesterase (GenBank #ABI18986), Madhuca longifolia fatty acyl-ACP thioesterase (GenBank #AAX51637), Brassica napus fatty acyl-ACP thioesterase (GenBank #ABH11710), Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase (GenBank #EAY86877), Oryza sativa (japonica cultivar-group) fatty acyl-ACP thioesterase (GenBank #NP_—

001068400), Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase (GenBank #EAY99617), and Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank #AAC49269).

10. The microalgae of claim 7, wherein the second exogenous gene encodes a sucrose invertase secreted into the extracellular space.

11. The microalgae of claim 7, wherein the second exogenous gene encodes a sucrose invertase targeted to the cytoplasm.

12. A microalgal culture containing:

(a) a population of microalgae containing at least 10% lipid; and

(b) a culture medium comprising xylose.

13. The microalgal culture of claim 12, wherein the microalgae is selected from Table 1.

14. The microalgal culture of claim 13, wherein the microalgae is selected from Table 17.

15. The microalgal culture of claim 12, wherein the microalgae are of the genus Prototheca.

16. The microalgal culture of claim 12, wherein the culture medium also contains depolymerized cellulosic material.

17. The microalgal culture of claim 16, wherein the depolymerized cellulosic material is selected from depolymerized sugarcane bagasse, rice hulls, corn fiber, 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, wood construction waste, switchgrass, hybrid poplar wood, miscanthus, fiber cane or fiber sorghum.

18. The microalgal culture of claim 15, wherein the microalgae is selected from the group consisting of Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamii, and Prototheca zopfii.

19. The microalgal culture of claim 12, wherein the culture medium is essentially free of carbon sources other than xylose.

20. The microalgal culture of claim 12, wherein the culture is axenic.

21. The microalgal culture of claim 12, wherein the microalgae are at least 50% lipid by dry weight.

22. A method of cultivating microalgae, comprising:

(a) providing a microalgae cell;

(b) placing the microalgae cell in culture medium comprising xylose as a carbon source; and

(c) culturing the microalgae cell under heterotrophic growth conditions in the presence of a polysaccharide degrading enzyme until the microalgae cell accumulates at least 10% of its dry cell weight as lipid.

23. The method of claim 22, wherein the microalgae cell is an obligate heterotroph.

24. The method of claim 22, wherein the microalgae cell is of the genus Prototheca.

25. The method of claim 24, wherein the microalgae cell is selected from the group consisting of Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamii, and Prototheca zopfii.

26. The method of claim 22, wherein the culture medium is axenic.

27. The method of claim 22, wherein the culture medium is essentially free of carbon sources other than xylose.

28. The method of claim 22, wherein the xylose is provided by a material selected from 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.

29. The method of claim 22, wherein the lipid comprises at least 50% C18:

1.

30. The method of claim 22, wherein the microalgae cell is at least 50% lipid by dry weight.

31. The method of claim 22, where the culture medium comprises at least 1% sucrose.

32. The method of claim 22, wherein the culturing step occurs primarily in an absence of light.

33. The method of claim 22, wherein the microalgae cell has a lipid profile comprising at least 3% C:

18;

0 and at least 8% C;

16;

0.

34. The method of claim 22, further comprising isolating the lipid from the microalgae cell.

35. The method of claim 34, further comprising subjecting the lipid to a chemical reaction that changes the chemical structure of the lipid.

36. The method of claim 35, wherein the chemical reaction is selected from cracking, transesterification, hydrogenation, deoxygenation, or isomerization.

37. The method of claim 36, wherein the lipid is subjected to hydrogenation, deoxygenation and isomerization to produce a renewable diesel fuel that meets ASTM D975 specifications.

38. The method of claim 36, wherein the lipid is subjected to transesterification to produce a biodiesel fuel that meets ASTM D6751 specifications.

39. The method of claim 22, wherein the culturing is continued until the microalgae cell accumulates at least 60% of its dry cell weight as lipid.

40. A method of cultivating microalgae, comprising culturing a recombinant microalgae cell of the genus Prototheca (a) in a culture medium containing xylose as a primary carbon source, (b) under heterotrophic growth and limited nitrogen conditions, and (c) until the microalgae cell accumulates at least 10% of its dry cell weight as lipid, wherein the xylose is provided to the culture medium in a feedstock selected sugarcane bagasse, rice hulls, corn fiber, 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, wood construction waste, switchgrass, hybrid poplar wood, miscanthus, fiber cane or fiber sorghum.

41. The method of claim 40, wherein the recombinant microalgae is cultured without selecting for a selectable marker other than xylose utilization.

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

43. The method of claim 41, wherein the culturing is continued until the microalgae cell accumulates at least 60% of its dry cell weight as lipid.